Biomedical subjects

L Orci

Publications and source records attributed to L Orci.

At least 73 records · Page 4Linked to original sources

PubMed1994

COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum.

In vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex, and Sec23p complex). Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP. However, GMP-PNP vesicles fail to target and fuse with the Golgi complex whereas GTP vesicles are functional. All the cytosolic proteins required for vesicle formation are retained on GMP-PNP vesicles, while Sar1p dissociates from GTP vesicles. Thin section electron microscopy of purified preparations reveals a uniform population of 60-65 nm vesicles with a 10 nm thick electron dense coat. The subunits of this novel coat complex are molecularly distinct from the constituents of the nonclathrin coatomer involved in intra-Golgi transport. Because the overall cycle of budding driven by these two types of coats appears mechanistically similar, we propose that the coat structures be called COPI and COPII.

Base Sequence↗

PubMed1994

Modulation of bovine microvascular endothelial cell proteolytic properties by inhibitors of angiogenesis.

A tightly controlled increase in extracellular proteolysis, restricted both in time and space, is an important component of the angiogenic process, while anti-proteolysis is effective in inhibiting angiogenesis. By focussing on the plasminogen activator (PA)-plasmin system, the objective of the present studies was to assess whether previously described inhibitors of angiogenesis modify bovine microvascular endothelial cell proteolytic properties. We demonstrate that although synthetic angiostatic steroids (U-24067 and U-42129), heparin, suramin, interferon alpha-2a, and retinoic acid are all inhibitors of in vitro angiogenesis, each of these agents has distinct effects on the plasminogen-dependent proteolytic system. Specifically, angiostatic steroids and interferon alpha-2a reduce urokinase-type PA (u-PA) and PA inhibitor-1 activity, while heparin and retinoic acid increase u-PA activity. Suramin reduces cell-associated u-PA activity and greatly increases PAI-1 production at doses which induce monolayer disruption. These findings demonstrate that a spectrum of alterations in extracellular proteolysis is associated with anti-angiogenesis, and that anti-angiogenesis and anti-proteolysis are not necessarily correlated. A reduction in extracellular proteolysis would be expected to reduce invasion, whereas an increase in proteolysis might modulate the activity of inhibitory cytokines, which in turn could reduce endothelial cell proliferation and migration and inhibit angiogenesis. The spectrum of effects on different elements of the PA system observed in response to the agents assessed suggests that the role of modulations in extracellular proteolytic activity in anti-angiogenesis is likely to be varied and complex.

Adrenal Cortex↗

PubMed1994

In vitro angiogenic and proteolytic properties of bovine lymphatic endothelial cells.

The aim of the present study was to determine whether angiogenic cytokines, which induce neovascularization in the blood vascular system, might also be operative in the lymphatic system. In an assay of spontaneous in vitro angiogenesis, endothelial cells isolated from bovine lymphatic vessels retained their histotypic morphogenetic properties by forming capillary-like tubes. In a second assay, in which endothelial cells could be induced to invade a three-dimensional collagen gel within which they formed tube-like structures, lymphatic endothelial cells responded to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in a manner similar to what has previously been observed with endothelial cells derived from the blood vascular system. Finally, since angiogenesis is believed to require extracellular proteolytic activity, we investigated the effects of bFGF and VEGF on lymphatic endothelial cell proteolytic properties by focussing on the plasminogen activator (PA) system. bFGF and VEGF increased urokinase, urokinase receptor, and tissue-type PA expression. This was accompanied by an increase in PA inhibitor-1, which is thought to play an important permissive role in angiogenesis by protecting the extracellular matrix against excessive proteolytic degradation. Taken together, these results demonstrate that with respect to in vitro morphogenetic and proteolytic properties, lymphatic endothelial cells respond to the previously described angiogenic factors, bFGF and VEGF, in a manner very similar to what has been described for endothelial cells derived from the blood vascular system.

