gravitational and space biology bulletinbaby.indstate.edu/asgsb/bulletins/v16n1/v16n1-p1-78.pdf ·...

Gravitational and Space Biology Bulletin 16(1), November 2002 1

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ABSTRACTS – SYMPOSIUM I

[1] [3] SYMPOSIUM ON SPACE RADIATION HEALTH: INTRODUCTORY REMARKS. Paul Todd, Space Hardware Optimization Technology, Inc., Greenville, IN

RADIATION DOSIMETRY AND DOSES FROM SPACE RADIATION. WHAT DOES IT MEAN? F. A. Cucinotta, H. Wu, M. Shavers, and K. George, NASA Lyndon B. Johnson Space Center, Houston TX The field of space radiation health has recently been singled out as one

of two major initiatives within the research programs of the U. S. National Aeronautics and Space Administration. The goals of space radiation health research are to understand qualitatively and quantitatively the ionizing radiations present in the space environment, identify qualitatively and quantitatively the risks associated with these radiations, and discover countermeasures to mitigate these risks. Speakers in the Symposium will address each of these three components of space radiation health research. It is helpful to understand some of the quantities and definitions used in this field. The SI unit of ionizing radiation dose is the Gray (1 Joule of energy loss in 1 kg of matter). A typical human on earth in a “civilized” culture experiences less than 10-3 Gy in a year; a cancer patient receives some 60 Gy in a treatment volume during a full course of therapy; and a total-body dose of about 2.5 Gy is the mean lethal dose for humans and many mammals. Space radiations consist of energetic protons from the sun, protons and electrons from the sun that are trapped in the earth’s magnetic field, and cosmic rays that include energetic nuclei of H, He, C. N. O and Fe atoms.. All of these produce secondary radiations such as neutrons and gamma rays when they interact with matter. The risks presented to space travelers by these radiations include cancer due to proton a cosmic-ray exposure, immune failure due to high-dose solar proton storms, and possible neurological effects caused by single tracks

A complicated mixture of high-energy protons and heavy ions, and secondary radiations produced by them, bombards astronauts during space travel. Developing a useful physical parameter to characterize the outcome of such exposures has been and remains a complicated problem because of lack of knowledge of the biological effects of heavy particles. We present an overview of past space measurements of radiation exposure including measurements of absorbed dose, linear energy transfer (LET) spectra, dose equivalent, or charge and energy spectra of particles. Expectations for future space exploration missions are also discussed. The approach used by NASA to answer problems related to space radiation risks is to pursue radiobiological research for understanding and mitigating potential health risks. One area of research has been in the development of models of radiation track structure, which provide a description of energy deposition events in biomolecules, cells, or tissues. We discuss progress in the biophysical description of heavy particle tracks that have direct application to the interpretation of space radiation dosimetry.

of cosmic-ray heavy nuclei. Potential countermeasures include shielding (which increases spacecraft up-mass), medication consisting of radical scavengers (that must be taken immediately before exposure), anti-oxidant consumption (which must be maintained continuously), cytokines (which may ameliorate immune and hematological effects after exposure), and cell transplants (which must be isologous).

[2] [4] OVERVIEW OF NASA'S RADIATION HEALTH PROGRAM. W. Schimmerling. NASA HQ, Code UB, Washington. The goal of radiation protection in space is to eventually enable a permanent human presence without incurring unacceptable health risks due to the unavoidable exposure to ionizing space radiation. The components of this radiation that are of concern are high-energy, charged particles, especially the components of galactic cosmic rays consisting of high-energy (high-E) nuclei of heavier (high atomic number Z) elements ("HZE particles"). Predictions about the nature and magnitude of these risks are limited by current scientific knowledge and are subject to large uncertainties. Prudent use of worst-case scenarios may impose unacceptable constraints on shielding mass for spacecraft or habitats, and on the radius and duration of sorties on planetary surfaces. NASA is developing the knowledge required to accurately predict and to efficiently manage radiation risk in space. The strategy employed has three components: (1) ground-based simulation of space radiation components to develop a science-based understanding of radiation risk; (2) space-based measurements of the radiation environment on planetary surfaces and interplanetary space, as well as use of space platforms to validate predictions; and, (3) implementation of countermeasures to mitigate risk. NASA intends to significantly expand its support of radiation research in line with completion of the Booster Applications Facility (BAF) at Brookhaven National Laboratory, expected in summer of 2003. A joint research solicitation with the Department of Energy is under way and other interagency collaborations are being considered. In addition, a Space Radiation Initiative has been submitted by the Administration to Congress that would provide answers to most questions related to the International Space Station within the next 10 years. The current aspects and status of this program will be discussed.

PROTON AND HEAVY ION FACILITIES FOR SPACE RADIO-BIOLOGY RESEARCH. J. Miller, Lawrence Berkeley National Laboratory Berkeley, California The electromagnetic and nuclear interactions of heavy charged particles—protons and heavier atomic nuclei—must be taken into account in order to understand and address the effects of solar particle events and galactic cosmic radiation on humans in space. Until relatively recently, most of the research into nuclear interactions in matter was driven primarily by the interests of the nuclear physics and radiotherapy research communities. However, many of the particles and energies used in radiobiology, radiotherapy and nuclear physics research on Earth are highly relevant for space radiation studies, and the experimental and theoretical methods and accelerator facilities developed for use in proton and heavy ion nuclear physics and radiotherapy are directly applicable to space radiobiology. I will survey the particle accelerator facilities available for ground-based space radiation research, and discuss the implications of some recent measurements.



[5] THE RADIOBIOLOGY OF SPACE RADIATION. G.A. Nelson.

Radiobiology Program, Loma Linda Univ., Loma Linda, CA. 92354 The unique feature of the space radiation environment is the dominance

of high energy charged particles (HZE or high LET radiation) emitted by the Sun and galactic sources, or trapped in the Van Allen radiation belts. These charged particles present a significant hazard to space flight crews and accelerator based experiments are underway to quantify the health risks due to unavoidable radiation exposure.

There are three principal properties of charged particles that distinguish them from conventional radiation, i.e. gamma rays and x-rays. First, they have a defined range in matter rather than an exponential absorption profile. Second, they undergo nuclear reactions to produce secondary particles. Third, and most important, they deposit their energy along well-defined linear paths or tracks rather than diffuse fields. The structured energy deposition pattern interacts on multiple scales with the biological structures of DNA, cells and tissues to produce correlated patterns of damage that evade repair systems. Traditional concepts of dose and its associated normalization parameter, RBE (relative biological effectiveness), break down under experimental scrutiny and probabilistic models of risk based on the number of particle traversals per cell may be more appropriate. Unique patterns of DNA damage, gene expression, mobilization of repair proteins, activation of cytokines and remodeling of cellular microenvironment are observed following exposure to high LET radiation. At low levels of exposure the communication of bioactive substances from irradiated to unirradiated “bystander” cells can amplify the damage and cause a significant deviation from linearity in dose vs. response relations. Under some circumstances, there is even a multigenerational delay in the expression of radiation-induced genetic damage (genomic instability) which is not strictly dose dependent. These issues and the experimental evidence derived from ground based experiments at particle accelerators will be discussed along with speculation about how modified inertial conditions might perturb homeostatic responses to radiation to further complicate risk assessment for space flight. (Supported by NASA: NCC9-149) [6] BIOLOGICAL COUNTERMEASURES IN SPACE RADIATION HEALTH. Ann R. Kennedy, Department of Radiation Oncology, University of Pennsylvania School of Medicine, Phil., PA Exposure to the types of ionizing radiation encountered during space travel may cause a number of health - related problems, but the primary concern is related to the increased risk of cancer induction in astronauts. The major types of radiation considered to be of importance during space travel are protons and particles of high atomic number and high energy (HZE particles). It is now clear that biological countermeasures can be used to prevent or reduce the levels of biological consequences resulting from exposure to radiation from protons or HZE particles, including the induction of cancer from these types of ionizing radiation. Research related to the dietary additions of agents to minimize the risks of developing health-related problems which can result from exposure to space radiation will be reviewed.



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ABSTRACTS – POSTER SESSION I–A

[7] [9] THE PROJECT AQUARIUS-XENOPUS ON THE SOYUZ FLIGHT ANDROMÈDE TO ISS WAS SUPPLEMENTED BY A EUROPEAN PUPIL PROJECT. E. Horn1, S. Böser1, Pupils and Teachers from Ulm2 and Nancy3, H. Membre4, and C. Dournon4. 1Gravitational Physiology, Univ of Ulm, Ulm, Germany; 2Schubart-Gymnasium, Ulm, Germany; 3Varoquaux High School, Tomblaine-Nancy, France; 4EA 3442 Genetic, Signaling, Differentiation, Université Henri Poincaré, Vandoeuvre-lès-Nancy cedex, France.

EFFECTS OF TEMPERATURE ON THE GROWTH RATE OF ERWINIA HERBICOLA. C.M. Draper1, J.L. Garland2, and M.S. Roberts2. 1Dept. of Food, Agricultural and Biological Engineering, The Ohio State University, Columbus and 2Life Support Group, Kennedy Space Center, FL. Microorganisms are an essential part of any biological system. Therefore, development of a passive experimental microbial system (PEMS) to research microbial growth and mutation in the space environment is necessary. The German-French biological experiment AQUARIUS-XENOPUS

which flew on the Soyuz taxi flight Andromède to the International Space Station ISS (launched October 21, 2001 in Baikonur/Kazakhstan) was extended by an outreach project. Pupils of class 10 to 12 from Ulm/Germany and Tomblaine-Nancy/France were involved in this actual space experiment. They recorded swimming behavior of Xenopus laevis tadpoles by video used as a 1g-ground control for similar observations in µg-exposed tadpoles on ISS. The pupils were instructed to perform all experimental steps following the protocol of the video recordings on ISS which were done by the French cosmonaut Claudie Haigneré. After the flight, they evaluated swimming activity of both ground controls and space animals using parameters such as type, velocity and acceleration of swimming, or the distribution patterns of tadpoles within the miniaquaria. The pupil project included theoretical components to introduce them to the field of gravitational biology. Nancy pupils established a homepage (www.xenope.com) about background and aim of their scientific project while Ulm pupils received an extended theoretical and practical education (1) about gravity effects on biological systems and what gravity means for life on Earth, and (2) on hardware used for biological research in Space. A feature of the project was the exchange of ideas between all pupils by internet and meetings which took place in Ulm (June 2001), Nancy (February 2002) and Paris (May 2002). We consider our approach as a successful way to include young people in space experiments on a cheap cost level and to bring ideas of gravitational biology into curricula of European schools. (Supported by the German Space Agency (DLR), the French Space Agency (CNES), the City of Ulm and local institutions of the Nancy region)

The purpose of this experiment is to attain proper growth conditions for the development of a suitable method for observing the behavior of microorganisms in a space environment. In this experiment, microbial growth is considered as a function of agar concentration, nutritive conditions, cell type, inoculation density, and temperature. Duplicates of each variable will be tested in order to collect a satisfactory amount of data without exceeding the number of growth cells allotted. Sixty-four growth chambers will be inoculated with a single strain of either Pseudomonas aeruginosa or Erwinia herbicola. Once results from the first trial are examined, the remaining 36 growth chambers will be used to improve the growth variables and the technique used to inoculate the growth chambers. Temperature impacts the growth capabilities of microorganisms significantly and is a necessary consideration when determining the optimal growth rate. Microbial growth will be monitored during the course of growth by visual observation, microscopy and, at the end of the growth period, by harvesting the cells.

[8] [10]

PSEUDOMONAS AERUGINOSA EXOTOXIN ASSAY FOR STS-107 EXPERIMENT BACTER. K.M. Hale and B.H. Pyle. Dept. of Microbiology, Montana State University, Bozeman.

SIMULATED MICROGRAVITY’S EFFECT ON THE CONTRACTILE ABILITY OF DEVELOPING CHICK HEART TISSUE EXPLANTS. Krista L. Todd and Darrell Wiens, Dept of Biology, University of Northern Iowa. An Exotoxin A (ETA) producing P. aeruginosa ATCC 29260 will Previous in vitro studies have shown that gravitational effects may disturb the cytoskeletal / extracellular matrix interaction, alter organo-genesis, and result in the development of a morphologically intact, yet non-contracting heart. Two of the proteins that play an important role in this interaction are paxillin and β1-integrin. We have examined the effect of simulated microgravity on the expression and distribution of these proteins in an attempt to explain the functional difference. Pre-cardiac tissue explants from HH stages 4 – 7+ were exposed to a zero sum vector force environment by vertical rotation in a 10.0 ml HARV bioreactor for 19 hours during development. We observed development of contractions in only 11 of 45 (24%), even less than in previous work. Forty-six of 54 (84%) of stationary or horizontally-rotated control explants contracted.

be flown on STS-107 to study their growth, physiology and virulence. In preparatory ground studies, ETA was analyzed by a 96-well ELISA in which standards and samples were bound to the microplate wells. After ETA binding, a blotto solution of skim milk and bovine serum albumin in Tris-Buffer Saline-Tween was used for blocking. A polyclonal rabbit primary antibody was added, followed by a secondary goat-HRP antibody and OPD for color development. Each binding step was followed by rinsing with TBS and TBS-T. Optical density was read at 490 nm on a 96-well plate reader. For ETA quantification, standards and samples were diluted in culture medium. The assay was optimized by adjusting standard and sample dilutions, incubation period, plating procedures, and by performing and analyzing ETA capture with the direct method of ELISA vs. an indirect method. Following optimization, the direct assay gave reliable results with samples from ground-based experiments,

We then immunostained the explants with paxillin and β1-integrin monoclonal antibodies to reveal areas of the explants expressing these proteins. It was observed in whole explants and 7µm sections that both proteins were localized to the perimeter, showing a solid staining area, a mottled area, and an area devoid of stain . Color threshold image analysis indicated no significant difference from control in percent total area of paxillin staining. However, initial results indicate integrin is less abundant in those exposed to microgravity during development.

with ETA concentrations in the range of 200-300 ng/ml. Results from simulated weightlessness experiments suggest that ETA will be produced in microgravity in detectable amounts. The flight data will assist in assessing the significance and risk of these bacteria to future space flight crew members. (Supported by NASA, ESA, Montana Space Grant Consortium and MSU Undergraduate Scholars Program.)



[11] [13] MEASURING RESPIRATORY ACTIVITY OF PSEUDOMONAS AERUGINOSA FOR STS-107 EXPERIMENT BACTER. S.A. Barton, S.C. Broadaway, and B. H. Pyle. Dept. of Microbiology, Montana State University, Bozeman.

IMAGE RESOLUTION ENHANCEMENT FOR THE REMOTE SENSING OF STRESS IN ARABIDOPSIS DURING SPACEFLIGHT. K.L. O’Brien1, A-L. Paul2 and R. J. Ferl2. 1Spaceflight and Life Sciences Training Program (SLSTP), Kennedy Space Center, 2University of Florida, Gainesville, FL. An Exotoxin A (ETA) producing P. aeruginosa ATCC 29260 will

be flown on STS-107 to study their growth, physiology, and virulence. Washed bacteria suspended in water will be inoculated into defined medium (MSDM2) and incubated at 37ºC for 24 hours. As defined by

A hardware prototype, the Transgenic Arabidopsis Gene Expression System (TAGES) Imager, has been designed to collect both white light and blue light images of Arabidopsis thaliana (Arabidopsis), that have been engineered with a reporter gene composed of the Adh gene promoter and GFP (Green Fluorescent Protein), during an upcoming spaceflight experiment. The data currently available through this prototype are low-resolution images at one continuous camera focal length. Without further modifications these images will not provide the information necessary for complete analysis of previously noted stresses in Arabidopsis. Due to spaceflight restrictions on hardware components it is prudent to pursue modifications in the following areas: spectral sensitivity, thermal signals, defect mapping, noise reduction, as well as storage formats. Each of these areas will be investigated separately, however, the most significant improvement will be a result of applying minor modifications pertaining

the flight timeline, the reagents must be stored for 9 days at 5ºC, 1 day at 37ºC, and incubated for 7 days at 5ºC before analysis. This study was aimed to determine if stored INT solutions would remain active during storage and be taken up by the cells during incubation. INT was dissolved in water at 4.9 mg/ml and either used immediately or stored for 7 days at 5ºC, followed by 24 hour storage at 37°C. P. aeruginosa was grown at 37ºC in defined medium to ca.109 CFU/ml, followed by the addition of INT with subsequent incubation at 5ºC for 7 days or at room temperature for 2 hours. INT formazan crystals could be seen as dark spots inside the SYBR Green stained cells during epifluorescent microscopy, with numbers comparable to enumeration by R2A agar plate counts. Incubation at room temperature for 2 hours produced a higher percent of INT positive cells compared to incubation at 5°C. INT dissolved in water can be stored

to each investigation area through software imaging enhancement package, Maxim DL, and optimizing the data storage format. Higher image quality is necessary to observe specific information that is fundamental in pursuing the cause of the stress signal perception and transduction observed in Arabidopsis in a previous flight experiment.

at 5oC without loss of activity. The flight experiment procedure gives reliable results, which permit detection of changes in the proportion of respiring cells that may be caused by microgravity. (Supported by NASA and ESA)

[12] [14] USER INTERFACE AND SOFTWARE CALIBRATION IMAGING OF THE ARABIDOPSIS THALIANA. I.F. Paderez1, A-L. Paul2 and R. J. Ferl2. 1Spaceflight and Life Sciences Training Program (SLSTP), Kennedy Space Center, 2 University of Florida, Gainesville, FL.

A QUANTITATIVE BIOCHEMICAL APPROACH FOR EVALUATION OF ONION QUALITY AND POTENTIAL VOLATILE EMISSION. Lisa Harris1, Jan Bauer2, and Lanfang Levine2. 1 Department of Chemistry, Southeast Missouri State University, Cape Girardeau, MO, 2 Dynamac Corporation, Mail Code: DYN-3, Kennedy Space Center, FL 32899.

Arabidopsis plants (Arabidopsis thaliana) have been engineered with a gene reporter composed of the Adh (alcohol dehydrogenase) gene promoter and the coding region of a fluorescent protein (GFP). The goal Plants are anticipated to play an important role in both short and long

term space missions as they provides fresh food to the crew members, is to use digital imaging hardware to collect images of the Arabidopsis under stress induced environments such as temperature, agar blankets to mimic root zone hypoxia, and CO2. Images of the transgenic Arabidopsis have been primarily taken with NASA computer software of a prototype Flight hardware called, “TAGES Imager” (TAGES – Transgenic Arabidopsis Gene Expression System). The TAGES imager uses two types of light to collect data on plant growth and stress responses in a controlled closed system. White light imaging records plant growth and development, while fluorescent light (488nm) imaging records where GFP is expressed in the plant. Research will consist of working with imaging system in its ability effectively collect images. The following areas will be emphasized: A 2-D coordinate plane system, efficiency to reside back to its place of origin specified by the user, and data collection of growth and development of Arabidopsis plants over time.

and revitalize water and air through transpiration and photosynthesis. Furthermore, their psychological benefits to the crews in an enclosed abiotic environment cannot be underestimated. Ideal plant species for space should have the characteristics of low space, power and processing requirements, and high nutritional value as well as low bio-emission of undesirable volatiles. Currently researchers at Kennedy Space Center are evaluating bunching onions (Allium fistulosum L.), one of the candidate salad crops, for use in near-term space missions. As a part of cultivar selection project, we attempted to develop quantitative metrics for onion’s nutritional and sensory value as well as their potential volatile emission. Onions are known for their characteristic flavor and health benefits due to the presence of organosulfur compounds, thiosulfinates. Thiosulfinates (flavor indicator) and pyruvic acid (pungency indicator) are formed

upon the disruption of onion cells due to the action of alliinase on flavor precursors (alk(en)yl cysteine sulphoxides). The flavor precursors and pyruvic acid were quantified in pseudobulbs and leaves of eight commercially available onion cultivars. Results demonstrated significant concentration differences between tissue types. Concentration differences were also detected between pseudobulbs from different cultivars but not between leaves of different cultivars. These results provide baseline information for cultivar selection based on their nutritional and sensory value, and potential volatile emission. They also provide a foundation

for future studies in which the influence of environmental factors on onion

quality can be evaluated.



[15] OMINDIRECTIONAL CLINOROTATION - A NEW SPIN ON

CLINOSTAT DESIGN. M.M. Gummere, J.E. Moses, A. Coleman, B. Cooperman, W. Darrah, P.A Rea1 and T.M. Lynch. Science

Department, William Penn Charter School, Philadelphia. 1Plant Science Institute, University of Pennsylvania, Philadelphia.

Clinorotation has been used since Dodart (1703) to help elucidate the

mechanisms living organisms use to detect gravity. Our new clinostat design offers an experimental platform that moves plants in three-dimensional space by randomly rotating a sphere housing experimental materials. To test the efficacy of this new device, affectionately referred to as the NotomyStat, wild type cultivars of Arabidopsis and Maize cv. Merit were clinorotated and root growth vectors were analyzed. Data from Arabidopsis cv. Columbia reveals random root growth with respect to the growth vector providing evidence that the new clinostat design was effective in simulating the microgravity environment. Maize cv. Merit was used as a control organism due to its inability to detect gravity in the absence of light. Therefore, Merit seeds clinorotated in darkness should provide the same result as those rotated in the presence of light. Further studies will be performed, using this new clinorotation instrument, to study the molecular mechanisms responsible for gravitropism.



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ABSTRACTS – POSTER SESSION I–B

[16] [18] EFFECTS OF HINDLIMB UNWEIGHTING ON THE MECHANICAL AND STRUCTURE PROPERTIES OF THE RAT ABDOMINAL AORTA. A. Papadopoulos and M.D. Delp.

BRIEF DAILY STRETCHING OF SOLEUS MUSCLES PREVENTS CENTRAL CORE-LIKE LESIONS IN HINDLIMB SUSPENSION UNLOADED RATS. D.V. Baewer, B.D. Curry, P.M. Reiser,

Departments of Health and Kinesiology and Medical Physiology, J.L.W. Bain, R.H. Fitts, D.A. Riley. Dept of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee WI, Dept of Biology, Marquette University, Milwaukee, WI.

Texas A&M University, College Station, TX. Previous studies have shown that hindlimb unweighting (HU) of rats, a model of microgravity, reduces evoked contractile tension of peripheral conduit arteries. It has been hypothesized that this diminished contractile tension is the result of alterations in the mechanical properties of these arteries (e.g., active and passive mechanics). Therefore, the purpose of this study was to determine whether the reduced contractile force of the abdominal aorta from 2-wk HU rats results from a mechanical function deficit resulting from structural vascular alterations. Both active and passive Cauchy stress-stretch relations were determined by incrementally increasing the uniaxial displacement of the aortic rings. There were no differences in the passive Cauchy stress-stretch response or gross vascular morphology (e.g., medial cross-sectional area: CON, 0.30 ± 0.02 mm2; HU, 0.32 ± 0.01 mm2) between groups. In addition, there were no differences in resting or basal vascular tone at the displacement which elicits peak developed tension (Lmax) between groups (resting tension: CON, 1.71 ± 0.06 g; HU, 1.78 ± 0.14 g). The active Cauchy stress-stretch response indicated that although maximum stress was lower in aortas from HU rats (CON, 8.13 ± 0.24 kPa; HU, 7.02 ± 0.44 kPa), it was achieved at

Hindlimb Suspension Unloading (HSU) is widely used to simulate the effects of microgravity unloading on the musculoskeletal system. In a standing rat, the ankle joint is dorsiflexed at ~30°during weight bearing. During suspension, gravity causes the hind feet to droop and open the ankle joint to ~90°. This postural change decreases the working range (length) of soleus by 15-20% [Riley et al. JAP 69:58, 1990]. After 12 days, soleus muscle fibers exhibit atrophy and central core-like (CCL) lesions, which are characterized by focal regions lacking myofibrillar ATPase activity and sarcomere disruption at the ultrastructural level. The present study sought to determine whether stretching the soleus muscle by bringing the ankle to 30° for 10 or 20 total min per day would prevent CCL lesion formation. The left legs of suspended, male Sprague Dawley rats (261±6 g) were fitted with external plastic splints to hold the ankle at a 30° and stretch the soleus. Four rats were splinted 10 min/day, and 5 animals were splinted 10 min twice a day. After 12 days, the left and right soleus muscles were removed from anesthetized rats and quick frozen for cryostat sectioning and histochemical staining for acid (pH 4.35) and alkaline (pH 9.4) myofibrillar ATPase for detection of fiber types and CCL lesions. Compared to the non-splinted side, the percentage of fibers with CCL lesions was less after treating 10 min (3.3±1.2% versus 27.5%±5) and 20 min (2.5±1.4% and 26.8±3.2%). Lesions were most common in slow fibers. These findings indicate that moving a muscle through its full working range is important for maintenance of sarcomere structure. (Supported by NASA NAG9-1156)

a similar uniaxial displacement and stretch. These results indicate that HU does not alter the functional mechanical or gross structural properties of conduit arteries. However, the significantly lower active Cauchy stress by HU aortas confirms a contractile deficit in these arteries. (Supported by NASA grant NAG2-1340 and NSBRI grant NCC9-58-42) SPCDVB02

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PREVENTIVE EFFECTS OF ANTIOXIDATIVE NUTRIENTS ON UNWEIGHTING-INDUCED PROTEIN UBIQUITINATION AND DEGRADATION IN RAT SKELETAL MUSCLE. K. Hirasaka1, M. Kano1, M. Ikemoto1, Y. Asanoma1, T. Ogawa2, K. Kishi1 and T. Nikawa1 1Dept of Nutrition and 2Dept of Orthopedic Surgery, School of Medicine, Univ of Tokushima.

THE EFFECTS OF LOW-DOSE PROTON RADIATION ON PREPULSE INHIBITION OF THE ACOUSTIC STARTLE REFLEX IN C57BL/6 MICE. C.N. Zuccarelli1,2, P.E. Haerich2, G.A. Nelson1, and M.J. Pecaut1. Depts of 1Radiation Medicine, Radiobiology Program and 2Department of Psychology. Loma Linda University and Medical Center, Loma Linda, CA.

We have previously reported that spaceflight and tail suspension enhanced degradation of rat myosin heavy chain (MHC) in association with activation of a ubiquitin-dependent proteolytic pathway [FASEB

Many studies have shown that radiation influences behavior. Roughly 85% of radiation in Solar Particle Events and Galactic Cosmic Rays are made up of high-energy protons. Because the long-term effects of proton radiation may have implications to the behavior of astronauts on extended missions in space, this study examines the effects of several doses of proton radiation on prepulse inhibition of the acoustic startle response (PPI), one of the classical measurements of behavior in rodent models.

J. 15 (2001) 1279-1281]. To elucidate whether the ubiquitination is accompanied by oxidative stress, we measured markers for oxidative stress, such as thiobarbituric acid-reactive substance (TBARS), glutathione disulfide (GSSG), and glutathione (GSH), in gastrocnemius muscle of tail-suspended rat. Tail-suspension reciprocally increased concentration 100 male C57BL/6 mice were irradiated with various doses of head-only

proton radiation (n=20/group, 0, 1, 2, 3 and 5 Gy, 250 MeV, unmodulated). Animals were then tested with the PPI protocol two days post irradiation. The protocol consisted of a five minute acclimation period, followed by 8 bins of 8 semi-randomized trials (two of each of four trial types). There was one trial with only a main burst (120 dB), and three trials with differing prepulse burst intensities (80, 85, and 90 dB). Prepulse onset to main pulse onset was 125 ms. Prepulse and main pulse burst lengths were 100 and 25 ms, respectively.

of TBARS and GSSG in parallel with enhancement of protein ubiquitination, suggesting that oxidative stress may play an important role in protein ubiquitination caused by tail-suspension. To prevent protein ubiquitination, we administered antioxidative nutrients, cysteine and α-tocopherol, to tail-suspension rats. Intragastric supplementation of 140 mg/rat of cysteine normalized the ratio of GSH to GSSG in the muscle and suppressed protein ubiquitination and MHC fragmentation, leading to the partial prevention of hindlimb muscle mass loss. In contrast, supplementation of 15 mg/rat of α-tocopherol did not suppressed protein ubiquitination and MHC fragmentation possibly due to its prooxidant action. Our results suggest that supplementation of antioxidative nutrients, such as cysteine, may be beneficial for preventing ubiquitination of muscle proteins caused by microgravity.

Results indicated that there were no significant differences in baseline startle reflex amplitude between 0, 1, 3, and 5 Gy. However, 2 Gy differed significantly from 0 Gy in producing a reliable reduction in baseline response as well as a decreased inhibition of the response due to the prepulse (p<0.05). This suggests some specific population of cells that influences startle behavior is activated at 2 Gy. At lower doses, these cells may not be activated at all, whereas at higher doses these cells may be compromised or in the process of recovery.

