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D Ferrari

Publications and source records attributed to D Ferrari.

At least 19 recordsLinked to original sources
PubMed1997

FGF-stimulated outgrowth and proliferation of limb mesoderm is dependent on syndecan-3.

The outgrowth of the mesoderm of the developing limb bud in response to the apical ectodermal ridge (AER) is mediated at least in part by members of the FGF family. Recent studies have indicated that FGFs need to interact with heparan sulfate proteoglycans in order to bind to and activate their specific cell surface receptors. Syndecan-3 is an integral membrane heparan sulfate proteoglycan that is highly expressed by the distal mesodermal cells of the chick limb bud that are undergoing proliferation and outgrowth in response to the AER. Here we report that maintenance of high-level syndecan-3 expression by the subridge mesoderm of the chick limb bud is directly or indirectly dependent on the AER, since its expression is severely impaired in the distal mesoderm of the limb buds of limbless and wingless mutant embryos which lack functional AERs capable of directing the outgrowth of limb mesoderm. We have also found that exogenous FGF-2 maintains a domain of high-level syndecan-3 expression in the outgrowing mesodermal cells of explants of the posterior mesoderm of normal limb buds cultured in the absence of the AER and in the outgrowing subapical mesoderm of explants of limbless mutant limb buds which lack a functional AER. These results suggest that the domain of high-level syndecan-3 expression in the subridge mesoderm of normal limb buds is maintained by FGFs produced by the AER. Finally, we report that polyclonal antibodies against a syndecan-3 fusion protein inhibit the ability of FGF-2 to promote the proliferation and outgrowth of the posterior subridge mesoderm of limb buds cultured in the absence of the AER. These results suggest that syndecan-3 plays an essential role in limb outgrowth by mediating the interaction of FGFs produced by the AER with the underlying mesoderm of the limb bud.

Animals↗
PubMed1997

Purinergic modulation of interleukin-1 beta release from microglial cells stimulated with bacterial endotoxin.

Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today, 16:524-528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R. A. North and G. Buell, 1996. Science (Wash. DC). 272:735-737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1 beta. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1 beta release triggered by LPS. Our data suggest that LPS-dependent IL-1 beta release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1 beta secretion.

Adenosine Triphosphate↗
PubMed1997

Reduction of protein disulfide bonds in an oxidizing environment. The disulfide bridge of cholera toxin A-subunit is reduced in the endoplasmic reticulum.

Following retrograde transport to the endoplasmic reticulum (ER) the A-subunit of cholera toxin (CTX-A) is partially cleaved into CTX-A1 and CTX-A2 by reduction of a disulfide bridge [Majoul et al. (1996) J. Cell Biol. 133, 777-789], although the redox state in the ER favors disulfide formation. We show here that the disulfide bridge of CTX-A is cleaved in vitro already at GSH/GSSG ratios between 1 and 3. Protein disulfide isomerase (PDI) exerts only a minor accelerating effect. Various mixed disulfide intermediates (CTX-A1-S-S-CTX-A1; PDI-S-S-A2; PDI-S-S-A1) appear during CTX-A reduction. These results indicate that in the ER protein disulfide formation and protein disulfide reduction can take place simultaneously.

Animals↗
PubMed1996

Mouse microglial cells express a plasma membrane pore gated by extracellular ATP.

We have investigated responses to extracellular ATP (ATPe) in the microglial cell lines N9 and N13 and in freshly isolated mouse microglial cells. Upon stimulation with this nucleotide, N9 and N13 cells underwent an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i), a sustained depolarization of the plasma membrane, and an uptake of extracellular markers such as ethidium bromide and lucifer yellow; increases in plasma membrane permeability were paralleled by striking morphologic changes. ATPe, as well as other nucleotides, activated a spiking Ca2+ release from intracellular stores; however, only ATPe was also able to cause a massive transmembrane Ca2+ influx. The ATP analogue 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) triggered a sustained Ca2+ influx accompanied by little release from stores. The ATP derivative oxidized ATP (oATP) strongly inhibited Ca2+ influx, minimally affecting Ca2+ release. From ATPe-sensitive microglial cell lines, we selected several ATPe-resistant clones that showed complete lack of ATPe-mediated plasma membrane permeability changes, although they retained the Ca2+ mobilization response from intracellular stores. ATPe-dependent plasma membrane permeability changes were also greatly reduced in growth-arrested microglial cells. Finally, ATPe triggered IL-1 beta release from wild-type but not ATPe-resistant microglial cells. These results show that microglial cells express at least two purinergic receptor subtypes, metabotropic (P2Y) and ionotropic (P2Z), and that the latter is modulated during cell cycle and coupled to IL-1 beta release.

