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Gravitational and Space Biology

Volume 20, Number 1

November 2006

Publication of the American Society for Gravitational and Space Biology ISSN 1089-988X

ASGSB EDITORIAL BOARD

Augusto Cogoli Zero-G LifeTec GmbH

Zürich, Switzerland

Luis Cubano Univ. Central del Caribe

Camuy, Puerto Rico

Emily Holton

NASA Ames Research Center

Moffett Field, CA

John Kiss Miami University

Oxford, OH

Patrick Masson University of Wisconsin

Madison, WI

Gloria Muday Wake Forest University

Winston Salem, CT

Anna-Lisa Paul University of Florida

Gainesville, FL

April Ronca Wake Forest University

Winston Salem, CT

Gerry Sonnenfeld SUNY Binghamton

Binghamton, NY

Paul Todd SHOT, Inc.

Greenville, IN

Sarah Wyatt Ohio University

Athens, OH

William Landis NE Ohio Univ College of Medicine

Rootstown, OH

PUBLISHING STAFF

Stan Roux Editor-in Chief

University of Texas

Austin, TX

Mary E. Musgrave Publishing Editor

University of Connecticut

Storrs, CT

Robert Blasiak Assistant Editor

Albert-Ludwigs-University of Freiburg

Freiburg, Germany

ii Gravitational and Space Biology 20(1) November 2006

GENERAL INFORMATION

Gravitational and Space Biology (ISSN 1089-988X) is a journal devoted to research in gravitational and space biology. It

is published by the American Society for Gravitational and Space Biology, a non-profit organization whose members share a

common goal of furthering the understanding of the biological effects of gravity and the use of the unique environment of

spaceflight for biological research. Gravitational and Space Biology is overseen by a steering committee consisting of the

Publications Committee, the Editor, the President, and the Secretary-Treasurer of the ASGSB.

The American Society for Gravitational and Space Biology was created in 1984 to provide an avenue for scientists

interested in gravitational and space biology to share information and join together to speak with a united voice in support of

this field of science. The biological effects of gravity have been acknowledged since Galileo’s time, but only since the 1970s

has gravitational biology begun to attract attention. With the birth of the space age, the opportunity for experimentation over

the full spectrum of gravity finally became a reality, and a new environment and research tool became available to probe

biological phenomena and expand scientific knowledge. Space and spaceflight introduced new questions about space

radiation and the physiological and psychological effects of the artificial environment of spacecraft.

The objectives of ASGSB are:

• To promote research, education, training, and development in the areas of gravitational and space biology and

to apply the knowledge gained to a better understanding of the effect of gravity and space environmental

factors on the flora and fauna of Earth.

• To disseminate information on gravitational and space biology research and the application of this research to

the solution of terrestrial and space biological problems.

• To provide a forum for communication among professionals in academia, government, business, and other

segments of society involved in gravitational and space biological research and application.

• To promote the study of concepts and the implementation of programs that can achieve these ends and further

the advancement and welfare of humankind.

MEMBERSHIP: The American Society for Gravitational and Space Biology welcomes individual, organizational, and

corporate members in all of the basic and applied fields of the space and gravitational life sciences. Members are active in the

fields of space medicine, plant and animal gravitational physiology, cell and developmental biology, biophysics, and space

hardware and life support system development. Membership is open to nationals of all countries. Members must have

education or research or applied experience in areas related to the Society’s purposes: i.e., Doctorate, Masters with 2 years

experience, Bachelors with 4 years experience (student members must be actively enrolled in an academic curriculum leading

toward a career related to the Society’s purposes), or special appointment by the Board of Directors. Membership

applications may be obtained by writing the American Society for Gravitational and Space Biology, P.O. Box 2581, Chapel

Hill, NC 27515, or at the society website (http://www.asgsb.org).

Gravitational and Space Biology is sent to all members of the American Society for Gravitational and Space Biology.

Requests for copies, information about subscriptions and membership, changes of address, questions on permission to

reproduce parts of this volume, and other correspondence should be sent to the American Society for Gravitational and Space

Biology P.O. Box 2581, Chapel Hill, NC 27515.

Copyright © 2006 by the American Society for Gravitational and Space Biology

Gravitational and Space Biology 20(1) November 2006 iii

American Society for

Gravitational and Space Biology

Program and Abstracts

for the

Twenty-second Annual Meeting

November 2-5, 2006

Arlington, VA

iv Gravitational and Space Biology 20(1) November 2006

Short Program…………………………………………………………………..………..v

Long Program………………………………………………………………...……...…..ix

Thursday…………………………………..…………...…………..……………..x

Friday Morning……………………….…...……………………………...……...x

Friday Afternoon………………….……………………………..………………xi

Friday Evening…………………………………………………………………..xv

Saturday Morning……………………………………………………..…….…xvi

Saturday Afternoon……………………………...……………….……..………xx

Saturday Evening………………………...………………………...…….……xxii

Sunday Morning……………………………...…………...………………...…xxii

Sunday Afternoon……………..……………………………….………….….xxiii

Abstracts…………………………………………………………………...……….…….1

Symposium I: Cell Mechanics……………………………….…...……..……….1

Posters I-A: Undergraduate Student Poster Competition………….….…...….3

Posters I-B: Graduate Student Poster Competition…………………...……….7

Posters II-A: Undergraduate Student Poster Competition…………………..11

Posters II-B: Graduate Student Poster Competition…………………...…….15

Symposium II: Genetic Adaptation for Stress………………………...……....19

Posters III-C: Cell Biology and Animal Development…….……………….....21

Posters III-D: Plant Development and Gravity Response…...……………… 25

Posters III-E: Education and Funding Opportunities…………….….………29

Oral Session I: Spaceflight Experiment Results………………...………….....31

Oral Session II: Plant Physiology and Gravity Response……...…….…….…35

Town Hall Meeting…………………………………………………………...…39

Symposium III: Integrated Physiology………………………………….…….41

Oral Session III: Space Physiology and Animal Development...….………....45

Oral Session IV: Biotechnology and Cell Biology………………...…..………49

Author Index……………………………………………………………………...……A1 T

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Gravitational and Space Biology 20(1), November 2006 v

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SHORT PROGRAM – 2006 ANNUAL MEETING

vi Gravitational and Space Biology Bulletin 20(1) November 2006

12:00 PM

2:00 PM - 5:00 PM

7:00 PM

Registration Begins

Pre-meeting workshop: Small Satellites as Platforms for

Science, Simon P. ("Pete") Worden, Director, NASA Ames

Research Center, Chair

ASGSB Governing Board meeting

7:30 AM

8:00 AM - 8:30 AM

8:30 AM - 12:00 noon

8:30 AM – 9:15 AM

9:15 AM – 10:00 AM

10:00 AM – 10:30 AM

10:30 AM – 11:15 AM

11:15 AM – 12:00 noon

12:00 – 1:00 PM

1:00 PM – 2:00 PM

Registration Opens

Opening remarks and welcome, John Z. Kiss, Chair

Symposium I: Cell Mechanics

Jack van Loon, Symposium Chair

Jack van Loon, Vrije Universiteit, Amsterdam

L. Romer, Johns Hopkins University School of Medicine

Break

Anja Geitmann, IRBV, Montreal

Patric Prendergast, Trinity College, Dublin, Ireland

Workshop: Alternate Funding Opportunities, Simon

Gilroy, Chair

Lunch, ASGSB committee meetings

Thursday, November 2 (Pre-Meeting)

Friday, November 3


Gravitational and Space Biology Bulletin 20(1) November 2006 vii

2:00 PM – 3:30 PM

3:30 PM

3:30 PM – 5:00 PM

6:30 PM – 9:30 PM

Concurrent Posters - Session I

A. Undergraduate Student Poster Competition

B. Graduate Student Poster Competition

Break Set Out

Concurrent Posters - Session II

A. Undergraduate Student Poster Competition

B. Graduate Student Poster Competition

Reception

8:00 AM - 10:30 AM

8:00 AM – 8:15 AM

8:15 AM – 9:00 AM

9:00 AM – 9:45 AM

9:45 AM – 10:30 AM

10:30 AM – 11:00 AM

11:00 AM – 12:30 PM

12:30 PM – 2:00 PM

2:00 – 4:00 PM

Symposium II: Genetic Adaptation for Stress

Karl Hasenstein, Symposium Chair

Karl Hasenstein, University of Louisiana, Lafayette

Hans Bohnert, University of Illinois, Urbana-Champaign

Alfred Goldberg, Harvard Medical School

Susan Rosenberg, Baylor College of Medicine

Break

Concurrent Posters III

Lunch and Committee Meetings

Concurrent Oral Sessions

I. Spaceflight Experiment Results, D. Marshall

Porterfield, Chair

II. Plant Physiology and Gravity Response, Elison

Blancaflor, Chair

Saturday, November 4


viii Gravitational and Space Biology Bulletin 20(1) November 2006

4:00

4:00 – 5:30 PM

6:30 - 9:00 PM

9:30 PM

Break Set Out

Special Session: Town Hall Discussion of ASGSB’s

Future, Chris Brown, Past President, Chair

Banquet and Business Meeting

Student Mixer

7:00 AM – 8:30 AM

8:30 AM – 12:30 PM

8:30 AM – 8:45 AM

8:45 AM – 9:25 AM

9:25 AM - 10:05 AM

10:05 AM – 10:30 AM

10:30 AM – 11:10 AM

11:10 AM – 11:50 AM

11:50 AM - 12:05 PM

12:05 – 1:30 PM

1:30 – 3:30 PM

ASGSB Governing Board Meeting

Symposium III: Integrated Physiology

Eberhard Horn, Symposium Chair

Eberhard Horn, Ulm University, Ulm

Rupert Gerzer (DLR-Flight Medicine, Cologne)

Charles Fuller, UC Davis

Break

Gilles Clement, CNRS-Universite, Toulouse

Stephen Moorman, University NJ, Piscataway

Eberhard Horn

Lunch

Concurrent Oral Sessions

III. Space Physiology and Animal Development,

Joseph Tash, Chair

IV. Biotechnology and Cell Biology

Peter Scherp, Chair

Sunday, November 5

Gravitational and Space Biology 20(1) November 2006 ix

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LONG PROGRAM – 2006 ANNUAL MEETING

x Gravitational and Space Biology 20(1) November 2006

12:00

2:00

7:00

Registration Begins

Pre-meeting workshop: Small Satellites as Platforms for Science, Simon P.

("Pete") Worden, Director, NASA Ames Research Center, Chair

ASGSB Governing Board meeting

7:30

8:00

Registration Opens

Opening remarks and welcome: John Z. Kiss, Program Chair

Start

8:30

9:15

10:00

10:30

11:15

12:00

1:00

Cell Mechanosensing and Gravity. Jack J.W.A. van Loon. [1]

Measuring Patterns, Regulation, and Biologic Consequences of Cellular

Traction Forces. L. Romer. [2]

Break

Quantitative Assessment of Cytomechanical Parameters in Plants - a

Challenge for Micromanipulation. A. Geitmann. [3]

Computational Modelling of Cell Responses to Mechanical Forces. P.J.

Prendergast and J. Klein-Nulend. [4]

Workshop: Alternate Funding Opportunities, Simon Gilroy, Chair

Lunch, ASGSB Committee Meetings

Page

2

2

2

2

Thursday, November 2

Friday Morning, November 3

Scientific Symposium I

Cell Mechanics

8:30 - 12:00 Jack van Loon, Symposium Chair


Gravitational and Space Biology 20(1) November 2006 xi

Poster

#

A01

A03

A05

A07

A09

A11

A13

A15

Engineering Design and Scientific Evaluation of a Novel Animal Support

Module for the Mars Gravity Biosatellite. E.S. Koksal, E.C. Guerra, A.M.

Heafitz, J.A. Hoffman, E.B. Wagner, P.L. Yang and T.R.F. Fulford-Jones. [5]

Effect of Hypergravity on Expression of Receptors Associated with

Lactogenesis in Rat Mammary Gland from Pregnancy to Lactation. M.K.

VanKlompenberg, O.V. Patel, H. Dover and K. Plaut. [6]

Examining Biological Variability: A Microarray Study. A.L. Kuntz, M.L.

Molas, and J.Z. Kiss. [7]

Coupled Effects of Temperature and Simulated Microgravity (Clinostat)

on E. coli Population. F.N. Ahmed, H.N. Howard and D.M. Klaus. [8]

Characterization of Gravity Sensing Mechanisms in Arabidopsis. A.

Lomax, C.Y. Hung, and I. Perera. [9]

Identification and Characterization of ARF9 as a GPS3-like Mutant. D.R.

Roberts, and S.E. Wyatt. [10]

Is Transcriptional Regulation of Gravitropism Conserved Between Dicots

and Monocots? R.L. Hammond, H. Myburg, I.Y. Perera, C.S. Brown and H.

Winter Sederoff. [11]

The Influence of Antibiotics on Bacterial Motility and its Implication for

Drug Efficacy in Microgravity. A.R. Stanczyk and D.M. Klaus. [12]

Page

4

4

4

4

5

5

5

5

Posters I-A

Undergraduate Student Poster Competition

Note: Presenters are to be next to their posters the entire time.

Friday Afternoon, November 3

Concurrent Poster Sessions I-A and I-B

2:00 – 3:30


xii Gravitational and Space Biology 20(1) November 2006

Poster

#

B01

B03

B05

B07

B09

B11

B13

B15

B17

Design and Validation of Atmospheric Life Support Systems for the Mars

Gravity Biosatellite. T.R.F. Fulford-Jones, A.M. Heafitz, E.B. Grosse, M.A.

Scott, J.C. Franklin and J.A. Hoffman. [13]

Seed Germination and Growth in Hypobaria. H.L. Gohil, J.R. Truett, A.J.

Stimpson, R.A. Bucklin and M.J. Correll. [14]

The Musculoskeletal Effects of Partial Weightbearing in Mice. E.B.

Wagner, N.P. Granzella. [15]

A MEMS Based In-Silico Cell Electrophysiology Device for Monitoring

Transcellular Calcium Currents in Ceratopteris richardii Fern Spores. A.

ul Haque, M. Rokkam, A.R. DeCarlo, S.T. Wereley, S.J. Roux, P.P. Irazoqui,

D.M. Porterfield. [16]

Cytoskeleton Changes in Mesenchymal Stem Cells Differentiate to

Osteoblasts and Chondrocytes and Effect of Electric Stimulation During

Three-dimensional Scaffold Culture. C. Umeda, Y. Kawahara, R.

Yoshimoto, T Kajiume, A. Sasaki, S.L. Wu, K. Naminohira and L. Yuge. [17]

Hypergravity and Hydrostatic Pressure Loading Resulted in Different

Modeled Stress and Strain but the Same Osteoblast Functional Response.

J.S. Alwood, E.A. Almeida, R.K. Globus and N.D. Searby. [18]

Proliferation of Hematopoietic Stem Cells Is Stimulated in 3D-clinostat

Culture. R. Yoshimoto, T. Kajiume, Y. Kawahara, C. Umeda, A. Sasaki, S.L.

Wu, K. Naminohira, K. Kataoka, and L. Yuge. [19]

The Effect of Combined Simulated Microgravity and Microgrooved

Surface Topography on Fibroblasts. W.A. Loesberg, X.F. Walboomers,

J.J.W.A. Van Loon, J.A. Jansen. [20]

Development of a Synthetic Seed Production Method for Long-Term

Transport and Advanced Life Support. J.E. Porter and T.P. West. [21]

Page

8

8

8

8

9

9

9

9

10

Posters I-B

Graduate Student Poster Competition



Gravitational and Space Biology 20(1) November 2006 xiii

Poster

#

B19

B21

B23

A02

A04

A06

A08

Characterization of Cytochrome P450 Proteins that Regulate Plant

Gravitropism. J.C. Withers and S.E. Wyatt [22]

Positive Phototropism in Roots: Who is Involved? M.L. Molas, M.J.

Correll, J.Z. Kiss. [23]

Dietary ROS Scavenger, alpha-Lipoic Acid, Prevents Testicular Atrophy

During 10 Wk Hindlimb Unloading (HLS) J. Zenisek, S. Wolfe, B.D.

Timmerberg, J.S. Tash. [24]

DNA-Based Life Detection on Earth and Mars: Polymerase Chain

Reaction Optimization Using Short Ribosomal Primers. N.M. Vahora, C.E.

Carr, M.T. Zuber, and G. Ruvkun. [25]

Analysis of Complex Biomarkers of Musculoskeletal Atrophy and Stress

in Preserved Mouse Urine for the Mars Gravity Biosatellite. V.Y. Chang,

E.B. Wagner. [26]

Neutrophil Activation in Transitional Gravity. A.W. von Deutsch, A.S.

Belton, R. Flowers, J. Wright, C. Williams, N.A. Silvestrov, D.F. Paulsen, B.J.

Klement and D.A. von Deutsch. [27]

Ultracellular Localization of ARG1 in Arabidopsis Columella Cells. A.K.

Spence, N.S. Kumar, R.E. Edelmann, and J.Z. Kiss. [28]

Page

10

10

10

12

12

12

12

Friday Afternoon, November 3

Concurrent Poster Sessions II-A and II-B

3:30 – 5:00

Posters II-A

Undergraduate Student Poster Competition



xiv Gravitational and Space Biology 20(1) November 2006

Poster

#

A10

A12

A14

B02

B04

B06

B08

B10

B12

The Effects of PHYC and PHYD on Tropistic Responses of Arabidopsis. C.E. Montgomery, P. Kumar and J.Z. Kiss. [29]

Elucidating Genes Involved in Early Gravity Signal Transduction in

Arabidopsis. J. Bascom and S.E. Wyatt. [30]

Morphological and Physiological Characterization of Transgenic Tomato

Lines Expressing Inositol Polyphosphate 5-phosphatase. C. Sword, M.

Khodakovskaya, I.Y. Perera, C.S.Brown, H. Winter Sederoff. [31]

Identifying the Neuronal Basis of Graviperception in Drosophila melanogaster. C.J. Johnson, M.T. Texada, R. Simonette, K.M. Beckingham.

[32]

Longitudinal Hindlimb Suspension Study Reveals Physical, Functional

and Biochemical Changes in Mouse Skeletal Muscle. A. Hanson, M.

Young, L. Stodieck and V. Ferguson. [33]

Effect of Local Iron Irradiation on Trabecular Bone. E.R. Bandstra, S.

Judex, M.E. Vazquez, T.A. Bateman. [34]

The Effect of Low-Shear Modeled Microgravity on Adherent Invasive

Escherichia coli Physiology and Virulence Potential. C.A. Allen, D.W.

Niesel, and A.G. Torres. [35]

Effects of Simulated Microgravity on Nitric Oxide Production and

Proteoglycan Synthesis by Chondrocytes Encapsulated in 3D PEG

Hydrogels. I. Villanueva, B. Klement, D. von Deutsch, and S.J. Bryant. [36]

Identification of Mechanosensitive Genes in Osteoblasts by Comparative

Microarray Studies Using the Rotating Wall Vessel and the Random

Positioning Machine. M.J. Patel, W. Liu, M.C. Sykes, N.E. Ward, S.A. Risin,

D. Risin, H. Jo. [37]

Page

13

13

13

16

16

16

16

17

17

Posters II-B

Graduate Student Poster Competition



Gravitational and Space Biology 20(1) November 2006 xv

Poster

#

B14

B16

B18

B20

B22

6:30

Simulated Microgravity Inhibits Neural Differentiation of Mouse Bone

Marrow Cells. M. Takeda, T. Okazaki, T. Magaki,

A. Sasaki, S.L. Wu, Y.

Kawahara, C. Umeda, R. Yoshimoto, T. Kajiume, L. Yuge, and K. Kurisu. [38]

Electrical Stimulation of Myoblasts Develops Spontaneously Contracting

Muscle Fibers. Y. Kawahara, K. Yamaoka, C. Umeda, R. Yoshimoto, T.

Kajiume, A. Sasaki, S.L. Wu, K. Kataoka, and L. Yuge. [39]

The Effect of Microgravity on the Arrangement of Amyloplasts in

Hypcotyl Endodermal Cells. D. Cirelli and J.Z. Kiss. [40]

Effect of arg1 Mutation on Plastid Sedimentation in Arabidopsis

Hypocotyls. N.S. Kumar and J.Z. Kiss. [41]

Microgravity Induced Changes in Aortic Stiffness and its Role in

Orthostatic Intolerance. E.C. Tuday, J.V. Meck, A.A. Shoukas, D.E.

Berkowitz. [42]

Reception

Page

17

17

18

18

18

Friday Evening, November 3


xvi Gravitational and Space Biology 20(1) November 2006

Start

8:00

8:15

9:00

9:45

10:30

Symposium: Genetic Adaptation for Stress. K.H. Hasenstein. [43]

Plant Adaptation To Abiotic Stress – From Physiology To Genomics. H.J.

Bohnert. [44]

Key Roles of the Proteasome in Cell Protection. A.L. Goldberg. [45]

Stress-induced Mutagenesis in Bacteria and the Regulation of

Evolvability. S. Rosenberg. [46]

Break

Page

20

20

20

20

Saturday Morning, November 4

Scientific Symposium II

Genetic Adaptation for Stress

8:00 - 10:30 Karl Hasenstein, Symposium Chair


Gravitational and Space Biology 20(1) November 2006 xvii

Poster

#

C01

C02

C03

C04

C05

C06

C07

ß-Actin Protein Concentration Fluctuates During Contraction of Loaded

and Unloaded Fibroblast Populated Collagen Gels. B.P. Johnson-Wint and

W. Heisler. [47]

Saccharomyces Cerevisiae Uses Gas Producing Anaerobic Metabolic

Pathways During Space Flight. T.G. Hammond, C.A. Nickerson, J. Freeman,

L.S. Stodieck, and P.L. Allen. [48]

Differences in Gene Regulation in E. coli Grown on Four Common

Modeled Microgravity Systems. E.A. Juergensmeyer, M.A. Juergensmeyer

and E.M. Mobley [49]

Utilization of NASA Managed Cold Stowage Resources for Optimal

Culture Incubation and Storage of SPEGIS Canisters. K. Sato, D. Connor,

J. Dean, D. Melendez, D.W. Niesel, N. Williams, U. Pandya, S. Ormsby, K.

Gibbs, K.E. Perkins and H.E. Teal. [50]

Gravitational Effects on Glucose Diffusion into Cartilage Tissue. R.E.

Flowers-Aime, C.M. Marshall, A.W. von Deutsch, A.A. Belton, J. Wright,

D.A. von Deutsch, D.F. Paulsen and B.J. Klement. [51]

Geotaxis of Infant Rodents: Think Positive. J.R. Alberts. [52]

Influence of Space Flight on the Genetics of Streptomyces lividans 66 –

pIJ702. T.L. Goins, V.G. Martinson, V.Yu. Tabakov, T.A. Voeikova, and

B.H. Pyle. [53]

Page

22

22

22

22

23

23

23

Posters III-C

Cell Biology and Animal Development


Concurrent Poster Sessions III C, D, E

11:00 – 12:30


xviii Gravitational and Space Biology 20(1) November 2006

Poster

#

D01

D02

D03

D04

D05

D06

D07

D08

D09

D10

D11

The SHL1 and SHL5 Genes Influence both Red- and Blue-light-based

Phototropism in Arabidopsis thaliana. P. Kumar and J.Z. Kiss. [54]

Metabolic and Genomic Profiling of Transgenic Tomato Lines Expressing

Inositol Polyphosphate 5-Phosphatase. M. Khodakovskaya, C-Y. Hung, I.

Perera, W. Boss, C. Brown and H. Winter Sederoff. [55]

Genetic Analysis of Gravity Signal Transduction in Arabidopsis Roots. P.H. Masson, B.R. Harrison and J. Stanga. [56]

Overexpression of Arabidopsis Fatty Acid Amide Hydrolase (FAAH)

Promotes Plant Growth and Modifies Sensitivity to Exogenous N-

acylethanolamines. Y.-S. Wang, R. Shrestha, K.D. Chapman, and E.B.

Blancaflor. [57]

Ethylene Regulation of Gravitropic Curvature in Arabidopsis Stems. M.A.

Harrison and M.L. Brown. [58]

Use of Rotato/Random Positioning Machine (R/RPM) Technology to

Investigate Gravity Sensing and the Gravitropic Motor Response of Maize

Roots. H. Ishikawa, E. Natori, and M.L. Evans. [59]

Analysis of the Triphasic Motor Response in Maize Root Gravitropism.

E. Natori, M.L. Evans, and H. Ishikawa [60]

Effect Of Solution Density On Growth And Gravitropic Response Of

Maize Roots. T.J. Mulkey. [61]

Seed Production in Hypergravity in Brassica and Arabidopsis. M.E.

Musgrave, A. Kuang, J. Allen, R. Darnell, R. Wagers-Hughes and J. Blasiak.

[62]

Inedible Biomass Biodegradation Kinetics for Advanced Life Support

Systems. J.C. Ramirez-Perez, P.F. Strom, and J. Hogan. [63]

Generation of Edible Biomass by the Deployable Vegetable Production

System (VEGGIE) in Unit Gravity. L.K. Tuominen, A.M. Rogney, and R.C.

Morrow. [64]

Page

26

26

26

26

27

27

27

27

28

28

28

Posters III-D

Plant Development and Gravity Response



Gravitational and Space Biology 20(1) November 2006 xix

Poster

#

E01

E02

E03

E04

12:30

Flies in Space Website. C.S. Elland, B.J. Navarro, J. Fernandez, B.H. Day, K.

Sato, S. Bhattacharya, J. Bulkowski. [65]

Radiation Biology Educator Guide. C.S. Elland, B.J. Navarro, J.C. Rask, Y.

Kovo, W.A. Vercoutere. [66]

Microbial Systems and the Space Flight Environment. D.M. Klaus. [67]

Grand Visions for Life and Living in Space. D.E. Jennings, R.E. Turner and

R.A. Cassanova. [68]

Lunch and Committee Meetings

Page

30

30

30

30

Posters III-E

Education and Funding Opportunities



xx Gravitational and Space Biology 20(1) November 2006

Start

2:00

2:15

2:30

2:45

3:00

3:15

3:30

3:45

Gravity Dependent Ca++

Signaling in Ceratopteris Measured Using a

MEMS Based In-Silico Cell Electrophysiology Sensor Device. D.M.

Porterfield, S.J. Roux, A. ul Haque, M. Salmi, W.T. McLamb, M. Rokkam,

A.R. DeCarlo, S.T. Wereley. [69]

Spaceflight-Induced Gene Expression Changes in the Mouse: Results from

STS-108. K. Schweighofer, T. G. Hammond, P. L. Allen, L. S. Stodieck, P. J.

Kostenuik, T. A. Bateman, S. Morony, D. Lacey, S.Y.C. Chang, and A.

Pohorille. [70]

Neuronal Excitability in Rat Hypothalamus Responding to Microgravity

Stimulus During Parabolic Flight. Y. Kumei, J. Zeredo, M. Ogasawara, H.

