Biomedical subjects

Marco Marchisio

Publications and source records attributed to Marco Marchisio.

12 recordsLinked to original sources

PubMed2007

Protein kinase Calpha is differentially activated during neonatal and adult erythropoiesis and favors expression of a reporter gene under the control of the (A)gamma globin-promoter in cellular models of hemoglobin switching.

PKCalpha was found to be expressed (mRNA and protein) throughout the in vitro maturation of primary human erythroblasts but its activity (phosphorylation levels and nuclear localization) was consistently higher in cells derived from human neonatal rather than adult blood. Since the gamma/gamma + beta globin expression ratio represented the major difference between neonatal and adult erythroblasts (58 +/- 12 vs. 7 +/- 3, respectively), we tested the hypothesis that PKCalpha might affect gamma-globin expression by measuring the levels of (A)gamma- or beta-promoter-driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual microLCRbetaprRluc(A)gammaprFluc reporter in the presence of transient expression of either the constitutively active (sPKCalpha) or catalytically inactive (iPKCalpha) PKCalpha. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 microM). In all the cells analyzed, sPKCalpha significantly increased (by two- to sixfold) the levels of luciferase activity driven by the (A)gamma-promoter and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the (A)gamma-driven luciferase activity and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic-specific functions in erythropoiesis and that modulation of PKCalpha might affect the activity of (A)gamma-promoter-driven reporters.

Acetophenones↗

PubMed2006

A flow cytometry procedure for simultaneous characterization of cell DNA content and expression of intracellular protein kinase C-zeta.

A selective involvement of protein kinase C-zeta (PKC-zeta) in the events regulating cell proliferation has been recently proposed. Here we report a flow cytometric method allowing the simultaneous association of intracellular PKC-zeta expression or phosphorylation with each cell cycle phase. Current methods for flow cytometry analysis were applied to several cell lines and compared to the method developed in our laboratory. The latter includes 2% paraformaldehyde (PFA), as fixing agent, a permeabilization/saturation step by means of a solution containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.4, 0.05% NP-40, 0.25% lambda-carrageenan and 0.02% NaN3, followed by labelling with a primary antibody (PKC-zeta or P-PKC-zeta) and with the appropriate FITC-conjugated secondary antibody. Cells processed by such a method disclosed no substantial modification of light scattering features with respect to live cells. In addition, stainability with anti-PKC-zeta or anti-P-PKC-zeta antibodies was well preserved while stoichiometric staining of DNA with PI enabled accurate cell cycle analysis. Results show that a distinct up-regulation of P-PKC-zeta in G2/M phase occurs. The method here described, therefore, represents a simple, reproducible and conservative assay for a simultaneous assessment of intracellular PKC or P-PKC modulations within each cell cycle phase.

Animals↗

PubMed2006

Parallel regulation of PKC-alpha and PKC-delta characterizes the occurrence of erythroid differentiation from human primary hematopoietic progenitors.

OBJECTIVE: Erythroid differentiation is a process characterized by modulation of different proteins including phosphoinositide-related enzymes such as protein kinase C (PKC) isoforms. Because in different cell lines PKC-alpha and PKC-delta have been reported to be involved in the mechanisms controlling proliferation and differentiation, the aim of this study was to examine the relative involvement of these PKC isoforms in the development of CD235a+ erythroid cells from human healthy hematopoietic progenitors. MATERIALS AND METHODS: Erythroid differentiation from human primary hematopoietic progenitor cells was achieved by adopting the human erythroblasts mass amplification culture. Expression and activity of PKC isoforms and their relationship with proliferation and differentiation were investigated by morphologic analysis, reverse-transcriptase polymerase chain reaction, Western blotting, multiparametric flow cytometry, and transfection experiments. RESULTS: PKC-alpha was found expressed and phosphorylated in cells undergoing both proliferation and differentiation, although PKC-delta, largely expressed and activated during proliferation, was evidently downregulated during differentiation. Overexpression of PKC-delta-CAT scarcely influenced the development of glycophorin-A (CD235a)+ erythroid cells from hematopoietic progenitors, although overexpression of PKC-alpha-CAT strongly induced the development of CD235a+ erythroid cells. On the other hand, in PKC-alpha-CAT-transfected cells, pharmacologic inhibition of PKC-delta further increased the number of CD235a+ cells, although inhibition of PKC-alpha resulted in an evident impairment of the development of CD235a+ erythroid cells. CONCLUSIONS: Our results indicate that the suppression or at least a strong downregulation of PKC-delta, concomitant to PKC-alpha expression and activity, might be a cofactor to be further investigated and might be involved in the events regulating erythropoietin-induced erythroid differentiation from human primary hematopoietic progenitor cells.

