Search PubMed⌕ Search

Biomedical subjects

N M Maraldi

Publications and source records attributed to N M Maraldi.

At least 55 records · Page 3Linked to original sources
PubMed1999

Phosphatidylinositol 3-kinase translocation to the nucleus is an early event in the interleukin-1 signalling mechanism in human osteosarcoma Saos-2 cells.

Interleukin 1 (IL-1) is a proinflammatory cytokine which can elicit proliferative, differentiative, or metabolic responses. The molecular mechanisms by which IL-1 signals are transduced from the plasma membrane to the nucleus, although extensively studied, have not been completely elucidated. We previously demonstrated that human osteosarcoma Saos-2 cells incubated with IL-1 presented a rapid and transient increase of phospholipase C activity exclusively at the nuclear level. Moreover, we presented evidence that not only the canonical inositol lipid signalling pathway was involved, but also the D3-phosphorylated lipids generated by phosphatidylinositol 3-kinase (PI 3-kinase) were affected. The results of this study indicate that in Saos-2 cells PI 3-kinase is recruited and activated by IL-1 receptor I (IL-1RI) through binding of the SH2 domains to the consensus sequence on the C-terminal tail of the receptor, and that Tyr-479 is essential for PI 3-kinase activation. Moreover, IL-1 treatment triggers PI 3-kinase translocation to the nucleus; this event is rapid and transient in cells expressing high levels of IL-1RI (Saos-2/IL-1R) as well as in untransfected cells, although to a lesser extent. The data, based on immunochemical and immunocytochemical quantitative methods, indicate that PI 3-kinase translocation to the nucleus depends on PI 3-kinase activation. In fact, inactivation by two independent mechanisms, addition of specific PI 3-kinase inhibitors, or overexpression of a mutant form of IL-1RI, resulted in a substantial inhibition of PI 3-kinase translocation to the nucleus. These data suggest that PI 3-kinase recruitment by the activated receptor is a limiting step in PI 3-kinase activation and nuclear translocation. This early event in the IL-1 signalling mechanisms confirms that D3 inositides, as well as canonical inositides produced by nuclear phospholipase C isoforms, are involved in this pathway of activation of transcription factors.

Biological Transport, Active↗
PubMed1999

Decreased expression of laminin beta 1 in chromosome 21-linked Bethlem myopathy.

Muscle biopsies of four patients affected by chromosome 21-linked Bethlem myopathy were investigated by means of immunohistochemistry, with monoclonal antibodies for laminin chains, dystrophin and dystrophin associated glycoproteins. The objective of this study was to determine whether an altered molecular structure of collagen type VI, characteristic of Bethlem myopathy, could influence the expression of the protein complex linking the extracellular matrix with the subsarcolemmal cytoskeleton. Normal expression of all proteins was found except for laminin beta 1, along with an age related progressive deficiency of this protein in the muscle fibre basal lamina. This study shows that Bethlem myopathy linked to chromosome 21 is associated with a secondary decrease in laminin beta 1 expression.

Adolescent↗
PubMed1999

The novel structural protein of human cytomegalovirus, pUL25, is localized in the viral tegument.

Human cytomegalovirus UL25 codes for a structural phosphoprotein of 85 kDa (C. J. Baldick and T. Shenk, J. Virol. 70:6097-6105, 1996; M. C. Battista et al., J. Virol. 73:3800-3809, 1999). In this study we analyzed the intracellular and intraviral localization of pUL25 by confocal and immunoelectron microscopy and found that pUL25 is a component of the viral tegument and the dense body matrix, acquired during the late cytoplasmic phase of virus maturation.

Cell Line↗
PubMed1999

Phosphatidylinositol 3-kinase translocation to the nucleus is induced by interleukin 1 and prevented by mutation of interleukin 1 receptor in human osteosarcoma Saos-2 cells.