Suzuki, T. Fujishima, K. Fukui, G. Fukushima, M.Kimoto, and K. Toda. [71]

Vestibular Otolith Development In Spaceflight and Hypergravity. J.D.

Dickman, A. Lysakowski, D. Huss, and S. Price. [72]

Skeletal Muscle Changes Following Six-Month Spaceflight. D.A. Riley,

J.L.W. Bain, R.H. Fitts, J.G. Romatowski and S.W. Trappe. [73]

Lessons and Recommendations from over a Decade of Microgravity Plant

Growth Experience. G.E. Bingham, S. B. Jones, B. Bugbee, M.A. Levinskih,

I.G. Podolski and V.S. Sychev. [74]

Countering Spaceflight Effects on C. elegans Biology. F. Selch, N.J.

Szewczyk, and C.A. Conley. [75]

POEMS: Passive Observatories for Experimental Microbial Systems. M.S.

Roberts, M.N. Birmele, D.W. Reed, and T.E. Mortenson. [76]

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Saturday Afternoon, November 4

Concurrent Oral Sessions I and II

2:00 – 4:00

Oral Session I

Spaceflight Experiment Results

D. Marshall Porterfield, Chair


Gravitational and Space Biology 20(1) November 2006 xxi

Start

2:00

2:15

2:30

2:45

3:00

3:15

3:30

3:45

4:00

4:00

Effects of Atmospheric Pressure on The Survival of Photosynthetic

Microorganisms During Simulations of Ecopoesis. D.J. Thomas, L.M.

Eubanks, C. Rector, J. Warrington and P. Todd. [77]

Aerotaxis in a Calcifying Alga does not Require Photosynthesis. J. Duke,

C. Auzenne and M. Marsh. [78]

Assessing the Role of Calcium Pumps in the Gravity Response in Single-

Celled Spores of Ceratopteris richardii. S.J. Roux, T.J. Bushart, A. ul Haque,

and D.M. Porterfield. [79]

Gravity and Light: The Role of Membrane Components in Tropic

Responses. H. Winter Sederoff, M. Khodakovskaya, J.M. Kimbrough, R.

Salinas-Mondragon, and C.S. Brown. [80]

An Arabidopsis Root Hair Mutant with Altered Growth Directionality

Displays Defective Cytoskeletal Dynamics in vivo. E.B. Blancaflor and C.-

M. Yoo. [81]

Effects of Gravity on Gene Expression in the Maize Pulvinus: Unraveling

the Roles of Transcription and Translation. H. Myburg, R. Salinas-

Mondragon, R.L. Hammond, I.Y. Perera, E. Davis, C.S. Brown and H. Winter

Sederoff. [82]

Optimizing the Growth Conditions of Arabidopsis thaliana from Seed-to-

Seed in the EMCS on ISS. A-I. Kittang, B.G. Solheim, H. Svare, G. Rakvaag,

A. Johannes, T-H. Iversen [83]

LEDs for Crop Production: Comparisons of Overhead vs. Intracanopy

Lighting. G.D. Massa, M.E. Mick, C.A. Mitchell. [84]

Break Set Out

Special Session: Town Hall Discussion of ASGSB’s Future, Chris Brown,

Past President, Chair

Page

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39

Oral Session II

Plant Physiology and Gravity Response

Elison Blancaflor, Chair


xxii Gravitational and Space Biology 20(1) November 2006

6:30

9:30

7:00

Start

8:30

8:45

9:25

10:05

10:30

11:10

11:50

12:05

Banquet and Business Meeting

Student Mixer

ASGSB Governing Board Meeting

Integrated Physiology - Its Competition and Cooperation with Molecular

and Genetic Research. E.R. Horn. [85]

Human Flight to Mars: Challenges for Integrative Human Physiology. R.

Gerzer, M. Heer, K. Ivanova. [86]

How Time Flies - The Effects of Spaceflight on the Circadian Timing

System. C.A. Fuller. [87]

Break

Sensory-Motor Function in Microgravity. G. Clément. [88]

Simulated-Microgravity Induced Changes in Gene Expression in

Zebrafish Embryos Suggest that the Primary Cilium is Involved in

Gravity Transduction. S.J. Moorman and N. Shimada. [89]

Remarks by Chairman, Eberhard Horn

Lunch

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Saturday Evening, November 4

Sunday Morning, November 4

Scientific Symposium III

Integrated Physiology

8:30 - 12:30 Eberhard Horn, Symposium Chair


Gravitational and Space Biology 20(1) November 2006 xxiii

Start

1:30

1:45

2:00

2:15

2:30

2:45

3:00

3:15

Studies of john glenn (jog) and alan shepard (alan), two Drosophila genes

with Roles in Gravitaxis. K. M. Beckingham, V. Konduri and S. Bjorum.

[90]

Effects of Spaceflight and Altered Gravity on Reproductive Processes of

Female Mammals. A.E. Ronca. [91]

Delayed Testis Development and Altered Gene Transcription in Male Rats

Exposed to Continuous Artificial Gravity from Gestational Day 9

Through Post-Natal Day 21. J.S. Tash, S. Wolfe, B.D. Timmerberg, L.A.

Baer, A.E. Ronca. [92]

Behaviour of Very Low- & Low- Frequency Components in Heart Rate

Variability Power Spectra During 6 Hours of Exposure to Microgravity

Simulated as Thermo-neutral, Dry, Supine Immersion. K. K. Tripathi,

M.D. Dangi, R. Kumar. [93]

Artificial Gravity as a Multi-System Countermeasure to Bed Rest

Deconditioning: Preliminary Results. L.E. Warren, W.H. Paloski, and L.R.

Young. [94]

Space Radiation and Bone Loss. T.A. Bateman, M.J. Pecaut, E.R. Bandstra,

J.S Willey, N.D. Travis, G.A. Nelson, D.S. Gridley. [95]

High-energy (HZE) Radiation Exposure Causes Delayed Axonal

Degeneration and Astrogliosis in the Central Nervous System of Mice. P.

Cummings, A. Obenaus, D. Heffron, G. Nelson, J.W. Mandell. [96]

Effects of Radiation on Macrophage Function. M.J. Pecaut, F. Baqai, E.

Bayeta and D.S. Gridley. [97]

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Sunday Afternoon, November 5

Concurrent Oral Sessions III and IV

1:30 – 3:30

Oral Session III

Space Physiology and Animal Development

Joseph Tash, Chair


xxiv Gravitational and Space Biology 20(1) November 2006

Start

1:30

1:45

2:00

2:15

2:30

2:45

3:00

3:15

Did Earth-Mars Meteoritic Exchange Spread Microbial Life? DNA-Based

Life Detection for Mars, with Human Spaceflight Applications. C.E. Carr,

M.T. Zuber, and G. Ruvkun. [98]

Detection Of Strand Cleavage And Oxidation Damage Using Model DNA

Molecules Captured In A Nanoscale Pore. V. DeGuzman, A. Solbrig, B.

Nogal, W. Vercoutere. [99]

High-Resolution Gene Expression Profiling in Single Cells and Small

Tissues P. Scherp and K.H. Hasenstein. [100]

Effect of Gravity Vector Changes on Intracellular Calcium Control in

Cultured Glial Cells. M.A. Masini, F. Strollo, F. Ricci, P. Prato, and B. Uva.

[101]

Hypergravity Increases Osteoblastogenesis and Osteoclastogenesis by

Bone Marrow Cells in Culture. H. Kondo, R.K. Globus, C. Limoli, E.

Almeida, D.J. Loftus, W. Vercoutere, R. Mojarrab, M. Atwal, N.D. Searby.

[102]

Gravity Dependence of Leukocyte Adhesion to a Simulated Blood Vessel

Wall. D.F. Kucik, R.L. Rouleau, L.W. Smith, and K.B. Gupta. [103]

Influence of 3D Clinorotation on the Activation of Rho Involved in Actin

Fiber Remodeling. A. Higashibata, M. Imamizo-Sato, M. Seki, T. Yamazaki

and N. Ishioka. [104]

Two Special Instruments for the Study of Simulated Altered Gravity.

M.A. Benjaminson, S. Lehrer, J.A. Gilchriest, L. Schonbrun and M.B.

Madigan. [105]

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Oral Session IV

Biotechnology and Cell Biology

Peter Scherp, Chair

ABSTRACTS – 2006 ANNUAL MEETING ISSUE

Gravitational and Space Biology 20(1) November 2006 1

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Cell Mechanics


2 Gravitational and Space Biology 20(1) November 2006

[1] CELL MECHANOSENSING AND GRAVITY.

Jack J.W.A. van Loon, Dutch Experiment Support Center (DESC), ACTA-

Vrije Universiteit van der Boechorststraat 7, 1081 BT Amsterdam, The

Netherlands. Web: www.desc.med.vu.nl, E-mail: [email protected].

Sensing gravity by ‘non-professional’ cells still puzzles many of us. Are

the effects direct or indirect. And if they were direct, what would be the

sensing organel or mechanism within a cell. These questions in

‘gravisensing’ are not much different from questions in general

mechanosensing. Various research groups are involved in probing cell

mechanicals. What are the mechanical properties of a cell. Are there

differences in mechanical properties between cell types and if so why.

With relatively novel techniques such as optical and magnetic tweezer,

atomic force microscopy but also computer modelling a whole field of

reseach has been opened up. A similar approach could also be applied for

specific gravity related research.

In this paper, as introductory part of the cell mechanics workshop, I will

address the current techniques used in mechanosensing and stress some

points in how a cell could sense the relatively insignificant force of

gravity.

[2] MEASURING PATTERNS, REGULATION, AND BIOLOGIC

CONSEQUENCES OF CELLULAR TRACTION FORCES. L. Romer, Departments of Anesthesiology and Critical Care Medicine,

Pediatrics, and Cell Biology, Johns Hopkins University School of

Medicine, Baltimore, Maryland.

The exchange of mechanical signals between mammalian cells and their

extracellular matrix microenvironment is a focus of keen interest for

biologists in the diverse fields of developmental organogenesis, vascular

wall function, tissue engineering, and oncology. The molecular machinery

of cellular mechanical signal response includes at least three major

components: transmembrane adhesion receptors for extracellular matrix;

the microfilament and microtubule cytoskeletal systems; and regulatory

elements including the Rho family of GTP-binding proteins that modulate

the density and contraction state of cytoskeletal elements. Complex

signaling pathways link these three arms of mechanical signal response,

and gravitational forces have been shown to alter some cytoskeletal

dynamics.

Arrays of microfabricated PDMS posts have provided a system for

reporting the traction force at individual cell-matrix interaction sites.

Volumetric imaging of posts with known spring constants allows the

efficient analysis of maps of cell traction force vectors and their

coincidence with both actin polymerization and signal molecule

localization. We have defined unique patterns of force generation that are

specific for cells of fibroblast, endothelial, epithelial, and smooth muscle

lineages. Additionally, the contributions of signaling molecules, including

non-receptor tyrosine kinases and GTPases, to the geometry and

magnitude of cell-matrix adhesion forces have been determined. Cell-

generated mechanical forces directly affect patterning of the matrix milieu

and the mechanical cues that are stored in it. These investigations have

important implications for understanding and optimizing cellular growth

behaviors during tissue development and repair.

(Supported by NIH and JHU-SOM FMD.)

[3] QUANTITATIVE ASSESSMENT OF CYTOMECHANICAL

PARAMETERS IN PLANTS - A CHALLENGE FOR MICROMANIPULATION. A. Geitmann, Institut de recherche en

biologie végétale, Département de sciences biologiques, Université de

Montréal, Montréal, Québec, Canada.

The mechanical behavior of organisms and organs is determined by the

physical properties of their building blocks, the cells and their components.

Cells act as mechanical structures whose morphogenesis is largely

determined by the physical properties of its components. Cells are able to

resist external physical forces such as gravity and to exert forces by way of

growth processes. They are also influenced by external physical forces

which play a fundamental role in the regulation of cell functions, including

gene induction, protein synthesis, cell growth, and death. To appreciate

and eventually model the mechanical aspect of cellular architecture and to

understand how cells are able to perceive and to react to applied

mechanical forces, we need to investigate and quantify their physical

properties.

The principal concept of quantitative mechano-physical studies consists

in the application of calibrated loads or deformations and the subsequent

quantification of the object's response. Assessing physical properties at the

cellular and subcellular level implies a technical challenge, however, since

experimental devices in the appropriate dimension - often making use of

micromanipulation - have to be developed.

All eukaryotic cells share at least two structural building elements that

determine cellular morphology: the cytoskeleton and the plasma

membrane. In plants, fungi and watermolds two additional features

contribute to cellular structure and mechanical behavior: the cell wall and

the turgor pressure. In this presentation, an overview will be given over the

various experimental approaches that have been developed to assess the

physical properties and the mechanical behavior of plant and fungal cells.

These include micro- and nano-indentation, atomic force microscopy,

instron techniques and tonometer approaches. Finally, devices will be

presented that are used to quantify dynamic cellular activities and the

generation of forces at cellular and subcellular level.

(Supported by NSERC, FCI, FQRNT, HFSP.)

[4] COMPUTATIONAL MODELLING OF CELL RESPONSES TO

MECHANICAL FORCES.

P.J. Prendergast1 and J. Klein-Nulend2 1Trinity Center for Bioengineering, School of Engineering, Trinity

College, Dublin, Ireland; 2Department of Oral Cell Biology, ACTA-Vrije

Universitiet Amsterdam, The Netherlands

The objectives of this research are to better understand the role of

mechanical forces in regulating expression of osteoblast-like cells. The

methods used in the investigation coupled computer modeling of the cell

using finite element analysis (FEA) with experimental approaches that

stimulated confluent layers of cells under fluid flow or substrate cyclic

strain (McGarry et al., FASEB Journal, 2005). The finite element model

represented the structural features of the cell, including the cell membrane,

cytoskeleton of actin filaments and microtubules (the latter formed into a

tensegrity network ), and the cytoplasm and nucleus. A spread

conformation was generated for the cell, and forces was applied of either

(i) shear force on the surface to model stimulation by fluid flow or (ii)

deformation at the substrate/cell boundary to model stimulation by

substrate stretch.

The experiments showed that fluid flow generating 0.6 MPa shear stress

had a 7.1-fold and 3.3- fold increase in NO and PGE2 production

respectively whereas the response to 0.1% substrate strain was only 1.65-

fold and 1.4-fold increase. On the other hand, the substrate strain had more

than double the amount of Collagen Type 1 production relative to fluid

flow. Differences in the outcome of mechanical stimulation by fluid flow

versus substrate strain may be explained by FEA. The model shows an

approximately 7.5-fold higher membrane stress due to fluid flow compared

to substrate strain; therefore membrane stressing may be correlated with

the release of these signaling molecules. The FEA model shows higher

stresses on the cell substrate attachments under substrate strain compared

to fluid flow indicating this is trans-membrane integrin may be involved in

regulating collagen I production to mechanical strain.

Supported by the Programme for Research in Third Level Institutions in

Ireland and the BITES project funded by the European Commission.



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[5] ENGINEERING DESIGN AND SCIENTIFIC EVALUATION OF A

NOVEL ANIMAL SUPPORT MODULE FOR THE MARS

GRAVITY BIOSATELLITE. E.S. Koksal1, E.C. Guerra2, A.M.

Heafitz3, J.A. Hoffman1, E.B. Wagner2,5, P.L. Yang4 and T.R.F. Fulford-

Jones1, 1MIT Department of Aeronautics and Astronautics, 2Payload

Systems, Inc., 3MIT Edgerton Center, 4MIT Department of Electrical

Engineering and Computer Science, 5Harvard-MIT Division of Health

Sciences and Technology, Cambridge, MA.

The Mars Gravity Biosatellite is a student-led spacecraft development

program. It will provide an artificial gravity testbed to study mammalian

adaptation to partial gravity. The 5-week mission profile specifies a launch

of fifteen female mice and uses both rotational and non-rotational time-

delayed ground controls.

Each Animal Support Module (ASM) is designed to house a single

mouse in a Mars-equivalent 0.38-g environment. The design includes the

specimen chamber and an underfloor waste collection zone, together with

a suite of sensors and actuators to gather scientific data in accordance with

Mars Gravity Biosatellite mission requirements. The ASM is designed to

interface with separate atmospheric and thermal control systems. Once

connected, the integrated systems perform all life support and animal

monitoring functions necessary for the 5-week mission.

The ASM successfully passed Critical Design Review in late 2005. Since

that time, Mars Gravity researchers have been engaged in constructing and

testing the module to verify engineering performance and to scientifically

validate the instrumentation. We present results from a comprehensive 6-

week test to demonstrate that the ASM meets requirements associated with

animal care and monitoring. The experiment simulates a one-week period

on the launch pad followed by 5 weeks in the standard orbital orientation.

We demonstrate capabilities and limitations of the sensor systems, while

confirming that the design is capable of adequately sustaining mice for the

duration of the Mars Gravity mission.

(Supported in part by NASA: SBIR Phase II Contract NNA05CP01C)

[6] EFFECT OF HYPERGRAVITY ON EXPRESSION OF

RECEPTORS ASSOCIATED WITH LACTOGENESIS IN RAT

MAMMARY GLAND FROM PREGNANCY TO LACTATION.

M.K. VanKlompenberg, O.V. Patel, H. Dover and K. Plaut; Dept. of

Animal Science, Michigan State Univ., E. Lansing, MI.

Prolactin (PRL), the key hormone responsible for lactogenesis, has

multiple receptor isoforms in rats; two of the principle forms are the long

(L) and short (S). Glucocorticoids (GC) that are essential for transition

from pregnancy to lactation also exert their cellular effect through their

specific receptor. Alteration in gravitational force has a detrimental effect

on lactogenesis and neonatal survival. Therefore, the objective of this

study was to see how hypergravity affects mRNA expression of the two

forms of PRL receptors and GC receptor in the rat mammary gland during

pregnancy and lactation.

Rats were placed on a centrifuge at 2 g (HG) starting at gestation day

11(G11) until parturition day 1(P1), while a group of stationary control

(SC) rats were housed in the same room at 1g. On G20 (SC:n=5, HG:n=5)

or P1(SC:n=4, HG:n=4), rats were euthanized and mammary tissue

collected and stored at -80oC. RNA was extracted from the mammary

tissue using Trizol®. RNA was reverse transcribed to cDNA, and receptor

abundance was quantified using qPCR. Relative expression among groups

was determined using the ∆∆ct method and statistics were run using SAS.

The PRL(L) receptor mRNA abundance was 7 fold less in HG G20

compared to SC G20 (p<.001). The same receptor increased 5 fold from

pregnancy to lactation in SC (p<.001) but only 2 fold in HG (p<.01). The

abundance of PRL(S) receptor was slightly less in HG than SC (P <.05).

PRL(S) receptor mRNA increased from pregnancy to lactation

approximately 4 fold (p<.001). The mRNA levels of GC receptor showed

a 4 fold decrease at HG G20 compared to SC G20 (p<.01). The latter

receptor increased 3 fold between pregnancy and lactation (p<.001).

Hypergravity decreases mRNA levels of both long and short form PRL

receptors and the GC receptor at days G20 and P1.

(Supported by NASA: NNA05CP91A)

[7] EXAMINING BIOLOGICAL VARIABILITY: A MICROARRAY

STUDY. A. L. Kuntz1, M. L. Molas1, and J. Z. Kiss1 1Department of

Botany, Miami University, Oxford, OH.

Since the introduction of microarray technology to analyze gene

expression, there have been concerns about the reproducibility of

experimental results obtained using different microarray platforms.

Reproducibility for most platforms within any laboratory is typically good,

but reproducibility between platforms and across laboratories is generally

poor. In addition, the variability of microarray results between

laboratories is associated with their use of different protocols. Thus, our

experiment addressed this problem by using separately four different types

of purification columns during the generation of an Arabidopsis cDNA

sample. After column purification, the Agilent 2100 bioanalyzer and RNA

6000 LabChip kit were implemented in order to assess the quality of the

cDNA sample before its usual hybridization onto a microarray platform.

From the bioanalyzer electropherogram, biological variability was found in

the single stranded cDNA population that resulted from the four different

purification columns. When different population of cDNA is hybridized to

a microarray platform, varying gene expression results will be produced.

Ultimately, with carefully designed and controlled experiments using a

standardized platform and protocol, microarray results can be better

compared across multiple laboratories; it is essential to facilitate

collaborative research efforts.

(Supported by Miami University DUOS program and NASA grant NCC 2-

1200)

[8] COUPLED EFFECTS OF TEMPERATURE AND SIMULATED

MICROGRAVITY (CLINOSTAT) ON E. COLI POPULATION. F.N.

Ahmed, H.N. Howard and D.M. Klaus, University of Colorado, Boulder,

CO 80309

In an attempt to reproduce previous results from Brown et al. (2002)

describing the effect of simulated microgravity (using clinorotation) on E.

coli population growth, our initial outcomes were inexplicably

inconsistent. Upon further analysis, however, an interesting trend was

identified between the temperature at which the cultures were grown and

the corresponding response to clinorotation that occurred. It was

consequently hypothesized that E. coli cell size and growth rate

concomitantly contributed to the net effect of simulated microgravity.

Lewis and Trueba (1981, 1982) showed that the temperature at which E.

coli are grown influences their size. Additionally, Woldringh and

Kubitschek (1981, 1984) showed that cell density and shape stay relatively

constant despite the changes in size with temperature. These relationships

imply that larger cells will sediment faster, hence, should exhibit a greater

response when sedimentation is prevented by clinorotation. In subsequent

experiments over a range of temperatures, we found that non-motile E. coli

cells grown on a clinostat experienced maximal final population

differences at 30ºC relative to static controls. These findings suggest that

E. coli cell size, as a function of temperature-dependent growth rate, plays

an important role in the overall influence of gravity on cell population.

Studies are now aimed at correlating sedimentation to clinostat results.

(Supported by UROP and BioServe)



[9] CHARACTERIZATION OF GRAVITY SENSING MECHANISMS

IN ARABIDOPSIS

Aaron Lomax, Chiu Yueh Hung, and Imara Perera

Dept. of Plant Biology, North Carolina State University, Raleigh NC

Plant gravitropism is a complex process involving different signaling

pathways. We have shown previously that inositol 1,4,5-trisphosphate

(InsP3), a second messenger generated in the phosphoinositide (PI)

pathway, increases in response to gravity in the pulvinus of inflorescence

stems of cereal grass stems and in inflorescence stems of Arabidopsis.

Furthermore, transgenic Arabidopsis plants expressing the human type I

inositol polyphosphate 5-phosphatase (InsP 5- ptase), an enzyme which

hydrolyzes InsP3, showed a delayed gravitropic response compared to wild

type plants (Perera et al., 2006 Plant Physiol. 140:746). This work is

focused on characterizing the role of InsP3 and its connection to other

events in the gravity signaling cascade such as amyloplast sedimentation

and auxin redistribution. To study the interaction between amyloplast

signaling and the PI pathway we will use the starchless (pgm) mutants.

The pgm mutants have reduced root and hypocotyl gravitropism; however,

the inflorescence response has not been well characterized (Caspar and

Pickard 1989, Plant 177:185). To compare pgm mutants to the wild type

plants we are monitoring inflorescence bending using time lapse

photography and InsP3 levels will be measured during the gravitropic

response. In addition, we are generating pgm/ InsP 5-ptase double mutants

to determine if the effects are additive. In order to look at the dynamics of

auxin redistribution we have generated transgenic plants expressing InsP

5-ptase and the auxin responsive promoter-reporter fusion; pIAA2-GUS.

Inflorescence bending and the appearance of asymmetric GUS staining in

the inflorescence stem will be compared in InsP 5-ptase plants and plants

expressing only the reporter gene.

(Supported by Undergraduate Research Fellowship funded by the NC

Space Grant)

[10] IDENTIFICATION AND CHARACTERIZATION OF ARF9 AS A

GPS3-LIKE MUTANT. D.R. Roberts1, and S.E. Wyatt1 1Dept. of

Environmental & Plant Biology, Ohio Univ., Athens, OH.

The gravity persistent signal gps3 mutant of Arabidopsis thaliana

displays an unusual phenotype when gravistimulated at 4ºC and returned to

room temperature. This GPS3 gene encodes a transcription factor that

belongs to the Auxin Response Factor (ARF) gene family (member

ARF23) and contains a B3 DNA binding domain. Using a bioinformatics

approach, five other genes have been identified that are similar to GPS3.

ARF9 (one of five) is most closely related to GPS3 , though the GPS3 gene

contains only the B3 DNA binding domain; ARF9 contains an Auxin

Response domain and an Auxin IAA binding domain in addition to the B3

DNA binding domain. Three SALK T-DNA insertion lines were obtained

for ARF9 to perform a phenotypic characterization which consisted of

measuring growth rates, phenotype and gravistimulation. Plants

homozygous for the SALK T-DNA insertion were gravistimulated at 4ºC,

one of the ARF9 insertion lines displayed a phenotype identical to that of

the gps3 mutant. The SALK T-DNA insertion line showed no abnormal

phenotype during development. Implying that GPS3 and ARF9 have

similar function, though the structure of the genes differ. The molecular

characterization and complementation of the ARF9 gene has the potential

to create a better understanding of the pathways being followed during

gravity stimulus.

(Partially Supported by Grasselli-Brown Undergraduate Research Award,

Ohio University, to DRR and NASA: NAG2-1608 to SEW.)

[11] IS TRANSCRIPTIONAL REGULATION OF GRAVITROPISM

CONSERVED BETWEEN DICOTS AND MONOCOTS? R.L.

Hammond1, H. Myburg1, I.Y. Perera1, C.S. Brown1,2 and H. Winter

Sederoff1 1Plant Sensory Genomics Group, Dept. of Plant Biology, and 2Kenan Institute for Engineering, Technology & Science, North Carolina

State University, Raleigh, NC, 27695.

Gravity is a constant force that directs the course of plant growth. Plants

sense the direction of the gravity vector and respond to changes in

orientation by redirecting their growth. This response to gravity requires

specific changes in gene expression to accomplish differential cell

elongation. Previous studies on Arabidopsis root tips (Kimbrough et al.

2004) and the maize pulvinus (Heilmann et al. 2001, Myburg,

unpublished) reveal that specific genes are regulated within minutes of

gravitropic stimulation. We are comparing gene expression patterns in

response to gravity between maize pulvini and Arabidopsis root tips to

identify possible conserved mechanisms. Conserved and tissue specific

gravity induced changes in transcript abundances will further be analyzed

for their temporal expression in maize root apices. In these experiments

we will monitor changes in transcript abundances in the maize root over a

time course during the first hour after reorientation using real-time PCR.

The expression profile information generated will then be compared

between Arabidopsis root tips and the maize pulvinus to assess the level of

conservation in transcriptional regulation between dicots and monocots.

