1,721,015 research outputs found
Searching the point of no return in Helicobacter pylori life: necrosis and/or programmed death.
No full textAims: Ultrastructural and molecular studies to support the hypothesis of programmed cell
death in Helicobacter pylori were conducted.
Methods and Results: Evidence of programmed death in H. pylori is provided through
electron microscopic detection and cytochemical labelling of electrondense bodies (EDB),
1 containing packaged DNA in coccoid cells, resembling micronuclei of apoptotic eukaryotic
cells. This morphological evidence is also supported by DNA cleavage in homogeneous
fragments of about 100 base pairs. Programmed cell death was observed in H. pylori cultures at
37 °C, with a maximum of 37á5% of EDB coccoid cells after 7 days. The non-permissive
temperature of 4 °C anticipated this process, with 40% of EDB coccoid forms within 3 days,
and it remained substantially unaffected during the observation time of 14 days.
Conclusion: In these experiments, deprivation of nutrients and a non-permissive temperature
acted as a powerful trigger for programmed cell death.
Significance and Impact of the Study: Helicobacter pylori bacterial populations, under
2 stressing stimuli, can respond with programmed cell suicide as a means of species preservation
Significance of nuclear phospholipase C signaling through type 1 IGF receptor.
No full textThe existence of a nuclear polyphosphoinositol metabolism independent from that at the plasma membrane is now widely recognized. Specific changes in the nuclear phosphatidylinositol (Ptdlns) metabolism have been implicated in cell growth, differentiation, and neoplastic transformation. Here we shall review the main features of nuclear inositol lipid signaling through type I IGF receptor, focusing the attention on the role of inositide-specific phospholipase C (PI-PLC) beta1 in cell proliferation and differentiation, given its peculiar localization in the nuclear compartment
Inhibition of phosphoinositide 3-kinase impairs pre-commitment cell cycle traverse and prevents differentiation in erythroleukaemia cells.
No full textDuring the early hours after exposure to differentiation inducing agents, Friend erythroleukaemia cells undergo alterations which commit them to cessation of growth and development of the characteristics of differentiation. Our current experiments have compared the expression and activity of phosphoinositide 3-kinase (PI 3-kinase) in control cells with cells undergoing differentiation which has been induced by dimethyl sulfoxide (DMSO). When the cultures were initiated with stationary phase cells and DMSO was added at the time of seeding, PI 3-kinase activity was stimulated in both treated and control cells during the first 3 h from seeding. This event appears to be a rate limiting step in commitment since pretreatment of cells with 10 microM LY294002 or down-regulation of p85 expression prior to adding DMSO completely prevents commitment to erythropoiesis. Accordingly, PI 3-kinase inhibition during the commitment period prevents DNA-binding of the transcription factor GATA-1, essential for erythroid differentiation. However, once cells are committed to differentiate, PI 3-kinase activity and expression dramatically decreases along with the differentiation programme, to become barely detectable after 96 h. Remarkably, LY294002 treatment leads to accumulation of cell in G1 phase and prevents DMSO-dependent cyclin D3 induction. Based on these data, we suggest that PI 3-kinase is rate limiting for the completion of the first round cycle of cell division required for initiation of erythrocytic differentiation. On the other hand, the late decrease of PI 3-kinase associated with the differentiation process seems to be part of the programmed shut off of genes not needed in mature erythrocytes
Nuclear lipid-dependent signal transduction in human osteosarcoma cells
No full textThe enzymes and substrates involved in phosphoinositide signal transduction which have been detected in the nucleus of several cell types have been demonstrated to be responsive to agonists. The complexity of this aspect of inositide function has been previously analyzed in some cell models characterized by a mitogenic or differentiating response to specific factors. An interesting experimental model is represented by human derived osteosarcoma Saos-2 cells, characterized by the expression of high affinity receptors for interleukin 1 alpha (IL-1 alpha), which is one of the most potent stimulators of bone resorption. In particular, we investigated the earliest intracellular events following the binding of IL-1 alpha to its receptor, involving