ANALYSIS OF EEG SIGNALS RECORDED AT DIFFERENT GRAVITY LEVELS DURING PARABOLIC FLIGHTS USING THE METHOD OF DIMENSIONALITY OF STRANGE ATTRACTORS

V. Pletser1, O. Quadens2
1Physical Sciences Section, Microgravity Division, ESTEC, Noordwijk, The Netherlands
2Belgian Institute of Space Aeronomy (BISA), Brussels, Belgium

The ability to measure the influence of different levels of gravity, or its absence, on the brain is important to explain the different ways human beings in space react to microgravity and to predict the capability of future astronauts to adapt to this condition.
As part of an on-going research, EEG signals were recorded on four healthy male volunteer subjects during low and high gravity phases of parabolic flights on board the Caravelle during the 12th ESA campaign of February 1991. One subject was recorded throughout the campaign on the morning, afternoon and morning of three consecutive days. This subjects' data were analyzed in depth and some results are presented here.
Only artifact free EEG signals recorded during waking rest over 46 parabolas were considered. Each parabola was divided in five periods of approximately 20 seconds corresponding to the different g phases: 1g (pre-parabola), 2g (pull-up), 0g, 2g (pull-out), 1g (post-parabola). EEG data were processed, based on the method used by Babloyantz et al. (Physics Letter 111A (3), 152-156, 1985), to calculate the value of the chaotic strange attractor dimension d and the corresponding dimension n of the phase space. The attractor dimension d is given by the slope in a log-log diagram of a correlation function C(r,n) computed by considering successively higher integer values of the dimension n of the phase space. The system possesses an attractor if the d versus n dependence is saturated beyond a value of n. If the dependence is not saturated for values of n above approximately 10, the system is said to be non converging. Only converging d values were considered further for statistical analyses as reported in (ESA-STM 259, 1999).
Results show in substance that there are clear differences between the three flights:
• the number of g phases with converging EEG signals decreases from Flight 1 to Flight 3 (respectively 84%, 78% and 55%).
• mean values of d over each flight for each g phases decrease from Flight 1 to Flight 3, (with ranges respectively 7.10 - 7.82, 6.60 - 7.51, and 5.06 - 5.95).
• means of d for each g phase decrease over the timely succession of periods at beginning, middle and end of the three flights. However, additional variations in d values, somehow oscillatory, are also observed.
To interpret these results, two hypotheses are introduced: the "fatigue/stress" hypothesis corresponding to long term fatigue accumulated over the three flights, and the "g stress" corresponding to short term fatigue in response to change in g levels.
The "fatigue/stress" hypothesis allows to explain the overall timely decrease of d values. Additional variations in the d values on shorter time scale are interpreted as being caused by the short term "g stress". However, d values vary differently at different moments in flight and the brain response to changing g levels is not univocal. The brain response degrades with time over the three flights and several degraded modes are observed: the "flat", "single event", "time shifted", "reversed", "cascading", and "home stable" brain responses, that can be explained by both short and long term fatigue hypotheses.
The non-linear dynamics method of calculating chaotic strange attractor dimensions d from recorded EEG signals provides meaningful information on the evolution of the state of the human brain when subjected to repeated regular change of gravity levels. Despite promising results, a lot of unknown factors remain and certain hypotheses, although acceptable, need to be confirmed. Additional experimental work needs to be conducted in the parabolic flight environment.


CONTINUOUS tridimensional vectocardiography and twelve leads EKG in humans during parabolic flights.

P. Vaïda¹, A. Capderou², O. Bailliart³, O. Atkov⁴, C. Kays¹, I. Desormes¹,
J. Maule⁴, P. Techoueyres¹, and J.L. Lachaud¹.
1)Médecine Aérospatiale, Université Bordeaux 2; 2)UPRES EA 2397 CHU Kremlin-Bicètre 3)CHU Lariboisière, Paris, Paris, 4)International Space University, Strasbourg, France.

