Search PubMed⌕ Search

Biomedical subjects

E Melloni

Publications and source records attributed to E Melloni.

At least 19 recordsLinked to original sources
PubMed2004

In vitro apoptotic cell death during erythroid differentiation.

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.

Antigens, CD34↗
PubMed2004

The response of human natural killer cells to interleukin-2.

Natural killer cells play a key role in the defence of organisms against virus infections and in the control of tumor onset. Interleukin-2 is a multifunctional inflammatory cytokine able to activate natural killer cells, essentially inducing cell proliferation, lymphokine-activated-killer cell generation and cytokine production. Here we discuss some signaling events generated by interleukin-2 in the cell nucleus of primary human natural killer cells, specifically focusing on the lipid signal transduction and the induction of the cyclic adenosine-5'-monophosphate response element binding protein transcription factor. The implications of these nuclear events in the response of natural killer cells to interleukin-2 are also discussed.

Animals↗
PubMed2003

Changes in calpastatin localization and expression during calpain activation: a new mechanism for the regulation of intracellular Ca(2+)-dependent proteolysis.

The amount of calpastatin directly available in cytosol is under the control of [Ca2+] and [cyclic AMP]. Prolonged calpain activation also promotes degradation of calpastatin. The fluctuation of calpastatin concentration in cell soluble fraction is accompanied by an initial decrease in calpastatin gene expression, followed by a fivefold increase in its expression when the inhibitor protein is degraded. This process can be conceptualized as a mechanism to regulate calpastatin availability in the cell. This conclusion is supported by the fact that calpain, the other component of this proteolytic system, undergoes changes in its levels of expression in a much more limited manner. Furthermore, this process can be observed both in cells exposed to different natural stimuli, or in other cell lines. Modification of calpastatin gene expression might represent a new tool for the in vivo control of the regulatory machinery required for the modulation of Ca(2+)-dependent proteolysis.

Calcium↗
PubMed2003

Synthesis of 3,3'-(1-piperidino)substituted methylene-bis-isoxazoles preventing stimulus-induced leukocytes activation.

Some 3,3'-(1-piperidino)substituted methylene-bis-isoxazoles were prepared via Mannich base and tested to verify their antiinflammatory-related activity. Human neutrophils stimulated with either PMA and f-MLP were used as the cellular model. The efficiency of eight differently substituted compounds (2-9) was established on their capacity to reduce the O(2)(-) production by activated human neutrophils. The rising hydrophobicity in the side-chain of methylene-bis-isoxazoles leads to a distinction in the neutrophil response against the two stimuli, favoring the inhibition of the PMA elicited cell activation and leaving inaffected the f-MLP induced cell responses. Compounds 8 and 9 are particularly active and abolish almost completely the neutrophil activation in the presence of PMA stimulus.

Humans↗
PubMed2002

Activation of PKC-beta isoforms mediates HNE-induced MCP-1 release by macrophages.

4-Hydroxynonenal (HNE) in the concentration range detectable in many pathophysiologic conditions is able to modulate signal transduction cascades and gene expression. Here, we report the stimulating effect of 1 microM HNE on the release of the monocyte chemotactic protein-1 (MCP-1) by murine macrophages. MCP-1-increased export following 1-h cell treatment with HNE proved to be comparable to that exerted by standard amounts of bacterial lipopolysaccharide (LPS). However, the key molecular event in HNE-induced secretion of MCP-1 appeared to be the increased activity of beta-PKC isoforms, which are recognized as playing a role in the regulation of cell protein transport and secretion. On the other hand, in LPS-stimulated cells, the delta isoform was seen to be involved and was probably related to LPS-mediated effects on MCP-1 expression and synthesis. In conclusion, HNE might interact with other pro-inflammatory stimuli, like LPS, in a concerted amplification of MCP-1 production and secretion.

Aldehydes↗
PubMed2001

Age-dependent degradation of calpastatin in kidney of hypertensive rats.

Hypertensive rats from the Milan strain show a significant decrease in calpastatin activity as compared with normotensive control animals. Calpastatin deficiency is age-related and highly relevant in kidney, heart, and erythrocytes and of minor entity in brain tissue. In normotensives the changes during aging in the levels of calpastatin activity and mRNA are consistent with an increase of calpastatin protein. In hypertensive rats such a relationship during aging is not observed, because a progressive accumulation of mRNA is accompanied by a lower amount of calpastatin protein as compared with control rats. Together with the low level of calpastatin in kidney of hypertensive rats, a progressive accumulation of an active 15-kDa calpastatin fragment, previously shown to represent a typical product of calpain-mediated calpastatin degradation, is also observed. Evidence for such intracellular proteolysis by Ca(2+)-activated calpain is provided by the normalization of the calpastatin level, up to that of control animals, in hypertensive rats treated with drugs known to reduce both blood pressure and intracellular Ca(2+) influx. Further evidence is provided by the disappearance, in these conditions, of the 15-kDa calpastatin fragment. These data allow the conclusion that calpastatin degradation is a relevant part of the overall mechanism for regulating calpain activity.