(Supported by NASA: Coop. Agreement NCC9-149.)



[20] [22] THE MARS GRAVITY BIOSATELLITE: A NEW PLATFORM FOR PARTIAL GRAVITY RESEARCH. E.L. Brown1,2

, C.E. Carr1,2,

and J.E. Keesee1. 1MIT Department of Aeronautics and Astronautics, 2Harvard-MIT Division of Health Sciences and Technology.

CONSEQUENCES OF ALTERED GAS DIFFUSION RATES FOR CELL DIVISION AND SECONDARY METABOLITE PRODUCTION BY SOYBEAN (Glycine max) CELLS GROWN IN TISSUE CULTURE. L.K. Tuominen1 and M.E. Musgrave2. 1Plant Biology Graduate Program and 2Biology Department, Univ. of Massachusetts, Amherst, MA 01003

The Mars Gravity Biosatellite, an international student-led program, will provide an artificial gravity testbed to study mammalian adaptation and development in partial gravity. The baseline mission profile specifies a launch of eleven female mice, including four pregnant dams, and utilizes both rotational and non-rotational time-delayed ground controls. The mission will last for seven weeks, allowing sufficient time for the young to be born and reared to maturity. Animals will be recovered rapidly after reentry to minimize the effects of reloading.

Observation of hypoxic responses in plants grown in spaceflight has led researchers to speculate that absence of buoyancy-driven convection in microgravity may limit gas exchange rates. The purpose of this study was to understand how the rate of availability of oxygen and carbon dioxide to plant cells influences cell division and secondary metabolite production. Callus culture of soybean (Glycine max (L.) Merr.) tissue provided a model system in which both processes could be readily quantified. Aliquots of the synthetic cytokinin, 6-benzylaminopurine (BA), were added to tissue culture medium to stimulate different rates of cell division, quantified by fresh weight gain of the callus. Similarly, liquid shake cultures derived from callus cells were induced to synthesize the plant estrogen diadzein (7,4’-dihydroxyisoflavone) by addition of BA. Following extraction of the cells at 4C with ethanol, diadzein was quantified spectrophotometrically using peaks at 262 and 270 nm. Synthetic atmospheres were devised as follows. The control atmosphere provided to the cultures was 21% oxygen, 400 ppm carbon dioxide, and the balance nitrogen. Replacement of nitrogen by helium in this system was used to increase the diffusion rate of oxygen, carbon dioxide and ethylene, thus making them more readily available to the plant tissues. Conversely, replacement of nitrogen by argon slowed the diffusion rates

Scientific priorities include evaluation of bone loss, muscular atrophy, neurovestibular adaptation, and changes in reproduction and development. Limited physiological and environmental parameters will be collected on board the satellite throughout the mission, but primary studies will take place pre- and post-flight. General studies will include a broad analysis of animal health and pathology. Principal investigators, chosen from the science community at large, will conduct detailed primary science investigations that focus on the musculoskeletal, vestibular, and developmental effects of the 0.38-g artificial gravity environment. Other studies will be selected on the basis of compatibility with the primary science investigations. Remaining flight and control animal tissues will be preserved and made available via a biospecimen sharing program. All studies will be carried out in accordance with NIH and NASA guidelines for ethical research and will be subject to peer review. Data from the Mars Gravity Biosatellite mission will provide insights into fundamental questions of gravitational biology and the use of artificial gravity as a countermeasure to microgravity deconditioning.

of the metabolic gases. Gases were sent through a humidifier before passing to test chambers. Flow rate was sufficient to maintain the desired atmospheric composition in the chambers, as confirmed by gas chromatography. For the cell division assay, treatment in the test gases lasted 28 days, while for the more rapid diadzein response, gas treatment lasted 72 h, with diadzein synthesis induced after 24 h. The results are significant for spaceflight applications because they model diffusion rate effects on basic plant growth and metabolism.

(Supported by NASA grant NAG2-1375.) [21] [23] CHEMICAL AND GRAVITY DEPENDENT FACTORS AFFECTING ESCHERICHIA COLI LAG PHASE TERMINATION. R.D. Elms1, T.A. Good1, D.M. Klaus2, M.V. Pishko3. 1Dept of Chemical Eng, Texas A&M Univ, 2Dept of Aerospace Science, Univ of CO, Boulder, 3Dept of Chemical Eng, Penn State Univ.

PLASTID SEDIMENTATION KINETICS IN ENDODERMAL CELLS OF ARABIDOPSIS. M. Palmieri and J. Z. Kiss. Department of Botany, Miami University, Oxford, Ohio. The endodermal cells of hypocotyls in flowering plants possess starch-filled organelles called amyloplasts, which appear to function in gravity perception. Following reorientation of seedlings, the amyloplasts settle The objectives of this study were to identify and quantify chemical

compounds suspected to induce and affect bacterial lag phase termination (LPT) and to characterize the role of gravity in LPT by correlating effects on chemical concentration gradients. The organism used for this study was E. coli ATCC 4157.

to the new bottom of the cell. The seedlings then exhibit a differential growth response in which the roots grow towards the gravity vector, and the hypocotyls grow away from gravity. The magnitude of this graviresponse has been correlated to plastid sedimentation in the columella cells of roots. Research in our laboratory has shown that a decrease in the amyloplast starch content causes less plastid sedimentation, resulting in reduced gravitropic curvature of starch-deficient Arabidopsis seedlings. This study aims to determine the sedimentation kinetics of amyloplasts

Acetic acid was not found in the extracellular environment of E. coli at the end of lag phase. Lactic acid (LA) was found in the extracellular environment of E. coli at the end of lag phase in small amounts. Evidence was not found to support the hypothesis that either LA or CO2 are critical by-products for LPT. At α = 0.05, LA (20 & 30 mg/L) supplementation increased E. coli lag phase length. At α = 0.10, CO2 supplementation via bubbling through 5% CO2 in air and equilibration of media with CO2 in

in the hypocotyl endodermal cells of Arabidopsis thaliana. Wild-type Arabidopsis seedlings were dark-grown and fixed at regular intervals following reorientation with respect to gravity. One micrometer median longitudinal sections were stained with Toluidine Blue and amyloplast position was visualized using light microscopy. Brightfield images were captured digitally, and image analysis was performed using Image Pro Plus software (Version 3.0; Media Cybernetics, Silver Spring, MD). The plastid positions and sedimentation rates in wild-type Arabidopsis provide important baseline data for future experiments involving disruption of the F-actin cytoskeleton and analysis of the effect of this disruption on plastid sedimentation kinetics and the graviresponse.

a flask increased lag phase length by 1.39 and 2.12 hours, respectively. Based on the mathematical analysis, gravity does not affect the mass transfer of LA and CO2 away from a single E. coli cell, but does affect the mass transfer of proteins having diffusivities on the order of 1 to10 µm2/s. Mass transfer for such proteins is greater when gravity is present. Evidence was not found to support the hypotheses that LA and CO2 are critical components for LPT. This conclusion was supported by the outcome of the supplementation experiments, and the relative movement and by-product concentration gradient analyses. The latter two analyses indicate by-products the size of LA (D = 2200 µm2/s) and CO2 (D = 2800 µm2/s) readily diffuse away from a single E. coli cell while by-products with D values closer to 1 to 10 µm2/s will remain at higher concentrations closer to the cell. Therefore, working with the supposition that critical by-products are necessary for LPT it is suggested that by-products having diffusivities on the order of 1 to 10 µm2/s are likely candidates to be critical by-products for LPT.

(Supported by NASA: NCC2-1200, and NIH: 1R15GM 57806-01.)




[24] THE EFFECT OF LACL3 ON ROOT GROWTH AND MICROTUBULE ORGANIZATION IN PRIMARY ROOTS OF ZEA MAYS L. M. Liu and K.H. Hasenstein, Biology Dept. UL Lafayette, Lafayette, Louisiana 70504-2451 The calcium antagonist LaCl3 was used to study the effect on primary root elongation and the organization of microtubules in maize roots. The tested concentrations varied from 1 µM to 10mM LaCl3. Growth was promoted ca. 45% at 1 µM La3+ but reduced by 40% at 1 mM or higher. Compared with controls (Mes/Tris buffer, pH 6.2), 100 µM and higher La3+ concentrations inhibited root elongation. Although growth inhibition was strongest in the first hour after application, it further increased over time. Immunostaining of microtubules (MT) showed that 1µM LaCl3 (5 hr) stabilized MT orientation perpendicular to the root axis. However, LaCl3 greater than100 µM caused disruption of MT in cortical and epidermal cells of the elongation zone and reduced mitotic activity and density of MT in the apical region. After 24 hrs, cortical MT in the elongation zone were severely depolymerized. Our results indicate that La3+-induced growth inhibition is concentration and time dependent. The MT reorganization after La-treatment suggests that La-mediated growth may interfere with calcium fluxes across the plasma membrane and affect calcium-dependent signal transduction and growth. (Supported by NASA grant NAG10-0190.) [25] THE USE OF RNAi TO INVESTIGATE GENE FUNCTION DURING POLARITY DEVELOPMENT IN CERATOPTERIS RICHARDII. S.C. Stout and S.J. Roux. Molecular Cell and Developmental Biology, University of Texas at Austin. We are developing RNA silencing as a technique to investigate gene function during gravity directed polarity development in the single celled spores of Ceratopteris richardii. In order to determine if RNA silencing, a post-transcriptional gene silencing mechanism, may be used in this system, we examined whether or not protein translation was required for spore germination. 35S labeled methionine incorporation revealed there was significant protein synthesis during the first 24 h of germination, and transient cycloheximide treatments delayed or prevented spore germination. These results indicate that new protein synthesis is needed for germination progression, and thus, that RNA silencing may be used to examine the role of specific genes during germination in Ceratopteris. We incubated germinating spores with various concentrations of double stranded RNA constructs derived from the sequences of three genes from Ceratopteris: a calmodulin, a calmodulin-domain protein kinase, and a profilin. dsRNA concentrations of 0.5 mg mL-1 to 1.0 mg mL-1 specifically suppress the expression of the target gene at the mRNA level. Additional experiments are necessary to determine the duration of suppression and the amount of suppression observed at the protein level. Nonetheless, these results indicate that RNA silencing is a promising approach for investigating the molecular basis of gravity perception and signal transduction in Ceratopteris richardii. (Supported by NAG10-0295, NAG2-1347 to SJR and NGT5-50371 to SCS.)


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UndergraduateStudent

Poster Session IICompetition


ABSTRACTS – POSTER SESSION II–A

[26] [28] ALTERATIONS IN BREATHING PATTERNS BY USE OF THE INSPIRATORY IMPEDANCE THRESHOLD VALVE (ITV). V.A. Convertino1, D.A. Ratliff2, S.D. Clah3. 1Research Physiologist, U.S. Army Institute of Surgical Research, 2Research Physiologist, Environmental Health, 3SLSTP trainee, Dine College, Shiprock.

EFFECTS OF SHAKING WITHOUT AERATION ON ESCHERICHIA COLI GROWN IN LIQUID MEDIA. Rachel N. Edes1, E.A. Juergensmeyer2, M.A. Juergensmeyer3, and E.S. Nelson4. 1 Illinois Math and Science Academy, Aurora, IL, 2 Judson College, Elgin, IL, 3 IIT Research Institute, Chicago, IL, and 4 NASA Glenn Research Center, Cleveland, OH. Approximately 50% of astronauts returning to Earth experience

orthostatic hypotension. Before spaceflight an astronaut’s blood is evenly distributed throughout his body due to adequate blood pressure regulation and the pressure of a 1-G environment. As the astronaut enters into space, the blood is re-directed to the chest and upper body due to lack of gravity. This shift causes excess fluid loss over time due to barorecptors signaling apparent increased blood pressure in the carotid bodies. When the astronauts reenter the earth’s gravitational pull, the blood and the fluids are again distributed throughout the body however, there is not enough blood. Therefore, astronauts could possibly faint due to lack of circulating fluids.

A common practice when growing aerobic bacterial cultures is to place liquid media on a shaker, with the assumption that shaking increases the amount of dissolved oxygen in the media. Space-flown cultures, however, are exposed to shaking in the form of vibration, without the increased exposure to air. We have investigated the effects of aeration vs. shaking without increased aeration to determine whether or not shaking alone can affect the growth rate of bacteria in liquid media. We have compared the growth of Escherichia coli with and without shaking in flasks (exposed to air), test tubes (with and without exposure to air) and syringes (no exposure to air). Shaking without aeration affects the growth curve In our experiment, we are testing a way to increase the blood pressure

upon reentry to the Earth to prevent fainting, by using a device called an Inspiratory Impedance Threshold Valve (ITV). We hypothesized that the ITV will increase venous return and lead to an increase in stroke volume. More importantly, we hypothesize breathing with the ITV will increase respiratory rate to maintain a steady state minute volume of airflow. This effect will be increased with increasing resistance of the ITV.

of E. coli. Failure to consider the effects of vibration on space flight experiments can lead to the attribution of effects to microgravity which are due to other causes. (Supported by NASA grant NAG2-1512)

Our hypothesis was tested with this protocol: Subjects were sensored for bioimpedance measurement of stroke volume. Subjects respiratory rate was measured by counting the number of baseline excursions of end tidal C02 in 1 minute. A minute volume of air inspired was also measured. After 3 minutes of breathing on the ITV respiration measurements were recorded. At minute 4, Stroke volume was sampled for 20 seconds.

We looked at the difference between the recorded measurements of the volume and rate using the 00 cm, 07 cm, or 12 cm valve. There has not been an experiment on how the ITV will affect respiration using the different size valves on the same subject. Another factor is that this will be the first time using the ITV on healthy human subjects. The results for breaths are given in bar graphs showing the increase of respiratory rate and minute volume of inspiration from breathing in three different size valves.

[27] [29]

OPTIMIZATION OF PSEUDOMONAS AERUGINOSA INOCULUM FOR STS-107 EXPERIMENT BACTER. A.C. Perez-Osorio, S.C. Broadaway, and B.H. Pyle. Microbiology Department, Montana State University, Bozeman.

PATTERNS OF LOCOMOTION ACTIVITY DURING HYPER-GRAVITY IN LARVAL DROSOPHILA MELANOGASTER. M.J. Potthoff and J.N. Thompson, jr. Dept. of Zoology, Univ. of Oklahoma, Norman. Adult Drosophila are highly mobile and can detect and respond to changes in gravity. Since larvae are slower and more limited in movement, it might be expected that their gravitational responses would be slight. In contrast, we hypothesize that hypergravity can be a stress that will impede their mobility significantly. A 22"-diameter centrifuge was constructed with a metal armature attached to a bearing assembly. A pulley system was used to reduce the rate of revolution. The drive wheel was attached to the power source, a variable-speed drill regulated

The growth, physiology and virulence of an Exotoxin A (ETA) producing isolate of P. aeruginosa will be studied in this flight experiment. A suspension of 5x108 cells/ml is required to ensure growth of defined medium cultures in ESA Phorbol cassettes with Type I containers. An initial culture was grown in Simple Defined Medium for18-24 hours. When the culture had reached between 60-100 Klett Units the Optical Density (OD) was also determined. After washing twice in PBS-glucose, the suspension was diluted to greater than 5x108 cells/ml based on the OD. The sample was then adjusted to 5x108 cells/ml according to SYBR Green staining with microscopic enumeration. The resulting suspensions were either enumerated immediately or stored at 50C for 7 days and analyzed to enumerate the surviving population and viability of the inoculum. Based on the results, SDM cultures for the space flight experiment will be grown to 0.5-0.6 OD prior to washing and inoculum preparation, rather than from the amount of time incubated. The inoculum survived the 7 day storage time, and subsequent inoculation of ETA medium produced growth as required.

by a rheostat. RPM was quantified with a photocontact tachometer, and a gravitational force of 4 g could be maintained for extended periods. Larval D. melanogaster were placed in a plastic arena where their behavior could be observed and videotaped using a miniature high resolution videocamera (ALM-2453 2.4 GHz Wireless Miniature Camera, 10× mag.). Larval locomotion rate was measured by digitizing the larval position coordinates continuously during experimental periods of 70 minutes each (10 min taped acclimation; 30 min at 4 g; 30 min at 1 g). Locomotion rate declined during exposure to 4 g (units of linear movement at 4 g: 1000.66 ± 884.47 s.d.; control: 1490.51 ± 1212.87; t150 = 2.68, P < 0.01), but began to return to normal when larvae returned to 1 g (for those that showed movement, 0-10 min post treatment = 938.29 ± 914.07; 20-30 post treatment = 1457 ± 1192.00; t66 = 2.03, P < 0.05). Larvae were frequently observed to exhibit an enhanced escape-like behavior (positive geotaxis) during and after exposure to hypergravity. Locomotion is reduced under hypergravity, but larvae are still able to move effectively and feed. Treatment at 4 g is stressful enough to stimulate escape reactions, and

Using an inoculum prepared as described, we anticipate reliable inoculation and growth of ETA medium cultures in the spaceflight hardware. The flight experiment will improve our understanding of the significance of P. aeruginosa to future spaceflight crews. (Supported by NASA, ESA, MSU Undergraduate Scholars Program, and Montana Space Grant Consortium) we predict that increased hypergravity and exposure time will make these

even stronger. (Supported by NASA: NAG2-1427; and UROP Undergraduate Research

Grant).



[30] [32] THE EFFECT OF TFIBA ON AUXIN TRANSPORT IN PRIMARY ROOTS OF ZEA MAYS L. G. Ku and K.H. Hasenstein, Biology Dept. UL Lafayette, Lafayette, Louisiana 70504-2451

NATIVE ALCOHOL DEHYDROGENASE EXPRESSION, GFP EXPRESSION, AND FLUORESCENT IMAGING OF TRANSGENIC ARABIDOPSIS THALIANA. C. Folmes 1, A-L. Paul 2, and R. J. Ferl 2. 1Spaceflight and Life Sciences Training Program (SLSTP), Kennedy Space Center, 2 University of Florida, Gainesville, FL

Previous research has shown that the promotion of root growth of 4,4,4-trifluoro-3-(indole-3-) butyric acid (TFIBA) is partially due to its effect as ethylene antagonist but it is also possible that TFIBA affects auxin transport. Therefore, we studied auxin transport in apical segments of 2-3 d old primary roots of corn in the presence of this compound. The root segments were sandwiched between an agar donor and receiver, and tritiated IAA (10-7 M) was applied to the cut surface. Auxin transport was measured in the presence of 100 µM TFIBA in the receiver, or donor, or both agar blocks for one and two hours. Based on the radioactivity of the receivers and uptake we found that compared to non-treated controls, TFIBA enhanced transport when applied to the receiver by 50%. In contrast, uptake was promoted regardless of the site of TFIBA application from 17 to > 25 %. After 2 h of transport the effect decreased. These patterns are consistent with TFIBA-enhanced acropetal auxin transport. Future work will study the rate and direction of TFIBA transport and its possible effect on IAA metabolism.

A better understanding of a plant’s metabolism and physiology is essential for long-term space flight missions. Transgenic Arabidopsis thaliana was exploited to determine the changes in gene regulation due to space flight stresses. Prior to flight, experiments will be performed to validate the methods that will be used. The transgenic plants contain a transgene composed of green fluorescent protein as a reporter gene, driven by the alcohol dehydrogenase gene promoter. Hypoxic stress was applied to plants by use of agar blankets, and the gene expression was determined with quantitative PCR (Taqman™ RT-PCR) and by fluorescent imaging. A correlation between native alcohol dehydrogenase gene expression, GFP gene expression and fluorescent intensity was determined within roots and shoots. In addition, the transduction of expression from the root to the shoot will be explored when the roots tunnel through agar blankets and in plants on a clinostat. Current data has shown an increase in GFP expression after 48 hours, with a decrease in expression after 96 hours. In addition the GFP expression is found specifically in the root tips and the root junctions.

(Supported by NASA grant NAG10-0190.)

[31] [33] MASS APPLICATION OF MECHANO-DWARFING STIMULI PROFILING THE STRESS RESPONSE OF ARABIDOPSIS USING

GREEN FLUORESCENT PROTEIN: A PRELUDE TO A FLIGHT EXPERIMENT. T. Do1, A-L. Paul2 and R. J. Ferl2. 1Spaceflight and Life Sciences Training Program (SLSTP), Kennedy Space Center, 2 University of Florida, Gainesville, FL

TO ARABIDOPSIS FOR MUTANT-SCREEN DEVELOPMENT. J. A. Montgomery, R.A. Bressan, C.A. Mitchell, Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN. Various forms of mechano-stimulation were applied to Arabidopsis thaliana (L.) Heynh. seedlings of the C-24 ecotype in effort to obtain uniform growth reduction compared to undisturbed controls. Preliminary work investigated effects of static impedance (perforated plates), dynamic compression and flexing (roller), dynamic frictional contact and flexing (brushes), or vibration in both greenhouse and controlled growth environments. Treatments were applied alone or in combination varying intensity, duration, and time of stress application. Treatment effects

Stress responses in the plant Arabidopsis were initiated by the application of four different stress types: flooding, increased ethylene, increased ethylene with KMnO4 to absorb the hormone, and a decreased temperature. An engineered reporter gene comprised of the alcohol dehydrogenase (Adh) promoter driving the Green Fluorescent Protein (GFP) reporter was incorporated into Arabidopsis to follow these stress responses in real time. The results were read visually with imagery from fluorescent microscopy and the gene expression was evaluated quantitatively with Real-Time Polymerase Chain Reaction (RT-PCR) “Taqman™” (ABI) chemistry. Images were read at 24 hr, 72 hr, and

in both locations were compromised due to interactions of PPF, media, temperature, and nutritional factors. Arabidopsis grown under low PPF (50-55 µmol m-2 s-1) using a rockwool growth medium hydrated with 1/4 X Hoagland’s solution grew upright with limited leaf expansion, which increased mechano-stress responsiveness. Low irradiance with different red/far-red wavelength ratios was tested for brush and plate treatments. Hypocotyl elongation and leaf expansion were reduced by twice daily brush or plate treatments. Under fluorescent radiation, brushed seedlings exhibited a 40% reduction in growth relative to undisturbed controls, whereas plate treatments produced a 30% reduction after 6 days of twice-daily treatment. Seedlings grown under fluorescent + incandescent lamps also exhibited stress-induced growth reductions, but stressed seedlings lodged to a greater extent due to more rapid hypocotyl elongation. Temperature and daylength optimization studies are underway. T-DNA insertional mutant lines will be screened for TCH-gene knockouts using a system that includes low PPF fluorescent lighting, and twice-daily brush treatment.

120 hr intervals, then compared to positive control plants transformed with a reporter gene that is constitutively expressed (CaMV35s/GFP). The fluorescent microscopy results showed the GFP expression primarily in the roots for the flooding and ethylene treatments while expression was shown principally in the leaves for the reduced temperature stress. Another important result showed no significant difference between the standard ethylene treatment and the sample subjected to KMnO4 for ethylene absorption.

(Supported in part by the Laurenz M. Greene Undergraduate Scholarship Program.)



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Graduate Student

Poster Session IICompetition


ABSTRACTS – POSTER SESSION II–B

[34] [36] MICRO-GRAVITY EXPOSURE RESULTS IN SIGNIFICANT ATROPHY OF MURINE SKELETAL MUSCLE WITHOUT ALTERATIONS IN MYOSIN HEAVY CHAIN GENE EXPRESSION. B.C. Harrison1, D.L. Allen1, L.S. Stodieck2,

ROLE OF THE ANTENNAE IN GRAVITY SENSING BY DROSOPHILA MELANOGASTER. G. Hetrick, C. S. Miller and K. Beckingham Dept. Biochemistry and Cell Biology, Rice University, TX.

P.J. Kostenuik3, T.A. Bateman2, S. Morony3 and L.A. Leinwand1. 1Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, 2BioServe Space Technologies, University of Colorado, Boulder, and 3Amgen, Inc., Thousand Oaks CA.

In many organisms, a clearly defined gravity sensing organ of the ‘statocyst’ type is present. In Dipteran insects, no such organ has been identified and thus gravity perception must entail other sensory devices. There is evidence that gravity sensing may involve a diverse array of sense organs including proprioceptors in the legs and body wall. Horn and Kessler (J. Comp. Physiol. 97, 189-203) have also provided evidence that the antennae are a major site of gravity perception in the Dipteran Calliphora erythrocephala. The Beckingham lab has identified a number of mutations that affect gravity perception during walking in the Diperan Drosophila melanogaster. The mutant flies were identified using gravitaxic mazes in which the flies make eight up/down choices and thus emerge at one of nine exits. Recent work to determine which tissues are affected by the mutations suggests that in many of these mutants the defective gene product is expressed in the antennae. Other sense organs

The purpose of this study was to investigate the effects of micro-gravity on murine skeletal muscle fiber size and myosin heavy chain (MyHC) isoform expression. As part of a larger study, female C57BL/6J mice, aged 64 days were divided into Animal Enclosure Module ground control (AEM) and Space Flight (SF). SF animals were flown on the Space Shuttle Endeavour (STS-108/UF-1) and subjected to 11 days and 17 hours of micro-gravity. Following the shuttle flight, both AEM control and SF animals were sacrificed, and the triceps surae (gastrocnemius, plantaris, and soleus) of each animal was dissected and frozen in liquid nitrogen-cooled isopentane. Immunohistochemical analysis of muscle fiber cross-sectional area (CSA) revealed that, in each of the muscles analyzed, mean muscle fiber CSA was significantly reduced (p < 0.0001) for all fiber types for SF versus AEM control. In the gastrocnemius, mean CSA was reduced by 29%, 35%, 15%, and 18% for type I, type IIA, type IIX, and type IIB fibers, respectively. In the plantaris, fiber CSA decreased by 22%, 20%, and 16% for types IIA, IIX, and IIB, respectively. In the soleus, type I CSA was reduced by micro-gravity exposure 31% while type IIA CSA was reduced by 26%. Immunohistochemical analysis of MyHC isoform expression revealed no significant change in the percentage of fibers expressing MyHC-I, -IIa, -IId/x, or –IIb across analyzed muscles. Overall, these data indicate that, in the mouse, exposure to micro-gravity results in significant muscle fiber atrophy. Furthermore, this muscle fiber atrophy is more pronounced in oxidative muscle fibers (Type I and IIA) than in glycolytic muscle fibers (Type IIX and IIB). Contrary to previously observed results in the rat, monkey, and human, however, micro-gravity exposure does not significantly alter MyHC isoform expression in mouse skeletal muscle.

on the head are also implicated. The mutant gene expression patterns will be presented. To address further the role of the antennae, experimental manipulation of the antenna is being performed and the effects on gravity perception subsequently assayed. These results will also be reported. (Supported by NIH grant DC05164-02, and the Century Scholars Program at Rice University).

(Supported by NIH GM 29090 to LAL) [35] [37] SYNAPTIC PLASTICITY IN RAT UTRICULAR MACULA UNDER CONTROLLED HYPERGRAVITY CONDITIONS. M. Ahmad and A. Lysakowski*. Dept. of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL 60612.