PURPOSE : following our studies on fluid shift in lung (J. Appl. Physiol. 1997), leg venous return (ASMA, Chicago 1997 J. Appl. Physiol. 1998) and cardiac output during parabolic flights (ICASM, London 1995) we have studied the vectorial changes of the cardiac electrical activity during the gravitational stresses induced by the parabolic flight. Preliminary results showed sensitive variation of the electrical axis curve. This could be attributed both to a spatial change of the heart and/or intrathoracic fluid shift. To counteract fluid shift during microgravity we submitted our subjects to Lower Body Negative Pressure (LBNP) at -60 mmHg.
METHODS : 8 subjects have been examined during parabolic flights performed with the Airbus A-300 Zero G managed by Novespace for French Space Agency (CNES) in Bordeaux. Each flight session lasted 3 hours, and incorporated 32 parabolas. Each parabola trajectory lasted 20 seconds during which vertical acceleration Gz relative to the plane was near zero G (0Gz). This microgravity phase was initiated by a 1.8 G acceleration (ascending pull-up) and completed by a near similar hypergravity phase (ending pull up). Subjects were studied in the upright position during ten to twelve parabolas using vectocardiography (Cardionics, Belgium).
RESULTS : 8 subjects have been examined ; for all subjects, there are statistically significant differences (ANOVA, p < 0.05) for HR (¯ ), QRS duration (­ ), T wave azimuth(¯ ) and QRS-T spatial angle (­ ). LBNP has significant effects upon HR (­ ), QRS duration (¯ ) and amplitude (¯ ), QRS azimuth (¯ ), T amplitude (¯ ), T azimuth (¯ ) as for T elevation (­ ) and QRS-T spatial angle (­ )CONCLUSIONS : ECG and VCG are useful to study cardiac adaptation during short gravity variations. When compared to 1 Gz values, the observed deviations during 0Gz phase are coherent with a cardiac overload decreased by LBNP application.


GENDER DIFFERENCES IN NEUROENDOCRINE REACTIVITY TO PASSIVE HEAD-UP TILT

F. Strollo*, M. Morè, L. Bollanti* A. Ciarmatori*, E. Longo*, A. Mambro*, S. Di Paolo*, G. Strollo* and A. Scano**
*INRCA, Endocrine Unit and **AIMAS – Rome - Italy

