Biomedical subjects

L Orci

Publications and source records attributed to L Orci.

At least 109 records · Page 6Linked to original sources

PubMed1991

ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: a novel role for a GTP-binding protein.

ADP-ribosylation factor (ARF) is an abundant and highly conserved low molecular weight GTP-binding protein that was originally identified as a key element required for the action of cholera toxin in mammalian cells, but whose physiological role is unknown. We report that ARF family proteins are highly concentrated in non-clathrin-coated transport vesicles and are coat proteins. About three copies of ARF are present on the outside of coated vesicles per alpha-COP (and thus per coatomer). ARF is highly enriched in coated vesicles as compared with parental Golgi cisternae, as shown both by biochemical and morphological methods, and ARF is removed from transport vesicles through uncoating during transport. Furthermore, ARF binds to Golgi cisternae in a GTP-dependent manner independently of coated vesicle budding. These observations strongly suggest a new role for GTP-binding proteins: ARF proteins may modulate vesicle budding and uncoating through controlled GTP hydrolysis.

ADP-Ribosylation Factors↗

PubMed1991

Mammalian Sec23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm.

The yeast Sec23 protein is required in vivo and in vitro for transport of proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Ultrastructural localization of the Sec23p mammalian homologue (detected by antibody cross-reaction) in exocrine and endocrine pancreatic cells shows a specific distribution to the cytoplasmic zone between the transitional ER cisternae and Golgi apparatus where it appears associated with the tubular protuberances of the transitional ER cisternae, as well as with a population of vesicles, and surrounding cytoplasm. When ER-Golgi transport is interrupted with an energy poison, protuberances and transfer vesicles markedly decrease but Sec23p immunoreactive sites remain in the transitional cytoplasm not apparently tethered by membrane attachment. This unanticipated degree of organization suggests that cytosolic proteins, such as Sec23p, may be retained in specialized areas of the cytoplasm. A structure within the transitional zone may organize the flux of transport vesicles and Sec proteins so as to ensure efficient protein traffic in this limb of the secretory pathway.

Animals↗

PubMed1991

Induction of epithelial tubular morphogenesis in vitro by fibroblast-derived soluble factors.

We have designed an in vitro system in which Madin-Darby canine kidney (MDCK) epithelial cells are cocultured in collagen gels with fibroblasts under conditions precluding heterocellular contact. Using this experimental approach, we have obtained evidence that fibroblast-derived soluble factors play a crucial role in the control of epithelial morphogenesis. First, MDCK cells suspended alone in collagen gels form spherical cysts, whereas in the presence of fibroblasts they form branching tubules. Second, MDCK cells grown as a monolayer on fibroblast-containing collagen gels invade the underlying matrix, within which they form a network of tubules. Third, fibroblast-conditioned medium mimics the effects of coculture by eliciting tubulogenesis by MDCK cells. These results demonstrate the involvement of diffusible paracrine factors in morphogenetic epithelial-mesenchymal interactions and provide a strategy for their molecular characterization.

Animals↗

PubMed1991

Plasminogen activator inhibitor-1 is induced in microvascular endothelial cells by a chondrocyte-derived transforming growth factor-beta.

We have previously demonstrated that a chondrocyte-derived TGF-beta inhibits spontaneous endothelial sprout formation in an in vitro model of angiogenesis (Pepper et al., J.Cell.Physiol. 146:170-179, 1991). We suggested that the inhibition might be mediated by an antiproteolytic effect. In this paper, we describe the induction of PAI-1 and PAI-1 mRNA in microvascular endothelial cells by chondrocyte conditioned medium. This effect can be significantly reduced by the addition of anti-TGF-beta antibodies to the conditioned medium, and can be mimicked by the addition of exogenous TGF-beta 1. Taken together with our previous observations, these results demonstrate that the inhibition of endothelial sprout formation occurs concomitantly with an increase in the production of PAI-1, a physiological plasminogen activator inhibitor. This suggests that TGF-beta-induced antiproteolysis is responsible for the inhibition of sprout formation.

Animals↗

PubMed1991

Brefeldin A, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on Golgi cisternae.