TENSION REDUCTION INDUCES SKELETAL MUSCLE ATROPHY IN A NOVEL IN VITRO GROUND BASED MODEL THAT CAN BE ATTENUATED BY HIGH LEVELS OF INSULIN. P.H.U. Lee, B.C. Creswick, M. Nackman, X. Wang and

The synaptic innervation of the crista ampullaris was examined in previous studies (Lysakowski, 1996; Lysakowski and Goldberg, 1997; Lysakowski, 2000). Regional variations in cytoarchitecture and synaptic innervation were found, which included variations in afferent bouton number per type II hair cell, in synaptic ribbon size and number per hair cell (especially in type II hair cells), and in the numbers and sizes of calyceal invaginations. The latter may be related to synaptic activity since they are greatest in the central zone, where activity is highest. Given this diversity of synaptic innervation, we were interested in the regional pattern of normal synaptic innervation in the adult rat, and in how this varies under controlled conditions of hypergravity stimulation. We chose the utricular macula for these experiments because of its morphology and response to linear forces. We are using a variable linear force paradigm

H.H. Vandenburgh. Dept. of Pathology, Brown Univ., Providence, RI Although tissue-engineered skeletal muscle constructs termed BioArtificial Muscles (BAMs) had demonstrated the direct effects of spaceflight on skeletal muscle atrophy on previous space shuttle missions (FASEB J 13, 1031; 1999), no ground based in vitro model of skeletal muscle atrophy currently exists. The objective of this study was to develop and test a ground based model for skeletal muscle atrophy using tension reduction in BAMs to complement spaceflight and ground studies and to test potential pharmacologic countermeasures. BAMs made from primary adult mouse myoblasts were tissue-engineered so that active forces generated in response to electrical stimuli can be measured. BAMs ranging from 2-4 weeks in age were reduced in length by 50% or released at one end for at least 7 days and incubated in regular growth medium (GM), or supplemented medium (SM) containing insulin (10 µg/ml) and FGF (0.2 ng/ml).

to prevent adaptation in the irregular afferents. Multiple samples were taken from each rat utricular macula. Each sample spanned the entire sensory epithelium and included material from all three regions: striola, juxtastriola, and extrastriola. Dissector counts

Compared to controls, after tension reduction in GM, maximum isometric tetanic forces decreased significantly by 21 to 50% in the four experiments, and in separate experiments, the mean myofiber cross-sectional area (CSA) was reduced by 10% (p<0.05), total non-collagenous protein content decreased by 8.7% (p<0.05), and the 6 hr incorporation of C14-phenylalanine decreased by 22% (p<0.05). Compared to tension reduced BAMs in GM, peak active forces increased 43 to 76%, protein content increased 4% (p=0.09), and myofiber CSA increased 20% (p<0.05) when incubated in SM.

of synaptic ribbons and calyceal invaginations were made as described previously (Lysakowski & Goldberg, 1997). Results are still preliminary, based upon a total of 118.5 hair cell equivalents (and only 75 in the invaginations data) from four samples of rat utricular macula. In particular, our numbers of type II cells are very low (striola = 7.5; extrastriola = 32), given the overall type I : type II hair cell ratio in rat (striola = 1.73, extrastriola = 1.19). Overall, synaptic ribbons are increased on the excited side of the striola and extrastriola, especially for type IC and type II hair cells, while calyceal invaginations are decreased on the excited side of the striola only, compared to the inhibited side. Synaptic ribbons in type II hair cells are larger and occur in clusters more often in the striola compared to the extrastriola, similar to our results in the crista.

These data demonstrate that reducing tension in BAMs induces skeletal muscle atrophy, possibly by decreasing protein synthesis rates. Additionally, high levels of insulin, likely interacting with IGF-1 receptors, may attenuate the induced atrophy. Further development and utilization of this novel in vitro model of skeletal muscle atrophy may reveal mechanisms of atrophy both on Earth and in spaceflight. (Supported by NASA NAG5-4593 and NIH R01 DC2521.)



[38] [40] GENE TRANSCRIPTION SIGNALING WITH A THICKNESS SHEAR MODE ACOUSTIC WAVE BIOSENSOR. C.N. Jayarajah and M. Thompson. Department of Chemistry, University of Toronto, Canada

TOUCH-INDUCED GENE EXPRESSION IN SOYBEAN. A.L. Santone, K.G. Raghothama and C.A. Mitchell. Dept. of Horticulture & Landscape Architecture, Purdue University, West Lafayette, IN.

An on-line acoustic wave biosensor for the detection of in-vitro gene transcription is presented. A piezoelectric crystal with a cold coating on which the DNA is immobilized functions as the sensor. The thickness shear mode acoustic wave device is used here to distinguish the binding

A growth system for isolating genes involved in cell expansion has been devised using etiolated soybean (Glycine max L. Merr. cv. Century 84) seedlings. Using an EST as a probe for Northern blots, a soy gene having homology to TCH3 of Arabidopsis has been shown to be up-regulated as early as 15 minutes after thigmic stimulation. The use of wind-shield/ humidification chambers has helped reduce spurious expression of the TCH3 homologue in highly sensitive control seedlings, thus improving the sensitivity of the system to detect changes in gene expression induced by controlled mechanical stress. Concurrently underway is a project utilizing cDNA-AFLP to discover novel touch-regulated genes in soybean.

of surface immobilized strong and weak, and closed and open promoter DNA sequences to the T7 RNA polymerase. Furthermore, the synthesis of mRNA on the sensor surface has been monitored. A two fold decrease in resonance frequency is observed in going from closed to open promoter in the absence of ribonucleotides. In the presence of ribonucleotides, a four fold decrease in resonance frequency is observed. This change is accompanied by a significant increase in motional resistance. The data obtained for the interaction of actinomycin-D with the promoter DNA

(Supported in part by NASA: NAG2-1389, NGT 2-52285)

in inhibiting transcription suggests that this drug molecule not only intercalates with double stranded DNA, but also binds strongly to single stranded DNA as demonstrated by a six fold decrease in resonance frequency. This biosensor offers several advantages over conventional methods, such as label free detection in real time, and the generation

of multidimensional data. The multidimensional data obtained includes resonance frequency and motional resistance, which afford us information other than mass loading, such as changes in viscoelastic properties, surface charge and free energy, and conformation. Moreover, the curves

of resonance frequency as a function of time contain information on association and dissociation kinetics. The results presented here suggest that this sensor is a viable technique to identify gravity dependent transcription factors, and to detect the effects of gravitational perturbation and other stress factors associated with space research on gene transcription. This biosensor is also useful in detecting specific gene expression levels in organisms with relevance to astrobiology.

(Supported by NSERC, Canada) [39] [41] RESULTS OF A LONG DURATION (72-DAY) SPACE FLIGHT EXPERIMENT ON MICROBIAL ANTIBIOTIC PRODUCTION. M.R. Benoit1, D.M. Klaus1 and W. Li2. 1BioServe Space Technologies, University of Colorado, Boulder, and 2Bristol-Myers Squibb Company, Wallingford, Connecticut.

EVALUATION OF AN ALTERNATIVE NUTRIENT DELIVERY SYSTEM FOR GROWING PLANTS IN MICROGRAVITY. J.F. Romagnano and P.J. Weathers. Dept. Biology/Biotechnology, Worcester Polytechnic Institute, Worcester, MA 01609. To grow plants in microgravity, porous tubes have been used to deliver liquid media to the root zone. Growth has, however, been less than desirable. The lack of a gravity vector likely leads to the formation of stagnant air pockets, and inadequate gas availability to the roots. We have compared an alternative nutrient delivery system using a mist with the porous tube system to feed germinating peas. The mist and porous tube systems were also compared against hypoxic and normoxic controls. After one week roots were harvested, weighed, and assayed for alcohol dehydrogenase (ADH) activity as an indicator of root hypoxia. Of the two experimental and two control systems, the mist system yielded the greatest fresh root mass per germinated seed. The hypoxic control supported the least amount of root mass. ADH activity of roots grown in the mist, porous tube, and normoxic controls were all about 10% of the activity measured in the hypoxic controls. When compared with the ADH activity of the hypoxic control, however, ADH activity of the mist system was significantly less than that of the porous tube. These results suggest that at least during germination, a mist system might be more successful than the porous tube for growing plants in microgravity. Work comparing fully developed plants grown using porous tubes to those grown in mist systems is underway.

Previous findings from three earlier pilot studies conducted aboard shuttle missions STS-77, -80 and -95 indicated that microbial antibiotic specific productivity in space was increased by up to 190% relative to comparable ground controls. Absolute yields of antibiotic obtained in the reactors used for these experiments, however, were substantially lower than those obtained in shake flasks typically used in the laboratory. Therefore, in conjunction with the primary research objectives, the hardware designs used to conduct the experiments in space were concurrently evolved to increase absolute production levels and allow autonomous, long duration operation. An experiment was flown aboard the International Space Station (ISS) for 72 days during the 8A Increment (April 8 to June 19, 2002). Cell cultures of Streptomyces plicatus that produce actinomycin D were grown with passive gas exchange in defined media maintained at 22° C. New automated flight hardware designed for this experiment (MOBIAS) provided the capability to feed and remove waste from the cultures and periodically remove samples for preservation at 4° C within the same middeck locker volume. Preflight baseline data collection indicated that actinomycin D production in MOBIAS (in 1g) reached a level comparable to that typically achieved in laboratory shake flasks. Samples from the ISS 8A experiment are currently being analyzed for time course of absolute and specific productivity of the antibiotic as well as cell viability and spore count of the viable cultures. Computational modeling is also being pursued in an attempt to identify which specific physical factors of weightlessness might be responsible for differences observed between the flight and ground experiments.

This work is supported by Bristol-Myers Squibb in collaboration with BioServe Space Technologies (NASA NCC8-131).




[42] UNRAVELLING TOUCH AND GRAVITY STIMULATION IN PLANT ROOTS. G.D. Massa and S. Gilroy. Dept of Biology, The Pennsylvania State University An initial characterization of primary and lateral root responses to obstacles led us to suspect that touching the obstacle modulates the graviresponse in these roots. Since, under natural conditions, roots would rarely reorient in the absence of mechanical stimulation, we propose that both touch stimulation and gravistimulation are integrated simultaneously in cells of the root cap, leading to the observed biphasic bending response. Experiments with roots that were touched and then gravistimulated and placed on a clinostat show that touching the root cap, but not elsewhere in the root, leads to an inhibition of subsequent gravicurvature. To identify possible signaling mechanisms, we have analyzed ionic signaling using Arabidopsis expressing GFP’s sensitive to Ca2+ and pH. In addition we have analyzed the touch response in mutants such as the Arabidopsis potassium channel mutant akt1-1, and the response of these roots to a barrier appears similar to that of wild-type seedlings. We have also applied a pharmacological approach, for example wild-type roots were treated with Gd3+, proposed to inhibit apoplastic calcium influx through mechanically gated channels at the plasma membrane. While this chemical did transiently reduce the growth rate of treated roots, the biphasic bending response to an obstacle appears unchanged. In conjunction with the results of Ca2+ imaging these results suggest sustained Ca2+ increase at the plasma membrane may not mediate the long-term response to an obstacle. The effects of touch on the cytoskeleton are being analyzed using plants transformed with GFP fusion protein complexes. (Supported by NSF: IBN9874445, NASA: NAG2-1366) [43] COMPARATIVE BIOLOGY OF THE PHENYLPROPANOID PATHWAY IN PRIMITIVE TRACHEOPHYTES. A.M. Patten, L.B. Davin, and N.G. Lewis. Institute of Biological Chemistry, Washington State University, Pullman. Plants evolved onto land more than 400 million years ago and initiated a terrestial environment which allowed for the continued evolution of flora as well as for the later evolution of fauna onto land. It is hypothesized that this crucial transition of plants to land was largely dependent on the evolution of supportive and conductive vascular systems via the phenylpropanoid pathway. Continued evolution of this pathway allowed plants to adapt to extreme environments and achieve structures of enormous height, as well as providing myriad means of defense. This study is the first comprehensive examination to elaborate the phenylpropanoid pathway in the most primitive extant taxa of the tracheophytes, i.e. in the cryptogams (orders: Lycopodiales, Psilotales, Equisetales, Isoetales, and Selaginellales) and the ferns (orders: Ophioglossales and Polypodiales). This was investigated as follows: via comparative examination of gene expression and anatomy of developing and mature tissues in the various taxonomic orders; cDNA library construction and screening for expression of genes encoding proteins central to phenylpropanoid synthesis; localization of said proteins and their encoding mRNAs by immunohistochemistry and mRNA in situ hybridization, respectively; examination of patterns of lignin deposition, an important product of both the early and extant phenylpropanoid pathway, as determined by light, epifluorescent, and electron microscopy techniques. The results so obtained are providing new insights into how the pathway has evolved during adaptation to land. Bolwell, G. P., Patten, A., Lewis, N. G., 2001. The Holy Grail of Wood Evolution. From Wood Anatomy to Tissue-specific Gene Expression: To What Extent do Molecular Studies of Biosynthesis of Cell Wall Biopolymers help the Understanding of the Evolution of Woody Species. Phytochemistry 57, 805-810. (Supported by NASA: NAG2-1513.)


Spec

ial E

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NASA Conceptsfor Free Flying Satellites

and Advanced Technologies tosupport Biological Research in Space


ABSTRACTS – SPECIAL EVENT

[44] OBPR ENTERPRISE FREE FLYER STUDY STATUS REPORT. B.D. Yost 1, K. A. Souza 2, M. L. Hines 3 1SETI Institute, NASA Ames Research Center, 2Girven Institute of Technology, NASA Ames Research Center, 3NASA Ames Research Center. The NASA Office of Biological and Physical Research (OBPR) has initiated a study activity to investigate the feasibility of providing an uncrewed, automated satellite (free flyer) program component to increase access to space for the OBPR Enterprise. The foundation for such a program must include that it is science driven and that it addresses unique scientific or programmatic problems and also that it complements and augments ongoing or planned research programs targeted for the International Space Station (ISS). This report summarizes the study activities to date, outlines study activities remaining, and discusses relevant known issues that must be resolved in order to enable increased access to space for the OBPR Enterprise.

[45] TECHNOLOGY FOR FUNDAMENTAL SPACE BIOLOGY. J. Hines1, B. Yost2, K. A. Souza3 NASA Ames Research Center1, SETI Institute2, Girvan Institute of Technology3, Moffett Field, CA A “Technology for Fundamental Space Biology Workshop” was conducted by NASA Ames Research Center to envision and brainstorm novel autonomous and miniaturized biological technologies and integrated systems for next-generation space biology research. The workshop focused on technologies that support research with a variety of well characterized high use species, from cells through small plants and animals. The science drivers for the workshop were derived from the goals and objectives of NASA’s Fundamental Space Biology (FSB) program. Workshop participants were instructed to consider technologies that could be flown on at least one of the following flight platforms, the ISS, Shuttle, and automated uncrewed satellites (free flyers). Participants included Nobel Laureates, members of the National Academy of Sciences, and scientists and engineers from NASA and other government, industry, academic and private laboratories. The workshop addressed the current span of equipment available for FSB research in space and evaluated the potential for making 21st century technologies available to FSB scientists. Representative fields of interest covered at the workshop included cell and molecular, radiation, developmental, and organismal biology. Ideally, technologies and integrated systems that support these science disciplines will enable in-situ monitoring and control, autonomous sampling, imaging, analysis, preservation, and remote modification of protocols as driven by interesting scientific results. The results of the workshop will be presented with the intent to encourage the evaluation and commentary of the ASGSB FSB community.



Sym

posi

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SignalingPathways


ABSTRACTS – SYMPOSIUM II

[46] [48] IMPACT OF SKELETAL UNLOADING ON BONE FORMATION. D.D. Bikle, T. Sakata, H. Elalieh, B.P. Halloran. University of California and Veterans Affairs Medical Center, San Francisco, CA 94121.

GRAVITATIONAL EFFECTS ON MECHANOTRANSDUCTION AND ADAPTATION IN SENSORY HAIR CELLS OF A MAMMALIAN VESTIBULAR ORGAN. Jeffrey R. Holt.

Skeletal unloading whether it occurs in space or in bed results in a decrease in bone formation. We have used the tail suspended rat model

Departments of Neuroscience and Otolaryngology, University of Virginia, Charlottesville, Virginia.

to study the mechanisms involved because this model simulates a number of physiologic effects of space travel including the cephalad fluid shift. This model also provides a built in control in that the forelimbs remain normally weighted. The tail suspended rat demonstrates a reversible inhibition of bone formation associated with decreased proliferation of osteoprogenitor cells and increased apoptosis of osteoblasts and osteocytes in the hindlimbs but not in the forelimbs. These results indicate that local factors are likely to dominate the response to unloading. We have focused on IGF-I as the local factor in that it is produced by bone and stimulates bone formation. Skeletal unloading results in resistance to the anabolic/proliferative actions of IGF-I. Recent data from our laboratory indicate that this resistance is at the receptor level. IGF-I receptors in osteoprogenitor cells are not activated by IGF-I. This leads to a failure of MAPK/ERK and PI3K/AKT activation explaining both the decreased proliferation and increased apoptosis of osteoblasts, osteocytes, and their precursors with skeletal unloading. The result is decreased bone formation with its attendant loss of bone.

The vestibular hair cells of the mammalian inner ear transduce head movements and gravitational stimuli into electrical signals that are transmitted to the brain. They are exquisitely sensitive, signaling deflections of their mechanosensitive hair bundles as small as 1-2 nanometers. Remarkably, they are able to preserve this level of sensitivity even when confronted with large tonic stimuli, such as gravity, which may impose hair bundle offsets as large as a micron. To accomplish this feat hair cells have devised an adaptation process that repositions the mechanotransduction apparatus on a millisecond time scale thus allowing high sensitivity over a broad operating range. We have been investigating mechanotransduction and adaptation in the hair cells of the mouse utricle, a gravity-sensitive, vestibular organ. The sensory epithelium is excised from neonatal mouse utricles and placed in a recording chamber. We use glass micropipettes mounted on piezoelectric bimorphs to deflect the hair bundles and whole-cell, tight-seal pipettes to record the current response. We find rapid sub-millisecond current activation with amplitudes up to several hundred picoAmperes. In response to sustained hair bundle deflections, the current decays over the subsequent 10-100 milliseconds. The current decay, or adaptation, has been hypothesized to result from the activity of an adaptation motor that continually adjusts its position regulating tension in the mechanotransduction apparatus.

(Supported by NASA NAG-2-1371) We have recently identified myosin Ic as a molecular component of the

adaptation motor. With our collaborators, we generated a transgenic mouse that carried a mutant form of the gene for myosin Ic. A substitution mutation (tyrosine to glycine at position 61) rendered myosin Ic sensitive to inhibition by an ADP analog. We found that addition of the analog abolished the hair cell’s ability to adapt to tonic stimuli in transgenic but not wild-type cells, confirming a role for myosin Ic in hair-cell adaptation. (Supported by NIH grant DC03279)

[49] [47] IONIC SIGNALLING IN PLANT GRAVITROPISM. S. Gilroy. Biology Department, The Pennsylvania State University

TROPISTIC RESPONSES IN ROOTS–HOW LIGHT AND GRAVITY INTERACT IN SHAPING PLANT FORM. J.Z. Kiss1, J.L. Mullen2, M.J. Correll1, R.E. Edelmann1. 1Dept. Bot., Miami Univ., Oxford, Ohio 45056. 2Dept. Biology, Indiana Univ., Bloomington, Indiana 47405.

Although gravitropism directs the downward growth of the root, other stimuli such as gradients in nutrients, water, and physical obstacles such as rocks all act on the root sensory systems to modify this gravitropic response and so optimize exploration of the soil. We have therefore investigated both the signaling events in the root cap that might regulate root tropic growth in response to the gravity signal and the interaction of this gravisensory system with responses to other stimuli such as touch. Laser ablation studies have mapped the most gravisensitive cells to the core of the columella region of the root cap. These cells produce a rapid cytoplasmic pH transient upon gravistimulation and blocking this transient using caged protons suggests the pH signal is required for gravisensing/signal transduction. Concomitantly, the cell wall of the root cap becomes acidified around the sensory cells implying regulation of the H+/ATPase at the plasma membrane is involved in generating these gravity-related pH signals. Consistent with a role for plasma membrane transport processes in gravity signaling, mutations in the AKT1 plasma membrane K+ channel reduce gravity sensitivity, perhaps through disruption of the systems that are needed to regulate membrane potential during gravity-induced H+ pump activation. Touch stimulation of the root cap inhibits both the cytoplasmic and wall pH-dependent gravity signaling events and does appear to reduce root gravity sensitivity and subsequent gravitropic response. These results suggest touch and gravity may interact at the level of signal transduction events in the columella cells. Touch stimulation of root cap peripheral cells elicits an increase in cytosolic that propagates from cell to cell throughout the cap. Touch of these peripheral cap cells also alters the dynamics of statolith movement in the columella cells implying a signal is passing from the surface cells to the gravity sensing cells at the core of the root cap. These observations suggest a model whereby touch and gravity coordinate root growth through the interaction of pH and Ca2+ signaling events in the columella cells. (Supported by NASA: NAG 1366 and NSF: MCB 98-74445).

To receive adequate light and nutrients for survival, plants orient stems and stem-like organs toward light and away from the gravity vector and, conversely, orient roots into the soil, away from light toward the direction of gravity. Therefore, both gravity and light can influence the differential growth of plant organs. To add to the complexity of the interactions between gravity and light, each stimulus can enhance or reduce the effectiveness of the other. On earth, the constant presence of gravity makes it difficult to determine whether plant growth and development is influenced by gravity or light alone or the combination of the two stimuli. In roots, gravitropism (i.e. the directed growth in response to gravity) is the predominant tropistic response, but recent studies have shown that phototropism also plays a role in the oriented growth of roots in flowering plants. In blue or white light, roots exhibit negative phototropism that is mediated primarily by the phototropin family of photoreceptors. In contrast, we recently have discovered a positive phototropism in response to red light in Arabidopsis roots. Since this red-light-based response is weak relative to both gravitropism and negative phototropism, we have utilized a novel device which combines image analysis of root growth with a feedback system in order to study phototropism without the complications of a counteracting gravitational stimulus. In positive root phototropism, sensing of red light occurs in the root itself and is not a result of a transfer of a signal resulting from light perception in the shoot. The differential growth response occurred at the basal end of the elongation zone, in contrast to the location of gravitropic curvature, which is initiated in the distal elongation zone. Mutants that lack the red-light-absorbing photosensitive pigment phytochrome A (phyA) or phyB were severely impaired in red-light-based phototropism while the phyD and phyE mutants were normal in this response. Thus, as in gravitropism, phyA and phyB play a key role in mediating red-light-based phototropism in roots. (Supported by NASA grant NCC 2-1200.)



[50] KISSING COUSINS: CALCIUM-REGULATED AND UNREGULATED KINASES IN THE CDPK/SNRK FAMILY OF PROTEIN KINASES IN ARABIDOPSIS. Alice C. Harmon, Dept. of Botany and the Program in Plant Molecular and Cellular Biology, Univ. of Florida. The CDPK/SnRK family of protein kinases is comprised of 84 members, 59 of which are thought to be regulated by calcium. The CDPKs (calcium-dependent protein kinases or calmodulin-like domain protein kinases) are directly regulated by the binding of calcium to a regulatory domain located at the C-terminus of these proteins. In contrast, members of subgroup 3 of the SNF1-related kinases (SnRK3) are regulated by the binding of calcium to a separate protein, which in turn binds to the kinase. The remaining 25 family members are divided among five groups: CDPK-related kinases (CRKs), PEP-Carboxylase kinases (PPCKs), PPCK-Related kinases (PEPRKs), SnRK1s, and SnRK2s. While these latter kinases are not regulated by calcium, they may overlap in substrate specificity with the calcium-regulated kinases. Current evidence indicates that family members play roles in the regulation of metabolism and gene expression in response to environmental and developmental cues. In a collaborative Arabidopsis 2010 research project, we are examining the function of 64 members of the CDPK/SnRK family. To identify substrates and interacting proteins, yeast-two hybrid screens are being performed with substrate-trap constructs. Results from a screen with CPK4 have revealed several potential substrates, and screens with PPCKs 1 and 2 are in progress. The substrate specificity of family members is being determined by mapping the phosphorylation sites of Arabidopsis proteins phosphorylated by the kinases in vitro. We are also determining the subcellular location of GFP-tagged constructs of the kinases. Preliminary results indicate that various CPKs are located in the cytoplasm, plasma membrane, endoplasmic reticulum, or with peroxisomes. (Supported by NSF MCB 0114769 to ACH et al. and DBI-9975808 to J.C. Walker et al.)



Ora

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I

SpaceflightExperiment

Results


ABSTRACTS – ORAL SESSION I

[53] [51] EFFECTS OF A FIVE-MONTH STAY ON BOARD THE MIR SPACE STATION ON IMMUNOGLOBULIN PRODUCTION IN THE URODELE AMPHIBIAN PLEURODELES WALTL. J.-P. Frippiat and C. Dournon. Lab. of Experimental Biology and Immunology, Univ. Nancy 1, Bvd des Aiguillettes, B.P.239. F-54506 Vandoeuvre-lès-Nancy FRANCE.

IS ANIMAL AGE A FACTOR IN THE RESPONSE OF BONE TO SPACEFLIGHT? E.R. Morey-Holton1, L. P. Garetto2, S.B. Doty3, B.P. Halloran4, and R.T. Turner5. 1NASA Ames Research Center, Moffett Field, CA, 2Indiana University School of Dentistry, Indianapolis, IN, 3Hospital for Special Surgery, New York, NY, 4VAMC, San Francisco, CA, and 5Mayo Clinic, Rochester, MN.

During the Perseus mission, two adult P. waltl did stay 5 months on board the MIR space station. Ten days after their return on Earth, animals were sacrificed and their spleen dissected. Total RNA was extracted from these tissues and used to analyze, by Northern blotting, the expression of IgM and IgY heavy chains. Our results indicated that the levels of IgM heavy chain transcripts in the spleen of animals which stayed in MIR are similar to those found in animals reared on Earth. However, the levels of IgY heavy chain transcripts were about three times higher than those found in ground control animals. We also analyzed the usage of the different VH families in the IgM heavy chain transcripts. Our experiments revealed important changes in the usage of the VH II and VH VI families. These families are used, respectively, in 28% and 60% of the IgM transcripts

The rodent bone response to spaceflight may be influenced by a multi- tude of factors including flight duration, strain, and housing. Review of bone formation rates during spaceflight suggests that age may also play a role in the response. Weanling rats show fewer bone changes than older rats. To determine if the long bones of weanling rats were insensitive to weight-bearing, a hindlimb unloading experiment was conducted simultaneously with a 9d shuttle flight in 34d old group-housed male rats. All animals were injected with bone markers 7d and 1d before flight and euthanized at landing, 24hr, and 72hr following recovery. If no differences in body weight, bone length, or bone formation at the tibiofibular junction were noted at the different time points, data were combined for each group. No significant differences in body weight were found at any time period among the groups. The humerus, tibia, and femur elongated significantly during the flight period with no difference in lengths between groups at the end of the flight period. The group-housed flight rats showed no change in cortical bone formation rate compared to preflight values, flight controls, or vivarium controls. However, the hindlimb unloading group showed a significant 30% decrease in bone formation rate compared to all other groups. Individually-housed 38d old animals flown for 14d showed ~10% suppression of cortical growth. Older singly-housed flight animals appear to show equal or greater bone changes compared to hindlimb unloaded rats. We speculate that the mechanical threshold required for cross-sectional bone growth is reached in group-house weanling rats during spaceflight, perhaps through physical interactions, and that weanling animals are sensitive to loading. However, the threshold is not fully reached in either singly-housed flight or hindlimb unloaded weanling rats. We conclude that age, housing, flight duration,

of ground control animals, and in 60% and 25% of the IgM transcripts of animals which stayed 5 months on board MIR. In conclusion, a prolonged stay on board a space station could affect the immunoglobulin production quantitatively and qualitatively. To confirm these first results, we will perform the same experiments on 30 P. waltl which stayed on board the International Space Station during the Andromede mission performed in October 2001. (Supported by CNES: DAR4800000024 and 8626) and strain have important roles in rodent skeletal responses to spaceflight. [54] [52] SPACEFLIGHT AND IMMUNITY IN THE MOUSE: PART I. M.J. Pecaut1, G.A. Nelson1, L.L. Peters5, P.J. Kostenuik6, T.A. Bateman3,4, S. Moroney6, L.S. Stodiek3, D.L. Lacey6, S.J. Simske3, and D.S. Gridley1,2. Depts of 1Radiation Medicine and 2Biochemistry & Microbiology, Loma Linda Univ. & Medical Center, Loma Linda, CA; 3Dept of Aerospace Engineering, BioServe Space Technologies, Univ of Colorado, Boulder, CO; 4Biomedical Engineering Program, Mech Engineering Dept, Colorado State Univ, Fort Collins, CO; 5The Jackson Laboratory, Bar Harbor, ME; & 6Amgen Inc., Thousand Oaks, CA.

SPACEFLIGHT-INDUCED CHANGES IN MOUSE BONE AND OSTEOPROTEGERIN THERAPY. T.A. Bateman1, S. Morony2, V.L. Ferguson1, S.J. Simske1, D.L. Lacey2, K.S. Warmington2, C.R. Dunstan2, L.S. Stodieck1 and P.J. Kostenuik2. 1BioServe Space Technologies, University of Colorado, Boulder. 2Amgen Inc., Thousand Oaks, CA. This experiment characterized the effects of spaceflight (SF) on the skeleton of mice treated with or without OPG, a protein that blocks bone resorption by osteoclasts. 10-week-old female C57BL/6J mice (n=12/group) received a single injection (SC, 24 h pre-launch) of either OPG (20mg/kg) or vehicle (VEH) and spent 12-days in orbit on Space Shuttle flight STS-108. Mass and age-matched ground control (GC) mice had similar treatments on a 48-hour delay.