Cell Cycle↗

PubMed2005

Implant surface roughness influences osteoclast proliferation and differentiation.

The osteoclast is a hematopoietic cell derived from CFU-GM and branches from the monocyte-macrophage lineage during the differentiation process. Biological environment appears to be crucial for osteoclast formation and activity. It has been reported that bone remodeling following implant placement requires a coordinated activity by osteoclasts and osteoblasts. The response of such cells at the bone-implant interface has been suggested to be affected by the structural and morphological features of the biomaterial surface. To shed more light on this topic we performed a multiparametric analysis of murine monocytes response to different titanium surfaces. These cells, RAW 264.7 type TIB-71, represent a very useful system because they differentiate into osteoclasts following treatment of definite doses of the osteoclast-differentiation factor RANKL and macrophage colony-stimulating factor (M-CSF). Cells, cultured on glass (control), on grade 3 machined and on titanium pull-spray superficial-TPSS surfaces disclosed profound different responses in terms of morphological rearrangements, adhesion, and differentiation abilities. Indeed, after 14 days, cells cultured on glass and machined surfaces were uniformly distributed, while, on the TPSS surface cells strictly aggregated into small isolated clusters were observed. In addition, cells cultured on the machined surface displayed a higher adhesion ability, while cells cultured on the rougher surface disclosed a more evident capability to differentiate. These results could explain the higher bone-implant contact percentage found around implants with rougher surfaces and suggest that osteoclasts may play an important role in the initial period after implant placement to prime or prepare the implant surface for the osteoblast activity.

Animals↗

PubMed2005

Erythroid cell differentiation is characterized by nuclear matrix localization and phosphorylation of protein kinases C (PKC) alpha, delta, and zeta.

Protein kinases C (PKC) zeta expression and phosphorylation at nuclear level during dimethyl sulfoxide (DMSO)-induced differentiation in Friend erythroleukemia cells have been previously reported, suggesting a possible role of this PKC isoform in the DMSO-related signaling. In order to shed more light on this tantalizing topic, we investigated PKC intracellular and sub-cellular localization and activity during DMSO-induced erythroid differentiation. Results indicated that at least PKC alpha, zeta, and delta are strongly and temporally involved in the DMSO-induced differentiation signals since their expression and phosphorylation, though at different extents, were observed during treatments. Intriguingly, while PKC alpha and zeta associate to the nuclear matrix during the differentiation event, PKC delta appears to be residentially associated to the nuclear matrix. Furthermore, an evident downregulation of the beta-globin gene transcription (differentiation hallmark) was detected upon a progressive inhibition of these PKC isoforms by means of specific inhibitors, indicating, therefore, that PKC alpha, zeta, and delta phosphorylation play a crucial role in the control of erythroid differentiation.

Animals↗

PubMed2005

Core-shell microspheres by dispersion polymerization as promising delivery systems for proteins.

Functional poly(methyl methacrylate) core-shell microspheres were prepared by dispersion polymerization. An appropriate selection of experimental parameters and in particular of the initiator and stabilizer amount and of the medium solvency power allowed a monodisperse sample as large as 600 nm to be prepared. To this purpose, low initiator concentration, high steric stabilizer amount and a low solvency power medium were employed. The microspheres present a core-shell structure in which the outer shell is constituted by the steric stabilizer which affords carboxylic groups able to interact with basic proteins, such as trypsin, whose adsorption is essentially driven by the carboxylic group density in the microsphere shell. Finally, fluorescent microspheres were prepared for biodistribution studies and shown to be readily taken up by the cells both in vitro and in vivo. These results suggest that these microspheres are promising delivery systems for the development of novel protein-based vaccines.

