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E Gouin

Publications and source records attributed to E Gouin.

At least 19 recordsLinked to original sources
PubMed2001

Listeria protein ActA mimics WASp family proteins: it activates filament barbed end branching by Arp2/3 complex.

Actin-based propulsion of the bacteria Listeria and Shigella mimics the forward movement of the leading edge of motile cells. While Shigella harnesses the eukaryotic protein N-WASp to stimulate actin polymerization and filament branching through Arp2/3 complex, the Listeria surface protein ActA directly activates Arp2/3 complex by an unknown mechanism. Here we show that the N-terminal domain of ActA binds one actin monomer, in a profilin-like fashion, and Arp2/3 complex and mimics the C-terminal domain of WASp family proteins in catalyzing filament barbed end branching by Arp2/3 complex. No evidence is found for side branching of filaments by ActA-activated Arp2/3 complex. Mutations in the conserved acidic (41)DEWEEE(46) and basic (146)KKRRK(150) regions of ActA affect Arp2/3 binding but not G-actin binding. The motility properties of wild-type and mutated Listeria strains in living cells and in the medium reconstituted from pure proteins confirm the conclusions of biochemical experiments. Filament branching is followed by rapid debranching. Debranching is 3-4-fold faster when Arp2/3 is activated by ActA than by the C-terminal domain of N-WASp. VASP is required for efficient propulsion of ActA-coated beads in the reconstituted motility medium, but it does not affect the rates of barbed end branching/debranching by ActA-activated Arp2/3 nor the capping of filaments. VASP therefore affects another still unidentified biochemical reaction that plays an important role in actin-based movement.

Actin-Related Protein 2↗
PubMed2001

A role for cofilin and LIM kinase in Listeria-induced phagocytosis.

The pathogenic bacterium Listeria monocytogenes is able to invade nonphagocytic cells, an essential feature for its pathogenicity. This induced phagocytosis process requires tightly regulated steps of actin polymerization and depolymerization. Here, we investigated how interactions of the invasion protein InlB with mammalian cells control the cytoskeleton during Listeria internalization. By fluorescence microscopy and transfection experiments, we show that the actin-nucleating Arp2/3 complex, the GTPase Rac, LIM kinase (LIMK), and cofilin are key proteins in InlB-induced phagocytosis. Overexpression of LIMK1, which has been shown to phosphorylate and inactivate cofilin, induces accumulation of F-actin beneath entering particles and inhibits internalization. Conversely, inhibition of LIMK's activity by expressing a dominant negative construct, LIMK1(-), or expression of the constitutively active S3A cofilin mutant induces loss of actin filaments at the phagocytic cup and also inhibits phagocytosis. Interestingly, those constructs similarly affect other actin-based phenomenons, such as InlB-induced membrane ruffling or Listeria comet tail formations. Thus, our data provide evidence for a control of phagocytosis by both activation and deactivation of cofilin. We propose a model in which cofilin is involved in the formation and disruption of the phagocytic cup as a result of its local progressive enrichment.

Actin Depolymerizing Factors↗
PubMed2001

In vitro co-incubation of pig islet cells with xenogeneic human blood mononuclear cells causes loss of insulin release during perifusion: involvement of non-T-cell- and T-cell-mediated mechanisms.