Animals↗

PubMed1994

A CD8+ T-lymphocyte-mediated and CD4+ T-lymphocyte-independent autoimmune diabetes of early onset in transgenic mice.

While transgenic mice expressing tumour necrosis factor-alpha under the control of the beta-cell-specific insulin promoter display a marked lymphocytic infiltration of the islets, they never develop insulin-dependent diabetes mellitus (IDDM). In striking contrast, "double" transgenic mice whose beta cells express both tumour necrosis factor-alpha as well as the co-stimulatory B7-1 molecule all develop IDDM at an early age. Furthermore, administration of anti-CD8 but not anti-CD4 immunoglobulins prevents diabetes onset. These results indicate that while tumour necrosis factor-alpha induced lymphocytic infiltration is not sufficient to effect beta-cell destruction, locally co-stimulated islet-infiltrating CD8+ T lymphocytes could play a critical role in the development of IDDM.

Animals↗

PubMed1994

ADP-ribosylation factor and coatomer couple fusion to vesicle budding.

The coat proteins required for budding COP-coated vesicles from Golgi membranes, coatomer and ADP-ribosylation factor (ARF) protein, are shown to be required to reconstitute the orderly process of transport between Golgi cisternae in which fusion of transport vesicles begins only after budding ends. When either coat protein is omitted, fusion is uncoupled from budding-donor and acceptor compartments pair directly without an intervening vesicle. Coupling may therefore results from the sequestration of fusogenic membrane proteins into assembling coated vesicles that are only exposed when the coat is removed after budding is complete. This mechanism of coupling explains the phenomenon of "retrograde transport" triggered by uncouplers such as the drug brefeldin A.

ADP-Ribosylation Factors↗

PubMed1994

En bloc incorporation of coatomer subunits during the assembly of COP-coated vesicles.

The cDNA encoding epsilon-COP, the 36-kD subunit of coatomer, was cloned from a bovine liver cDNA library and sequenced. Immunoblotting with an anti-epsilon-COP antibody showed that epsilon-COP exists in COP-coated vesicles as well as in the cytosolic coatomer. Using the cloned cDNA, recombinant His6- tagged epsilon-COP was overexpressed in cultured Chinese hamster ovary (CHO) cells, from which metabolically radiolabeled coatomer was purified by taking advantage of the His6 tag. Radiolabeled coatomer was employed to establish that all the subunits of the coatomer enter coated vesicles as an intact unit.

Amino Acid Sequence↗

PubMed1994

Sar1 promotes vesicle budding from the endoplasmic reticulum but not Golgi compartments.

Two new members (Sar1a and Sar1b) of the SAR1 gene family have been identified in mammalian cells. Using immunoelectron microscopy, Sar1 was found to be restricted to the transitional region where the protein was enriched 20-40-fold in vesicular carriers mediating ER to Golgi traffic. Biochemical analysis revealed that Sar1 was essential for an early step in vesicle budding. A Sar1-specific antibody potently inhibited export of vesicular stomatitis virus glycoprotein (VSV-G) from the ER in vitro. Consistent with the role of guanine nucleotide exchange in Sar1 function, a trans-dominant mutant (Sar1a[T39N]) with a preferential affinity for GDP also strongly inhibited vesicle budding from the ER. In contrast, Sar1 was not found to be required for the transport of VSV-G between sequential Golgi compartments, suggesting that components active in formation of vesicular carriers mediating ER to Golgi traffic may differ, at least in part, from those involved in intra-Golgi transport. The requirement for novel components at different stages of the secretory pathway may reflect the recently recognized differences in protein transport between the Golgi stacks as opposed to the selective sorting and concentration of protein during export from the ER.

Amino Acid Sequence↗

PubMed1994

pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle.