Age Factors↗
PubMed2001

Extracellular processing of amphoterin generates a peptide active on erythroleukaemia cell differentiation.

The release of amphoterin by murine erythroleukaemia cells exposed to the chemical inducer hexamethylenebisacetamide represents an essential step for the process of their terminal differentiation. Once exported in the culture medium, amphoterin undergoes limited proteolysis, catalysed by a serine proteinase also secreted by stimulated cells. The isolated proteinase is responsible for degradation of amphoterin, with the production of a 10-amino-acid-residue fragment, specifically retaining the cell-differentiation-stimulating activity of the native protein molecule. This peptide does not express other properties of amphoterin, such as protein kinase C-stimulating activity or systemic toxicity. These findings define a selective mechanism accounting for extracellular amphoterin functional maturation.

Animals↗
PubMed2001

Supravital exposure to propidium iodide identifies apoptosis on adherent cells.

BACKGROUND: Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.

Annexin A5↗
PubMed2001

Activation of Ca(2+)-dependent proteolysis in skeletal muscle and heart in cancer cachexia.

Cachexia is a syndrome characterized by profound tissue wasting that frequently complicates malignancies. In a cancer cachexia model we have shown that protein depletion in the skeletal muscle, which is a prominent feature of the syndrome, is mostly due to enhanced proteolysis. There is consensus on the views that the ubiquitin/proteasome pathway plays an important role in such metabolic response and that cytotoxic cytokines such as TNFalpha are involved in its triggering (Costelli and Baccino, 2000), yet the mechanisms by which the relevant extracellular signals are transduced into protein hypercatabolism are largely unknown. Moreover, little information is presently available as to the possible involvement in muscle protein waste of the Ca(2+)-dependent proteolysis, which may provide a rapidly activated system in response to the extracellular signals. In the present work we have evaluated the status of the Ca(2+)-dependent proteolytic system in the gastrocnemius muscle of AH-130 tumour-bearing rats by assaying the activity of calpain as well as the levels of calpastatin, the natural calpain inhibitor, and of the 130 kDa Ca(2+)-ATPase, both of which are known calpain substrates. After tumour transplantation, total calpastatin activity progressively declined, while total calpain activity remained unchanged, resulting in a progressively increasing unbalance in the calpain/calpastatin ratio. A decrease was also observed for the 130 kDa plasma membrane form of Ca(2+)-ATPase, while there was no change in the level of the 90 kDa sarcoplasmic Ca(2+)-ATPase, which is resistant to the action of calpain. Decreased levels of both calpastatin and 130 kDa Ca(2+)-ATPase have been also detected in the heart of the tumour-bearers. These observations strongly suggest that Ca(2+)-dependent proteolysis was activated in the skeletal muscle and heart of tumour-bearing animals and raise the possibility that such activation may play a role in sparking off the muscle protein hypercatabolic response that characterizes cancer cachexia.

Animals↗
PubMed2001

Changes in intracellular calpastatin localization are mediated by reversible phosphorylation.

We have previously reported that, in neuroblastoma LAN-5 cells, calpastatin is in an aggregated state, close to the cell nucleus [de Tullio, Passalacqua, Averna, Salamino, Melloni and Pontremoli (1999) Biochem. J. 343, 467-472]. In the present paper, we demonstrate that aggregated calpastatin is predominantly in a phosphorylated state. An increase in intracellular free [Ca2+] induces both dephosphorylation of calpastatin, through the action of a phosphoprotein phosphatase, and its redistribution as a soluble inhibitor species. cAMP, but not PMA-induced phosphorylation, reverses calpastatin distribution favouring its aggregation. This intracellular reversible mechanism, regulating the level of cytosolic calpastatin, could be considered a strategy through which calpain can escape calpastatin inhibition, especially during earlier steps of its activation process.

Calcium-Binding Proteins↗
PubMed2001

Extracellular Tat activates c-fos promoter in low serum-starved CD4+ T cells.

The regulatory human immunodeficiency virus-1 (HIV-1) Tat protein shows pleiotropic effects on the survival and growth of both HIV-1-infected and uninfected CD4+ T lymphocytes. In this study, we have demonstrated that low concentrations (10 ng/ml) of extracellular Tat protein induce the expression of both c-fos mRNA and protein in serum-starved Jurkat CD4+ lymphoblastoid T cells. Using deletion mutants, we demonstrates that the SRE, CRE and, to a lesser extent, also the SIE domains (all placed in the first 356 bp of c-fos promoter) play a key role in mediating the response to extracellular Tat. Moreover, the ability of Tat to activate the transcriptional activity of c-fos promoter was consistently decreased by pretreatment with the ERK/MAPK kinase inhibitor PD98058. Activation of c-fos is functional as demonstrated by induction of the AP-1 transcription factor, which is involved in the regulation of critical genes for the activation of T lymphocytes, such as interleukin 2. The Tat-mediated induction of c-fos and AP-1 in uninfected lymphoid T cells may contribute to explain the immune hyperactivation that characterizes the progression to autoimmuno deficiency syndrome and constitutes the optimal environment for HIV-1 replication, occurring predominantly in activated/proliferating CD4+ T cells.