Adenosine Triphosphate↗
PubMed1996

An ATP-activated channel is involved in mitogenic stimulation of human T lymphocytes.

We investigated the effect of pharmacologic modulation of the ATP receptor on intracellular ion changes and proliferative response of human peripheral blood lymphocytes (PBLs) and purified T lymphocytes. Extracellular ATP (ATPe) triggered in these cells an increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization. Whereas both Ca2+ release from intracellular stores and influx across the plasma membrane were detected in the whole PBL population, only Ca2+ influx was observed in T cells. In the presence of near physiologic extracellular Na+ concentrations (125 mmol/L), Ca2+ permeability through the ATPe-gated channel was very low, suggesting a higher selectivity for monovalent over divalent cations. The selective P2Z agonist benzoylbenzoic ATP (BzATP) increased [Ca2+]i in the presence but not the absence of extracellular Ca2+ and also caused plasma membrane depolarization. The covalent blocker oxidized ATP (oATP), an inhibitor of P2X and P2Z receptors, prevented Ca2+ influx and plasma membrane depolarization, but had no effect on Ca2+ release from stores. Stimulation with ATPe alone had no significant effects on PBL 3H-thymidine incorporation. On the contrary, ATPe or BzATP had a synergistic effect on DNA synthesis stimulated by selective T-cell mitogens such as phytohemagglutinin, anti-CD3 monoclonal antibody, or allogenic PBLs (mixed lymphocyte cultures). Treatment with oATP inhibited mitogenic stimulation by these receptor-directed agents but not by the combined application of the Ca2+ ionophore ionomycin and phorbol myristate acetate. Interleukin-2 partially relieved inhibition by oATP. These results suggest that human T lymphocytes express a plasma membrane channel gated by ATPe that is involved in mitogenic stimulation.

Adenosine Triphosphate↗
PubMed1996

Role of the purinergic P2Z receptor in spontaneous cell death in J774 macrophage cultures.

J774 mouse macrophages express an ionotropic receptor gated by extracellular ATP. Activation of this receptor, currently named purinergic P2Z, causes transmembrane ion fluxes, plasma membrane depolarization, cell swelling and eventual cell death. The physiological role of this receptor is as yet unknown. In the present report we show that macrophage cell clones that hypo-express the P2Z receptor showed a very low degree of spontaneous cell death in culture, while hyper-expressing clones were exceedingly susceptible to cell death. To further support a role for ATP receptors in spontaneous cell death, addition to the macrophage cell cultures of oxidized ATP, a selective inhibitor of ionotropic purinergic receptors, or the ATP-hydrolysing enzyme apyrase, also reduced spontaneous death.

Adenosine Triphosphate↗
PubMed1996

P2 purinoceptors in the immune system.

Immune cells express plasma membrane receptors for extracellular nucleotides. Both G protein-linked metabotropic and channel-forming ionotropic receptors have been described, although no P2 receptor subtype has been cloned from the immune system thus far. Metabotropic receptors have been described in human B but not T lymphocytes; they have not been found in mouse B and T cells. Ionotropic receptors seem to be ubiquitously expressed in the immune system; however, their functional properties, if not their pharmacology, appear to be different in different immune cells. Human T normal and B leukaemic lymphocytes, human macrophages, mouse B and T lymphocytes, mouse microglial and macrophage cells, and rat mast cells express ionotropic receptors that recognize ATP4- as the preferred ligand, are activated by 3'-O-(4-benzoyl)benzoyl ATP and inhibited by oxidized ATP. The pharmacological profile of ionotropic receptors expressed by different immune cells is similar, but their permeability properties may be different: the pore formed by receptors expressed by macrophages, microglial cells and mast cells is typically permeable to charged molecules of molecular mass up to 900 Da; on the contrary, that expressed by lymphocytes has a molecular cut-off of 200-300 Da. The ionotrpic receptor of immune cells is modulated by inflammatory cytokines (e.g. interleukin [IL]-2 and gamma-interferon) and is also modulated during monocyte to macrophage differentiation. Transient stimulation of the ionotropic receptor of macrophages and microglial cells elicits IL-1 beta release. Sustained activation leads to cell death, either by necrosis or apoptosis, depending on the given cell type.