Because the different steps of the human cellular immune rejection of pig islets are still poorly understood, our previous work concerned the intensity and mechanisms of the proliferation of human peripheral blood mononuclear cells (PBMC) to adult pig islet cells (PIC). As lymphocyte proliferation is not indicative of alteration of PIC, the present in vitro study evaluated cell-mediated immune effectors possibly involved in impairment of adult PIC. A test was thus developed, based on perifusion analysis of the alteration of insulin release from PIC incubated with different human cells. Compared to PIC incubation alone or with autologous pig splenocytes, seven-day co-incubation with whole human peripheral blood mononuclear cells (PBMC) (n = 18) led to almost complete abolition of basal and stimulated insulin releases (p < 0.0001). This effect could not be reversed by extensive sequential washes before perifusion of PIC, and the number of PIC was decreased by 78% after seven-day co-incubation with PBMC. PBMC are a complex mixture of cells involved in different xenogeneic mechanisms, and two components of this PIC impairment were then detected separately. First, the effect of PBMC against PIC was decreased (p < 0.0001) after removal of either MHC class II+ or CD14+ cells from PBMC. On the contrary, decreasing effect (p < 0.001) on insulin secretion was observed when only plastic-adherent or CD14+ cells were co-incubated with PIC. Additionally, alteration of insulin release from PIC cultured with PBMC or plastic-adherent cells was abolished dose-dependently (p < 0.0001 and p < 0.04, respectively) by gadolinium chloride (which inhibits macrophages), but not modified by cyclosporin A or mycophenolate mofetil which did not alter insulin release from PIC but blocked the proliferation of PBMC against PIC. A second mechanism was also detected, since co-incubation of PIC with purified human T cells remixed with antigen-presenting cells led to a decrease (p < 0.0001) of insulin release. This model based on the alteration of dynamic basal and stimulated insulin secretion provides detailed account of in vitro human cell-mediated impairment of PIC. It shows that the xenogeneic effect of whole mononuclear cells was strong and rapid. A crucial role was played by MHC class II+, CD14+, and plastic-adherent cells. Two mechanisms appear to be responsible for the role of these cells: 1) early direct effect, potentially involved in vivo in primary nonfunction of islets aggressed by monocytes/macrophages; and 2) the presentation of PIC xenoantigens leading to impairment by T lymphocytes, which may be involved in in vivo specific cellular rejection.

Animals↗
PubMed2001

Co-incubation of pig islet cells with spleen cells from non-obese diabetic mice causes decreased insulin release by non-T-cell- and T-cell-mediated mechanisms.

In vitro studies were conducted in the non-obese diabetic (NOD) mouse, prone to Type 1 autoimmune diabetes, to investigate the mechanisms involved in cell-mediated rejection of pig islet xenografts. Our previous work concerning the mechanisms of proliferation of xenogeneic lymphocytes to pig islet cells (PIC) was not indicative of PIC impairment. Consequently, a test was developed based on perifusion analysis of the alteration of basal and stimulated insulin release from adult PIC incubated with mouse splenocytes or subsets. Compared with PIC incubation alone or with syngeneic pig splenocytes, co-incubation with mouse whole spleen cells resulted in a decrease of basal and stimulated insulin release (P < 0.001). Two components of this alteration were detected separately: PIC impairment was decreased (P < 0.01) after removal of plastic-adherent cells from spleen cells, but maintained (P < 0.01) when plastic-adherent cells alone were co-incubated with PIC. The increase of murine interleukin-1 beta when mouse plastic-adherent spleen cells were cultured with PIC (P < 0.04) was indicative of macrophage activation. Soluble factors produced during co-incubation of mouse splenocytes or plastic-adherent cells with PIC were involved in the impairment process, since supernatant fluids collected during previous PIC-mouse cell co-incubations directly altered (P < 0.01) insulin release from PIC. Moreover, impairment of PIC by mouse spleen cells was abolished (P < 0.01) by gadolinium chloride (which inhibits macrophages), but not by cyclosporin A. Another mechanism was apparent, since co-incubation of PIC with purified mouse T cells or CD4+ T cells, re-mixed with antigen-presenting cells, led to a decrease (P < 0.01) of insulin release. This model, based on the alteration of dynamic basal and stimulated insulin release, is indicative of in vitro cell-mediated alteration of PIC in the NOD mouse. The effect of whole spleen cells was rapid, and a crucial role was played by plastic-adherent cells. Two mechanisms were responsible for the behaviour of these cells: an early direct effect (at least in part via soluble products); and the indirect presentation of PIC xenoantigens (leading to impairment by CD4+ T lymphocytes).

Animals↗
PubMed2000

Listeria monocytogenes ActA protein interacts with phosphatidylinositol 4,5-bisphosphate in vitro.

The N-terminal region of the Listeria monocytogenes ActA protein, in conjunction with host cell factors, is sufficient for actin polymerization at the bacterial surface. Previous data suggested that ActA could protect barbed ends from capping proteins. We tested this hypothesis by actin polymerization experiments in the presence of the ActA N-terminal fragment and capping protein. ActA does not protect barbed ends from capping protein. In contrast, this polypeptide prevents PIP(2) from inhibiting the capping activity of capping protein. Gel filtration and tryptophan fluorescence experiments showed that the purified ActA N-terminal fragment binds to PIP(2) and PIP, defining phosphoinositides as novels ligands for this functional domain of ActA. Phosphoinositide binding to the N-terminal region of ActA may induce conformational changes in ActA and/or facilitate binding of other cell components, important for ActA-induced actin polymerization.