Although interleukin 1 (IL-1) functions have been extensively characterized, the mechanisms by which IL-1 signals are transduced from the plasma membrane to the nucleus are less known. Recent evidence indicates that phosphatidylinositol 3-kinase (PI3-kinase) could be activated by a direct association with the activated IL-1 receptor. In this study we analyzed the effects of IL-1 on the intracellular distribution of PI3-kinase in wild-type Saos-2 human osteosarcoma cells, and in cell clones overexpressing type I IL-1 receptor (IL-1RI). PI3-kinase intracellular distribution displays two distinct patterns. In quiescent cells, PI3-kinase is distributed through the cytoplasm, although a portion is present in the nucleus; following stimulation with IL-1, PI3-kinase is redistributed, increasing in the nuclear compartment. Both immunoblotting and immunofluorescence data indicate that IL-1 causes a rapid and transient translocation of PI3-kinase from the cytoplasm to the nucleus. This phenomenon is prevented by PI3-kinase inhibitors, suggesting that the maintenance of PI3-kinase activity is essential for IL-1-induced translocation. Indeed, in cell clones stably transfected with Y479F receptor mutant, in which the binding of the enzyme to the activated receptor is blocked, IL-1-induced PI3-kinase translocation to the nucleus is completely prevented. These data suggest that PI3-kinase translocation to the nucleus upon IL-1R activation is an early event in IL-1 signaling mechanism, and may be involved in transcriptional activation.

Base Sequence↗
PubMed1999

P-glycoprotein subcellular localization and cell morphotype in MDR1 gene-transfected human osteosarcoma cells.

Efflux of chemotherapy agents by P-glycoprotein at the plasma membrane is thought to be a major cause of cancer multidrug-resistance (MDR). However, the mechanism underlying the cellular accumulation and distribution of cytotoxic drugs is still poorly defined. We have recently found that P-glycoprotein is expressed also in the nucleus of MDR cell lines selected in doxorubicin (DXR), suggesting the possible involvement of this protein in the direct extrusion of the drug from the nucleus of resistant cells. In this study, we analyzed the subcellular localization of P-glycoprotein, in a series of U-2 OS osteosarcoma cell clones transfected with MDR1 gene in order to verify whether the nucleus is a constant site for the localization and functional activity of P-glycoprotein, and in which way some aspects of cell morphology related to MDR depend on the subcellular P-glycoprotein localization rather than on the exposure to the selective drug. Our results indicate that to achieve a subcellular drug distribution prevailing in the cytoplasm but not in the nucleus, a significant increase in the expression of P-glycoprotein at the different cellular compartments, including the plasma membrane, the cytoplasm, and the nucleus, is needed, although the in vitro drug resistance appears to be mainly dependent on the expression of P-glycoprotein at the cell surface. With regard to the morphological characteristics of MDR cells involving the cell surface and the chromatin arrangement, the influence of DXR appears to be prevalent, although P-glycoprotein overexpression cannot be excluded.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Phosphatidylinositol 3-kinase is recruited to a specific site in the activated IL-1 receptor I.

Interleukin 1 (IL-1) delivers a stimulatory signal which increases the expression of a set of genes by modulating the transcription factor NF-kappaB. The IL-1 receptors are transmembrane glycoproteins which lack a catalytic domain. The C-terminal portion of the type I IL-1 receptor (IL-IRI) is essential for IL-1 signalling and for IL-1 dependent activation of NF-kappaB. This portion contains a putative phosphatidylinositol 3-kinase (PI 3-kinase) binding domain (Tyr-E-X-Met), which is highly conserved between the human, mouse and chicken sequences, as well as the related cytoplasmic domain of the Drosophila receptor Toll. This observation prompted us to investigate the role of PI 3-kinase in IL-1 signalling. Here we report evidence that PI 3-kinase is recruited by the activated IL-IRI, causing rapid and transient activation of PI 3-kinase. We also show that the receptor is tyrosine phosphorylated in response to IL-1. Expression of a receptor mutant lacking the putative binding site for p85 demonstrates that Tyr479 in the receptor cytoplasmic domain is essential for PI 3-kinase activation by IL-1. Our results indicate that PI 3-kinase is likely to be an important mediator of some IL-1 effects, providing docking sites for additional signalling molecules.

Binding Sites↗
PubMed1998

Increase of neuronal nitric oxide synthase in rat skeletal muscle during ageing.

Nitric oxide synthases (NOS) are different widely expressed enzymes which produce the molecular messenger nitric oxide. The neuronal isoform of NOS (nNOS) is involved in several processes of the cell metabolism, most of which are, at present, not fully understood (neurotransmission, smooth muscle motility, myoblast and myocyte biology and others). In skeletal muscle nNOS is present mainly at the plasmalemma, where it is attached to the dystrophin-related proteins; in fact, in pathologies involving dystrophin, nNOS is altered as well. We report that in aged rats the nNOS amount in skeletal muscle increases both in the soluble and microsomal fractions and that an additional intracytoplasmic localisation appears.