On December 5th, 2001, the Space Shuttle Endeavor (STS-108/UF-1) launched for a 12-day mission to examine the effects of spaceflight on physiology in the C57BL/6 mouse model. Mission-related psychological stress, low-dose/low-dose-rate radiation, and changes in inertial condition have all already been shown to influence immunity in humans and rat models. However, this is the first time the mouse model has been utilized to examine these systems. Within 3-5 hours of their return, the mice were euthanized and dissected. Immunological assays were performed on the spleen, blood, and bone marrow. Exposure to spaceflight did not significantly alter general circulating leukocyte percentages. However, splenic lymphocyte percentages increased while granulocyte percentages decreased. Flow cytometric analysis of splenic lymphocytes indicated there was a shift away from CD3+ T cells toward B220+ B cells. Decreases in splenic CD3+/CD4+ helper T cells resulted in an overall decrease in the CD4:CD8 ratio. In contrast, there were increases in bone marrow T cells, with decreases in B cells. Additionally, although the total blast population decreased, CD34+ cells increased. As this is the first time these analyses have been conducted in a mouse model after spaceflight, there is little to which the results can be directly compared. However, many of the results are similar to those of past flights with rat models and humans. Rather than isolated peripheral changes, the shift in bone marrow populations is more consistent with a shift in hematopoiesis. Because there also appear to be shifts in populations involved in cellular and humoral immune defense mechanisms, responses to bacterial and/or viral pathogens may be less than optimal.

Upon landing, SF/VEH femur dry mass was lower compared to GC/VEH mice. OPG significantly increased femoral dry mass in both SF and GC conditions. Femur whole bone mineral mass (Min-M) and percent mineral composition (%Min) were lower in SF/VEH mice compared to GC/VEH. SF/OPG had a greater Min-M and %Min than SF/VEH. SF reduced elastic strength at the femoral midshaft in VEH mice, while OPG increased elastic strength in SF mice. pQCT analysis of the lumbar vertebra (L5) and proximal tibia revealed a deficit in BMD of SF/VEH mice compared to GC/VEH, indicating a systemic decline in bone mass. OPG blocked these SF-induced BMD changes. Serum and mRNA (humeral diaphysis) analyses suggest changes in both bone formation and resorption contributed to the SF-induced osteopenia. A significant decline in mRNA expression of osteocalcin combined with a decline in serum alkaline phosphotase levels indicate a reduction in bone formation. Additionally, SF reduced periosteal and endocortical bone formation rates in the femoral diaphysis. Increased bone resorption in SF mice was suggested by a trend towards increased mRNA expression of the pro-resorptive cytokine RANK ligand and significantly elevated serum TRAP levels. OPG treatment reduced osteoclast surfaces in the proximal tibia by >95% in all groups. . This experiment demonstrates that the mouse is an appropriate model for SF-induced ostepenia, and that OPG is an effective countermeasure. (Supported by Amgen Inc. and NASA CA# NCC8-242) (Supported by NASA: Coop. Agreement NCC9-149)



[55] [57] GENE EXPRESSION PROFILING OF ARABIDOPSIS THALIANA GROWN ON THE INTERNATIONAL SPACE STATION. Link B, Zhou W, Stankovic B. University of Wisconsin-Madison, Wisconsin Center for Space Automation and Robotics

VENTRAL ROOT RECORDINGS DURING FICTIVE SWIMMING IN TADPOLES (XENOPUS LAEVIS) WITH MICROGRAVITY OR HYPERGRAVITY EXPERIENCE. S. Böser1, C. Dournon2, L. Gualandris-Parisot3, and E. Horn1. 1Gravitational Physiology, Univ of Ulm, Ulm, Germany; 2EA 3442 Genetic, Signaling, Differentiation, Univ Henri Poincaré, Vandoeuvre-lès-Nancy cedex, France; 3Centre de Biologie du Développement, Univ Paul-Sabatier, Toulouse cedex, France.

Many early space experiments with plants were conducted in sealed containers to prevent the experiment’s contents from leaking into the crew cabin damaging the crews, or the orbiters’ sensitive electronic systems. These studies suffered from poor, or no gas exchange, uncontrolled moisture, humidity and temperature levels. The International Space Station (ISS) is opening a door for real science to be done. Instead

Tadpole swimming, a stereotyped rhythmical activity, is a model to examine effects of altered gravity on the motor system since the movement simplicity makes it easy to detect basic changes. High speed camera recordings have shown that tadpoles (Xenopus laevis) raised of short shuttle missions, plants can be grown from seed to seed in space. in microgravity exhibited a lower tailbeat frequency than 1g-controls (Fejtek et al. 1998, J Exp Biol 201:1917-26). Swimming pattern is generated by a central oscillator. It is possible to observe the rhythmical, burstlike activity of motoneurons by extracellular recordings from ventral roots (VR) of the spinal cord in paralysed animals (fictive swimming). Typical for the swimming pattern is the rostrocaudal delay of activity in ipsilateral myotomes of different segments. - Experiments were performed with Xenopus tadpoles exposed either to 10- to 11-day 3g-hypergravity on ground or to 10-day microgravity during the Andromède mission to ISS

The Wisconsin Center for Space Automation and Robotics (WCSAR) designs, builds, and flies plant growth chambers on the ISS. In partnership with Space Explorers Inc., we produced a crop of Arabidopsis thaliana on the ISS. The plants were grown in the Advanced Astroculture (ADVASC) hardware, which controls temperature, humidity, minimum CO2 level, light levels, and has an adjustable soil moisture level. In the second flight of ADVASC on the ISS A. thaliana plants were collected at two different times by the astronauts and placed in RNAlater (Ambion Inc.). RNA was successfully isolated and used as probes for the Affymetrix A. thaliana gene chips. We will show the ten genes having the highest statistical change in expression level as well as changes for known gravitational mutants. In addition we will show that microgravity alters floral stem branch angles.

in October 2001. At onset of altered gravity, they were younger than stage 28. VR-recordings started 4 hrs after the end of 3g- and 48 hrs after the end of µg-exposure. Fictive swimming was induced by a mechanical stimulus. We determined burst duration, rostrocaudal delay, episode duration and cycle length of VR-activity. – Results: The 3g-exposure increased significantly the VR burst duration. After µg-exposure,

the duration of episodes of fictive swimming was increased and the rostrocaudal delay was decreased compared to ground-reared 1g-controls; in addition, the burst duration of fictive swimming was slightly decreased. Readaptation of VR-acitivity was completed within 8 days for both

3g- and µg-animals. – Conclusions: (1) There are evidences that 3g- or µg-induced changes in fictive swimming have a vestibular origin. (2) The µg-induced reduction of macular activity may affect the development of descending reticulospinal and raphespinal projections to the spinal cord

in a reversible manner. (Supported by DLR, grant 50WB0140 to Horn) [56] [58] THE STATIC VESTIBULOOCULAR REFLEX AND ITS RELATION TO FICTIVE SWIMMING IN TADPOLES (XENOPUS LAEVIS) AFTER MICROGRAVITY EXPOSURE. E. Horn1, L. Gualandris-Parisot2, C. Dournon3, and S. Böser1. 1Gravitational Physiology, Univ of Ulm, Ulm, Germany; 2Centre de Biologie du Développement, Univ Paul-Sabatier, Toulouse cedex, France; 3EA 3442 Genetic, Signaling, Differentiation, Univ Henri Poincaré, Vandoeuvre-lès-Nancy cedex, France.

MICROGRAVITY EFFECTS ON WHEAT GROWTH AND PHOTOSYNTHESIS: PRELIMINARY RESULTS FROM PESTO EXPERIMENT. G.W. Stutte, G.M. Monje, G.C. Goins, and D.C. Chapman. Dynamac Corporation, Mail Code DYN-3, Kennedy Space Center, FL. The Photosynthesis Experiment Subsystem Testing and Operations (PESTO) experiment was conducted onboard the International Space Station (ISS) in the Biomass Production Chamber (BPS) during for Sensory deprivation during early periods of life affects the development

of sensory systems and causes long-lasting morphological and/or physiological modifications. Experiments with the amphibian Xenopus laevis which flew on the German D-2 mission (1993) and the Shuttle-to-Mir mission SMM-06 (1997) revealed that µg-exposure affects the development of the roll-induced vestibuloocular reflex (rVOR). During the recent Soyuz taxi flight Andromède to ISS (October 2001), we extended these studies (1) by comparing embryos (stage 25-28) which had not yet developed their rVOR at launch with those (stage 45) which had developed it, and (2) by correlating the extent of rVOR modifications in the young group with modifications within their rhythmic activity patterns of spinal ventral roots (VR). - Results: (1) In young Xenopus tadpoles, µg induced a depression of the rVOR, in particular in animals which developed upward bended tails during the space flight; in older ones, the rVOR was depressed in tadpoles with upward-bended tails while it was augmented in tadpoles with a straight body shape. (2) Significant correlations between the extent of the rVOR and VR activity existed either only in µg-exposed animals (burst duration and rostrocaudal delay) or only in 1g-tadpoles (episode duration). In particular, the VOR-amplitude was significantly correlated with the VR-parameters burst duration, episode duration and rostrocaudal delay. In the µg-group, there was also a significant correlation between cycle length and rVOR gain during the back-down posture. - Conclusions: (1) Microgravity sensitizes or desensitizes the otolithic vestibular system to static roll-stimulation depending on pre-flight rVOR-experience; (2) the development of the rVOR network and the spinal motor system reveals features which might be based on an intrinsic overall-relation or on a dependency of these subsystems mediated by descending pathways from the brainstem to the spinal cord. (Supported by DLR, grant 50WB0140)

73 days during Increment IV. Nine plant growth cycles (18 to 23 days in duration) were completed during this experiment. Excellent germination (>97%) was obtained on each of six on orbit plantings. Growth rates of germinated wheat of microgravity and ground plants were similar on each of the planting. A total of over 300 plants were frozen on orbit for dry mass and chemical characterization. Closed system and semi-closed system measurements of canopy level carbon exchange rates were obtained daily, and detailed characterization of CO2 and light response curves were obtained at three stages of development for each cycle. Preliminary analysis indicates that canopy level CER were similar between plants grown on ISS and on Earth under comparable environmental conditions. (This research was supported by grant from NASA Fundamental Biology Progroms (NCC10-0027)).



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Cell Biology


ABSTRACTS – ORAL SESSSION II

[59] [61] EFFECT OF SPACEFLIGHT ON EXPRESSION OF GENES REGULATING CELL GROWTH, CELL CYCLE, SIGNAL TRANSDUCTION, AND TUMOR SUPPRESSION IN T LYMPHOCYTES. M.L. Lewis1, L.A. Cubano2, and M. Hughes-Fulford3. 1Department of Biological Sciences, University of Alabama, Huntsville, 2Dept of Nephrology, Tulane University, 3Laboratory for Cell Research, VAMC, San Francisco, CA.

WEIGHTLESSNESS-INDUCED ALTERATIONS IN THE GLIAL CELL MACHINERY. B.M. Uva1, M.A. Masini1, M. Sturla1, G. Tagliafierro 1 and F. Strollo2. 1Dipartimento di Biologia Sperimentale, Università di Genova, Italy, 2 “Unità Endocrinologica” INRCA & Università “La Sapienza” Roma, Italy. The Central Nervous System is made up of neurons and glia, the glia being by far the most abundant. In their response to injury, glial cells act as a vast network; disturbances at any one point of the net are rapidly reflected throughout the entire system. As glia is essential for the correct functioning of neurons, any damages to the glia impair Central Nervous System activities. Our previous researches on cultured glial cells showed that simulated microgravity alters the cytoskeleton and causes programmed cell death of glial cells; the phenomenon is transient and, after 32h in simulated microgravity, new glial cells are born and normal cycling is restored. The integrity of intracellular machinery is essential to macromolecular transfer and ion transport; moreover, the integrity of inner and outer cell membranes is needed for ion channels to function properly and thus to go on playing their crucial roles in prevention of brain oedema. In fact, in the absence of any suitable compensatory mechanisms, brain oedema would be the obvious consequence of the typical cephalad blood shift experienced by humans during spaceflight. Aim of the present work was to investigate upon the integrity of intracellular organelles and of the inner and outer cell membranes in glial cells submitted to simulated microgravity using a Fokker Random Positioning Machine. The expression of the sodium pump and ion cotransport proteins was also studied. The results indicate that as early as 15 min in simulated microgravity alterations occur in cell cycling, in intracellular organelles and in the expression of the enzymes correlated to ion transport. The present investigation was carried out by immunohistochemistry using antibodies to Na+/K+ATPase, to the inner mitochondrial membrane and to Na+/K+/Cl- cotransport proteins. Transmission Electron Microscopy was used to investigate upon the integrity of various microstructure elements.

We demonstrated on STS-76, 80 and 95 that leukemic T lymphocytes (Jurkat) do not grow in microgravity. Conversely, ground controls doubled and after an initial loss of 30%, cells subjected to simulated shuttle launch vibration doubled between 24 and 48 hours. Thus growth arrest in microgravity is not a direct result of shuttle launch vibration. We also reported (Lewis and Cubano, Gravitational and Space Biology Bulletin, 2001 and Lewis et al., The FASEB Jour., 2001) that microgravity per se or other orbit-related factors alter cytoskeletal gene expression. We now describe spaceflight effects on expression of genes that regulate growth, cell cycle, signal transduction and tumor suppression. Gene expression in Jurkat cells flown on STS-95 and ground controls was evaluated by cDNA microarray at 24 and 48 hours (4,324 and >20,000 genes respectively). We found marked differences in gene expression for tuberin, a tumor suppressor effecting arrest at G1/S; Cyclin-dependent kinase 6 (critical for G1/S progression); Prohibitin, an inhibitor of G1/S cycle traverse; an apoptosis-associated cleavage stimulating factor involved in cycle arrest at G0/G1; Endoglin, a cell surface antigen in the TGF-beta family; a TGF-beta superfamily protein that induces cytoskeletal reorganization and signaling via receptor complexes associated with serine and threonine kinases; and genes involved in dephosphorylation of inositol diphospate (IP2). Other signal transduction and transcription factor genes were also expressed differently in flown cells compared to ground. In conclusion, these data provide information, at the gene expression level, that is critical to the understanding of growth arrest in spaceflown lymphocytes. (Supported in part by NASA: NAG2-985 and NAGW-2-1286) [60] [62] CHARACTERISTICS OF TUMOR-DERIVED CELLS IN 3-D ANALOGUE MICROGRAVITY ENVIRONMENT. V. Chopra1, T. Reed1, T. V. Dinh1, T. G. Wood2, N. R. Pellis3, and E. V. Hannigan1. Department of Ob-Gyn1, and Molecular Science Center2, UTMB, Galveston, TX and Cellular Biotechnology, NASA, JSC3, Houston, TX.

DENSITY DEPENDENT MODULATION OF DIFFERENTIATION IN HUMAN RETINAL PROGENITORS. Kamla Dutt, T. Lindsay, R. Kumar, I. Ezeonu, Department of Pathology, Morehouse School of Medicine, Atlanta, GA Purpose: To examine the role of cell density in cell commitment and

Co-cultures of three-dimensional (3-D) constructs of one cell type with dispersed cells of a second cell type in low-shear rotating suspension cultures in analogue microgravity environment have been used to investigate invasive properties of normal and malignant cells. Immunohistochemical staining procedures of cocultured harvests of

differentiation in retinal progenitors. Specifically to investigate the response to exogenously added growth factors and RPE secreted growth factors in progenitors. Methods: Non-transformed human retinal progenitors (Ret 1-4, 208) plated at the density of 1x104 cell/cm2 (low), 3x104 cells/cm2 (medium) and 1 x 105/cells/cm2 (high) in serum free

tumor-derived cells from gynecological cancer patients along with fibroblasts and endothelial cells demonstrate various markers of interest.

medium were either exposed to bFGF, TGFα, BDNF, CNTF (100 ng/ml) or retinal pigment epithelium (RPE) conditioned medium (100%, 50%, 25% 12.5%). Emerging phenotypes were scored by morphological analysis, immunophenotyping (for opsin, D4, D2 D3 receptors, tyrosine hydroxylase and Protein Kinase C). RT-PCR and western analyses were performed for identification of secreted products. Results: At low density 90% of precursors differentiated into photoreceptors in response to bFGF, TGFα in serum free conditions. At medium and high cell densities 30% and 25% of the cells, respectively, acquired photoreceptor phenotype.

Human umbilical vein-derived endothelial cells (HUVEC) were used to study the mitogenic response of the conditioned medium collected from 3-D monocultures and cocultures using proliferation and migration assays. The production of interleukin-2, -6, and -8, vascular endothelial cell growth factor (VEGF), and basic fibroblast growth factor were studied by using ELISA and RT-PCR. The conditioned medium collected from 3-D cocultures showed increased expression of 1) the message level of VEGF and its receptor flt-1 and KDR and 2) intracellular and vascular In contrast, BDNF and CNTF enhanced both multipolar (neuronal) and

photoreceptor cell differentiation in low-density cultures. Conditioned medium from RPE at 50% concentration induced photoreceptor phenotype in low-density cultures on day 2 and 3 in 67% and 87% of cells, respectively, whereas 100% conditioned medium induced multipolar neuronal differentiation in 84% and 87% of the cells on day 2 and 3, respectively. Brdu labeling confirms that cell commitment occurs in the latter part of the cell cycle. Conclusions: Cell density serves as a mechanism by which cells sense their external environment and control their response to growth factors. bFGF and TGFα predominantly induce photoreceptor phenotype in low-density culture. On the other hand, BDNF and CNTF promote both multipolar ganglionic and photoreceptor phenotype. RPE cells secrete multiple factors, with varying effects on cell commitment at different concentrations and densities.

cell adhesion molecules, when measured by using Live cell ELISA assays and immunofluorescence staining as compared with 3-D monocultures. There was an increase in production of 1) enzymatic activity that could generate bioactive angiostatin from purified human plasminogen, and 2) fibrin, mucin, and elastic fiber by cell aggregates of 3-D cocultures of patient-derived cells as compared with 3-D monocultures We have shown that the epithelial and endothelial cells undergo a switch in characteristics when grown in an in vitro 3-D environment, that mimics the in vivo host environment as compared with conventional two-dimensional cultures. This coculture provides new insights into the invasive process and its effects on both invading and invaded cells and can be used to study the effectiveness of various antiangiogenic agents. This project was funded by NASA, contract # NCC8-170. (Supported by NASA growth NCC-9-112 (KD) NAG 9-96-4-(KD) and

NAG 9-1169 (KD).


ABSTRACTS – ORAL SESSION II

[63] [65] RESISTANCE TO PSEUDOMONAS AERUGINOSA IS DECREASED IN HINDLIMB UNLOADED MICE.

INTEGRIN-MEDIATED BONE CELL RESPONSE TO HYPERGRAVITY W. Vercoutere1, C. Roden1, M. Parra1, C. Damsky2, E. Holton1, N, Searby1, R. Globus1,2, E. Almeida1,2 1NASA Ames Research Center, and 2 University of California, San Francisco.

H.O. Aviles, T. Belay, K. Fountain, M. Vance, and G. Sonnenfeld. Dept. of Microbiology, Biochemistry, & Immunology, Morehouse School of Medicine, Atlanta, GA. When vertebrates are placed in altered gravity environments they show

physiological responses, including loss or gain of bone and muscle tissue. The underlying cellular mechanisms, however, are still poorly understood. Existing studies of cellular mechanical stimulation by other forces such as flow and substrate deformation suggest that integrin adhesion to extra-cellular matrix (ECM) may play a key role in mechano-transduction. Bone and muscle cells may also respond to gravity via ECM/integrin-initiated signaling pathways. Using cell culture centrifugation as a model for hypergravity we found that primary rat osteoblast and fibroblast adhesion to fibronectin at 50-g promotes increased integrin focal adhesion number, focal adhesion kinase phosphorylation, cell survival and proliferation.

In humans and laboratory animals, immune function is modulated during and after exposure to space flight conditions. However, it is not clear whether space flight induced immunomodulation results in altered resistance to infection. Ground-based models have proven to be very useful for study of this problem. The hindlimb unloading rodent has been extensively used for creating conditions similar to those that occur during space flight. The objective of this study was to asses the effect of hindlimb unloading on resistance to Pseudomonas aeruginosa. P. aeruginosa, an increasingly prevalent opportunistic human pathogen, is one of the most common gram-negative bacterium found in nosocomial infections and has great potential to colonize immunocompromised hosts. It has also been isolated from infections in astronauts. Hindlimb unloaded and control (restrained and normally housed) mice were subcutaneously infected with a dose of P. aeruginosa lethal for 50% (LD50) of normally housed mice. Survival was significantly decreased in the hindlimb unloaded group compared to controls (p=0.009). The mean day of death was also decreased in the hindlimb unloaded group (4.7±0.8) compared to the control group (8.9±0.9) (p<0.01). The results of this study suggest that hindlimb unloading enhanced dissemination leading to decreased survival. The mechanism of induction of altered resistance are not yet completely defined. However, the production of critical cytokines involved in the control of bacterial growth, such as tumor necrosis factor-�may be impaired. The National Aeronautics and Space Administration supported this study under NASA cooperative agreement NCC 9-58 with the National Space Biomedical Research Institute.

To determine if the proliferative response of osteoblasts to hypergravity is mediated by particular integrins, we examined osteoblast growth in various ECMs. The NASA-developed cell culture centrifuge consists of a modi-fied 1-ft-diameter vented Eppendorf 5804 centrifuge inside an environ-mental cell culture chamber with heating, refrigeration, humidity and CO2 controls. We hypothesized that specific ECMs but not uncoated tissue-culture plastic would stimulate gravity-induced hyperproliferation. If true, hyper-proliferation response is likely to be mediated by specific integrins and their signaling complexes. Conversely, if cells subjected to hyper-gravity all exhibit the same increase in proliferation on various ECMs and plastic, then the process is unlikely to be mediated by specific integrins. In our current experiments, proliferation of primary osteoblasts grown on fibronectin, laminin, collagen Type I, and collagen Type IV was enhanced by continuous 24 hour 50-g hypergravity stimulus as compared with the 1-g control. No such increase was observed for cells grown on uncoated surfaces. Furthermore the proliferative response was greatest for cells on collagen Type I (1.4 fold increase), suggesting that αβ, α2β1, α3β and ανβ integrins may be significantly involved. These results suggest that specific ECM-integrin signaling in hypergravity conditions upregulate cell survival and proliferation pathways. (Supported by NASA: 00-OBPR-01-066).

[66] [64] BACTERIAL BIOFILMS UNDER MICROGRAVITY CONDITIONS. P.W. Baker and L. Leff. Dept. of Biological Sciences, Kent State Univ., Kent, OH 44242.

CHARACTERIZATION OF THE PARALLEL PLATE FLOW CHAMBER FOR MECHANICAL STIMULATION OF BONE CELLS UNDER MICROGRAVITY. CELLULAR RESPONSE TO FLUID FLOW BY NO PRODUCTION IS FLUID SHEAR RATE DEPENDENT. van Loon, JJWA*,**, Bacabac, RG**; Dijks, SJ**; Mullender, M**; Smit, TH***; Klein-Nulend, J**. *Dutch Exp. Support Center (DESC), Vrije Universiteit (VU), Amsterdam, NL, **Dept. Oral Cell Biology, ACTA-VU, Amsterdam, NL. ***Dept. Clinical Physics & Informatics, VU Medical Center, Amsterdam, NL.

Bacteria and other microorganisms commonly form communities embedded in an extracellular polysaccharide matrix on surfaces (i.e., biofilms) exposed to water. The purpose of this study was to examine the formation of bacterial biofilms under simulated microgravity conditions using a modified rotary cell culture system (RCCS). The results obtained were compared to biofilms formed under normal gravity conditions. Three types of bacteria were used in the experiments, Xanthomonas maltophila, Sphingobacterium thalpophilium and Burkholderia picketti, that were originally isolated either from condensated water (hot or cold) or the SVO storage tank from the Mir space station. Prior to the experiments, the bacteria were grown under nutrient limited conditions and incubated under low nutrient conditions during the experiments. Biofilms were formed on round stainless steel disks held within the RCCS that were periodically removed and sampled. Planktonic cells were also sampled. Cell numbers were determined using several different methods that revealed cell number and physiological state. Methods of enumeration used included: colony forming units (CFU),

The catabolic effects of microgravity on the skeleton of astronauts might be explained as resulting from an exceptional form of disuse. The mechanical adaptation of bone is a cellular process. Loading-induced flow of interstitial fluid through the osteocyte lacuno-canalicular network is a likely signal for bone cell adaptive responses. It is possible that the mechanosensitivity of bone cells is modulated by microgravity. An in vitro model to test bone cell mechanosensitivity utilizes dynamic fluid flow between parallel plates to simulate in vivo fluidic shear stress in the mineralized skeleton. To test whether microgravity decreases bone cell mechanosensitivity, the in vitro fluid flow model needs to be downscaled. We describe the characteristics of parallel flow chambers associated size reduction for an upcoming spaceflight experiment (Biopack on Shuttle). the BacLight Live-Dead stain (Molecular Probes), DAPI and 16S rRNA

fluorescent in situ hybridization. Different methods of enumeration yielded different results as the number and activity of the cells changed. There were also differences among the three species examined. Initial results suggest that there were subtle differences between planktonic cells grown under microgravity conditions compared to gravity conditions. Under microgravity conditions, the bacteria appeared to have difficulty attaching to the metal disks used as the biofilm substrate as cell numbers were low; however, these disks were readily colonized under other experimental conditions. In conclusion, the bacteria studied have the ability to persist under low nutrient conditions and differed in their responses to starvation and gravitational conditions. This research was supported by a grant from NASA.

In this study we also address the nature by which fluid shear stress activates bone cells by comparing variations in fluid shear stress amplitude and frequency, using nitric oxide (NO) production as a parameter for bone cell activation. This study employed precise design conditions by which the parallel-plate flow chamber (PPFC) can be utilized for dynamic flow regimes for on ground as well as (future) in flight experiments. In the present study, we tested whether fluid shear stress with varying frequencies and amplitudes affects the nitric oxide (NO) production by MC3T3-E1 osteoblast-like cells.


ABSTRACTS – ORAL SESSSION II



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Animal Development and Gravity

Sensing


ABSTRACTS – POSTER SESSION III–C

[67] [69] THE ROLE OF THE NERVOUS SYSTEM IN MODULATING GRAVITY-DEPENDENT SWIMMING BEHAVIOUR IN THE LARVAE OF MYTILUS EDULIS. J. T. Plummer1, D. L. Jackson2

INTRACELLULAR SIGNAL-TRANSDUCTION PATHWAYS IN CULTURED SKELETAL MUSCLE CELLS IN RESPONSE TO HEAT AND MECHANICAL STRESSES DURING HYPERTROPHY. K. Goto1, T. Kobayashi1, K. Uehara1, M. Honda1, T. Sugiura2, S. Yamada3, T. Akema1, and T. Yoshioka1,4. 1Dept of Physiol, St. Marianna Univ Sch of Med, Kawasaki; 2Faculty of Edu, Yamaguchi Univ, Yamaguchi; 3Dept of Life Sci, Graduate Sch of Arts and Sci, Univ of Tokyo, Tokyo; 4Aomori Univ of Health and Welfare, Aomori, Japan

and R. P. Croll1,1 Dalhousie University and 2Department of Fisheries and Oceans, Halifax, N.S., Canada. Previous flight studies in which bivalve veliger larvae deviated from their normal helical swimming in a micro-gravity environment prompted our investigation into the possible role of the larval nervous system in controlling this behaviour. Immunocytochemical techniques coupled Mechanical stretching could induce muscular hypertrophy. It has been

reported that heat stress facilitates satellite cell proliferation and differentiation in chicks. However, the mechanism responsible for

with pharmacological studies have provided evidence for a direct control of swimming behaviour by the larval nervous system in in bivalve molluscs. Both serotonin- and catecholamine-containing axons innervate the velum, which serves as tthe primary swimming organ in the larvae.

the effects of heat stress on muscular hypertrophy remains unclear. To elucidate this issue, we investigated the extent to which heat and mechanical stresses induced the mitogen-activated protein kinase (MAPK) cascades, a series of signal-transduction pathways, in skeletal muscle cells. Cultured mouse skeletal muscle cells (C2C12) were plated on collagenized Silastic membranes and incubated at 41° for 60 minutes. Following the incubation, the cells were subjected to a continuous cycle of two-second stretching followed by four-second releasing for three days at 37°. The cellular protein concentrations in the homogenates of the cultured skeletal muscle cells increased under heat shock and/or mechanical stretching. Extracellular regulated kinase (ERK)1/2, p38MAPK, c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK) were activated by heat and/or mechanical stresses. Protein kinase B (PKB) α/Akt1 transiently decreased following heat stress. Our results suggest that mechanical and heat stresses may promote gene expression and differentiation via separate signal-transduction pathways. Heat stress may be a useful tool for a countermeasure for muscoskeletal deconditioning.