Actin Depolymerizing Factors↗
PubMed2000

[Acceptability of xenograft in type 1 diabetic patients and by the general French population].

OBJECTIVE: The possibility of using pig xenografts raises the questions of their acceptability and the reasons for reluctance by patients and society, which have not been clearly investigated in Europe. RESEARCH DESIGN AND METHODS: A survey using a multiple-choice questionnaire was conducted to quantify the acceptability of pig xenografts in type 1 diabetic patients potentially concerned by xenografts (n = 377) as compared to a sample of the French population (n = 697). RESULTS: Willingness to accept a xenograft was significantly greater among diabetic patients than the general population (64% vs 54%, P < 0.001). The notion of using pig xenografts appears to be rather well accepted by the general population, and more information might improve acceptability. The acceptance of xenografts in general and pig tissues in particular was higher in diabetic patients. CONCLUSIONS: Because the general population and type 1 diabetic patients are not aware of the sanitary risks specifically related to a xenograft, the decision to use xenografts cannot be based simply on the expectations of possible recipients. The sanitary risks need to be assessed before further xenografts are performed, particularly in diabetic patients whose risk/benefit ratio is not particularly favourable.

Animals↗
PubMed1999

Characterisation of a new operon encoding a Zur-like protein and an associated ABC zinc permease in Listeria monocytogenes.

Metal ions uptake is mainly studied for iron, as it has often been implicated in bacterial virulence. Although Listeria monocytogenes virulence is expected to be controlled by the iron availability, little is known about such an uptake and its regulation. We describe the analysis of the first operon involved in metal ions uptake in L. monocytogenes. Its three ORFs encode respectively (1) an ABC protein, likely implicated in zinc uptake, (2) a hydrophobic membrane protein, generally associated with ABC proteins and (3) a ferric uptake regulator-like protein, that we named zinc uptake regulator, as it shows strong homologies with the zinc uptake regulator, a regulator of the zinc homeostasis in Bacillus subtilis. The expression of this operon is regulated by the zinc concentration.

Amino Acid Sequence↗
PubMed1999

Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation.

BACKGROUND: Cdc42, a GTP-binding protein of the Rho family, controls actin cytoskeletal organization and helps to generate actin-based protruding structures, such as filopodia. In vitro, Cdc42 regulates actin polymerization by facilitating the creation of free barbed ends - the more rapidly growing ends of actin filaments - and subsequent elongation at these ends. The Wiskott- Aldrich syndrome protein, WASP, which has a pleckstrin-homology domain and a Cdc42/Rac-binding motif, has been implicated in cell signaling and cytoskeleton reorganization. We have investigated the consequences of local recruitment of activated Cdc42 or WASP to the plasma membrane. RESULTS: We used an activated Cdc42 protein that could be recruited to an engineered membrane receptor by adding rapamycin as a bridge, and added antibody-coupled beads to aggregate these receptors. Inducible recruitment of Cdc42 to clusters of receptors stimulated actin polymerization, resulting in the formation of membrane protrusions. Cdc42-induced protrusions were enriched in the vasodilator-stimulated phosphoprotein VASP and the focal-adhesion-associated proteins zyxin and ezrin. The Cdc42 effector WASP could also induce the formation of protrusions, albeit of different morphology. CONCLUSIONS: This is the first demonstration that the local recruitment of activated Cdc42 or its downstream effector, WASP, to a membrane receptor in whole cells is sufficient to trigger actin polymerization that results in the formation of membrane protrusions. Our data suggest that Cdc42-induced actin-based protrusions result from the local and serial recruitment of cytoskeletal proteins including zyxin, VASP, and ezrin.

Actins↗
PubMed1999

Intensity and mechanisms of in vitro xenorecognition of adult pig pancreatic islet cells by CD4+ and CD8+ lymphocytes from type I diabetic or healthy subjects.