Heilmann, I. Shin, J., Huang, J., Perera, I.Y., Davies, E. (2001) Transient

Dissociation of Polyribosomes and Concurrent Recruitment of Calreticulin

and Calmodulin Transcripts in Gravistimulated Maize Pulvini.. Plant

Physiol. 127:1193-1203.

Kimbrough, J.M., Salinas-Mondragon, R., Boss, W.F., Brown, C.S.,

Sederoff, H.W. (2004) The Fast and Transient Transcriptional Network

of Gravity and Mechanical Stimulation in the Arabidopsis Root Apex.

Plant Physiol. 136:2790-2805.

(Supported by NCSU RNA Biology fellowship to R.L.H. and NASA

grants NAAG2-1566 to CSB, and NNA04CC56G)

[12] THE INFLUENCE OF ANTIBIOTICS ON BACTERIAL

MOTILITY AND ITS IMPLICATION FOR DRUG EFFICACY IN

MICROGRAVITY. A.R. Stanczyk1 and D.M. Klaus2 1University of Michigan, Flint, MI, 48502 and 2University of Colorado,

Boulder, CO 80309.

Understanding in vitro bacterial response to antibiotics in a microgravity

environment is an important step toward the goal of minimizing astronaut

health risks. Previous studies have shown that bacteria cultured in space

are able to proliferate in normally inhibitory concentrations of antibiotics.

Identifying the cause of this reported behavior has proven difficult,

however, due to the complex interactions of gravitational influence on

living organisms. Research aimed at identifying and isolating independent

variables can help to elucidate the responsible mechanisms. This project

focuses on the study of one such variable, cell motility. It is hypothesized

that antibiotic effectiveness in space is reduced as an indirect consequence

of bacterial motility inhibition. The rationale behind our hypothesis is

based on related research suggesting that less motile bacteria exhibit more

pronounced responses to microgravity in general than highly motile ones,

purportedly due to extracellular mass transport factors. Motility of E. coli

(ATCC 4157) was evaluated as a function of radial growth from a stab

culture inoculated on semi-solid agar using different nutrient sources, both

with and without Streptomycin. Results are correlated to literature

describing antibiotic experiments conducted in space. (Supported by UM-

Flint Honors Scholar Program and BioServe Space Technologies, NASA

NCC8-242)



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Session I



[13] DESIGN AND VALIDATION OF ATMOSPHERIC LIFE SUPPORT

SYSTEMS FOR THE MARS GRAVITY BIOSATELLITE. T.R.F.

Fulford-Jones1, A.M. Heafitz2, E.B. Grosse1, M.A. Scott3, J.C. Franklin4

and J.A. Hoffman1 1MIT Department of Aeronautics and Astronautics, 2MIT Edgerton Center, 3Cambridge University Department of Electrical

Engineering, 4MIT Department of Mechanical Engineering.

The atmospherics control subassembly of the Mars Gravity Biosatellite

will cleanse and continuously recirculate breathable air throughout the

spacecraft’s fifteen habitat modules. The design incorporates canisters of

lithium hydroxide, silica gel desiccant and activated carbon to control

contaminants and regulate humidity.

Generation of ammonia through interaction of waste products is a

particular concern because of the dangers associated with mycoplasmosis.

We present results from an extended-duration experiment to investigate the

rate of ammonia production in a closed-loop system with a single mouse in

a flight-like habitat module. This experimental data suggests that an

appropriately-sized activated carbon bed will adequately control ammonia

buildup while also eliminating trace contaminants.

We additionally present data which shows effective control of humidity

and carbon dioxide. The laboratory system incorporates silica gel and

lithium hydroxide canisters together with computer-activated valves and

variable-speed pumps. Combining this data with theoretical values for

mouse contaminant production rates generates accurate sizing predictions

for the atmospherics control hardware on board the biosatellite.

(Supported in part by NASA grant NCC2-1369)

[14] SEED GERMINATION AND GROWTH IN HYPOBARIA. H.L. Gohil, J.R. Truett, A.J. Stimpson, R.A. Bucklin and M.J. Correll

Dept. of Agricultural & Biological Engineering, Univ. of Florida,

Gainesville, FL.

For long-term human space missions, plants will be needed for food, for

air and water purification, for biofuels and for materials such as paper

products. Therefore, chambers for growing plants in the harsh conditions

during spaceflight or on Mars need to be developed. Low Pressure Growth

Chambers (LPGCs) have been proposed as a possible option to grow

plants in conditions where the environment has low atmospheric pressure

such as on Mars (≈1 kPa). We have constructed LPGCs that can monitor

and/or control humidity, pressure, temperature, light, and gas composition.

In preliminary experiments, plants exhibited significant wilting within 15

minutes when exposed to hypobaria of 10 kPa (PO2 = 2.1 kPa; PCO2 =

0.0003 kPa; PN2 = 7.9 kPa), although the plants recovered after few hours

once atmospheric pressure was returned to earth-normal (101 kPa). This

suggests that plants undergo stress during hypobaria. In addition, no seeds

germinated at 15 kPa (PO2 = 3.15 kPa; PCO2 = 0.00045 kPa; PN2 = 11.85

kPa) for either Arabidopsis (Arabidopsis thaliana) or Tomato

(Lycopersicon esculentum cv. Micro-Tom). When the atmospheric pressure

was raised to 25kPa (PO2 = 5.25 kPa; PCO2 = 0.00075 kPa; PN2 = 19.75 kPa),

approximately half of the Arabidopsis seeds germinated while none of the

tomato seeds germinated. This poor germination may be a result of

hypoxia (low oxygen) or limited water uptake and not a direct pressure

effect. Studies on germination and growth of seedlings at low pressure

with supplemental oxygen are currently being performed to clarify this

response (support provided by UCF-UF Space Research Initiative

20040006). (Supported by NASA NNA04CK29G)

[15] THE MUSCULOSKELETAL EFFECTS OF PARTIAL

WEIGHTBEARING IN MICE. E.B. Wagner1,2, N.P. Granzella3, 1MIT

Department of Aeronautics and Astronautics, 2Harvard-MIT Division of

Health Sciences and Technology, Cambridge, MA; 3Pepperdine

University, Departments of Social Science and Natural Science, Malibu,

CA.

The effects of partial gravity environments on mammalian physiology are

currently poorly characterized. Understanding whether such an

environment prevents or reduces the levels of deconditioning seen in

microgravity is critical to determining the viability of extended human

missions beyond low earth orbit. With a payload of fifteen mice, the Mars

Gravity Biosatellite aims to study the effects of, and adaptation to, a 0.38-g

environment.

In preparation for the flight, a ground-based model of partial

weightbearing has been developed to study adult mice undergoing reduced

musculoskeletal loading. This full-body suspension model allows for

manipulation of load levels to simulate various gravitational environments,

permitting investigation of how deconditioning scales across the 0-g to 1-g

continuum.

This novel hardware draws on the heritage of rodent tail suspension and

human partial weightbearing studies. It uses spring-suspended forelimb

jackets and tail wraps to provide balanced unloading of both the front and

hind limbs. Strain gaged animal support flexures, in vivo tibial strain

gages, and treadmill-based gait testing enable validation of the

musculoskeletal loading environment.

Adult female BALB/cByJ mice have been successfully supported for

periods of up to three weeks in both hypodynamic experiments and fully-

weightbearing harnessed controls. The musculoskeletal effects of partial

weightbearing will be quantified using histomorphometry, serum

biochemistry, microscale imaging, and biomechanical testing.

(Supported by NASA GSRP NNG 04-GN71H)

[16] A MEMS BASED IN-SILICO CELL ELECTROPHYSIOLOGY

DEVICE FOR MONITORING TRANSCELLULAR CALCIUM CURRENTS IN CERATOPTERIS RICHARDII FERN SPORES. A. ul

Haque1, 2, M. Rokkam1, 3, A.R. DeCarlo1, 2, S.T. Wereley4, S.J. Roux5, P.P.

Irazoqui6, D.M. Porterfield1, 2, 6, 1Bindley Bioscience Center: Physiological

Sensing Facility, 2Dept. of Ag. & Bio. Eng., 3Dept. of Elec. & Comp. Eng., 4Dept. of Mech. Eng., Purdue University, West Lafayette, IN, 5Molecular,

Cellular & Developmental Biol., Univ. of Texas, Austin, TX, 6Dept. of

Horticulture & Landscape Architecture, Purdue University, West

Lafayette, IN.

Gravity is known to direct the polarity of development of Ceratopteris

richardii fern spores, and this process involves the activation of a trans-

cellular calcium ion current through the individual cells. This has been

studied in the past using a self referencing calcium ion probe, but does

have inherent limitations in the spatial and temporal resolution of the

probe, which has impeded our ability to further study the system. Now we

have developed an in-silico Cell Electrophysiology Lab-on-a-Chip device

(CEL-C) using advanced micro-fabrication techniques, and this has

enabled us to monitor Ca++ ionic gradients around multiple fern spores in

real time. Each chip has 16 pyramidal pores on it with four Ag/AgCl

electrodes leading into them at the four poles. An SU-8 layer is used to

insulate the electrodes and the electrodes are coated with a Ca++ selective

membrane to impart ion selectivity. A custom amplification PCB designed

for signal amplification and noise reduction is used to interface the CEL-C

device with a 32 channel 18 bit data acquisition unit. A state of the art

software, designed in LabView 7.0, allows continuous data measurement

and recordings. The CEL-C device has been calibrated and has

demonstrated expected Nernstian characteristics with excellent

repeatability. Initial ground measurements on the fern spores have been

conducted and very promising results have been obtained. While this

version of the in-silico CEL-C device has been designed targeting analysis

of transcellular ionic calcium currents in Ceratopteris fern spores, the

device is actually much more versatile and can be adapted to a variety of

cell physiology and other biomedical applications to become an

indispensable tool for biology. (Supported by the NASA and the Lilly

foundation) (Supported by NASA: NCC8-242)



[17] CYTOSKELETON CHANGES IN MESENCHYMAL STEM CELLS

DIFFERENTIATE TO OSTEOBLASTS AND CHONDROCYTES

AND EFFECT OF ELECTRIC STIMULATION DURING THREE-

DIMENSIONAL SCAFFOLD CULTURE. C. Umeda1, Y. Kawahara1,3,

R. Yoshimoto1, T Kajiume2, A. Sasaki1, S.L. Wu, 1 K. Naminohira1 and L.

Yuge1,3. 1Graduate School of Health Sciences, 2Graduate School of

Biomedical Sciences, 3Space Bio-Laboratories Y.K., Hiroshima

University, Japan.

The Rho GTPase has been influence cell morphogenesis through

remodeling of the actin cytoskeleton and has been implicated lineage

commitment of hMSCs. Here, we showed actin stress filaments formation

and Rho activity when MSCs differentiate into osteoblasts and

chondrocytes. Human MSCs were cultured in osteogenic induction

medium and chondrogenic induction medium. Histological examination by

light microscope showed that elongated shape cell has long process in

osteoblast, while round or polygonal morphology in chondrocyte. The

organization of actin filaments and stress fiber were examines by

immunostaining with Rhodamine conjugated phalloidin and anti vinculin

antibody for focal contact form. A remarkable difference in cytoskeleton

organization was observed between in human MSCs during osteogenic and

chondrogenic. In osteoblast, random and thin actin filaments and a few

focal contact. While, in chondrocyte, parallel and thick actin filament as

stress fiber was observed and a large number of focal contact. The activity

of Rho analyzed by pull-down assay was no distinct difference in

osteoblast and chondrocyte. Balance of mDia and ROCK activities decides

to induce stress fiber and focal contact formation. And we examined that

three-dimensional scaffolds seeds with cultured MSCs were applied to

electrical stimulation.

[18] HYPERGRAVITY AND HYDROSTATIC PRESSURE LOADING

RESULTED IN DIFFERENT MODELED STRESS AND STRAIN

BUT THE SAME OSTEOBLAST FUNCTIONAL RESPONSE. J.S.

Alwood1, E.A. Almeida2, R.K. Globus2 and N.D. Searby2. 1Aeronautics

and Astronautics, Stanford University, Stanford, CA, 2NASA Ames

Research Center, Moffett Field, CA.

Reduced mechanical stress during microgravity exposure is known to

result in bone loss, yet the role gravity (acceleration) plays in

mechanotransduction at the cellular level is unclear. In this study, we

computationally and experimentally investigated the respective

contribution of hydrostatic pressure (HP) and hypergravity to cellular

mechanical stress to understand gravity’s role at the cellular level. A 1000-

element axisymmetric finite element model of a subconfluent cell attached

to a substrate was developed, and its elastic response was analyzed using

ANSYS software. For experiments, MC3T3-E1 preosteoblast cells were

plated on collagen coated dishes and allowed to adhere for 1 hour in α-

MEM supplemented with 1% FBS, then centrifuged at 10-g (98 m/s2) or

loaded in a pressure chamber at 1.2 kPa for 24 hours and compared with

unloaded cells (1-g and 0.12 kPa). Cell number was determined by DNA

quantity. For our experimental load values, the finite element model

indicated maximum displacement and von Mises stress/strain due to HP

was three orders of magnitude greater than corresponding acceleration

values, yet hypergravity resulted in a unique stress/strain contour

compared to HP. Maximum stress occurred at the peripheral cell-substrate

contact under HP and below the nucleus during acceleration. Reduction of

HP to a stress magnitude on the order of gravity resulted in maximum

stress occurring simultaneously at the peripheral cell-substrate contact and

below the nucleus. Experimentally, both hypergravity and HP increased

cell number by 10-15% compared to unloaded controls, with no statistical

difference in cell number between hypergravity and HP. In conclusion,

modeling suggested the hypergravity elastic response was dominated by

HP and increased cell numbers due to centrifugation are reproduced by

HP. The effect of gravity on cellular mechanotransduction appeared to be

less relevant than the external HP. (Supported by NASA GSRP grant

NNA04CK68H and NASA DDF grant 02-02)

[19] PROLIFERATION OF HEMATOPOIETIC STEM CELLS IS STIMULATED IN 3D-CLINOSTAT CULTURE. R. Yoshimoto1, T.

Kajiume2, Y. Kawahara1,3, C. Umeda1, A. Sasaki1, S.L. Wu1, K.

Naminohira1, K. Kataoka2, and L. Yuge1,3 1Graduate School of Health

Sciences, 2Graduate School of Biomedical Sciences, 3Space Bio-

Laboratories Y. K., Hiroshima University, Japan.

Hematopoietic stem cells (HSCs) are multipotent stem cells, which have

abilities for self-renewal and differentiation. Different cytokines and

growth factors have been used to expand stem cells. The well-established

strategies for ex vivo expansion of HSCs include culture with cytokine

combination and co-culture with bone marrow stromal cells. A 3D-

clinostat is a multi-directional gravity device for simulating microgravity.

By controlled rotation of two axes, a 3D-clinostat minimizes the

cumulative gravity vector in cells cultured at the center of the device and

makes 10-3 G average over time. In this study, we investigated the

application of microgravity to HSC culture using a 3D-clinostat.

Mouse bone marrow cells were cultured for 3 to 7 days in a 3D-clinostat

(group CL), or in control cultures at 1G (group C) in the presence of

optimal cytokine combination. Then the cultured cells were transferred

into semisolid culture for colony-forming cell assay (CFC assay). The

number of cells was assessed by surface antigen analysis on day 3 and day

7. After 1-wk culture in, CFCs were collected, and then 2nd CFC assay

was performed. Highly purified HSCs were defined as lineage marker

depletion and expression of the cell surface markers, Sca1 and c-Kit cells

(Lin(-) Sca1(+) c-Kit(+) [LSK]), such cells were established by 2nd CFC.

The fold increase of cells expanded from day 3 to day 7 was significantly

greater in group CL than in group C. Furthermore, group CL included

larger proportion of LSK than group C at day 7. The number of CFCs in

2nd CFC assay was also more abundant in group CL. In conclusion, we

demonstrated that HSCs were well proliferated and maintained their

multipotential in culture in simulated microgravity by 3D-clinostat.

[20] THE EFFECT OF COMBINED SIMULATED MICROGRAVITY

AND MICROGROOVED SURFACE TOPOGRAPHY ON

FIBROBLASTS. WA Loesberg1, XF Walboomers1, JJWA Van Loon2,

JA Jansen1 1Dept. Periodontology & Biomaterials, Radboud Univ.,

Nijmegen, The Netherlands, 2DESC OCB-ACTA, Vrije Univ.,

Amsterdam, The Netherlands.

This study evaluated in vitro the differences in morphological behaviour

between fibroblast cultured on smooth and microgrooved substrata (groove

depth: 0.5 µm, width: 1, 2, 5, 10 µm), which undergo simulated

microgravity by means of RPM. The aim of the study was to clarify which

of these parameters was more dominant to determine cell behaviour.

Morphological characteristics were investigated using scanning electron

microscopy and fluorescence microscopy in order to obtain qualitative

information on cell alignment and area. Confocal laser scanning

microscopy visualised distribution of actin filaments and vinculin

anchoring points through immuno-staining. Finally, expression of collagen

type I, fibronectin, and α1- and β1-integrin were investigated by PCR.

Microscopy and image analysis showed that the fibroblasts aligned along

the groove direction on all textured surfaces. On the smooth substrata cells

spread out in a random fashion. The alignment of cells cultured on grooved

surfaces decreased under simulated microgravity, especially after 24 hours

of culturing. Cell surface area on grooved substrata were significantly

smaller than on smooth substrata. Simulated microgravity on the grooved

groups resulted in an enlargement of cell area. ANOVA was performed on

all main parameters: topography, gravity force, and time. In this analysis,

all parameters proved significant. In addition, gene levels were reduced by

microgravity particularly those of β1-integrin and fibronectin. From our

data it is concluded that the fibroblasts primarily adjust their shape

according to morphological environmental cues like substratum surface

whilst a secondary, but significant, role is played by simulated

microgravity.

(Supported by NWO-SRON: MG-057 & MG-063)



[21] DEVELOPMENT OF A SYNTHETIC SEED PRODUCTION

METHOD FOR LONG-TERM TRANSPORT AND ADVANCED

LIFE SUPPORT. J.E. Porter and T.P. West Davis College of

Agriculture, Forestry and Consumer Sciences, West Virginia Univ.,

Morgantown, WV.

A system to produce viable synthetic seeds of select crops is desirous for

the long-term storage and transport of clonal plant material into space.

Current limitations of flight experiments and germplasm transport allow

only for the use of sexually-produced seeds, introducing genetic variability

and the possibility for seed-borne diseases.

This study will strive to evaluate the use of synthetic seed technology to

create synseeds of Arabidopsis thaliana (L.) and Lycopersicon

escuelentum (Mill.) tomato F1 hybrids. A comparison of gene expression

of A. thaliana (L.) synseeds produced by both somatic embryos and

encapsulated nodes will be used to evaluate differences in both production

techniques for genetic variability (due to somaclonal variation in somatic

embryos) and stability during storage. This study will also evaluate the

ability of synseeds to be reintroduced into tissue culture and direct

introduction into hydroponic production systems for crop production.

Four tomato F1 hybrids (‘Celebrity’, ‘Big Beef’, ‘Sweet Olive’, and ‘Sun

Cherry’) along with A. thaliana (L.) will be evaluated for ability to

produce somatic embryos and axillary shoot cultures for encapsulation in

order to produce synthetic seeds. Components of the encapsulation

medium will be evaluated to determine the appropriate formula for each

propagule. The synseeds will be stored for various time periods to

determine effect of storage on viability and vigor. Gene expression will be

compared between synseeds of A. thaliana (L.) from both production

methods and plants grown under standard conditions using microarray

technology. Synseeds from both propagule sources will be evaluated for

success in direct introduction to a hydroponic system for differences in

growth and yield. The synseeds will also be placed back into tissue culture

to determine if a perpetual propagation cycle can be obtained to regenerate

more synseeds.

(Supported by NASA WV Space Grant Consortium)

[22] CHARACTERIZATION OF CYTOCHROME P450 PROTEINS THAT REGULATE PLANT GRAVITROPISM. J.C. Withers1 and

S.E. Wyatt1 1Dept. of Environmental and Plant Biology, Ohio University,

Athens, OH.

An understanding of gene expression that occurs during the gravity

response is of central importance to studying the cellular mechanisms

linking the physical perception of gravity to the biochemical activities

governing the growth response. Plants with a mutation in the GRAVITY

PERSISTENCE SIGNAL (GPS)1 locus of Arabidopsis thaliana show a

“No Response” phenotype during gravistimulation experiments. Cloning

of GPS1 revealed that it encodes CYP705A22 (A22), a cytochrome P450

protein (P450) of unknown function. Microarray data collected from

Arabidopsis root tips indicated that the expression of a closely related

family member, CYP705A5 (A5), is increased following a gravity

stimulus in roots.

An expression profile was generated for A5 using real-time quantitative

PCR, and the data indicate that A5 is up-regulated nearly five fold within

the first five minutes of gravity stimulation. Reporter gene constructs that

link the A5 gene to the green fluorescent protein (GFP) have shown that

A5 is expressed in the root zones of elongation and maturation. To further

investigate the role of P450s in regulating gravitropism, plants containing a

T-DNA insertion at the A5 locus were obtained and characterized with

respect to their gravity response. Homozygous mutants showed an

attenuated rate of curvature that was able to be rescued by the addition of

dihydroquercetin, a flavonol known to be the product of a P450

hydroxylation event.

Computer modeling of the catalytic domain and screening of potential

substrates has generated a list of 130 compounds that may have the ability

to bind to A22 and A5, and nearly 50% of the compounds are derivatives

of the phenylpropanoid biosynthetic pathway. Protein expression

constructs were created using the cDNA sequence encoding each of the

proteins and expressed in cell culture in order to isolate the proteins and

determine the specific biological substrate for each.

(Supported by NASA: NAG2-1608 and NSF: 0618506 to SEW)

[23] POSITIVE PHOTOTROPISM IN ROOTS: WHO IS INVOLVED?

M.L. Molas1, Correll MJ2, Kiss JZ (1) Dept. of Botany, Miami University,

Oxford. OH 45056 (2) Dept. of Agricultural and Biological Engineering,

University of Florida, Gainesville. FL 32611-0570

Red light, acting through the phytochromes, controls numerous aspects

of plant development. Previously, we had identified a positive

phototropism in roots induced by red light, which is mediated by PHYA

and PHYB. To investigate the set of genes involved in this novel

phototropic response, we performed gene profiling studies using cDNA

microarrays and quantitative Real-Time PCR in roots of Arabidopsis

seedlings exposed to 1 h of red light. Some of the genes found to be

differentially expressed in this study were PHYTOCHROME KINASE 1

(PKS1), LONG HYPOCOTYL 5 (HY5), EARLY FLOWERING 4 (ELF4),

and GIGANTEA (GI), all significantly upregulated in roots of seedlings

exposed to 1 h of red light. In addition, the upregulation of SUPPRESSOR

OF PHYTOCHROME A RESPONSES 1 (SPA1) and CONSTITUTIVE

PHOTOMORPHOGENIC 1-like (COP1-like) genes suggests that the

PHYA-mediated pathway was attenuated by red light. Interestingly,

members of the RPT2/NPH3 (ROOT PHOTOTROPIC 2/NON

PHOTOTROPIC HYPOCOTYL 3) family, which have been shown to

mediate blue-light-induced phototropism, also were differentially regulated

in roots in red light. Therefore, these results suggest that red and blue light

pathways interact in roots. Currently, experiments are in progress testing

rpt2, hy5 and pks1 mutants to investigate their involvement in positive

phototropism in roots induced by red light. These studies utilize a

computer-based feedback system to obtain high resolution data on this

response in the mutants compared to the response in the wild type.

(Supported by Miami University DUOS program and NASA grant NCC 2-

1200)

[24] DIETARY ROS SCAVENGER, ALPHA-LIPOIC ACID, PREVENTS TESTICULAR ATROPHY DURING 10 WK HINDLIMB UNLOADING (HLS) Joseph Zenisek1, S. Wolfe1, B.D. Timmerberg1, J.S. Tash1, 1Dept. of Molecular & Integrative Physiology, University of Kansas Medical Center, Kansas City, KS. The Bioastronautics Roadmap risks include unacceptable levels of tissue

degeneration caused by occupational radiation exposure or the combined

effects of radiation and other space flight factors. Hindlimb unloading in

rats (HLS) is widely used to mimic many physiologic changes that occur

during space flight. Previous work from our lab has demonstrated that long

term HLS causes severe testicular atrophy and male sterility. More recent

studies suggest that there is an inverse relation between age and the

duration of HLS necessary to cause testicular degeneration. Reactive

oxygen species (ROS) are a major factor in producing radiation and non-

radiation induced apoptotic loss in the testis. We examined whether

administration of the dietary ROS scavenger, alpha-lipoic acid (LA), could

ameliorate HLS-induced testicular atrophy. HLS animals, and TO controls

(tail harnessed and elevated, but with all limbs remaining in contact with

the cage floor, and pair fed to HLS) were prepared as previously

published, including partial ligation of the inguinal canal to prevent the

testes from becoming abdominal during HLS. Animals were 12-13 wks old

at the start of the study. After 10 wks of HLS, a 35% decline in testis

weight and testis atrophy ranging from Sertoli cell-only like morphology

with apparently normal interstitial histology, to total seminiferous and

severe interstitial cell pyknosis were noted. On the other hand, LA (50

mg/day in the drinking water) resulted in retention of normal testicular

histology and no decline in testis weight in HLS relative to TO and free

roaming controls (FRC). LA had no significant effect on body weight gain

during the 10 wk experiment within each treatment group. A problem

noted in the long 10 wk duration of the HLS study, was the low n’s yielded

in the HLS groups from loss of animals due to animals becoming

unharnessed. Future studies will require re-harnessing at 5 wks while

maintaining lack of hindlimb contact during re-harness for the HLS

animals. (Supported by NASA and NIH).



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Session II



[25] DNA-BASED LIFE DETECTION ON EARTH AND MARS:

POLYMERASE CHAIN REACTION OPTIMIZATION USING

SHORT RIBOSOMAL PRIMERS. N.M. Vahora1, C.E. Carr2, M.T.

Zuber2, and G. Ruvkun3,4

1Department of Civil and Environmental Engineering,

Massachusetts Institute of Technology, Cambridge, MA 2Department of Earth, Atmospheric and Planetary Sciences, Massachusetts

Institute of Technology, Cambridge, MA. 3Department of Genetics, Harvard University, Cambridge, MA. 4Department of Molecular Biology, Massachusetts General Hospital,

Boston, MA.

