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F Gremo

Publications and source records attributed to F Gremo.

At least 19 recordsLinked to original sources
PubMed2001

Intra- and intercellular distribution of mitochondrial probes and changes after treatment with MDR modulators.

Fluorescent probes are currently used to evaluate the mitochondrial transmembrane potential in situ. However, in parallel experiments using the probes JC-1 and TMRM in different cell types (human astrocytes, HEp-2, Vero, KB, and HeLa cells), we found that the distribution of JC-1 and TMRM is highly variable not only in different cell types but also in different cells of the same cell type, a condition that has never been documented until our work. This phenomenon depends on a hidden, widespread multidrug resistance (MDR) phenotype that can be recognized only by comparative assays with MDR inhibitors (progesterone, verapamil, and cyclosporin A) and represents a serious risk of error in the evaluation of the mitochondrial potential.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Homogeneous longitudinal profiles and synchronous fluctuations of mitochondrial transmembrane potential.

This study reports for the first time (a) the longitudinal profile of the transmembrane potential (mDeltapsi) of single mitochondria using a Nernstian fluorescent probe and (b) the distribution of mDeltapsi fluctuations of mitochondria undergoing permanent depolarization. Our findings show that (1) mitochondria in different energetic conditions coexist in the same cell, (2) mDeltapsi is rather homogeneous along the entire length of single mitochondria, (3) mDeltapsi is not influenced by the surrounding cytoplasmic environment and (4) mDeltapsi fluctuations occur simultaneously in groups of mitochondria connected in a network. Taken together, these findings provide further evidence for a functional relationship between mitochondrial arrangement and energetic condition.

Astrocytes↗
PubMed1999

Subcellular heterogeneity of mitochondrial membrane potential: relationship with organelle distribution and intercellular contacts in normal, hypoxic and apoptotic cells.

The subcellular heterogeneity of mitochondrial membrane potential (mDelta psi) was investigated in confluent and sub-confluent cultures of four cell types (human astrocytes, HEp-2, MDCK and Vero cells) in normal growth conditions, hypoxia and apoptosis. The distribution of high-polarized mitochondria, detected by the potential-sensitive probe JC-1, was found to depend on: (1) the proximity to the cell edge; (2) the local absence of cell-cell contacts; and (3) the local absence of acidic vesicles. Both hypoxia and apoptosis produced a general mDelta psi increase with different redistributions of high-polarized mitochondria. Hypoxic cells maintained high-polarized mitochondria for over 24 hours, until cells underwent necrosis. On the other hand, apoptotic cells showed an unexpected convergence of high-polarized mitochondria into an extremely packed mass at one side of the nucleus, in a stage preceding nuclear condensation, but correlated to the retraction of cell-cell contacts.

Animals↗
PubMed1998

Developmentally regulated expression and localization of fibroblast growth factor receptors in the human muscle.

Fibroblast growth factors (FGFs) are believed to play a key role in tissue differentiation and maturation. Thus, the expression of the four members of the high-affinity tyrosine kinase FGF receptor family (FGFRs) and of the low-affinity heparan sulphate proteoglycan binding sites, syndecan-1 and perlecan, was studied in the human skeletal muscle during development. Northern blot analysis demonstrated a developmentally regulated expression of the mRNAs for FGFR-1, FGFR-3, FGFR-4, whereas only traces of FGFR-2 mRNA were found. Each receptor type had a different developmental pattern, suggesting an independent regulation. Signal for FGFR-3 was retained only in the adult muscle. Among the low-affinity FGF binding sites, perlecan was absent, whereas RNA transcript for syndecan-1 peaked at week 13 of gestation, after which a significant decrease was observed. Immunohistochemistry for FGFRs revealed that their localization changed with muscle maturation. At early embryonic stages, FGFR-3 and FGFR-4 had a scattered distribution in the tissue, and FGFR-1 was found on myotube and myofiber plasma membranes. At later stages, FGFR-1 positivity decreased and was found in a few areas of the muscle, FGFR-3 was concentrated in the nuclei of some, but not all, muscle fibers, and FGFR-4 maintained an association with plasma membrane. In adult tissue, weak positivity for FGFR-3 and FGFR-4 was observed in the connective tissue only. When immunocytochemistry was performed on human fetal myoblasts in culture, confocal microscope analysis revealed a nonhomogeneous cell membrane distribution of FGFRs. Taken together, the data strongly suggest that developmentally regulated expression and cell distribution of FGFRs play a role during muscle maturation.