The intensity and mechanisms of cell-mediated rejection of pig islet cells were studied in 49 Type I diabetic and 34 healthy subjects. Human peripheral mononuclear cells proliferated strongly in response to pig islet cells (p<0.001), though with notable interindividual variations (stimulation index 2 to 215). The variance of stimulation index was higher in diabetic than healthy subjects (p<0.0001). The response to islet cells was stronger (p<0.01) than that to pig splenocytes. Proliferation in response to islet cells was strongly decreased (p<0.01) when CD4+ T cells were blocked with monoclonal antibodies, whereas the blocking of CD8+ cells or NK cells gave less pronounced effects. The response to islet cells was decreased (p<0.01), but not abolished, after antigen-presenting cells were removed. Purified CD4+ cells alone did not proliferate in response to islet cells but recovered their proliferative ability when mixed with antigen-presenting cells, whereas CD8+ cells alone proliferated in the presence of interleukin-2 in response to islet cells. Proliferation was blocked (p<0.01) by anti-DR monoclonal antibodies. During proliferation in response to islet cells, interleukin-10 increased 43-fold (p<0.01) but interferon-gamma increased only slightly. No statistical differences were detected between diabetic and control subjects with respect to lymphocyte subsets and the recognition mechanisms or to interferon-gamma/interleukin-10 production in response to islet cells. These results provide the first detailed information on human cell-mediated xenoreaction to pig islet cells. This situation involves a dominant CD4 class II-restricted Th2 response, with an indirect recognition pathway, as well as a CD8 T-cell response resulting from direct recognition. This strong reaction constitutes a serious obstacle which may vary in degree among subjects.

Adult↗
PubMed1999

A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conorii.

Listeria monocytogenes, Shigella flexneri, and Rickettsia conorii are three bacterial pathogens that are able to polymerize actin into 'comet tail' structures and move within the cytosol of infected cells. The actin-based motilities of L. monocytogenes and S. flexneri are known to require the bacterial proteins ActA and IcsA, respectively, and several mammalian cytoskeleton proteins including the Arp2/3 complex and VASP (vasodilator-stimulated phosphoprotein) for L. monocytogenes and vinculin and N-WASP (the neural Wiskott-Aldrich syndrome protein) for S. flexneri. In contrast, little is known about the motility of R. conorii. In the present study, we have analysed the actin-based motility of this bacterium in comparison to that of L. monocytogenes and S. flexneri. Rickettsia moved at least three times more slowly than Listeria and Shigella in both infected cells and Xenopus laevis egg extracts. Decoration of actin with the S1 subfragment of myosin in infected cells showed that the comet tails of Rickettsia have a structure strikingly different from those of L. monocytogenes or S. flexneri. In Listeria and Shigella tails, actin filaments form a branching network while Rickettsia tails display longer and not cross-linked actin filaments. Immunofluorescence studies revealed that the two host proteins, VASP and (&agr;)-actinin colocalized with actin in the tails of Rickettsia but neither the Arp2/3 complex which we detected in the Shigella actin tails, nor N-WASP, were detected in Rickettsia actin tails. Taken together, these results suggest that R. conorii may use a different mechanism of actin polymerization.

Actins↗
PubMed1998

In vitro xenorecognition of adult pig pancreatic islet cells by splenocytes from nonobese diabetic or non-diabetes-prone mice.