Acrylic Resins↗

PubMed2004

Novel biocompatible anionic polymeric microspheres for the delivery of the HIV-1 Tat protein for vaccine application.

Two novel classes of biocompatible core-shell anionic microspheres, composed of an inner hard insoluble core, either made of poly(styrene) (PS) or poly(methyl methacrylate) (PMMA), and a soft outer tentacular shell made of long soluble negatively charged arms derived from the steric stabilizer, hemisuccinated poly(vinyl alcohol) or Eudragit L100/55, respectively, were prepared by dispersion polymerization and characterized. Five types of these novel microspheres, two made of poly(styrene) and hemisuccinated poly(vinyl alcohol) (A4 and A7), and three made of poly(methyl methacrylate) and Eudragit L100/55 (1D, 1E, H1D), differing for chemical composition, size, and surface charge density were analyzed for the delivery of the HIV-1 Tat protein for vaccine applications. All microspheres reversibly adsorbed the native biologically active HIV-1 Tat protein preventing Tat from oxidation and maintaining its biological activity, therefore increasing the shelf-life of the Tat protein vaccine. The microspheres efficiently delivered Tat intracellularly, and were not toxic in vitro nor in mice, even after multiple administrations. These results indicate that these novel microparticles are safe and represent a promising delivery system for vaccination with Tat, as well as for other subunit vaccines, particularly when a native protein conformation is required.

AIDS Vaccines↗

PubMed2004

Association of PI 3-K with tyrosine phosphorylated Vav is essential for its activity in neutrophil-like maturation of myeloid cells.

The importance of the Vav family of signal transduction molecules in hematopoietic cells has long been acknowledged, even though its role and its regulatory mechanism are not completely understood. We have previously demonstrated that tyrosine-phosphorylated Vav, also located inside the nucleus of myeloid cells, is up-regulated during maturation of promyelocytic precursors induced by all-trans-retinoic acid (ATRA). Here, we report that the tyrosine phosphorylation of Vav during granulocytic maturation is dependent on the tyrosine kinase Syk and is essential for the morphological changes of the cell nucleus. These ATRA-induced events are independent on the guanine nucleotide exchange activity of Vav. We also found that, in differentiating cells, and in both cytoplasmic and nuclear compartments, tyrosine phosphorylated Vav associates with the regulatory subunit of phosphoinositide 3-kinase (PI 3-K). The Vav/p85 interaction is essential for the ATRA-induced PI 3-K activity and for association of PI 3-K with actin, particularly in the nucleus. Our data indicate an unprecedented crucial function for Vav in modulating the morphological maturation process of myeloid cells in a GDP-GTP exchange factor (GEF)-independent manner and suggest a role of Vav as an adaptor protein responsible of targeting PI 3-K to its intranuclear substrates.

Actins↗

PubMed2003

Mitigation of tumor necrosis factor alpha cytotoxicity by aurintricarboxylic acid in human peripheral B lymphocytes.

The aims of this study were to ascertain whether aurintricarboxylic acid (ATA), an endonuclease inhibitor, known to interfere, with the actions of cytokines such as interferons, is able to antagonize the toxic effects produced by tumor necrosis factor alpha (TNF-alpha) in human healthy peripheral B lymphocytes and try to elucidate the molecular machinery through which this possible antagonism takes place. Results evidenced that the balance of survival signals of human B lymphocytes in the presence of TNF-alpha was altered by the interaction of TNF-alpha with a salicylate compound, ATA. Apoptosis effected by TNF-alpha alone was suppressed in the presence of ATA, and this effect appeared essentially characterized by: (i) phosphorylation of phosphatidylinositol-3 kinase (PI-3K), influencing in turn protein kinase B/Akt (Akt) and Bad phosphorylation; (ii) nuclear translocation of the nuclear factor kappa B (NF-kappaB) and (iii) nuclear translocation of protein kinase C zed (PKCzeta). Reversal of TNF-alpha/ATA effects occurred in the presence of the PI-3K specific inhibitors wortmannin or LY294002 in the culture medium and was coincident with inhibition of the translocation of PKCzeta in the nucleus, while NF-kappaB was less affected. These results indicate, therefore, that PI-3K-mediated activation and nuclear transfer of PKCzeta might be essential steps of ATA antagonism against TNF-alpha, suggesting that possible ATA pharmacological applications might be taken into account for staving off systemic or local toxic effects produced by TNF-alpha.

