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Biomedical subjects

S Paris

Publications and source records attributed to S Paris.

At least 91 records · Page 5Linked to original sources
PubMed1992

Activation of G proteins by (Rp) and (Sp) diastereomers of guanosine 5'-[beta-thio]triphosphate in hamster fibroblasts. Differential stereospecificity of Gi, Gs and Gp.

The effects of guanosine 5'-[beta-thio]triphosphate (GTP beta[S]) on G proteins have been examined in Chinese hamster lung fibroblasts (CCL39 line) permeabilized with alpha-toxin from Staphylococcus aureus. Although much less effective than guanosine 5'-[gamma-thio]triphosphate (GTP gamma[S]), both (Rp) and (Sp) diastereomers of GTP beta[S] were found to activate three G protein-mediated pathways: inhibition of forskolin-stimulated adenylate cyclase (mediated by Gi), potentiation of receptor-mediated activation of adenylate cyclase (mediated by Gs), and activation of phosphoinositide breakdown (mediated by Gp). Activation of Gi and Gs occurred above 3 microM-GTP beta[S], but activation of Gp only occurred above 100 microM-GTP beta[S]. Moreover, the order of effectiveness of the two diastereomers was not the same for the three G protein-mediated processes. Whereas both Gi and Gs were more effectively activated (about 5-fold) by (Sp)-GTP beta[S] than by (Rp)-GTP beta[S], Gp showed a marked preference for the (Rp) isomer. Indeed, (Rp)-GTP beta[S] induced the formation of inositol phosphates with a shorter latency and was a better competitor of GDP for binding to Gp than the (Sp) isomer. These results point to different guanine nucleotide-binding properties for Gi and Gs on the one hand and Gp on the other. At least two distinct Gp proteins, differing by their sensitivity to pertussis toxin, are present in CCL39 cells. Since pretreatment of cells with pertussis toxin completely suppressed the effects of (Rp)-GTP beta[S] on Gi, while only slightly attenuating its effects on Gp, we believe that it is the pertussis toxin-insensitive Gp which prefers the (Rp) isomer. Therefore (Rp)-GTP beta[S] may be a valuable tool for the selective activation and the biochemical characterization of this pertussis toxin-insensitive Gp.

Adenylate Cyclase Toxin↗
PubMed1992

Alternaria spore and mycelium sensitivity in allergic patients: in vivo and in vitro studies.

Extracts from Alternaria spores and mycelia were prepared to evaluate their allergenic potencies in allergic patients. Twelve Alternaria-sensitive patients, with histories of rhinitis or asthma, were submitted to skin prick tests and five of 12 patients received nasal challenges with spores and mycelia. In vitro, the allergenic activity of each extract was determined by RAST, basophil histamine-release and RAST-inhibition. All patients demonstrated skin reactivity to both extracts while skin reactivity to mycelia was higher than that induced by the spore extract (P < .005). Of the 12 patients, 11 had positive mycelia-RAST and 9/12 had positive spore-RAST. It was found that mycelium-IgE antibody levels were higher than spore-IgE antibody levels (P < .005). Nine RAST-positive patients had positive histamine release tests (> 50%) and basophils challenged with mycelia appeared 10-fold more sensitive compared to the spore challenge. In four of five patients subjected to nasal provocation tests, immediate-type rhinitis was elicited either after spore or mycelium challenge. The patients exhibited a higher nasal reactivity with the mycelium challenge than with spores. RAST-inhibition studies demonstrated that mycelial extracts shared common allergens with spore. These results indicated that Alternaria spore and mycelium were potent allergens in allergic patients and there was a variability in the pattern of in vivo and in vitro reactions between the patients for each allergen.

Adolescent↗
PubMed1991

The 31 kd major allergen, Alt a I1563, of Alternaria alternata.

A component of Alternaria extract, previously identified as the major allergen, Alt a I1563, was purified to homogeneity from Alternaria mycelium by means of acetone precipitation and ion-exchange chromatography. The homogeneity of Alt a I1563 was assessed by one single band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by one single radiolabeled band after transfer to nitrocellulose, and by one single peak after size-exclusion chromatography by high-performance liquid chromatography. Alt a I1563 was isolated as a heat-stable acidic glycoprotein (carbohydrate content, 20%; pI, 4.0 to 4.5; 31 kd). The role of the carbohydrate moiety in the allergenicity is suggested. This major allergen is located in the cytoplasm of mycelium and spore.