BACKGROUND: In vitro studies of the nonobese diabetic (NOD) mouse prone to type 1 autoimmune diabetes were conducted in order to investigate the mechanisms possibly involved in cell-mediated rejection of adult pig islet xenografts. Mouse cellular proliferation in discordant situations was previously investigated only with stimulator lymphocytes and found to be low in intensity and due to an indirect recognition mechanism involving murine antigen-presenting cells (APC). It was also important to characterize murine anti-pig islet response. METHODS AND RESULTS: In the present study, mouse splenocytes responded to pig islet cells since primary proliferations were detected in non-diabetes-prone Balb/c (P<0.04) or NOD (P<0.001) mice. Moreover, NOD mice displayed a higher (P<0.003) splenocyte response to pig islet cells (stimulation index: 5.8+/-0.7) than did Balb/c mice (stimulation index: 2.3+/-0.3), whereas responses to pig stimulator splenocytes were similar in both strains. The proliferation of NOD splenocytes to pig islet cells was lower (P<0.0001) than the allogeneic response to Balb/c islet cells but similar to syngeneic proliferation to NOD islet cells. In both NOD and Balb/c mice, splenocyte proliferation to pig islet cells was abolished (P<0.01) when CD4+ cells were blocked with antibodies, whereas the blocking of CD8+ cells had a nonsignificant effect. The main T-splenocyte subsets involved were restricted to mouse MHC class II molecules as they did not proliferate in the presence of monoclonal antibodies directed at I-A molecules. NOD and Balb/c splenocyte proliferation to pig islet cells was abolished after removal of plastic-adherent APC, which indicates that the major activation pathway was indirect. Purified CD4+ or CD8- cells alone did not proliferate in response to pig islet cells but recovered a proliferative ability when mixed with APC. CD4- cells, alone or in the presence of APC, were not capable of responding to pig islet cells. Both Th1 and Th2 splenocytes were involved in response to pig islet cells since interferon-gamma (IFN-gamma) and interleukin (IL-)-4 production increased significantly (300-fold and 11-fold, respectively, P<0.02 for both), whereas the increase in IL-10 production was much lower (only 1.5-fold). The IFN-gamma/IL-4 and IFN-gamma/IL-10 ratios stimulated by pig islet cells were not different with NOD and Balb/c splenocytes. CONCLUSION: In conclusion, mouse cell-mediated reaction against xenogeneic adult pig islet cells mainly involves class II-restricted CD4+ T lymphocytes of Th1 and Th2 subtypes, with an indirect pathway for the recognition. Although of low intensity, this cell-mediated reaction constitutes an obstacle to pig islet engraftment in the mouse, although one not necessarily more insurmountable than alloreactivity. The peculiarity of NOD mouse splenocytes, in terms of proliferation against pig islets, suggests that the study of islet xenograft rejection should take the immunogenetic context of diabetes into account, in which case the use of non-diabetes-prone mice has its limitations.

Animals↗
PubMed1998

Spleen cells of non-obese diabetic mice fed with pig splenocytes display modified proliferation and reduced aggressiveness in vitro against pig islet cells.

A new means of modifying xenogeneic reaction to pig islet cells, which involves pre-feeding with pig spleen cells, was investigated for the first time in the non-obese diabetic (NOD) mouse. Compared with controls, mice fed with pig spleen cells displayed much higher splenocyte proliferation in response to pig spleen and islet cells (p < 0.0001). This enhanced proliferation was specific for the species providing the fed cells. Positive relationships (p < 0.01) were found between increased splenocyte proliferation in response to pig spleen or islet cells and the number of cells per feeding or the number of daily feedings. Concomitantly, while co-incubation with splenocytes from control mice led to inhibition of both basal and stimulated insulin releases from pig islet cells (p < 0.001), this aggressiveness was abolished (p < 0.001) after co-culture with splenocytes from mice fed with pig spleen cells. The proliferative responses of splenocytes from fed or control mice to pig islet or spleen cells were abolished after removal of plastic-adherent cells, indicating that the major indirect pathway of T-cell activation was unchanged by pig spleen cell feeding. The main T-splenocyte subsets involved were restricted to MHC class II as they did not proliferate in the presence of monoclonal antibodies (mAbs) directed at I-A molecules. In mice fed with pig spleen cells, as well as in control mice, the blocking of CD4 + T cells with mAbs led to abolition of proliferation (p < 0.002), while the blocking of CD8 + led to a less marked effect. However, an increase in the blocking effect of anti-CD8 mAbs was noted in mice fed with pig spleen cells (p < 0.02). In control mice, the main splenocyte subset involved during proliferation in response to pig islet cells was Thl, since interferon gamma (IFNgamma) production increased significantly (p < 0.01) while that of interleukin-10 (IL-10) increased only slightly. The main change observed in mice fed with pig spleen cells was a marked increase in basal IL-10 production (p < 0.01) and the basal IL-10/IFNgamma ratio (p < 0.001). It seems likely that feeding with pig spleen cells shifted the Th1/Th2 balance towards a dominance of Th2-type class II-restricted CD4 + T cells, which may have been conducive to activating CD8 + suppressor T cells. In any event, oral administration of pig cells modified xenogeneic cellular response, which may have implications for xenografts of pig islets. In a more general sense, physiological feeding of cells from xenogeneic species would appear to have certain effects on the immune system.

