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Biomedical subjects

M A Meloni

Publications and source records attributed to M A Meloni.

At least 19 recordsLinked to original sources
PubMed2006

Cytoskeleton changes and impaired motility of monocytes at modelled low gravity.

Investigations performed in space have shown that gravity changes affect important cellular mechanisms like proliferation, differentiation, genetic expression, cytoskeletal architecture, and motility in lymphocytes, monocytes, and other mammalian cells. In particular, a dramatic depression of the mitogenic in vitro activation of human peripheral blood lymphocytes was observed at low gravity. The hypothesis of the present work is that a reduced interaction between T lymphocytes and monocytes, essential for the second signalling pathway, might be one of the reasons for the observed depression of the in vitro activation of human lymphocytes. Cell motility and with it a continuous rearrangement of the cytoskeletal network within the cell is essential for cell-to-cell contacts. Whereas nonactivated lymphocytes in suspension are highly motile at low gravity, no data are available so far on the motility of adherent monocytes. It thus can be argued that impaired monocyte locomotion and cytoskeletal changes could be responsible for a reduced interaction of monocytes with T lymphocytes. In this study, the locomotion ability of J-111 cells, an adherent monocyte cell line, attached to colloidal gold particles on coverslips and exposed to modelled low gravity in the random positioning machine was found to be severely reduced compared with that of controls and the structures of actin, tubulin, and vinculin were affected.

Actin Cytoskeleton↗
PubMed2005

Key gravity-sensitive signaling pathways drive T cell activation.

Returning astronauts have experienced altered immune function and increased vulnerability to infection during spaceflights dating back to Apollo and Skylab. Lack of immune response in microgravity occurs at the cellular level. We analyzed differential gene expression to find gravity-dependent genes and pathways. We found inhibited induction of 91 genes in the simulated freefall environment of the random positioning machine. Altered induction of 10 genes regulated by key signaling pathways was verified using real-time RT-PCR. We discovered that impaired induction of early genes regulated primarily by transcription factors NF-kappaB, CREB, ELK, AP-1, and STAT after crosslinking the T-cell receptor contributes to T-cell dysfunction in altered gravity environments. We have previously shown that PKA and PKC are key early regulators in T-cell activation. Since the majority of the genes were regulated by NF-kappaB, CREB, and AP-1, we studied the pathways that regulated these transcription factors. We found that the PKA pathway was down-regulated in vg. In contrast, PI3-K, PKC, and its upstream regulator pLAT were not significantly down-regulated by vectorless gravity. Since NF-kappaB, AP-1, and CREB are all regulated by PKA and are transcription factors predicted by microarray analysis to be involved in the altered gene expression in vectorless gravity, the data suggest that PKA is a key player in the loss of T-cell activation in altered gravity.

Astronauts↗
PubMed2004

Effect of simulated microgravity on PGE2-induced edema and hyperalgesia in rat paws: pharmacological data and biochemical correlates.

The aim of the present study has been to investigate the effect of conditions of modeled microgravity using a three-dimensional clinostat (Random Positioning Machine, RPM) on edema and thermal hyperalgesia induced by prostaglandin E2 (PGE2) in the hind paw of rat. Our results showed that RPM reduced PGE2-evoked edema associated to a significant decrease in hyperalgesia compared to ground control animals. To further characterize the mechanisms by which RPM induces anti-inflammatory and anti-hyperalgesic action, we performed biochemical assays of PGE2 and Western immunoblot experiments to assess whether the intraplantar administration of exogenous PGE2 modifies the expression of the iNOS. These results showed that RPM diminished the levels of PGE2 in exudates of paws previously treated with PGE2, but did not influence the iNOS expression.

Animals↗
PubMed2004

Modeled microgravity affects motility and cytoskeletal structures.