By quantitative immunoelectron microscopy and HPLC, we have studied the effect of disrupting pH gradients, by ammonium chloride, on proinsulin conversion in the insulin-producing B-cells of the islets of langerhans. Proinsulin content and pH in single secretory vesicles were measured on consecutive serial sections immunostained alternately with anti-proinsulin or anti-dinitrophenol (to reveal the pH-sensitive probe DAMP) antibodies. Radioactivity labeled proinsulin, proinsulin cleavage intermediates, and insulin were quantitated by HPLC analysis of extracts of islets treated in the same conditions. Cleavage at the C-peptide/A-chain junction is significantly less sensitive to pH gradient disruption than that of the B-chain/C-peptide junction, but the range of pH and proinsulin content in individual vesicles indicate that both cleavages occur in the same vesicle released from the TGN.

Ammonium Chloride↗

PubMed1994

TGF-beta 1 influences the relative development of the exocrine and endocrine pancreas in vitro.

Pancreatic rudiments from E12.5 mouse embryos undergo extensive development and differentiation when cultured in three-dimensional gels of extracellular matrix proteins for up to 12 days. Whereas collagen gels promote the formation of numerous exocrine acini and relatively small clusters of endocrine cells, in basement membrane (EHS) matrices the development of endocrine cells is dramatically favoured over that of acinar tissue. Buds embedded in a collagen gel contiguous to an EHS gel also fail to develop acini, suggesting the involvement of diffusible factor(s). Addition of cytokines to cultures of pancreatic buds in collagen gels modifies the relative proportions of the epithelial components of the gland. In the presence of EGF the proportion of the tissue occupied by ducts overrides that of acinar structures, whereas the endocrine portion of the tissue is not significantly modified. TGF-beta 1 partially mimicks the effect of EHS matrix in inhibiting the development of acinar tissue without decreasing the amount of ducts and mesenchyme; TGF-beta 1 also promotes the development of endocrine cells, in particular of insulin-containing beta cells and of cells expressing genes of the PP-fold family. These results show that cytokines can modulate the development of the pancreas and suggest a role for TGF-beta 1 in regulating the balance between the acinar and endocrine portions of the gland in vivo. More generally, they are compatible with the notion that, during organogenesis, cytokines act as paracrine factors responsible for the development and maintenance of appropriate proportions of different tissue constituents.

Animals↗

PubMed1994

Expression of neural cell adhesion molecule (N-CAM) in rat islets and its role in islet cell type segregation.

Endocrine cell types are non-randomly distributed within pancreatic islets of Langerhans. In the rat, insulin-secreting B-cells occupy the core of the islets and are surrounded by A-, D- and PP-cells, secreting glucagon, somatostatin and pancreatic polypeptide, respectively. Furthermore, dissociated islet cells have the ability in vitro to form aggregates with the same cell-type organization as native islets (pseudoislets). These observations suggest that a differential expression of cell adhesion molecules (CAMs) might characterize B- and non-B-cells (A-, D- and PP-cells), and be in part responsible for the establishment and maintenance of islet architecture. Indirect immunofluorescence using antibodies against CAMs and islet hormones was performed on serial sections of the splenic and duodenal parts of the rat pancreas. Staining for the Ca(2+)-dependent CAM E-cadherin was detected on both exocrine and endocrine tissue and was uniform over the entire islet section, in both pancreatic regions. By contrast, staining for the Ca(2+)-independent neural CAM (N-CAM) was restricted to endocrine tissue and nerve endings. Furthermore, N-CAM staining of endocrine cells was stronger in the islet periphery, a region composed mostly of non-B-cells. Serial sections demonstrate that cells staining strongly for N-CAM in the splenic part correspond to glucagon cells and in the duodenal part to pancreatic polypeptide cells. Within pseudoislets in vitro a stronger staining for N-CAM was also observed on peripheral cells, corresponding to non-B-cells.

Animals↗

PubMed1993

Stepwise assembly of functionally active transport vesicles.