Administration, Oral↗
PubMed1998

Identification of cofilin, coronin, Rac and capZ in actin tails using a Listeria affinity approach.

Actin assembly is involved in cell motility and intracellular movement of Listeria monocytogenes. Induction of Listeria actin tails is mediated by the surface protein ActA. The N-terminal domain of ActA is sufficient for this function. Cell components known to play a role in the actin-based motility of Listeria are VASP (vasodilatator-stimulated phosphoprotein), the multiprotein Arp2/3 complex and cofilin. VASP interacts with the central domain of ActA. Proteins interacting with the N-terminal domain of ActA have not been identified. To identify novel host cell components of ActA-induced actin tails, we used bovine brain extracts and an affinity approach with Listeria as matrix. Several known components of Listeria tails were isolated including VASP, Arp3 and cofilin. Cofilin was identified by peptide sequencing, and cofilin recruitment and Listeria tail length were found to be pH-dependent, in agreement with its recently reported role in enhancing actin filament turnover. In addition, three proteins not previously known to be associated with Listeria tails, coronin, Rac and capZ, were identified in our affinity approach. In infected cells, the localization of the identified proteins was studied by immunofluorescence. Our findings suggest that these latter proteins, which are known to play critical roles in cellular actin rearrangements, may also be involved in the dynamics of Listeria-induced actin assembly.

Actin Depolymerizing Factors↗
PubMed1998

Perifusion analysis of insulin secretion from specific pathogen-free large-white pig islets shows satisfactory functional characteristics for xenografts in humans.

Though the pig appears to be the islet donor of choice for grafts in diabetic patients, there may be a risk of transmission of infectious agents. In this context, we adopted a strategy of islet isolation from pigs raised and killed in specific pathogen-free (SPF) conditions as a minimum with regard to the concept of quality assurance. Accordingly, the present study investigated the function of SPF pig islets to determine whether they react qualitatively and quantitatively to nutriments, hormones and neuromediators with which they would be confronted in man and could therefore provide effective regulation during physiologic or physiopathologic situations. beta cells from 18 Large-White SPF pigs were functionally intact after 7 days in culture. Insulin stimulation indexes (SI) of 3.1 +/- 0.2, 2.2 +/- 0.1, and 4.4 +/- 0.3 were found respectively for 30 mmol/l K+, 100 mumol/l tolbutamide and 10 mmol/l theophylline. Basal insulin secretion (72.2 +/- 7.6 muU/min) had already increased significantly (p < 0.001) with 5.5 mmol/l glucose (184.2 +/- 25.5 muU/min, SI: 2.5 +/- 0.6), indicating that the threshold stimulatory concentration was comparable to that of human islets. Insulin secretion increased in a glucose dose-dependent manner (p < 0.001): SI: 3.1 +/- 0.3 and 3.6 +/- 0.2 with 11.0 mmol/l and 22.0 mmol/l glucose, which showed a satisfactory magnitude with reference to human islets. Even the subtle phenomenon of "glucose memory" was apparent in these pig islets. Arginine stimulated (p < 0.001) insulin secretion dose-dependently (SI: 2.2 +/- 0.3 with 5 mmol/l and 2.9 +/- 0.2 with 10 mmol/l). The ketone body beta-hydroxybutyrate (10 mmol/l) also induced insulin secretion (SI: 4.3 +/- 0.3). Insulin release was stimulated by 4 mumol/l gastric inhibitory peptide, revealing sensitivity to the hormonal enteroinsular axis, and by 2 mumol/l glucagon. Parasympathetic cholinergic influence was studied using 500 mumol/l carbamylcholine, which increased insulin secretion. The influence of orthosympathetic control and of stress situations was also studied. As in human islet response, epinephrine and the alpha 2-agonist clonidine (50 mumol/l) inhibited insulin secretion. Finally pre-culture of islets may be beneficial for graft outcome, provided that no deterioration in islet function occurs. A prolonged 21-day culture of SPF pig islets showed no decrease in insulin response to glucose, arginine and potassium, even with an unaltered threshold stimulatory glucose concentration. Thus, Large-White SPF pigs and the application of our isolation procedure provided islets with functional characteristics reproducibly compatible with potential utilisation for effective regulation of glycaemia under physiologic and physiopathologic situations in humans.