A reasonable case can be made that life (past or present) on Mars may be

ancestrally related to life on Earth because of significant meteoritic

exchange between Earth and Mars. On Earth, the DNA-based polymerase

chain reaction (PCR) provides a simple, standard, and powerful method to

detect life. A soil sample from an extreme environment can be surveyed

for the signature of life, a DNA fragment of a gene that is universal to life

on Earth. Of the approximately 500 “universal genes” carried in the DNA

of every known living organism, the 16S small subunit ribosomal RNA

gene shows some of the highest levels of conservation at the base pair

level. While some regions are conserved across all known life, other

regions vary significantly, making 16S a good chronometer by which to

build a tree of life and to classify organisms on the basis of their 16S

sequences. By targeting the most conserved regions of 16S we can create

“universal primers” able to amplify DNA from any known living

organism; however, these primers must be short enough to achieve

universal sensitivity but long enough to primarily target 16S. Using

primers identified from analysis of base pair conservation, we are

validating candidate primers against metagenomic libraries to identify

primers with the highest signal to noise ratio. This optimization may result

in a protocol able to detect and classify new and interesting organisms,

whether on Earth or Mars.

(Supported by NASA grant NNG05GK27G.)

[26] ANALYSIS OF COMPLEX BIOMARKERS OF

MUSCULOSKELETAL ATROPHY AND STRESS IN PRESERVED

MOUSE URINE FOR THE MARS GRAVITY BIOSATELLITE. V.Y. Chang1,2, E.B. Wagner3,4, MIT Departments of 1Biology, 2Chemistry, 3Aeronautics and Astronautics; 4Harvard-MIT Division of Health Sciences

and Technology, Cambridge, MA.

The Mars Gravity Biosatellite Program is developing an uncrewed partial

gravity research platform capable of carrying fifteen mice in a 0.38-g

environment for periods of up to five weeks. In order to understand the

timecourse of adaptation to a partial gravity environment, we have

developed a method for autonomously collecting and preserving rodent

urine. Time-stamped weekly samples will be analyzed post-flight for

evidence of musculoskeletal atrophy and systemic stress. Earlier research

with this system examined preservation of urea and simple ionic moieties.

We have since advanced to more robust and specific biomarkers, selecting

a combination of ELISA and HPLC for small-sample reliability and high-

specificity detection of these analytes.

Corticosterone measurements using ELISA confirm the degree to which

musculoskeletal degradation is due to low gravity conditions, rather than

acute and chronic stresses of flight. Muscle atrophy is measured with a

combination ELISA/HPLC method to determine the Creatine/Creatinine

ratio, which increases with muscle breakdown. ELISA kits are currently

being evaluated for deoxypyridinoline and n-telopeptides, bone-specific

collagen components whose presence indicates an increase in bone

turnover.

Five week validation studies used pure urine adsorbed on a

polyvinylidene difluoride microporous membrane (Durapore, Millipore

Corp.) with and without applied Chlorhexidine n-Propyl-Gallate (CPG)

preservative. The biomarker analysis method provides feedback on

preservation while minimizing conflicts resulting from major urinary

protein effects, Durapore absorption, chlorhexidine-protein binding, and

dilution factors. (Supported by NASA SBIR NNA05CP01C)

[27] NEUTROPHIL ACTIVATION IN TRANSITIONAL GRAVITY.

AW. von Deutsch3, AS. Belton3, R Flowers4, J Wright8, C Williams9, NA.

Silvestrov3,6, DF. Paulsen4, BJ. Klement4 and DA. von Deutsch3,6. Depts.

of 3Pharmacology & Toxicology, 4Anatomy & Neurobiology and; 6Clinical

Research Center, Morehouse School of Medicine, Atlanta, GA; 8Auburn

University, 9UT Houston Health Sciences Center.

The release of myeloperoxidase (MPO) from neutrophils represents one

possible pathway for increasing the damage associated with space flight

and recovery. Oxidative damage that occurs because of space flight might

arise from many sources including radiation and the gravitational changes

defined as transitional gravity (TG). The relationship existing between TG,

oxidative stress and tissue damage is unclear. In particular, neutrophils

generate reactive oxygen species in respond to stressful challenges such as

those presented by TG. The purpose of this study was to determine

whether TG would activate human neutrophils and thereby induce

increased levels of oxidative stress. TG experiments were conducted

aboard the NASA C-9 aircraft during parabolic flights. A flight-certified

apparatus was used that consisted of a cell module (holding 5 cell

cartridges) chamber and a multi-channeled peristaltic pump (N=5). Human

neutrophils were isolated preflight from blood drawn from the faculty and

student investigators (N=5). Neutrophils were counted, loaded into the cell

cartridges and maintained at 37°C prior to flight. Half of the loaded

cartridges were flown while the remainder served as the ground controls.

Lipid peroxidation levels were significantly higher (248.1±50.6%) in the

flight group than in the ground controls. However, MPO levels in flight

neutrophils (6.7±5.8%) and that released into the media (19.6±11.3%)

were both significantly less than levels observed in ground controls.

Although the data only half supported our hypothesis-increased levels of

cellular oxidative stress, it supported the literature with respect to

decreased neutrophil function following space flight. This work was

supported, in part, by NASA Grants NCC9-112 and NAG3-2611, NCRR

5P20RR11104, and MBRS Grant 506GM08248.

[28] ULTRACELLULAR LOCALIZATION OF ARG1 IN ARABIDOPSIS COLUMELLA CELLS A.K. Spence, N.S. Kumar, R.E. Edelmann, and

J.Z. Kiss. Dept. of Botany, Miami Univ., Oxford, OH.

Gravitropism is the process by which plants direct organ growth in

response to gravitational stimuli. In the roots, gravity is initially signaled

by the movement of starch-containing amyloplasts located within the

gravity perceiving cells. These cells, known as statocytes, collectively

make up the columella located within the root tip. The peripheral

membrane protein ARG1 has been found to play a role in the early phase

of gravitropism (signal-transduction) through its presence in the columella

of the root. Previous studies at the confocal level have found ARG1 to be

associated with cellular compartments of the elongation zone and root cap

columella cells including the Golgi stacks, vesicles, endoplasmic

reticulum, and the plasma membrane. This association with compartments

of the secretory pathway supports the idea that ARG1 is involved in

gravitational signal-transduction, possibly aided by its interaction with the

cytoskeleton of the statocytes.

Our current research aims to localize ARG1 at the ultracellular level

using higher resolution transmission electron microscopy. Both light and

dark grown Arabidopsis seedlings of an ARG-GFP line were prepared for

TEM using two different chemical fixation protocols. The first procedure

fixed the specimens in paraformaldehyde and glutaraldehyde while the

second procedure utilized a mixture of glutaradehyde and osmium

tetroxide as to fix and provide en bloc staining simultaneously. The

seedlings were then infiltrated and flat-embedded with LR White resin.

Ultra-thin sections of the embedded specimens were immuno-labeled with

primary anti-GFP antibody and secondary gold anti-Rabbit IgG antibody

and then imaged with a JOEL transmission electron microscope. Using the

data obtained from the immuno-labeling, we hope to determine the specific

membranes and organelles with which ARG1 interacts and to ascertain the

nature of the protein’s association with the cytoskeleton. Localizing ARG1

at the ultracellular level will provide further insight into the signal-

transduction phase of gravitropism.

(Supported by the Miami U. Undergraduate Summer Scholars Program

and NASA.)



[29] THE EFFECTS OF PHYC AND PHYD ON TROPISTIC

RESPONSES OF ARABIDOPSIS. C.E. Montgomery, P. Kumar and J.Z.

Kiss Miami University, Oxford, OH.

Plants constantly adjust their architecture in order to optimize their

growth in response to a variety of environmental stimuli including light

and gravity. In seedlings, primary roots are positively gravitropic and

negatively phototropic while shoots are positively phototropic. Plants

sense light using the red-light-absorbing phytochromes and the blue-light-

absorbing cryptochromes and phototropins. The five members of the phy

family (PHYA to PHYE) have been shown to be involved in the

orientation of the shoots and roots of Arabidopsis. As the phyC mutant has

been isolated only recently, studies on the role of PHYC on tropistic

responses are incomplete. Thus in this project we studied the role of PHYC

in growth and tropisms by using the mutants phyCD and phyD and

compared them to the wild type Wassilewskija (WS WT). Four-day-old

seedlings were grown on half-strength MS salt in 1.2% agar, and

experiments were conducted on seedlings grown either in continuous white

light (70 µmol m-1s-1) or darkness. In general, growth of the mutants was

robust and comparable to the WT. The most significant differences were

found in experiments with light-grown seedlings. Roots of both the phyCD

and phyD mutants were impaired in negative blue-light induced

phototropism compared to the WT. Thus, our results suggest that both

PHYC and PHYD play a role in root phototropism.

(Supported by Miami University Undergraduate Summer Scholar

Program.)

[30] ELUCIDATING GENES INVOLVED IN EARLY GRAVITY

SIGNAL TRANSDUCTION IN ARABIDOPSIS. J. Bascom and S.E.

Wyatt Dept. of Environmental and Plant Biology, Ohio University,

Athens, OH.

Few genes are known to function early in the gravity signal transduction

pathway. Cold treatments have been used previously to identify mutants

in early gravity signal transduction (the gravity signal persistence (gps)

mutants of Arabidopsis). Therefore, a cold treatment, in conjunction with

microarray analysis, was performed on wild-type Arabidopsis to identify

additional genes involved in early signal transduction. Wild-type

Arabidopsis were gravistimulated at 4°C and this treatment was compared

to a simple cold (4°C) treatment in which plants were kept vertical as a

control. The inflorescence stem tissue in the region of elongation was

collected five minutes after the plants in each treatment were returned to

vertical at room temperature. RNA was extracted from these populations

and used to make the target population for hybridization to Affymetrix

ATH1 Arabidopsis gene chips. Analysis of the microarray data was

performed using GeneSpring software (Silicon Genetics, CA) and the

Cross-Gene Error Model. Differentially expressed genes were sorted

based on function, fold change, and up- vs. down-regulation. These

include several transcription factors, as well as kinases and other signal

transduction molecules. Several auxin-responsive proteins were also

strongly upregulated, as were a number of proteins whose function is

unknown and several cytochrome p450 proteins. Promising genes were

confirmed with Quantitative RT-PCR over a time course of cold

gravistimulation. These results present many favorable gene candidates

for further study as components of early gravity signal transduction in

Arabidopsis. (Partially supported by NASA: NAG2-1608 to SEW and the

Honors Tutorial College at Ohio University).

[31] MORPHOLOGICAL AND PHYSIOLOGICAL

CHARACTERIZATION OF TRANSGENIC TOMATO LINES

EXPRESSING INOSITOL POLYPHOSPHATE 5-PHOSPHATASE.

Courtney Sword1, Mariya Khodakovskaya1, Imara Y. Perera1, Christopher

S.Brown1, 2, Heike Winter- Sederoff 1 1Department of Plant Biology, and 2Kenan Institute for Science, Technology, and Engineering, North Carolina

State University, Raleigh, NC

Plants are adapted to the physical conditions they encounter on earth in

their specific environment. Most of the environmental factors such as

temperature, water availability, light, atmospheric pressure, and radiation

will be dramatically different in space and on other planets. The only

possible approach to increase the tolerance of crop plants to multiple and

extreme environmental condition is genetic engineering. It has been shown

that inositol-1,4,5-trisphosphate (InsP3) is involved in several signal

transduction pathways. Plant responses to salt, drought, cold, and osmotic

stresses as well as tropic responses to light and gravity are mediated by

inositolphosphate metabolism (Meijer and Munnik, 2003, Perera et al.

2006 and unpublished data). We generated transgenic tomato plants

expressing the human inositol polyphosphate 5-phosphatase (InsP3 5-

ptase), an enzyme that hydrolyzes InsP3. These plants have an enhanced

tolerance to drought stress (Khodakovskaya et. al. 2006, unpublished data).

To understand the mechanisms involved in stress tolerance of these

transgenic tomato plants we characterized morphological and

physiological parameters of independent homozygeous transgenic lines in

comparison with wild type and vector control lines. The average leaf

thickness was significantly increased in the transgenic lines and the

diameter of the main stems increased. Even though their tolerance to

drought stress dramatically increased, no significant physiological

differences were observed between wild type and transgenic tomato lines

in stomatal conductance, electron transport rate, and maximum quantum

yield. We are presenting data of detailed morphological characterization

and discussing possible mechanisms of the basis of those observed

morphological changes in the genetically modified lines.

(Project supported by NASA grant NAG2-1566 to Christopher S. Brown

and North Carolina State Grant Consortium grant 526294 to Mariya

Khodakovskaya)



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[32] IDENTIFYING THE NEURONAL BASIS OF GRAVIPERCEPTION

IN DROSOPHILA MELANOGASTER. C.J. Johnson1 , M.T. Texada1, R.

Simonette1, K.M. Beckingham1 1Dept. of Biochem. And Cell Biol., Rice

Univ., Houston, T.X.

The antenna of Drosophila is used to detect smell and sound. A

mechanosensory organ in the second antennal segment called Johnston's

Organ (JO) is responsible for audition. The array of 200 chordotonal

organs (CHO's) in this structure contains stretch responsive neurons.

However, in addition to sound perception, data from our laboratory

suggest that the JO also plays a role in graviperception.

A genetic screen in the Beckingham lab using a gravitaxic maze, has

identified mutant flies with altered gravitaxic behavior. A mutant of the

novel gene yuri gargarin (yuri) identified in this screen indicates that

effects on the JO are responsible for its aberrant gravitaxis. Thus, the

mutation appears to alter gravitaxic behavior through effects in a minor

subset of the JO CHO's. We are testing the hypothesis that there are two

functionally distinct classes of CHO's in the JO specialized for either

sound or graviperception. There are two anatomical classes of CHO's in

the JO that could serve these two different functions. We are beginning

with studies of the yuri gravitaxic mutant. If the CHO's affected in this

mutant are specialized for graviperception, we hypothesize i) that these

CHO's will correspond to only one of the two anatomical classes

previously identified in the JO and ii) the mutant will suffer no hearing

loss. Audition will be studied in collaboration with Dr. M Gopfert, Univ.

Koeln Germany. Detailed studies of the CHO's affected by the yuri

mutation are in progress. Several further mutants isolated from our screen

also affect the JO. Studies are in progress to identify those mutants that

affect subsets of CHO's in the JO so that these can also be used to test the

hypothesis that the JO serves as a graviperceptor.

[33] LONGITUDINAL HINDLIMB SUSPENSION STUDY REVEALS

PHYSICAL, FUNCTIONAL AND BIOCHEMICAL CHANGES IN MOUSE SKELETAL MUSCLE. A. Hanson, M. Young, L. Stodieck

and V. Ferguson

BioServe Space Technologies, University of Colorado, Boulder, CO.

Prolonged hindlimb suspension is a common method used to study

disuse muscle atrophy; however, the early stages of skeletal muscle

atrophy are poorly understood. A longitudinal analysis was performed to

examine changes in physical, functional and biochemical parameters

throughout a two week period. Thirty-five 12-week old male mice were

assigned to five equal groups that were suspended for 0, 1, 3, 11 or 14

days. At sacrifice, electrophysiology was performed to examine functional

strength of the soleus. Lean body mass and wet muscle weights were

recorded and muscles were cryo-preserved for future analysis. Protein

content was quantified for the soleus muscles. The soleus, a fast twitch

muscle, significantly decreased in wet mass throughout the study. The

soleus mass, normalized to day 0 body mass values in muscle (mg)/body

weight (g), decreased 13.9% (NS) by day 1, 17.1% (NS) by day 3, 32.8%

(p<0.05) by day 11 and 32.9% (p<0.05) by day 14. These data are

consistent with published results in a similar study performed in rats (Isfort

et. al., 2002). A similar trend in soleus electrophysiology data was

observed with an overall decrease by day 14. The gastrocnemius, a mixed

fiber type muscle, was less affected by hindlimb suspension. Normalized

gastrocnemius values decreased 1.5% (NS) by day 1, 7.7% (NS) by day 3,

11.0% (p<0.05) by day 11 and 13.4% (p<0.05) by day 14. Total protein

content of the soleus decreased to 57.7±4.4% of control values over the

course of the study. Compared to controls, protein concentrations dropped

from 58.6±4.3 to 43.6±4.4% and 49.1±1.7 to 30.0±1.4%, for soluble and

myofibrillar protein respectively; thus indicating substantial muscle

atrophy. In summary, these data provide an improved understanding of

longitudinal changes in physical, functional, and biochemical parameters

due to disuse muscle atrophy.

(Supported by NASA: NCC8-242)

[34] EFFECT OF LOCAL IRON IRRADIATION ON TRABECULAR

BONE. E.R. Bandstra1, S. Judex2, M.E. Vazquez3, T.A. Bateman1

1Dept. of Bioengineering, Clemson Univ., Clemson, SC, 2Dept. of

Biomedical Engineering, State Univ. of New York at Stony Brook, 3Medical Dept., Brookhaven National Laboratory, Upton, NY.

On long-duration exploratory missions, astronauts will be exposed to

higher doses of ionizing radiation from both solar and cosmic sources.

While NASA has been concerned with radiation exposure’s effect on the

central nervous and immune systems, we have recently identified that

spaceflight-relevant types of radiation, approaching doses astronauts will

be exposed to on exploratory missions, cause profound trabecular bone

loss. This study aims to investigate the effects of local (head only)

irradiation on regional and systemic trabecular bone. Male C57BL/6 mice

were irradiated with 1GeV/n HZE 56Fe at 16 weeks of age at the NASA

Space Radiation National Laboratory. The radiation was collimated such

that the head received 2.4 Gy and the rest of the body, including humerus

and tibia, received 14% (33.6 cGy) of the total dose delivered. The mice

were sacrificed 8 weeks after irradiation. MicroCT analysis was performed

on the trabecular bone of the proximal humerus and tibia. The humerus

showed a significant 15.1% decline in trabecular volume fraction

compared to the control group. However, the tibia did not show any

changes compared to control (insignificant decline in trabecular volume

fraction of 4%). The bone loss in the humerus, without corresponding loss

in the tibia, indicates either a differential response of these bones or a

greater loss in bones proximal to higher doses of radiation. Another

important contribution of this study is the demonstration of bone loss in

mice near peak bone mass. Our previous studies examined mice irradiated

at 9 weeks of age, when the skeletal system is rapidly growing. Radiation-

induced bone loss in the mature skeleton further demonstrates that this is a

potential concern for astronauts on long-duration exploratory missions and

should be studied to further characterize the bone loss, identify causal

mechanisms and develop countermeasures. Supported by South Carolina

Space Grant, Procter and Gamble, the NSBRI (NASA NCC9-58),

BioServe Space Technologies (NASA NCC 8-242), and NASA NAG 9-

1499 (S.J.).

[35] THE EFFECT OF LOW-SHEAR MODELED MICROGRAVITY ON

ADHERENT INVASIVE ESCHERICHIA COLI PHYSIOLOGY AND

VIRULENCE POTENTIAL. C.A. Allen, D.W. Niesel, and A.G. Torres. Department of Microbiology &

Immunology, University of Texas Medical Branch, Galveston, TX.

Adherent-invasive E. coli (AIEC) exhibit intrinsic adherent and invasive

characteristics during host cell interactions and have been found to

colonize the lesions of Crohn’s Disease (CD) patients. To date, little is

known about the environmental triggers which induce changes in AIEC

allowing it to persist in the gut and contribute to the chronic inflammation

associated with CD. AIEC strain O83:H1 was cultured under low-shear

modeled microgravity (LSMMG) using high-aspect ratio vessels (HARVs)

to examine the impact of this environment on AIEC physiology and

virulence potential. Physiological assays revealed increases in both thermal

stress resistance and in adherence to Caco-2 monolayers by AIEC O83:H1

after growth under LSMMG. The alternative sigma factor and global

stress response regulator RpoS was examined under LSMMG using an

isogenic rpoS mutant. The mutant exhibited similar characteristics as

AIEC O83:H1 but with significantly increased thermal stress resistance

and adherence. Western blot analysis revealed no significant differences

in RpoS expression in AIEC O83:H1 under LSMMG suggesting a

potential regulatory role at the transcriptional level. Transposon

mutagenesis of the rpoS mutant produced two isolates with diminished

adherence capabilities. These results suggest a regulatory role for RpoS in

AIEC adhesion under LSMMG conditions. Characterization of the

transposon mutants is currently underway to identify putative adhesins/and

or adherence-associated genes which play a key role in AIEC adherence

under LSMMG conditions.

(Supported by NASA: 98-HEDS-02-291 and GSRP Fellowship 424490)



[36] EFFECTS OF SIMULATED MICROGRAVITY ON NITRIC OXIDE

PRODUCTION AND PROTEOGLYCAN SYNTHESIS BY

CHONDROCYTES ENCAPSULATED IN 3D PEG HYDROGELS.

I. Villanueva1, B. Klement2, D. von Deutsch3, and S.J. Bryant1. 1Dept. of

Chemical and Biological Engineering, University of Colorado 2Dept. of

Anatomy and Neurobiology, and 3Dept. of Pharmacology and Toxicology,

Morehouse School of Medicine,.

Overproduction of nitric oxide (NO) due to shear stress and disruptive

loading has been implicated in cartilage degeneration within the

articulating joints. Rotating wall vessel (RWV) bioreactors provide tissues

with a rotation about a horizontal axis, which is depicted by extremely low

fluid shear stresses and turbulence. The constructs placed in the RWV

remain suspended within a constantly moving body of nutrient medium.

In this study, neutral polyethylene glycol (PEG) hydrogels with varying

crosslinking densities (ρx) were used as 3D scaffolds for chondrocyte

(cartilage forming cells) culture. These cartilage models were used to

study chondrocyte activity in the absence of loading and high shear

stresses. A spinner flask was used as the control. Chondrocyte response

was measured by NO production and proteoglycan production, both

normalized to total DNA content. PEG gels were fabricated with two ρx’s,

0.1 and 0.5 mol/L, by varying the PEG concentration to yield gels with the

same chemistry but different network structures. Chondrocytes were

encapsulated in the gels 24 hours prior to loading in the RWV. Gel

constructs were cultured in the RWV bioreactors for 2 and 5 days with an

average rotational speed of 22rpm. Mean proteoglycan content increased

and mean NO production decreased compared to controls at each time

point for each ρx. However, signficance was only observed at day 2 in the

low ρx gel with NO inhibition in the RWV compared to controls.

Crosslinking density did not affect chondrocyte response over the 5 days

of culture. Overall, an inhibition in NO was correlated with upregulation in

PG synthesis (r= 0.52 , p<0.001).This study demonstrates that PEG

hydrogel constructs cultured within the RWV culture provide a 3D

environment that inhibits NO production and has positive effects on the

formation of matrix components, which is essential for healthy cartilage

maintenance..

(Supported by NASA NAG2-1274 and NIH: Grant # K22 DE 016608)

[37] IDENTIFICATION OF MECHANOSENSITIVE GENES IN

OSTEOBLASTS BY COMPARATIVE MICROARRAY STUDIES

USING THE ROTATING WALL VESSEL AND THE RANDOM POSITIONING MACHINE. Patel, MJ1,2, Liu, W3, Sykes, MC1, Ward,

NE2, Risin, SA4, Risin, D2, Jo, H1,5. 1Wallace H. Coulter Department of

Biomedical Engineering, Georgia Institute of Technology and Emory

University, 2Space Life Sciences Directorate, Human Adaptation and

Countermeasures Office, NASA/Johnson Space Center, 3Department of

Molecular and Human Genetics and Microarray Core Facility, Baylor

College of Medicine, 4Department of Pathology and Laboratory Medicine,

University of Texas-Houston Medical School, 5Division of Cardiology,

Emory University School of Medicine

Microgravity of spaceflight induces bone loss due in part to decreased

bone formation by osteoblasts. Recently we have shown that simulated

microgravity changes gene expression profiles in 2T3 preosteoblasts using

the Random Positioning Machine (RPM). Here, we hypothesized that

exposure of preosteoblasts to an independent microgravity simulator, the

Rotating Wall Vessel (RWV) bioreactor, induces similar changes in

differentiation and gene transcript profiles, resulting in a more confined list

of mechanosensitive genes that may play a role in bone formation. In

comparison to static 1g controls, exposure of 2T3 cells to the RWV for 3

days inhibited alkaline phosphatase activity, a marker of differentiation,

and downregulated 61 and upregulated 45 genes by more than two-fold as

shown by microarray analysis. The microarray results were confirmed by

real-time PCR for downregulated genes osteomodulin, bone morphogenic

protein 4 (BMP4), runx2, and parathyroid hormone receptor 1 and by

Western blots for 3 downregulated genes, BMP4, peroxiredoxin IV, and

osteoglycin, and 1 upregulated gene peroxiredoxin I. Comparison of the

microarrays from the RPM and RWV studies identified 14

mechanosensitive genes that changed in the same direction. Further

comparison of our results to a published microarray data with

mechanically loaded mouse tibias revealed 16 genes upregulated by the

loading that were downregulated by RWV and RPM.

[38] SIMULATED MICROGRAVITY INHIBITS NEURAL

DIFFERENTIATION OF MOUSE BONE MARROW CELLS. M.

Takeda1, T. Okazaki1, T. Magaki1, A. Sasaki2, S.L. Wu2, Y. Kawahara2, 3,

C. Umeda2, R. Yoshimoto2, T. Kajiume1, L. Yuge2,3, and K. Kurisu1. 1Graduate School of Biomedical Sciences, 2Graduate School of Health

Sciences, 3Space Bio-Laboratories Y. K., Hiroshima University, Japan.

A 3D-clinostat is a multi-directional gravity device for simulating

microgravity. By controlled rotation of two axes, a 3D-clinostat minimizes

the cumulative gravity vector in cells cultured at the center of the device

and makes 10-3 G average over time. Bone marrow-derived cells (BMCs)

are reported to differentiate into the cells of neural lineages as well as

mesenchymal cells such as bone, cartilage, and muscles etc. in a media

(neural differentiation medium). Culture of BMCs under the state of

simulated microgravity may inhibit differentiation into these various types

of cells. Therefore, we investigated the effect of simulated microgravity

on differentiation of mouse BMCs using the 3D-clinostat, which special

reference to neural differentiation.

BMCs were isolated from BALB/cBy mice and allowed to proliferate in

normal 1G condition. Proliferated cells were, then, cultured under the

neural differentiation conditions in 3D-clinostat or normal 1G environment

for 7 days. The cells were examined for their growth pattern, morphology

and expression of neuronal marker mRNA (neurofilament). The cell

number was decreased 1.6 times more in 3D-clinostat than 1G

environment. However, differentiation of the cells into neural lineages

was inhibited: the cells extending processes and expressing neural maker

mRNA were scarcely found. Here we show that mouse BMCs proliferate

better and remain more undifferentiated in culture in a 3D-clinostat.

It is essential to obtain large number of neural cells for use in Cell

Therapy, since the proliferation is limited in cells of neural lineage. Thus

the culture in a 3D-clinostat and then transfer of the cells to neural

differentiating condition may contribute this field of medicine.

[39] ELECTRICAL STIMULATION OF MYOBLASTS DEVELOPS

SPONTANEOUSLY CONTRACTING MUSCLE FIBERS.