The hypothesis to be tested is that reduced cell-cell interactions between T cells and monocytes are one of the reasons for the observed depression of the "in vitro" activation of human lymphocytes in microgravity. Locomotion is essential for cell-cell contacts. Lymphocytes in suspension are highly motile in microgravity, whereas no data are available so far on the motility of adherent monocytes. It can be argued that an impaired locomotion of monocytes and cytoskeletal changes, both linked to cell contacts, could be responsible for their reduced interaction with T lymphocytes. This study is aimed at revealing how locomotion as well as cytoskeletal structures of adherent monocytes are modified under modeled microgravity conditions using the Random Positioning Machine (RPM, Dutch-Space) as earth based model of spaceflight.

Actins↗
PubMed2002

Effects of microgravity and hypergravity on early development stages of Xeonopus laevis.

The aim of this work is to evaluate the development of X.l. in modified gravity conditions. The simulation of hyper and microgravity was performed utilizing: an hyperfuge, a Clinostat and later on a Random Positioning Machine (RPM, 3d Clinostat). The effect of hypergravity on embryos is significantly higher than that of microgravity; the exposure of embryos to 3xg for 3 days before and after hatch causes an activation of HSP-60 and HSP-70. Embryos exposed to 3xg during the first 3 days of development are very sensitive and show a retard of development, with a lower content of DNA, neutral glycolipids and gangliosides compared to controls.

Journal Article↗
PubMed2002

Signal transduction in T lymphocites under simulated microgravity conditions: involvement of PKC isoforms.

Previous data obtained from experiments either in space or in clinostats have shown that: a) human T lymphocytes activation is strongly inhibited; b) the distribution of protein kinase C (PKC) in human leukocytes is altered; c) expression of IL-2 and IL-2-R-alpha is altered. In this study we focus our attention on different isoforms of PKC to determine whether microgravity directly affects the activity and subcellular distribution of PKC. This work was carried out with Con A and anti-CD 28 activated human T cells in simulated microgravity conditions in the Random Positioning Machine (RPM). The cellular fractions (nuclear, cytosolic and membrane) extracted were subjected to Western blotting and RT-PCR analysis.

Journal Article↗
PubMed2002

Preliminary study of gene expression levels in human T-cells exposed to cosmic radiations.

Several experiments demonstrated the influence of microgravity on mitogenic activation of T cells at molecular level. To discriminate between effects of microgravity and cosmic radiations, in this work we studied the effects of high cosmic radiations on the genetic expression in human T cells boarded in a stratospheric balloon (BIRBA-1 mission, 22 hours of flight). The genetic expression was analyzed by the cDNA microarray hybridization technology, which allows the comparative and simultaneous estimate of hundreds of mRNAs Activated cells react to the ionizing stress by activating genes involved in cell cycle check-point, oxidative stress response, heat shock proteins production or by repressing genes involved in antigen recognition.

Journal Article↗
PubMed2002

Effects of simulated microgravity conditions on carrageenin-induced oedema in rat.

The effects of simulated microgravity conditions, using a three-dimensional clinostat (Random Positioning Machine, RPM), on carrageenin-induced paw oedema in rats as a model of local inflammation were evaluated. RPM-exposed animals showed a significant reduction of oedema and a more pronounced decrease in body weight with respect to control groups. Moreover, aspirin (ASA) treatment, an anti-inflammatory agent, on RPM-exposed rats did not exhibit any activity after carrageenin challenge with respect to RPM control animals on the ground. ASA activity on RPM could be prevented by RPM-induced anti-oedematous effect. RPM-induced anti-oedematous effect did not reversed by pre-treatment with the non-selective glucocorticoid receptor antagonist, mifepristone ruling out the supposed influence of an of cortisol release during the RPM treatment.

Journal Article↗
PubMed2001

Effects of simulated microgravity on metabolic activities related to DNA damage and repair in lymphoblastoid cells.