We report that brefeldin A prevents the assembly of non-clathrin-coated vesicles from Golgi cisternae in a cell-free system. This finding provides a simple molecular explanation for the primary effect of this remarkable compound in blocking constitutive secretion. We further report that when coated vesicle assembly is blocked, extensive tubule networks form that connect previously separate cisternae and stacks into a single topological unit, allowing the intermixing of contents of Golgi cisternae, presumably by lateral diffusion. Formation of the tubule networks requires ATP, cytosol, and the general fusion protein NSF. Tubule networks may be related to the membrane tubules mediating retrograde transport in vivo.

Animals↗

PubMed1991

A coat subunit of Golgi-derived non-clathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein beta-adaptin.

Four high-molecular-weight proteins form the main subunits of the coat of Golgi-derived (non-clathrin) coated vesicles. One of these coat proteins, beta-COP, is identical to a Golgi-associated protein of relative mass 110,000 (110K) that shares homology with the adaptin proteins of clathrin-coated vesicles. This connection, and the comparable molecular weights of the coat proteins of Golgi-derived and clathrin-coated vesicles, indicates that they may be structurally related. The identification of beta-COP as the 110K protein explains the blocking of secretion by the drug brefeldin A.

Adaptor Protein Complex beta Subunits↗

PubMed1991

Chondrocytes inhibit endothelial sprout formation in vitro: evidence for involvement of a transforming growth factor-beta.

Using a quantitative in vitro model of spontaneous endothelial sprout formation, we have attempted to define physiological inhibitors of angiogenesis from hyaline cartilage, a tissue whose antiangiogenic properties have been well described. The model consists of embedding bovine microvascular endothelial cell aggregates into fibrin or collagen gels, which results in the formation of radially growing sprouts. When chondrocytes derived from the permanent cartilagenous region of the chick embryo sternum are cocultured with the endothelial cell aggregates, sprout formation is markedly inhibited. Addition of anti-TGF-beta antibodies to the cocultures significantly reduced the inhibitory effect of chondrocytes on sprout formation. Chondrocyte-conditioned medium or exogenously added TGF-beta 1 have a similar albeit transient inhibitory effect. Depletion of TGF-beta from chondrocyte conditioned medium with anti-TGF-beta antibodies and solid-phase protein-A significantly decreases the inhibition of sprout formation. These results demonstrate that a chondrocyte-derived TGF-beta-like molecule inhibits capillary sprout formation in vitro and suggest that the antiangiogenic properties of cartilage may at least in part, be mediated by TGF-beta.

Animals↗

PubMed1991

Intracellular membrane fusion.

Protein trafficking and membrane assembly are accomplished in eukaryotes by the specific targeting and fusion of vesicles. In this review we describe some of the molecules implicated as components of the fusion apparatus, and evidence that suggests the same factors are recruited for a variety of intracellular fusion events.

Carrier Proteins↗

PubMed1991

Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers.

Human proinsulin and insulin oligomerize to form dimers and hexamers. It has been suggested that the ability of prohormones to self associate and form aggregates may be responsible for the sorting process at the trans-Golgi. To examine whether insulin oligomerization is required for proper sorting into regulated storage granules, we have constructed point mutations in human insulin B chain that have been previously shown to prevent formation of insulin hexamers (Brange, J., U. Ribel, J. F. Hansen, G. Dodson, M. T. Hansen, S. Havelund, S. G. Melberg, F. Norris, K. Norris, L. Snel, A. R. Sorensen, and H. O. Voight. 1988. Nature [Lond.]. 333:679-682). One mutant (B10His----Asp) allows formation of dimers but not hexamers and the other (B9Ser----Asp) prevents formation of both dimers and hexamers. The mutants were transfected into the mouse pituitary AtT-20 cells, and their ability to be sorted into regulated secretory granules was compared to wild-type insulin. We found that while B10His----Asp is sorted somewhat less efficiently than wild-type insulin as reported previously (Carroll, R. J., R. E. Hammer, S. J. Chan, H. H. Swift, A. H. Rubenstein, and D. F. Steiner. 1988. Proc. Natl. Acad. Sci. USA. 85:8943-8947; Gross, D. J., P. A. Halban, C. R. Kahn, G. C. Weir, and L. Villa-Kumaroff. 1989. Proc. Natl. Acad. Sci. USA. 86:4107-4111). B9Ser----Asp is targeted to granules as efficiently as wild-type insulin. These results indicate that self association of proinsulin into hexamers is not required for its targeting to the regulated secretory pathway.