BACKGROUND: One of the major concerns in space medicine is post-mission postural hypotension. Since the beginning, most of the studies have been aimed at the identification of mechanical and neuroautonomic mechanisms within the cardiovascular system potentially involved in this phenomenon. Some gender differences in the cardiovascular reactivity to changing posture have been described so far, which still need better clarification. Moreover, the neuroendocrine contribution to postflight readaptation deserves more indepth studies. AIM OF THE STUDY: Sudden passive head-up tilting is considered to be a good simulation method for hemodynamic changes occurring during transition from 0xg to 1xg. The aim of this study was to verify whether cardiovascular or neuroendocrine changes occurring after sudden passive +70° head-up tilt (HUT) became already evident within 10 min and were similar in healthy young-adult men and women. MATERIALS AND METHODS: 12 young-adult healthy subjects volunteered for the study. They were divided in two groups depending on sex, which were proved to be statistically homogeneous for age and body mass index (BMI) based upon One Way ANOVA: 6 males were 28 to 42 years (mean 36.3 years), with BMI 22.0 to 27.1 Kg/m² (mean 24.9 Kg/m²) and 6 females were in the follicular phase of their menstrual cycle, 22 to 50 years (mean 35.0 years), with BMI 22.0 to 31.2 Kg/m² (mean 27.3 Kg/m²). After an overnight fast, between 9:00 and 10:30 in the morning, all subjects were securely fastened to a horizontally oriented tilt table and kept lying there for 30 minutes, ready for R-R interval monitoring through a standard ECG recorder. A standard manually operated sphygmomanometer cuff (Riva-Rocci) was fixed to the right arm for blood pressure measurements. The tilt table was specially meant for true passive tilting: in fact the angular speed and final tilt angle were under our direct control and the lower limb proprioceptive inputs were prevented from interfering with test results by keeping subjects’ foot plants suspended (subjects were sustained by a bicycle saddle). In order to avoid the masking effect of needle stress upon hormonal levels, an indwelling catheter was inserted in the antecubital vein for subsequent blood draws; blood clotting was prevented by low-rate (10 drops/min) constant saline infusion. At the end of the 30 minutes resting period the table was tilted up to +70° within 3 seconds and subjects were kept in that position under our continuous supervision for 10 minutes. At the end of this session, the table was tilted back to the horizontal position and the test ended. The ECG was recorded from 5 minutes before to 5 min after the end of the HUT. The first 15 RR intervals immediately before HUT start (which was defined as " 0’ ") and immediately after HUT end (which was defined as " 10’ ") were recorded for the study. The mean level of each of those sequences were used for subsequent analysis. Three systolic and diastolic blood pressure (SBP and DBP) determination were quickly made at 0’ and 10’ and the mean of the three was used for subsequent analysis. At 0’ and 10’, 10 ml blood were drawn into a standard whole blood tube and 5 ml into an ice-cold EDTA plasma tube for subsequent centrifugation and serum/plasma storage at –20°C. Samples were then thawed and assayed in duplicate during a single session (in order to take rid of interassay coefficient of variation) of adrenocorticotrophin (ACTH), cortisol (CORT), biorenin (R), aldosterone (ALDO), growth hormone (GH) and prolactin (PRL) by very sensitive and accurate commercial RIA or IRMA methods commonly used in our research lab. Statistics: One way ANOVA was used to test gender differences in post-HUT to pre-HUT ratios for all parameters under study and the Student’s t test for paired samples was used to test post-HUT changes within the same gender group. Results are given as means+S.E.M’s. RESULTS: (1) Gender differences. No significant differences were found in post- to pre-HUT ratios for RR, SBP or DBP. The same applies to all neuroendocrine parameters, but for ACTH and PRL, which proved to be more reactive in the male gender. In fact, in men ACTH ratio was 3.06+0.42 vs 1.50+0.27 in women (p=0.011) and PRL ratio was 1.20+0.04 vs 1.01+0.03 in women (p=0.002). (2) Cardiovascular HUT effects. In men no significant changes were found in SBP or DBP after 10’ HUT (110.0+3.4 and 90.0+3.2 mmHg, respectively) relative to baseline (114.5+1.7 and 82.5+2.5 mmHg) while RR interval significantly dropped from 984.9+58.9 to 780.0+54.1 msec (p=0.001). The same considerations apply to women: no significant changes were found in SBP or DBP after 10’ HUT (111.3+9.2 and 82.7+5.7 mmHg, respectively) relative to baseline (114.0+8.8 and 76.0+4.2 mmHg) while RR interval significantly dropped from 949.4+31.3 to 721.0+28.4 msec (p=0.001). (3) Neuroendocrine HUT effects. In men ACTH increased from 10.8+2.1 to 29.0+3.1 pg/ml (p<0.0001) while CORT only showed an increase trend from 111.0+20.3 to 154.5+22.8 ng/ml (n.s.). R and ALDO did not increase significantly either (165.3+42.6 to 198.0+41.0 ng/dl and from 26.3+5.6 to 34.3+4.3 ng/dl, respectively). Two neuroendocrine markers of stress and intracerebral catecholaminergic tone, GH and PRL behaved different from each other and opposite to what observed already during head-down tilt. In fact, PRL increased from 110.5+12.2 to 131.3+13.4 mU/L (p=0.001), while GH showed a non-significant decrease trend from 0.68+0.29 to 0.50+0.09 ng/ml. In women ACTH kept almost unchanged (from 19.9+5.4 to 26.8+5.6 pg/ml (n.s.) while CORT only showed an increase trend from 75.0+12.7 to 89.2+17.2 ng/ml (n.s.). R and ALDO did not increase significantly either (112.3+34.9 to 164.0+43.2.0 ng/dl and from 41.6+8.1 to 54.5+1.2 pg/ml, respectively). GH and PRL behaved in a slightly different way from what observed in men: PRL did not increase significantly (changing from.216.5+9.3 to 219.7+10.7 mU/L), while GH showed a significant decrease from 1.61+0.45 to 0.99+0.25 ng/ml (p=0.042). CONCLUSIONS: Our results indicate that +70° HUT is able to induce a significant decrease in RR interval but to evoke only minor SBP and DBP changes at 10 min in both sexes. Gender differences are apparent, instead, at the level of neuroendocrine response to HUT, which appears to be prompter in terms of ACTH and PRL in men than in women. Our group is further investigating these differences by longer duration studies with shorter interval monitoring in a larger number of young volunteers in order to better elucidate the time course of described changes.


CELL CYCLE IN THE PRIMARY LENTIL ROOT DURING GERMINATION

F. Yu, D. Driss-Ecole and G. Perbal
Laboratoire CEMV, Université Pierre et Marie Curie, 4 Place Jussieu F-75252
Paris Cedex O5

Cell cycle was investigated in the cortical cells of lentil roots grown on the ground or in space in the frame of the IML1 and lML2 Missions of Spacelab. Ground experiments have shown that in seeds the majority of cortical cells had a 2C DNA content and that there were a clear peak of DNA synthesis and a clear peak of mitosis after 17 h and 25 h of hydration. At 25 h all cells had left the GO/G1 phase and had entered at least one S phase. After 29 h, the meristematic cells were either still In their first cell cycle or had at most reached the S phase of the second cycle.
Lentil seedlings were grown in space for 28 h (IML1) or 29 h (lML2) in microgravity (Fm g samples) or on the centrifuge (F1g). The Fm g sample (in both missions) had less cells in the S phase of the second cycle and more cells in the G2 phase of the first cycle than the F1g sample (or the G1g control on the ground). This result meant that cell cycle was slower in microgravity than in the controls. The controls (F1g and G1g) were similar in IML1 but different in the lML2. However, in the latter mission the 1g centrifuge sample was subjected to a period of at least 15 mm in m g (for a photography session). This result indicated that cell cycle could be perturbed by a very short period of microgravity.