Animals↗
PubMed1998

Long-term culture of free or encapsulated islets isolated from specific pathogen-free (SPF) pigs.

As the risk of recipient contamination is a limiting factor for xenotransplantation, the use of specific pathogen-free (SPF) pigs is mandatory. This study investigated the long-term culture of SPF pig islets and evaluated their insulin production when encapsulated in AN69 hollow fibres. Insulin secretion was studied after 3 weeks (n = 10), 2 months (n = 8) and 3 months (n = 3) by 45-min incubation in the presence of 2.8, 5.5, 11 and 16.5mM glucose. Although a decrease in the amount of secreted insulin occurred (1385 +/- 421 and 4323 +/- 1068 microns U/ml at 3 weeks for 2.8 and 16.5 mM glucose respectively; 702 +/- 261 and 2397 +/- 1047 microU/ml at 2 months; 59 +/- 23 and 154 +/- 34 microU/ml at 3 months), glucose-dependent insulin secretion was observed in all cases, i.e. stimulation indices of 8.1 +/- 3.1 (p < 0.05 vs the presence of 5.5 mM glucose) at 3 weeks, 3.3 +/- 1.1 at 2 months and 3.0 +/- 0.7 at 3 months. The insulin secretion of encapsulated SPF pig islets, cultured for 1 or 3 weeks, was evaluated under perifusion conditions using a stimulus of 10mM glucose plus 5.5 mM theophylline. Glucose stimulation resulted in a significant two-fold increase in insulin secretion (p < 0.05), which was maintained over culture time. These results indicate that SPF-isolated islets remained functional when cultured for several weeks either as free or encapsulated islets, although the magnitude of insulin secretion decreased dramatically after three months of culture.

Animals↗
PubMed1997

ActA is a dimer.

ActA, a surface protein of Listeria monocytogenes, is able to induce continuous actin polymerization at the rear of the bacterium, in the cytosol of the infected cells. Its N-terminal domain is sufficient to induce actin tail formation and movement. Here, we demonstrate, using the yeast two-hybrid system, that the N-terminal domain of ActA may form homodimers. By using chemical cross-linking to explore the possibility that ActA could be a multimer on the surface of the bacteria, we show that ActA is a dimer. Cross-linking experiments on various L. monocytogenes strains expressing different ActA variants demonstrated that the region spanning amino acids 97-126, and previously identified as critical for actin tail formation, is also critical for dimer formation. A model of actin polymerization by L. monocytogenes, involving the ActA dimer, is presented.

Bacterial Proteins↗
PubMed1997

Identification of two regions in the N-terminal domain of ActA involved in the actin comet tail formation by Listeria monocytogenes.

The ActA protein of Listeria monocytogenes induces actin nucleation on the bacterial surface. The continuous process of actin filament elongation provides the driving force for bacterial propulsion in infected cells or cytoplasmic extracts. Here, by fusing the N-terminus of ActA (residues 1-234) to the omega fragment of beta-galactosidase, we present the first evidence that this domain contains all the necessary elements for actin tail formation. A detailed analysis of ActA variants, in which small fragments of the N-terminal region were deleted, allowed the identification of two critical regions. Both are required to initiate the actin polymerization process, but each has in addition a specific role to maintain the dynamics of the process. The first region (region T, amino acids 117-121) is critical for filament elongation, as shown by the absence of actin tail in a 117-121 deletion mutant or when motility assays are performed in the presence of anti-region T antibodies. The second region (region C, amino acids 21-97), is more specifically involved in maintenance of the continuity of the process, probably by F-actin binding or prevention of barbed end capping, as strongly suggested by both a deletion (21-97) leading to 'discontinuous' actin tail formation and in vitro experiments showing that a synthetic peptide covering residues 33-74 can interact with F-actin. Our results provide the first insights in the molecular dissection of the actin polymerization process induced by the N-terminal domain of ActA.

Actins↗