Biomedical subjects

O Trubiani

Publications and source records attributed to O Trubiani.

36 records · Page 2Linked to original sources

PubMed1994

Protein kinase C modulation in apoptotic rat thymocytes: an ultrastructural analysis.

Numerous events in the cell, such as gene expression, cell growth and metabolism are regulated by signal transduction pathways involving protein kinase C (PKC). Recent data indicate that a PKC-dependent mechanism also underlies the apoptotic death of cells induced by glucocorticoid hormones. In this report we have analysed the changes of PKC during dexamethasone-induced apoptosis in thymocytes by means of immunocytochemical and immunochemical analysis. The data obtained show an increase and intracellular movement of protein kinase C, which is translocated to the nucleus and linked to the nuclear matrix during the apoptotic process.

Animals↗

PubMed1994

Human leukemic pre-B line (KM-3) treated with phorbol-ester: trend of polyamines during cell differentiation.

Aliphatic polyamines, putrescine, spermine and spermidine present in bacteria and eukaryotic cells are essential for cell growth. Generally, polyamine levels are elevated in rapidly growing normal and pathological systems. Since polyamines belong to the category of molecules whose synthesis is strongly activated during the G1 period they have been implicated in the cell's preparation for DNA replication. In our experiments, we have differentiated by phorbol-ester, the human pre-B leukemic cell line KM-3. Biochemical and flow cytometric analysis show an increase, in treated cells, of polyamines pathway related to G1 and early S-phase of cell cycle during B cell differentiation.

B-Lymphocytes↗

PubMed1993

Phorbol ester-induced effects on cell cycle progression and terminal deoxynucleotidyltransferase (TdT) activity in KM-3 pre-B cell line.

Phorbol myristic acetate (PMA) is a tumor-promoting agent that has been shown to induce differentiation of human leukemia cells and of normal lymphoid cells. We have investigated the ability of PMA to induce inhibition of cell growth of the human KM-3 pre-B leukemic cell line by multiparametric analysis. Our results show that PMA treatment induces cell differentiation with the disappearance of terminal deoxynucleotidyltransferase and a decrease of cell growth, as evaluated by [3H]thymidine uptake. Flow cytometric analysis of BrdU incorporation shows that PMA is able to induce a modification of the cell cycle with a sharp decrease of the percentage of S-phase cells, which is more evident after 24 h of treatment. Comparison between the cell growth kinetics and TdT synthesis and activity shows that differentiated cells are still able to proliferate to a certain extent and that the TdT disappearance and the initial decrease of cell proliferation are two independent effects of PMA.

B-Lymphocytes↗

PubMed1993

Phorbol ester induces changes in the synthesis of nuclear polyphosphoinositides and expression of terminal deoxynucleotidil transferase (TdT) in nuclei of KM-3 cells.

Terminal deoxynucleotidyl Transferase (TdT) play an essential role in the immune system differentiation. KM-3 cells are lymphoblastoid cells expressing the TdT and when induced to differentiate by phorbol ester (PMA) they loose this enzyme. Therefore, because of the suggested involvement of polyphosphoinositide in controlling the nuclear events it has been analyzed the phosphorylation of nuclear polyphosphoinositides during KM-3 differentiation. When the differentiated state is reached the phosphorylation level of PIP2 increases in isolated nuclei and this is accompanied by a concomitant decrease of PIP and PA, hinting at a correlation between polyphosphoinositide metabolism and TdT expression.

Cell Differentiation↗

PubMed1992

Intracellular localization of terminal transferase during the cell cycle.

Changes in the localization of terminal transferase during the cell cycle in random cultures of human pre-T leukemia line RPMI-8402 were examined by light and electron microscopy on immunoperoxidase-stained preparations. Paraformaldehyde-fixed and saponin-permeabilized human cells were used with a monoclonal anti-human terminal deoxynucleotidyl transferase (TdT) primary reagent to demonstrate changes in enzyme distribution occurring between interphase and mitosis. Nuclear localization is found uniformly during interphase. At metaphase, however, the majority of TdT staining appears randomly distributed in the cytoplasm and traces of TdT staining remain associated with mitotic chromatin. At later phases, when the daughter cells are forming, the enzyme again appears to be restricted to the new nuclear structure.

Cell Cycle↗

PubMed1992

Interferon affects cell growth progression by modulating DNA polymerases activity.

