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Biomedical subjects

P Stahl

Publications and source records attributed to P Stahl.

At least 19 recordsLinked to original sources
PubMed2000

Prevalence and pathogenicity of retroviruses in wildcats in France.

Feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) are frequently encountered in domestic cats (Felis catus) and in wild felids, but only FeLV has been previously identified in wildcats (Fellis silvestris). Thirty-eight wildcats, either captured alive or found dead, were sampled in eastern and central France. Nine of them (23.7 per cent) carried the FeLV p27 antigen, and three (7.9 per cent) had antibodies to FIV. There was a significant relationship between two measures of body condition and FeLV status; the FeLV-positive cats being in poorer condition than the FeLV-negative cats. The results suggest that FeLV is common in wildcats and may increase mortality in this species. The FIV-positive results constitute the first indication of a FIV-related virus in wildcats.

Animals↗
PubMed1999

Asymmetric Synthesis of the Nakijiquinones-Selective Inhibitors of the Her-2/Neu Protooncogene.

A Wieland-Miescher type ketone and a tetramethoxyaryl derivative are the key building blocks for the enantioselective total synthesis of nakijiquinone C (1). The nakijiquinones are the only natural products known that selectively inhibit the Her-2/Neu tyrosine kinase, a protooncogene product that is vastly overexpressed in about 30 % of primary breast, ovary, and gastric carcinomas.

Journal Article↗
PubMed1997

Identification of naturally processed T cell epitopes from glutamic acid decarboxylase presented in the context of HLA-DR alleles by T lymphocytes of recent onset IDDM patients.

Glutamic acid decarboxylase (GAD) has been defined as a major target antigen in insulin-dependent diabetes mellitus (IDDM). To identify the molecular ligands triggering a T cell response to GAD, a panel of human GAD65-specific T lymphocyte lines was generated from peripheral blood of three recent onset IDDM patients. All lines derived from a patient expressing the high-risk-conferring HLA-DR*0301/ *0401 haplotypes recognized a single epitope localized between amino acid positions 270 and 283 of GAD65, a stretch that is located in close proximity to the homology region shared with Coxsackie virus P2-C protein. All lines with this specificity were restricted to the DRA, B1*0401 product of the DR4 haplotype. Analysis of the GAD-specific T cell response in a second patient homozygous for DR4 haplotypes demonstrated that the same DRA, B1*0401 allele selected T cells specific for a different determinant. The T cell response profile in a third patient showed that DR*1501/ *1601-encoding haplotypes could present at least three different epitopes to GAD65-specific T lymphocytes. One of these epitopes was presented by a DR allele associated with the resistance-conferring DRB1*1501 haplotype. GAD-specific T cell lines could not be isolated from HLA class II-matched normal individuals. Our data reveal that (a) the T cell response to GAD65 is quite heterogenous in recent onset IDDM patients; (b) HLA-DR, not DQ, seems to be the principal restriction element used by T cells present at the onset of the disease; and (c) T cells responding to epitopes containing identical sequences to Coxsackie virus P2-C protein were not detected.

Alleles↗
PubMed1995

Bee venom phospholipase A2 is recognized by the macrophage mannose receptor.

A high affinity and a specific binding site for bee venom PLA2 was found on the surface of J774E macrophages. The binding sites for bee venom PLA2 are entirely different from the binding sites for pancreatic and snake venom PLA2 as revealed by competition experiments. Binding and uptake of bee venom PLA2 by J774E macrophages was shown to be competed by mannose-BSA, glucose-BSA, N-acetylglucosamine-BSA, but not by galactose-BSA, indicating that the binding of bee venom PLA2 is probably mediated by macrophage mannose receptor. An affinity labeling experiment revealed that the bee venom PLA2 specifically binds to a single polypeptide with a mass of approximately 180 kDa. Moreover, the affinity labeled protein component, i.e., the binding site, was not detected in the presence of excess mannose-BSA, suggesting that mannose-BSA and the bee venom PLA2 bind to the same site on macrophages. These observations were further supported by the binding of bee venom PLA2 to cells which are known to express the mannose receptor and by specific binding of bee venom PLA2 to the purified mannose receptor. These data confirm that bee venom PLA2 binding to macrophages is mediated through the mannose receptor.