Aging↗
PubMed1998

Immunofluorescence study of a muscle biopsy from a 1-year-old patient with Walker-Warburg syndrome.

A previous study of two patients with Walker-Warburg syndrome (WWS) showed a severe deficiency of the extracellular matrix protein laminin beta2 chain and alpha-sarcoglycan (adhalin) in skeletal muscle fibers. More recently, however, other researchers have shown that in their WWS patients the expression of the laminin beta2 chain and alpha-sarcoglycan was normal. Here we describe a 1-year-old boy affected with WWS. We performed immunohistochemical studies on a muscle biopsy from this patient using monoclonal antibodies against dystrophin, dystrophin-associated glycoproteins and several proteins of the extracellular matrix. We confirm previously reports as far as the diminished expression of laminin beta2 chain and alpha-sarcoglycan is concerned. The expression of some other laminins was unusual, whereas the expression of collagen IV and VI was normal. These results suggest that complex syndromes like WWS are quite heterogeneous, although they might represent variant expressions of a single pathological entity.

Biopsy↗
PubMed1998

Inositides in the nucleus: taking stock of PLC beta 1.

The nucleus was shown to be a site for inositol lipid cycle which can be affected by treatment of quiescent cells with growth factors such as IGF-I. In fact, the exposure of Swiss 3T3 cells to IGF-I results in a rapid and transient increase in nuclear PLC beta 1 activity. In addition, several other reports have shown the involvement of PLC beta 1 in nuclear signalling in different cell types. Indeed, PLC beta 1 differs from the PLC gamma and della isozymes in that it has a long COOH-terminal sequence which contains a cluster of lysine residues that are critical for association with the nucleus. Although the demonstration of PtInsP and PtdInsP2 hydrolysis by nuclear PLC beta 1 established the existence of nuclear PLC signalling, the significance of this autonomous pathway in the nucleus has yet to be thoroughly clarified. By inducing both the inhibition of PLC beta 1 expression by antisense RNA and its overexpression we show that this nuclear PLC is essential for the onset of DNA synthesis following IGF-I stimulation of quiescent Swiss 3T3 cells. Moreover, using a different cell system, i.e. Friend erythroleukemia cells induced to differentiate towards erythrocytes, it has been evidenced that there is a relationship between the expression and activity of nuclear PLC beta 1 and the association of PI-PT alpha with the nucleus in that, when PLC activity ceases, in differentiated and resting cells at the same time there is a dramatic decrease of the association of PI-PT alpha with the nucleus.

3T3 Cells↗
PubMed1998

Oral exfoliative cytology for the non-invasive diagnosis in X-linked Emery-Dreifuss muscular dystrophy patients and carriers.

Emery-Dreifuss muscular dystrophy (EMD) is an inherited myopathy characterised by muscle contractures, progressive muscle wasting and weakness, with humeroperoneal distribution. Cardiac arrhythmia and heart conduction block are also important characteristics of this disease. The X-linked form of EMD is caused by the absence of emerin, encoded by the STA gene (Xq28). Emerin is normally localized in muscle and other tissues at the nuclear rim. Currently, muscle and skin biopsies are used for the immunohistochemical diagnosis. We demonstrate that emerin is present in the cheek oral mucosa, in the exfoliating epithelial cells, and we propose the collection of these cells as a new method for the diagnosis of X-linked EMD patients and the detection of carriers by immunofluorescence techniques: smears from healthy subjects contained about 98% emerin-positive cells, those from X-linked EMD patients contained none and those from carriers contained about 45%. The technique is completely non-invasive, simple, repeatable and inexpensive.

Adolescent↗
PubMed1998

Hereditary motor and sensory neuropathy Lom type in an Italian Gypsy family.

We describe a form of hereditary motor and sensory neuropathy (HMSN) affecting four siblings in an Italian family of Gypsy ethnic origin with both clinical and pathological findings very reminiscent of the HMSN Lom type (HMSNL), recently described in a group of Bulgarian Gypsies. Genetic analysis demonstrated linkage to chromosome 8q24 and conserved haplotypes in the HMSNL region, thus confirming that this is the first Gypsy family outside the Balkans suffering from the same disorder.

Adolescent↗
PubMed1998

Immunocytochemical detection of emerin within the nuclear matrix.