DYNAMIC REDISTRIBUTION OF CYTOSKELETAL PROTEINS IN TIP-GROWING CELL TYPES

M. Braun
Botanisches Institut, University of Bonn, Venusbergweg 22, D-53115 Bonn, Germany

Tip-growing cells are characterized by a strong polar cytoplasmic organization which is reflected by the arrangement of the cytoskeletal elements. Axially oriented bundles of actin filaments and microtubules are commonly found during the elongation phase in these cell types. The initiation and the termination of tip growth, the strongly localized exocytosis at a very limited area of the cell membrane, however, is accompanied by a dramatic reorientation of cytoskeletal elements and associated proteins.
In root hairs, the actomyosin-dependent polarity of the newly assembling tip-growth machinery is established at discrete plasma membrane domains of the trichoblasts which continue to elongate by microtubule-dependent diffuse growth. Profilin, a small G-actin binding protein, which is a potent regulator of actin dynamics has been specifically localized at the tip of root hairs co-distributed with a diffusely fluorescing apical cap of actin, but not with actin MF bundles. Profilin and actin caps are present in the bulge of emerging and the apex of fast-growing root hairs; both disappear when root hairs terminate tip growth indicating a tip-growth mechanism involving profilin-actin interactions for the delivery and localized exocytosis of secretory vesicles. Phosphatidylinositol-4,5-bisphosphate (PIP₂), a ligand of profilin which regulates, or is regulated by, profilin, was localized in the outgrowing bulge and in form of a weak tip-to-base gradient in growing root hairs. When tip growth was eliminated by cytochalasin D, the apical profilin and actin caps disappeared. After mastoparan treatment, which is known to affect the PIP₂ cycle, a fine meshwork of distinct actin filaments replaced the diffuse fluorescing cap in the apex. This suggests that mastoparan has an impact on the profilin-actin interactions which seem to be essential for tip growth in root hairs.
In contrast to root hairs, the gravitropic tip-growth of rhizoids and protonemata of Chara requires a complex organization of actin filaments in the apex and accordingly, profilin has not been detected. Mainly axially oriented actin filaments focus towards an apical spot which is colocalized with a dense aggregation of endoplasmic reticulum membranes representing the structural center of the Spitzenkörper. The actin filament system is involved in the organization of the Spitzenkörper and in the positioning and transport of the statoliths. After termination of tip growth, the complex arrangement of actin filaments is replaced by a homogeneous meshwork.
The differences in the arrangement of the actin cytoskeleton and the distribution of profilin indicate fundamental differences in the mechanism of tip growth in cells which respond and those which do not respond to external or internal stimuli.

This work was supported within the AGRAVIS project by Deutsches Zentrum für Luft- und Raumfahrt (DLR), Bonn and the Ministerium für Wissenschaft und Forschung, Düsseldorf.


RHA1, A NEW ARABIDOPSIS MUTANT SHOWING REDUCED ROOT GRAVITROPISM TOGETHER WITH INCREASED RESISTANCE TO AUXIN AND ETHYLENE.

F. Migliaccio, S. Piconese, Z. El Gawhary
Institute of Plant Biochemistry and Ecophysiology, Consiglio Nazionale delle Ricerche, via Salaria Km 29.300, 00016 Monterotondo (Rome) Italy.

We recently isolated a new root mutant from the Feldmann T-tagged insertional mutagenesis collection of Arabidopsis lines that was named rha1. It is characterized by primary roots showing a notable reduction of positive gravitropism and of right-handed slanting, with respect to the wild-type Wassilevskija ecotype. In addition, the mutant shows increased root resistance to auxin (given as 2.4D), auxin transport inhibitors (TIBA and NPA) and ethylene. Even though the ensemble of these characteristics are also present in different already isolated mutants, such as aux1, axr1, axr2, agr1, and eir1, complementation tests between rha1 and these mutants showed that it is not allele to them. The mutation thus was ascribed to a new complementation group. Apart of the reduced root gravitropic response, we consider of particular interest the resistance to ethylene shown by the mutant roots, because so far the role of this important hormone in root gravitropism has been notably neglected. The mutation has been preliminarily mapped to chromosome 5. The cloning of the mutated gene is in progress through the technique of TAIL-PCR, taking advantage of the fact that the mutation appears to be tagged by a single T-DNA insert.