Y. Kawahara1,3, K. Yamaoka2, C. Umeda1, R. Yoshimoto1, T. Kajiume2, A.

Sasaki1, S.L. Wu1, K. Kataoka2, and L. Yuge1,3. 1Graduate School of

Health Sciences, 2Graduate School of Biomedical Sciences, 3Space Bio-

Laboratories Y. K., Hiroshima University, Japan.

Electric stimulation has been clinically used for treatment of muscle

atrophy in space flights. However, the effect of electrical stimulation, or

an ability to induce morphological, physiological, and molecular biological

effects on myoblasts during cell differentiation, has remained to be

elucidated. L6 rat myoblast cells (IFO50364) were seeded in 100-mm

culture dishes and maintained in a high-glucose Dulbecco modified

Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS).

When L6 cells reached confluence on culture day 6, they were transferred

into FBS-free medium and then exposed to electrical stimulation via field

electrodes (5min) on days 6, 8, 10, and 12 (group E) using a stimulator to

pass rectangular current pulses (2.0 ms, 50 V, 0.5 Hz) between two

platinum wires placed in the culture medium. The electrical stimulation

accelerated the appearance of myotubes, and subsequently produced

spontaneously contracting muscle fibers. Measurement of membrane

potential showed that the contracting L6 cells had functional ion channels

and gap junctions. In the electrically stimulated cells, expression of MyoD

family, myogenin and Myf-6, and myosin proteins was enhanced.

Expression of gap junction protein, connexin 43, was increased and

maintained at a high level in the electrically stimulated cells by western

blot analysis and immunostaining. The differentiation of myoblasts was

accelerated, and even striated muscle cells were obtained. These results

suggest the possibility that electric stimulation, effectively used in clinical

therapy of muscle atrophy, facilitates not only the development of existing

muscle fibers but also the differentiation of myoblasts.



[40] THE EFFECT OF MICROGRAVITY ON THE ARRANGEMENT

OF AMYLOPLASTS IN HYPCOTYL ENDODERMAL CELLS. D.

Cirelli and J.Z. Kiss, Department of Botany, Miami University, Oxford,

OH 45056.

The overarching goal of this project is to study the effects of

microgravity (µg) on the development of plant cells. Specifically, the

structure of gravity-perceiving endodermal cells of hypocotyls will be

studied by morphometric analyses in wild-type, a reduced-starch mutant,

and a starchless mutant of Arabidopsis thaliana grown in µg (F-µg) and

compared to ground 1g (G-1g) and flight 1g (F-1g) controls. These

experiments were performed as part of the Shuttle-to-Mir Biorack

missions, where an incubator with an on-board reference centrifuge was

included. Studies are in progress to assess the appropriate software for

modeling the position of statolithic amyloplasts in endodermal cells during

the various gravity treatments provided during the spaceflight experiments.

Our results with hypocotyls will be compared to studies on the columella

cells of the root cap. In addition, we plan to test the hypothesis that

changing gravity stimulation can elicit feedback control over statolith mass

by changing the size, number, and grouping of amyloplasts. Plastid

grouping and position is also of particular interest since some organisms

display a programmed pre-grouping while others show a seemingly

random disposition in µg.

[41] EFFECT OF ARG1 MUTATION ON PLASTID SEDIMENTATION

IN ARABIDOPSIS HYPOCOTYLS. N.S. Kumar and J.Z. Kiss,

Department of Botany, Miami University, Oxford, OH.

The ARG1 (altered response to gravity) protein is hypothesized to be

involved in signal transduction phase of gravitropism, and ARG1 is

essential for normal gravitropism in roots and hypocotyls of Arabidopsis.

Previous studies have characterized gravitropic curvatures in roots and

hypocotyls of dark-grown seedlings and found that they are impaired in

gravitropism. The arg1 mutants demonstrated lower curvature angles

compared to the wild-type. We wanted to investigate whether arg1

mutation affects plastid movement in the gravity-perceiving endodermal

cells of hypocotyls. For this, we conducted light microscopic studies to

analyze plastid sedimentation rates in hypocotyls of light-grown WS and

arg1 seedlings. Movement of plastids was studied in vertical and 90°

reoriented seedlings that were processed by cryofixation and freeze

substitution techniques. The hypocotyls were sectioned, stained and

observed using light microscopy. Results show that arg1 mutation affects

plastid movement in hypocotyls. Plastid movement is reduced in the

endodermal cells of arg1 compared to those of the wild-type. Our data also

indicate that arg1 mutation reduces gravitropism in hypocotyls by

modulating plastid sedimentation and that arg1 mutants are likely to be

altered in the actin cytoskeleton. Therefore, these studies support the

hypothesis that ARG1 is involved in the early events of gravitropic signal

transduction in shoots.

(Supported by NASA grant: NCC2-1200).

[42] MICROGRAVITY INDUCED CHANGES IN AORTIC STIFFNESS

AND ITS ROLE IN ORTHOSTATIC INTOLERANCE. E.C. Tuday,

J.V. Meck, A.A. Shoukas, D.E. Berkowitz 1Dept. of Biomedical

Engineering, 2Dept. of Anesthesiology and Critical Care Medicine, Johns

Hopkins University, Baltimore MD and 3Human Adaptation and

Countermeasures Office, NASA Johnson Space Center, Houston, TX

Microgravity (µG) induced orthostatic intolerance (OI) in astronauts, a

common consequence of manned spaceflight, is characterized by a marked

decrease in cardiac output (CO) in response to an orthostatic stress. Since

CO is highly dependent on venous return, alterations in the resistance to

venous return (RVR) may be important in contributing to OI. The RVR is

inversely dependent on arterial compliance (CA), where aortic compliance

(Ca) contributes up to 60% of CA. We tested the hypothesis that µG

induced changes in Ca may represent a protective mechanism against OI. A

retrospective analysis on hemodynamic data collected from astronauts

after 5-18 day spaceflight missions revealed that orthostatically tolerant

(OT) astronauts showed a significant decrease in Ca after spaceflight

(1.996±0.09741 ml/mmHg to 1.707±0.08313 ml/mmHg; p=0.0011; N

=40) while OI astronauts showed a slight increase in Ca (1.888±0.1269

ml/mmHg to 2.280±0.3868 ml/mmHg; p=0.3607; N=17). A ground based

animal model simulating µG, hindlimb unweighted (HLU) rats, was used

to explore this phenomenon. Two independent assessments of Ca, in vivo

pulse wave velocity (PWV) of the thoracic aorta and in vitro pressure-

diameter squared relationship (PDSR) measurements of the excised

thoracic aorta were determined. PWV showed a significant increase in

aortic stiffness compared to control (PWV: 7.227±0.1375 m/s vs.

4.074±0.1879 m/s, p<0.0001, N=6) despite unchanged blood pressures.

This increase in arterial stiffness was confirmed by the PDSR analysis

(PDSR slopes: 176.5 ± 22.47 mmH2O/mm^2 vs. 113.7 ± 5.409

mmH2O/mm^2, p= 0.0216, N=6). Thus both actual µG in humans and

simulated µG in rats induces changes in Ca. The difference in Ca in OT and

OI astronaut suggests that the µG induced decrease in Ca is a protective

adaptation to spaceflight that reduces the RVR and allows for the

maintenance of adequate CO in response to an orthostatic stress.



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Genetic Adaptation for Stress



[43] SYMPOSIUM: GENETIC ADAPTATION FOR STRESS. K.H.

Hasenstein, Biology Dept. University of Louisiana Lafayette, LA 70504-

2451

Exploration of new environments is associated with many stresses.

Genetic stress is perhaps best described by the ‘Founder Effect’: few

individuals colonize a new habitat and have to cope with their genetic

predispositions, limited gene pool and thus limited genetic response to

their new environment. In preparing for the colonization of the moon and

planets, the challenge for scientists is to recognize, counteract, and

possibly exploit the increased levels of mutations expected to occur in

humans, microbes and life support organisms. The modification of genetic

material by high-energy radiation is probably the most deleterious

condition encountered by space travelers, resulting in various forms of

cancer and immune diseases. While ethical concerns prevent changing

human genetics, genetic modifications have the potential to reduce

virulence of viruses or bacteria, improve the functionality of life support

systems and, perhaps most relevant, lead to the discovery of new methods

to minimize adverse effects of new environments on humans. It is

therefore important to develop gene therapy to counteract radiation

problems, discover how plants can be adapted to extreme conditions such

as unknown mineral compositions or substrates, low light, low pressure,

and high energy radiation. We must also prepare to understand the effect

of closed environments on the propagation, mutagenic effects and

virulence of microbes. Lastly the benefit of learning how the human body

deals with damaging environmental influences and recycles or disposes of

altered proteins and modified cellular tools is important for the success of

future space missions and improvement of life on Earth.

[44] PLANT ADAPTATION TO ABIOTIC STRESS – FROM

PHYSIOLOGY TO GENOMICS. Hans J. Bohnert, Department of

Plant Biology & Department of Crop Sciences, University of Illinois at

Urbana-Champaign, Urbana, IL 61801, USA

Physiological balance in plants is often challenged by abiotic factors,

such as temperature extremes, osmotic and ionic imbalances, light quantity

or quality, or nutrient deficit. Several decades of studies have resulted in a

body of knowledge that described stress-dependent changes in overall

plant development, and the physiology and biochemistry of photosynthetic

tissues, organs and cells. Genetic and molecular studies provided

information on individual components of the plant stress response but

genomics-type studies have truly revolutionized our approach to

understanding what constitutes stress and stress tolerance or sensitivity.

Based on established physiological protocols, experiments can now be

designed and the results correlated with gene complement, transcript

profiles, protein amount and dynamics, and metabolite changes. A

common set of close to 30,000 genes in all angiosperms has evolved

through genome and gene duplications and changes in the control of gene

and protein expression. Thus, stress-adaptive evolutionary diversity in

species, and in ecotypes and lines of one species, appears to be based

mainly on how (fast) any stress is perceived and signaled to a common set

of downstream genes and the modification of constitutively expressed

proteins. Examples will highlight hormonal abiotic stress responses that,

transmitted through reactive oxygen species and calcium-dependent

signaling, determine plant tolerance capacity (supported by NSF, DOE,

USDA and institutional funds).

[45] KEY ROLES OF THE PROTEASOME IN CELL PROTECTION

Alfred L. Goldberg, Department of Cell Biology, Harvard Medical School,

Boston, MA

Most proteins in mammalian cells are degraded by the ubiquitin-

proteasome pathway, where protein substrates are linked to ubiquitin

molecules by one of the cell’s many ubiquitin ligases (E3s). This

modification marks the protein for rapid degradation by the 26S

proteasome. This large complex uses ATP to unfold the proteins and to

inject them into its 20S core particle, where they are digested to small

peptides. This system selectively destroys abnormally folded proteins as

result from mutations and postsynthetic damage by heat or oxygen

radicals. Such proteins accumulate in various neurodegenerative diseases,

and this pathway is critical in homeostasis and rapidly destroys many

regulatory proteins, important in the control of gene expression and

growth.

Peptides released by proteasomes range from 2-24 residues in length.

Although most are rapidly digested to amino acids, some are transported

through the ER to the cell surface, where they are presented to the immune

system on MHC Class I molecules. This process enables circulating

cytotoxic T cells to screen for and eliminate virally infected cells and

cancers.

With denervation, inactivity (e.g. bed rest or in space personnel due to lack

of gravity) in many systemic diseases (e.g. cancer) and fasting, muscles

atrophy due to a general activation of the ubiquitin-proteasome pathway in

muscles. The atrophying muscles show a common pattern of changes in

expression of specific genes (which we term “atrogenes”). The two

proteins induced most dramatically are muscle-specific ubiquitin ligases,

atrogin-1 and MuRF-1, which are essential in the atrophy process and the

accelerated proteolysis. In atrophying muscles, the Foxo family of

transcription factors transcribes atrogin and triggers muscle wasting.

Therefore, inhibitors of Foxo activation or the ubiquitin-proteasome

pathway are an attractive approach to combat muscle wasting.

Much has been learned about the functions of this ubiquitin-

proteasome system by the use of inhibitors of the proteasome that enter

cells and inhibit intracellular proteolysis. Blocking proteasome function

eventually induces apoptosis, especially in cancer cells. One such

inhibitor (Velcade PS341) has been approved by the FDA for treatment of

multiple myeloma, but it is now in many phase II trials against diverse

cancers.

[46] Abstract Not Available



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Cell Biology and Animal

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[47] ß-ACTIN PROTEIN CONCENTRATION FLUCTUATES DURING

CONTRACTION OF LOADED AND UNLOADED FIBROBLAST

POPULATED COLLAGEN GELS. B. P. Johnson-Wint and W.

Heisler. Dept. of Biological Sciences, Northern Illinois University,

DeKalb, IL.

Fibroblasts use a cytoplasmic actin-myosin force generating machinery

to contract type-I collagen gels. ß-actin is the major actin expressed in

fibroblasts and exists as monomers or microfilaments capable of force

generation. In the present study we ask if total ß-actin protein

concentration changes in fibroblasts during contraction of unloaded and

loaded fibroblast populated collagen gels (FPCGs).

Fibroblasts were isolated from adult rat tail tendon and maintained in cell

culture. FPCGs were formed in wells of 48-well culture plates and

maintained in 0.5 mls of culture medium. Two glass beads weighing 19

mg were placed on top of each loaded FPCG. FPCGs were harvested and

subject to SDS-PAGE and quantitative ß-actin Western blotting. A DNA

assay was used to determine the cell number in each FPCG. FPCGs were

photographed to track gel contraction.

FPCGs contracted 80% in the first 24 hours. Loaded FPCGs contracted

faster than unloaded FPCGs. Cell number in the FPCGs stayed at 3.7 X

105 for 48 hours and gradually fell to 3 X 105 cells at 96 hours. The ß-

actin protein content of the fibroblasts in FPCGs rose and fell twice during

the 96 hour culture period. The first elevation of ß-actin concentration

occurred during the first 24 hours of contraction where it went from 100

million molecules/cell to 240 million molecules/cell. ß-actin protein levels

rose and peaked 6 hours earlier in loaded than in unloaded FPCGs. From

24 to 58 hours ß-actin levels fell back down to 100 million molecules per

cell. This was followed by a second rise in ß-actin protein levels to 300

million molecules/cell at 74 hours and fall to 125 million molecules/cell by

96 hours.

In conclusion, we have shown that ß-actin protein content of fibroblasts

in contracting FPCGs rises and falls twice during a 96 hour culture period

spanning a range of 100 million to 300 million ß-actin molecules/cell. The

first rise in ß-actin concentration coincides with early reduction of FPCG

area and is accelerated in loaded FPCGs. Supported by NIU and the NIU

Foundation.

[48] SACCHAROMYCES CEREVISIAE USES GAS PRODUCING

ANAEROBIC METABOLIC PATHWAYS DURING SPACE FLIGHT. Timothy G Hammond1,2, , Cheryl A Nickerson3, Jake Freeman4,

Louis S Stodieck4, and Patricia L Allen1. 1Department of Medicine, Tulane

University School of Medicine, & VAMC, New Orleans, LA, 3Center for

Infectious Diseases and Vaccinology, Arizona State Univ., Tempe AZ, ;

and 4BioServe Space Technologies, University of Colorado, Boulder, CO.

As part of a large-scale effort to examine the function of all yeast genes,

a consortium of investigators created a series of heterozygous and

homozygous diploid strains, each containing a deletion of a single gene

allele. A selectable marker that carries adjacent gene specific

oligonucleotide sequences replaces each gene. These sequences serve as

an identifier of the locus essentially acting as a barcode. The individual

strains can then pooled so that all are represented in equimolar amounts,

and grown en mass. The result of this genomic approach to a selective

growth condition has been coined "fitness profiling". We dried down

multiple identical aliquots of these heterozygous and homozygous diploid

strain pools on filters, and loaded them into 3 compartment glass tubes

(Fluid Processing Apparatus - FPA). The first compartment in each tube

contained the dried yeast with growth media and fixative loaded in the

other two compartments, respectively. Eight FPAs were loaded into hand

cranked containers (Group Activation Packs –GAPs) for simultaneous

activation. Two GAPs were held for ground based activation in the

investigators lab at Tulane in New Orleans, two shipped to Moscow, and

two more launched to the International Space Station (Mission 13P) and

grown for 72 hours. All samples grew robustly. However, there were

dramatic differences in gas production as the homozygous yeast pool flight

samples produced 2.73+0.39 ml of gas (mean + standard error, n=6), while

both the Russian controls (0.16+0.07) and the Tulane growth controls

(0+0.02) produced significantly less gas (p<0.01, ANOVA and Tukey’s

post hoc test). The pattern in the heterozygous yeast pool was similar but

less marked. This demonstrates a near complete switch from anaerobic

carbon dioxide producing pathways in flight, to aerobic carbon dioxide

free metabolic pathways on the ground.

[49] DIFFERENCES IN GENE REGULATION IN E. COLI GROWN ON

FOUR COMMON MODELED MICROGRAVITY SYSTEMS. E.A.

Juergensmeyer1, M.A. Juergensmeyer2 and E.M. Mobley2 1Judson College, Elgin, IL, and 2IIT Research Institute, Chicago, IL.

Bacterial responses to spaceflight are mimicked on the ground using

modeled microgravity (MMG). A variety of different MMG systems exist,

and cultures grown in each system are capable of demonstrating

physiological responses similar to those seen in flight culture, such as

increased growth rate and changes in antibiotic resistance. However, these

systems are rarely compared to each other, and it is not known which

MMG is the most similar to spaceflight. We have grown E. coli in various

MMG systems, isolated the RNA from these cultures at mid-log phase, and

analyzed the gene expression patterns of the cultures using Affymetrix

microarrays. While there are differences between the various growth

systems, certain pathways seem to be uniformly down-regulated by MMG,

including sugar transport and the nitrogen metabolism pathways. Slow

clinorotation is extremely different from the other treatments, resulting in

over 300 genes differentially regulated, the majority of which were up-

regulated. These results indicate that a profound difference in response

exists to various modeled microgravity systems and that the choice of a

modeled microgravity system for ground research may be dictated by the

aspect of the microgravity environment in question.

[50] UTILIZATION OF NASA MANAGED COLD STOWAGE

RESOURCES FOR OPTIMAL CULTURE INCUBATION AND STORAGE OF SPEGIS CANISTERS. K. Sato1, D. Connor2, J. Dean3,

D. Melendez3, D.W. Niesel4, N. Williams4, U. Pandya4, S. Ormsby1, K.

Gibbs1, K.E. Perkins1 and H.E. Teal5 1Lockheed Martin Space Operations, NASA Ames Research Center,

Moffett Field, CA, 2Center for Biophysical Sciences and Engineering, The

University of Alabama at Birmingham, Birmingham, AL, 3Jacobs

Sverdrup, Jacobs Engineering Group Inc., Houston, TX, 4Department of

Microbiology and Immunology, University of Texas Medical Branch,

Galveston, TX, and 5NASA Ames Research Center, Moffett Field, CA.

Providing optimal on-orbit heating, cooling, and low temperature storage

of biological flight samples is a critical component for successful

spaceflight investigations. The NASA managed Cold Stowage Working

Group, NASA Ames Research Center, and the University of Texas

Medical Branch are cooperatively fine-tuning the experimental parameters

for use of the Cold Stowage Resources with biological specimens

requiring specific incubation and storage temperatures. The space biology

experiment, SPEGIS- Streptococcus pneumoniae Gene Expression and

Virulence Potential in the Space Environment, entails S. pneumoniae being

launched in vials that are contained within the SPEGIS Canisters aboard

the Space Shuttle at 4 °C. While in orbit, the cultures will be activated

upon reaching 37 °C and later stored at –20 °C (or colder) until recovery.

In order to assess the time required for the SPEGIS Canisters to reach 37

°C from 4 °C, and then –20 °C (or colder), a number of testing scenarios

have been performed utilizing the Cold Stowage Minus Eighty Laboratory

Freezer for ISS (MELFI), Microgravity Experiment Research

Locker/INcubator (MERLIN), and simulated incubator/cold-room models

at UTMB to mimic the Cold Stowage results. Determination of the time

required for temperature equilibration of the SPEGIS Canisters will

establish the necessary parameters for starting culture concentrations and

optimization of sample return at designated collection time points.

(Supported by NASA: NCC2-1160)



[51] GRAVITATIONAL EFFECTS ON GLUCOSE DIFFUSION INTO

CARTILAGE TISSUE. R.E. Flowers-Aime1, C.M. Marshall1, A.W. von

Deutsch2,3, A.A. Belton2, J. Wright4, D.A. von Deutsch2, D.F. Paulsen1 and

B.J. Klement1 Depts. of 1Anatomy & Neurobiology and 2Pharmacology &

Toxicology, 3Clinical Research Center, Morehouse School of Medicine,

Atlanta GA, 4Auburn University.

Cartilage has the ability to bear mechanical stress without distortion. It is

avascular, so nutrients and oxygen must diffuse through the extracellular

matrix to reach the cells. Changes in nutrient availability could alter cell

metabolism and ultimately cartilage structure and function. The objective

of this project was to determine if glucose diffusion into cartilage was

altered in a microgravity (MG) or hypergravity (HG) environment

compared to controls at standard 1x gravity (1xG). The experiments were

conducted in parabolic flight aboard the NASA C-9 aircraft. A flight-

certified apparatus consisting of three interconnected syringes contained

each experiment on the aircraft. Bovine articular cartilage was exposed to

a fluorescent labeled glucose solution for 20 seconds during the MG, HG

or 1xG episodes during flight. The amount of glucose that diffused into the

tissue was quantitated. There was no significant difference in the wet or

dry weights of the samples. The total amount of glucose that diffused into

cartilage during MG and HG was significantly less than at 1xG by 23%

and 27%, respectively. The amount of glucose per tissue wet weight was

reduced in the samples exposed to MG and HG compared to 1xG controls,

but they were not significant. However, the amount of glucose per tissue

dry weight was significantly less in the MG (26%) and HG (41%) samples

than at 1xG. Tissue fluid volume may not be as important for diffusion in

MG and HG as the structural components of the matrix. Since diffusion is

reduced in both MG and HG, two different mechanisms, such as matrix

conformation and tissue fluid flow, may be responsible. This suggests that

cartilage nutrition may be impaired in different gravitational environments.

Supported by NASA grants NCC9-112, NAG3-2611 and RGSFOP at JSC,

as well as RR03034 and NCRR 5P20RR11104.

[52] GEOTAXIS OF INFANT RODENTS: THINK POSITIVE. J.R.

Alberts. Department. of Psychological and Brain Sciences, Indiana

University, Bloomington, IN.

Traditionally, infant rodents have been characterized behaviorally as

displaying “negative geotaxis”, i.e., a reflex-like tendency to orient and

move directionally against gravity, such as up an inclined plane. Recent

re-evaluations and experiments indicate that this tradition is patently

incorrect. Previous reports of negative geotaxis by young rodents are

unreliable or, in many cases, appear to describe postural adjustments or

other compensatory responses to prevent falling. If rat pups are geotaxic

at all, they display positive, not negative geotaxis. 10-day-old rat pups

placed on modest inclines (e.g, 4o) moved downhill (positive geotaxis), but

their downward orientation required contact with a wall. Such wall

contact was subsequently associated with greater velocity of movment. A

broader and more integrative view of a young rodent’s gravity-guided

responses is needed, because thigmotaxis and orthokinesis may also be

involved. These findings will be discussed in the context of research and

testing in gravitational and space studies.

[53] INFLUENCE OF SPACE FLIGHT ON THE GENETICS OF

STREPTOMYCES LIVIDANS 66 – PIJ702.

T. L. Goins1, V.G. Martinson1, V.Yu. Tabakov2, T. A. Voeikova2, and B.

H. Pyle1. 1Department of Microbiology, Montana State University,

Bozeman, Montana, and 2GosNIIGenetika, 1 Dorozhnyi proezd, Moscow

117545, Russia.

Gram-positive bacteria belonging to the genus Streptomyces are

characterized by a high level of genetic instability in response to external

factors. Streptomyces lividans is a typical representative species with a

complex life cycle including vegetative mycelia and sexual spores. The

strain selected to study specific spaceflight factors (SSF) was S. lividans

66 harboring the multi-copy plasmid pIJ702. The possible effects of

radiation and microgravity on genetic stability were assessed using the

plasmid marker genes for melanin production and thiostrepton antibiotic

resistance. Cultures of S. lividans 66 [pIJ702] were flown on Foton-M2

(May 31 – June 16, 2005) with on-board radiation and temperature

monitors. An asynchronous ground control (AGC) mimicked the growth

temperature of the flight cultures, while an additional laboratory control

(LC) grew the cultures under optimal maturation temperature (26°C to

28°C). Unfortunately, the temperature aboard Foton-M2 was 15°C to

20°C during flight and did not permit maturation of the culture. Post-

flight, the flight and AGC cultures were maintained at 2°C to 10°C until

being permitted to differentiate at 28°C for five days to facilitate

sporulation. Clones recovered under thiostrepton selection were also

melanin negative indicating a loss of pIJ702. Additionally, non-melanized

clones recovered on non-selective media proved to be thiostrepton

sensitive upon transfer to media containing the antibiotic. The rate of

pIJ702 loss was significantly higher in the flight and asynchronous ground

control clones than in the laboratory control clones, indicating a

temperature effect. Neither microgravity nor radiation effects were

detected as there was no significant difference between flight and AGC

clones. However, lack of differentiation may have obscured the effects of

radiation exposure (200-300 mRad) or microgravity on genetic instability.

(Supported by the Institute for Biomedical Problems, Moscow, Russia, and

NASA: NCC2-1143.)



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Plant Development and Gravity

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[54] THE SHL1 AND SHL5 GENES INFLUENCE BOTH RED- AND

BLUE-LIGHT-BASED PHOTOTROPISM IN ARABIDOPSIS

THALIANA. P. Kumar and J.Z. Kiss.

Plants receive sensory input from a large number of environmental

stimuli including light, gravity, and touch. Of these, light is one of the

most important factors in the development throughout the life cycle of a

plant. As a consequence, plants have evolved several families of

photoreceptor molecules. The major groups of photoreceptors in

flowering plants include the phytochromes (for red, far-red),

cryptochromes (blue), and phototropins (blue). While considerable

progress has been made in understanding the photobiology of light

sensing, relatively little is known about the events downstream to

perception. The shl (seedlings hypersenstive to light) mutants exhibit an

inhibition in hypocotyl length even under low fluence rates, and the SHL

gene family is hypothesized to be a negative regulator of

photomorphogenesis. In this report, we studied tropistic responses of

seedlings of shl1 and shl5 and compared these responses to those of wild-

type seedlings. Roots of both mutants have an enhanced positive

phototropic response to red light, and hypocotyls of shl5 have an enhanced

positive phototropism relative to blue light. However, roots of shl1 and

shl5 seedlings exhibit a diminished negative blue-light phototropism. In

contrast, both shl mutants have little to no alteration in gravitropism and in

growth rates of roots and hypocotyls. Thus, SHL1 and SHL5 appear to be

part of the signaling pathway downstream to the phototropins as well as to

the cryptochromes and the phytochromes. Our results support the

hypothesis that the SHL genes act at the juncture of red and blue light

signaling networks.