Amino Acid Sequence↗

PubMed1991

Extent and modulation of junctional communication between pancreatic acinar cells in vivo.

To assess whether junctional communication may be of physiological relevance in the control of exocrine pancreas secretion, we have studied acinar cell coupling by microinjecting Lucifer Yellow CH in the intact pancreas of anesthetized rats. Reconstructions from serial sections showed that, under control conditions, pancreatic cells are extensively coupled within each acinus but do not communicate with centroacinar cells, duct cells, and cells of neighboring acini. Intravenous infusion of acetylcholine and caerulein, or electrical stimulation of the vagus nerve, increased pancreatic secretion (P less than 0.02-0.001). Under these stimulatory conditions, the extent of acinar cell communication was decreased (P less than 0.001) by 40%. The acetylcholine-induced uncoupling was prevented by treating rats with atropine. Thus, in the intact pancreas, acinar cells intercommunicate extensively within each acinus under resting conditions and reduce their coupling during stimulation. These data support the view that modulation of cell coupling is a physiologically relevant mechanism for the regulation of exocrine pancreas secretion in vivo.

Acetylcholine↗

PubMed1991

Molecular heterogeneity and cellular localization of carboxypeptidase H in the islets of Langerhans.

The intracellular distribution and molecular heterogeneity of carboxypeptidase H was studied in rat insulinoma tissue and isolated islets of Langerhans by a combination of immunohistochemical, ultrastructural, subcellular fractionation, and immunoblotting analyses. Immunofluorescence microscopy of islets demonstrated the presence of carboxypeptidase H in both insulin-containing B cells and glucagon-containing A cells. Quantitative ultrastructural analyses of islet B cells indicated that the enzyme was concentrated in mature insulin secretory granules, clathrin-coated condensing granules, and to a lesser extent the Golgi apparatus. Carboxypeptidase H activity was localized principally to secretory granule subfractions of insulinoma tissue, where it was present for the major part (70%) as a form which is readily solubilizable at pH values prevailing in the granule interior (5.5). This species migrated as a diffuse band of 53-57 kilodaltons (kDa) on immunoblot analysis using antisera raised against the purified native enzyme. In contrast, the insoluble form which was associated with the granule membrane at pH 5.5, migrated as a relatively compact band of 55-57 kDa. Carboxypeptidase H activity was also present in subcellular fractions which contained Golgi membranes together with elements of the endoplasmic reticulum, and in a low density secretory granule fraction which may represent immature granules. The enzyme in these compartments, like the granule membrane species, migrated as a compact 55-57 kDa band on immunoblots. Two-dimensional electrophoretic immunoblot analysis of secretory granules suggested that both membrane and soluble forms of the enzyme were glycoproteins and that the terminal glycosylation was similar in both instances. Antiserum raised against the deduced C-terminal 11 amino acids of the cloned carboxypeptidase H sequence recognized the 55-57 kDa membrane component in granules but did not react with the 53-57 kDa soluble species. A major difference between the soluble and membrane forms therefore appears to be a structural modification or proteolytic removal of the C-terminal domain in the trans-Golgi or early secretory granule compartment. The concept that proteolysis is involved is further supported by the observation that the relative proportion of the high and low mol wt forms of the enzyme in different subcellular fractions correlated with that of proinsulin and insulin, respectively. The membrane association of the 55-57 kDa form of carboxypeptidase H is disrupted at pH values of 9 and is dependent on ionic strength. This further suggests that the C-terminus of the protein may have an important role in the sorting or concentration of the enzyme in vesicular elements of the regulated pathway of secretion.

Animals↗

PubMed1991

Embryogenesis of the murine endocrine pancreas; early expression of pancreatic polypeptide gene.