Adult↗
PubMed1997

Effect of dystrophin antisense oligonucleotides on cultured human neurons.

Antisense oligonucleotides offer the potential to block the expression of specific molecules within the cell, thus providing a useful tool in cell function studies. In this paper, we tested the possibility to block dystrophin expression in in vitro cultured neurons with antisense oligonucleotides administration. Human fetal neuronal cultures were treated with different doses of antisense oligonucleotides against dystrophin, the protein coded by the Duchenne muscular dystrophy gene. Results showed that labelled oligonucleotides rapidly accumulated into cultured neurons, but were discarded 15-24 h after treatment. However, no effects could be observed until 3-4 days after treatment, when immunocytochemical staining for dystrophin was significantly decreased in treated neurons. This result was confirmed by polymerase chain reaction assay which showed a significantly lower expression of the dystrophin specific mRNA. Electron microscope observations confirmed that neurons were affected. Large inclusions or packed granules were detectable in their cytoplasm and in terminal endings. Neuronal nuclear membrane was sometimes shredded, so that nuclear shape was altered. These phenomena were dose-dependent, further substantiating the hypothesis of a specific effect of antisense treatment. This interpretation was supported by the absence of alterations when cultures were treated with mismatch or non specific antisenses. Since the function of dystrophin is still unknown, these data might help in understanding the role played by this protein in the developing brain.

Cells, Cultured↗
PubMed1996

On the presence of DNA polymerase alpha in human lymphocyte nuclei and chromosomes.

Experiments were carried out to correlate the cytological localization of DNA polymerase alpha with the presence of its specific mRNA in human lymphocytes studied at different times after phytohaemagglutinin stimulation. Our data indicated that in resting cells it is not possible to detect DNA polymerase alpha protein or mRNA by Northern hybridization. By contrast, in stimulated cells the detection of mRNA specific for DNA polymerase alpha synthesis is possible after 16 h phytohaemagglutin stimulation, whereas immunolocalization is possible after only 4 h stimulation. Observation of cytological preparations from cells stimulated for times long enough to obtain mitoses surprisingly showed an intense immunoreaction in mitotic chromosomes treated with monoclonal antibodies to DNA polymerase alpha.

Antibodies, Monoclonal↗
PubMed1995

Human ramified microglial cells produce nitric oxide upon Escherichia coli lipopolysaccharide and tumor necrosis factor alpha stimulation.

This study shows that human ramified microglial cells derived from fetal brain primary cultures, are able to produce nitric oxide (NO). In fact, stimulation with Escherichia coli lipopolysaccharide (LPS) (1 microgram ml-1) or tumor necrosis factor alpha (TNF alpha) (500 U ml-1) enhances nitrite release in cell supernatants, as determined by the Griess reaction. A synergistic effect is achieved following treatment with LPS plus TNF alpha, this effect being inhibited by pretreating cells with NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME). Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis, we also found that LPS/TNF alpha produce an increase of inducible NO synthase (iNOS) mRNA expression.

Base Sequence↗
PubMed1995

Expression of basic fibroblast growth factor and its receptors in human fetal microglia cells.