DIRECT EFFECT OF MICROGRAVITY ON T LYMPHOCYTE SIGNAL TRANSDUCTION

A. Cogoli, I. Walther, F. Turrini, F. Mannu, M.A. Meloni, P. Pippia
Space Biology, ETH Zurich, ^* Department of Physiological, Biochemical and Cellular Sciences, University of Sassari

One of the most crucial questions in gravitational and space biology is whether the effects observed in single cells under altered gravity are due to a direct and specific interaction of the g forces with cellular structures and functions or rather to metabolic effects arising from changes of convection, sedimentation, cell-cell contacts, vibrations and other. In this paper we describe an approach that could contribute to discriminate between the two alternatives. Such approach is based on the assumption that a metabolic effect will influence indiscriminately several cellular functions whereas a direct effect will touch specifically only few elements of the complex cell machinery.
We have focused our attention on the genetic expression of the interleukin-2 receptor (IL-2R) in T lymphocytes. Previous experiments in space and clinostats have shown that T lymphocyte activation by mitogens is strongly impaired in microgravity. Thereby the data indicated that the expression of the IL-2R might be strongly inhibited. Expression and anchorage of IL-2R in the cell membrane is a key step of the signal transduction pathway to full T cell activation. The IL-2R is made of three subunits, the a -, b - and g -chains. The a -subunit is responsible, in association with the b - and g -chains, of the high affinity of the receptor toward IL-2, but only the b - and g -chains are involved in the intracellular signal transduction. While the latter is constitutive, the other two are increasingly expressed during the activation process.
We have asked the questions: is the genetic expression of IL-2R really inhibited in microgravity? If yes, are both a - and b -chains affected or only one of them?
Simulated microgravity was attained in the random positioning machine and in the fast rotating clinostat. Both instruments are suitable to reproduce the effects observed on T lymphocytes in true microgravity in space.
We focused our attention on the a - and b -chains only, because the g -chain is constitutively expressed. In simulated 0 g, the expression of the a -chain is significantly inhibited whereas the expression of the b -chain is not influenced. Such interpretation is based on the assumption that the expression of b -actin is not influenced by altered gravity. This has to be verified in further investigations. Other internal standards may result to be more appropriate.
Based on our findings, those of Cooper and Pellis and of Schmitt et al. we suggest that different events contribute synergistically to the loss of activity of T cells in microgravity such as a dysfunction of the transcription of IL-2Ra gene and of the signal transduction upstream of PKC. An important conclusion is that the differential effect of simulated microgravity on IL-2Ra and IL-2Rb , respectively, points to a direct effect of microgravity. We suggest that a similar approach might be applied to other cell systems to distinguish between direct and indirect effects.
The random positioning machine has proven to be a useful tool to simulate microgravity in the ground laboratory and to prepare space investigation. In fact, we plan to continue our study of the genetic expression during T lymphocyte activation with an experiment on a space shuttle flight scheduled in the year 2000.


PRE-EXPOSURE TO SIMULATED MICROGRAVITY DOES NOT INFLUENCE MITOGENIC RESPONSE OF T LYMPHOCYTES AT 1 g

G. Cossu ¹, M. Schwarzenberg ², M.A. Meloni ¹, F. Turrini ¹, F. Mannu ¹, M. Cogoli-Greuter², P. Pippia ¹ and A. Cogoli ^2
1) Department of Physiological, Biochemical and Cellular Sciences, University of Sassari, Italy
²⁾ Space Biology, ETHZ, Zurich, Switzerland

There are two reasons to investigate whether exposure to 0 g prior to addition of an activator (inducing for instance cell differentiation or genetic expression) may affect signal transduction in mammalian cells. One is to answer the fundamental question on "memory effects" that may influence their behavior when a signal is transmitted later to the cell; the other is to assess the validity of 1 g controls in space. In fact, most of the experiments hitherto conducted where activated several hours after launch. Thus the in-flight control samples in the 1 g centrifuge experienced a prolonged exposure to 0 g. This was the case in the Biorack facility and it will be in several experiments in future missions, included the International Space Station.
The purpose of the work presented here was to determine the activation at 1 g by the T cell mitogen concanavalin A, Con A, in human lymphocytes after exposure for 1, 2 and 3 days, respectively, to simulated microgravity in the random positioning machine, RPM. This experimental system is particularly suitable for such studies because T lymphocytes are known to lose their capacity to respond to mitogens almost completely at true 0 g in space and by more than 50% in simulated microgravity in the clinostat. In addition, lymphocytes are isolated from human peripheral blood as "resting" cells, i.e. in the G₀ phase, and undergo differentiation to "activated" cells that begin to proliferate and to produce several cytokines within 3 d after exposure to mitogens like Con A.
The data show that the cultures pre-incubated without Con A for 24, 48 and 72 h at simulated microgravity, followed by incubation for 72 h at 1 g in the presence of Con A have the same level of activation as their respective controls kept for 24, 48 and 72 h at 1 g without Con A, followed by incubation for 72 h at 1 g with Con A. As expected, the activation was remarkably inhibited in a second set of control cultures kept 24, 48 and 72 h in the RPM without Con A, followed by incubation for 72 h in the RPM with Con A.
These findings permit us to conclude that despite prolonged exposure to simulated microgravity, T cells do not lose their capacity to react to mitogens. The existence of "windows of sensitivity" after delivery od the activation signal cannot be excluded, however. Experimental data obtained in an earlier experiment in Spacelab IML-2 are supporting this notion. In fact, due to a failure of the 1 g control centrifuge for 40 min immediately after addition of Con A, followed by nominal 1 g centrifugation for the remaining three days of incubation lead to a strong inhibition of the activation.
The search for windows of sensitivity is now in progress in our laboratory.