Allergens↗
PubMed1991

Antigens of diagnostic value in three isolates of Paracoccidioides brasiliensis.

Yeast cellular extracts of three isolates of Paracoccidioides brasiliensis were tested by the Western-blot technique against 53 sera from patients with paracoccidioidomycosis (PCM). Numerous antigens were recognized by the sera but only five were specific for (58, 57, 48, 44 and 23 kDa). These specific antigens had the same relative molecular mass in the different isolates but their reactivity with sera from PCM patients was variable. An antigen in isolate 688, which was specific for PCM, had an apparent molecular mass of 48 kDa. Although a 48 kDa antigen was also present in isolates B339 and 1789.88 it was not specific for PCM, demonstrating antigenic variability among isolates. The 44 kDa antigen in isolate B339 and the 44 and 48 kDa antigens in isolate 688 reacted with a rabbit antiserum raised against a 43 kDa glycoprotein, a specific antigen used in the diagnosis of PCM. There was no correlation between the specific antigen(s) detected and the clinical form of the disease.

Antibodies, Fungal↗
PubMed1991

Mitogenic effects of fibroblast growth factors in cultured fibroblasts. Interaction with the G-protein-mediated signaling pathways.

We have shown that FGF (basic or acidic) is mitogenic for quiescent hamster lung fibroblasts (CCL39 line). It is active alone but is much more efficient in synergistic combinations with G-protein-activating agents. When used alone, FGF appears to exert its mitogenic effects without involving any of the major G-protein-mediated signaling pathways. It causes no significant hydrolysis of phosphoinositides, it does not alter the activity of adenylate cyclase, and its mitogenicity is insensitive to pertussis toxin. It therefore seems likely that all pleiotropic actions of FGF are primarily mediated by the intrinsic protein tyrosine kinase of its receptors. However, FGF, acting through its receptor tyrosine kinase, and thrombin, acting through G-protein-coupled receptors, induce a common set of early responses detected within seconds or minutes at the level of membranes, cytoplasm, and nuclei. Typical examples of early responses are activation of Na/H antiporter and Na/K/Cl cotransporter, phosphorylation of ribosomal protein S6, and increased transcription of early-immediate genes (c-fos, c-jun, and c-myc). Not only various classes of growth factors acting via distinct transducing mechanisms activate common targets, but also their synergistic effects on reinitiation of DNA synthesis is reflected on the early responses. How does the coordination of these signaling events take place? A partial answer to this question is illustrated in Figure 6 in which "switch kinases" play the role of integrators of multiple extracellular signals. Raf and, perhaps more convincingly, MAP kinases that are activated by dual phosphorylation on tyrosine and threonine residues are potential good candidates for this integration. This hypothetical scheme could therefore explain, in part, the coordination and the synergy commonly observed in the mitogenic response. The synergy could be generated at the level of MAP kinases simply by dual activating phosphorylations. With the recent cloning of MAP kinases, these questions will be more easily addressed. Another important gap that will have to be filled in future studies is the identification of all the members of the kinase cascade. When used in synergistic combinations with G-protein-activating agents, FGF does exert in contrast some effects on the G-protein-mediated pathways. It potentiates the G-protein-mediated activations of both PIP2-PLC and adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylate Cyclase Toxin↗
PubMed1990

Guanosine 5'-O-(3-thiotriphosphate) and guanosine 5'-O-(2-thiodiphosphate) activate G proteins and potentiate fibroblast growth factor-induced DNA synthesis in hamster fibroblasts.

Addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to intact Chinese hamster lung fibroblasts (CCL39) depolarized by high K+ concentrations results in activation of phosphoinositide-specific phospholipase C (PLC) (at GTP gamma S concentrations greater than 0.1 mM), inhibition of adenylate cyclase (between 10 microM and 0.5 mM), and activation of adenylate cyclase (above 0.5 mM). Since GTP gamma S-induced activation of PLC is dramatically enhanced upon receptor-mediated stimulation of PLC by alpha-thrombin, we conclude that in depolarized CCL39 cells GTP gamma S directly activates various guanine nucleotide-binding regulatory proteins (G proteins) coupled to PLC (Gp(s)) and to adenylate cyclase (Gi and Gs). Pretreatment of cells with pertussis toxin strongly inhibits GTP gamma S-induced activation of PLC and inhibition of adenylate cyclase. GTP gamma S cannot be replaced by other nucleotides, except by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which mimics after a lag period of 15-20 min all the effects of GTP gamma S, with the same concentration dependence and the same sensitivity to pertussis toxin. We suggest that GDP beta S is converted in cells into GTP beta S, which acts as GTP gamma S. Since cell viability is not affected by a transient depolarization, these observations provide a simple method to examine long-term effects of G protein activation on DNA synthesis. We show that a transient exposure of G0-arrested CCL39 cells to GTP gamma S or GDP beta S under depolarizing conditions is not sufficient by itself to induce a significant mitogenic response, but markedly potentiates the mitogenic action of fibroblast growth factor, a mitogen known to activate a receptor-tyrosine kinase. The potentiating effect is maximal after 60 min of pretreatment with 2 mM GTP gamma S. GDP beta S is equally efficient but only after a lag period of 15-20 min. Mitogenic effects of both guanine nucleotide analogs are suppressed by pertussis toxin. Since the activation of G proteins by GTP gamma S under these conditions vanishes after a few hours, we conclude that a transient activation of G proteins facilitates the transition G0----G1 in CCL39 cells, whereas tyrosine kinase-induced signals are sufficient to mediate the progression into S phase.

Adenylate Cyclase Toxin↗
PubMed1990

Comparison of different extraction methods of Alternaria allergens.

Spore and mycelial allergens of two species of Alternaria (A. brassicicola and A. alternata) extracted under two different conditions were analyzed by radiorocket immunoelectrophoresis, crossed radioimmunoelectrophoresis, and Western blotting with a pool of sera from Alternaria-allergic patients. More allergens were extracted after disruption of the cells if protease inhibitors and a phenol-binding component were included in the homogenization buffer. However, the 31 kDa major allergen was extracted in about the same amount, either by incubation or by cell breakage of the cells. This 31 kDa allergen was present in higher concentration in the mycelium than in the spore. The difference between the two species studied is less than between spore and mycelium from the same species.

Allergens↗
PubMed1990

Comparison of conidial and mycelial allergens of Alternaria alternata.

Alternaria allergens, separated by SDS polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane, were identified using sera from Alternaria-allergic patients. 30 Alternaria components of spore and mycelium, ranging in molecular weight from more than 150 to 12 kD, bound IgE antibodies. 15 were detected by more than 25% of the sera and only 4 (85, 56, 42, 31 kD) by more than 50% of the sera. Among these four major allergens, the 56-kD component was present mostly in the spore extract, the 85- and 42-kD fractions in the mycelium extract and the 31-kD in both extracts. The 31-kD component showed the highest IgE binding and the highest frequency of binding (95% with mycelium extract and 79% with spore extract). It was composed of two subunits.

Allergens↗
PubMed1989

Cyclic AMP inhibits mitogen-induced DNA synthesis in hamster fibroblasts, regardless of the signalling pathway involved.

Mitogen-induced initiation of DNA synthesis in quiescent Chinese hamster lung fibroblasts (CCL39) is strongly inhibited by 8-Br cAMP and cAMP-evaluating agents (prostaglandin E1, cholera toxin, isobutylmethylxanthine). This inhibition is reversible and occurs very early in G0/G1. As exponential growth is much less affected by increased cAMP, we propose that cAMP inhibits an early signal essential for the exit from G0. CCL39 cells can be stimulated by alpha-thrombin, which activates phosphoinositide (PI) breakdown, as well as by mitogens (FGF or FGF + serotonin) which do not involve the PI pathway. Here we show that the action of both classes of mitogens is likewise inhibited by cAMP. Therefore, although PI breakdown is inhibited by cAMP in CCL39 cells, this effect cannot entirely account for th antimitogenic activity of cAMP. Other early steps of the mitogenic response must be also affected.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Fibroblast growth factor potentiates receptor- and nonreceptor-mediated stimulation of adenylate cyclase in hamster fibroblasts.