(Supported by NASA Grant NCC2-1200).

[55] METABOLIC AND GENOMIC PROFILING OF TRANSGENIC

TOMATO LINES EXPRESSING INOSITOL POLYPHOSPHATE 5-

PHOSPHATASE Mariya Khodakovskaya1, Chiu-Yeh Hung1, Imara

Perera1, Wendy Boss1, Christopher Brown1,2 and Heike Winter Sederoff 1 1Department of Plant Biology, 2Kenan Institute for Science and

Engineering, North Carolina State University, Raleigh, NC

Inositol-1,4,5-trisphosphate (InsP3) is secondary messenger involved in

plant signal transduction pathways. Rapid, transient increases in InsP3 have

been demonstrated in plant tissues in response to a variety of

environmental stresses. Expression of inositol polyphosphate 5-

phosphatase (InsP 5-ptase), an enzyme which hydrolyzes InsP3,

dramatically reduced steady-state levels of InsP3 in Arabidopsis plants and

resulted in a decreased and delayed response to gravity and increased

tolerance to drought stress (Perera et al. 2006; and unpublished data). We

transformed tomato plants (v. Micro-Tom) with InsP 5-ptase to study the

effect of dampened InsP3 levels in a crop plant. Constitutive expression of

InsP 5-ptase decreased InsP3 levels in leaves and fruits analyzed.

Preliminary data indicate correlating increases in ABA levels and

dramatically increased tolerance to drought stress. Some transgenic lines

exhibited phenotypical and morphological differences in leaf and stem

thickness. We are currently analyzing the effects of the transgene

expression on global gene expression using Affymetrix Microarrays. We

are suggesting that decreasing of InsP3 levels affect not only plant stress

response but also the metabolic profile and nutritional characteristics in the

transgenic lines. Using realtime PCR, we found several fold increases in

the transcript abundance of chalcone synthase (LECHS1), a key enzyme in

the flavonoid biosynthetic pathway, in five independent transgenic tomato

lines compared to wild type. Because primary and secondary metabolite

levels play important roles in plant stress responses, composition and level

of basic metabolite groups should be different in cells expressing genes

involved in stress signal transductional pathway. We are integrating

genomic and metabolomic data with to understand the mechanism by

which InsP3 metabolism regulates vegetative and generative tissues of

transgenic tomato lines.

(Supported by NASA grant NAG2-1566 to CSB and NC Space Grant

526294 to MVK)

[56] GENETIC ANALYSIS OF GRAVITY SIGNAL TRANSDUCTION

IN ARABIDOPSIS ROOTS. P.H. Masson, B.R. Harrison and J. Stanga

Laboratory of Genetics (room 3262), University of Wisconsin-Madison,

425G Henry Mall, Madison, WI 53706, USA

Arabidopsis roots respond to gravistimulation by developing a curvature

that is modulated by a lateral gradient of auxin. This gradient originates in

the columella statocytes, and is associated with a lateral repositioning of

the PIN3 auxin efflux facilitator in these cells. We used genetics to identify

proteins that contribute to gravity signal transduction in the statocytes.

ARG1 and ARL2 are needed for lateral auxin transport across the cap.

ARG1 is associated with the vesicular trafficking pathway, suggesting it

regulates PIN3 function or trafficking. Indeed, immunolocalization studies

confirm a lack of PIN3 relocalization in gravistimulated statocytes of

arg1-2 and arl2-1 mutant root caps. Interestingly, arg1-2 and arl2-1

mutant seedlings still show significant gravitropic responses, as do starch-

deficient mutants like pgm. However, arg1-2 pgm and arl2-1 pgm double

mutants display strongly enhanced gravitropic defects relative to each

single mutant, suggesting a novel genetic approach to isolate new gravity

signal transducers that function in the PGM pathway. Accordingly, we

isolated and characterized two genetic enhancers of arg1-2, called mar1-1

and mar2-1. mar1-1 and mar2-1 mutant seedlings display almost wild type

gravitropism in an ARG1 background. However, arg1-2 mar1-1 and arg1-

2 mar2-1 double mutant seedlings are almost completely agravitropic

while retaining wild-type root-growth responses to phytohormones and

polar auxin transport inhibitors, remaining phototropism-competent,

accumulating starch like wild type, and displaying seemingly wild-type

amyloplast sedimentation in their statocytes. Hence, MAR1 and MAR2

appear to function in early phases of gravity signal transduction. The MAR

loci were cloned and shown to encode proteins that are targeted to the

plastid outer envelope, suggesting a more direct involvement of plastid-

borne proteins in gravity signal transduction than originally anticipated by

the classical starch-statolith hypothesis.

(Supported by NSF: Grant #MCB-0240084).

[57] OVEREXPRESSION OF ARABIDOPSIS FATTY ACID AMIDE

HYDROLASE (FAAH) PROMOTES PLANT GROWTH AND

MODIFIES SENSITIVITY TO EXOGENOUS N-

ACYLETHANOLAMINES. Y.-S. Wang1, R. Shrestha2, K. D.

Chapman2, and E.B. Blancaflor1 1Plant Biology Division, The Samuel

Roberts Noble Foundation, Ardmore, Oklahoma, 2Center for Plant Lipid

Research, Department of Biological Sciences, Univ of North Texas, USA

N-acylethanolamines (NAEs) have emerged as a group of lipid

mediators involved in a wide range of animal physiological processes. One

of the polyunsaturated NAEs, namely anandamide (NAE20:4), is an

important component of the endocannabinoid signaling pathway that

modulates a variety of physiological and behavioral processes in

vertebrates. Its signaling activity within the endocannabinoid pathway is

terminated by a fatty acid amide hydrolase (FAAH) that catalyzes NAEs

into their corresponding free fatty acids and ethanolamines. Despite the

lack of the complete endocannabinoid machinery in plants to date, several

NAE types have been identified and quantified in plants. Moreover,

bioinformatic approaches led to the identification of a FAAH homolog in

Arabidopsis (AtFAAH; At5g64440) that is capable of hydrolyzing NAEs

in vitro. We generated plants overexpressing AtFAAH to better understand

the physiological role (s) of NAEs in plant development. Here we report

the localization and functional characterization of the AtFAAH. An

AtFAAH-GFP fusion localized to an ER-like compartment, which is

consistent with its endomembrane localization in mammals. Constitutively

overexpressing the AtFAAH in Arabidopsis made seedlings more tolerant

to elevated levels of exogenous NAEs, which typical is inhibitory to

seedling development and led to plants with significantly larger organs,

partly due to increased cell size. Moreover, RT-PCR suggested constitutive

AtFAAH expression in all tissues, whereas a promoter study revealed its

preferential expression in rapidly elongating cells such as expanding

hypocotyls cells in the dark and the elongation zone of primary roots.

Taken together, we propose that overexpression of AtFAAH promotes cell

enlargement/elongation, possibly through the depletion of endogenous

NAEs

(Supporter by DOE Biosciences grant DE-FG02-05ER15647 to KDC and

EBB).



[58] ETHYLENE REGULATION OF GRAVITROPIC CURVATURE IN

ARABIDOPSIS STEMS. M. A. Harrison and M. L. Brown. Dept. of

Biological Sciences, Marshall University, Huntington, WV.

Horizontal placement of a plant stem causes the redistribution of the

soluble hormone auxin and stimulates biosynthesis of the gaseous hormone

ethylene. While auxin is the primary plant hormone engaged in gravitropic

responses through stimulation of growth, ethylene plays a modulating role

in regulating the kinetics of this process. Ethylene often acts as an inhibitor

of shoot and root growth, slowing gravitropic curvature, but has been

reported to stimulate growth under certain conditions. Ethylene is

produced by the oxidation of 1-aminocyclopropane-1-carboxylic acid

(ACC), which is formed from S-adenosyl methionine. The regulation of

ACC synthesis by ACC synthase (ACS) serves as the rate-controlling step

in ethylene biosynthesis. ACS enzymes are encoded by a gene family

whose expression is differentially regulated in various tissues. Our major

research objective is to evaluate individual ACS forms in the regulation of

gravitropism in dark-grown Arabidopsis seedlings. Potential changes in

expression of the various ACS forms were evaluated in transgenic plants

carrying ACS promoter-GUS fusions and by RT-PCR. Preliminary results

do not reveal distinct changes in ACS expression in curving hypocotyls in

wild type plants. The role of each ACS member in hypocotyl growth and

gravitropic curvature was determined by comparing wild-type responses

with those of mutants that do not express specific ACS forms. Gravitropic

curvature and growth rate were measured from digital images taken at 0, 3,

5, and 7 hours after horizontal placement. Compared to wild-type

seedlings, mutants with increased ethylene production showed

significantly increased curvature by 7 hours after horizontal placement.

Other acs mutants that did not exhibit increased ethylene production did

not show changes in curvature kinetics. Overall, these results indicate a

stimulatory role for ethylene in gravitropic curvature for Arabidopsis

hypocotyls. (Supported by grants from the USDA National Research

Initiative Competitive Grants Program, the American Society of

Gravitational and Space Biology, and the WV Space Grant Consortium.)

[59] USE OF ROTATO/RANDOM POSITIONING MACHINE (R/RPM)

TECHNOLOGY TO INVESTIGATE GRAVITY SENSING AND

THE GRAVITROPIC MOTOR RESPONSE OF MAIZE ROOTS.

H. Ishikawa, E. Natori, and M.L. Evans

Dept. of Plant Cellular and Molecular Biology, The Ohio State University,

Columbus, OH.

We have merged the capabilities of ROTATO (an imaging/mechanical

rotation system capable of maintaining a target such as a root tip in a fixed

position during a gravitropic response, Mullen et al. Plant Physiol 123:

665-670) with a 3-D clinostat (Random Positioning Machine, RPM). The

combined system (R/RPM) is capable of automated recording of root

growth rate patterns and localized shape changes while simultaneously

exposing a selected target region of the root to simulated zero g or

simulated hypo-g (e.g. 0.5 g). The approach to simulation of hypo-g is to

program the RPM so that the 3-D rotation, instead of being completely

random (simulated zero g), favors net orientation of a gravity-sensing

region (e.g. root tip) in a particular direction. Thus partial g simulation is

achieved through integration of the stimulus over time. The ROTATO (

R) component of the R/RPM is used to maintain the stimulation target

tissue in a fixed orientation so that the integrated stimulus provided by the

RPM can be applied to that region. An early application of the this system

is to investigate the effects of simulated zero g and hypo-g on the two key

components of the gravitropic response of maize roots (Phase 1 and Phase

2 curvature, see Natori et al., this meeting). The R/RPM is being used to

examine the effects of prestimulation at simulated zero g or hypo g on

Phase 1 and Phase 2 curvature with and without maintenance of a constant

gravitational stimulus by ROTATO and to examine the effect of transient

loss (zero g) or reduction (hypo-g) of the gravistimulus midcourse during a

gravitropic response.

(Supported by NASA: NNA04CC65G)

[60] ANALYSIS OF THE TRIPHASIC MOTOR RESPONSE IN MAIZE

ROOT GRAVITROPISM. E. Natori, M.L. Evans, and H. Ishikawa

Dept. of Plant Cellular and Molecular Biology, The Ohio State University,

Columbus, OH.

Maize (Zea mays, L.) root gravitropism displays several phases of

curvature. There is an early phase (Phase 1) of downward curvature

followed by a period of curvature reversal or straightening (S) and then a

second phase of downward curvature (Phase 2). When the root apex is

maintained at a constant angle, the same phases appear, indicating that

they are not induced by change in angle as the response proceeds. Under

conditions where the root apex is held at a constant angle of

gravistimulation, Phase 2 curvature continues indefinitely. The

straightening phase of the gravitropic response is initiated in the distal

elongation zone (DEZ). A comparison of the dependence of the rate of

straightening on fixed angle of orientation of the DEZ with the dependence

of Phase 1 and Phase 2 curvature on this angle showed the following: 1.

Both Phase 1 and S are induced even at very low angles of stimulation,

while the threshold for Phase 2 is high (30o). 2. The rate of straightening

(loss of curvature, deg/min) declines at higher angles of stimulation while

the rates (curvature development, deg/min) of Phases 1 and 2 increase. 3.

Straightening is not observed at angles of 70o or higher. Instead the roots

progress from Phase 1 to Phase 2 without an intervening S phase. The

prolonged steady curvature of Phase 2 under constant stimulus input is

especially intriguing since it indicates that there is steady signal output to

the curving region long after the initial dynamic events of reorientation.

These observations have implications for the statolith model of

gravisensing.

(supported by NASA: NNA04CC65G)

[61] EFFECT OF SOLUTION DENSITY ON GROWTH AND

GRAVITROPIC RESPONSE OF MAIZE ROOTS. Timothy J.

Mulkey. Life Science Dept., Indiana State University, Terre Haute, IN

47809.

The ability of roots to perceive and respond to gravity has been

investigated for many years. The sensory mechanism for the detection of

gravity in roots has been suggested as the sedimentation of amyloplasts in

the root cap. An alternative model suggests that plants sense the

gravitational vector via a pressure differential exerted on the cell

membrane. Thus the entire cell, instead of specific organelles, functions as

statoliths. Elongation and gravitropic response by intact primary roots of

Zea mays L. is examined under various solution densities. Ten percent

sucrose solution with 5% polyethylene glycol (PEG) inhibits elongation of

roots by approximately 80% for 2 hrs after the root is submerged in the

solution. After 2 hr the elongation rate recovers to within 10% of the

control rate. Comparisons of roots in humid air (RH >98%), distilled

water and sucrose/PEG solutions (up to 10%; after recovery) are made to

determine the effect of solution density/protoplast buoyancy on

gravicurvature. Horizontally oriented roots in humid air exhibit 80±5°

positive curvature within 90 min. Horizontally-oriented roots in distilled

water exhibit weak positive gravicurvature (40±15°) after 3 hr.

Horizontally oriented roots in 10% sucrose/PEG solutions exhibit no

significant curvature after 8 hrs. Microscopic examination of the root cap

indicate that in all treatments the amyloplast sediment normally. These

results support the hypothesis that roots respond to gravity by sensing

gravitational pressures exerted by the protoplast.



[62] SEED PRODUCTION IN HYPERGRAVITY IN BRASSICA AND

ARABIDOPSIS. M.E. Musgrave1, A. Kuang2, J. Allen1, R. Darnell1, R.

Wagers-Hughes2 and J. Blasiak1. 1Department of Plant Science, University

of Connecticut, Storrs, CT 06269, 2Biology Department, University of

Texas Pan American, Edinburg, TX 78541.

To investigate potential effects of hypergravity on seed development, we

conducted a series of experiments on the 8-ft and 24-ft centrifuges at Ames

Research Center during summer 2006. Brassica rapa L. cv. Astroplants

and Arabidopsis thaliana L. var. Columbia were grown in the Plant

Growth Facility (PGF) and Plant Growth Unit (PGU) respectively,

according to the experiment conditions on STS-87 (B-STIC) and STS-68

(CHROMEX-5). Brassica siliques in tissue culture were also grown in

chambers 1-3 of the PGF in a new experiment (B-POD), designed to give

information about late stages of seed development not covered in the B-

STIC experiment timecourse. During a 16-d timecourse beginning 11-d

after pollination, these siliques were destructively sampled to determine

the composition of their internal atmosphere and the biochemical

composition of the developing seeds. Our results show that pollen formed

in hypergravity had normal viability in both species. Arabidopsis flowers

are self-pollinating, and seed development proceeded normally at

hypergravity in this species, with full-sized siliques present at the end of

the 11-d hypergravity treatment. Brassica rapa flowers were manually

pollinated during daily stops of the centrifuge, resulting in siliques ranging

in age from 8-15 days after pollination at the end of the 16-d experiment.

Ultimately the full data set from these new experiments at 2-g and 4-g will

be compared with our existing data on seed development in microgravity

and at 1-g to understand how aspects of seed development vary across a

gravity continuum. We have hypothesized that the changes in seed

development occurring in microgravity may be caused by lack of

convective air movement within the developing seed pod. Because

buoyancy-driven convection is gravity dependent, we expect to enhance

internal gas mixing by subjecting plants to hypergravity treatments. This

is the first report of seed production in hypergravity. Supported by NASA

grant NAG-10-329.

[63] INEDIBLE BIOMASS BIODEGRADATION KINETICS FOR

ADVANCED LIFE SUPPORT SYSTEMS. Javier C. Ramirez-Perez1,

Peter F. Strom1, and John Hogan2. 1Department of Environmental Science,

Rutgers University, New Brunswick, NJ, and 2National Space Grant

Foundation,

Aerobic biodegradation is a biological technology candidate in

Advanced Life Support (ALS) systems for solid wastes processing and

resource recovery (SWPRR) for long term manned space missions and

potential planetary base on the Moon or Mars. Aerobic biodegradation has

to be integrated to other ALS ecosystem compartments to treat

biodegradable organic solid wastes generated in space and recycling

nutrients and organic matter for the development of the technology for a

future bioregenerative ALS system aimed towards progressive self

sufficiency. Important questions are how long should wastes be treated,

and what is the quality (stability/maturity) of the product.

To address these questions inedible crop biomass collected from

hydroponically plant growth systems at NASA research centers, amended

with food and human waste simulant were composted in a pilot scale

reactor, aeration pattern in conjunction with temperature feedback process

control were tested. Reactor characteristics were: volume 330 L, 7 days

retention time, the product, compost was assessed over a 162 day period

with a range of physical, chemical, microbiological analyses, along with

the kinetics of the biodegradation process.

Maximum temperatures greater than 55oC were maintained for more than

40 days. Based on observed temporal and spatial temperature variations,

the system can be represented as a process with one-dimensional (axial)

spatial variation. Volume/mass reductions achieved were 79%/67%. Fecal

streptococci, used as an indicator of sanitation, were reduced by 7.8 log-

units. The biodegradation followed first order kinetics (k = 0.0367 kg/kg-

day, and dry mass remaining/initial = 0.29).

(Supported by NASA-NJNSCORT)

[64] GENERATION OF EDIBLE BIOMASS BY THE DEPLOYABLE

VEGETABLE PRODUCTION SYSTEM (VEGGIE) IN UNIT GRAVITY. L.K. Tuominen, A.M. Rogney, and R.C. Morrow. Orbital

Technologies Corporation, Madison, Wisconsin.

To minimize the need for resupply missions in accordance with an

increasingly limited number of flight opportunities to the International

Space Station, ORBITEC has developed a deployable vegetable

production system (VEGGIE) that can be stowed in a small volume and

deployed as a large growth system as needed. While demonstration tests

have shown the capability of the VEGGIE to grow a wide variety of

vegetable crops and herbs, we also wished to clarify whether the system

could produce biomass at levels consistent with hydroponic growth

systems in unit gravity.

Several growth trials of lettuce (Lactuca sativa cv. Grand Rapids) were

conducted to determine the optimal fertilizer regime and root mat design as

well as to compare growth in the VEGGIE to growth in an established

hydroponic system. Growth trials were also conducted for radish

(Raphanus sativus) to establish optimal planting density and cultivar,

although no comparisons to previously published growth systems were

made. Results for lettuce revealed that a looped mat design allowed for

the best plant growth, with productivity as high as 8.0 g dry mass/m2/d in

an early prototype mat and exceeding 5.4 g dm/m2/d in a flight-like

prototype mat. The latter productivity level approximated that observed in

hydroponically-grown lettuce watered four times daily, but plants grown in

VEGGIEs required watering on average only once every 3.8 d. While

VEGGIE-grown lettuce plants were on average smaller than those grown

hydroponically, high planting densities were achievable in the mats

without loss of growth uniformity, thus accounting for the similar

productivity between the two systems. Radish cultivar comparisons

suggested that Cabernet radishes were the best of the available options for

growth in VEGGIE mats; they produced on average 36.6 g edible fresh

mass/m2/d over at a planting density of 16 plants per mat as compared to

the 21.5 g edible fm/m2/d produced by the cultivar Champion. Plant

productivity in the VEGGIE is sufficiently close to that under hydroponic

conditions to warrant its consideration as a means of supplementing crew

meals on long-term missions.

Supported by NASA SBIR Phase II Grant NNK04OA5C.



Post

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-E

Education and Funding

Opportunities



[65] FLIES IN SPACE WEBSITE. C.S. Elland1, B.J. Navarro2, J. Fernandez3,

B.H. Day4, K. Sato1, S. Bhattacharya2, J. Bulkowski1. 1Lockheed Martin

Space Operations, Moffett Field, CA, 2NASA Ames Research Center,

Moffett Field, CA, 3Education Associates Program, Moffett Field, CA &

San Francisco State University, 4Planners Collaborative.

NASA’s Flies in Space Web site (http://quest.nasa.gov/projects/flies/) is

designed for middle school students (grades 5-8). It provides information

about current NASA space biology research, the scientific method, fruit

flies, and the immune system. Students can also communicate with expert

fly researchers by asking questions of the scientists on-line, and making

predictions about the Flies in Space experiment. The Flies In Space

website was designed to reflect features of the FIT (Drosophila)

experiment, which will fly on the space shuttle in the near future. The

goals of the FIT experiment include characterizing the effects of space

travel (including weightlessness and radiation exposure) on fruit flies’

immune systems. Fruit flies have long been used by scientists worldwide

because their genome has been completely mapped, their short life cycle

enables multiple generations to be studied in a short amount of time, and

they have many analogous processes to humans. The student materials

include pre- and post-tests. The educator documents will assist teachers to

use this website. The Flies in Space Website in the Classroom provides

information about the national standards addressed in the site and includes

a worksheet (with answer key) that students can complete as they explore

the site. The Flies in Space Activity Guide enables educators to conduct

hands-on activities relating to the Flies in Space experiment in the

classroom. Both the student and educator pages are available in both

English and Spanish.

[66] RADIATION BIOLOGY EDUCATOR GUIDE. C.S. Elland1, B.J.

Navarro2, J.C. Rask3, Y. Kovo1, W.A. Vercoutere2. 1Lockheed Martin

Space Operations, Moffett Field, CA, 2NASA Ames Research Center,

Moffett Field, CA, 3E.A.S.I., Moffett Field, CA

The Radiation Biology Professional Educator Guide is designed for pre-

service and practicing high school science teachers. The goal of this guide

is to enable teachers to bring authentic NASA Exploration Systems

Mission Directorate (ESMD) research into the classroom. Teachers will

learn background information and current research in radiation biology

through use of the NASA Radiation Biology Educator Guide. The

Radiation Biology Educator Guide, accessible on the web

(http://radiationbiology.arc.nasa.gov/), provides learning activities for

inquiry-based radiation biology and genetics studies related to research on

a variety of radiation sources, including ultraviolet and cosmic radiation.

Existing life science standards-based curricula will be enhanced with links

to relevant research conducted by NASA scientists. The modules can be

used for week-long to semester-long lesson plans. Students, guided by

teachers in their classrooms, will conduct experiments analogous to the

research conducted at NASA. These activities are aligned with national

science, technology, and mathematics standards. The web-based Educator

Guide will provide educators with access to the lesson modules, activity

guides, information about current NASA research, and opportunities to ask

questions about classroom implementation. A critical goal of NASA

scientists is to identify and mitigate the effects of radiation on travelers to

the Moon, Mars, and beyond. The goal of this Educator Guide is to

facilitate hands-on activities in the classroom based on NASA radiation

research, apply research-based theories of education in their classrooms

(including inquiry-based science instruction), highlight NASA research’s

impact on the quality of life on Earth, and grow professionally by

increasing science content knowledge.

[67] MICROBIAL SYSTEMS AND THE SPACE FLIGHT

ENVIRONMENT. D.M. Klaus, University of Colorado, Boulder, CO

80309

Bacteria have been studied in the context of space flight under a variety

of objectives ranging from understanding basic sciences to developing

engineering applications. Reports pertaining to growth kinetics, crew

health implications, antimicrobial issues, fermentation technology,

biophysical interface phenomena, biological waste treatment, energy

production, astrobiology and planetary protection concerns can be found in

space-related literature. Each of these topics can, in turn, be used to

exemplify a key aspect of systems engineering - characterizing direct and

indirect relationships between the individual, isolated components of a

system and with their surrounding environment. The implementation of

this analytical approach is broad-reaching across diverse science and

engineering interests. For example, examining gravity’s influence on

bacteria can be used to address questions relevant to general microbiology,

as well as concerns specific to space flight. And from an engineering

perspective, incorporating microbial processes into a spacecraft life

support system can provide beneficial functions that enable more efficient

and sustainable long-term space habitation.

An academic framework for teaching aerospace science and engineering

courses on a foundation of microbial systems is defined under the auspices

of Bioastronautics, the study and support of life in space. (Supported by

BioServe Space Technologies, NASA NCC8-242)

[68] GRAND VISIONS FOR LIFE AND LIVING IN SPACE. 1D.E.Jennings, 2R.E.Turner, and 1R.A.Cassanova. 1NASA Institute for

Advanced Concepts, 75 5th Street NW, Suite 318, Atlanta, GA

30308;2ANSER Analytic Services, Inc., 2900 South Quincy Street, Suite

800, Arlington, VA 22206.

The NASA Institute for Advanced Concepts (NIAC) has, since 1998,

sought exciting proposals that address grand visions for future possibilities

in aerospace, including that of a self-sustaining human presence

throughout the solar system. This presentation will briefly describe the

NIAC and survey recent NIAC-funded studies that engage life scientists

and interdisciplinary collaborators in defining our future possibilities

Some NIAC-funded investigations exploit biological mechanisms as

tools to meet aerospace challenges. For example, researchers have

examined the feasibility of employing biological molecules such as

proteins and ion channels in the development of sensor networks for

exploration and radiation protection. Others presently examine the use of

mechanosensitive proteins for power production.

This presentation will also survey NIAC-funded projects that look at

ways to create environments hospitable to life. Sometimes this requires the

modification of life forms, such as plants; sometimes this requires the

selection of organisms that might tolerate harsh conditions, such as

extremophiles.

Finally, we will briefly survey projects that “bring home with us”,

including habitat construction and life support.

(Supported by NASA: NAS5-03110 to the Universities Space Research

Association)



Ora

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I

Spaceflight Experiment

Results

D. Marshall Porterfield, Chair



[69] GRAVITY DEPENDENT CA++ SIGNALING IN CERATOPTERIS

MEASURED USING A MEMS BASED IN-SILICO CELL

ELECTROPHYSIOLOGY SENSOR DEVICE. D.M. Porterfield1,2,3,

S.J. Roux4, A. ul Haque1,2, M. Salmi4, W.T. McLamb5, M. Rokkam1,6, A.R.

DeCarlo1,2, S.T. Wereley7.