EFFECTS OF MICROGRAVITY CONDITIONS ON APOPTOSIS IN PERIPHERAL BLOOD LYMPHOCYTES

D. Risin¹ and N. R. Pellis²
1) Wyle Life Sciences, Systems and Services, Houston, USA
2) NASA/Johnson Space Center, Houston, USA

Our studies in simulated microgravity (SMG) and in real space conditions have demonstrated that microgravity directly affects numerous lymphocyte functions: it interferes with expression of cell surface molecules, causes inhibition of lymphocyte locomotion and suppresses polyclonal and antigen-specific lymphocyte activation (Pellis et al.,1994,1997; Risin et al.,1995, 1998, Cooper and Pellis, 1998). This suggests that microgravity interferes with fundamental biological processes associated with functional and structural changes in cell surface membranes and cell surface molecules. Apoptosis or programmed cell death (PCD) plays an important role in sustaining homeostasis in lymphocyte populations. Since PCD is surface-receptor-dependent we hypothesized that it is affected by microgravity. In this study spontaneous, activation- and radiation-induced PCD was examined in peripheral blood mononuclear cells (PBMC) exposed to simulated microgravity. Approximation of microgravitational conditions at ground level has been attained by using the rotating wall vessel bioreactor (RWV), developed at NASA JSC, which approximates the real microgravity conditions by sustaining cells in continuous free fall. PCD was assessed by FACS based on DNA content measurement (Nicoletti et al., 1991) and by DNA fragmentation assay.
Maintaining of PBMC in simulated microgravity conditions inhibited apoptosis induced by gamma irradiation of the cells prior to placing them into the RWV. Rescue from apoptosis in SMG was also observed in activated T lymphocytes when PCD was triggered by surface antigen engagement with PHA or by PMA+ionomycin stimulation. We did not find any difference in the expression of Fas antigen and membrane-bound FasL on the surface of activated T lymphocytes between the cells cultured in stationary and simulated microgravity conditions when programmed cell death was triggered by PHA. Inhibition of apoptosis in activated T lymphocytes was not related to changes in expression of bcl-2 or bax antigen. Microgravity did not affect apoptosis triggered by exogenous soluble Fas ligand. This suggests that MG most likely interferes not with the expression of Fas antigen or Fas ligand but rather with the interaction of membrane-bound Fas Ag and Fas ligand on the surface of the same or neighboring cells. In summary, our findings demonstrate for the first time a novel biological phenomenon - inhibition of apoptotic cell death in simulated microgravity conditions.


DIRECT EFFECT OF MICROGRAVITY (MG) ON HUMAN LYMPHOCYTES: FUNCTIONAL AND MORPHOLOGICAL ASPECTS

N. R. Pellis¹, D. Risin², A. Sundaresan² and D. Cooper³
NASA/Johnson Space Center, Houston, TX, USA
Wyle Life Sciences, Systems and Services, Houston, TX, USA
Div. Immunochemistry, LaJolla Inst. for Allergy and Immunology, San Diego, CA, USA