Basic fibroblast growth factor (FGF) has no effect alone on the basal cAMP synthesis in Chinese hamster fibroblasts (CCL39) but it potentiates (by up to 50%) the stimulation of adenylate cyclase by prostaglandin E1, cholera toxin or forskolin. This potentiating effect is not abolished by pretreatment of the cells with pertussis toxin, which indicates that it is not due to the withdrawal of a tonic inhibition of adenylate cyclase by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi). Therefore, we conclude that FGF enhances the activation of adenylate cyclase by the stimulatory GTP-binding protein (Gs). Although activation of protein kinase C in CCL39 cells results in a similar potentiation of cAMP production, we provide evidence that the effect of FGF is not mediated by protein kinase C, since (1) the potentiating effects of FGF and phorbol esters are additive and (2) FGF effect persists after down-regulation of protein kinase C. A role of FGF-induced rise in cytoplasmic Ca2+ can also be ruled out because the FGF effect is not mimicked by a Ca2+ ionophore and it persists in Ca2(+)-free medium. Since a similar potentiating effect on cAMP production is elicited by epidermal growth factor, a mitogen known to activate a receptor tyrosine kinase, we suggest that the FGF effect on adenylate cyclase might be mediated by the tyrosine kinase activity that is very likely to be associated with FGF receptors.

Adenylate Cyclase Toxin↗
PubMed1988

Tyrosine kinase-activating growth factors potentiate thrombin- and AIF4- -induced phosphoinositide breakdown in hamster fibroblasts. Evidence for positive cross-talk between the two mitogenic signaling pathways.

Basic fibroblast growth factor (FGF) and alpha-thrombin can stimulate DNA synthesis in Chinese hamster fibroblasts (CCL39) by two separate signaling pathways (Chambard, J.C., Paris, S., L'Allemain, G., and Pouysségur, J. (1987) Nature 326, 800-803) but can also act synergistically. We have examined whether this synergism might depend upon changes in inositol lipid metabolism. Indeed, FGF, which has no effect on its own on phosphoinositide hydrolysis, potentiates (by up to 2-fold) thrombin-induced formation of inositol phosphates. This enhancing effect is also observed upon direct activation by AIF4- of the GTP-binding protein coupled to phospholipase C, and is best revealed when phospholipase C is weakly stimulated. With low thrombin concentrations or with AIF4-, the formation of inositol phosphates is immediately increased with a marked reduction of the initial lag, whereas at high thrombin concentrations, the stimulation by FGF becomes pronounced only after desensitization of phospholipase C to thrombin. FGF-induced potentiation is not mimicked by calcium ionophores, but is likewise elicited by epidermal growth factor, platelet-derived growth factor, and to a lesser extent by insulin, other growth factors known to activate receptor tyrosine kinases. We therefore propose that the tyrosine kinase-activating growth factors enhance the coupling between GTP-binding protein and phospholipase C, presumably through the phosphorylation of one of these two proteins. Treatment of cells with pertussis toxin attenuates thrombin-induced phospholipase C activity but does not impede the potentiation by FGF. Comparison of the potentiating effects of FGF on inositol phosphate formation and on DNA synthesis suggests than an increased production of second messengers by the inositol lipid pathway in the first hours of stimulation might be, at least in part, responsible for the synergistic actions of FGF and thrombin on DNA synthesis.

Aluminum↗
PubMed1988

Homologous desensitization of thrombin-induced phosphoinositide breakdown in hamster lung fibroblasts.