The presence of basic fibroblast growth factor (bFGF) and FGF receptors was investigated in microglia cells derived from human fetal brain long-term cultures. Production of bFGF was suggested through the capability of microglial extracts to stimulate plasminogen activator (PA) synthesis in endothelial cells. The identity of PA-stimulating activity with bFGF was confirmed by its high affinity for heparin and its cross-reactivity with polyclonal antibodies to human recombinant bFGF. These antibodies recognized a cell-associated M(r) 18,000 protein as well as trace amounts of the M(r) 24,000 bFGF isoform in Western blot. All microglial cells showed bFGF immunoreactivity in the cytoplasm and, sometimes, in the nucleus. Scatchard plot analysis of 125I-bFGF binding data revealed the presence of low affinity heparansulphate proteoglycans (380,000 +/- 60,000 sites/cell; Kd = 730 +/- 200 nM) and of high affinity tyrosine-kinase receptors (10,300 + 2500 sites/cell; Kd = 30 +/- 9 pM). Immunocytochemistry confirmed the presence of FGF receptor (1/flg) on the cell surface of some, but not all microglial cells, with prevalent association to ameboid microglia. Transcripts for FGF receptors 1, 2, 3 and 4 were found in microglia by Northern blot analysis. Co-expression of bFGF and its receptors in human fetal microglia suggests an autocrine role of bFGF in these cells.

Cells, Cultured↗
PubMed1995

Human microglia cultures: a powerful model to study their origin and immunoreactive capacity.

In this paper, we report that pure cultures of human microglia were obtained from long-term astrocytic cultures of human fetal brain. After five to six months and repeated cell passages, macrophage-like cells started to spontaneously form in vitro, so that in two to three weeks the whole culture was populated by them. These cells were grown up to over 50 passages in culture and analyzed for morphology, specific marker positivity, growth rate and major histocompatibility complex (MHC) antigen expression with or without gamma-interferon (IFN) stimulation. We found that, regardless of embryonic age of original cultures (10-15 weeks of gestation), cultures showed a remarkable homogeneity and purity and over 90 stained for typical microglial markers. Under basal conditions, two cell subpopulations similar to those described in vivo, we observed: the reactive 'ameboid' type and the resting 'ramified' one, the latter increasing with time in vitro and cell passages. Both cell subpopulations were capable of active phagocytosis and of high-rate proliferation. They spontaneously expressed low levels of MHC class II antigens, but were negative for MHC class I. Stimulation with gamma-interferon lymphokine upregulated the MHC class II expression as well as the MHC class I heavy chain form in ameboid, 'reactive' cells but not in the ramified ones. We also found that beta 2 microglobulin, already expressed in basal conditions, was dissociated from HLA A-B-C molecules in lymphokine-stimulated cells at early passages. The physiological significance of these data, as well as the possible correlation with in vivo ontogenetic modifications, are also discussed.

Brain↗
PubMed1995

Immunolocalisation of dystrophin in the immature human neurons and muscles.

Dystrophin, the product of the Duchenne muscular dystrophy gene, has been shown to be developmentally regulated in both human muscle and brain tissues. We consequently performed an immunocytochemical study using electron microscopy to localise the protein in the immature human fetal muscle and neurons. Results demonstrated that, even if dystrophin was partially associated to the plasma membrane in both tissues, some product was also linked to the neurofilaments network in neurons and to microfilaments in muscle. An intense staining was also found in satellite cells.

Astrocytes↗
PubMed1994

Differential expression of fibroblast growth factor receptors by human neurones, astrocytes and microglia.

Expression of fibroblast-growth factor receptors (FGFRs) was studied in human fetal neurones, astrocytes and microglia in culture. Northern blot analysis showed that neurones and microglia expressed the mRNAs for FGFR-1, FGFR-2, FGFR-3, FGFR-4 at different levels, whereas astrocytes expressed only FGFR-1 and FGFR-4 mRNAs. Immunocytochemical localization of FGFR-1 revealed that this receptor was predominantly localized on the axon hillock membrane in neurones, and was associated with the plasma membrane of ameboid, activated microglia and of glial-fibrillar acidic protein positive astrocytes. The expression of various members of the FGFR family in all the cell types investigated implicates FGFs in human brain development and functions.