In addition, serotonin and its reuptake inhibitor, fluoxetine, appeared to have a cilio-excitatory effect on larvae causing them to swim at a greater velocity. Conversely, the serotonin antagonist, mianserin, had the opposite effect. The catecholamines, dopamine and norepinephrine, appeared to directly decrease ciliary activity. These results were confirmed by treatment with spiperone, an antagonist to catecholamines which increased swimming velocities. The cilio-inhibitory effect of catecholamines caused the animals to not only decrease their swimming speeds, but also to deviate from their normal helical upward swimming pattern. The compilation of these results suggest a specific role of the larval nervous system in mediating patterns in bivalve larval swimming, and are consistent with the hypothesis that the alterations observed in micro-gravity may possibly result from underlying changes in neuronal development. (Funding provided by the Canadian Space Agency). [68] [70] EFFECTS OF MICROGRAVITY ON OXIDATIVE AND ANTIOXIDANT ENZYMES IN MOUSE HINDLIMB MUSCLE. B. Girten1, R. Hoopes2, M. Steele2, S. Morony3, T.A. Bateman4. 1NASA Ames Research Center, Moffett Field, CA, 2Lockheed Martin, Moffett Field, CA, 3Amgen Pharmaceticals, CA, 4BioServe Space Technologies, University of Colorodo.

EFFECT OF AGE ON INACTIVITY IN RESPONSE TO INCREASED BODY WEIGHT INDUCED BY EXPOSURE TO HYPER-GRAVITY. C.E. Wade1, M.M. Moran1, P.A. Fuller2, T.M. Hobin-Higgins2, L.A. Baer1, T.P. Stein3 and C.A. Fuller2. 1Life Sciences Division, NASA Ames Research Center, Moffett Field, CA; 2Section of Neurobiology Physiology and Behavior, University of California, Davis, CA; and 3Department of Surgery, University of Medicine and Dentistry of NJ Stratford, NJ.

Gastrocnemius muscle of mice were analyzed in order to examine the effects of 12-days of microgravity on the oxidative enzyme citrate synthase (CS) and the antioxidant enzyme superoxide dismutase (SOD). The female C57BL/6J mice utilized for this study were part of the Commercial Biomedical Testing Module (CBTM) payload that flew aboard STS-108. Mice were housed in Animal Enclosure Modules (AEMs) provided by NASA Ames. The flight (FLT) group and the ground control (CON) group each had 12 mice per group. The AEMs that held the CON group operated on a 48-hour delay from the FLT group and were located inside the Orbital Environmental Simulator (OES) at Kennedy Space Center. The temperature, CO2 and relative humidity inside the OES was regulated based on downlinked information from the shuttle middeck. Student T tests were used to compare groups and a p < 0.05 was used to determine statistical significance. Results indicated that CS levels for the FLT group were significantly lower than the CON group while the SOD levels were significantly higher. The CS FLT mean was 19% lower and the SOD FLT mean was 17% higher than the respective CON group means. Although these findings are among the first muscle enzyme values reported for mice from a shuttle mission, these results are similar to some results previously reported for rats exposed to microgravity or hindlimb suspension. The changes seen during the CBTM payload are reflective

We have previously demonstrated a reduction in activity related to the increase in body weight induced by centrifugation. The decrease in activity allows energy balance to be sustained. This work was conducted in young growing animals. With age there is a reduction in activity and energy expenditure. We hypothesized that mature animals would not adjust their activity and thus would have to increase energy intake to sustain balance. Young (Y; 1.5 mo.) and Mature (M; 8 mo.) male rats were divided into control (C) and hyper-gravity (2 g; HG) exposure groups (n= 8/group) and studied over 14 days. Body mass was reduced in both ages in response to HG: Y-C- 310 ± 4; Y-HG- 277 ± 3; M-C- 500 ± 9; M-HG- 438 ± 8 g. Energy balance and activity measurements were made over days 8-14. Energy intake and expenditure were greater in Y animals. Energy balance was 56± 16.7 and 10± 5.1 for Y and M. There was no effect of HG. Activity was increased in the young rats compared to mature, 16 ± 0.5 vs. 7 ± 0.8 counts/min. In HG activity was reduced to 54 and 68% of control. The reduction in activity was inversely proportional to the increase in body weight (mass x g-level), 178% and 175%, to sustain energy balance. The reduction in activity was not effected by the age of the animals. (Supported by NASA Grants: 121-10-30, 121-10-40, 121-10-50.)

of the deconditioning that takes place with disuse of the hindlimbs and indicate that muscle enzyme changes induced by disuse deconditioning

are similar in both rodent species.



[71] CHANGES OF MOOD STATES DURING THE PERIOD OF SLEEP DEPRIVATION UP TO 48 HOURS. S.Zhang, X.Y. Wu, X.Y. Li and L.P. Han. Department of Aerospace Biodynamics, The Fourth Military Medical University, Xi’an 710032, P.R.China Introduction: Manned space flights have shown it is possible to sleep in microgravity. However, some sleep disturbances, i.e. sleep deprivation, sleep reversal, naps, and circadian rhythm alternation, have been reported which influence performance of the crew and safety of space flight. Moreover, psychosocial factors are also likely to play an increasingly important role in determining mission success. The main purpose of the research was to study the effects of sleep deprivation up to 48 h on human mood state. Method: Fourteen healthy male subjects (aged 19～21) underwent sleep deprivation up to 48 hours. During which their mood states were evaluated by self-assessment questionnaire, profile of mood state (POMS). The POMS consists of 65 adjective words with 5-point scale degrees of agreement, resulting in 6 types of mood states, i.e. T: tension-anxiety; D: depression-dejection; A: anger- hostility; V: vigor-activity; F: fatigue-inertia and C: confusion- bewilderment. Results: The positive mood state factor points (V) changed to a remarkably lower level at the 20 h of sleep deprivation (P<0.01) and declined to the 30% level of control at the end of sleep deprivation. The negative mood state factor points (T, F, C) increased significantly from the 24 h of sleep deprivation (P<0.05) and reached their highest level at the 48 h of sleep deprivation (P<0.01). Meanwhile, the points of A and D had a tendency to increase but not to a significant extent. Conclusions: Sleep deprivation up to 48 hours can disturb human mood state. This suggests that sleep deprivation may be detrimental psychologically for the individual as well as for the operational success and safety of the missions.

[72] LACK OF EFFECTS OF HYPERGRAVITY ON PREGNANCY MAINTENANCE AND CONNEXIN 26 AND 43 IN THE PREGNANT AND POST PARTUM UTERUS OF RATS. H.Burden1, J. Zary1, L. Baer2, and A. Ronca2. 1Dept. of Anatomy & Cell Biology, Brody School of Med., East Carolina Univ., Greenville, NC and 2Developmental Gravitational Biology Laboratory, NASA Ames Research Center, Moffett Field, CA. In a previous study using pregnant rats, we showed that space flight with its associated microgravity decreased connexin 43, the major gap junction protein in the myometrium, but did not affect connexin 26, localized primarily in epithelial cells of the endometrium (Burden et al., J. Reprod. Fertil. (1999) 116: 229-234.) Since the biological effects of altered gravity may vary across the continuum from microgravity to 1-g to hypergravity, the objective of the present study was to examine the effects of hypergravity on these uterine connexins in a parallel experimental design. Pregnant rats were subjected to three levels of hypergravity (1.5, 1.75 and 2.0-g) produced by centrifugation during gestation days 11-20. At day 20 of pregnancy, rats were removed from the centrifuge. Half the animals were euthanized for analysis and the other half was allowed to go to term and deliver. The rotational control and all hypergravity groups showed significantly less (p<0.05) weight gain during the gestation days 11-20 interval but the number of live fetuses was not different. Animals allowed to go to term had similar body mass by day 22 and delivered comparable numbers of pups on days 22 or 23. Myometrial connexin 43 was not altered by hypergravity at day 20 or three hours after delivery. Also, the degree of phosphorylation of connexin 43 was not different at day 20 or three hours after delivery in any of the treatment groups. Lastly, connexin 26, localized primarily in uterine epithelium, was similar in all groups. Thus, hypergravity, initiated after pregnancy is well established, and terminated prior to delivery, is compatible with pregnancy maintenance, and normal delivery. In addition, uterine gap junction proteins, which help mediate the delivery process, are not altered. (Supported by NASA grant 121-1040 and NASA-Ames Cooperative Agreement No. NCC 2-1165.)



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Cell Biology


ABSTRACTS – POSTER SESSION III–D

[73] HUMAN MESENCHYMAL STEM CELLS CULTURED IN A 3D-CLINOSTAT DIFFERENTIATE TO CHONDROCYTES AFTER TRANSPLANTATION. Louis Yuge1, Atsuko Okubo1, Takashi Miyashita1, Katsuko Kataoka2, and Masamoto Kanno3 (1Inst. of Health Sciences, 2Dept. of Anatomy, 3Dept. of Immunology: Graduate School of Biomedical Sciences, Hiroshima Univ.) A 3D-clinostat is a multi-directional gravity device for simulating microgravity (10-3G). We cultured human mesenchymal stem cells (hMSC) in Dulbecco`s MEM for 3 days in the 3D-clinostat (group CL) or in 1G environment (group C), and the cell pellet was obtained. The pellet of hMSC was 2-times lager in group CL than in group C. The pellet of both groups was further cultured in a chondrocyte differentiation medium for 2 weeks in 1G. Type II collagen production was increased in group C, while it was not expressed in group CL. Phosphorylation of MAPK/ERK was repressed in group CL, while total MAPK/ERK was not changed. Then we transplanted the pellets into the cartilage defect made in the intercondylar fossa of the mouse femur. After 2 weeks, necrosis of the transplanted cells from group C were often seen. When the transplants were from group CL, necrosis was scaresely seen, and the transplants formed hyaline cartilage. These results suggest that hMSC remain more undifferentiated in culture in a 3D-clinostat, which makes better differentiation after transplantation. (Supported by “Ground Research Announcement for Space Utilization” and Ministry of Education, Science, Sports, Culture and Technology of Japan) [74] GRAVITY VECTOR VARIATION AFFECTS CYTOSKELETAL ORGANIZATION OF ENDOTHELIAL CELLS. A. Higashibata1, M. Imamizo-Sato2 and N. Ishioka1. 1Space Utilization Research Center, Space Utilization System, National Space Development Agency of Japan, 2Space Development Division, Advanced Engineering Services Co., Ltd, Tsukuba, Ibaraki, JAPAN. The effect of microgravity environment on cellular events has been observed in past space experiments, but the gravity-sense mechanism of cells is unclear. The goals of our study are to define the signaling pathway of gravity-sense and to reveal the gravity sensor in cells. We have investigated morphologically the cytoskeletal changes of endothelial cells cultured under clinorotated condition. Under that condition, microtubule organizing centers were poorly defined, and the clusters of MAP2 (microtubule associate protein 2) were not observed. Furthermore, RhoA, small G protein, was widely located in clinorotated cells, but the organizing of actin stress fiber was not promoted compared with those of static cultured. These results indicated that gravity vector variation affects the cytoskeleton organizing in cells, especially signaling pathway that is related to Rho protein. The results of mRNA level analysis support those morphological data. The images of differential display showed that gene expression level of cytoskeletal proteins and of cell-adhesion proteins responded to gravity vector variation.

[75] EXPRESSIONS OF INTEGRIN SUBUNITS IN OSTEOBLASTS DURING WEIGHTLESSNESS SIMULATION USING CLINOSTAT. Z. Shu1, 2, B. Wang2, J.L. Nie1, Y.H. Li1. 1Lab of Space Cellular and Molecular Biology, ISME, Beijing, 100094 and 2Dept of Aerospace Medicine, FMMU, Xi’an, 710032, P.R.China. Space flight experiments and studies carried out in altered gravity environments have revealed that exposure to altered gravity conditions results in (mal) adaptation of cellular function. In the present study, we used a clinostat to generate a vector-averaged gravity to simulate weightlessness environment. We then observed the responses of rat calvarial osteoblasts subsequent to rotation at 30 revolutions per minute (rpm) for 72 h. We found that the protein expressions of three integrin subunits started to change from 24 h of rotation in clinostat but not in stationary cultures. The decreased percent changes of integrin α5 protein at 24, 48 and 72 h were 13.1 +/- 3.4%, 20.3 +/- 6.7% and 11.9 +/- 2.5%, respectively. The same tendency was saw in the expression of integrin αv protein as 7.4 +/- 4.1%, 18.2 +/- 5.3% and 25.2 +/- 7.5%, respectively. Moreover, the expressions of integrin β1 protein in different periods were also declined with the percent changes of 18.6 +/- 3.3%, 25.9 +/- 4.7% and 27.5 +/- 6.5%, respectively. We all know that most cell-matrix interactions are mainly mediated by receptors of the integrins family, heterodimeric molecules made of an extracellular domain connected through a transmembrane sequence to an intracyto- plasmic tail. Our results suggest that vector-averaged gravity causes alterations of signal transduction and integrin-mediated cell adhesion in osteoblasts by altering the protein expressions of several crucial integrin subunits. These alterations might contribute to the pathogenesis of osteoporotic bone loss as observed in actual space flights. [76] EFFECTS OF VERY HIGH CO2 ATMOSPHERES ON THE CYANOBACTERIAL PHOTOSYNTHETIC APPARATUS. D. J. Thomas, S. L. Sullivan and S. M. Zimmerman. Science Division, Lyon College, Batesville, Arkansas, USA. According to current theories of early biosphere evolution, cyanobacteria and similar organisms produced most of the oxygen found in Earth's atmosphere. Early photosynthetic organisms would have adapted to an atmosphere that was rich in CO2 and poor in O2. In many ways (except for the pressure), Earth's early atmosphere may have resembled Mars' current atmosphere. However, many modern cyanobacterial species do not grow under these conditions. We are currently investigating the tolerance of several cyanobacterial species to very high (>20%) concentrations of atmospheric CO2. Cultures of Synechococcus, Synechocystis, Plectonema boryanum and Anabaena were grown in liquid culture and bubbled with CO2-enriched air. Culture growth was monitored by measuring optical density. Photosystem II activity was monitored by measurements of variable chlorophyll fluorescence (FV/FM). Synechococcus, Plectonema, and Anabaena tolerated CO2 concentrations up to 100% when the CO2 content was gradually increased from ambient by 10-15% per day. However, Synechocystis did not tolerate high CO2. Additional experiments to differentiate between CO2, O2 and pH effects showed that strains that were sensitive to high CO2 were also sensitive to low initial pH (pH 5-6), indicating that the formation of carbonic acid is probably responsible for the inhibited growth in high CO2 environments. Research in progress to determine the effects of high CO2 environments on photosystem II will be presented. In addition to providing insight as to the adaptations necessary on the early Earth, this research has applications for Mars exploration (e.g. a martian exploratory base or greenhouse). Also, this research provides insight into the possibilities, however remote, of forward-contamination of Mars by robotic and human exploration, and the survival of such contaminants. This research was supported by grants from the Arkansas Space Grant Consortium.



Post

er S

essi

on II

I–E

Plant Growth

Development and Gravity Sensing


ABSTRACTS – POSTER III–E

[79] [77] MODIFICATION OF GRAVITROPIC RESPONSE AND GROWTH IN MAIZE ROOTS BY PHORBOL ESTERS. Timothy J. Mulkey Life Science Dept., Indiana State University, Terre Haute, IN 47809

METABOLIC NETWORKS IN MONOLIGNOL (RADICAL RADICAL) COUPLING IN ARABIDOPSIS. K.W. Kim, M.K. Kim, L.B. Davin and N.G. Lewis. Institute of Biological Chemistry, Washington State University, Pullman. Phorbol esters have been shown to be activators and inhibitors of the

second messenger signal transduction system in animal cells. These compounds exhibit possible interactions with auxin and ethylene during elongation and the motor response of gravitropism in maize roots. The tumor promoting agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and diacylglycerol (DAG) are known activators of Protein Kinase C (PKC) in a variety of animal models. Staurosporine (STA) and phorbol 12-myristate 12-acetate-4-O-methyl ether (mTPA) inhibit or block activation of PKC

The arabidopsis genome consists of at least sixteen isovariants of the monolignol radical radical coupling proteins, defined as dirigent and dirigent-like proteins, respectively (Latin: dirigere, to guide and/or align). One objective of the ongoing study is to define the physiological roles of each of the dirigent protein isovariants, including those of any related homologues awaiting discovery. Together, the 16 isovariants appear to constitute a comprehensive monolignol radical radical coupling network operative in different cells and tissue types during the life cycle of arabidopsis. Some of the genes are anticipated to encode proteins involved in lignan formation for plant defense, whereas others are presumed to harbor arrays of multiple dirigent (monolignol binding) sites considered

in a wide variety of systems. Treatment of primary roots of maize with PKC-activating agents results in a biphasic increase in elongation rates over the control by 100-200%; these treatments result in an increase in the degree of the gravitropic motor response and in the pattern of curvature. The increase in elongation rate induced by PKC activating agents is blocked by pretreatment or reversed by post-treatment with STA. Application of TPA (10-6 M) to primary roots inhibited by high auxin concentrations (10-6-10-9 M) results in a recovery (20-100%) from inhibition of elongation. STA (10 nM) inhibits promotion of elongation and slows the motor response in roots pretreated with AVG (aminoethoxyvinylglycine; 10-6M) and exposed to auxin (10-9-10-11 M); these treatments do not prevent curvature but alters the resulting pattern

to be involved in lignin biosynthesis. The 16 isovariants have 25-60% sequence identity to the Forsythia dirigent gene (GenBank AF210061). The experimental strategy employed thus far was designed to establish the spatial and temporal expression pattern of each of the 16 isovariants. This was achieved by constructing two sets of different expression plasmids consisting of 1) 16 equivalent dirigent promoter::gus plus::3′flanking sequence::T-nos and 2) 16 equivalent dirigent promoter::mgfp::3′flanking sequence::T-nos. Transformation of each fusion plasmid into arabidopsis was carried out using hygromycin for selection. Molecular, biochemical and histochemical analyses of gus and gfp expression reveal further the expression patterns of the different isovariants during arabidopsis growth and development, i.e., in accordance with the existence of a complex network controlling monolignol radical radical coupling.

of curvature. Electrophoretic and autoradiographic patterns of soluble and membrane proteins from roots indicates that TPA and IAA treatment results in similar alterations of protein profiles and protein phosphorylation. (Supported by NASA: NAG2-1513)

[78] [80] INCREASED GRAVISENSITIVITY OF ROOTS WITH A DISRUPTED CAP ACTIN CYTOSKELETON. E.B. Blancaflor, G.C. Hou, and D.R. Mohamalawari. Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma.

PHOSPHOINOSITIDE SIGNALING AND PLANT GRAVITROPISM. I.Y. Perera, C.Y. Hung, B. Stapperfenne and W. F. Boss, Dept of Botany, North Carolina State University, Raleigh, NC The signaling processes linking gravity sensing to the initiation of a differential growth response in plants are not well understood. Using the pulvinus of cereal grasses as a model system, we have shown previously that both rapid and long term increases in inositol 1,4,5-trisphosphate (InsP3) correlate positively with the gravitropic bending response of both oat and maize stems (Perera et al., 1999, Proc Nat Acad Sci 96:5838-5843; Perera et al., 2001, Plant Physiol 125:1499-1507). As an approach towards further delineating the role of InsP3 in plant gravitropism, we have generated Arabidopsis plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes InsP3. The transgenic plants have no obvious phenotype under normal growth conditions. Strong expression of the InsP 5-ptase transgene is detectable in all tissues of the transgenic plants and basal InsP3 levels are greatly reduced compared to wild type Arabidopsis. Significantly, the transgenic seedlings exhibit a reduced gravitropic response compared to wild type seedlings. Our results are consistent with the involvement of InsP3 signaling in the gravitropic response of plants. This work is funded by NASA (# NAG 2-1502 to IYP and WFB).

The actin cytoskeleton has been proposed to be a major player in the gravitropic response of plants. However, there are still major gaps in our understanding of the role of actin in this process. To further address this problem, we analyzed the effect of various cytoskeletal inhibitors on root gravitropism. Latrunculin B (LB), an actin-disrupting drug promoted root curvature in a variety of plant species tested. More significantly, the gravitropic sensitivity of maize and Medicago truncatula roots was enhanced after LB-treatment as evident from the comparison of presentation times in LB-treated versus untreated roots. The microtubule inhibitor, oryzalin, did not have any significant effect on gravitropic sensitivity or root curvature. Interestingly, roots treated with LB consistently showed hyper-gravitropic responses even with short induction times. LB-treated roots gravistimulated for 5-10 min followed by rotation on a clinostat displayed extensive bending responses and often times the direction of root growth in the clinostat was characterized by 180 degree loops. These extensive bending responses occurred despite the strong growth inhibitory effects of LB. Application of LB specifically to maize root caps resulted in localized disruption of cap actin and this was sufficient to induce the increased gravitropic sensitivity of roots. On the other hand, disruption of actin in the elongation zone with LB did not affect gravisensitivity. Application of napthyphthalamic acid (NPA) to LB-treated roots abolished these extreme curvature responses of roots. Work on labeling of actin filaments in the columella is in progress to determine if gravistimulation results in actin reorganization. Our results indicate that while an intact actin network in the cap is not necessary for root curvature to occur, the increased sensitivity and response of roots treated with LB are consistent with a role for actin in modulating the activity of signaling molecules originating from the cap during root gravitropism.

(Supported by NASA grant NAG 2-1518).



[81] [83] DIRECT EVIDENCE OF ASSOCIATION BETWEEN PROTON EFFLUX AND BENDING OF ARABIDOPSIS ROOT UNDER GRAVISTIMULATION. Y. Xu, E. Johannes and N.S. Allen. Department of Botany, North Carolina State University, NC.

IN VITRO GROWTH OF BRASSICA SILIQUES – A NOVEL MODEL FOR THE STUDY OF SEED DEVELOPMENT AND MATURATION IN SPACEFLIGHT CONDITIONS. A. Kuang1, J. Blasiak2, M. E. Musgrave2, 1Department of Biology, University of Texas – Pan American, Edinburg, TX; 2Biology Department, University Rapid changes in cytosolic pH within specific columella cells in the

Arabidopsis root tip have been demonstrated and these changes clearly play a role in early perception of changes in the gravity vector. Proton fluxes surrounding the distant elongation zone of gravistimulted Arabidopsis seedlings have been investigated using the Non-Invasive Ion Selective Probe. 3~4-day old sterilized seedlings were mounted in a 3D Measurement Chamber (3DMC), which allowed the seedlings to be moved in a 3D fashion and to be viewed in both top view (x/y axis) and side view (y/z axis) so that the relative position between the probe and the seedling can be precisely defined. The seedlings were mounted on the 3DMC in such a way that the main axis of their root was perpendicular to the ground level. Gravistimulation was achieved by turning the seedlings 90 degree. The roots started to bend after ca. 30 mins. Two proton probes were used simultaneously to measure the proton fluxes of both upper and lower sides of the bending region. The results indicate that proton effluxes occur on both sides of the root with a flux two times larger on the upper side. This implies that a more acidic environment is associated with the more active elongation areas the upper part of the root.

of Massachusetts, 221 Morrill Science Center, Amherst, MA 01003 Our previous study reported that cell ultrastructure and storage reserves in Brassica seeds had changed during maturation in spaceflight on Mir. Understanding how reserve deposition is affected during seed development in the spaceflight environment is our goal. However, study of seed development and maturation in microgravity is impeded by limitation of supply of materials caused by the small size of plant growth chambers currently available and limited access to long-duration spaceflight exposure. To solve this problem of limited study materials, we use a “tissue culture” method for seed development in vitro and collect developing seeds from siliques cultured on agar medium instead of siliques from growing plants. Siliques of Brassica are removed from growing plants 10-12 days post-pollination. After surface sterilization of the siliques with 10% Chlorox solution, their cut ends are inserted 3-4 mm into the sterilized MS agar medium containing basic nutrients. Time to silique ripening in culture was comparable to time to ripening on the plant. Seeds from those siliques grown in agar medium and from the plants were fixed for light and electron microscopy observation. The microscopy observation of cell structure and storage reserves shows no differences between seeds from siliques cultured on agar medium and from growing plants. Dry seeds collected from siliques grown on medium geminate normally and seedlings are vigorous. There is no difference between

(Supported by NASA grant NAGW 4984.) the germination rate of these seeds and that of the seeds harvested from

growing plants. The tissue culture method could provide plenty of seeds at late developmental stages for both microscopy observation and

biochemical analysis, thus solving the problem of the limitation of material supply caused by the current lack of facilities needed to grow plants

full-term under spaceflight conditions. (Supported by NASA grant NAG2 – 1375.) [82] [84] OXYGEN DEPENDENCY OF FLAX SEED GERMINATION AND ROOT CURVATURE. O.A. Kuznetsov and K.H. Hasenstein, UL Lafayette, LA 70504-2451

GERMINATION OF LENTILS IN ALTERED MICROGRAVITY ENVIRONMENT. D. Woodard1, T. Reed2, D. Bulgher3, D. Richardson4, G. Vogt5, and V. Chopra2. 1NASA, MSFC, Huntsville, AL, 2Dept. Ob-Gyn, UTMB, Galveston, TX; 3NASA, JSC, Houston, TX; 4Mississippi State University, Starkville, MS; 5University of Colorado, Fort Collins, CO.

Respiration provides the energy requirements for the breakdown of storage compounds and germination. Therefore, oxygen content is a limiting factor for seed germination. In weightlessness the formation of Experiments were designed to study the effect of microgravity on the

germination of lentils in three different bioreactors. Two of the bioreactors are commercially available (Slow Turning Lateral Vessel and High Aspect Ratio Vessel). The third type, Classroom Bioreactor (CB) a simplified version of STLV, was designed by the authors to be used by the high school biology students. The CB was shown to favorably reproduce the experiments, and generated results as that of the NASA-designed bioreactors, thus enabling the students and teachers to perform experiments aseptically or under non-aseptic conditions. Our results indicated that the lentils germinated in all three bioreactors at a constant speed of 9-12 rpm over a period of 2-6 days in comparison with lentils germinated under Earth’s gravity conditions in regular tap water at room temperature. There was a variation in the length and weight of the lentils after sprouting them in the STLV, HARV and CB as compared with lentils sprouted under Earth’s gravity conditions in the control experiments.

a water layer around seeds and the lack of buoyancy-driven gas exchange further limits oxygen availability. In preparation for an upcoming shuttle experiment (MICRO on STS-107) we studied the oxygen dependency of germination and growth of flax (Linum usitatissimum L.) seedlings in the experimental hardware. We tested between 4 to 32 seeds glued to germination paper by 10 µl of 1% (w/v) gum guar in hermetically sealed experimental chambers (gas volume = 14 mL, O2 = 2.9 mL). For imbibition a previously optimized amount of distilled water was dispensed. The seedlings were allowed to grow for 36 h at 27C (previously determined optimal temperature) before their root length was measured. With 32 seeds per chamber, the germination rate decreased from 94 to 69%, and the root length was reduced by 20%. The inhibition was independent of ethylene. Elimination of the guar glue improved germination and growth. Experiments in controlled oxygen levels (3.6, 7.2, 9.2, 10.5, 13.9 and 21.6% O2) showed that oxygen concentrations above 10% are able to support germination. At 3.6% O2 no germination occurred but the seeds remained viable and 85% germinated within 24 h after transfer to 21% O2. Interestingly, within 3 h of gravistimulation the curvature of primary roots increased at lower O2 concentrations. At 9.2% O2 partial removal of the seed cover improved germination from 59%

A change in length of sprouts was also observed in lentils sprouted in the HARV as compared with those sprouted using the STLV, CB and Earth’s gravity conditions. The sprouting conditions were similar in the STLV and CB when the sprout lengths were measured as a function of time as compared with control lentils. There was also a change in the growth pattern of the plants cultivated from the lentils germinated in three different microgravity conditions (STLV, HARV and CB) as compared with growth patterns of lentils sprouted under Earth’s gravity conditions.

to 81% and root growth (from 3.6±0.4 to 7.9±1.0 mm), suggesting that at low O2 concentrations seed coat and glue impede oxygen diffusion to the embryo. However, the gas volume in the fligh hardware is sufficient to support normal seed germination and growth for the duration of the experiments.

This project is further being developed for a Teachers’ Guide for grades 9-12, in collaboration with NASA and NABT. (Supported by funding from NASA for Educational Brief).