The fundamental question which we are addressing in our studies is whether MG directly affects lymphocyte morphology and functions, and if it does, what are the possible mechanisms of such effects. To examine the possible impacts of MG we are using the rotating wall vessel (RWV) bioreactor, developed at NASA JSC, which approximates the MG conditions by sustaining cells in continuous free fall. We previously showed that simulated and true MG (U.S. Space Shuttle missions STS-54 and STS-56) inhibits lymphocyte locomotion in type 1 collagen. By applying video microscopy and digital scanning, we observed, both in lymphocyte samples from the RWV and from the Shuttle missions, changes in cell shape suggestive of a decreased ability to polarize. Activation of PBL prior to placement into RWV partially or completely abrogated the inhibitory effect of MG on lymphocyte motility. Simulated MG (SMG) suppressed polyclonal activation of peripheral blood mononuclear cell (PBMC) by PHA, secretion and/or production of T-cell-associated cytokines, IL-2 and IFN-g . At the same time secretion of costimulatory cytokines, IL-1b and IL-6, was substantially enhanced in the RWV. SMG affected the expression of the cell surface molecules critical for lymphocyte activation (CD71, CD25 and CD69). There was substantial inhibition of antigen-specific activation of lymphocytes: a) in mixed-lymphocyte culture, b) in specific recall of human lymphocytes to tetanus toxoid and c) in specific recall of murine T lymphocyte cell lines to Borrelia burgdorfferi. We studied the possible impacts of MG on cell-to-cell and cell-substrate interactions. Results showed that suppression of T cell activation in the RWV bioreactor is unlikely to be due to the lack of cell-to-cell contact. By using FITC-labeled PHA we showed that suppression of activation is not related to the defect in PHA interaction with surface receptors. Adding of exogenous recombinant IL-2 to RWV cultures (even at concentrations as high as 100 U/ml) did not restore polyclonal lymphocyte activation. At the same time PMA and ionomycin added together were able to almost fully activate PBMC and purified T cells in SMG. This suggests that the cellular mechanisms involved in T cell activation downstream of PKC and calcium ion flux are not affected by SMG. The defect has to be located either upstream or at the level of PKC activation. Direct activation of PKC by PMA was also able to restore the SMG-inhibited lymphocyte locomotion as well as their polyclonal activation by PHA, whereas ionomycin by itself was unable to restore these functions. This indicates that the observed suppression of lymphocyte activation and motility in MG is not related to the impairment in the calcium flux. Thus, simulated and potentially true MG causes a fundamental defect in signal transduction, namely in the PKC pathway, that results in blunted locomotion and loss of proliferative response to activation signals. Preliminary data suggest that SMG may affect selectively the expression of novel Ca2+ independent isoforms, in particularly PKC e .


MICROTUBULE SELF-ORGANISATION DEPENDS UPON GRAVITY.

Papaseit, N. Pochon, J. Tabony
Laboratoire R.M.B.M, Département de Biologie Moléculaire et Structurale,
D.S.V, C.E.A. Grenoble, 17, rue des Martyrs, 38054 Grenoble Cedex 9, France

The molecular processes by which gravity is transduced into biological systems are poorly, if at all, understood. Under equilibrium conditions, chemical and biochemical structures do not depend upon gravity. It has been proposed that biological systems might show a gravity dependence by way of the bifurcation properties of certain types of non-linear chemical reactions that are far-from-equilibrium. We have found that in-vitro preparations of microtubules, an important element of the cellular cytoskeleton, show this type of behaviour. On earth, the solutions show macroscopic self-ordering, and the morphology of the structures that form depend upon the orientation of the sample with respect to gravity at a critical moment prior to the progressive appearance of the self-organised state. An experiment carried out in a MAXUS sounding rocket, showed that as predicted by theories of this type, no self-organisation occurs when the microtubules are assembled under microgravity conditions. The same preparations formed on a 1g on-board centrifuge showed the morphologies that form on earth. This is an experimental demonstration of how a very simple biochemical system, containing only two molecules, can be gravity sensitive.


OSTEOGENESIS OF EMBRYONIC LONGBONES IN SIMULATED MICRO-GRAVITY

J. Maroothynaden
Centre for Tissue Regeneration and Repair
Imperial College of Science, Technology and Medicine, London SW7 2BP, jm2@ic.ac.uk

The cost of spaceflight severely limits access of experimental investigations in space. Load bearing bones have been found to demineralise at much faster rates in space than on the Earth. Even though this phenomenon has been quantified, the fundamental biological mechanisms remain unresolved.
The aim of this study was to see if a commercial rotating culture vessel system could be used to develop a low cost and easy to operate experimental model for microgravity induced demineralisation.
Embryonic mice femora were cultured in a supplemented alpha-MEM medium in a "simulated microgravity" environment, provided by the rotating culture vessel, for 4-days. A simultaneous 1-gravity control was performed. All sections were terminated, sectioned and stained. Image analysis was also used for histomorphometric analysis. Inductive Coupled Plasma (ICP) analysis was also used to analyse any changes in culture solution chemistry.
Femora cultured in the "simulated microgravity" environment were found to follow similar histomorphometric trends as longbones cultured, in-vitro, during spaceflight. Decreased mineralisation was observed along with a change in growth-plate kinetics. ICP analysis showed dramatic changes between the 1-gravity and "simulated microgravity" culture solutions thus providing an incite into gravity dependant changes in the biochemistry of mineralisation.
A low cost, easy to use and repeatable model has been developed for the ground based investigation of microgravity related bone demineralisation.