A multiparametric analysis of the effects of human recombinant interferon alpha type A on Daudi cells involving flow cytometry and in vitro analysis of alpha and beta DNA polymerase activities has been performed. Results have disclosed (within 60 min of interferon treatment) a decrease of alpha polymerase driven DNA synthesis persisting to at least 24 h, while beta polymerase was poorly affected. Moreover, after 24 h of interferon treatment, a reduction of BrdUrd incorporation per cell, assessed by flow cytometry, was observed suggesting that DNA synthesis in S phase cells is almost completely abolished. The analysis of the effect of interferon on the distribution of cell cycle phases indicated that the G1/S transition is not inhibited by the treatment. These results support the hypothesis that interferon generates a transient initiating signal which quickly reaches the nucleus and produces a rapid inhibition of alpha polymerase activity, leading finally to the slowing of cell cycle progression.

Cell Cycle↗

PubMed1992

Immunocytochemical study of recombinant terminal deoxynucleotidyl transferase (TdT) synthesized by baculovirus-infected insect cells.

The ability to overproduce terminal transferase through recombinant DNA technology should provide alternate means for generating sufficient quantities for structural and mechanistic study of this creative DNA polymerase. In this work we have investigated, at electron microscope level, the morphological modification and ultrastructural localization of synthesized human terminal transferase occurring in Sf-9 cells during recombinant baculovirus infection time. The results obtained showed that TdT is localized and stored only at the cytoplasmic level; the nucleus did not show any specific site able to link the neosynthesized TdT. The amount of the enzyme, estimate by immunostaining analysis, increased with the viral infection time. Morphological changes occurring during viral infection consist mainly of variations of cellular surface, different size and shape of cytoplasmic organelles and modification of nuclear components.

Animals↗

PubMed1992

Ultrastructural localization of Terminal deoxynucleotidyl Transferase (TdT) in rat thymocytes.

TdT positive cells in rat thymus belong to distinct subsets as shown by light and electron microscopic immunocytochemistry. Using polyclonal antibodies to calf TdT and peroxidase labeled goat anti-rabbit IgG it has been possible to identify several subpopulations of TdT positive thymocytes. Large blasts corresponding to thymocytes at early maturational stages, are strongly positive for TdT which is diffusely distributed in both the nucleus and the cytoplasm. Smaller cells which correspond presumably to more advanced stages of maturation display nuclear TdT only, or are negative. Ultrastructural analyses of TdT indicate that the localization of the enzyme is related to the morphological features of the cells and TdT expression corresponds to maturational stages of T-cells.

Animals↗

PubMed1991

Association between nuclear matrix and terminal transferase: an electron microscope immunocytochemical analysis.

Nuclear matrix extracted from KM-3, a human pre-B leukemia cell line, appears to have a site of linkage for terminal deoxynucleotidyl transferase (TdT). The immunocytochemical analysis of the distribution of TdT using a rabbit polyclonal antibody which recognizes human terminal transferase, shows that the nuclear framework of these cells contains sites of immunoreactivity that appear uniformly distributed on the matrix fibres, while the nucleolar region is unreactive. This evidence points out the possibility that TdT could reside in the proteinaceous scaffold of the nucleus defined as nuclear matrix, thus strengthening the evidence for the metabolic and regulatory roles ascribed to this nuclear framework.

Cell Line↗

PubMed1991

Nuclear metabolic changes induced by tumor necrosis factor in Daudi lymphoma cells; a multiparametric analysis.

The effects of r-TNF alpha on cell cycle progression and DNA polymerase activity in Daudi lymphoma cells have been analyzed. Cytofluorimetric analysis of the cell cycle after 6 to 24 hr of treatment revealed both a decrease of BrdU incorporation per cell and a light inhibition of S phase as assessed by the analysis of the percentual distribution of cell cycle compartments. The reduction of BrdU incorporation can be related to the early decrease in the rate of DNA synthesis that follows r-TNF alpha treatment. These results suggest that one of the early events induced by r-TNF alpha at nuclear level is the slowering of DNA synthesis leading to a reduced cell cycle progression.

Bromodeoxyuridine↗

PubMed1990

Nuclear lipids in Friend cells. Shifted profile of diacylglycerol during erythroid differentiation induced by DMSO.

Friend cells were labelled for 90 min. with [3H]-Glycerol and the radioactivity in DAG# and TG was measured. The relative percentage of radiolabelled DAG differs in isolated nuclei as compared to intact cells. Moreover the level of newly synthesized DAG decreases in nuclei isolated from cells treated with DMSO for 24 hrs. as well as in nuclei from cells treated for 96 hrs. and terminally differentiated in the erythroid pathway. Since these changes are not seen in intact cells, the results are consistent with the hypothesis that nuclear lipids and namely the products of polyphosphoinositide hydrolysis, such as DAG, are involved in the regulation of the processes leading to cell differentiation.

Animals↗

PubMedDate not supplied

The c-myc gene regulates the polyamine pathway in DMSO-induced apoptosis.