the inositide signal transduction pathway. Saos-2 cells present a partitioning of the phosphoinositidase (PLC) isoforms; in fact, the nucleus contains both PLC beta 1 and gamma 1, while the cytoplasm contains almost exclusively the gamma 1 isoform. IL-1 alpha evokes a rapid and transient increase of the PLC beta 1 activity in the nucleus, which causes the hydrolysis of phosphatidylinositol mono- and bis-phosphate. In response to IL-1 alpha, not only the canonical inositol lipid pathway appears to be involved; also the 3'-phosphorylated lipids generated by phosphatidylinositol 3-kinase (PI 3-K), which may act as second messengers, appear to be affected. In fact, Saos-2 cells present a nuclear PI 3-K activity which can be enhanced by the IL-1 alpha treatment. Among the possible targets of the second messengers released by the nuclear PLC beta 1 activation, we found that some protein kinase C isoforms, namely the epsilon and zeta, which are present within the nucleus, are activated after IL-1 alpha exposure. These activated PKC isoforms, in turn, could modulate the activity of the transcription factor NFkB, which, 5 min after IL-1 alpha treatment, has already translocated to the nucleus and bound to DNA to promote gene activation. The actual role of the inositide pathway in the Saos-2 cell function has also been investigated by utilizing cell clones transfected with the mouse sequence of the PLC beta 1
Inositol lipid cycle and autonomous nuclear signalling
No full textThe involvement of phospholipids and in particular polyphosphoinositides in cellular signalling has been documented in detail in the last 20 years. In addition to the plasma membrane localization also the nucleus is shown to be a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. Previous observation have established that the nucleus possesses a specific PLC for inositol lipids, i.e., the PLC beta 1 isoform, which undergoes rapid and transient activation after IGF-I stimulation of quiescent Swiss 3T3 cells and is down-regulated after treatment of Friend erythroleukemia cells with DMSO. Here we have reviewed: (i) the potential of nuclear PLC beta 1 to be a target for anti-cancer drug, (ii) the capability of this PLC isoform, when activated by IGF-I, to be a key signalling molecule in the onset of DNA synthesis, via DAG generation and PKC alpha translocation to the nucleus, (iii) the chromosome mapping of PLC beta 1 gene. The differentiation program of Friend cells can be activated by other agents besides DMSO including tiazofurin, an anti-tumor drug, also capable of affecting the nuclear inositol lipid cycle. Tiazofurin induces a lowering of the activity of PLC beta 1 due to down regulation of this isoform as revealed by both Western blotting and Northern blotting analyses. Using Swiss 3T3 cells stably transformed with an antisense PLC beta 1 construct, the knock-out of the PLC beta 1 gene induces both a loss of PLC beta 1 expression, as determined by Western blots, and a loss of the mitogenic responsiveness to IGF-I. These events show a direct relationship between nuclear PLC beta 1 evoked signals and IGF-I induced cell proliferation. Finally, the assignment of the PLC beta 1 gene to the band q35-36 of rat chromosome 3 paves the way for further genetic studies given the fact that the region where PLC beta 1 gene maps is a hot spot for genetic alterations in a number of experimentally induced rat tumors. Taken as a whole, these results assign a key role to the regulation of nuclear PLC activity and expression both in growth-factor activated mitogenesis and in in vitro erythroid differentiation
Genetic alterations at the nuclear localization signal of the RB2/p130 gene occur in lymphoid tumor but not in osteosarcoma cell lines
No full textThe retinoblastoma gene (RB/p105) is the prototypic tumor suppressor gene, whose inactivation has been related to several human neoplasia. Based on structural and functional similarity to pRb/p105, the pRb2/p107 and the pRb2/p130 proteins are considered to form the Rb protein family. These nuclear proteins display a cell-cycle-related phosphorylation, form complexes with the E2F family of transcription factors, and act as negative regulators of cell cycle progression, blocking cells in the G1 phase. The Rb proteins present cell-type-specific growth suppressive properties, so that they are considered not functionally redundant, but complementary, pRb2/p130 expression in lung cancer appears inversely related to tumor malignancy and RB2/p130 maps to a chromosome area in which loss of heterozygosity has been found in several human tumors, suggesting an involvement of RB2/ p130 mutation in human