By immunofluorescence on cytospin preparations and on semithin sections of mouse pancreatic buds, we have found glucagon and pancreatic polypeptide (PP)-containing cells at embryonal day 10.5 (E 10.5) in dorsal buds and at E 11.5 in ventral buds. Insulin-containing cells appear in dorsal buds at E 11.5, and one to two days later in ventral buds. Somatostatin-containing cells are detectable from E 13.5 in both dorsal and ventral buds. A quantitative analysis shows that up to E 15.5, PP-containing cells are relatively abundant in both buds. By PCR amplification of oligo(dT)-primed cDNAs prepared from total pancreatic RNA, we also detect PP mRNA from E 10.5 onwards, thus confirming the early expression of the PP gene in the developing mouse pancreas. Analysis of endocrine cells in situ suggests three major patterns of cell distribution in embryonic pancreas. First, individual hormone-containing cells are located within the epithelium of pancreatic ducts. In both dorsal and ventral buds, the majority of these endocrine cells contain PP, but many also contain glucagon, insulin or somatostatin. Secondly, clusters of endocrine cells are found in the pancreatic interstitium. Many of these cells contain both glucagon and PP which, by immunogold labelling of consecutive thin sections, can be shown to co-exist within individual secretory granules. Finally, starting on E 18.5, typical islets are formed with centrally located B cells and with the adult 'one cell-one hormone' phenotype. These results suggest an intriguing ontogenic relationship between A- and PP-cells, and also indicate that PP-containing cells may occupy a hitherto unexpected place in the lineage of endocrine islet cells.

Animals↗

PubMed1990

Underexpression of beta cell high Km glucose transporters in noninsulin-dependent diabetes.

The role of defective glucose transport in the pathogenesis of noninsulin-dependent diabetes (NIDDM) was examined in Zucker diabetic fatty rats, a model of NIDDM. As in human NIDDM, insulin secretion was unresponsive to 20 mM glucose. Uptake of 3-O-methylglucose by islet cells was less than 19% of controls. The beta cell glucose transporter (GLUT-2) immunoreactivity and amount of GLUT-2 messenger RNA were profoundly reduced. Whenever fewer than 60% of beta cells were GLUT-2-positive, the response to glucose was absent and hyperglycemia exceeded 11 mM plasma glucose. We conclude that in NIDDM underexpression of GLUT-2 messenger RNA lowers high Km glucose transport in beta cells, and thereby impairs glucose-stimulated insulin secretion and prevents correction of hyperglycemia.

3-O-Methylglucose↗

PubMed1990

Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene.

Expression of the polyoma virus middle T (mT) oncogene in vivo is associated with a profound subversion of normal vascular development, which results in the formation of endothelial tumors (hemangiomas). In an attempt to understand the molecular mechanisms responsible for this phenomenon, we have investigated, in an in vitro system, the morphogenetic properties of endothelial cells expressing this oncogene. mT-expressing endothelioma (End) cells grown within fibrin gels formed large hemangioma-like cystic structures. All End cell lines examined expressed high levels of fibrinolytic activity resulting from increased production of urokinase-type plasminogen activator and decreased production of plasminogen activator inhibitors. Neutralization of excess proteolytic activity by exogenously added serine protease inhibitors corrected the aberrant in vitro behavior of End cells and allowed the formation of capillary-like tubules. These results suggest that tightly controlled proteolytic activity is essential for vascular morphogenesis and that physiological protease inhibitors play an important regulatory role in angiogenesis.

Animals↗

PubMed1990

Compensatory capabilities of islets of BB/Wor rats exposed to sustained hyperglycemia.

To determine if discordance for autoimmune diabetes in genetically homogeneous animals might reflect differences in the compensatory capacity of their beta cells, the glycemic responses of diabetes-prone BB/Wor rats during a high rate infusion of 50% glucose were compared with normal and with 40% pancreatectomized Wistar rats similarly infused. In all three groups, the initially severe hyperglycemia declined after the first 48 hours to below the target level of 300 mg/dL despite an increasing rate of glucose infusion. The glycemic profile did not differ from controls and was lower than that of the partially depancreatized rats. Five of 20 hyperglycemic BB/Wor rats became diabetic during the 12-day infusion of 50% glucose; there was no difference between their glucose profiles and those of the 15 prediabetic BB/Wor rats that remained nondiabetic throughout the period of hyperglycemic infusion. The latter group of BB/Wor rats, many of which would ultimately have become diabetic, exhibited a 2.4-fold increase in the volume density of their beta cells, compared with a 2.1-fold increase in the Wistar controls. This clinical and morphologic evidence of beta-cell compensation in diabetes-prone rats, even in on the verge of overt diabetes, excludes the possibility that subnormal compensation by beta cells contributes to diabetes in the BB/Wor rat.