Emerin, the protein whose production is altered in the X-linked form of Emery-Dreifuss muscular distrophy, has been hypothesized to be associated with the nuclear matrix on the basis of biochemical studies. In addition, immunocytochemical data reported its localization at the nuclear periphery, on the nuclear lamina, in sections of several normal tissues. We investigated the association of emerin with the nuclear matrix, by using cultured cells (SaOS-2, MG63 and HeLa-S3) and their in situ extracted matrix as a model, and immunocytochemical methods, both at the light and electron microscope level. Our results show a normal presence of emerin in the cultured cells and the specific persistence of emerin on the lamina of the in situ extracted nuclear matrix. This suggests a tight binding between emerin and the nuclear lamina independently from the interactions between the C-terminal hydrophobic domain of the protein and the inner nuclear membrane.

Cell Nucleus↗
PubMed1998

Ataxin-3 is transported into the nucleus and associates with the nuclear matrix.

It has been reported that the ataxin-3 protein containing a polyglutamine sequence in the pathological range (61-84Q) is localized within the nucleus of neuronal cells, whereas ataxin-3 with a normal repeat length (12-37Q) is predominantly a cytoplasmic protein. In this study, the subcellular localization of the full-length ataxin-3 protein with a glutamine sequence in the normal range (Q3KQ22) was analysed in two mammalian cell lines. Using two affinity-purified polyclonal antibodies raised against the N- or C-terminal portion of ataxin-3, the protein was detected predominantly, but not exclusively, in the nucleus of COS-7 as well as neuroblastoma cells by immunofluorescence and confocal laser scanning microscopy (CLSM). The distribution of the protein in these cellular compartments was confirmed by biochemical subcellular fractionations. Furthermore, CLSM revealed that the ataxin-3 protein present in the nucleus of neuroblastoma cells is associated with the inner nuclear matrix. Our results taken together with the finding of a nuclear localization signal in ataxin-3 indicate that the ataxin-3 protein per se translocates to the nucleus and that an expanded glutamine repeat is not essential for this transport.

Animals↗
PubMed1998

Cytochemistry of the functional domains of the nucleus in normal and in pathologic conditions.

By means of ultrastructural cytochemistry significant advances have been made in understanding the functional roles of many nuclear domains. This review gives schematic information about the main nuclear domains involved in replication, transcription, processing and transport of the transcripts in normal and in pathologic conditions. Particular attention is paid to a functional domain that appears to be involved in signal transduction. Data are reported on the intranuclear specific localization of key elements of the polyphosphoinositide signal transduction system in different cell types including human osteosarcoma cell lines. Compared with the compartmentalization of the cytoplasm, the nucleus has long been considered as relatively unstructured. On the other hand, fundamental nuclear functions, such as DNA replication and RNA transcription, can be molecularly characterized also in cell-free systems, suggesting that supramolecular organization is not so strictly required as for other cell functions occurring within intact cytoplasmic organelles. Nevertheless, a stringent organization is required for packing about 200 cm of DNA in the about 30 micron 3 of the nucleus. In the absence of membrane-delimited organelles, the nuclear organization is based on functional compartments, or domains, whose spatial localization involves the nuclear matrix, which shares many properties with the cytoskeleton. The nuclear domains are defined as structural compartments, not necessarily stable but dynamically variable, which perform specific metabolic functions through the partitioning of molecular complexes. Their identification has been made possible in the last few years by the development of specific nuclear probes for confocal and electron microscope immunocytochemistry. Therefore, the complex network of structures and enzymatic functions that make up the nucleus is in several cases yielding to molecular analysis, but a large part remains unknown (Strouboulis and Wolffe, 1996; Laemmli and Tjian, 1996). Rapid advances in understanding the functional role of the nuclear domains have been made recently: in particular, of the nuclear envelope, of the nucleolus, and of RNA splicing. In other cases, e.g. the precise localization of the nuclear domains involved in signal transduction, much remains to be clarified (Forbes and Johnson, 1997). It is conceivable that in the near future unexpected new nuclear domains will come to light and new nuclear functions may emerge, especially in field of post-transcriptional processing and transport of RNAs, and in the relationships between the nucleo-skeleton and enzymic fixed sites involved in replication, transcription and signal transduction. The aim of this review is to provide information about the morphological characteristics, the associated functions and the molecular composition of the main nuclear domains found to date. To simplify the exposition, the main data on each nuclear domain are reported in Tables, together with the principal references on the subject. Figures refer to original findings on some aspects of nuclear domain organization.