IN VITRO SPICULOGENESIS OF SEA URCHINS: AN IN VITRO MODEL SYSTEM FOR STUDYING BIOMINERALIZATION PROCESSES.

H. -J. Marthy and R. Bacchieri
Observatoire Océanologique, CNRS (UMR 7628) - UPMC
66650 Banyuls sur mer, France

The so-called micromeres, located at the vegetal pole of early cleavage stages of sea urchin eggs, differentiate, when isolated and cultured in vitro, into squeletogenic cells (= primary mesenchyme cells (pmcs)) as they do, normally, in situ , inside the gel filled blastocoelic cavity (1). In situ, these cells produce an organic matrix (2,3,4) on which endoskeletal spicules of CaCO3 are deposited (4). Size and shape of the spicules, thus the architectural pattern of the skeleton, are determined by the amount of the calcareous deposit, the space available inside the blastocoelic cavity and, not least, the "influence" of the ectodermal wall on the skeletogenic cells (5). In vitro too, as shown by several authors and for different sea urchin species (e.g. for review:5), pmcs, differentiated from micromeres, which were isolated at the 16-cells stage, produce spicules, thus revealing clearly their determination and capacity for a normal mineralization process. However, in the absence of in vivo conditions and other necessary developmental informations (in particular of the lacking interaction with the ectodermal substrat), the spicules sizes and shapes appear more or less arbitrary, following the (arbitrary) arrangement in a cluster of pmcs in suspension or in a group of pmcs fixed on the artificial substrate. In the latter case, fusiform rods and longer than in vivo, are most frequently observed. Within suspended clusters, a great variety of spicule forms and even pseudo-skeletons is obtained.
Within the context of our past (6,7), on-going and future studies on skeletogenesis in sea urchin embryos/larvae under varied gravity conditions, particularly under microgravity conditions, we developed suitable methods for isolating and in vitro culturing of micromeres of early cleavage stages of the sea urchin species Sphaerechinus granularis. By means of photographs taken with a polarization microscope of such spicules and skeletal parts, completed by Scanning Electron Microscopy observations, this remarkable "model system for in vitro biomineralization" will be presented and evaluated in view of its use in fundamental and applied research under real and simulated microgravity conditions.
References:
1) R. R. Strathmann (1989). Existence and Functions of a Gel Filled Primary Body Cavity in Development of Echinoderms and Hemichordates. Biol. Bull. 176: 25-31).
2) Okazaki, K. (1960). Skeleton Formation of sea urchin larvae. II. Organic matrix of the spicule. Embryologia, 5 (3): 283-320.
3) Benson S.C., N.C. Benson and F. Wilt (1986). The organic matrix of skeletal spicule of sea urchin embryos. J. Cell Biol. 102 (5): 1878-1886.
4) Kitajima T. and R. Matsuda (1982). Specific protein synthesis of sea urchin micromeres during differentiation. Zool. Mag. 91: 200-205.
5) Armstrong, N., J. Hardin and D.R. McClay (1993). Cell-cell interactions regulate skeleton formation in the sea urchin embryo. Development 119: 833-840.
6) Marthy, H.-J. et al. (1996). The sea urchin larva, a suitable model for biomineralisation studies in space (IML-2 ESA Biorack Experiment "24-F Urchin). J. of Biotechnology, 47: 167-177.
7) Marthy, H.-J. (1999). Sea urchin development in space as revealed by skeletogenesis. ESA SP-1222, ESA Publications Division, Noordwijk, in press.


EFFECT OF GRAVITY AT MACROMOLECULAR LEVEL

P. Boncinelli^1 F. Ranaldi^2 E. Giachetti^2 P. Vanni^2
1 Dipartimento di Energetica ``Sergio Stecco'' - Universita' di Firenze
2 Dipartimento di Scienze Biochimiche - Universita' di Firenze

The aim of the present work is to prove that the effects due to altered gravity (g>g_{earth} or g<g_{earth}) can be observed not only in cells and other complex organisms, but also in simpler biological systems, such as substances with high molecular weight and complex over-molecular structures (membranes, associations between macromolecules, and any type of colloidal systems).
The force of gravity (macro-gravity or micro-gravity) is comparable, in terms of the energy of the interaction per mole, with other fundamental physical and chemical interactions (intermolecular electrostatic forces, thermic agitation), which are known to influence the chemical behaviour of such systems.