Animals↗
PubMed1996

Topical oxatomide: an alternative approach for the treatment of vulvar lichen sclerosus.

OBJECTIVE: The treatment of vulvar lichen sclerosus has greatly improved in recent years, with the introduction of new pharmacological approaches and reconsideration of the traditional ones. Oxatomide is a molecule with both antihistamine and inhibiting activities for the inflammatory response, which may have potential use against this disease. METHODS: We enrolled 22 patients affected by vulvar lichen sclerosus in a double-blind, cross-over, controlled trial. They were administered a 5% oxatomide-based gel formulation and a placebo (petrolatum ointment) topically. At the beginning and the end of the investigation the severity and duration of symptoms, clinical appearance and tolerability were recorded. Statistical analysis of data was performed by Fisher's exact test and the Student-Newman-Keuls test. RESULTS: The results indicate that both regimens are significantly correlated with vulvar pruritus improvement and that 5% oxatomide gel has better anti-itching effects compared to the placebo. Other symptoms responded poorly to the treatment. The rate of complete-partial regression of pruritus was significantly higher (P < 0.05) in the oxatomide group than in controls, while no significant improvement was obtained neither with oxatomide or with placebo in terms of clinical appearance. CONCLUSIONS: These results, adequately confirmed, could suggest the introduction of oxatomide, in selected patients, as an alternative for the treatment of vulvar lichen sclerosus.

Administration, Topical↗
PubMed1996

Vascular endothelial growth factor. Expression in human vulvar neoplastic and nonneoplastic tissues.

OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF) in human vulvar neoplastic and nonneoplastic tissues. STUDY DESIGN: Specimens were collected at the Vulvovaginal Clinic, Department of Obstetrics and Gynecology, University of Milan. Human vulvar neoplastic and nonneoplastic tissues were dissected and frozen immediately at -80 degrees C until RNA extraction. Five micrograms of total RNA from each sample was denatured and transferred to nitrocellulose and nylon membranes for dot blot hybridization with labeled [alpha-32P]dCTP cDNA probe for VEGF. RESULTS: Messenger RNA encoding VEGF was detected in all tissues studied. VEGF mRNA was highly expressed in vulvar epithelial neoplasia (VIN) associated with human papillomavirus infection and minimally expressed in invasive squamous cells carcinoma of the vulva. Nonneoplastic lesions, such as chronic inflammation, lichen sclerosus, lichen planus, squamous hyperplasia and squamous papilloma, were also assessed, and none had a significant difference in VEGF mRNA expression. CONCLUSION: The prominence of VEGF mRNA levels in particular cases of VIN demonstrated that VEGF may be involved in promoting a new vascular network as a basic condition for the progression or at least self-maintenance of those lesions.

Blotting, Northern↗
PubMed1995

Ionic regulation of endonuclease activity in PC12 cells.

We have investigated the Ca2+ dependency of DNA degradation into nucleosome-sized fragments in intact chromaffin-like PC12 cells and PC12 nuclear fractions. In intact cells we were unable to trigger DNA fragmentation by inducing either transient or sustained elevations of cytoplasmic Ca2+ ([Ca2+]i) with the Ca2+ ionophore ionomycin. On the contrary, DNA fragmentation was induced in intact cells by the intracellular Zn2+ chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). To characterize further PC12 cell endonuclease activity, we then investigated digestion by purified PC12 cell fractions of exogenously added plasmids. In nuclear fractions two endonuclease activities were identified: an acidic (pH 5.0) endonuclease activity that was fully Ca2+- and Mg(2+)-independent; and a neutral (pH 7.6) endonuclease activity that was Ca(2+)-independent but Mg(2+)-dependent. Both endonuclease activities were inhibited by Zn2+. Nuclear membrane permeabilization greatly enhanced plasmid digestion at pH 7.6, but not at pH 5.0. This suggests that neutral endonuclease was located in a membrane-bound compartment, whereas acidic endonuclease was freely accessible to the substrate even in the presence of an intact nuclear membrane. In intact nuclei, digestion of genomic DNA could not be triggered by increasing the bivalent cation composition of the medium. On the contrary, in hypotonic medium we observed a large spontaneous nucleolytic DNA degradation that was increased by Zn2+ chelation. However, an acidic pH shift was a potent stimulus for DNA fragmentation in isotonic as well as hypotonic medium.