Animals↗
PubMed1993

Control of protein traffic between distinct plasma membrane domains. Requirement for a novel 108,000 protein in the fusion of transcytotic vesicles with the apical plasma membrane.

We have developed a cell-free system that reconstitutes the last step in transcytosis, i.e. the fusion of transcytotic transport vesicles with the apical plasma membrane (PM). Subcellular fractions containing transcytotic vesicles (the donor) or apical PM (the acceptor) were prepared from rat liver by sucrose density centrifugation. Fusion between the donor and acceptor fractions was measured by the conversion of the 120,000 transmembrane form of the polymeric IgA receptor (pIgA-R), an endogenous protein of transcytotic vesicles, to a processed fragment by a protease endogenous to the apical PM. Fusion occurred only at 37 degrees C and was critically dependent on the presence of ATP and cytosol. Fusion was inhibited by treating the in vitro fusion reaction with N-ethylmaleimide or by adding antibodies against N-ethylmaleimide-sensitive factor (NSF). We have previously identified a specific transcytotic vesicle-associated protein (TAP) and here show that TAP exists in both cytosolic and membrane-associated pools. Because of its exclusive interaction with transcytotic vesicles, we tested the involvement of TAP in distinct fusion processes. Removal of TAP inhibited fusion in an in vitro transcytotic fusion reaction but had no inhibitory effect in an in vitro endosome-endosome fusion system or in an in vitro intra-Golgi transport reaction. We propose that TAP represents part of the molecular machinery specifically involved in targeting and/or fusion of transcytotic vesicles with the apical PM.

Adenosine Triphosphate↗
PubMed1991

Characterization of trypsin-sensitive factor(s) required for endosome-endosome fusion.

Fusion of endosomes appears to be required at early steps of receptor-mediated endocytosis. These fusion events have been reconstituted using a cell-free assay and have been shown to require both cytosolic and membrane-associated proteins. We report here that trypsinization of endosomes completely inhibited fusion. Addition of untreated cytosol cannot restore fusion of trypsinized endosomes. However, fusion activity is restored by the addition of either untreated vesicles or a high salt extract containing peripheral membrane proteins (KE). KE contains both the membrane-associated factor(s) required for the reconstitution of fusion using trypsinized endosomes and the factors that are normally provided by the cytosol. The restorative activity of KE was sensitive to trypsin treatment or incubation at 100 degrees C, but was largely N-ethylmaleimide (NEM)-resistant. This and other criteria demonstrated that the trypsin-sensitive factor is distinct from N-ethylmaleimide-sensitive factor (NSF), an NEM-sensitive protein involved in vesicular fusion, and from other known factors that may participate in membrane fusion events. Preliminary fractionation studies indicate that the restorative activity of KE is associated with one or more high molecular weight proteins. The present study indicates that a novel trypsin-sensitive protein(s) is involved in endosome-endosome fusion. This factor is membrane-associated and is not found in an active form in cytosol as prepared.

Biological Transport↗
PubMed1991

Contact formation and transfer of mannose BSA gold from macrophages to cocultured fibroblasts.

When macrophages were cocultured with fibroblasts many of the cells formed firm contacts. In some of these contacts both cell types were closely apposed and in others they were more clearly separated with numerous pseudopodia extending from macrophages toward the fibroblasts. Many small vesicles similar in structure to caveoli were observed immediately beneath the plasma membrane of some fibroblasts in regions immediately adjacent to areas of contact with macrophages. The membrane integrity of both cell types was always maintained and no connecting cytoplasmic strands were observed between contacting cells. Junctions were freely permeable to ruthenium red and less permeable to the larger cationized ferritin. Gold conjugated to mannose BSA was taken up readily by macrophages but not by fibroblasts. When fibroblasts were cocultured with macrophages that had been labeled with endocytosed gold, increasing amounts were transferred to them. Gold was observed within gaps formed between cocultured cells and within recipient fibroblasts in vesicles anatomically similar to lysosomes. These points of contact thus appear to provide a series of specialized protected clefts into which directed exocytosis of ligands from donor cells can take place and from which endocytosis into recipient cells is facilitated.