Adaptation, Physiological↗

PubMed1990

Processing of prosomatostatin.

Several peptides are generated from prosomatostatin (proSS) in addition to somatostatin-14 (SS-14) and somatostatin-28 (SS-28). These are SS-28(1-12), proSS(1-76) and, as shown more recently, proSS(1-63) and antrin. Important variations in the proportion of these molecular forms are seen among different tissues and among different species. Processing of the precursor in the human brain yields minimal quantities of SS-28(1-12) and high levels of proSS(1-76), namely in cortical areas and in the bed nucleus of the stria terminalis. This is in contrast with findings in rat brain, where SS-28(1-12) is a predominant molecular form. Antrin, which corresponds to proSS(1-10), reaches its highest concentration in the antral portion of the stomach (117 +/- 13 pmol/g wet weight), where it is found in secretory granules of delta cells. We observed an inverse relationship between levels of antrin and proSS(1-63) after chromatography of various tissue extracts. This suggests a precursor-product relationship between these two peptides.

Amino Acid Sequence↗

PubMed1990

Evidence that down-regulation of beta-cell glucose transporters in non-insulin-dependent diabetes may be the cause of diabetic hyperglycemia.

Non-insulin-dependent diabetes mellitus (NIDDM) is attributed to a failure of pancreatic beta cells to maintain insulin secretion at a level sufficient to compensate for underlying insulin resistance. In the ZDF rat, a model of NIDDM that closely resembles the human syndrome, we have previously reported profound underexpression of GLUT-2, the high-Km facilitative glucose transporter expressed by beta cells of normal animals. Here we report that islets of diabetic rats exhibit a marked decrease in the volume of GLUT-2-positive beta cells and a reduction at the electron-microscopic level in the number of GLUT-2-immunoreactive sites per unit of beta-cell plasma membrane. The deficiency of GLUT-2 cannot be induced in normal beta cells by in vivo or in vitro exposure to high levels of glucose nor can it be prevented in beta cells of prediabetic ZDF rats by elimination of hyperglycemia. We conclude that this dearth of immunodetectable GLUT-2 in NIDDM is not secondary to hyperglycemia and therefore that it may well play a causal role in the development of hyperglycemia.

Animals↗

PubMed1990

Transforming growth factor-beta 1 modulates basic fibroblast growth factor-induced proteolytic and angiogenic properties of endothelial cells in vitro.

Tightly controlled proteolytic degradation of the extracellular matrix by invading microvascular endothelial cells is believed to be a necessary component of the angiogenic process. We have previously demonstrated the induction of plasminogen activators (PAs) in bovine microvascular endothelial (BME) cells by three agents that induce angiogenesis in vitro: basic FGF (bFGF), PMA, and sodium orthovanadate. Surprisingly, we find that these agents also induce plasminogen activator inhibitor-1 (PAI-1) activity and mRNA in BME cells. We also find that transforming growth factor-beta 1 (TGF-beta 1), which in vitro modulates a number of endothelial cell functions relevant to angiogenesis, also increases both PAI-1 and urokinase-type PA (u-PA) mRNA. Thus, production of both proteases and protease inhibitors is increased by angiogenic agents and TGF-beta 1. However, the kinetics and amplitude of PAI-1 and u-PA mRNA induction by these agents are strikingly different. We have used the ratio of u-PA:PAI-1 mRNA levels as an indicator of proteolytic balance. This ratio is tilted towards enhanced proteolysis in response to bFGF, towards antiproteolysis in response to TGF-beta 1, and is similar to that in untreated cultures when the two agents are added simultaneously. Using an in vitro angiogenesis assay in three-dimensional fibrin gels, we find that TGF-beta 1 inhibits the bFGF-induced formation of tube-like structures, resulting in the formation of solid endothelial cell cords within the superficial parts of the gel. These results suggest that a net positive proteolytic balance is required for capillary lumen formation. A novel perspective is provided on the relationship between extracellular matrix invasion, lumen formation, and net proteolytic balance, thereby reflecting the interplay between angiogenesis-modulating cytokines such as bFGF and TGF-beta 1.

Amino Acid Sequence↗