Animals↗
PubMed1995

Activation of microglial cells by beta-amyloid protein and interferon-gamma.

Alzheimer's disease is the most common cause of progressive intellectual failure. The lesions that develop, called senile plaques, are extracellular deposits principally composed of insoluble aggregates of beta-amyloid protein (A beta), infiltrated by reactive microglia and astrocytes. Although A beta, and a portion of it, the fragment 25-35 (A beta (25-35)), have been shown to exert a direct toxic effect on neurons, the role of microglia in such neuronal injury remains unclear. Here we report a synergistic effect between A beta and interferon-gamma (IFN-gamma) in triggering the production of reactive nitrogen intermediates and tumour-necrosis factor-alpha (TNF-alpha) from microglia. Furthermore, using co-culture experiments, we show that activation of microglia with IFN-gamma and A beta leads to neuronal cell injury in vitro. These findings suggest that A beta and IFN-gamma activate microglia to produce reactive nitrogen intermediates and TNF-alpha, and this may have a role in the pathogenesis of neuronal degeneration observed in ageing and Alzheimer's disease.

Aging↗
PubMed1995

The expression pattern of the Distal-less homeobox-containing gene Dlx-5 in the developing chick limb bud suggests its involvement in apical ectodermal ridge activity, pattern formation, and cartilage differentiation.

Here we report the isolation from a chick limb bud cDNA library of a cDNA that contains the full coding sequence of chicken Dlx-5, a member of the Distal-less (Dlx) family of homeobox-containing genes that encode homeodomains highly similar to that of the Drosophila Distal-less gene, a gene that is required for limb development in the Drosophila embryo. The expression pattern of Dlx-5 in the developing chick limb bud suggests that it may be involved in several aspects of limb morphogenesis. Dlx-5 is expressed in the apical ectodermal ridge (AER) which directs the outgrowth and patterning of underlying limb mesoderm. During early limb development Dlx-5 is also expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the mid-proximal posterior margin that corresponds to the posterior necrotic zone. These mesodermal domains of Dlx-5 expression roughly correspond to the anterior and posterior boundaries of the progress zone, the group of highly proliferating undifferentiated mesodermal cells underneath the AER that will give rise to the skeletal elements of the limb and associated structures. The AER and anterior and posterior mesodermal domains of Dlx-5 expression are regions in which the homeobox-containing gene Msx-2 is also highly expressed, suggesting that Dlx-5 and Msx-2 might be involved in regulatory networks that control AER activity and demarcate the progress zone. In addition, Dlx-5 is expressed in high amounts by the differentiating cartilaginous skeletal elements of the limb, suggesting it may be involved in regulating the onset of limb cartilage differentiation.

Amino Acid Sequence↗
PubMed1995

Increased sensitivity of DMD lymphoblastoid cell to low doses of X-irradiation.

Several cell membrane abnormalities affecting various cell populations have been reported in Duchenne muscular dystrophy (DMD) by different investigators. In peripheral blood lymphocytes intrinsic cellular membrane defect evidentiated by impairment of capping capacities has been repeatedly obtained, suggesting that DMD product could act in such cellular phenotype at the cytoskeletal compartment. It has been previously reported that lymphoid cells are characterized by high radiosensitivity. On the assumption that DMD phenotypes could increase this susceptibility, we have compared the radiosensitivity of normal and DMD lymphoblastoid cell lines (LCLs) to small doses (0-2Gy) of x-irradiation. The results obtained suggest an increased sensitivity of DMD cells without Ca++ uptake or apoptotic phenomena, associated with an effect upon cell cycle length.

Calcium↗
PubMed1995

The purinergic P2Z receptor of human macrophage cells. Characterization and possible physiological role.