Astrocytes↗
PubMed1992

Dystrophin immunoreactivity in normal and Duchenne human fetal neurons in culture.

Dystrophin, the protein product defective in Duchenne muscular dystrophy (DMD), is present in all types of muscle and in the brain. The function of the protein is unknown and its role in the brain is unclear, although 30% of DMD patients show nonprogressive mental retardation. We have therefore studied the localisation of dystrophin in cultures of normal and DMD human fetal neurons using antibodies raised to different regions of the protein. Dystrophin immunoreactivity was demonstrated in the soma and axon hillock of normal neurons and appeared to be associated with the inner part of the cell membrane, although some intracellular staining was also observed. Positive dystrophin staining was present only in cells with fully developed neuronal features, although not all the neurons were positive. Glial cells were always negative for the antigen. Immunostaining with antibodies to the brain spectrins indicate that the dystrophin antibodies did not crossreact with these proteins. The possibility of cross-reactivity with other proteins is discussed. Studies of cells cultured from a DMD fetus also showed specific dystrophin immunostaining in neurons, although the muscle was generally negative for dystrophin. However, the localisation of dystrophin immunostaining and that of the brain spectrins and neurofilaments appeared abnormal, as did the overall morphology of the cells. This suggests that dystrophin may play a role during brain development and dystrophin deficiency results in abnormal neuronal features. This would be consistent with the nonprogressive nature of the mental retardation observed in DMD patients.

Blotting, Western↗
PubMed1992

Immunocompetent cell markers in human fetal astrocytes and neurons in culture.

During the past few years, evidence has accumulated that interaction with peripheral immune cells as well as immunoregulatory functions in the central nervous system (CNS) can be played by several types of brain resident cells. Since very little information is available in man, however, we investigated the presence of markers so far considered typical of immunocompetent cells in in vitro cultures of human fetal brain. Immunocytochemistry at the light, scanning, and transmission electron microscopic levels revealed positivity for a very restricted range of macrophage antigens in astrocytes, which, however, were incapable of phagocytosis. In particular, expression of the major histocompatibility complex-class II antigen HLA-DR was observed in the cytoplasm and on the cell surface of GFA-P+ astrocytes and increased with time in culture and cell passages. Among the T-lymphocyte markers tested, Thy.1 and CD4 were positive. Both neurons and astrocytes carried Thy.1 from early cell passages. Noteworthy was the presence of CD4, which serves as the receptor for AIDS virus, in neurons from the first 2 weeks, whereas astrocytes became positive after only 4-6 weeks. Even if most staining was in the cytoplasm, some was exposed on cell surface. Astrocytes were found positive for the B-lymphocyte marker CD21, the cellular receptor for Epstein-Barr virus, whereas CD24 was detected in both neurons and astrocytes. Both antigens are related to B-cell proliferation. Results are in favour of the hypothesis of human brain cells being actively involved in CNS immunological events.

Astrocytes↗
PubMed1992

Human astroglial but not microglial cells synthesize alpha 2-macroglobulin in vitro.

Alpha 2-macroglobulin (alpha 2M) is a serum proteinase inhibitor with a broad specificity. At present its role in human brain is unknown, but recent data report its presence in the CNS, particularly at glial level. Previous studies from our group demonstrated the synthesis and secretion of alpha 2M in different glial cultures derived from an astrocytoma and a glioblastoma. In the present study a human fetal astroglial cell line and two microglial established cell lines are examined for the presence of alpha 2M by using polyclonal antibodies in ELISA and immunofluorescence assays. While we observed a strong specific positivity in the cytoplasm and in the culture medium of the GFAP, vimentine positive cells, no positivity was detected in FcR, lysozyme positive microglial cells. Since interaction of proteinases and proteinase inhibitors appear to play a crucial role in the development of neuroimmunological competence, these data suggest a dissociation of macro and micro-glia immune functions.

Astrocytes↗