(Supported by NASA grant NAG10-0190).




[85] MICROGRAVITY EFFECTS ON WHEAT GROWTH AND PHOTOSYNTHESIS: PRELIMINARY RESULTS FROM PESTO EXPERIMENT. G.W. Stutte, G.M. Monje, G.C. Goins, and D.C. Chapman. Dynamac Corporation, Mail Code DYN-3, Kennedy Space Center, FL. The Photosynthesis Experiment Subsystem Testing and Operations (PESTO) experiment was conducted onboard the International Space Station (ISS) in the Biomass Production Chamber (BPS) during for 73 days during Increment IV. Nine plant growth cycles (18 to 23 days in duration) were completed during this experiment. Excellent germination (>97%) was obtained on each of six on orbit plantings. Growth rates of germinated wheat of microgravity and ground plants were similar on each of the planting. A total of over 300 plants were frozen on orbit for dry mass and chemical characterization. Closed system and semi-closed system measurements of canopy level carbon exchange rates were obtained daily, and detailed characterization of CO2 and light response curves were obtained at three stages of development for each cycle. Preliminary analysis indicates that canopy level CER were similar between plants grown on ISS and on Earth under comparable environmental conditions. (This research was supported by grant from NASA Fundamental Biology Progroms (NCC10-0027)). [86] UNIQUE CHARACTERISTICS OF THE SIX SUPER DWARF CROP PLANTS UNDER STUDY AT UTAH STATE UNIVERSITY. Bruce Bugbee, Jonathan Frantz, Shryl Clawson, and Steve Klassen. Utah State University. Logan. Two decades ago, we began a wheat breeding program to develop a dwarf wheat line that was adapted to controlled environment conditions. This work led to the release of USU-Apogee in 1996, which was the first example of a genetic change for the spaceflight environment. However, at 50 cm tall, USU-Apogee is too tall for long-term studies on the ISS. We recently released USU-Perigee (25 cm tall), a super-dwarf successor to USU-Apogee. We have continued to search the world germplasm collection to identify extremely dwarf lines of the major crop species. To date, we have identified and characterized super-dwarf germplasm of rice (cv. Super-Dwarf), tomato (cv. Micro-Tina), pepper (cv. Triton), and soybeans (cv. Hoyt). Here we review important environmental responses for each of these unique crop species. Our studies include temperature and photoperiod effects on time to flowering and duration of the life cycle, productivity in extremely low light, intumescence susceptibility, and ethylene sensitivity.


Post

er S

essi

on II

I–F

Hardware andSpaceflightExperiment

Development


ABSTRACTS – POSTER SESSION III–F

[87] [89] MISSION OVERVIEW: BIOMASS PRODUCTION SYSTEM’S INAUGURAL FLIGHT TO THE INTERNATIONAL SPACE STATION. K.M. Stolp1, K.Y. Sato2, W.T. McLamb3, K.E. Lagel2, R.C.Morrow1. 1Orbital Technologies Corporation (ORBITEC), Madison, WI. 2Lockheed Martin, NASA-Ames Research Center, Moffett Field, CA. 3Bionetics Corporation, NASA-Kennedy Space Center, FL.

STS-107 EXPERIMENT BACTER: GROUND SIMULATION STUDY RESULTS. E. dL. Pulcini and B.H. Pyle. Dept of Microbiology, Montana State University, Bozeman. A proteomic analysis of differential protein expression in Pseudomonas aeruginosa ATCC 29260 under conditions of simulated microgravity was performed. Comparative control cultures of P. aeruginosa were grown under normal gravity (1 x G) conditions and maintained under identical conditions of temperature, oxygen, and nutrient availability. Simulated microgravity was accomplished using a clinostat system rotating at 80 rpm. The bacteria were grown in a medium to stimulate exotoxin A virulence factor production during growth. The analysis of variations in exotoxin A production was accomplished using ELISA, Western Blots

The Biomass Production System (BPS) is an environmental control unit that allows the user to independently control humidity, light levels, nutrient supply, CO2, and temperature in each of its four chambers. BPS flew to the International Space Station (ISS) aboard Atlantis on STS-110 (08 April 2002). The BPS remained aboard the ISS until it was returned to Earth 19 June 2002 via the Orbiter Endeavour, leaving the BPS in orbit for 73 days and allowing BPS to complete a hardware validation and experiments. BPS was occupied by two experiments, Photosynthesis Experiment Subsystem Testing and Operation (PESTO) and the Technology Verification Test (TVT). The project team consisted

and proteomic methodologies. In order to better identify the physiologic changes that take place in simulated microgravity, a comparison of protein expression patterns using P. aeruginosa signaling mutants with mutations in the las, rhl, and las/rhl systems was made. Results indicate variations in protein expression do occur in P. aeruginosa under conditions of simulated microgravity representing changes in metabolic and physiologic functions as well as changes in putative pathways for the production of virulence factors. These results will allow for a better understanding of the factors involved in crew health and safety during space flight.

of personnel spanning three research and support centers (ORBITEC; NASA-ARC; and NASA-KSC), combining knowledge and experience that pulled the team together during the mission. Mission support from these centers was based on nearly uninterrupted monitoring of BPS Health and Status via down-linked data to the Communication and Data System (CDS [NASA-ARC developed]). The CDS was integral in obtaining (Supported by NASA NAS2-14263 and Montana Space Grant Consortium

NASA EPSCoR) real-time data and project information directly from the hardware while it was on Station. Included in the information that was received was real-time health and status of the hardware, each chamber’s environmental conditions and photos of the plants in each chamber. The health and status reports allowed the team to make decisions based on real-time information, greatly enhancing science. The photos allowed the team to assess the health and growth stage of the plants and to determine their interaction with the hardware and micro-gravity, and were used in an outreach effort that was based at ORBITEC. Selected photos and data were placed on the ORBITEC website (orbitec.com) and were used by students to compare orbiting plants to their own plants.

[88] [90] INITIAL ASSESSMENT OF ON-ORBIT PLANT DEVELOPMENT USING THE BPS IMAGING SYSTEM. R.C. Morrow, K.M. Stolp, M.C. Lee. Orbital Technologies Corporation, Madison, WI 53717.

RODENT BIOCOMPATIBILITY TEST USING THE NASA FOODBAR AND EPOXY EP21LV. J. Tillman1, M. Steele1, P. Dumars1, M. Vasques2 and B. Girten2. 1Lockheed Martin Space Operations, NASA Ames Research Center, Moffett Field, CA; 2Code SLO, NASA Ames Research Center, Moffett Field, CA.

The Biomass Production System (BPS) was flown on the ISS for a total of 74 days as part of the Increment 4 mission. A ground control was conducted as part of this experiment using a second BPS unit. During this mission, a total of 11 sets of plants were grown, seven sets of dwarf wheat (Triticum aestivum cv. Apogee) and two sets of Brassica rapa cv. ASTROPLANTS.

Epoxy has been used successfully to affix NASA foodbars to the inner walls of the Animal Enclosure Module for past space flight experiments utilizing rodents. The epoxy used on past missions was discontinued, making it necessary to identify a new epoxy for use on the STS-108 and STS-107 missions. This experiment was designed to test the basic biocompatibility of epoxy EP21LV with male rats (Sprague Dawley) and mice (Swiss Webster) when applied to NASA foodbars. For each species, the test was conducted with a control group fed untreated foodbars and an experimental group fed foodbars applied with EP21LV. For each species, there were no group differences in animal health and no statistical differences (P<0.05) in body weights throughout the study. In mice, there was a 16% increase in heart weight in the epoxy group; this result was not found in rats. For both species, there were no statistical differences found in other organ weights measured. In rats, blood glucose levels were 15% higher and both total protein and globulin were 10% lower in the epoxy group. Statistical differences in these parameters were not found in mice. For both species, no statistical differences were found in other blood parameters tested. Food consumption was not different in rats but water consumption was significantly decreased 10 to 15% in the epoxy group. The difference in water consumption is likely due to an increased water content of the epoxy-treated foodbars. Finally, both species avoided consumption of the epoxy material. Based on the global analysis of the results, the few parameters found to be statistically different do not appear to be a physiologically relevant effect of the epoxy material. We conclude that the EP21LV epoxy is biocompatible with rodents.

Part of the standard complement of each of the four BPS chambers is a color CCD camera. These cameras were used with a digitizing circuit card to collect images every two hours. These images were downlinked daily and provided an initial evaluation of plant growth and development on-orbit. The images allowed evaluation of approximate plant height and general plant health, and tracking of developmental stages (e.g. germination, flowering, and seed pod development). A total of over 3000 images were collected during the mission, not including images taken separately by the crew. The BPS cameras were also used to collect full video of wheat and Brassica plants to evaluate movement imparted by chamber air circulation fans. Developmental stages for Brassica grown in µg, from germination through flowering, seed production and senescence appeared to track well with plants grown in at 1g, at least in terms of gross morphology. Wheat growth also appeared outwardly similar for plants grown in µg and 1g. Other interesting observations were made with the imaging system, including a reorientation of plants probably due to a vehicle reboost maneuver. Future improvements to the imaging system will include providing a deeper field of focus, improved resolution and color reproduction, and use of cameras with infrared and low light capabilities.

(Project support provided by Phase I & II SBIR grants through NASA KSC and a Phase III SBIR grant through NASA ARC.)



[91] DEVELOPMENT OF A HIGH-RESOLUTION REMOTE SENSING IMAGER SYSTEM FOR THE MONITORING OF GFP REPORTER GENES FOR SPACE BIOLOGY EXPERIMENTS. A.C. Schuerger1, A-L. Paul2, J.T. Richards1, and R.J. Ferl2. 1Dynamac Corp., Kennedy Space Center, FL and 2Hort. Sci. Dept., Univ. of Florida, Gainesville, FL. Green fluorescent protein (GFP) reporter genes have been proposed as non-destructive means of monitoring gene expression in plants grown in space biology experiments. In order to accurately monitor the GFP-tagged reporter genes a remote sensing imaging (RSI) system was developed based on off-the-shelf hardware and software components. The RSI system was composed of a high-resolution 16-bit astronomical CCD camera with a pixel array of 2184 (H) x 1472 (V) (CCD chip was a Kodak model KAF-3200). Individual pixels measured 6.8 x 6.8 µm and the CCD chip could be cooled to –13 C using an internal peltier cooler. The RSI system also was configured with a set of eight narrow bandpass filters centered at 440, 510, 550, 685, 700, 720, 740 and 760 nm (all filters with 10 nm FWHM) mounted in a PC-controlled filter wheel. A broad-spectrum xenon-arc lamp was used to generate a set of eight spectral reflectance images in the visible and near-infrared regions. A second light source centered at 470 nm (20 nm FWHM) was used to excite the GFP reporter genes (510 nm) and to excite chlorophyll fluorescence (685 and 740 nm) in plant leaves. All images were calibrated and processed using Maxim DL (Diffraction Ltd., Nepean, Ontario) or Mira Pro 6.0 (Axiom Res., Tucson, AZ). The RSI system could image features on leaves of transgenic Arabidopsis thaliana plants at the sub-trichome level. Green fluorescence of expressed GFP reporter genes was clearly observable in small groups of cells throughout the plant canopies. Spectral reflectance images were used to estimate leaf biomass, chlorophyll concentration in leaves, and overall health of plant canopies. The RSI system can be modularized or miniaturized for space biology experiments depending on the mission objectives and constraints. (Supported by NASA: AO-99-HEDS-01-032 and NAG10-291.) [92] CREW TRAINING FOR THE INTERNATIONAL SPACE STATION: EXPERIENCES WITH THE BIOMASS PRODUCTION SYSTEM (BPS) PAYLOAD. J.J. Stadler1 and W. T. McLamb2. 1Orbital Technologies Corporation, Madison, WI and 2The Bionetics Corporation, KSC. The recent flight of the Biomass Production System (BPS) to the International Space Station (ISS) can provide valuable insights to payload developers with regard to the process of training ISS crewmembers. There are many challenges when training a crewmember for an ISS payload. The complexity of the BPS hardware and the crew-intensive operations on orbit demanded significant time and resources during the pre-flight crew training flow. Changes to ISS mission manifests, the extreme length of missions, and crew proficiency issues must also be considered. ISS crew training flow differs in many respects from Space Shuttle crew training, but integration of these two processes was necessary when flying a payload to the International Space Station. The ISS crew training process provided many advantages to the payload. Interactions with crewmembers and crew representatives provided a valuable astronaut perspective to hardware designers, as well as recommendations that could be incorporated into BPS hardware design, procedures, and stowage. The importance of hands-on crew training should not be underestimated. Training sessions provided the ISS crewmembers with access to the hardware to be used on orbit and the subject matter experts to supply them with a sound understanding of the hardware and science objectives. This proved invaluable during the course of the mission in allowing the crewmember to respond as anomalies and unique opportunities arose.



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[93] [95] CHANGES IN MICROTUBULE DISTRIBUTION IN GRAVISTIMULATED CAULONEMAL TIP CELLS OF THE MOSS PHYSCOMITRELLA PATENS. P. Chattaraj, E. Johannes and N.S. Allen. Department of Botany, North Carolina State University, NC.

CALCIUM/CALMODULIN-MEDIATED GRAVITROPIC RESPONSE IN PLANTS. B.W. Poovaiah, 1 T. Yang1 and J.J.W.A. van Loon.2 1Washington State University, Pullman, WA, and 2DESC, OCB-ACTA-VU, Amsterdam, Netherlands.

Cytoskeletal elements play an important role in the early steps of gravity signaling [Kiss. (2000) Crit Rev Plant Sci 19: 551-573]. Whereas higher plants are likely to use actin filaments to sense changes in the gravity vector, microtubules appear to have a dominant role in mosses [Schwuchow et al. (1990) Protoplasma 159:60-69]. In this study, we use the moss Physcomitrella patens as a model system to investigate microtubules dynamics following gravistimulation. The tip-growing apical cells of dark-grown caulonemal filaments of P. patens respond to gravity with upward growth. Six-day-old filaments were embedded in low melting point agarose under dim green light and allowed to recover in darkness overnight. Filaments were then rotated 90° and microtubule distribution in the tip cells was monitored over time by indirect immunofluorescence microscopy. Microtubules accumulated on the lower flank of the tip cell 30 minutes after gravistimulation. This implies that microtubules are involved in gravity perception and early signal transduction in P. patens.

Calcium and calmodulin (CaM) play an important role in gravity signal transduction. However, the molecular and biochemical mechanisms involved in gravity signal transduction are not clearly understood. It is becoming clear that hydrogen peroxide and auxin play important roles in gravity signal transduction. Recent results indicate that Ca2+/CaM is involved in hydrogen peroxide homeostasis by regulating catalase activity in plants (Yang and Poovaiah, Proc. Natl. Acad. Sci. USA 99: 4097-4102, 2002). Results also indicate that an auxin-responsive gene family (SAURs) encodes for Ca2+/CaM-binding proteins (Yang and Poovaiah, J. Biol. Chem. 275: 3137-3143, 2000). In addition, this laboratory has identified and characterized a family of signal responsive genes that encode for CaM-binding proteins in Arabidopsis. To investigate the effects of gravity on the expression of these genes, as well as other genes involved in Ca2+/CaM-mediated signaling, Arabidopsis seedlings were subjected to simulated microgravity using the Random Positioning Machine, and hypergravity using the MidiCAR centrifuge. The changes in mRNA levels were studied. Selective and significant differences in gene expression were observed in simulated microgravity- and hypergravity-treated plants. The relevance of these genes in gravity signal perception and transduction will be discussed.

(Supported by NASA grant NAGW 4984.)

(This work was supported by NASA grant NAG5-4841 to Poovaiah and SRON/NIVR combined grant #MG-051 to van Loon.)

[94] [96] REGULATION OF AUXIN TRANSPORT BY FLAVONOIDS DURING GRAVITROPISM. Buer, C.S., Muday, G.K. Dept. of Biology, Wake Forest University, Winston Salem, NC.

DIFFERENTIAL SUPPRESSION OF TWO DIFFERENT ANNEXINS IN CERATOPTERIS USING RNA INTERFERENCE. S. J. Roux, G. B. Clark, S. C. Stout, and S. Archer-Evans. Sect. of Molecular Cell & Developmental Biology, Univ. of Texas, Austin. Plants respond to changes in the gravity vector by differential growth

across the gravity-stimulated organ. Auxin is normally basipetally transported but changes its direction in response to changing vectors of gravity or light, and this auxin redistribution has been suggested to drive the resulting differential growth. The mechanisms by which auxin transport directionality changes in response to a change in the gravity vector are largely unknown. Using Arabidopsis thaliana, we are exploring regulatory mechanisms that may control auxin transport. Transport may be regulated during gravity response by changes in concentration or localization of an endogenous auxin efflux inhibitor. Flavonoids, such

Annexins, which are calcium-dependent, membrane-binding proteins, have been implicated in a variety of cellular functions in plants and animals, including secretion and calcium channel activity. We have isolated and sequenced two different full-length cDNAs, AnnCr1 and AnnCr2, that are both expressed in germinating spores of the fern Ceratopteris. The proteins encoded by these two genes are 78% identical in sequence. To test the possible role of these genes in spore polarity development (which is directed by gravity), we suppressed their expression using RNA interference (RNAi). Gene-specific primers were used to amplify double-stranded (ds) annexin constructs of 200 base pairs from the 3’ regions of AnnCr1 and AnnCr2. Intact spores readily take up dsRNA during their hydration. As judged by sensitive RT-PCR assays, treatment of spores with annexin dsRNA significantly and selectively reduced the level of each annexin mRNA, but not other messages, indicating that the effects of the dsRNA treatments are relatively specific. The effectiveness of these constructs in suppressing the expression of annexin genes was concentration- and sequence-dependent. Sequences of dsRNA based on ORF regions were effective; sequences based on 3’ untranslated regions were not. Suppression was effective for more than 24 h. Resultant phenotypes from dsRNA treatments ranged from inhibition of germination to delayed and altered development, although initial results indicate these effects occur only when high concentrations of dsRNA are used, and the specificity of RNA suppression at these concentrations is not yet clear. Spores can also take up single-stranded antisense constructs, and these are also effective in selectively depressing annexin mRNA levels, but the sequence dependency of these effects is different from that required for dsRNA effectiveness, suggesting a possibly different mode of action.

as quercetin and kaempferol, have been implicated in regulating auxin transport in vivo and in vitro. Plants with the tt4 mutation make no flavonoids and have elevated auxin transport (Brown et al., 2001 Plant Phys. 126: 524-535). The tt4 mutant also has reduced root gravitropic bending, which is due to a lag in initiation of gravitropic curvature. This difference in the gravity response in tt4 plants is enhanced when plants are grown under conditions that induce flavonoid synthesis in wild-type plants. Furthermore, changes in auxin-induced gene expression and flavonoid accumulation have been observed during gravity stimulation. Current studies are examining whether there are spatial and temporal changes in flavonoid accumulation that correlate with the timing and position of gravitropic bending and whether the absence of these changes is the cause of the altered gravity response in plants with the tt4 mutation blocking flavonoid synthesis. These results support the idea that auxin transport may be regulated during gravity response by the synthesis of ligands that control the activity of auxin transport proteins. (This work is supported by NASA grant NAG2-1507.) (Supported by NAG2-1347 and NAG10-295 to S.J.R.)



[97] [99] MICROINJECTED MAGNETIC BEADS INDUCE CURVATURE IN CHARA RHIZOIDS. P. Scherp and K.H. Hasenstein, Biology Department, UL Lafayette, Lafayette, LA 70504-2451

HYPOXIC ROOT METABOLISM IS NOT INDUCED BY DISRUPTION OF GRAVITY SENSING IN ARABIDOPSIS THALIANA. D.M. Porterfield1,2, and J. Liao1. 1Dept. of Biological Sciences and 2Dept. of Electrical and Computer Engineering, Univ. In the rhizoid of the green algae Chara, sedimenting statoliths and the

actin network determine spatial information and control the response to reorientation with altered tip growth. To study the statolith behavior and influence of cytoskeleton, we injected 5 to 20 magnetic beads (~1 µm diameter) into the tip region of rhizoids after reducing turgor pressure by 0.16 M sorbitol. During ca. 30 min after delivery of the beads, the injection pipette (6 µm outer diameter) was slowly removed to allow closure of the puncture by deposition of wound callose. Immediately after injection, the beads could be manipulated with sub-mm sized magnets. After transfer of the injected rhizoids onto solidified Chara medium (1.5% agar), the rhizoids grew and responded to reorientation like non-injected controls. The injected beads initially aggregated with the endogenous statoliths and when displaced laterally by a magnet (10×10×4 mm), they induced transient curvature/bulging toward the magnet. The curvature induced by interaction between injected beads and the magnet was a function of the distance between magnet and rhizoid, and the amount of beads injected. Growth studies on sorbitol treated, injected and non-injected rhizoids showed that only the osmoticum but not injection itself reduced growth. The rhizoids fully recover and grow at the same rate as controls. The data indicate a non-linear and adaptive response of the rhizoids to the injection of beads. The relationship between the number of injected beads, the magnetic field strength and the induced curvature indicate that the endogenous statoliths and magnetic beads control the distribution of wall material and that the position but not the displacement of statoliths controls the growth angle.

of Missouri-Rolla. Previous spaceflight experiments that have monitored alcohol dehydrogenase (ADH) have shown that this hypoxically responsive gene is induced, and is associated with increased ADH enzyme activity in microgravity. The two hypotheses that have been proposed to explain these changes in gene expression and metabolic activity are: 1) ADH is induced by spaceflight hypoxia caused by the inhibition of gravity mediated O2 transport, and 2) the induction of ADH is a non-specific stress response due to inhibition of gravisensing. We tested these hypotheses in a series of clinostat experiments to determine if disruption of gravisensing can induce a non-specific ADH response. In these experiments we utilized transgenic Arabidopsis plants (ADH promoted GUS marker gene) and monitored gene induction and ADH activity in clinostat rotated (1 rpm), control, and waterlogged exposed plants. The plants were grown on agar slats in a growth chamber before being exposed to the experimental treatments. The plants were stained for GUS activity localization, and subjected to biochemical tests for ADH, and GUS enzyme activity. These tests showed that the waterlogging treatment induced significant increases in GUS and ADH enzyme activities, while the control and clinostat treatments showed no response. This work demonstrates that the perturbation of gravisensing by clinostat rotation does not induce a non-specific stress response involving ADH and supports the microgravity convection inhibition model for explaining changes in root metabolism during spaceflight. (Supported by funding from the Missouri Research Board, and the USDA/NRICGP (2001-35100-10751) to DMP.) (Supported by NASA grant NAG 2-1423.)

[98] [100]

ECONOMICS OF GROWING PLANTS IN SPACE. Alan Drysdale1

and Ray Wheeler2. 1 The Boeing Company. 2 NASA Kennedy Space Center

ANALYSIS OF THE PHASES OF MAIZE ROOT GRAVITROPISM USING ROTATO, A SEEDLING MANIPULATION DEVICE. H. Ishikawa1, E. Natori1, C. Wolverton2, and M.L. Evans1. Dept of Plant Biology, Ohio State University, Columbus and Dept of Botany-Microbiology, Ohio Wesleyan University, Delaware, OH.

Life support systems using bioregeneration offer the possibility of a substantial increase in autonomy over fully physico chemical systems using shipped food. However, the cost of growing plants in space is much higher than the cost of growing them on the Earth, where sunlight, water, air, and many minerals are generally free. The economics of growing plants in space was investigated. A rationale has been presented previously for comparing the cost of growing plants in space to the cost of shipping similar products. Cost estimates are developed here for a number of crop plants of interest to the Advanced Life Support program and cost factors are evaluated to identify what our options are for reducing the costs. A number of plants were found to be cost effective for Mars missions with current plant system designs, and the number of species did not increase substantially with longer missions. The mass of plant waste produced by these assumptions was calculated for a range of missions.

Maize roots show at least three phases in their gravitropic motor response, early rapid downward curvature (Phase I), a period of curvature reversal sometimes referred to as auto-straightening (Phase II) and a period of slower downward curvature (Phase III). When we used ROTATO, an automated seedling manipulation device, to maintain the tip of maize roots at a pre-determined angle during the entire response, the same three phases appeared, indicating that they are inherent to the motor response and not determined by the change in angle of stimulation as the response proceeds. We examined the dependence of induction of all three phases on the angle of stimulation. Phase I was induced at angles of a few degrees or less while the threshold for induction of Phase III was significantly higher (15 degrees). The rate of curvature for both Phase I and Phase III increased with increasing angle of stimulation. Phase II was also induced at very low stimulation angles, but in contrast to the other phases, the magnitude of Phase II declined at higher angles with the result that Phase II was not observable at angles of 70 degrees or higher. Under these high angles of stimulation, the roots progressed smoothly from Phase I to Phase III without passing through Phase II. It is possible that, at the higher angles, the magnitude of Phases I and III (positive curvature) are sufficiently high to mask the negative curvature of Phase II.

(Support was provided by NAG 9 1311.)

The slow steady curvature of Phase III is particularly striking in that it continues for many hours even though the root tip is held at a steady shallow angle. This indicates that there is steady signal output from the root apex to the curving region with little or no adaptation long after the initial dynamic events of reorientation. This has implications for the statolith model of gravisensing.

(Supported by NASA: NAG2-1411)



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ABSTRACTS – ORAL SESSION IV

[101] [103] THE GRAVITATIONAL CONTINUUM: A REVIEW. R.W. Phillips, Department of Biomedical Sciences, Colorado State University, Fort Collins.

LOSS OF SPERMATOGENESIS IN LONG TERM SIMULATED MICROGRAVITY: RAPID BLOODFLOW CHANGES IN THE RAT TESTIS DURING HINDLIMB SUSPENSION. JS Tash1, JD Pierce2, MJ Wulser1, and S McDonald1, 1Dept Molecular & Integrative Physiology, 2School of Nursing, Univ of Kansas Medical Center, Kansas City, KS.

Gravitational effects have been described during exposure and adaptation to both hypergravity (hG) and microgravity (uG) environments. It should not be surprising that opposite effects occur as organisms adapt to increases or decreases in the gravitational field. The musculoskeletal system tends to atrophy in uG and hypertrophy in hG. The same opposite effects can be seen in the cardiovascular and pulmonary systems. Heart rate, stroke volume and cardiac output decrease in uG and increase in hG. In the lungs vital capacity is increased in uG and decreased in hG. Hematocrit presents the same picture, being higher in uG and lower in hG although this response is more directly related to blood volume changes. The ratio of white and red precursor cell types in the bone marrow of rats is altered in opposite directions above and below unit gravity. Vestibular type II hair cells have increased numbers of synapses in uG. Synapse numbers decrease soon after returning from space flight. Following exposure to centrifugation there are less synapses present. Developing cichlid fish had decreased succinic dehydrogenase activity in vestibular nuclei after exposure to uG and increased after 3G exposure. Humans, with eyes closed, tend to point low at previously visualized targets during uG and high during hG. Following spaceflight growth hormone secreting cells have increased cell size, cytoplasmic granules and staining intensity. The reverse is seen following hG exposure. Sperm seem to function more efficiently during uG than during hG. That is, forward motility and egg fertilization are enhanced during space flight.

Previous studies demonstrated that short duration (<=21days) microgravity (µG) or hindlimb suspension (HLS) has minor effects on spermatogenesis. While long-term µG studies must await implementation on the International Space Station, we found that long-term (6 wk) exposure to simulated µG using HLS showed complete inhibition of production of mature sperm by the rat (J. Appl. Physiol. 92:1191-1198, 2002). The spermatogenic loss was not associated with changes in male reproductive hormones. Experiments were initiated to examine whether altered testicular bloodflow and/or intra-testicular temperature could be a primary factor in the loss of spermatogenesis in the long-term HLS model. Catheters for injection of fluorescent microspheres and removal of blood were inserted into the left carotid and femoral arteries, respectively, of anesthetized 6-month old male NIH Fisher rats. The Inguinal Canals were partially Ligated with a purse-string stitch (HLS and ICL control groups) to prevent the testes from becoming abdominal during HLS. Bloodflow was measured at 0 min, then at 25 min and at subsequent 1 hr intervals during HLS. In HLS animals there was a rapid and time-dependent decline in bloodflow by 25 min after suspension that continued for the next 3 hrs (down to 30% of the 0 min flow rate). The ICL (but not suspended) control group also showed a decline (but less severe) in bloodflow by 25 min. Sham operated animals (anesthetized, no HLS, no ICL) showed no significant change in bloodflow by 25 min post “surgery”. The reduced level of bloodflow measured in the HLS animals was found to be sufficient to cause spermatogenic loss in other mammalian models and could explain the long-term loss of spermatogenesis in HLS.