Animals↗
PubMed1991

Endosomal density shift is related to a decrease in fusion capacity.

Dinitrophenol (DNP)-beta-glucuronidase and mannosylated anti-DNP IgG, which are endocytosed by the mannose receptor and delivered to lysosomes, were previously developed as probes for examination of fusion between early endosomes in a cell-free system. In this study, these probes were found to be transported by intact cells to endocytic vesicles with heavy buoyant density at different rates, as determined by Percoll gradient fractionation of cell homogenates. There was a concomitant loss of in vitro fusion activity as the ligands moved to dense compartments. In monensin-treated cells, DNP-beta-glucuronidase was retained in a light compartment corresponding to intracellular vesicles capable of fusion in vitro. Pulse-chase studies using a DNP-derivatized transferrin-alkaline phosphatase conjugate showed that a recycling ligand was always found in light intracellular vesicles that were capable of fusion to early endosomes in vitro. In contrast to cell-free systems, intact cells sequentially labeled with DNP-beta-glucuronidase and then mannosylated anti-DNP IgG showed ligand mixing in both early and late endocytic compartments. Treatment with nocodazole or colchicine did not affect the rate of DNP-beta-glucuronidase transport to heavy vesicles in intact cells, however, the extent of ligand mixing in late endosomes was decreased by microtubule disruption. Using sequentially labeled cells split into two groups, we directly compared ligand mixing in vitro to mixing by intact cells. Fusion alone does not mediate increases in vesicle density, since DNP-beta-glucuronidase/anti-DNP IgG complexes formed in vitro were found in light vesicles, while intact cells showed immune complexes predominantly in heavy vesicles. These results suggest that the density shift is an initial step in targeting to lysosomes.

Animals↗
PubMed1991

[Behavioral ecology of the transmission of rabies].

Red fox behavioural ecology was studied in a rabies-enzootic area in order to determine how population size is balanced despite rabies-induced mortality. The results suggest that the red fox rabies virus equilibrium evolves, and is due to the solitary behaviour pattern of the fox which reduces the risk of virus transmission from on territory to another; and to the subsequent autumn dispersal, which allows the local fox population to recover in the space of under a year. The hypothesis is put forward that rabies does not seem to regulate fox population size. There would therefore be no reason to fear a population explosion after oral immunisation of foxes against rabies.

Animals↗
PubMed1989

In vitro clustering and multiple fusion among macrophage endosomes.

Early steps of receptor-mediated endocytosis appear to require the fusion of endosomes with each other. Recently, these fusion events have been reconstituted in vitro using vesicle preparations from J774 macrophages which have internalized ligands via the mannose receptor (Diaz, R., Mayorga, L., and Stahl, P. (1988) J. Biol. Chem. 263, 6093-6100). The present studies indicate that endosomes first form clusters when incubated under fusogenic conditions. Aggregation state was determined by electron microscopy using vesicles containing ligand-coated colloidal gold of different sizes previously internalized via the mannose receptor. Aggregation required cytosol and ATP. Afterwards, the limiting membranes of the vesicles composing these aggregates undergo multiple fusion and bring about the formation of large diameter vesicles that maintained the same density as endosomes when analyzed by Percoll gradient sedimentation. These large diameter vesicles were no longer fusogenic in the fusion assay. Multiple fusion was determined morphologically by the co-localization of three different size colloidal gold vesicles inside endocytic vesicles and biochemically by the fusion-dependent formation of triple immune complexes between three endocytic ligands internalized by receptor-mediated endocytosis: anti-dinitrophenol mouse IgG and dinitrophenol-derivatized beta-glucuronidase, ligands for the mannose receptor, and aggregated rabbit anti-mouse IgG, a ligand for the macrophage Fc receptor.

Animals↗
PubMed1989

The use of permeabilized cells to study the ion requirements of receptor-ligand dissociation in endosomes.