Aurintricarboxylic Acid↗

PubMed2002

Micellar-type complexes of tailor-made synthetic block copolymers containing the HIV-1 tat DNA for vaccine application.

A novel class of cationic block copolymers constituted by a neutral hydrophilic poly(ethylene glycol) (PEG) block and a positively charged poly(dimethylamino)ethyl methacrylate block was prepared for delivery of DNA. These block copolymers spontaneously assemble with DNA to give in aqueous medium micellar-like structures. Five of these novel block copolymers (K1-5), differing in the length of both the PEG chain and the linear charge density of the poly(dimethylamino)ethyl methacrylate block, were prepared and analyzed for gene delivery, gene expression and safety. All five block copolymers protected DNA from DNAse I digestion and delivered the DNA into the cell. However, only three of them (K1, K2 and K5) released the DNA at level allowing efficient gene expression into cells. No toxic effects of both the copolymers alone or their DNA complexes were observed in vitro or in mice. In addition, copolymers were scarcely immunogenic. These results indicate that this novel class of cationic block copolymers is safe and possesses the biological characteristics required for DNA delivery, thus, representing promising vehicles for DNA vaccination.

AIDS Vaccines↗

PubMed2002

Tumor necrosis factor alpha (TNF-alpha) activates Jak1/Stat3-Stat5B signaling through TNFR-1 in human B cells.

The biological actions of tumor necrosis factor alpha (TNF-alpha) are mediated by two cell surface receptors, TNFR-1 and TNFR-2. These receptors do not display protein tyrosine kinase activity. Nevertheless, an early TNF-induced activation of specific tyrosine kinases has been reported as an important cue to the cellular response to this cytokine. Here we present evidence that TNF-alpha induces the activation of the cytoplasmic Janus tyrosine kinases Jak1 and Tyk2 in both human healthy peripheral and lymphoma B cells. This event was accompanied by the recruitment of a specific set of latent cytosolic transcription factors, Stat3 and Stat5b. Furthermore, Jak1 coprecipitated with TNFR-1 after TNF-alpha treatment. These data suggest that at least in human B cells this cytokine can exert its biological effects through the Jak-Stat signaling pathway and that such signals are initiated through an interaction between TNFR-1 and Jak 1.

Antigens, CD↗

PubMed2002

Selective up-regulation of phospholipase C-beta2 during granulocytic differentiation of normal and leukemic hematopoietic progenitors.

In this study, we have investigated the expression of phospholipase C-beta2 during the course of granulocytic differentiation of normal and malignant progenitors. As a model system, we used the NB4 cell line, a reliable in vitro model for the study of acute promyelocytic leukemia (APL), a variety of acute myeloid leukemia (AML) that responds to pharmacological doses of all trans-retinoic acid (ATRA) by differentiating in a neutrophil-like manner. We found that PLC-beta2, virtually absent in untreated NB4 cells, was strongly up-regulated after ATRA-induced granulocytic differentiation. Remarkably, using primary blasts purified from bone marrow of patients affected by APL successfully induced to remission by treatment with ATRA, we showed a striking correlation between the amount of PLC-beta2 expression and the responsiveness of APL blasts to the differentiative activity of ATRA. An increase of PLC-beta2 expression also characterized the cytokine-induced granulocytic differentiation of CD34+ normal hematopoietic progenitors. Taken together, these data show that PLC-beta2 represents a sensitive and reliable marker of neutrophil maturation of normal and malignant myeloid progenitors. Moreover, PLC-beta2 levels can predict the in vivo responsiveness to ATRA of APL patients.

Acute Disease↗