It is accepted that apoptosis is a gene-controlled process of cellular self-destruction. It occurs during physiological regulation and in pathological situations in the life of a cell. In the immune system, several different intracellular and extracellular factors have been associated with the induction of apoptosis, and the final responses depend on the cell system and the acquired signals. In lymphoid cells, dexamethasone-induced apoptosis is associated with c-myc downregulation in cells that remain in G0-G1 until the point of death. Ornithine decarboxylase (ODC), a key enzyme involved in polyamine biosynthesis, is regulated by c-myc, which is a transcriptional activator implicated not only in the control of cell proliferation and differentiation but also in programmed cell death. As dimethylsulphoxide (DMSO) induces apoptosis in the RPMI-8402 human pre-T cell line, the present study analysed the involvement of the c-myc proto-oncogene and polyamine pathway as mediators of apoptosis. Cell growth, programmed cell death, c-myc expression, ODC activity and intracellular polyamine content were detected after DMSO and difluoromethylornithine (DFMO) treatment. DMSO-treated cells exhibit a decrease in ODC activity and polyamine levels associated with cell growth arrest and programmed cell death induction. The expression of c-myc proto-oncogene, as its mRNA or protein, is specifically down-regulated. DFMO, a well defined polyamine biosynthesis inhibitor, completely blocks ODC activity, resulting in growth inhibition but not apoptosis. Moreover, in these samples no evidence of changes of c-myc expression were found. The results obtained suggest that, in RPMI-8402 cells, DMSO provokes a c-myc-dependent decrease of ODC activity followed by a depletion of intracellular polyamine levels, associated with programmed cell death and cell growth arrest.

Apoptosis↗

PubMedDate not supplied

DMSO modifies structural and functional properties of RPMI-8402 cells by promoting programmed cell death.

Apoptosis in lymphoid cells can be induced in different ways depending on cell type and acquired signal. Biochemical modifications occur at an early phase of cell death while at late times the typical morphological features of apoptosis can be visualized. The aim of this study is to verify by multiparametric analyses the plasma membrane fluidity, the intracellular Ca2+ concentration and the nitric oxide synthase (NOS) activity during cell death progression induced by DMSO treatment. The RPMI-8402 human pre-T lymphoblastoid cell line was induced to cell death by DMSO. Analyses rescued at early times of treatment prove a substantial modification of plasma membrane fluidity associated with an increase of intracellular Ca2+. Moreover, these modifications are associated with an up regulation of NOS activity. Our results are consistent with the hypothesis that programmed cell death can be induced by up regulation of the intracellular Ca2+ associated with an increase of cell membrane fluidity. The apoptotic mechanisms seem to involve not only membrane damage and increased intracellular calcium levels but also production of nitric oxide.

Apoptosis↗

PubMedDate not supplied

Sphingolipid microdomains mediate CD38 internalization: topography of the endocytosis.

Plasma membranes of several cell types contain specialized microdomains (or lipid rafts) enriched in sphingolipids, cholesterol, sphingomyelin, and glycosyl-phosphatidylinositol-anchored proteins. These membrane domains are characterized by detergent insolubility at low temperatures and low buoyant density. Human CD38 is the prototype of a gene family encoding surface molecules endowed with multiple functional activities. The endocytosis of the human CD38 molecule has been investigated in normal lymphocytes and in a number of leukemia- and lymphoma-derived cell lines demonstrating that internalization after CD38 ligation is a reproducible event involving only a fraction of the whole amount of the surface molecule. This study reports the results obtained by conventional, confocal, and electron microscopy on the effects induced by the engagement of the molecule with agonistic mAb, reproducing the signals mediated by its natural ligand. The results demonstrate that the endocytosis induced as consequence of CD38 ligation is preceded by a thorough rearrangement of the cell surface with formation of glycosphingolipid- and cholesterol-rich plasma membrane microdomains. These data suggest that specialized raft microdomains might be the plasma membrane structure through which CD38 translocates at intracellular level. The CD38/lipid interactions during the coated pit formation trigger a process that generate membrane curvature, considered as the first step of CD38 endocytosis. Moreover, ultrastructural studies show that early CD38(+) endosomes are pleiomorphic and contain cisternal and vesicular regions. Late endosomes exhibit a complex organisation, containing uncoupled CD38-ligand multivesicular- or multilamellar-regions.

ADP-ribosyl Cyclase↗

PubMedDate not supplied

Morphological and cytofluorimetric analysis of adult mesenchymal stem cells expanded ex vivo from periodontal ligament.