Budding of COP-coated vesicles (the likely carriers of newly synthesized proteins from the endoplasmic reticulum through the Golgi stack) from Golgi cisternae requires ADP-ribosylation factor (ARF), coatomer proteins from the cytosol, GTP, and fatty acyl-coenzyme A (CoA). The assembly of coated buds on the membranes requires coatomer, ARF, and GTP. When palmitoyl-CoA is added, membrane fission occurs at the coated bud, releasing coated vesicles. We show that COP-coated vesicles can be generated stepwise in vitro and isolated in a functionally active form, demonstrating that the minimal set of cytosolic components required for their formation as well as principal steps in their assembly have been identified.

ADP-Ribosylation Factors↗

PubMed1993

"BFA bodies": a subcompartment of the endoplasmic reticulum.

A specialized region of the endoplasmic reticulum--the BFA body--is defined by the site of accumulation of coatomer when nonclathrin coat protein (COP)-coated vesicle assembly is prevented by the drug brefeldin A (BFA). BFA bodies are formed by part smooth, part rough domains of endoplasmic reticulum that are cis to the classical transitional endoplasmic reticulum and to BFA-induced Golgi remnants.

Animals↗

PubMed1993

Binding of coatomer to Golgi membranes requires ADP-ribosylation factor.

Coatomer, a complex of seven proteins, appears to be the precursor of the coat structure of non-clathrin-coated Golgi-derived vesicles. Another component of this vesicle coat is the cytosolic protein ADP-ribosylation factor (ARF). Like coatomer, ARF appears to reversibly associate with Golgi membranes. We now report that ARF is required for coatomer binding to Golgi membranes and that myristoylated, but not non-myristoylated, ARF is the required species. We utilize an antibody directed against the beta-subunit of coatomer (beta-COP) to follow coatomer binding. ARF and beta-COP bind stoichiometrically to Golgi membranes. ARF-dependent beta-COP binding requires a membrane-associated protein, is saturable, and is enhanced in the presence of stable GTP analogues like guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). ARF and beta-COP bind sequentially to Golgi membranes, since beta-COP can be bound to reisolated membranes that had been previously incubated with ARF and GTP gamma S. We conclude that membrane-bound ARF confers to Golgi membranes all of the requirements for specific beta-COP binding.

ADP-Ribosylation Factors↗

PubMed1993

Budding from Golgi membranes requires the coatomer complex of non-clathrin coat proteins.

Do the coats on vesicles budded from the Golgi apparatus actually cause the budding, or do they simply coat buds (Fig. 1)? One view (the membrane-mediated budding hypothesis) is that budding is an intrinsic property of Golgi membranes not requiring extrinsic coat proteins. Assembly of coats from dispersed subunits is super-imposed upon the intrinsic budding process and is proposed to convert the tips of tubules into vesicles. The alternative view (the coat-mediated budding hypothesis) is that coat formation provides the essential driving force for budding. The membrane-mediated budding hypothesis was inspired by the microtubule-dependent extension of apparently uncoated, 90-nm-diameter membrane tubules from the Golgi apparatus and other organelles in vivo after treatment with brefeldin A, a drug that inhibits the assembly of coat proteins onto Golgi membranes. This hypothesis predicts that tubules will be extended when coat proteins are unavailable to convert tubule-derived membrane into vesicles. Here we use a cell-free system in which coated vesicles are formed from Golgi cisternae to show that, on the contrary, when budding diminishes as a result of immunodepletion of coat protein pools, tubules are not formed at the expense of vesicles. We conclude that coat proteins are required for budding from Golgi membranes.

ADP-Ribosylation Factors↗

PubMed1993

beta'-COP, a novel subunit of coatomer.