Activation of phosphoinositide breakdown is thought to be an important signaling pathway involved in the mitogenic effects of alpha-thrombin in Chinese hamster lung fibroblasts. We have previously shown that the initial strong stimulation of inositol phosphate formation induced by thrombin in quiescent hamster cells (CCL39 line) is rapidly attenuated. We now report that this desensitization of phospholipase C to thrombin 1) is independent of protein kinase C activation, because thrombin-induced desensitization normally occurs in cells that have been depleted in protein kinase C by a prolonged treatment with a phorbol ester, and 2) is even independent of phosphoinositide hydrolysis because the desensitization still occurs, although at a lesser degree, at 4 degrees C, in the absence of any phospholipase C activity. Furthermore, phospholipase C desensitization to thrombin is homologous. It does not affect the response to thrombin-free serum or the direct activation by A1F-4 of the GTP-binding protein (G-protein) coupled to phospholipase C. We therefore conclude that the desensitization of phospholipase C to thrombin does not result from an impairment of the G-protein-phospholipase C complex, or from a depletion in phosphoinositides, but rather from a modification of thrombin receptors leading to their uncoupling from G-protein. This modification is slowly reversible because, upon thrombin removal, a prolonged incubation (approximately 2 h) restores responsiveness of the cells to thrombin. Although the desensitization seems to depend on thrombin receptor occupancy, it cannot be accounted for by an internalization of the occupied receptors, because it is not blocked at 4 degrees C. The exact mechanism underlying this homologous desensitization of thrombin receptors remains to be elucidated.

Aluminum↗
PubMed1988

Thrombin exerts a dual effect on stimulated adenylate cyclase in hamster fibroblasts, an inhibition via a GTP-binding protein and a potentiation via activation of protein kinase C.

Previous studies in Chinese-hamster fibroblasts (CCL39 line) indicate that an important signalling pathway involved in thrombin's mitogenicity is the activation of a phosphoinositide-specific phospholipase C, mediated by a pertussis-toxin-sensitive GTP-binding protein (Gp). The present studies examine the effects of thrombin on the adenylate cyclase system and the interactions between the two signal transduction pathways. We report that thrombin exerts two opposite effects on cyclic AMP accumulation stimulated by cholera toxin, forskolin or prostaglandin E1. (1) Low thrombin concentrations (below 0.1 nM) decrease cyclic AMP formation. A similar inhibition is induced by A1F4-, and both thrombin- and A1F4- -induced inhibitions are abolished by pertussis toxin. (2) Increasing thrombin concentration from 0.1 to 10 nM results in a progressive suppression of adenylate cyclase inhibition and in a marked enhancement of cyclic AMP formation in pertussis-toxin-treated cells. A similar stimulation is induced by an active phorbol ester, and thrombin-induced potentiation of adenylate cyclase is suppressed by down-regulation of protein kinase C. Therefore, we conclude that (1) the inhibitory effect of thrombin on adenylate cyclase is the direct consequence of the activation of a pertussis-toxin-sensitive inhibitory GTP-binding protein (Gi) possibly identical with Gp, and (2) the potentiating effect of thrombin on cyclic AMP formation is due to stimulation of protein kinase C, as an indirect consequence of Gp activation. Our results suggest that the target of protein kinase C is an element of the adenylate cyclase-stimulatory GTP-binding protein (Gs) complex. At low thrombin concentrations, activation of phospholipase C is greatly attenuated by increased cyclic AMP, leading to predominance of the Gi-mediated inhibition.

Adenylate Cyclase Toxin↗
PubMed1988

Effect of mutations affecting Na+: H+ antiport activity on tumorigenic potential of hamster lung fibroblasts.

Mutants unable to regulate intracellular pH through the Na+: H+ antiport system were found to evolve tumors less frequently than wild-type CCL39 hamster lung fibroblasts, after transplantation in athymic nude mice. When rare tumors arose, they comprised cells which were transformed in vitro, but which upon retransplantation grew at a lower rate than tumor cells originating from CCL39 cells. Both parental and mutant cells became transformed after transfection of the activated Harvey ras oncogene, but transfectants derived from the mutants had a weaker tumorigenic potential. These results suggest that transformed characteristics can be acquired independently from the Na+: H+ antiporter. However, the presence of this system provides a selective growth advantage when cells are confronted with natural environments, as it occurs during the expansion of tumors in a host.

Animals↗
PubMed1988

Nasal allergy to fungi in guinea pigs.

