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.

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.

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.

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.

Chiral and non-chiral nutations in Arabidopsis roots grown on the random positioning machine.

Arabidopsis thaliana roots grown on a vertically set plate do not elongate straight down the gravitational vector, but by making waves and coils, and by conspicuously slanting towards the right-hand. This behaviour, in a previous paper, was ascribed to the simultaneous effect of three processes: circumnutation, positive gravitropism and negative thigmotropism. However, when the plants are grown on the Random Positioning Machine (RPM), in conditions that are believed to simulate space microgravitational conditions closely, the roots do not show the usual pattern. In the wild type, the roots make large loops to the right-hand side, whereas in the gravitropic and auxinic mutants aux1, eir1, rha1, they just move randomly around the initial direction. Therefore, if the movements made on the RPM are those produced by the exclusion of gravitropism and negative thigmotropism, as is apparent, the conclusion is that Arabidopsis roots are animated by a form of chiral circumnutation, that is lacking in the auxinic and gravitropic mutants aux1, eir1 and rha1. In addition, the 1 g condition appears to reduce the scatter among the circumnutating tracks produced by the roots of the wild types, but not among those of the mutants. Because there is a scarcity of literature regarding circumnutation in roots, it is not known how widely root chiral circumnutation is spread, but it is known that, in some previously studied species, just random nutations are observed. Two kinds of nutating movements seem to exist in plant roots and, whereas the random process does not seem to be connected with auxin physiology and transport, the chiral process appears to be connected in the same way as gravitropism is.

Anti-inflammatory activity of linalool and linalyl acetate constituents of essential oils.

Linalool and linalyl acetate are the principal components of many essential oils known to possess several biological activities, attributable to these monoterpene compounds. In this work, we evaluated individually the anti-inflammatory properties of (-) linalool, that is, the natural occurring enantiomer, and its racemate form, present in various amounts in distilled or extracted essential oils. Because in the linalool-containing essential oils, linalyl acetate, is frequently present, we also examined the anti-inflammatory action of this monoterpene ester. Carrageenin-induced edema in rats was used as a model of inflammation. The experimental data indicate that both the pure enantiomer and its racemate induced, after systemic administration, a reduction of edema. Moreover, the pure enantiomer, at a dose of 25 mg/kg, elicited a delayed and more prolonged effect, while the racemate form induced a significant reduction of the edema only one hour after carrageenin administration. At higher doses, no differences were observed between the (-) enantiomer and the racemate; a further increase in the dose of both forms did not result in an increased effect at any time of observation. The effects of equi-molar doses of linalyl acetate on local edema were less relevant and more delayed than that of the corresponding alcohol. These finding suggest a typical pro-drug behavior of linalyl acetate. The results obtained indicate that linalool and the corresponding acetate play a major role in the anti-inflammatory activity displayed by the essential oils containing them, and provide further evidence suggesting that linalool and linalyl acetate-producing species are potentially anti-inflammatory agents.

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.

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.

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.

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.

Simulated microgravity conditions affect poly(ADP-ribose) polymerase activity in cultured human lymphocytes.

NASA: Human peripheral blood lymphocytes (PBL), activated with concanavalin A (ConA), were used to determine the effects of simulated microgravity on poly(ADP-ribose) polymerase (PARP) activity. Results indicate that the ConA stimulation of human cultured PBL induces a partial but signitficant inhibition of PARP-1 acitvity (-30%). In control PBL, not exposed to ConA, after 24 hours, there was a clear decrease in PARP-1 acitivty (-40%). In PBL exposed to ConA and simulated weightlessness, activity decreased by -37%.^ieng

Effect of simulated microgravity conditions on poly(ADP-ribose) polymerase activity in primary cultures of adult rat hepatocytes.

The possible involvement of poly(ADP-ribose) polymerase [PARP; E.C. 2.4.2.30] in the adaptive response to low-g conditions was studied in cultured adult rat hepatocytes exposed to simulated microgravity produced by the random positioning machine (RPM-3D-clinostat). Four different poly(ADP-ribose) polymerases (PARPs) have been identified recently. The best-studied member of this family is PARP-1, a highly conserved, multimodular 113 kDa protein. In multicellular organisms PARPs catalyze poly(ADP-ribose) synthesis from NAD+ to a number of structural and catalytic proteins. Moreover, PARP-1 can control its protein and DNA interactions by catalyzing its automodification with poly(ADP-ribose) molecules that can include up to 200 ADP-ribose residues and several branching points; by these polymers, PARP-1 may nocovalently interact with other proteins and alter their functions. PARP-1 binds to DNA and is activated by free ends interacting with several other DNA damage checkpoint proteins. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions. Poly(ADP-ribosyl)ation plays a central role in genome stability and is involved in DNA replication and repair, gene expression, cell differentiation and transformation. We have shown that a loss of PARP-1 activity is a critical event in the early molecular steps of the hepatocarcinogenesis process. Moreover, a prompt increase in this enzymatic activity is linked not only to the presence of DNA free ends but is linked also to the start of DNA synthesis. More recently, we have reported that PARP-1 is involved in hormone-mediated gene expression in vitro and in vivo during rat liver regeneration.

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.

Gravitational effects on the response to different stimulatory signals in T cells.

The purpose of this paper is to present new data on the in vitro activation of purified T cells with three different mitogenic agents in conditions simulating low-gravity.

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.

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.

Influence of microgravity on mitogen binding and cytoskeleton in Jurkat cells.

The effects of microgravity on Jurkat cells--a T-lymphoid cell line--was studied on a sounding rocket flight. An automated pre-programmed instrument permitted the injection of fluorescent labelled concanavalin A (Con A), culture medium and/or fixative at given times. An in-flight 1 g centrifuge allowed the comparison of the data obtained in microgravity with a 1 g control having the same history related to launch and re-entry. After flight, the cells fixed either at the onset of microgravity or after a or 12 minute incubation time with fluorescent concanavalin A were labelled for vimentin and actin and analysed by fluorescence microscopy. Binding of Con A to Jurkat cells is not influenced by microgravity, whereas patching of the Con A receptors is significantly lower. A significant higher number of cells show changes in the structure of vimentin in microgravity. Most evident is the appearance of large bundles, significantly increased in the microgravity samples. No changes are found in the structure of actin and in the colocalisation of actin on the inner side of the cell membrane with the Con A receptors after binding of the mitogen.

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.

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.

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.