Many adult tissues contain a population of stem cells that have the ability of regeneration after trauma, disease or aging. Recently, there has been great interest in mesenchymal stem cells and their roles in maintaining the physiological structure of tissues, and their studies have been considered very important and intriguing, after having shown that this cell population can be expanded ex vivo to regenerate tissues not only of the mesenchymal lineage, such as intervertebral disc cartilage, bone, tooth-associated tissue, cardiomyocytes, but also to differentiate into cells derived from other embryonic layers, including neurons. Currently, different efforts have been focused on the identification of odontogenic progenitors from oral tissues. In this study we isolated and characterized a population of homogeneous human mesenchymal stem cells proliferating in culture with an attached well-spread morphology derived from periodontal ligament, a tissue of ectomesenchymal origin, with the ability to form a specialized joint between alveolar bone and tooth. The adherent cells were harvested and expanded ex vivo under specific conditions and analysed by FACScan flow cytometer and morphological analysis was carried out by light, scanning and transmission electron microscopy. Our results displayed highly evident cells with a fibroblast-like morphology and a secretory apparatus, probably indicating that the enhanced function of the secretory apparatus of the mesenchymal stem cells may be associated with the secretion of molecules that are required to survive and proliferate. Moreover, the presence in periodontal ligament of CD90, CD29, CD44,CD166, CD 105, CD13 positive cells, antigens that are also identified as stromal precursors of the bone marrow, indicate that the periodontal ligament may turn out to be a new efficient source of the cells with intrinsic capacity to self-renewal, high ability to proliferate and differentiate, that can be utilized for a new approach to regenerative medicine and tissue engineering.

Adult↗

PubMedDate not supplied

Adult mesenchymal stem cells in dental research: a new approach for tissue engineering.

Many adult tissues contain a population of stem cells that have the ability to regenerate after trauma, disease or aging. Recently, there has been great interest in mesenchymal stem cells and their roles in maintaining the physiological structure of tissues. The studies on stem cells are thought to be very important and, in fact, it has been shown that this cell population can be expanded ex vivo to regenerate tissues not only of the mesenchymal lineage, such as intervertebral disc cartilage, bone and tooth-associated tissues, but also other types of tissues. Several studies have focused on the identification of odontogenic progenitors from oral tissues, and it has been shown that the mesenchymal stem cells obtained from periodontal ligament and dental pulp could have similar morphological and phenotypical features of the bone marrow mesenchymal cells. In fact a population of homogeneous human mesenchymal stem cells derived from periodontal ligament and dental pulp, and proliferating in culture with a well-spread morphology, can be recovered and characterized. Since these cells are considered as candidates for regenerative medicine, the knowledge of the cell differentiation mechanisms is imperative for the development of predictable techniques in implant dentistry, oral surgery and maxillo-facial reconstruction. Thus, future research efforts might be focused on the potential use of this cell population in tissue engineering. Further studies will be carried out to elucidate the molecular mechanisms involved in their maintenance and differentiation in vitro and in vivo.

Adult Stem Cells↗

PubMedDate not supplied

Programmed cell death of peripheral myeloid precursor cells in Down patients: effect of zinc therapy.

Hemopoietic stem cell differentiation represents the primary rule of self-renewal, proliferation, and specialization modulated by several mechanisms, including growth factors, cell interactions, and bioavailability of various ions, especially Ca2+ and Zn2+. Apoptotic death, during normal cell turnover, has been widely studied and is recognized as an important pathway for clonal deletion in the hemopoietic system. Multiparametric analyses have shown that subjects with Down syndrome show low levels of plasmic zinc associated with the presence of immature myeloid cells in the peripheral blood. This arrangement is repaired by in vivo zinc therapy. This study presents morphological and biochemical analyses to show that ZnSO4 therapy induces the disappearance of peripheral myeloid precursor cells by a programmed cell death mechanism. The programmed zinc-therapy-induced cell death presumably provides a simple way to regulate the myeloid differentiation selecting appropriate cells.

Adolescent↗

PubMedDate not supplied

TCR and immunophenotype changes in dimethyl sulfoxide-dependent programmed cell death.

In the thymus most deleted cells are immature thymocytes and the high rate of cell death within the thymus is involved in the development of the initial T-cell receptor repertoire. Functional T-cell receptor recognition units are created by somatic rearrangements of gene segments, and the expression of successfully assembled TCR complex is the key to molecular events that culminate in T-cell activation, growth and differentiation. Previously, we reported that DMSO induces apoptosis in RPMI-8402 human pre-T cells. Here we examine the fate of pre-T cells undergoing negative selection analysing the responsiveness to DMSO-enforced TCR expression and immunophenotype modulation. Our results demonstrate that DMSO induces cell growth inhibition, cell phenotype changes, with down-regulation of CD2 and CD7, and increases in alpha/beta or gamma/delta TCR chains led by TdT, RAG-1 and RAG-2 activity. These modifications are associated with an apoptotic program. Taken together, these data suggest the existence of an early checkpoint that ensures in vivo the effective intrathymic differentiation supported from another point of view, the linkage between immunophenotypes and TCR regulation in T-cell differentiation and programmed cell death.

Antigens, CD↗