Several lines of evidence favour the hypothesis that intracellular biosynthetic protein transport in eukaryotes is mediated by non-clathrin-coated vesicles (for a review see Rothman and Orci, 1992). The vesicles have been isolated and a set of their surface proteins has been characterized as coat proteins (COPs). These COPs exist in the cytosol as a preformed complex, the coatomer, which was prior to this study known to contain six subunits: four (alpha-, beta-, gamma- and delta-COP) with molecular weights between 160 and 58 kDa, and two additional proteins of approximately 36 and 20 kDa, epsilon- and xi-COP. Here we describe a novel subunit of the coatomer complex, beta'-COP. This subunit occurs in amounts stoichiometric to the established COPs both in the coatomer and in nonclathrin-coated vesicles and shows homology to the beta-subunits of trimeric G proteins.

Amino Acid Sequence↗

PubMed1993

Biphasic effect of transforming growth factor-beta 1 on in vitro angiogenesis.

Although the existence of an increasing number of angiogenesis-regulating cytokines is well documented, the response elicited by combinations of these cytokines is largely unknown. Using an in vitro model in which microvascular endothelial cells can be induced to form capillary-like tubes within three-dimensional collagen or fibrin gels, we have investigated the effect of transforming growth factor-beta 1 (TGF-beta 1) on basic fibroblast growth factor (bFGF)-induced and vascular endothelial growth factor (VEGF)-induced angiogenesis. Endothelial cell invasion and capillary lumen formation were inhibited by TGF-beta 1 at relatively high concentrations (5-10 ng/ml), while lower concentrations (100 pg/ml-1 ng/ml) of TGF-beta 1 potentiated the effect of bFGF- and VEGF-induced invasion. The optimal potentiating effect was observed at 200-500 pg/ml TGF-beta 1. At invasion-potentiating doses of TGF-beta 1, lumen size in fibrin gels was markedly reduced compared to that in cultures treated with bFGF alone. These results show that TGF-beta 1 exerts a biphasic effect on bFGF- and VEGF-induced angiogenesis in vitro. Our studies support the notion that the nature of the angiogenic response elicited by a specific cytokine is contextual, i.e., depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell.

Adrenal Cortex↗

PubMed1993

Upregulation of urokinase receptor expression on migrating endothelial cells.

One of the phenotypic hallmarks of migrating endothelial cells, both in vivo and in vitro, is expression of the urokinase-type plasminogen activator (u-PA), a key mediator of extracellular proteolysis. In the study reported here, we have used an in vitro model of endothelial cell migration to explore the mechanism of this phenomenon. We have found that wounding of an endothelial cell monolayer triggers a marked, rapid and sustained increase in expression of a specific high-affinity receptor for u-PA (u-PAr) on the surface of migrating cells. Migrating cells displayed an increase in the levels of u-PA and u-PAr mRNAs, and this increase was mediated by endogenous basic fibroblast growth factor (bFGF). We also show that the increase in u-PA activity on migrating cells can be accounted for by an increase in receptor-bound u-PA, and that the increase in activity is also dependent on endogenous bFGF. These results demonstrate that the expression of plasmin-mediated proteolytic activity by migrating endothelial cells is a consequence of increased production of both u-PA and its receptor, and that this in turn is mediated by endogenous bFGF. This suggests that u-PA, produced at increased levels by migrating cells, binds to u-PAr whose expression is upregulated on the same cells. These observations are in accord with the postulated role of u-PAr in mediating efficient and spatially restricted extracellular proteolysis, particularly in the context of cell migration.

Animals↗

PubMed1993

Two steps of insulin receptor internalization depend on different domains of the beta-subunit.

The internalization of signaling receptors such as the insulin receptor is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin receptor and to establish which receptor domains are implicated in each of these by the use of receptors possessing in vitro mutations. We find that kinase activation and autophosphorylation of all three regulatory tyrosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the COOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the receptor from microvilli where initial binding occurs to the nonvillous domain of the cell. Early intracellular steps in insulin signal transduction involving the activation of phosphatidylinositol 3'-kinase are not required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin-coated pits. In contrast to the first ligand specific step, this step is common to many receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but requires a juxta-membrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent mechanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit.

Amino Acid Sequence↗