1Physiological Sensing Facility, 2Dept. of Ag. & Bio. Eng., 3Dept. of Hort.

& Landscape Arch., 4Molecular, Cell. & Dev. Biol., Univ. of Texas,

Austin, TX, 5Dynamac Corporation, KSC, FL. 6Dept. of Elec. & Comp.

Eng., 7Dept. of Mech. Eng., Purdue Univ., W. Lafayette, IN,

Polar ion currents driven by Ca++ have been studied in numerous

developmental systems. These studies have shown that ion currents can

direct polarity in cellular development, and in Ceratopteris this polar

current correlates with gravity sensing. Critical questions that still need to

be answered in this system require spatial and temporal resolutions that

exceed that available using the self-referencing microsensor technique. We

have developed an in-silico Cell Electrophysiology Lab-on-a-Chip (CEL-

C) device that has enabled us to monitor Ca++ ion currents through multiple

fern spores in real time. Using an advanced physiological sensing

modality, called dual electrode differential coupling, we can directly

measure trans-cellular ion currents across the cell. In static ground studies

we have been able to replicate earlier published reports that show Ca++

currents through the cell correlate with gravity responsiveness. We have

also been able to continuously measure Ca++ currents in dynamic

experiments where the CEL-C device and the immobilized cells are being

rotated. Full 180o rotation was done in 5 seconds and the polar current

reversed to full magnitude in 20-25 seconds. Microgravity flight

experiments were flow on the NASA’s reduced gravity DC-9 and allowed

us to measure cellular responses as gravity changed from microgravity to

2g. The top-bottom Ca++ current changed in phase with the accelerometer

data, whereas side-side controls did not show any predictable pattern

related to the reduced gravity flight. These data obtained using the CEL-C

device have allowed us to analyze the dynamics involved with gravity

sensing in this single cell system, at a level of resolution not previously

possible.

(Supported by the NASA and the Lilly Foundation)

[70] SPACEFLIGHT-INDUCED GENE EXPRESSION CHANGES IN

THE MOUSE: RESULTS FROM STS-108. K. Schweighofer,6 T. G.

Hammond,1,2 P. L. Allen,1,2 L. S. Stodieck,3 P. J. Kostenuik,4 T. A.

Bateman,5 S. Morony,4 D. Lacey,4, S.Y.C. Chang6, and A. Pohorille7.1

Nephrology Section, and Tulane Environmental Astrobiology Center,

Tulane University Medical Center and 2VA Medical Center, New Orleans,

LA, USA; 3BioServe Space Technologies, University of Colorado,

Boulder, Colorado 80309; 4Metabolic Disorders Research, Amgen

Incorporated, Thousand Oaks, California 91320; and 5Department of

Bioengineering, Clemson University, Clemson, South Carolina 29634; 6RIACS, Mountain View California 94041; 7NASA Ames Research

Center, Moffett Field, California 94035

Several aspects of spaceflight may lead to changes in liver and kidney

function including changes in hemodynamics and gravitational loading.

C57BL/6 mice were flown on a 12 day space shuttle mission and hepatic

and renal tissue harvested immediately upon their return. Gene array

analysis suggest tissue specific changes in expression as a result of

spaceflight factors, with liver tissue exhibiting a 20% increase over kidney

in the number of significant observations. Gene expression profiles for

liver versus kidney are markedly different, suggesting that the response to

short-duration spaceflight is tissue specific in the mouse. Both tissues,

however, show significant up-regulation in metalothioneins 1 and 2 (MtI

and MtII), and insulin-like growth factor binding protein 1 (IGFBP1). In

addition, many genes involved in cell cycle, signal transduction, and

cellular damage are altered. Notably absent are changes in the major drug-

metabolizing enzymes, and in central metabolism. This demonstrates that

space flight factors (at least on short duration) modulate biological

processes in subtle, but potentially significant ways.

[71] NEURONAL EXCITABILITY IN RAT HYPOTHALAMUS

RESPONDING TO MICROGRAVITY STIMULUS DURING PARABOLIC FLIGHT. Y. Kumei1, J. Zeredo2, M. Ogasawara3, H.

Suzuki4, T. Fujishima4, K. Fukui4, G. Fukushima5, M.Kimoto6, and K.

Toda2. 1Graduate School Tokyo Medical and Dental University, Tokyo, 2Nagasaki University Graduate School, Nagasaki, 3Physiotech. Co. Ltd.,

Tokyo, 4Japan Space Forum, Tokyo, 5Diamond Air Service, Inc., Aichi, 6Japan Women’s University, Tokyo, Japan

We examined the neuronal activities in the rat limbic system during

parabolic flights. Male Wistar rats were exposed to microgravity (less than

0.1 G) for 7 seconds in each parabolic flight. The rats floated during

microgravity exposure but remained calm for the rest of the flight. The

firing spikes were recorded by telemetric measurement through chronically

implanted electrodes into the hippocampus, hypothalamus, amygdala, and

dentate gyrus. Upon microgravity exposure, the firing frequency in the

hypothalamus increased momentarily to a level three-times as high as the

basal 1G level, and increased again to the same level upon recovery from

microgravity. No significant changes were observed in the neuronal

activities of hippocampus, amygdala and dentate gyrus. Hypothalamus

might be the primarily sensitive site in the rat limbic system that responds

to microgravity environment.

(Supported by Japan Space Forum Foundation)

[72] VESTIBULAR OTOLITH DEVELOPMENT IN SPACEFLIGHT

AND HYPERGRAVITY. J.D. Dickman1, A. Lysakowski2, D. Huss1, and

S. Price2. Dept. Anatomy & Neurobiology1, Washington Univ., St. Louis,

MO and Dept. Anatomy & Cell Biol.2, Univ. Illinois - Chicago, Chicago,

IL

The goal of the present study was to determine how gravity affects the

development of the vestibular otolith organs. The receptors, otoconia, and

afferent innervation, and regulatory genes of the otolith maculae were

examined in embryonic quails raised in one of three gravity environments,

including normal 1g, microgravity (0g), or hypergravity (2g). Fertilized

eggs were first arrested from development by cooling, then placed into one

of three gravity conditions and allowed to develop for 12 days at 37deg C.

1G and 0G embryos were developed in low Earth-orbital spaceflight (STS-

108) in a specially designed incubator (ADF - SHOT, Inc) that contained a

microgravity carousel and a 1g centrifuge carousel. 2g embryos were

developed during constant centrifugation in a laboratory incubator. We

found that the mean saccular epithelium area was smaller in 0g and larger

in 2g embryos, than 1g controls. Hair cells showed normal stereocilia

polarizations for all gravity conditions. We also found that no significant

differences in otoconial formation nor the expression of Otop1 or Oc90

(two genes regulating otoconial growth) were present between 1g and 2g

embryos. However, neural tracings (HRP) of macular afferents revealed

strong differences due to gravity exposure. Zero g fibers had smaller

axons, were less branched, and had fewer terminals as compared to 1g

controls. Two g afferents were significantly larger, contained more

arborizations, larger terminals and more growth cones as compared to 1g

controls. Electron microscopic observations reveal that the ribbon

synapses were more numerous for type I but not type II hair cells in both

1g and 2g maculae, as compared to 1g controls. The results show that

vestibular otolith development is dependent upon gravity exposure for

synaptogenesis and afferent innervation.

Supported in part by funds from NASA NNA04CC52G, NIH

DC006913.



[73] SKELETAL MUSCLE CHANGES FOLLOWING SIX-MONTH

SPACEFLIGHT. DA Riley1, JLW Bain1, RH Fitts2, JG Romatowski2 and

SW Trappe3. 1Dept. Cell Biol., Neurobiol. & Anatomy, Medical College

of Wisconsin and 2Dept. Biology, Marquette University, Milwaukee, WI

and 3Human Performance Lab, Ball State University, Muncie, IN.

Skeletal muscle wasting raises concern for the health and performance of

humans undertaking long term spaceflight. The present study examined

soleus and gastrocnemius muscle biopsies from 5 astronauts and 5

cosmonauts who participated in 6-month, Expedition missions on the

International Space Station during 2000-2005. Tissues were analyzed by

light and electron microscopy. The intersubject variation in the percentage

of muscle fiber atrophy was striking; atrophy ranged from 3 to 64% in

soleus and 5 to 46% in gastrocnemius. Mitochondrial content and

cytochrome oxidase activity and oil red O-positive intramyofiber lipids

were retained in the muscles exhibiting the least atrophy. PAS-positive

glycogen and putative glycogen granules were increased in all muscles,

most dramatically in fibers showing the greatest atrophy. Immunostaining

for a battery of metabolic markers, including carnitine palmityol

transferase, phosphofructokinase, glycogen transporter 4, and uncoupling

protein 3, revealed no major changes during spaceflight. For soleus and

gastrocnemius, on the average only a small percentage (5-7%) of slow

fibers transitioned into slow/fast (I/IIA) and fast (IIA) fibers. In normal

skeletal muscle fibers, myonuclei are peripherally located, and central

myonuclei indicate muscle fiber degeneration/regeneration. Following

spaceflight, the percentages of fibers with central myonuclei (soleus

4.62±.02%, gastrocnemius 2.01±.01%) were not significantly different

from preflight values (soleus 3.76±.01%, gastrocnemius 1.75±.01%). The

large increase in glycogen after long term spaceflight indicates shifts in

muscle fibers toward utilizing lipids and reduced glycogen breakdown.

The 5% or less atrophy is an acceptable risk for long term spaceflight.

However, atrophy of 46% or greater is of concern because countermeasure

exercise was performed. (Supported by NASA grant EC400 NCC 9-116.)

[74] LESSONS AND RECOMMENDATIONS FROM OVER A DECADE

OF MICROGRAVITY PLANT GROWTH EXPERIENCE. G.E.

Bingham1, S. B. Jones1, B. Bugbee1, M.A. Levinskih2, I.G. Podolski2 and

V.S. Sychev2

1Space Dynamics Laboratory and Utah State University, Logan, UT, and 2Institute of Biomedical Problems, Moscow, RU.

The past decade has seen the culture of plants in microgravity (µg)

evolve from a chancy, low probability exercise to a rather routine

experience. This paper will review the key landmarks along this road and

discuss lessons learned in the process. It will also discuss options that face

engineers and scientists as exploration turns beyond LEO. The discussion

will include development requirements for plants, equipment and

procedures. We claim that the most important area required for good plant

growth is the management of water and oxygen in the root zone. We

discuss the history and development of the science in this area and

describe an upcoming experiment to expand our knowledge. We have

verified the fears of a great Russian scientist that cabin air contamination

will limit viability, and we review the impact of ethylene on plants and

discuss the choices that face future explorers. We will also discuss the true

value of plants as partners with our explorers and share initial work being

done to assure food safety. Finally, we will describe the difference

between plants as partners in exploration and plants as research subjects.

[75] COUNTERING SPACEFLIGHT EFFECTS ON C. ELEGANS

BIOLOGY. F. Selch1, N.J. Szewczyk2,3, and C. A. Conley2

1 Department of Molecular Cell Biology, University of Vienna 2 NASA Ames Research Center 3Department of Biological Sciences, University of Pittsburgh

Despite common study of C. elegans, there is little evidence that worms

sense or respond to gravity. We find C. elegans grown on solid or in liquid

chemically defined medium display reproducible gene expression changes

(Stanford microarray), likely due to the difference in externally-applied

surface tension, a force roughly equivalent to 10,000 x unit Gravity. When

plotted on the C. elegans gene expression self organizing map, the data

indicate that mounts 8 (intestinal), 12 (neuronal), 15, 22 (collagen), and 36

(heat shock) are down regulated and 4 (sperm), 19 (amino acid

metabolism), 21 (lipid metabolism), and 24 (fatty acid oxidation) are up

regulated in response to increased mechanical load (p<.0001). Conversely,

the gravitational unloading of a 10 day spaceflight increased expression of

mounts 8 and 36 and decreased expression of mounts 19 and 21 (p<.001).

Additionally, spaceflight decreased expression of mounts 1 (muscle) and

17 (collagen) and produced a movement defect. Together these results

show that C. elegans sense gravity and suggest that 70% of the genes

induced and 40% of the genes repressed by spaceflight can be blocked by

increased mechanical load alone. Increased expression of mount 36 can be

reversed while decreased expression of muscle or skeletal (collagen) genes

cannot be reversed by mechanical load alone in C. elegans. Strikingly in

accordance with these predictions, the movement defect is not rescued by

growth on solid medium during spaceflight (i.e. increased mechanical

load). These results demonstrate C. elegans can be used to study the

effects of altered gravity and suggest that artificial gravity alone is

sufficient to only partially counter the biological effects of spaceflight.

(Supported by NASA: NNA04CK22A)

[76] POEMS: PASSIVE OBSERVATORIES FOR EXPERIMENTAL

MICROBIAL SYSTEMS

M.S. Roberts1, M.N. Birmele1, D.W. Reed2, and T.E. Mortenson2. 1

Dynamac Corporation, and 2 Bionetics Corporation, Space Life Sciences

Lab, Kennedy Space Center, FL.

Passive Observatories for Experimental Microbial Systems (POEMS) is

a NASA spaceflight experiment to the International Space Station

launched on shuttle mission STS-121. POEMS utilizes the BRIC-Opti

(Biological Research in Canisters for OptiCells™) payload hardware to

explore the relationship between recombination (horizontal gene transfer

or HGT) and sequence divergence in the genetic transformation of a

naturally competent population of Bacillus subtilis cultivated in

microgravity on ISS and shuttle in the presence of DNA from different

donor strains. Previous ground-based studies in B. subtilis and other

bacteria have demonstrated that the frequency of recombination is

negatively correlated with the level of sexual isolation at a locus, i.e.

recombination is reduced by a factor of 10 for every 1% of sequence

divergence between donor and recipient loci. Recombination is not a

significant force of cohesion within bacterial species, but HGT is the

primary mechanism for acquisition of new functions in bacteria. In the

space environment where reduced gravity and elevated radiation have

multiple effects upon cellular and community-level processes, both the

extent and rate of horizontal gene transfer among bacteria are affected. By

extension, the ability of bacteria to acquire new functions, exploit novel

environments, and form new ecological species may be accelerated in

space because of elevated rates of genetic transfer, relaxed constraints in

mismatch repair, or other responses to microgravity. We will present an

operational assessment of BRIC-Opti hardware performance and

preliminary phenotypic and genotypic data from the POEMS experiment

that reports on the effect of microgravity on the growth, diversity,

recombination, and evolution of B. subtilis in an HGT permissive

environment.



Ora

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Plant Physiology and Gravity

Response

Elison Blancaflor, Chair



[77] EFFECTS OF ATMOSPHERIC PRESSURE ON THE SURVIVAL

OF PHOTOSYNTHETIC MICROORGANISMS DURING

SIMULATIONS OF ECOPOESIS. David J. Thomas1, L. Michelle

Eubanks1, Carl Rector1, Jaime Warrington1 and Paul Todd2. 1Science Division, Lyon College, 2300 Highland Road, Batesville, AR

72501, USA; 2Space Hardware Optimization Technology, Inc., 7200

Highway 150, Greenville, IN 47124, USA.

Three cyanobacteria (Anabaena sp., Plectonema boryanum,

Chroococcidiopsis CCMEE171) and an alga (Chlorella ellipsoidea) were

grown under simulated martian ecopoesis conditions. A xenon arc lamp

with a solar filter provided simulated martian sunlight, and temperature

cycled diurnally from -80°C to 26°C. A Mars-like atmosphere of 100%

CO2 was provided at 25, 100, 300, 500 and 1000 mbar. The cyanobacteria

and alga were inoculated into JSC Mars-1 soil simulant and exposed to

each atmospheric pressure for five weeks. Survival and growth were

determined via extractable chlorophyll a and total esterase (fluorescein

diacetate hydrolysis) activity. Maximum survival occurred at 100-300

mbar. At 25, 500 and 1000 mbar, esterase activity was near zero, and

extractable chlorophyll a was less than 10% of control samples. Overall,

the cyanobacteria survived better than the alga. Low survival at 25 mbar

was probably due to desiccation. Low survival at 500 and 1000 mbar may

have been due to CO2 toxicity. This research was supported by the NASA

Institute for Advanced Concepts and by the Arkansas Space Grant

Consortium.

[78] AEROTAXIS IN A CALCIFYING ALGA DOES NOT REQUIRE

PHOTOSYNTHESIS. J. Duke1, C. Auzenne3 and M. Marsh2. 1Dept. of

Orthodontics, Dental Branch, 3Dept. of Biochemistry and Molecular

Biology, Medical School, University of Texas Health Science Center at

Houston, 3DeBakey High School for the Health Professions, Houston.

Previously, we determined that the calcifying alga Pleurochrysis

cartarae is aerotaxic during the light portion of the light-dark cycle. The

relationship between locomotion and photosynthesis led us to the objective

of our current experiment, to determine if aerotaxis occurs in the dark as

well. Four experimental devices were constructed using 35 ml T-flasks and

Silastic “bubbles” from the 1992 CELLS experiment on IML-1 (Duke and

Montufar-Solis, 1999, Adv. Space Res.). The two part bubble was halved

and one-half (one bubble) inserted into a hole made in the side of the T-

flask, then sealed into place with Silastic glue. Flasks were cured for a

week and rinsed extensively prior to use. Cells from a stock cell culture of

P. carterae, Plymouth strain 136, were grown overnight in F/2 medium

(18oC), then divided into the 4 T-flasks and placed in the incubator.

Movement of gases through the membranes was confirmed by appearance

of gas bubbles in the flask. Aerotaxis in the light was positive in 3 of 4

flasks as assessed by the accumulation of cells at a particular distance from

the gas exchange bubble, forming a graded green arc around a clear zone

next to the bubble. Cells were dark adapted for 48 hrs, then placed in the

experimental containers and checked for the presence of a gradient every

10 minutes. After 10 minutes, a gradient had been established in 2 of the 4,

and by 30 minutes in 3 of the 4. Over the hour of observation, the gradient

did not disperse, and no gravity-related bioconvection was observed in

dark cultures. The experiment was repeated with the same result and with

the same flask not demonstrating aerotaxis. This we attributed to limited

gas exchange due to age-related cross-linking of the membrane polymer.

This study shows that photosynthesis is not necessary for aerotaxis to

occur in P. carterae cultures, and demonstrates how air bubbles in

solutions in spaceflight experiments could alter results of gravitaxis

experiments. Support: Michael E. DeBakey High School for the Heath

Professions; Dept of Orthodontics, UT Dental Branch.

[79] ASSESSING THE ROLE OF CALCIUM PUMPS IN THE GRAVITY

RESPONSE IN SINGLE-CELLED SPORES OF CERATOPTERIS

RICHARDII. S.J. Roux1, T.J. Bushart1, A. ul Haque2, and D.M.

Porterfield2 1Molecular Cell and Developmental Biology, The University

of Texas at Austin, and 2Department of Agriculture and Biological

Engineering, Purdue University, West Lafayette, IN.

Previous investigations into how gravity directs the polarity of growth in

fern spores of Ceratopteris richardii revealed a key role for a trans-cell

calcium current during the period gravity fixes the polarity. This calcium

current reorients 180o when the spores are rotated 180o. Blocking calcium

channels with nifedipine disrupts the calcium flux and disorients the

subsequent rhizoid emergence, indicating that calcium uptake is required

for gravity to orient the direction of rhizoid emergence. Here we report on

the role that calcium-pumping ATPases play in the calcium flux and

polarity establishment.

Spores grown in a fixed orientation were exposed to various

concentrations of BHQ, an inhibitor of endomembrane Ca2+-ATPases, or

Eosin Y, an inhibitor of plasma membrane Ca2+-ATPases, and assayed for

changes in germination and polarity establishment. Both 100 µM BHQ and

10 µM Eosin Y inhibit 100% of rhizoid emergence if applied continuously.

Eosin Y also depresses the calcium efflux of the spores. However, limited

exposure to either drug from 0-24 hrs, the period of polarity fixation, does

not disrupt the ability of gravity to orient rhizoid emergence. In a few

cases this short-term exposure to BHQ appears to lead to a very small

(<5%) but statistically significant increase in downward emerging rhizoids.

Taken together, the inhibitor data indicate that Ca2+ pump activity is

required for the initiation of polarized growth, but is not specifically

required for gravity to orient the direction of that growth. Because calcium

channel activity is required for the gravity response in Ceratopteris, our

data are consistent with the hypothesis that the intake of calcium at the

bottom of the spore, possibly through stretch-activated calcium channels,

is the key event for initiating the gravity response. A model has been

developed based on this and other evidence. (Supported by NAG2-1586

and NAG10-295)

[80] GRAVITY AND LIGHT: THE ROLE OF MEMBRANE

COMPONENTS IN TROPIC RESPONSES. Heike Winter Sederoff1, Mariya Khodakovskaya1, Jeffrey M. Kimbrough1,

Raul Salinas-Mondragon1, and Christopher S. Brown1,2 1Dept. Plant Biology, and 2Kenan Institute for Science, Technology&

Engineering, North Carolina State University, Raleigh, NC 27695-7612.

Plant roots use light and gravity as directional signals to grow down into

the soil. The response to light and gravity requires an integration and

prioritization of a growth response. We have shown that the transcriptional

response in Arabidopsis root tips occurs within less than 2 min and is

distinct from the transcriptional response to mechanical stimulation

(Kimbrough et al. 2004). Inositol 1,4,5 triphosphate (InsP3) is an early

signal transduction elements in gravitropism (Perera et al. 2006). We show

here that InsP3 mediates specific activation and repression of transcript

abundance changes in response to gravity and light stimulation in root

apices of the transgenic Arabidopsis plants. These gene-specific effects

can be mimicked by inhibition of Phospholipase C. We have identified one

of the fast InsP3-mediated light and gravity regulated genes to be a

cholesterol and cardiolipin binding protein, possibly involved in

cholesterol transport between organells. Using One-Hybrid analysis to

identify possible cis-regulatory elements involved in transcriptional

regulation of light and gravity responsive genes, we fished a C2-domain

DNA-binding protein. In vitro binding studies showed that the C2 domain

binds to galactosylceramide-3-O-sulfate. We will present a hypothetical

model for the possible roles those membrane components in the signal

transduction and response pathways to light and gravity in roots.

(Supported by NASA grant NAG2-1566).

Kimbrough, J. M., R. Salinas-Mondragon, W. F. Boss, C. S. Brown and H.

W. Sederoff (2004). Plant Physiol. 136(1): 2790-2805

Perera, I. Y., C. Y. Hung, S. Brady, G. K. Muday and W. F. Boss (2006)

Plant Physiol 140(2): 746-60.



[81] AN ARABIDOPSIS ROOT HAIR MUTANT WITH ALTERED

GROWTH DIRECTIONALITY DISPLAYS DEFECTIVE

CYTOSKELETAL DYNAMICS IN VIVO. E.B. Blancaflor and C.-M.

Yoo, Plant Biology Division, The Samuel Roberts Noble Foundation,

Ardmore, OK 73401

Because of their rapid growth and accessibility, root hairs have been

widely used as a model system for studies of plant cell biology,

particularly with regard to the mechanisms underlying tip growth. We

screened a population of activation-tagged Arabidopsis seedlings for

defects in root development and have identified a number of root hair

mutants with altered morphology and growth directionality. Phenotypes

resulting from this screen included root hairs with multiple tips, short root

hairs, root hairs with swollen bases, root hair branching and exploding tips.

Some of these mutants appear to be disrupted in genes that have not

previously been implicated in root hair growth. The root hairs of one

mutant displayed a wavy and/or spiral growth habit with no apparent

impact on the diameter or the length of the root hair. It appeared that the

wavy growth pattern in this mutant is likely caused by differential

deposition of the cell membrane and wall materials to the tip. Since the

cytoskeleton plays a major role in controlling vesicle trafficking important

for transporting cell wall materials to the expanding cell, we crossed this

mutant and several of the other root hair morphology mutants with

transgenic plants expressing green fluorescent protein (GFP) reporters for

microtubules, F-actin, and Golgi. The behavior of the GFP reporters in the

wavy root hair mutant was different from that of wild-type. In particular,

F-actin bundles, microtubules and Golgi occasionally protruded into the

growing tip of the wavy root hair mutant. This could result in differential

transport of vesicles carrying cell wall precursors to the tip, which could

explain the wavy/spiral growth habit of the mutant (Supported by the

Noble Foundation and NSF DBI-0400580).

[82] EFFECTS OF GRAVITY ON GENE EXPRESSION IN THE MAIZE

PULVINUS: UNRAVELING THE ROLES OF TRANSCRIPTION AND TRANSLATION. H. Myburg1, R. Salinas-Mondragon1, R.L.

Hammond1, I.Y. Perera1, E. Davis1, C.S. Brown1,2 and H. Winter Sederoff1.

Plant Sensory Genomics Group, 1Dept. of Plant Biology, 2Kenan Institute

for Science, Technology & Engineering, North Carolina State University,

Raleigh, NC, 27695.

Gravitropic bending in cereal grass stems is brought about by differential

growth of the pulvinus, a specialized tissue that senses and responds only

to gravity. Previous work has shown that translational regulation of

specific transcripts is correlated with the extent of asymmetric cell

elongation in the maize pulvinus and may be a central controlling step in

the initiation of a growth response during gravitropism (Heilmann et al.

2001). Global changes in the steady-state levels of transcripts

(transcriptional control) and transcript recruitment into polyribosomes

(translational control) were assessed by DNA microarray analysis. We

compared transcript abundance changes in response to 90º reorientation

during the first hour between upper (slow elongation) and lower (fast

elongation) halves of the most gravity competent pulvini. For each sample

we purified and fractionated total mRNA and polyribosome-associated

mRNA and analyzed transcript profiles using Affymetrix GeneChip®

Maize Genome Arrays. A total of 13584 transcripts were screened in a

time course ranging from 2 minutes up to one hour. More than 540

transcripts showed a response to gravity. Within this group the majority of

the transcripts seem to be transcriptionally regulated while a smaller group

of transcripts show regulation at the translational level. We will discuss

the implications of translational regulation on gravitropic responses and

compare possible functional roles for translationally regulated genes.

Heilmann, I., J. Shin, J. Huang, I.Y. Perera and E. Davies (2001) Transient

Dissociation of Polyribosomes and Concurrent Recruitment of Calreticulin

and Calmodulin Transcripts in Gravistimulated Maize Pulvini. Plant

Physiol. 127(3): 1193-1203.

(Supported by NASA grant NNA04CC56G to HWS and NSCU RNA

biology fellowship to RLH.)

[83] OPTIMIZING THE GROWTH CONDITIONS OF ARABIDOPSIS

THALIANA FROM SEED-TO-SEED IN THE EMCS ON ISS. * A.-I.

Kittang, B.G., Solheim, H. Svare, G. Rakvaag, A. Johannes, T.-H.