We have investigated responses of human monocyte/macrophage cells to extracellular ATP (ATPe). Freshly isolated peripheral blood monocytes showed responses linked to P2Y but not P2Z purinergic receptors; however, during in vitro macrophage differentiation, these cells also exhibited responses suggestive of the presence of the membrane-permeabilizing P2Z receptor. In fact, in human macrophages a brief (15-min) exposure to ATPe, but not other nucleotides, caused (1) a rapid and long-lasting plasma membrane depolarization; (2) a large increase in intracellular Ca2+ concentration followed by efflux of the Ca2+ indicator; (3) uptake of low molecular weight hydrophilic molecules such as Lucifer yellow and ethidium bromide; and (4) cell rounding, swelling, and eventual release of the cytoplasmic enzyme lactate dehydrogenase. rIFN-gamma enhanced both membrane-permeabilizing and cytotoxic ATPe effects. Membrane permeabilization and cytotoxicity were fully blocked by pretreatment of the cells with oxidized ATP, a compound recently shown to block P2Z receptors covalently in macrophages. Blocking of the P2Z receptor by oxidized ATP also inhibited multinucleated giant cell generation stimulated by concanavalin A or rIFN-gamma without decreasing monocyte migration or membrane adhesion molecule expression. These data suggest that human macrophages express rIFN-gamma-modulated purinergic P2Z receptors in vitro and hint at a role for these plasma membrane molecules in the generation of macrophage polykarions.

Adenosine Triphosphate↗
PubMed1994

Limb mesenchymal cells inhibited from undergoing cartilage differentiation by a tumor promoting phorbol ester maintain expression of the homeobox-containing gene Msx1 and fail to exhibit gap junctional communication.

Tumor promoting phorbol esters are potent inhibitors of the chondrogenic differentiation of limb mesenchymal cells, but the mechanism by which these agents elicit their antichondrogenic effect is unknown. Here we report that limb mesenchymal cells inhibited from undergoing chondrogenesis by a tumor promoting phorbol ester exhibit deregulated expression of the homeobox-containing gene Msx1, a gene implicated in suppressing differentiation of limb mesenchymal cells, and fail to exhibit the extensive gap junctional intercellular communication that normally occurs at the onset of chondrogenesis. These results suggest that tumor promoting activity of phorbol esters may relate to their ability to modulate the expression of regulatory genes involved in controlling terminal differentiation, as well as to their ability to disrupt the intercellular communication involved in initiating the differentiated phenotype of cells.

Animals↗
PubMed1994

Macrophages loaded with doxorubicin by ATP-mediated permeabilization: potential carriers for antitumor therapy.

In many cell types extracellular ATP (ATPe) has been shown to cause reversible plasma membrane permeabilization to low molecular weight (< 900 Da) water-soluble compounds. In the present report we have exploited this technique to incorporate the anticancer drug doxorubicin (DXR), molecular mass 543 Da, into the cytoplasm of two mouse cell lines that had previously been shown to express the ATPe-gated pore, J774 macrophages and tumor necrosis factor (TNF)-resistant L929 fibroblasts. Compared to passively loaded cells, ATPe-mediated reversible permeabilization allowed an at least 4-fold increase in DXR intracellular trapping (0.5 pg/cell versus 2 pg/cell). Analysis of the release kinetics at 37 degrees C showed that about 40% of total intracellular DXR was discharged during the first hour from both ATPe-permeabilized and passively loaded cells; about 15% further release was observed upon incubation up to 4 h. DXR release profiles were similar in ATPe-permeabilized and passively loaded cells. ATPe-permeabilized, DXR-loaded (ATPe-DXR) cells strongly inhibited the proliferation of K562 tumor cells. Taken together these results indicate that ATPe-mediated reversible plasma membrane permeabilization can be effectively used to load cells of different histotypes with high concentrations of DXR. This approach could permit to vehicle high doses of anticancer agents by using living cells while reducing systemic toxic effects.

Adenosine Triphosphate↗
PubMed1994

Responses to extracellular ATP of lymphoblastoid cell lines from Duchenne muscular dystrophy patients.

We have observed a striking difference in the response to extracellular ATP in lymphoblastoid cell lines established from Duchenne muscular dystrophy patients and normal subjects. Duchenne muscular dystrophy cells stimulated by extracellular ATP underwent a large increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization, while normal cell lines were little or not at all responsive. These changes in intracellular ion homeostasis were due to activation of an ATP-gated membrane channel permeable to Na+ and Ca2+, with little or no contribution of Ca2+ release from intracellular stores. The channel was selectively activated by ATP, since other purine/pyrimidine nucleotides were ineffective, and it was inhibited by pretreatment with oxidized ATP, a compound previously reported to irreversibly inhibit P2 purinergic receptors. In the presence of extracellular ATP, lymphoblastoid cells established from Duchenne muscular dystrophy patients, but not from healthy controls, underwent rounding and swelling and eventually lysed. The results of this study suggest that lymphoblastoid cells isolated from Duchenne muscular dystrophy patients are eminently sensitive to stimulation by extracellular ATP.

Adenosine Triphosphate↗