The mannose receptor mediates the transport of high-mannose glycoproteins from the cell surface to lysosomes in macrophages. The binding of ligand to the receptor is dependent on both pH and Ca2+. Upon internalization, ligands enter an acidic pre-lysosomal compartment where receptor-ligand dissociation takes place. Acidification is driven by an endosomal proton pump and anion transport is coupled to this acidification step. A permeabilized-cell assay has been designed to characterize the ionic requirements for receptor-ligand dissociation in endosomes. The plasma membrane of macrophages has been permeabilized selectively with digitonin without affecting endosomal membranes. Receptor-ligand dissociation in permeabilized cells required ATP and was blocked by proton ionophores. Di-isothiocyanostilbene-disulphonic acid and N-ethylmaleimide also blocked dissociation, but mitochondrial ATPase inhibitors and vanadate were ineffective. To explore the nature of the anion requirement for acidification, the ability of different anions to compensate for Cl- was tested. For the halide series, Br- was as equally effective as Cl- in supporting receptor-ligand dissociation, but I- was inhibitory. Citrate and gluconate were only partially effective, while SO4(2-), NO3- and PO4(2-) blocked dissociation. Addition of Ca2+ to permeabilized-cell preparations impaired ATP-dependent dissociation without affecting endosome acidification. These results suggest that the endosomal membrane has a Ca2+ conductance that would permit the rapid efflux of Ca2+ from endosomes during acidification, and this would appear to be a necessary step for efficient sorting of Ca2+-dependent receptors from their ligands.

Adenosine Triphosphate↗
PubMed1988

In vitro fusion of endosomes following receptor-mediated endocytosis.

Receptor-mediated endocytosis and receptor recycling involve a series of intracellular membrane fusion events that appear to play an important role in the regulation of the overall rate and efficiency of the process. An endosome-endosome fusion assay is described using two ligands that (i) rapidly and efficiently enter the endosomal compartment via the macrophage mannose receptor and (ii) that mutually recognize each other. Dinitrophenol-derivatized beta-glucuronidase (DNP-beta-glucuronidase), a ligand for the mannose receptor, was endocytosed by one population of J774 E clone cells, and mannose-derivatized monoclonal anti-DNP IgG (Man-IgG) was internalized by a second set of cells. Both ligands were localized in endosomes as determined by fractionation on Percoll gradients. Incubation of vesicles prepared from the two set of cells resulted in vesicle fusion as indicated by the formation of DNP-beta-glucuronidase-Man-IgG complexes. Under standard conditions, fusion was time-, ATP-, and temperature-dependent. KCl was required for fusion. Fusion required both cytosolic- and membrane-associated proteins. N-Ethylmaleimide treatment of cytosol inhibited fusion. Proton ionophores and amines had no effect on the fusion reaction. ATP-dependent fusion was only observed between early endocytic compartments. While in the presence of a Ca2+ chelator fusion was ATP-dependent, in its absence fusion was also observed in an ATP-independent fashion.

Adenosine Triphosphate↗
PubMed1988

Cell contact and direct transfer between co-cultured macrophages and fibroblasts.

Mouse peritoneal macrophages formed attachments with beta-glucuronidase deficient human fibroblasts within an hour after co-cultures were initiated. Some of these attachments were transitory, while in others macrophages remained in firm contact with fibroblasts for many hours. Attachment of one macrophage did not prevent attachment of others, since many fibroblasts made firm contact with four or five other cells. Not all macrophages, however, attached themselves to fibroblasts. Macrophages injected with Lucifer yellow did not transfer the dye to fibroblasts with which they had made contact, nor was there any reverse transfer from injected fibroblasts to macrophages. Lucifer yellow was, however, transferred rapidly from injected fibroblasts to other adjacent fibroblasts with which they had formed gap junctions. Macrophages whose lysosomes had been pre-loaded with FITC-dextran did transfer this ligand to recipient fibroblasts, where it became localised in a perinuclear pattern with many bright punctate patches adjacent to donor macrophages. Transfer of FITC-dextran was blocked when cells were separated by nucleopore membranes in an analogous manner to transfer of endogenous lysosomal beta-glucuronidase.

Cell Adhesion↗