Cell Nucleus↗
PubMed1997

Increase of nuclear phosphatidylinositol 4,5-bisphosphate and phospholipase C beta 1 is not associated to variations of protein kinase C in multidrug-resistant Saos-2 cells.

The multidrug resistance (MDR) phenotype that is mediated by an overexpression of P-glycoprotein, has been suggested to be related also to an increased activity of protein kinase C (PKC) and to changes in phospholipid pattern. By electron microscope quantitative immunocytochemistry, we investigated whether PKC and other elements of the polyphosphoinositide signal transduction system are affected in an MDR variant of the human osteosarcoma cell line Saos-2. These cells, which are characterized by an increased expression of P-glycoprotein not only at the plasma membrane but also at the nuclear level, showed increased intranuclear amounts of phosphatidylinositol 4,5-bisphosphate and of phospholipase C beta 1, while both the amount and activity of both nuclear and cellular PKC were not modified with respect to sensitive cells. These results suggest that, in this model, the changes observed in the elements of nuclear signal transduction could be related to previously reported modifications of the MDR phenotype, but that P-glycoprotein phosphorylation is not dependent from increased PKC activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Redistribution of DNA topoisomerase II beta after in vitro stabilization of human erythroleukemic nuclei by heat or Cu++ revealed by confocal microscopy.

Using confocal laser scanning microscope and a monoclonal antibody we have examined by means of indirect immunofluorescence techniques the distribution of DNA topoisomerase II beta (the 180-kDa nucleolar isoform of topoisomerase II) following stabilization of isolated nuclei by exposure to moderate heat (37 degrees or 42 degrees C) or Cu++. In intact cells the antibody specifically decorated the nucleoli. The same pattern was maintained if nuclei were incubated at 0 degree C in a buffer containing spermine/spermidine/KCl or stabilized by means of 0.5 mM Cu++ for 10 minutes at 0 degree C in the same buffer. On the contrary, if stabilization was performed by incubating the nuclei either at 37 degrees or 42 degrees C, the immunoreactivity dispersed all over the nucleus, forming numerous speckles. This phenomenon was not detected if, in addition to spermine/spermidine/KCl, the incubation buffer also contained 5 mM Mg++ and the temperature was 37 degrees C. If the stabilization was performed at 42 degrees C, Mg++ failed to maintain the original distribution of DNA topoisomerase II beta, as seen in intact cells. The analysis on 2-D optical section showed the alteration of the nucleolar profile, particularly at 37 degrees C, even when the samples were treated with Mg++. The 3-D reconstruction figured out the irregularity of the surface at 37 degrees C and the variations of the volume occupied by the fluorescent figures. These were in close proximity to each other both in intact cells and in 0 degree C incubated nuclei; they showed a certain degree of shrinkage in 0 degree C plus Cu++ exposed samples (-20% of the volume), and, on the contrary, the labeled structures were scattered in a volume increased two- or threefold when exposed to 37 degrees or 42 degrees C, respectively. The addition of Mg++ restored the original spatial relationship and volume at 37 degrees C, but not at 42 degrees C, where the volumetric analysis showed an increase of about 50%. Our results demonstrate that heat stabilization of isolated nuclei in a buffer without Mg++ (i.e., a technique often employed to prepare the nuclear matrix or scaffold) cannot be considered an optimal procedure to maintain the original distribution of protein within the nucleus.

Antibodies, Monoclonal↗
PubMed1997

Intramembrane protein distribution in cell cultures is affected by 50 Hz pulsed magnetic fields.

Intramembrane proteins (IMP) represent a class of proteins located in the lipid bilayer of the cell membrane which function as ion channels, enzymes or receptors. Since it has been argued that biological effects of extremely low frequency (ELF) electromagnetic fields are mediated by plasma membrane. this work was designed to study the possible effects of 50 Hz pulsed magnetic fields (PMF) of the type used to stimulate bone repair, on the distribution of IMP in the plasma membrane of Swiss NIH 3T3 fibroblasts. Evaluations were based on the calculation of a distribution factor, which allows discrimination between random, regular and clustered distribution of IMP, in electron microscope images of freeze-fractured membranes. The results indicate that cells exposed to PMF for more than two hours have a significant clustering of the IMP distribution compared to control unexposed cells.

3T3 Cells↗