We adopted a simple experimental framework to follow the dependence of structural aberrations and the modifications in selected metabolic processes correlated with the exposure of cells to microgravity. Alterations to the cellular metabolism induced by exposure to microgravity are evidentiated in the modification of PARP activity (strongly dependent to the presence of DNA damages and to the altered gene expression), in the modification of the repair ability and in the cell's energy homeostasis (NAD and ATP). Cells are exposed continuously to microgravity in a Random Positioning Machine (RPM) in complete medium for 48 hours. At the end of this period a part of these cells are immediately analysed for the parameters reported above and the remaining were furtherly incubated in standard laboratory conditions to document eventual defects during the phases of the recovery process. A part of cells, just after exposure to microgravity, were also subjected to treatment with a strong damaging agent, KBrO3, and these cells were subsequently analyzed. This final treatment was meant to amplify the eventual deficiencies experienced by microgravity-exposed cells in the DNA repair process also in dependence with the alterated metabolic conditions resulting after the exposure to microgravity.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Modifications of glycosphingolipid profile and synthesis in normal rat fibroblasts and in syngeneic neoplastic cells at different subculture stages.

Glycosphingolipids are plasma membrane macromolecules involved in diversified recognition functions on the cell surface resulting in modulation of cell adhesion and differentiation. As the in vitro cellular system of the neoplastic cell line SGS/4A and syngeneic normal fibroblasts (FG) represents a useful tool for studies on molecular mechanisms regulating cell adhesion, neoplastic transformation and cellular ageing, we studied the changes of glycosphingolipid and of the enzymes involved in their metabolism in both cultured cells at different subculture stages. The FG subculture progression induces a drastic decrease of total glycosphingolipid content with consistent alterations in the molecular composition. In particular, a significant decrease of GM(3), a slight increase of GD(1a), the disappearance of 'b'-series gangliosides and the drastic reduction of triosylceramides were observed. On the contrary, the increasing number of SGS/4A subcultures, characterized by a specific and different glycosphingolipid composition as compared with FG cells, does not cause modifications. Although glycosyltransferase activity levels quite well parallel the glycosphingolipid patterns and can account for the noted variations, the mRNA expression analysis of two glycosyltransferases suggests that the in vitro cell ageing of normal rat fibroblasts causes drastic changes in the glycosphingolipid profile through the regulation, at either the transcriptional or post-translational level, of some biosynthetic enzymes.

Animals↗
PubMed1999

Human immune cells as space travelers.

Experiments in space have shown that T lymphocyte function is altered in more than 50% of space crew members. There is strong evidence that such effect is due to stress rather than to weightlessness per se. However the health of astronauts was never threatened so far. Experiments in-vitro with cultures of human peripheral blood lymphocytes (not from astronauts) have shown that T cell function is dramatically reduced. Recent work with the random positioning machine, a new instrument to simulate conditions similar to microgravity, indicate that there are direct gravitational effects on the genetic expression of interleukin-2 and of its receptor in T lymphocytes.

Actins↗
PubMed1999

Signal transduction in T lymphocytes--a comparison of the data from space, the free fall machine and the random positioning machine.

In this paper we discuss the effect of microgravity on T cells and we present the data of studies with two new machines for 0 g simulations. Several experiments in space show that mitogenic T cell activation is lost at 0 g. Immunocytochemistry indicates that such effect is associated with changes of the cytoskeleton. Biochemical studies suggest that the lack of expression of the interleukin-2 receptor is one of the major causes of the loss of activity. In fact, interleukin-2 is the third signal required for full activation. In order to deepen our investigations we are now working with the free-fall machine, FFM, invented by D. Mesland, and with the random positioning machine, RPM, or three-dimensional clinostat, developed by T. Hoson. The FFM produces periods of free-fall lasting approximately 800 ms followed by bounces of 15-30 g lasting 45-60 ms. The RPM eliminates the effect of gravity by rotating biological specimen randomly around two orthogonal axes. While the FFM failed to reproduce the results obtained with T lymphocytes in space, the data from the RPM are in good agreement with those in real microgravity. In fact, the inhibition of the mitotic index in the RPM is 89% compared to static controls. The RPM (as the FFM) can carry markedly larger specimen than the fast rotating clinostat and thus allows to conduct comprehensive studies to select suitable biological objects for further investigations in space.