carcinogenesis. Taking advantage of the knowledge of the complete genomic sequence of the RB2/p130 gene, the expression and the functional role of pRb2/p130 have been investigated in two clusters of tumor lymphoid and osteosarcoma cell lines. Since all the examined lymphoid tumor cell lines showed pRb2/ p130 localized at the cytoplasm level, while normal lymphocytes and the osteosarcoma cell lines all exhibited an exclusive nuclear localization, possible mutations occurring at the nuclear localization signal (NLS) in the C-terminus of the protein have been screened. The mutations, mainly found at exons 21 and 22, all resulted in the alteration of NLS in lymphoid tumor cell lines, as well as in EBV-positive primary Burkitt's lymphomas. This ruled out the possibility that mutations were a product of cell growth in culture. On the other hand, the point mutations found in exons 19-21 in the osteosarcoma cell lines did not alter the NLS, in agreement with the nuclear localization of pRb2/p 130 in these cells. Besides the loss of functional NLS, the mutations occurring in lymphoid tumor cell lines caused a premature stop codon and a shorter transcription product, as confirmed by Western blot analysis of pRb2/p130 protein, which showed a 116 kD mutant form. The mutations observed in osteosarcoma cells, causing alterations in the two putative phosphorylation sites, resulted in either unphosphorylated or persistent hypophosphorylated forms of the protein. The functional consequences of NLS disruption were analyzed by recreating the NQ point mutations by PCR-based site-directed mutagenesis of the full-length pRb2/p130 protein in an expression vector that drives gene expression by the constitutive CMV promoter. The EGFP fluorescent chimerical protein has been found to be localized at the cytoplasm in transiently transfected osteosarcoma Saos-2 cells that normally show an exclusive nuclear localization when the pRb2/p130 protein has been mutated at both NLS. The effect on cell proliferation of these mutant NLS was determined in transfected Saos-2 cells by FACS analysis and colony assay. The results indicated that only the disruption of both NLS almost completely abolished the growth suppressive activity of pRb/p130 protein and that this effect was not due to the toxicity of the over-expressed protein. In conclusion, the genetic alterations of the RB2/p130 gene found in some primary tumors suggest that, as in case of the RB2/p105 gene, pRb2/p130 is involved in cell cycle control and that it can act as a tumor suppressor gene. Moreover, the non-redundancy of growth-suppressive properties of the Rb protein family is further demonstrated by the presence of specific mutations in the NLS sequences of the pRb2/p130 in lymphoid tumor cells that prevent a nuclear localization of the protein with an obvious loss of its function, while in osteosarcoma cells this can occur by mechanisms affecting the phosphorylation of the protein or its interaction with viral oncoproteins
Immunocytochemical detection of the specific association of different PIC isoforms with cytoskeletal and nuclear matrix compartments in PC12 cells
No full textThe increasing evidence of discrete roles of phosphoinositidase C (PIC) isoforms and the assessment of their localization in the cytoskeleton and in the nucleus support the involvement of particular isotypes of this enzyme in signal transduction at multiple levels. PC12 rat pheochromocytoma is one of the few cell lines expressing three immunologically distinct isoforms of PIC. We have analyzed the subcellular distribution of the PIC β2, γ1 and δ1 isoforms using confocal and electron microscope immunocytochemistry. PIC β1 is mainly found in the nucleus and is associated with interchromatin domains. On the other hand, the PIC γ1 isoform is found in the nucleus and in the cytosol, while PIC δ1 is exclusively cytoplasmic. Immunoblot and immunocytochemical experiments indicate that the various PIC isoforms are differently bound to structural cell compartments, such as cytoskeletal and nuclear matrix elements. In fact, PIC β1 and PIC γ1 isoforms are tightly associated with the nuclear matrix, while only about 50% of PIC γ1 is associated with the cytoskeleton after DNase I and high salt extractions. PIC γ1 is almost completely soluble under these conditions. These results further confirm the complexity of the inositide signal transduction mechanism, which involves several PIC isoforms, specifically localized in different cell compartments and support the existence of a membrane-unrelated inositol lipid-dependent signalling in the nuclear interior
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