CD4-Positive T-Lymphocytes↗
PubMed2000

Human neuroblastoma cell differentiation requires protein kinase C-theta.

Neuroblastoma LAN-5 cells exposed to retinoic acid cease to multiply and extend neurite outgrowths acquiring a neuronal phenotype. We now report that protein kinase C-theta; (PKC-theta;) isozyme is involved in this differentiation process due to the following findings: (i) PKC-theta; is expressed by LAN-5 cells as a nuclear and perinuclear protein; (ii) cell stimulation with retinoic acid promotes in a large increase in the expression level of the kinase and its intracellular redistribution; and (iii) a PKC-theta; antisense oligonucleotide reduces at the same time the expression level of the kinase and the cell response to retinoic acid. Altogether these data are consistent with a specific role played by PKC-theta; in the differentiation program of neuronal cells.

Cell Differentiation↗
PubMed2000

Neuronal differentiation of PC12 cells involves changes in protein kinase C-theta distribution and molecular properties.

In this study we demonstrate that the rat pheochromocytoma PC12 cell line expresses the novel protein kinase C isozyme designated PKC-θ. The isozyme is almost completely localized in the nuclear compartment of proliferating cells. Following stimulation with the nerve growth factor, PKC-θ is redistributed into the cytoplasm and the outgrowing neurite processes, mostly as a cytoskeletal associated kinase. This event is accompanied by an eightfold increase in the expression level and by the appearance of specific modifications of PKC-θ molecule. Conversely, the kinase is down-regulated once cells reach the terminally differentiated state displaying a neuron-like phenotype. These data suggest a functional role for the kinase in the regulation of cytoskeletal modeling along the multistage differentiation process of PC12 cells.

Animals↗
PubMed2000

Differential degradation of calpastatin by mu- and m-calpain in Ca(2+)-enriched human neuroblastoma LAN-5 cells.

In neuroblastoma LAN-5 cells during calpain activation, in addition to the two expressed 70 kDa and 30 kDa calpastatin forms, other inhibitory species are produced, having molecular masses of 50 kDa and 15 kDa. At longer times of incubation, both native and new calpastatin species disappear. The formation of these new calpastatins as well as the decrease in intracellular total calpastatin activity are mediated by calpain itself, as indicated by the effect of the synthetic calpain inhibitor I, which prevents both degradative processes. Analysis of the calcium concentrations required for the two processes indicates that the first conservative proteolytic event is mediated by micro-calpain, whereas the second one is preferentially carried out by m-calpain. The appearance of the 15 kDa form, containing only the calpastatin repetitive inhibitory domain and identified also in red cells of hypertensive rats as the major inhibitor form, can be considered a marker of intracellular calpain activation, and it can be used for the monitoring of the involvement of calpain in pathological situations.

Animals↗
PubMed2000

Properties and intracellular localization of calpain activator protein.

In this paper, we have further analyzed the properties of calpain activator (CA) in order to better define its physiological function. The activator shows a pH optimum approximately 7.8-8.0, independently of the nature of the buffer used. Although the maximal activity is observed with human acid-denatured globin, the effect of CA is detectable with other protein substrates, such as casein and insulin. A comparable activating effect is observed also with the synthetic substrate Succ-Leu-Tyr-AMC. The activatory effect has been evaluated in a reconstructed system, using plasma membrane Ca(2+)-ATPase as substrate. CA is localized in erythrocyte precursor cells on the inner surface of the plasma membrane in very high amount and its level profoundly decreases up to 10% of the original value when cells reach the terminal differentiated state.

Animals↗
PubMed2000

Acyl-CoA-binding protein is a potent m-calpain activator.

Acyl-CoA-binding protein, a 20-kDa homodimer that exerts many physiological functions, promotes activation of the classic calpain forms, most markedly that of the m-isozyme. This protein factor was purified from rat skeletal muscle and was also expressed in Escherichia coli. Both native and recombinant acyl-CoA-binding proteins show the same molecular properties and an identical capacity to decrease the [Ca(2+)] required for m-calpain activity. The binding of long-chain acyl-CoAs to acyl-CoA-binding protein does not modify the activating effect on calpains. Acyl-CoA-binding protein seems to be involved in the m-calpain regulation process, whereas the previously identified UK114 activator is a specific modulator of micro-calpain. Acyl-CoA-binding protein is proposed as a new component of the Ca(2+)-dependent proteolytic system. A comparative analysis among levels of classic calpains and their activator proteins is also reported.

Amino Acid Sequence↗