Cells, Cultured↗
PubMed1998

Simulated microgravity inhibits the genetic expression of interleukin-2 and its receptor in mitogen-activated T lymphocytes.

Experiments conducted in space in the last two decades have shown that T lymphocyte activation in vitro is remarkably reduced in microgravity. The data indicate that a failure of the expression of the interleukin-2 receptor (measured as protein secreted in the supernatant) is responsible of the loss of activity. To test such hypothesis we have studied the genetic expression of interleukin-2 and of its receptor in concanavalin A-activated lymphocytes with the RT-PCR technology. Microgravity conditions were simulated in the fast rotating clinostat and in the random positioning machine. The latter is an instrument introduced recently to study gravitational effects on single cells. Our data clearly show that the expression of both IL-2 and IL-2Ralpha genes is significantly inhibited in simulated O X g. Thus full activation is prevented.

Biophysics↗
PubMed1998

Cellular adhesion in neoplastic and syngeneic normal cells under altered gravitational conditions.

The major objective of several experiments performed in space in the last 15 years was to establish whether single cells are sensitive to gravity. It was found in certain cells that reduced gravity leads to profound changes of a number of physiological functions like genetic expression, cell proliferation, signal transduction and cytoskeleton structure. In cell biology studies microgravity can be simulated on Earth in the clinostat. Nearly all data on experiments in the clinostat are related to cells cultured in suspension and, therefore, to adhesion-independent cells. In contrast, several biological phenomena as neoplastic transformation, cell differentiation, in-vitro cellular aging, contact inhibition and cellular adhesion require mainly cellular systems that are adhesion-dependent. The purpose of this work was: a) to study the behaviour of two rat cell strains (neoplastic SGS/4A and syngeneic fibroblasts FG) in order to test whether adhesion-dependent cells are suitable for clinorotation and b) to investigate cell-cell and cell-substratum adhesion in these cells kept under simulated low-g in the fast rotating clinostat and in hypergravity at l0g in the centrifuge.

Animals↗
PubMed1998

Microgravity simulations with human lymphocytes in the free fall machine and in the random positioning machine.

The purpose of this paper is to present the results obtained in our laboratory with both instruments, the FFM [free fall machine] and the RPM [random positioning machine], to compare them with the data from earlier experiments with human lymphocytes conducted in the FRC [fast rotating clinostat] and in space. Furthermore, the suitability of the FFM and RPM for research in gravitational cell biology is discussed.

Cells, Cultured↗
PubMed1997

Channelling of deoxyribose moiety of exogenous DNA into carbohydrate metabolism: role of deoxyriboaldolase.

In bacteria, the addition of (deoxy)nucleosides or (deoxy)ribose to the growth medium causes induction of enzymes involved in their catabolism, leading to the utilisation of the pentose moiety as carbon and energy source. In this respect, deoxyriboaldolase appears the key enzyme, allowing the utilisation of deoxyribose 5-P through glycolysis. We observed that not only deoxynucleosides, but also DNA added to the growth medium of Bacillus cereus induced deoxyriboaldolase; furthermore, the switch of the culture from aerobic to anaerobic conditions caused a further increase in enzyme activity, leading to a more efficient channelling of deoxyribose 5-P into glycolysis, probably as a response to the low energy yield of the sugar fermentation. In eukaryotes, the catabolism of (deoxy)nucleosides is well known. However, the research in this field has been mainly devoted to the salvage of the bases formed by the action of nucleoside phosphorylases, whereas the metabolic fate of the sugar moiety has been largely neglected. Our results indicate that the deoxyriboaldolase activity is present in the liver of several vertebrates and in a number of cell lines. We discuss our observations looking at the nucleic acids not only as informational molecules, but also as a not negligible source of readily usable phosphorylated sugar.

Aldehyde-Lyases↗