There does appear to be a gravitational continuum in biological response to altered gravitational fields. More prolonged studies on the ISS will be needed to establish adaptive endpoints. (Supported by NASA NCC 2-1099) ( Supported by NASA NAG 2-1491 and the NIH Center for Reproductive

Sciences HD-33994.)

[104] [102] EFFECTS OF HYPERGRAVITY REARING ON GROWTH HORMONE (GH) SECRETION IN PREWEANLING RATS. L.A. Baer1, J.H. Chowdhury2, R.E. Grindeland3, C.E. Wade3 and A.E. Ronca3. 1Lockheed Martin, Moffett Field, CA, 2 University of Alberta, Alberta, CANADA, 3Life Sciences Division, NASA Ames Research Center, Moffett Field, CA.

SPONTANEOUS MINERALIZATION OF EMBRYONIC MOUSE LIMB CELL AGGREGATES GROWN IN A ROTATING BIOREACTOR. P.J. Duke, N. Horn, and D. Montufar-Solis. The Univ. of Texas Dental Branch, Houston, TX. Longitudinal bone growth is dependent on a normal endochondral ossification process, which itself depends on maintenance of growth plate function. Abnormal arrangement of chondrocytes within the growth plate is associated with various chondrodysplasias and exostoses, but the cause of disarrangement is not known. An in vitro system that allows cells to grow and differentiate into an organized three-dimensional unit may provide clues to understanding organization of a growth plate and how this organization may be disrupted by mutations or teratogens. The Synthecon bioreactor, which has low shear, enhanced nutrient diffusion and gas exchange, is a system of choice for growing three-dimensional tissues representative of in vivo tissue construction. In this study, we evaluated the ability of the rotating bioreactor to support the growth and differentiation of E10.5 murine limb mesenchymal cells. Cells from limb buds of ICR mice, were aggregated, and cultured in the bioreactor for three weeks. Histology showed well-differentiated cartilage nodules with a perichondrium-like outer layer, and areas of growth plate-like organization (regions characteristic of reserve, proliferative and hypertrophic zones). Goldner's trichrome staining revealed a matrix containing collagen, and Von Kossa staining showed areas of calcification in the lower hypertrophic zone. These results show that the bioreactor can support the growth and differentiation of organized growth plate-like structures, and that mineralization of this endochondral cartilage occurs without addition of β-glycerol phosphate.

We previously reported that rat pups reared at 1.5-g, 1.75 or 2.0-g hypergravity weigh 6-15% less than 1.0-g controls (Baer et al., JGP, in press). To account for these findings, we measured the lactational hormones, prolaction (Prl) and oxytocin (OT), in the pups’ mothers. Gravity related differences in Prl were not observed whereas OT of lactating dams was significantly reduced relative to controls. Milk transfer from dam to pup was not impaired in hypergravity-reared litters tested at 1-g. Together, these findings suggest that impaired lactation and milk transfer do not account for reduced body masses of postnatal rats reared in hypergravity. In the present study, we analyzed growth hormone (GH) secretion and maternal licking in pups reared in hypergravity and in 1.0-g controls. Recent reports using dwarfing phenotypes in mouse mutants (Lupu et al., 2001) have provided evidence for postnatal dependence on GH and insulin-like growth factors (IGFs). Beginning on Gestational day (G)11 of the rats’ 22 day pregnancy, rat dams and their litters were exposed to either 1.5-g, 1.75-g or 2.0-g. On Postnatal day (P)10, we measured plasma GH using enzyme immunoassay (EIA). Contrary to our hypothesis, GH was significantly elevated in pups reared at 2.0-g relative to 1.0-g controls (mean +/- s.d., ng/ml: 2.0-g, 10.6 [3.0], 1.5-g 8.9 [4.0], 1.0, 7.95[3.1]). Pup-oriented behaviors of the hypergravity dams were also changed, possibly accounting for the increase in pup GH. GH alone does not appear to play a role in reduced body weights of hypergravity-reared pups.

(Supported in part by NIH/NIDCR Training Grant T35DE07252.)

(Supported by NIMH Grant MH46485 and NASA Grants 121-10-50, 121-40-10, 121-10-30.)



[107] [105] VESTIBULAR RECEPTOR DEVELOPMENT DURING SPACEFLIGHT AND HYPERGRAVITY. J.D. Dickman,

ACUTE WOUND HEALING AND REPAIR OF RODENT MCL DURING HINDLIMB UNWEIGHTING IN RESPONSE TO RHGH AND IGF-I. COUNTERMEASURES. D.A. Martinez1, R. Vanderby, Jr.2, R.E. Grindeland3, A.K. Lee1, H.Y. Hoang1, P. Le1, A. Lust1, N. Garcia1 and A.C. Vailas1. 1Connective Tissue Physiol. Lab, Univ.

A. Lysakowski, D. Huss, S Price. Dept. of Research, Central Institute for the Deaf, Washington University, St. Louis, MO Vestibular receptor development of the otolith organs was examined in embryonic quails raised from fertilization in either normal 1g, microgravity (0g), or hypergravity (2g) conditions. Fertilized eggs were arrested from development by cooling, placed into an incubator

of Houston, TX, 2Dept. of Surgery, Univ. of Wisconsin, Madison, WI, 3NASA-Ames, Moffett Field, CA. Microgravity and simulated microgravity have pronounced detrimental effects on the strength and composition of dense fibrous connective tissues. Previously we have reported that the time course of wound healing in the rodent medial collateral ligament (MCL) during hindlimb unweighting (HLS) is significantly delayed, which was demonstrated by reduced ligament strength and stiffness, decreased ECM organization , elevated MCL cellularity, altered collagen characteristics. rhGH and IGF-I are known to be potent anabolic endocrine modulators of collagen metabolism. Therefore, our primary purpose of this study was to use rhGH, IGF-I and rhGH+IGF-I as countermeasure therapies to ameliorate the “delayed” injury repair response of the MCL during HLS. Ten rats in each group were randomly assigned to HLS or ambulatory (AMB) groups receiving either bilateral MCL lesions or sham surgeries. Each group and their respective controls received twice daily injections (s.c.) of rhGH, rhIGF-I, rhGH+IGF-I or saline for 3 weeks. MCL biochemical analyses showed an increase in DNA concentration (P<0.05) in lesion MCL’s that were partially restored by IGF-I. There was an increase in proteoglycans

(ADF -SHOT, Inc), and flown on STS108 for 12 days in space. Group 1 eggs were exposed to 0g and group 2 eggs underwent constant velocity centrifugation at 1g in space. Four 0g quail embryos and six 1g embryos were recovered alive at E12. Seven additional quail embryos were centrifuged on Earth at 2g and ten embryos raised in normal ground incubator conditions. The utricle, saccule and lagena otolith organs were harvested from all E12 embryos for study. We found that the saccule otoconia stones increased by 40% in weight for the 0g and 80% for the 2g as compared to both ground and flight 1g controls. Examination of the hair cell stereocilia polarizations of 0g embryos suggest that deviations in organization from normal 1g otoliths occur, but data were still being analyzed at time of submission. Neural tracing (HRP) experiments were performed on all E12 embryos, with vestibular nerve afferent innervation of otolith organs being examined. Excellent fiber tracing occurred in all animals, regardless of gravity condition. Group 1 0g fibers were highly branched with dense arborizations and numerous terminals as compared to 1g controls. Quantitative reconstructions of afferents from all animals in the lesion groups, however there was no effect by hormone

countermeasures (P>0.05). MCL hydroxyproline (Hyp) concentration is under study. (Supported in part by funds from NASA NCC2-1159.) was reduced in both the lesion and unweighted groups. rhGH or IGF-I

supplementation did not alter MCL collagen concentrations. The maturation of the MCL collagen measured by the content of HP cross-links showed that unweighting by itself increased HP cross-link content, while rats exposed to both surgical lesions and unweighting had greater HP contents indicative of mature un-remodeled collagen. rhGH treatment increased MCL HP content in HLS+lesion animals, whereas IGF-I had no effect.

(Supported by NASA Grant: NAG9-1152)

[106] [108] HISTAMINE IS THE PUTATIVE NEUROTRANSMITTER USED BY GRAVIRECEPTOR CELLS OF THE STATOCYSTS IN MOLLUSCS. R.P. Croll1,2, O. Braubach1, J. Borycz2, J. Nason1 and C. Evans1. 1Dept. Physiol. & Biophys. and 2Neurosci. Inst., Dalhousie Univ., Halifax, Canada

NEURONAL RESPONSE TO GRAVITY CHANGE: HYPOTHALAMUS VS. HIGHER CNS. Y. Kumei1, R. Shimokawa1, H. Shimokawa1, T. Miyamoto2 and K. Toda2. 1Graduate School Tokyo Medical and Dental University, Tokyo 113-8549, and 2Nagasaki University Graduate School, 852-8588 Nagasaki , Japan

Both bivalve and gastropod molluscs have flown on Space Shuttle missions (STS-77 and STS-90, respectively) and yet large gaps exist

We previously reported that 2G hypergravity exposure modulated the nociceptive response of rats, being mediated through induction of

in our understanding of the physiology of the gravireceptor organs β-endorphin in the arcuate, paraventricular, suprachiasmatic and supraoptic nuclei of the hypothalamus. However, it is unclear whether these hypothalamic areas are the primarily sensitive sites of gravity change

(the statocysts) in these organisms. For example, while several neurotransmitters have previously been detected in statocysts, it remains unclear which one(s) function to relay information to second order neurones involved in gravity-oriented behaviours. Our research exploited the pond snail, Lymnaea stagnalis, as a primary model for studying such problems. Using immunohistochemistry and confocal microscopy, we were able to localize histamine to both the somata of the receptor cells which line the statocysts and to their axons which project to the cerebral ganglion via the statocyst nerve. Histamine was also localized in several other cells scattered throughout the central ganglia, thus suggesting that it is a widespread transmitter involved in diverse functions. The presence

or the secondarily affected areas via neurons that are projected from the higher CNS, including limbic system. In the present study, we compared 2G-induced neuronal activity in the hypothalamic slices vs. decapitated whole brain. We found that the hypothalamus contains not only primarily responding sites to gravity change, but also secondarily responding areas that are regulated by the higher CNS. These hypothalamic areas in decapitated whole brain show 2- to 3-fold higher sensitivity to gravity change than in the isolated hypothalamic slice. (The present study was suppored by Grant from Ministry of Science, Education, Culture of Japan, and properly performed under the control of the Guideline for Animal Experimentation in Tokyo Medical and Dental University.)

of histamine in the nervous system was confirmed using high performance liquid chromotography (HPLC). Next, the role of histamine was examined in a behavioural assay of geotactic behaviour. Uninjected snails and snails injected with saline both exhibited direct locomotory paths up an inclined plane. Snails injected with histamine or with pyrilamine (an antagonist

of H1 receptors in vertebrates) exhibited significantly more circuitous pathways. Finally, in a series of ongoing experiments we are examining the generality of these findings across the molluscs.

(Supported by a contract from the Canadian Space Agency and by a grant from the Natural Sciences and Engineering Research Council of Canada.)



Post

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Animal Development and Gravity

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ABSTRACTS – POSTER SESSION IV–C

[109] [111] HYPERGRAVITY EFFECTS ON RODENT PREGNANCY AND PARTURITION. A.E. Ronca1, L.A. Baer2, N.A. Mills3 and C.E. Wade1. 1Life Sciences Division, NASA Ames Research Center, Moffett Field, CA. 2Lockheed Martin, Moffett Field, CA., 3University of California, Davis, CA.

INFLUENCE OF 2G HYPERGRAVITY ON GROWTH OF THE MOUSE EMBRYO. P. McCaffery, L. Ma. Dep’t Cell Biology, UMass/E.K. Shriver Center, Waltham, MA. It has been reported that hypergravity can alter the rate of growth of cell lines and embryonic tissues. The mechanism of this effect however is unknown. To investigate this phenomenon in greater detail pregnant CD-1 mice were centrifuged at 2G in the 24-foot centrifuge at NASA-Ames. Centrifugation was applied between embryonic days 4 to 14 (E4-E14) and at E10, E12 and E14 the embryos were dissected for analysis by Northern blotting and gene array. As expected, there was a significant decrease in the size of these embryos. To determine whether the embryos underwent

No mammal has yet undergone birth, or parturition, in the microgravity of space. Previous studies (Ronca & Alberts, 2000) have shown that mid-pregnant rat dams exposed to spaceflight (0-g) and landed 48-72 hrs before term successfully delivered robust, healthy offspring. Microgravity-exposed dams exhibited twice the expected numbers of labor contractions whereas length of pregnancy, duration of labor, fetal wastage, number of neonates born and litter gender ratios were identical to controls. In the present study, we report the results of rodent pregnancy and parturition

a general stress response 23 stress related genes were analyzed at the earliest time point after centrifugation. All genes were normalized by comparison to a set of control “housekeeping” genes. None of the genes exhibited a significant alteration in expression. Given the growth inhibitory effects of centrifugation, cell cycle regulatory genes were also investigated. Two negative growth regulatory genes were found to decline at E10, p15INK4b, which fell 2-fold, and Gadd45a, which declined

at the opposite end of the gravity spectrum, in hypergravity. Dams exposed to either: 1.0-g, 1.5-g, 1.75-g or 2.0-g from Gestational day (G) 11and throughout the births of their litters had comparable durations of pregnancy and labor, fetal wastage, numbers of neonates born and litter gender ratios. During parturition, hypergravity-exposed dams exhibited significantly fewer labor contractions as compared to 1.0-g controls. Dams that underwent birth in hypergravity had fewer offspring surviving the immediate postpartum period (P1: 1.0-g, 11.92 +/- 2.84; 1.5-g,

14-fold. In conclusion, these initial results indicate that the centrifugation does not lead to a generalized stress response but instead initiates a decline in several growth suppressor genes suggesting that this may be a response to reverse the centrifugation induced decline in growth. This is reflected by the eventual equalization in size of the centrifuged and control embryos by E14.

10.88 +/- 2.17; 1.75-g, 9.22 +/- 1.99; 2.0-g, 8.83 +/- 3.31). Thus, following birth in hypergravity, postnatal but not prenatal survival was compromised. Together, these findings identify in pregnant and parturient rodents similarities and differences across the gravity continuum. Maternal and neonatal factors that contribute to peri-parturitional vulnerability to altered gravity environments will be discussed.

(Supported by NASA: NAG2-1438 and NIH: H05515)

(Supported by NIMH Grant MH46485 and NASA Grants 121-10-50, 121-40-10, 121-10-30.)

[110] [112]

PRELIMINARY STUDY OF AQUATIC ANIMAL EXPERIMENT FACILITY, AQUATIC HABITAT (AQH) , FOR INTERNATIONAL SPACE STATION. T.Sakimura1, S.Uchida1, Y.Kono1, T.Ochiai2, M.Masukawa2, N.Ishioka2. 1Mitsubishi Heavy Industries (MHI), Kobe Shipyard & Machinery Works. 2National Space Development Agency of Japan (NASDA), Space Utilization Research Center.

SPACE SHUTTLE FLIGHT (STS-90) ENHANCES DEGRADATION OF RAT MYOSIN HEAVY CHAIN IN ASSOCIATION WITH ACTIVATION OF UBIQUITIN-PROTEASOME PATHWAY. T. Nikawa1, M. Ikemoto1, K. Hirasaka1, M. Kano1, S. Takeda2, K.M. Baldwin3, R. Izumi4, T. Ogawa5, I. Nonaka2 and K. Kishi1. 1Dept of Nutrition, School of Med, Univ of Tokushima, Japan, 2National Center of Neurology and Psychiatry, Tokyo, Japan, 3Dept of Physiology and Biophysics, Univ of California, Irvine, 4NASDA, Tokyo, Japan and 5Dept of Orthopedic Surgery, School of Med, Univ of Tokushima.

NASDA and MHI have developed aquatic animal experiment facilities for NASA Space Shuttle use and have conducted many experiments in space with various aquatic animals. Currently, we are studying the next-generation aquatic animal experiment facility or Aquatic Habitat (AQH) for both of the Space Shuttle and the Space Station use. The AQH will have the capabilities to accommodate three-generations of small freshwater fish, medaka and zebrafish, and egg through metamorphosis

The elucidate the mechanisms of microgravity-induced muscle atrophy, we focused on fast-type myosin heavy chain (MHC) degradation and expression of proteases in atrophied gastrocnemius muscles of neonatal rats exposed to 16-d spaceflight (STS-90). The spaceflight stimulated ubiquitination of proteins, including a MHC molecule, and accumulation of MHC degradation fragments in the muscles. Semi-quantitative RT-PCR revealed that the spaceflight significantly increased mRNA levels of cathepsin L, proteasome components, polyubiquitin, and ubiquitin-conjugating enzyme in the muscles, compared with those of ground control rats. The levels of µ-calpain, m-calpain cathepsin B, and cathepsin H mRNAs were not changed by the spaceflight. We also found that tail-suspension of rats for 10 d or longer caused the ubiquitination and degradation of MHC in gastrocnemius muscle, as was observed in the spaceflight rats. In the muscle of suspended rats, these changes were closely associated with activation of proteasome and up-regulation of expression of mRNA for the proteasome components and polyubiquitin. Administration of a cysteine protease inhibitor, E-64, to the suspended rats did not prevent the MHC degradation. Our results suggest that spaceflight induces the degradation of muscle contractile proteins, including MHC, possibly through a ubiquitin-dependent proteolytic pathway.

of Xenopus (African clawed frog). A prototype breeding system was developed and preliminary tests were performed with medaka and Xenopus. The medaka mating and spawning was performed successfully in the small specimen chamber with 600ml water volume. And two sets of the hatched 10 larvae were put in two sets of the same chamber for next generation breeding. The first spawning was observed at the 45th day from the hatching in one chamber, and at the 47th day in the other chamber. Xenopus metamorphosis was also completed in the same specimen chamber. These test results gave us the feasibility of the expected AQH experiments, although some minor problems that should be resolved were recognized. Right now, we are performing preliminary spawning and breeding tests of zebrafish by using the same prototype breeding system, and also fabricating a partial engineering model of the AQH , reflecting the above test results.



[113] SPACEFLIGHT AND IMMUNITY IN THE MOUSE: PART II. D.S. Gridley1,2, G.A. Nelson1, L.L. Peters5, P.J. Kostenuik6, T.A. Bateman3,4, S. Moroney6, L.S. Stodiek3, D.L. Lacey6, S.J. Simske3, and M.J. Pecaut1. Depts of 1Radiation Medicine and 2Biochemistry & Microbiology, Loma Linda Univ. and Medical Center, Loma Linda, CA;

3Dept. of Aerospace Engineering, BioServe Space Technologies, Univ. of Colorado, Boulder, CO; 4Biomedical Engineering Program, Mechanical Engineering Dept, Colorado State Univ., Fort Collins, CO; 5The Jackson Laboratory, Bar Harbor, ME; and 6Amgen Inc., Thousand Oaks, CA. The goal of this portion of the study was to quantify in the C57BL/6 mouse the effects of a 12-day shuttle mission (STS-108/UF-1) on: body and lymphoid organ masses, activation marker expression, cytokine secretion and red blood cell (RBC) and thrombocyte characteristics. Mice aboard the shuttle (flight or FLT group) had 10-12% lower body mass compared to ground controls housed in either Animal Enclosure Modules (AEM) or under standard vivarium (VIV) conditions (P<0.001). Thymus and spleen masses were lowest in FLT animals, although some atrophy was noted in AEM mice. Flow cytometry showed that the percentages of CD25+ lymphocytes, CD3+/CD25+ T cells, and NK1.1+/CD25+ NK cells in spleens from FLT mice were higher compared to both control groups (P<0.05). In contrast, CD71 expression was depressed in FLT and AEM mice compared to VIV animals (P<0.001). Secretion of interferon-γ, interleukin-2 (IL-2) and IL-4, but not tumor necrosis factor-a or IL-5, by PHA-activated splenocytes from FLT mice was decreased relative to either one or both ground control groups (P<0.05). FLT mice also had high RBC and thrombocyte counts compared to both controls; low RBC volume and distribution width, percentage of reticulocytes, and platelet volume were also noted (P<0.05 or less). The data indicate that relatively short exposure to the spaceflight environment can induce profound changes that may become especially significant during long-term missions in space. (Supported by NASA: Coop. Agreement NCC9-149)



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Cell Biology


ABSTRACTS – POSTER SESSION IV–D

[114] EFFECTS OF VIBRATION ON BACTERIAL GROWTH RATE AND SIMILARITIES TO SPACE-FLOWN CULTURES. E.A. Juergensmeyer1, M.A. Juergensmeyer2, Rachel Edes3 and E.S. Nelson4. 1 Judson College, Elgin, IL, 2 IIT Research Institute, Chicago, IL, 3 Illinois Math and Science Academy, Aurora, IL, and 4 NASA Glenn Research Center, Cleveland, OH. A popular misconception is that space experiments are exposed to a constant value of 1x10-6 times earth gravity when in the microgravity environment on the Space Shuttle or the International Space Station. Instead, vibrational accelerations of varying values dominate the acceleration environment in space labs. These accelerations are due to structural response, crew exercise, and equipment operation, and are well characterized, although the effects of these accelerations are not, particularly in biological experiments. We have compared stationary E. coli cultures to cultures shaken by a single-frequency, constant-magnitude linear acceleration over a range of magnitude and frequency. The stroke length (which is correlated to the acceleration magnitude) and frequency of the shaking were chosen to correspond with vibrational accelerations typically experienced during periods designated as microgravity on the Space Shuttle and ISS. Two different culture devices were used to mimic both hard-sided and soft-sided flight hardware. Vibration of the cultures at certain frequencies induced a reduced lag phase and an increased total cell number similar to the results seen during flight. We believe that improved mixing and homogeneity induced by vibration is responsible for these results. It is therefore crucial that biological experiments bound for space consider the effects of vibration-induced mixing due to the background environment in their analysis and design for microgravity. (Supported by NASA grant NAG2-1512) [115] ACTIVATION OF CYCLIC AMP SYNTHESIS BY FULL AND PARTIAL BETA-ADRENERGIC RECEPTOR AGONISTS IN CHICKEN SKELETAL MUSCLE CELLS. R.B.Young and K.Y. Bridge. Biotechnology Sciences Group, SD46, Marshall Space Flight Center, Huntsville, AL 35812. Several beta-adrenergic receptor (βAR) agonists are known to cause hypertrophy of skeletal muscle tissue. Accordingly, five βAR agonists encompassing a range in activity from strong to weak were evaluated for their ability to stimulate cAMP accumulation in embryonic chicken skeletal muscle cells in culture. Two strong agonists (epinephrine and isoproterenol), one moderate agonist (albuterol), and two weak agonists known to cause hypertrophy in animals (clenbuterol and cimaterol) were studied. Dose response curves were determined over six orders of magnitude in concentration for each agonist, and values were determined for their maximum stimulation of cAMP synthesis rate (Bmax) and the agonist concentration at which 50% stimulation of cAMP synthesis (EC50) occurred. Bmax values decreased in the following order: isoproterenol, epinephrine, albuterol, cimaterol, clenbuterol. Cimaterol and clenbuterol at their Bmax concentrations were approximately 15-fold weaker than isoproterenol in stimulating the rate of cAMP synthesis. When cimaterol and clenbuterol were added to culture media at concentrations known to cause significant muscle hypertrophy in animals, there was no detectable effect on stimulation of cAMP synthesis. Finally, these same levels of cimaterol and clenbuterol did not antagonize the stimulation of cAMP by either epinephrine or isoproterenol.

[116] OSTEOBLAST DIFFERENTIATION DECREASES HYPERGRAVITY-STIMULATED RELEASE OF PGE2. N.D. Searby1, C. R. Steele2, and R.K. Globus1. 1NASA Ames Research Center, Moffett Field, CA, 2Mechanics and Computation Division, Stanford University, Stanford, CA. We determined if progressive differentiation of osteoblasts influences their sensitivity to gravitational loading. Osteoblasts were cultured for 4 days (confluent monolayer), 6 days (prenodules), 9 days (nodules) and 19 days (mineralized nodules), then centrifuged at 10 times gravity (g) or 50-g for 3 hours using the NASA Ames 1-ft. Diameter Centrifuge. Stationary controls were placed in an adjacent incubator. Following centrifugation, conditioned media were collected and analyzed for PGE2 by ELISA. Microtubules were fluorescently labeled and analyzed by confocal microscopy to determine microtubule network morphology and height. Centrifugation at 10-g reduced microtubule network height by 15% on d4 and 10% on d6, with variable changes in more mature cultures. No major changes in microtubule morphology were observed. PGE2 release by d4 cultures increased in a dose-dependent fashion (3-fold at 10-g and 6-fold at 50-g relative to controls). D6 cultures produced a 5-fold increase for both 10-g and 50-g. PGE2 increased only 1.5-fold by d9, and by d19, PGE2 was not detectable in either the control or hypergravity-stimulated cells. Thus, as osteoblasts differentiate in culture, responsiveness of the microtubule cytoskeleton and the PGE2 pathway to hypergravity declines. (Supported by NASA: NAGW4625 and 99-HEDS-02)



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Plant Growth

Development and Gravity Sensing


ABSTRACTS – POSTER SESSION IV–E

[117] [119] BASIPETAL PROPAGATION OF GRAVITY-INDUCED SURFACE pH CHANGES ALONG PRIMARY ROOTS OF LEPIDIUM SATIVUM L. Gabriele B. Monshausen1, Andreas Sievers2 1Botanisches Institut I, Universität Karlsruhe (TH), Germany, and 2Botanisches Institut, Universität Bonn, Germany.

CRITICAL ROLE OF ACTIN AND ACTIN-BINDING PROTEINS IN GRAVITROPIC TIP GROWTH. A. Sievers and M. Braun, Botanisches Institut, Universität Bonn, Bonn, Germany. The arrangements and multiple functions of actin in characean rhizoids and protonemata, including cytoplasmic streaming, polarized growth and gravitropic orientation, are regulated by the concerted action of numerous actin-binding proteins. Actin microfilaments form a mainly axially oriented meshwork of fine bundles in the subapical zone and focus in a spherically shaped, central area close to the very tip, where they organize a unique aggregation of endoplasmic reticulum, the structural center of the Spitzenkörper. We found a set of actin-binding proteins expressed specifically in this center of the Spitzenkörper. Immunolocalization of actin-binding proteins failed and the ER aggregate rapidly disintegrated when tip growth was arrested by the application of actin-depolymerizing cytochalasins or latrunculin B. Spectrin- and α-actinin-like proteins are likely to be involved in stabilizing the cisternae of the ER aggregate by crosslinking them to actin. They may also provide a mechanism for recruiting a specific subset of membrane proteins which help to create the particular apical conditions for gravity-oriented tip growth. The presence of actin-severing ADF and G-actin-binding profilin in the center of the Spitzenkörper indicates an actin dynamizing and actin polymerizing function of this area. The complexly arranged apical actin array is required for defining the center of maximal growth and for the positioning and the directing of sedimenting statoliths to specific graviperception sites at the plasma membrane. This statolith-sensitive membrane area is confined to the statolith region (10-35 µm) in positively gravitropic rhizoids, whereas in negatively gravitropic protonemata, this area is limited to the apical plasma membrane (5-10 µm). It is concluded that the apical actin cytoskeleton is essentially involved in susception, the early phase of the gravitropic pathway that leads to perception and the molecular mechanisms of the opposite graviresponses in characean rhizoids and protonemata.