Sensitized guinea pigs produced specific IgG and IgE antibodies toward Cladosporium and Alternaria. In presence of fungal extracts, nasal mast cells degranulate. Ultrastructural modifications of the cells during degranulation have been established. The ciliary epithelium and the ciliary beating are not affected by fungal allergens.

Alternaria↗
PubMed1987

Further evidence for a phospholipase C-coupled G protein in hamster fibroblasts. Induction of inositol phosphate formation by fluoroaluminate and vanadate and inhibition by pertussis toxin.

We have previously reported that alpha-thrombin induces in resting hamster fibroblasts (CCL39) the formation of inositol phosphates (IP) by activating a GTP-binding protein (G protein) sensitive to pertussis toxin (Paris, S., and Pouysségur, J. (1986) EMBO J. 5, 55-60). Here we show that IP formation in CCL39 cells can also be induced by NaF with AlCl3 and by vanadate. In the presence of Li+, IP accumulation is linear over 30 min with no detectable lag and is concentration-dependent. NaF alone is slightly stimulatory, but a marked potentiation is observed in the presence of AlCl3, by itself without effect. Maximal stimulation is obtained with 10 mM NaF and 3 microM AlCl3, and with vanadate half-maximal effect is achieved at 0.3 mM. Both stimulations are markedly inhibited (up to 80%) by pertussis toxin (half-maximal inhibition at 1-2 ng/ml). We therefore conclude that phospholipase C is stimulated by NaF plus AlCl3 (presumably acting as AlF-4) and by vanadate by direct activation of the regulatory G protein. In addition, NaF inhibits the inositol-1-phosphatase, but this effect is not potentiated by AlCl3. Similarly, vanadate inhibits inositol trisphosphate degradation. Maximal stimulations of phospholipase C by AlF-4 and vanadate are not additive, whereas they are both additive with thrombin effects. Pretreatment of cells for 15 min with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate nearly completely abolishes induction of IP formation by AlF-4 and vanadate, suggesting that protein kinase C exerts a feedback negative control either on the G protein or on phospholipase C itself. An increase in cellular cyclic AMP similarly results in a marked attenuation of AlF-4-induced IP formation, indicating that activation of phospholipase C can be controlled also by cyclic AMP. However, the stimulatory effect of AlF-4 on phospholipase C is clearly dissociated from its effect on the adenylate cyclase system.

Adenylate Cyclase Toxin↗
PubMed1987

Coupling between phosphoinositide breakdown and early mitogenic events in fibroblasts. Studies with fluoroaluminate, vanadate, and pertussis toxin.

In the preceding paper (Paris, S., and Pouysségur J. (1987) J. Biol. Chem. 262, 1970-1976), AlF4- and vanadate have been shown to induce inositol phosphate formation in resting hamster fibroblasts (CCL39). In this study, we show that these two phosphate analogs are good tools to explore the causal relationship between phosphoinositide breakdown and early mitogenic events. AlF4- can activate, very similarly to the mitogen alpha-thrombin: the amiloride-sensitive Na+/H+ antiport, the bumetanide-sensitive Na+/K+/Cl- co-transport, and the expression of c-myc mRNA. The link between phospholipase C activation and these early events of the mitogenic response is demonstrated by the similarity of all dose-response curves for NaF and AlCl3 and by the common sensitivity of the four events to pertussis toxin. Vanadate likewise stimulates the Na+/H+ antiport through a pertussis toxin-sensitive pathway. On longer incubations, both fluoride and vanadate were found to be toxic and failed to induce DNA synthesis. Therefore, we have used pertussis toxin to investigate the link between phospholipase C activation and commitment to DNA synthesis. We show that pertussis toxin strikingly inhibits thrombin-induced reinitiation of DNA synthesis but does not affect the stimulation by the epidermal or fibroblast growth factors, two mitogens that do not stimulate phosphoinositide breakdown in CCL39 cells. In conclusion, these studies demonstrate that activation of phospholipase C, if not an obligatory step in the action of all growth factors, plays a crucial role in the mitogenic signaling pathway of alpha-thrombin.

Aluminum↗