While there is ample evidence for a role of auxin in root gravitropism, the seeming rapidity of gravi-induced changes in electrical parameters has so far been an argument against auxin being a primary signal in gravitropic signal transmission. Here, we present new evidence for a slower signal propagation along gravistimulated Lepidium sativum roots. Using multiple proton-selective microelectrodes to simultaneously measure surface pH on opposite root flanks at different distances from the root tip, we observed gravi-induced asymmetric pH changes at the surface of all investigated root zones. Upon gravistimulation, the surface pH decreased on the physically upper root flank and increased on the lower flank. The pH asymmetry appeared first (2.1±0.4 min after tilting) at the root cap and then – with incrementing lag times – at the meristem (after 2.5±0.3 min at 300 µm distance from root tip; after 3.7±0.4 min at 700 µm) and apical elongation zone (4.8±0.5 min at 1000 µm), suggesting a basipetal propagation of differential surface acidification at a rate of 250-350 µm min-1, consistent with reported velocities of polar auxin transport. (Supported by DLR on behalf of BMBF (50WB99998)) [118] [120] CHANGES IN AUXIN TRANSPORT IN RESPONSE TO GRAVITY AND LIGHT ARE MEDIATED BY REVERSIBLE PROTEIN PHOSPHORYLATION. S.R. Brady, A.M. Rashotte, N. Kirpalani and G.K. Muday. Dept. of Biology, Wake Forest University, Winston-Salem, NC

GENETICS OF THE GRAVITROPIC SET-POINT ANGLE IN LATERAL ORGANS OF ARABIDOPSIS. J.L. Mullen and R.P. Hangarter. Dept of Biology, Indiana University, Bloomington, IN. Research on gravity responses in plants has mostly focused on primary roots and shoots, which typically orient to a vertical orientation. However, the distribution of lateral organs and their typically non-vertical growth orientation are critical for the determination of plant form. For example,

In response to gravitropic stimulation, auxin is redistributed from its normal polar transport stream. This resulting lateral auxin gradient then drives the differential growth that reorients plants relative to the new gravity vector. The mechanisms that control the activity of the auxin transport proteins responsible for the changes in direction of transport are largely unknown. We are currently testing the hypothesis that reversible protein phosphorylation may regulate auxin transport during gravitropism and phototropism and that in hypocotyls, this regulation is modulated by photomorphogenesis. We have previously reported that the Arabidopsis mutant rcn1, which exhibits reduced phosphatase 2A activity, has increased basipetal transport in the roots and a reduced gravity response (Rashotte, DeLong, and Muday (2001) Plant Cell 13, 1683). Our current studies are focused on characterization of gravitropic and phototropic responses in the hypocotyls of rcn1. Light grown hypocotyls of rcn1 have a phenotype similar to wild-type plants, but etiolated rcn1 hypocotyls are shorter and have an open hook. Phototropism and gravitropism are also normal in light grown rcn1 hypocotyls, but etiolated rcn1 hypocotyls exhibit a faster gravitropic response. Similarly, auxin transport is altered in etiolated but not light grown hypocotyls of rcn1 and the regulation of auxin transport also differs between etiolated and light grown wild-type plants. Together, these results suggest that protein phosphorylation plays a role in regulation of auxin transport and may account for changes in auxin transport in response to light and gravity stimulation.

in Arabidopsis, when lateral roots emerge from the primary root, they grow at a nearly horizontal orientation. As they elongate, the roots slowly curve until they eventually reach a vertical orientation. The regulation of this lateral root orientation is an important component affecting the overall root system architecture. We found that this change in orientation is not simply due to the onset of gravitropic competence, as non-vertical lateral roots are capable of both positive and negative gravitropism. Thus, the horizontal growth the new lateral roots is determined by what is called the gravitropic set-point angle (GSA). This developmental control of the GSA of lateral roots in Arabidopsis provides a useful system for investigating the components involved in regulating gravitropic responses. Using this system, we have identified several Arabidopsis mutants that have altered lateral root orientations but maintain normal primary root orientation. Two of these mutants also have altered orientation of their rosette leaves, indicating some common mechanisms in the positioning of root and shoot lateral organs. Rosette leaves and lateral roots also have in common a regulation of orientation by red light that may be due to red-light-dependent changes in the GSA. Further molecular and physiological analyses of the GSA mutants will provide insight into the basis of GSA regulation and, thus, a better understanding of how gravity controls plant architecture.

(This work was supported by the National Aeronautics and Space Administration grant NAG2-1507).

(Supported by NASA: NCC2-1200.)



[121] [123] PHOTOTROPISM IN ROOTS OF ARABIDOPSIS ON A FEEDBACK SYSTEM. C. Wolverton1, H. Ishikawa2, M.L. Evans2 1Dept. of Botany-Microbiology, Ohio Wesleyan University, Delaware

MEASURING THE HYDRAULIC CONDUCTIVITY OF POROUS MEDIA USED FOR PLANT GROWTH IN MICROGRAVITY. S. L. Steinberg1, and G. J. Kluitenberg2. 1Universities Space Research Association, Houston TX and 2Department of Agronomy, Kansas State University, Manhattan KS.

and 2Dept. of Plant Biology, Ohio State University, Columbus. Although plants are nonmotile organisms, they are extremely sensitive to environmental stimuli. Cues such as light, touch, wind, temperature, Control of water and air in the root zone of plants has been problematic

in microgravity. This is largely due to a lack of fundamental information about water transport through porous media in microgravity. One of the fundamental properties controlling water flow in porous media is the hydraulic conductivity function, which characterizes how hydraulic conductivity varies with degree of saturation. Flight-compatible methods for measuring the conductivity function are needed in order to evaluate the influence of microgravity on conductivity. The objective of the study was to develop and evaluate flight-compatible methods for measuring hydraulic conductivity. Evaluation of candidate methods was achieved using earth-based experiments that simulate flight-like conditions. These included the use of membranes to control water delivery to media, and horizontal water flux through thin sections of media. One of the principle challenges encountered in developing flight-compatible methods has been the measurement and control of relatively small hydraulic gradients. During one-step transient outflow the greatest pressure drop occurred between the membrane and media, with the horizontal section of media draining nearly uniformly. Hydraulic gradients of less than 1.43 kPa m-1 were common during steady state measurement of saturated conductivity. The magnitude of hydraulic gradients will impact the development

and gravity combine to influence the morphology of plants. To date, relatively little is known about how plants perceive these environmental cues and transduce them into signals that regulate the timing and localization of cell expansion and other response pathways. In an attempt to understand how plants integrate multiple external signals into a coherent growth response, we have begun to study the interaction of phototropism and gravitropism in Arabidopsis roots. Past investigations of phototropism have been complicated by the obvious drawback of inducing gravitropic stimulation as a result of phototropic response. A second problem results from displacement of the sensing region from its original position during the response, resulting in reduced stimulation. We have overcome both drawbacks through the use of a feedback system based on real-time image analysis. Here we show that, as previously reported for maize roots, the response to unilateral illumination in Arabidopsis roots occurs in the central elongation zone (CEZ). This contrasts with the site of initial response to gravitropic stimulation, which occurs in the distal elongation zone (DEZ) in both species. The latent period for phototropism in Arabidopsis (approx. 40 min) is much longer than for gravitropism (approx. 10 min). This is also the case for maize root phototropism and gravitropism. Arabidopsis roots respond to differing fluence rates of methods to measure hydraulic conductivity of porous media by modulating their rate of curvature, indicating a dose-dependent response relationship. By eliminating gravitropic stimulation through

in microgravity. (Supported by NASA: NRA 01-OBPR-01-009)

a feedback system, the phototropic response continues much longer than the response under non-feedback conditions, indicating a lack of sensory adaptation at the fluence rates we tested. Feedback control coupled to real-time image analysis represents an informative new tool for dissecting plant responses to multiple directional cues.

(Supported by NASA: NAG2-1411)

[122] [124] PHYSIOLOGICAL RESPONSES OF ARABIDOPSIS THALIANA TO REDUCED ATMOSPHERIC PRESSURES. J.T. Richards1, A.C. Schuerger1, and K. Corey2. 1Dynamac Corporation, Kennedy Space Center, FL, and 2Science Consultant, Interior Sancta, Miller Falls, MA.

MODIFICATION OF METABOLIC GAS BIOAVAILABILITY DURING SEED DEVELOPMENT: CONSEQUENCES FOR SPACEFLIGHT M.E. Musgrave1, J. Blasiak1 and A. Kuang2. 1Biology Department, University of Massachusetts, Amherst, MA 01003 and 2Biology Department, University of Texas Pan American, Edinburg, TX Understanding fundamental physiological responses of plants to reduced

atmospheric pressures is an important precursor to greenhouse design and plant growth on Mars. A low-pressure plant growth chamber (LPGC)

Results of previous spaceflight experiments suggest that changes in gas movement in microgravity may adversely affect seed development. Seeds of Brassica rapa maturing in microgravity were smaller and had less developed storage reserves than their 1-g counterparts. A series of ground-based experiments was conducted to understand how changes in metabolic gas levels, resulting from differences in diffusion resistance, influence seed development in the loculate fruits: Arabidopsis, Brassica and Capsicum. Using green pepper as a model system, we manipulated its internal atmosphere and found that seed size and storage reserve deposition were affected by small changes in internal oxygen and carbon dioxide levels. Similar alterations in internal gas concentrations would be expected to occur in the absence of convective air movement in microgravity. In a third set of experiments, synthetic atmospheres were created in which He or Ar were used to replace N, while oxygen and carbon dioxide were maintained at atmospheric concentrations. Diffusion rates of oxygen and carbon dioxide were inversely proportional to the density of the balance gas. When Brassica rapa seeds were produced in an atmosphere of decreased density, development was greatly accelerated, while patterns

was constructed that maintained low-pressure environments capable of supporting basic physiological studies. Short-term studies of net canopy CO2 assimilation and transpiration rates were measured on A. thaliana plants enclosed in the LPGC with total atmospheric pressures set at either 101, 75, 50, 25 or 10 kPa. CO2 levels were maintained at a ppCO2 of 0.1 kPa prior to initiation of gas exchange measurements. Studies were conducted in order to assess whether acclimation to the reduced pressures was required to accurately measure CO2 exchange. Plant canopies acclimated for either 1 hr or 16 hrs resulted in similar CO2 draw-down responses over time. Furthermore, results from this study indicated that CO2 exchange was identical for all atmospheric pressures when the ppCO2 levels decreased from the initial 0.1 kPa to 0.06 kPa. Below 0.06 kPa ppCO2, net CO2 exchange measurements at the various pressures began to diverge, with the greatest changes occurring at lower pressures. This suggested a threshold exists above which photosynthesis was unaffected by pressure. From the resultant CO2 draw-down curves at 101, 75, 50, 25 and 10 kPa total pressure, CO2 compensation points were calculated of embryo development were altered. The results illustrate that what is

perceived as "normal" embryonic development only occurs under ambient atmospheric conditions and that altering these conditions, intentionally or inadvertently, has profound effects on embryonic development and maturation.

and values decreased from 101 kPa to 10kPa total pressure, suggesting a possible effect of reduced pressure on photorespiration. In addition, water loss data was collected and an inverse relationship resulted between total atmospheric pressure and total water loss due to transpiration. (Supported by NASA:AO-99-HEDS-01-032.) (Supported by NASA NAG2-1375).




[125] ENVIRONMENTAL AND GENETIC INTERACTIONS WITH ETHYLENE SENSITIVITY IN WHEAT. Steve Klassen, Tim Hudelson, and Bruce Bugbee. Utah State University, Logan, www.usu.edu/cpl. Elevated levels of C2H4 gas cause a variety of abnormal responses in plants including shortened height, leaf rolling, premature senescence and sterility. Levels as high as 1000 ppb (1 ppm) have been measured in space studies and were implicated as the cause of sterility in wheat grown on MIR (Campbell et al., 2001). We have since found that levels as low as 25 ppb significantly inhibited seedling growth in salad crops and 50 ppb reduced seed set in USU-Apogee wheat by 40%. Catalytic air scrubbing systems will reduce C2H4 levels in space environments but will not likely be sufficient to maintain levels low enough for normal plant growth and reproduction. Genetic screening and control of environmental factors affecting ethylene sensitivity and production are needed to reduce C2H4 induced problems in space. In a previous study, the C2H4 sensitivity of wheat was not influenced by elevated CO2 but was significantly different between cultivars (Klassen and Bugbee, 2002). A comparison of the C2H4 sensitivity of wheat between studies suggested a possible interaction with temperature. In this study we compared the sensitivity of USU-Apogee and USU-Perigee wheat to 100 ppb C2H4 at 15, 19 and 23 oC. Yield was significantly reduced in both cultivars at all three temperatures but C2H4 sensitivity dramatically decreased with increasing temperature. USU-Apogee was less C2H4 sensitive than USU-Perigee at all three temperatures and both cultivars had twice the yield reduction at 15 oC than at 23 oC as compared to controls. Temperature treatments were limited to a 10 d period bracketing anthesis indicating that short term environmental manipulations can significantly reduce C2H4 induced sterility in wheat. (Supported by NASA Advanced Life Support Program)


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Hardware andSpaceflightExperiment

Development


ABSTRACTS – POSTER SESSION IV–F

[126] [128] SCIENCE INVESTIGATIONS WITH THE PLANT RESEARCH UNIT. Mark C. Lee, Robert C. Morrow, Katherine M. Stolp.

STS-107 EXPERIMENT BACTER: GROUND SIMULATION STUDY RESULTS. E. dL. Pulcini and B.H. Pyle. Dept of Microbiology, Montana State University, Bozeman. Orbital Technologies Corporation, Madison, WI. A proteomic analysis of differential protein expression in Pseudomonas aeruginosa ATCC 29260 under conditions of simulated microgravity was performed. Comparative control cultures of P. aeruginosa were grown under normal gravity (1 x G) conditions and maintained under identical conditions of temperature, oxygen, and nutrient availability. Simulated microgravity was accomplished using a clinostat system rotating at

The Plant Research Unit (PRU) is the Space Station Biological Research Program plant growth facility being developed for the International Space Station. The plant habitat is designed for experiments in near-zero gravity, or it can be rotated by the ISS Centrifuge for experiments at any gravity level from microgravity to twice Earth’s gravity. Plant experimentation will be possible in multiple Plant Research Units at one time, isolating the effect of gravity on the biological specimens. The PRU will provide and control all aspects of a plant’s needs in a nearly closed system. The PRU will interface with the Habitat Holding Rack and Centrifuge Rotor. The PRU will provide continuous data logging of the environment including video images of the plants. The Plant Research Unit will provide a significant new science capability that could accelerate biotechnology and controlled agriculture applications on Earth. For experimenters, ORBITEC has developed a Science Evaluation Unit (SEU) that uses nearly the same plant chamber that will be developed for PRU. The SEU will be at the conference for demonstrations and can be used by scientists now to develop protocols and become familiar with the hardware. The paper will review the status of the program. Discussion will encompass the prototype testing that has taken place and expected capabilities of the flight unit, and research preparations that could be conducted. In addition, recent updates to the Science Technical Requirements document will be covered along with expected science data and protocols that could be supported by the PRU.

80 rpm. The bacteria were grown in a medium to stimulate exotoxin A virulence factor production during growth. The analysis of variations in exotoxin A production was accomplished using ELISA, Western Blots and proteomic methodologies. In order to better identify the physiologic changes that take place in simulated microgravity, a comparison of protein expression patterns using P. aeruginosa signaling mutants with mutations in the las, rhl, and las/rhl systems was made. Results indicate variations in protein expression do occur in P. aeruginosa under conditions of simulated microgravity representing changes in metabolic and physiologic functions as well as changes in putative pathways for the production of virulence factors. These results will allow for a better understanding of the factors involved in crew health and safety during space flight. (Supported by NASA NAS2-14263 and Montana Space Grant Consortium NASA EPSCoR)

[127] [129]

DESIGNING FOR VARIABLE GRAVITY GROWTH OF YEAST DEVELOPMENT OF BIOCOMPATIBILITY PROCEDURES FOR ASSESSMENT OF PLANT GROWTH IN GROUND TEST HARDWARE FOR THE EMCS. R. Bowman and M. Steele,

IN THE CELL CULTURE UNIT. F. Donovan1, L. Sun2, H. Park2, G. Vunjak-Novakovic2, S. Walker3, E. Nelson4, J. Kizito5, C. Krebs6, K. Slater6, D. Vandendriesche3, P Larenas1, C. Havens1, N. Searby3. 1Lockheed Martin, Moffett Field, CA; 2MIT, Cambridge, MA; 3NASA ARC, CA; 4NASA GRC, Cleveland, OH, 6Payload Systems Inc, Cambridge, MA.

Lockheed Martin, NASA Ames Research Center, CA. The European Modular Cultivation System (EMCS) is an European Space Agency-developed facility designed to support plant research in microgravity on the ISS. NASA is responsible for providing US specific hardware to use within the EMCS. In preparation for flight, research will be developed and tested at Ames Research Center in the EMCS ground test hardware, the Experiment Reference Module (ERM). In order to determine the acceptability of the ERM for such purposes, biocompati-bility tests will be performed to determine that the hardware functions

Yeast (S. cerevisiae) cultures require homogeneous suspensions for representative cell sampling and optimal gas and nutrient exchange, whether in microgravity (µ-g), on earth (1-g), or in a space centrifuge (hypo-, 1-, and hyper-g). The Cell Culture Unit being developed for the International Space Station utilizes independent control of mixing (via stir bars), gas exchange (via medium recirculation through a gas exchanger), and nutrient supply (via perfusion with periodic partial medium replacement). We empirically derived an algorithm for 1-g: (a) stirring

as intended and successfully supports the growth of plants. In this report, we describe the development of procedures and the collection of baseline data against which to compare ERM function, e.g. biocompatibility testing. A simple and robust system was developed to grow whole Arabidopsis thaliana plants within the confined volumes characteristic of spaceflight hardware. Our system for growing plants eliminated the need for a water/nutrient delivery system and allowed for quantifiable assessment

at 90 rpm with a change in direction every minute, (b) forward/reverse medium recirculation with a net forward flow rate of 0.83 ml/min, and (c) continuous gas exchange to supply oxygen (O2) and remove carbon dioxide (CO2). During four subcultivations, the growth rate, suspension uniformity, and medium concentrations of O2, CO2 and pH were comparable to control flask cultures. The flow patterns were characterized experimentally (by flow visualization and residence time distribution),

of individual plants, as well as population dynamics. To insure uniform germination, seeds were started in small straw segments and transplanted into modified scintillation vials. Seedlings were selected prior to transplantation to decrease genetic variability. Plants were grown for and numerically (by computational fluid dynamics and scaling analysis).

The flow was dominated by stirring and characterized by high dispersion numbers and good mixing. The scaling analysis predicted maximum rotational velocities several orders of magnitude greater than yeast sedimentation and perfusion velocities. Since microgravity may require less stirring, the effects of no and low-rate stirring were evaluated. In the absence of stirring, flow perfused from one end of the chamber to the other with uniform concentration fields. Low rate stirring (5 rpm) generated qualitatively similar flow patterns as stirring at 90 rpm but with weaker rotational structures. Combined cell and flow studies can define protocols that minimize forces while maintaining suspension and a well-mixed nutrient supply at any g-level.

a total of 24 days in standard laboratory plant growth chambers under controlled conditions. Sequential digital still images were taken on a daily basis. Analysis of these images allowed for the quantification of even minute environmental effects on growth dynamics of whole plants. The data collected provide reliable growth curves against which to compare plants grown in the ERM. (Supported by NASA: NAS2-96001)



[130] PROTOTYPE TESTING OF SPACE FLIGHT GREEN FLUORESCENT PROTEIN IMAGING SYSTEM FOR PLANTS. A-L. Paul1, T. Murdoch2, E.J. Ferl3 and R.J. Ferl1. 1Horticultural Sciences Department, Univ. of Florida, Gainesville, FL, 2Bionetics Corp., Kennedy Space Center, FL and 3Buchholz High School, Gainesville, FL The use of Green Fluorescent Protein (GFP) as a reporter gene system is an accurate and non-destructive means for generating organ, tissue and cell-specific data on gene expression in plants. While many commercial imaging systems are available for standard terrestrial laboratory imaging of GFP, none of these systems begin to meet the requirements for a flight unit that could be used to monitor plant gene expression within the confines of current space flight habitats. We present here data derived from ground testing of a prototype GFP flight imaging unit, the TAGES Imaging System (TIS). The TIS uses a system of blue-light LEDs and narrow bandpass filters for excitation, coupled with a CCD camera capturing images at 640x480 pixels resolution, under computer control for camera position, lighting control and imaging scheduling. Experiments conducted with Arabidopsis thaliana expressing various levels of GFP confirm that the system is capable of monitoring GFP expression over time, without disrupting plant growth in any way. The TIS is being developed as an integrated part of the payload hardware design of the next generation Plant Growth Facility (PGF). (Supported by NASA: NAG 10-291)



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ABSTRACTS – PANEL DISCUSSION

[131] PANEL DISCUSSION: LESSONS LEARNED DURING A LONG DURATION LIFE SCIENCE EXPERIMENT ON THE ISS. K. Lagel1, R.C. Morrow2, G. Stutte3, K. Sato1, B.W. McLamb4, K. Stolp3, D. Chapman4, D. Heathcote1, J. Stadler2, R. Berthold5, M. Lee2, D. Cox6. 1Lockheed Martin, NASA Ames Research Center, Moffett Field, CA; 2Orbital Technologies Corporation, Madison, WI; 3Dynamac Corporation, NASA Kennedy Space Center, FL; 4Bionetics Corporation, NASA Kennedy Space Center, FL; 5NASA Ames Research Center, Moffett Field, CA; 6NASA Kennedy Space Center, FL . The Biomass Production System recently returned from a 73-day mission aboard the ISS (Increment 4). The mission consisted of two experiments; the BPS Technology Validation Test and the PESTO wheat photosynthesis experiment. A total of eleven plantings were grown as part of these experiments, resulting in collection of over 320 specimens. Significant crew effort was involved in on-orbit activities such as plant harvesting, priming root modules, pollination, collection of gas and fluid samples, and resupply of water and CO2. A large number of automated operations were also conducted, requiring careful coordination between replan activities, crew activities, and payload command files. BPS was one of the first complex payloads through the new ISS process and resulted in a number of “lessons learned” of use to future flights. The mission duration, level of crew involvement, participation of multiple groups from several NASA centers and corporations, and the evolution of a new template for payload integration combined to make the BPS experiment one of the more complex biological payloads flown to-date. Areas to be discussed by the panel include support of mission operations, crew activities, handling of large data sets, communication between teams and between ground personnel and the ISS, special issues related to biological payloads, the payload integration process, opportunities to expand data collection, and interacting with school outreach programs. This panel discussion will also provide an opportunity for participants in other payloads to share experiences and recommendations for future payloads.



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ABSTRACTS – SYMPOSIUM III

[132] [134] VISIONARIES AND VISIONS OF THE FUTURE IN AERONAUTICS AND SPACE. R. Cassanova1, P. Russell1, R. Turner2. 1NASA Institute for Advanced Concepts, 555-A 14th Street NW, Atlanta, Georgia 30318 and 2ANSER, 2900 South Quincy Street Suite 800, Arlington, Virginia 22206..

SPACE ELEVATOR DESIGN AND DEVELOPMENT. B.C. Edwards, Eureka Scientific, 1904 3rd Ave, Seattle Washington, USA. The objective of our effort has been to develop a viable scenario for the construction, deployment and operation of a space elevator using current or near future technology. This effort has been primarily a paper study with several experimental tests of specific systems. Computer simulations, engineering designs, literature studies and inclusion of existing programs have been utilized to produce a design for the first space elevator. The results from this effort illustrate a viable design using current and near-term technology for the construction of the first space elevator. The timeline for possible construction is within the coming decades and estimated costs are less than $10B. The initial elevator would have a 5 ton/day capacity and operating costs near $100/lb for payloads going

The basic human desire to understand the forces of nature, the mysteries of the cosmos and the role of life in the vastness of the universe has been fascinating scientists and artists for thousands of years. Only within the last hundred years have the level of knowledge and the availability of astronomical instruments and space exploration technology enabled humans to begin a significant leap towards an in-depth understanding of our planet Earth, the solar system and cosmological phenomena. This paper will briefly review the evolution of scientific and artistic discovery that has been the inspiration for the on-going physical exploration of space with robots and humans and the gathering of cosmological data through astronomy. This historical perspective will serve as a background for a description of recent activities of the NASA Institute for Advanced Concepts (NIAC) will be described. NIAC is inspiring, funding and nurturing visionary architectures and systems for space-related endeavors that may revolutionize space travel and human habitation of space.

to any Earth orbit or traveling to the Moon, Mars, Venus or the asteroids. Further engineering studies are required to confirm these designs (This work is supported by a grant from the NASA Institute for Advanced Concepts (NIAC).)

(Supported by NASA Contract NAS5-98051) [133] [135] A PLANT GENETIC ASSESSMENT AND CONTROL SYSTEM FOR SPACE ENVIRONMENTS. T.L. Lomax and W.E. Winner. Dept. of Botany & Plant Pathology, Oregon State University, Corvallis. [email protected]

THE CHAMELEON SUIT – A LIBERATED FUTURE FOR SPACE EXPLORERS? Edward W. Hodgson, Hamilton Sundstrand Space Systems International, 1 Hamilton Road, Windsor Locks, CT 06096 Mankind’s future as a spacefaring species demands the ability to work freely and frequently in space environments. Traditional space suit system design approaches burden both the spacefarer and and the total space mission limiting the extent to which this is possible. The spacefarer is encumbered by the weight and bulk of a pressure suit designed for nearly complete isolation from the environment and a life support system designed to replace everything our environment normally provides.

Plants will play an essential role in providing life support for any long-term space exploration. Plant growth in space environments requires an adaptable system that measures the response of plants to any unique space condition, optimizes plant performance under those conditions, and provides cues for changing environmental factors. We are exploring designs for a flexible module that will monitor plant responses to environmental changes by combining emerging technologies in the fields of plant genomics, microarray analysis of gene expression, bioinformatics, and whole plant physiology.

The space mission is also burdened by this equipment and by all of the expendable materials required for its operation and maintenance. As a result, we are not presently free to work in space as frequently, as long,

The key to our approach is to use microarray analysis to monitor genetic activity for plants growing in defined environments. The project exploits the completed sequences of two plant genomes (Arabidopsis and rice) that are now available, and the many plant genome sequences that will be known for other species in the future. The expression of plant genes can be evaluated using microarray technology that simultaneously measures the responses of thousands of plant genes to environmental change. Expression of specific plant genes will increase and decrease as plants regulate physiological processes to compensate for environmental changes. By systematically monitoring changes in gene expression and environmental factors, the physiological status of plants is defined. Environmental adjustments can then be made to alter gene expression, thereby controlling plant processes needed for growth and other life support processes.

or in all of the locations envisioned in NASA’s long range plans. Under NASA Institute for Advanced Concepts (NIAC) sponsorship (Research grant 07600-082), Hamilton Sundstrand Space Systems International (HSSSI) has been studying an alternative concept, “The Chameleon Suit” that seeks to liberate future exploreers and missions from these limitations. In this concept, the space suit system is made to work with the environment in an adaptive fashion to achieve life support functions with minimum hardware and expendable materials requirements. To achieve this, traditional functions of the life support system are integrated with the pressure suit using emerging materials and design technology. A range of advanced materials and biomimetic technologies and system design options is under study. These include use of shape change polymers and electroemissive materials to modify heat transfer characteristics of the space suit “skin” achieving thermo-regulation analogous to that in natural biological systems. This approach has been shown to be feasible for many space missions during the completed Phase I study program. The current Phase II program is investigating more aggressive technology applications aimed at eliminating the need for almost all of the hardware systrems currently included in the space suit’s life support backpack. This paper will describe the design concept and its possible impact on future human space exploration. The results of its investigation during the completed Phase I study program and the initial stages of the Phase II study that is currently under way will be discussed.

Future additions to the architecture will include the technical advances necessary for remote collection and evaluation of data. In addition, we are studying the major feasibility issues associated with cost, performance, development time, and key technology issues for developing the module for NASA. The research is being carried out in Phase II of a project funded by the NASA Institute for Advanced Concepts.


mailto:[email protected]


[136] HUMAN UTILIZATION OF SUBSURFACE EXTRA-TERRESTRIAL ENVIRONMENTS. P.J. Boston1,2, R.D. Frederick3, J. Werker1, S.L. Thompson4, S.M. Welch,1 Caves have been used in the ancient past as habitat and shelter by many organisms (including hominids). Since antiquity, humans have explored caves for the minerals they contain and sometimes for ceremonial purposes. Over the past century, caves have become the target of increasing exploration, scientific research, and recreation. The use of caves on extraterrestrial bodies for human habitation has been suggested by several investigators. Lunar lava tube bases received early attention because lava tubes were clearly visible in early lunar images. More recently, MOC data shows clear evidence of large tubes visible in a number of volcanic regions on Mars. The budding field of cave geomicrobiology has direct application to questions about subsurface life on other planets. This makes caves and other subsurface habitats a prime target for astrobiological missions to Mars and possibly other bodies. We present the results of a completed Phase I and on-going Phase II NASA Institute for Advanced Concepts (NIAC) study that intensively examines the possibilities of using extraterrestrial caves as both a resource for human explorers and as a highly promising scientific target for both robotic and future human missions to Mars and beyond.. 1Complex Systems Research, Inc. Boulder, CO, 2New Mexico Inst. Min. & Tech., Socorro, NM 3Oregon Public Education Network, Silverton, OR 4Lawrence Livermore Nat. Lab., Livermore, CA


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