Iversen.The PlantBio Center, Dep. of Biology, NTNU, Trondheim,

Norway*Correspondence: [email protected]

In the MULTIGEN-1 experiment the model plant, Arabidopsis thaliana,

will be grown from seed-to-seed in the European Modular Cultivation

System (EMCS) on the ISS. The experiment is planned to be launched on

flight 13A.1 (currently scheduled for June 11, 2007) and will be carried

out in Increment 15 as the first long duration experiment in EMCS. The

facility is commissioned during Europe’s first long-duration mission to the

ISS (“Astrolab”), which began with flight STS 121. By monitoring the

plants using the EMCS cameras, a comparative study of plants grown on a

static centrifuge (microgravity) and on a rotating centrifuge (1xg) is

performed. The scientific objective is to obtain basic knowledge about the

growth patterns and the development of Arabidopsis thaliana in the

microgravity environment.

The plants are supplied with nutrients and water during the whole life

cycle via a Plant Cultivation Chamber (PCC). The PCCs monitor the

relative humidity in the growth media and supply water automatically. The

flowerpots of the PCCs are filled with zeolite that is enriched with

nutrients. A poly-propylene-felt is placed on top of the zeolite to support in

the seed germination phase. The EMCS water reservoirs supply the water

to the PCC. The same reservoir is also used to obtain the requested relative

humidity in the atmosphere and for this reason water economy is

important. Another parameter that has to be considered in the same context

is the air flow, which if running continuously at the nominal rates of

EMCS, has a drying effect on the seedlings. Prevention of this effect and

optimisation of growth media will be discussed in this presentation.

Furthermore testing during the integrating phase of MULTIGEN-1 into the

EMCS using the Ground Models is outlined from an investigator’s point of

view.

[84] LEDS FOR CROP PRODUCTION: COMPARISONS OF

OVERHEAD VS. INTRACANOPY LIGHTING. G.D. Massa, M.E.

Mick, C.A. Mitchell. ALS NSCORT, Purdue Univ., West Lafayette, IN.

Electric lighting for crop production using red and blue LEDs is being

investigated as a component of human life-support systems. Energy

savings drives the development of crop-growth subsystems. Two

reconfigurable LED arrays have been developed and are being tested in a

side-by-side arrangement, with one array configured into a single overhead

(OH) plane of light engines and the second array suspended vertically as

separate intracanopy (IC) “lightsicles” among plants within the crop stand.

Cowpea (Vigna unguiculata L. Walp PI IT87D-941-1 (D-941)) and dwarf

pepper (Capsicum annuum cv. Triton) have been grown hydroponically

using the two LED lighting arrays. Oedema has been observed on both

crops. On cowpea, symptoms seem to be directly related to the amount of

blue light received by the leaves, as well as to leaf age. For pepper,

increasing the level or proportion of blue light did not reduce oedema. In

spite of oedema, excellent cowpea vegetative growth has been observed

under or within both arrays. Also, oedema in peppers did not

detrimentally affect flower or fruit formation. This is our first observation

of reproductive development using the IC configuration with LEDs as the

sole source of crop lighting. Electrical power usage per unit edible

biomass produced was greater in the OH configuration, where mutual

shading of lower leaves by upper leaves occurs. In addition, the amount of

water lost due to transpiration was higher with OH lighting. These results

indicate that, for planophile crops, IC lighting is a more efficient lighting

geometry. Work with a crop-canopy gas-exchange cuvette to accurately

measure real-time canopy photosynthesis with IC LEDs is underway. This

will allow faster optimization of lighting preferences by a given crop and

more detailed calculations to model mass flow through the biomass-

production subsystem of a life-support system. The LED lighting system

is being developed jointly by NSCORT and Orbitec with input and

assistance from R. Morrow, M. Bourget, and J. Emmerich of Orbitec. This

research was partially supported by NASA: NAG5-12686.



Tow

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Special Meeting on the Future

of the American Society for

Gravitational and Space

Biology



TOWN HALL MEETING ON THE FUTURE

OF THE AMERICAN SOCIETY FOR

GRAVITATIONAL AND SPACE BIOLOGY.

By the end of FY06, proposed cuts in microgravity life and physical

sciences will have eliminated entire research communities in the physical

and life sciences. Society members engaged in exploration-related

research for NASA in the microgravity life, biomedical and physical

sciences are very concerned about the future of research and research

funding at NASA. Across the disciplines formerly sponsored by the

Office of Biological and Physical Research, the downsizing over the last

2-3 years has negatively affected some 3000 graduate students, and

research communities that have taken a decade or more to form now face

dissolution. These events have a direct effect on the ASGSB. This

"town hall" meeting will provide an open forum for the discussion of the

future direction that we want our society to take.

The ASGSB Executive Committee invites your participation in this

discussion



Sym

posi

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III

Integrated Physiology



[85] INTEGRATED PHYSIOLOGY - ITS COMPETITION AND

COOPERATION WITH MOLECULAR AND GENETIC

RESEARCH. E.R. Horn. Gravitational Physiology, Ulm University, Ulm,

Germany

Organisms are composed of organs and tissues that were formed and

connected during development according to genetic programs and

environmental factors. During the last two decades, molecular and genetic

aspects have pushed integrated (systemic) approaches into the background,

leading sometimes to a feeling that organisms are only the sum of cells or

molecules. However, central integration between all structures and

compartments of a body is mandatory for the functional stability of the

organism. Thus, the general requirements for modern life science research

are (1) to describe facts related to the whole body, and (2) to analyse

underlying molecular and genetic mechanisms in a combined manner.

Space Life Science Research offers the unique possibility to keep thinking

about INTEGRATED PHYSIOLOGY alive due to the driving force of

making spaceflight safe for men, and to develop not only physical but also

pharmacological countermeasures in case of microgravity-induced

disturbances of the integrated system (i.e., the organism). - Some flight and

ground-based studies will be discussed in the frame of INTEGRATED

PHYSIOLOGY and MOLECULAR BIOLOGY. Pompeiano and his team

(Pisa) have identified changes in early gene expression in specific brain

areas of microgravity-exposed rats that might be important in adaptive

gain changes of vestibular and vegetative function. Ground-based studies

in birds by Anokhin and colleagues (Moscow) postulated that acquisition

of new experience is related to early gene expression in the brain which

might be basis of adaptation to new environmental conditions such as

microgravity. Own studies with tadpoles after knock-down of a

transcription factor of the wnt-pathway revealed similarities in their

phenotype with that of microgravity exposed tadpoles leading to the

conclusion that growth factor gradients in the embryo are modified by

microgravity. Thus, integrated physiology will benefit from molecular

approaches, and most important, vice versa. - Space projects of Horn were

supported by DLR.

[86] HUMAN FLIGHT TO MARS: CHALLENGES FOR

INTEGRATIVE HUMAN PHYSIOLOGY. R. Gerzer, M. Heer, K.

Ivanova. DLR Institute of Aerospace Medicine, Cologne, Germany

The effects of microgravity and of radiation in space on the human body

are still incompletely understood. Being exposed to these conditions and

being isolated during flights to Mars for over a year makes it necessary to

understand these influences and to prepare countermeasures. A holistic

approach including molecular analyses and systems interplay is

mandatory.

Results from the recent years, e.g., suggest that sodium homeostasis is

altered in microgravity. Sodium exchange with hydrogen on

glycosaminoglycans appears to be important and may also have important

roles in physiology and pathophysiology on earth. This newly discovered

mechanism not only influences our understanding on fluid and electrolyte

balance in space, but also on acid balance and bone and muscle turnover

and on immune function, to name just a few of the systems involved. This

indicates that countermeasure development is a multidisciplinary task that

has to involve knowledge on the interplay and relative importances of a

multitude of body systems.

Since gravity affects all cells, some general mechanisms of cell functions

appear also altered in microgravity. Thus, in melanocytes hypergravity

alters the expression of MRP4 and 5 (“drug resistance proteins”), enzymes

involved in the functions of malignant tumors. If also microgravity alters

the expression profiles of these enzymes, then it can be expected that

astronauts may have altered sensitivity to carcinogenic effects, e.g., to

space radiation.

A concerted “digital human” approach in which the interplay of body

systems is studied based on molecular analyses may be more important for

the future of (space) physiology and for countermeasure development than

the reductionistic approach of only studying isolated molecular or

functional systems.

[87] HOW TIME FLIES - THE EFFECTS OF SPACEFLIGHT ON THE

CIRCADIAN TIMING SYSTEM. C.A. Fuller. University of

California, Davis, CA.

Altered circadian function has been documented in species ranging from

fungi to humans during exposure to the microgravity of spaceflight. Such

observations include changes in all of the fundamental properties of the

circadian clock, including: endogenous period, internal and external

phases, and photic sensitivity. One of the most extensive demonstrations

of such changes occurred during NASA STS-90 Neurolab Spaceflight ,

which provided a unique opportunity to evaluate the direct impact of

microgravity on the circadian timing system and the circadian pacemaker.

In this study, rats were exposed to 16 days of microgravity. These rats

were chronically implanted with biotelemetry transmitters to record body

temperature (Tb) and heart rate (HR). Six of the rats were exposed to

constant light (LL) and 18 of the rats were exposed to a 24-hr light-dark

cycle (LD 12:12). The ability of light to induce c-Fos in the

suprachiasmatic nucleus (SCN), the mammalian circadian pacemaker, was

examined in animals sacrificed on flight day 2 (FD2), FD14, recovery day

2 (R1) and R13. The flight rats in LL exhibited persisting free-running

rhythms, with a change in circadian period relative to ground controls.

The period changes returned to control values one week after landing. The

flight animals in LL maintained internal phase angle relationships between

rhythms compared with ground controls. The flight rats in LD remained

entrained to the LD cycle; however, they evidenced a pronounced phase

delay in Tb, but not HR compared to controls. The flight rats also

demonstrated a decrease in Tb and a change in the daily waveform

compared to controls. Finally, early in-flight, the flight animals

demonstrated a reduced sensitivity to light as evidenced by a highly

attenuated c-Fos immunoreactivity compared to controls. These findings

suggest and further, corroborate with previous space-flight data, that

microgravity affects the circadian clock, including the clock’s ability to

maintain temporal organization and to properly entrain to an external LD

cycle. More recent studies, conducted in ground-based experiments,

suggest that most, if not all, of these circadian responses are a result of

altered vestibular signaling.

[88] SENSORY-MOTOR FUNCTION IN MICROGRAVITY G. Clément

CNRS-Université Paul Sabatier, Toulouse, France

The human sensory-motor system allows us to ascertain the status of our

body, sense our environment, and make relevant adjustments in relation to

this environment to achieve various goals. The sensory part, relying on our

body’s physiological sensors, detects the motion or position of body parts

relative to each other or to the environment. The motor part refers to our

movement within and relative to our environment.

Spaceflight creates a challenge for those sensory-motor functions that

depend on gravity, which include postural balance, locomotion, and eye-

head or hand coordination. The sensory system, and in particular the

vestibular system, must adapt to microgravity when entering orbit and

normal gravity upon re-entry to Earth. The motor system is also affected:

the mass and strength of the “antigravity” muscles decrease because they

are less used in microgravity. Until adaptation is complete, which takes

much longer than the actual g-transition itself, these systems can be

considered maladjusted, resulting in disturbed sensory-motor functioning.

The vestibular system also plays a role in spatial cognition and

navigation, i.e., the knowledge of directional heading and place in the

environment. Recent research has demonstrated that this system also

adjusts heart rate, blood pressure, immune responses, and arousal.

Astronauts experience dizziness and disorientation during their first days

in weightlessness. Upon returning to Earth after spaceflight, they

frequently have difficulties standing upright, stabilizing their gaze, and

walking or turning corners in a coordinated manner. Their sense of balance

and spatial orientation take time to re-adapt to Earth-normal conditions.

Something about the vestibular system obviously adapts to changing

conditions, but what? Why? How? Might a better understanding of this

microgravity-induced vestibular function help people back on Earth

prevent the dizziness, disorientation, and susceptibility to falling that some

patients and older people experience? Answers to these important and

interesting questions require us to know more about the physiology of the

human vestibular system on Earth as well as in space.



[89] SIMULATED-MICROGRAVITY INDUCED CHANGES IN GENE

EXPRESSION IN ZEBRAFISH EMBRYOS SUGGEST THAT THE

PRIMARY CILIUM IS INVOLVED IN GRAVITY TRANSDUCTION. S.J. Moorman and N. Shimada Department of

Neuroscience and Cell Biology, Robert Wood Johnson Medical School,

Piscataway, NJ

Gravity has been a constant physical factor during the evolution and

development of life on Earth. We have been studying effects of simulated-

microgravity on gene expression in transgenic zebrafish embryos

expressing gfp under the influence of gene-specific promoters. We have

looked at a number of different genes expressed in a variety of different

organ systems. For instance, we have looked at beta-actin expression in

the heart, eye, notochord and rohon beard neurons, hsp70 expression in the

lens, alpha-A1 and beta-B1 crystallin expression in the lens, and fli1

expression in the heart and blood vessels. Different organs and cell types

show periods of maximum susceptibility during developmental periods

that coincide with specific developmental events. The organ-specific

developmental events correlate with periods when primary cilia are

playing organ-specific developmental roles. In the notochord, each

primary cilium is positioned to function as a ‘strain gauge’ to monitor the

stresses associated with bending of the notochord in response to forces

such as gravity. Unloading the notochord by placing the embryos in a

simulated-microgravity environment causes more dramatic changes in

gene expression than those seen in any other tissue. The developing

cardiovascular system looses its susceptibility to simulated-microgravity

induced changes in gene expression as the primary cilium of the

endothelial cell becomes a flow sensor in the lumen of the blood vessels.

Rohon beard neurons show simulated-microgravity induced changes in the

variability of gene expression levels that can be explained by a change in

the balance between the canonical and non-canonical Wnt pathways,

pathways that are influenced by the primary cilium. The ubiquitous nature

of the primary cilium as a cell organelle suggests that gravity sensing

might be a general feature of all vertebrate cells where the primary cilium

has not been co-opted for another sensory function. Supported by NASA

NAG2-1591



Ora

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III

Space Physiology and Animal

Development

Joseph Tash, Chair



[90] STUDIES OF JOHN GLENN (JOG) AND ALAN SHEPARD (ALAN),

TWO DROSOPHILA GENES WITH ROLES IN GRAVITAXIS. K.

M. Beckingham, V. Konduri and S. Bjorum.

Dept. Biochemistry and Cell Biology, Rice Univ., Houston, TX

A large genetic screen was performed to identify mutants of the fruit fly

Drosophila melanogaster that show altered behavior in a gravitaxic maze

assay. For 18 of the mutant lines isolated, it proved possible to identify the

affected gene. A subset of these genes has been chosen for further study.

All novel genes are being named after astronauts to recognize our support

from NASA. We will present our findings to date on the genes john glenn

(jog) and alan shepard (alan). jog encodes a protein indicated to play a

role in receptor tyrosine kinase -related signaling. Our gravitaxic

mutation to the gene specifically interrupts an exon of the gene that is

unique to one of the two isoforms of the protein. We have used the

original mutation to the gene to generate further mutations that delete

much of this first exon. These more severe mutations display even greater

behavioral abnormalities including in one case a complete failure to

traverse the gravitaxic maze. We hypothesize that the isoform of the

protein affected by these mutations is largely dedicated to behavioral

responses. alan encodes a RNA binding protein. Our gravitaxic mutation

of alan also appears to influence a subset of the transcripts of the affected

gene. However, some of the additional mutations to this gene that we have

generated show developmental defects particularly in the eyes and head

bristles. We hypothesize that this gene has roles in development as well as

behavioral responses.

[91] EFFECTS OF SPACEFLIGHT AND ALTERED GRAVITY ON

REPRODUCTIVE PROCESSES OF FEMALE MAMMALS. A.E.

Ronca, Departments of Obstetrics & Gynecology, Neurobiology &

Anatomy, Wake Forest University School of Medicine, Winston-Salem,

NC, 28157.

In recent years, the numbers of female astronauts has grown immensely.

As we move forward with the Vision for Space Exploration, women are

expected to increase their involvement in the Space Program and

particularly, long-duration missions. The new space initiative calls for

completing and utilizing the International Space Station (ISS), establishing

an extended human presence on the moon, and preparing for the Mission

to Mars, all of which require sustained habitation of space. In view of these

plans, it is vital that we improve our understanding of acute and enduring

effects of space travel on the female reproductive system. There is limited

knowledge of the effects of space and altered gravity effects on the human

female reproductive system, however animal studies have provided some

understanding of these changes. In this presentation, I will describe the

current state of knowledge of spaceflight and gravity-induced changes in

reproductive function in female mammals, including estrus cycling,

ovarian function, conception, pregnancy, birth, maternal-offspring

interactions and postnatal development.

Supported by NIH Grant HD50201 and NASA Grant NNA04CK83.

[92] DELAYED TESTIS DEVELOPMENT AND ALTERED GENE TRANSCRIPTION IN MALE RATS EXPOSED TO CONTINUOUS ARTIFICIAL GRAVITY FROM GESTATIONAL DAY 9 THROUGH POST-NATAL DAY 21. J.S. Tash1, S. Wolfe1, B.D. Timmerberg1, L.A. Baer2, A.E. Ronca3, 1Dept. of Molecular & Integrative Physiology, Univ of Kansas Medical Ctr, Kansas City, KS; 2Wyle Laboratories, NASA/Ames Research Ctr, Moffett Field, CA; 3Dept. Obstetrics & Gynecology, Wake Forest Univ. School of Medicine, NC. The Bioastronautics Roadmap risks include “unacceptable levels of

increased hereditary, fertility, or sterility risk caused by occupational

radiation exposure or the combined effects of radiation and other space

flight factors.” One potential countermeasure to the effects of microgravity

(µG) is artificial gravity (AG) using centrifugal forces. We examined the

effect of AG (2G) on testis development and fertility in male rats. Pregnant

dams were exposed to continuous 2G from gestational day 9 (G9), parallel

pregnant dam controls were Rotational controls (RC) and stationary

controls (SC). At birth, liters were adjusted to 8 male and 2 female pups

per litter and maintained for an additional 21 days of post-natal

development in continuous AG, RC or SC conditions, respectively. Testes

were harvested at 21, 45, and 60 days of age, and mating trials were run at

45 and 60 days of age. Testis and whole body weight were significantly

lower in HG vs RC and SC animals at all times. Histology of the testes

showed a delayed onset of spermatogenesis in the 45 day old HG rats vs

RC and SC. Gene array analysis of 21 day old testis RNA revealed

significant upregulation of Hsp90-beta (critical for late stage

spermatogenesis), and ATRX (a gene critical for developmental

conversion of the female to male gonad), and significantly lower

transcription of Hsp70t (a testis-specific gene critical for late stage

spermatogenesis) and Hsp70BP and Hsp40 (both complexed with Hsp’s

for function during meiosis). Spermatogonial (SG) apoptosis was also

significantly higher in the 21 day old HG testis vs RC and SC, which

corresponds to a delay in the normal apoptotic reduction in SG populations

that occurs ~5 days earlier during normal testis development. All HG, SC

and RC animals failed to produce litters and 45 days, however all animals

in all groups produced normal litters by 60 days of age. (Supported by

NASA and NIH).

[93] BEHAVIOUR OF VERY LOW- & LOW- FREQUENCY

COMPONENTS IN HEART RATE VARIABILITY POWER

SPECTRA DURING 6 HOURS OF EXPOSURE TO

MICROGRAVITY SIMULATED AS THERMO-NEUTRAL, DRY,

SUPINE IMMERSION K. K. Tripathi, Mona Dahiya Dangi, Ranjit Kumar

Dept of High Altitude Physiology & Hyperbaric Medicine, Institute of

Aerospace Medicine, Vimanapura PO, Bangalore- 560 017 (INDIA)

Interpretation of very low frequency (VLF) and low frequency (LF)

components in the power spectrum of heart rate variability (HRV) is

controversial. Recent reports suggest susceptibility of both LF and VLF

power to parasympathetic influences. We examined behaviour of VLF and

LF components of HRV power spectra in 7 healthy male volunteers during

6 hours of dry, thermo-neutral (33-34°C), supine immersion, a

manipulation known to increase vagal outflow and attenuate renin-

angiotensin mechanisms. Indices of HRV were estimated from ECG

records of 8.5 minutes during immersion (at 20 minutes, 2 hours, 4 hours

& 6 hours) and compared with ground-based supine values. Thoracic

impedance, BP & Systolic Time Intervals (STI’s) were also measured.

Statistical tools included Friedman’s non parametric ANOVA for HRV

indices (due to departures from the assumptions of normality and

homogeneity of variance) and a repeated measure parametric ANOVA

with Greenhouse Geisser correction for other data. During immersion, all

the time & frequency domain indices of HRV exhibited a significant

increase [p=0.002, 0.021, 0.002 & 0.022, for SDNN, CV, pNN50 &

RMSSD, respectively; p=0.007, 0.000 & 0.010 for VLF, LF & HF power,

respectively]. The centre frequencies of the spectra did not change

[p=0.771, 0.153 & 0.125 for VLF, LF & HF, respectively]. Increase in

HRV was much more pronounced than that in R-R intervals (at 6 hours, R-

R interval increased by ~15% but the total power in HRV spectrum

increased ~four-fold) and the increase in spectral indices was appreciable

even after normalisation with the square of mean R-R interval. However,

variation in VLF, LF and HF was not significant when normalised to total

power.



[94] ARTIFICIAL GRAVITY AS A MULTI-SYSTEM

COUNTERMEASURE TO BED REST DECONDITIONING:

PRELIMINARY RESULTS. L.E. Warren1, W.H. Paloski2, and L.R.

Young3 1Universities Space Research Association, Houston, TX, 2Human

Adaptation & Countermeasures Office, NASA Johnson Space Center,

Houston, TX, and 3Man Vehicle Laboratory (37-219), Department of

Aeronautics and Astronautics, Massachusetts Institute of Technology,

Cambridge, MA.

Artificial gravity paradigms may offer effective, efficient, multi-system

protection from the untoward effects of adaptation to the microgravity of

space or the hypogravity of planetary surfaces. Intermittent artificial

gravity (AG) produced by a horizontal short-radius centrifuge (SRC) has

recently been utilized on human test subjects deconditioned by bed rest.

This presentation will review preliminary results of a 41 day study

conducted at the University of Texas Medical Branch, Galveston, TX bed

rest facility. During the first eleven days of the protocol, subjects were

ambulatory, but confined to the facility. They began a carefully controlled

diet, and participated in multiple baseline tests of bone, muscle,

cardiovascular, sensory-motor, immunological, and psychological

function. On the twelfth day, subjects entered the bed rest phase of the

study, during which they were confined to strict 6˚ head down tilt bed rest

for 21 days. Beginning 24 hrs into this period, treatment subjects received

one hour daily exposures to artificial gravity which was produced by

spinning the subjects on a 3.0 m radius SRC. They were oriented radially

in the supine position so that the centrifugal force was aligned with their

long body axis, and while spinning, they “stood” on a force plate,

supporting the centrifugal loading (2.5 g at the feet, 1.0 g at the heart). The

subject station allowed free translation over approximately 10 cm to ensure

full loading of the lower extremities and to allow for anti-orthostatic

muscle contractions. Control subjects were positioned on the centrifuge

but did not spin. Following the bed rest phase, subjects were allowed to

ambulate again, but remained within the facility for an additional 9 days

and participated in multiple follow-up tests of physiological function.

(Supported by NASA and NIH GCRC).

[95] SPACE RADIATION AND BONE LOSS. T.A. Bateman1, M.J. Pecaut2,

E.R. Bandstra1, J.S Willey1, N.D. Travis1, G.A. Nelson2, D.S. Gridley2

1Department of Bioengineering, Clemson University, Clemson, SC and 2Department of Radiation Medicine, Loma Linda University Medical

Center, Loma Linda, CA.

Microgravity causes rapid bone loss in astronauts on extended stays

aboard the International Space Station (Lang et al., JBMR 2004). On

exploratory missions, (beyond low-Earth orbit) in addition to microgravity

astronauts will be exposed to low-dose-rate radiation from cosmic and

solar sources. This radiation exposure has proven to be of significant

concern for the central nervous system and for causing increased incidence

of cancer. However, the effect of space radiation on the skeletal system

has not been studied. Cancer patients receiving radiation therapy

experience increased fracture risk (Baxter et al., JAMA 2005). However,

astronauts will be exposed to lower doses and dose rates that of cancer

patients. Space radiation is also fundamentally different in that protons

and heavy ions predominate rather than gamma radiation. We have

recently demonstrated, in the five studies summarized here, profound bone

loss in murines exposed to space relevant sources of radiation approaching

doses that astronauts will be exposed to on a mission to Mars. Data will be

presented that demonstrates profound loss of trabecular bone (up to 29-

39% loss of trabecular volume fraction and 46-64% decline in trabecular

connectivity) from radiation types that model both solar and cosmic

sources. The hypothesized time course of this bone loss, the relative

changes in bone formation and bone resorption, and the effect of dose rate

(modeling a solar flair) will be discussed.

[96] HIGH-ENERGY (HZE) RADIATION EXPOSURE CAUSES

DELAYED AXONAL DEGENERATION AND ASTROGLIOSIS IN THE CENTRAL NERVOUS SYSTEM OF MICE. Peter Cummings,

M.Sc., M.D.1, Andy Obenaus, Ph.D.2, Dan Heffron1, Greg Nelson, Ph.D.2,

James W. Mandell, M.D., Ph.D.1 1Department of Pathology, University of Virginia, Charlottesville, VA; 2Department of Radiation Medicine, Loma Linda University Medical

Center,Loma Linda, CA.

Limiting the biological effects of cosmic radiation will be a key factor in

determining the success of long duration space missions. The purpose of

this study is to determine if exposure to high-energy radiation (HZE) leads

to central nervous system (CNS) injury and astrogliosis. Astrogliosis, the

ubiquitous response of astrocytes to CNS injury, can prevent regeneration

and neurite outgrowth due to the formation of a glial scar. Ionizing

radiation is known to be a potent stimulus of CNS injury and results in the

activation of astrocytes accompanied by neuronal degeneration. It is

unknown at this time if similar processes occur in the CNS following HZE

exposure. Animals received whole-body irradiation with a 600

MeV/nucleon 56Fe beam in a single fraction of 0 or 4 Gy. Rats were

euthanized at 1, 6, and 12 months post-exposure and brain tissue was

stored for histological analysis. Antibodies directed against glial fibrillary

acidic protein (GFAP) were used to characterize the astrocytic response. A

modified Gallyas silver stain was performed to identify neuronal and

axonal degeneration. When compared to controls, irradiated rats showed

evidence of axonal degeneration in white matter with coinciding gliosis.

The axonal degeneration was more pronounced at 12 months (882

degenerating axons per whole-brain section, 16 for controls) compared to 1

month post-exposure (120 degenerating axons per whole-brain section, 0

for controls), suggesting a delayed injury mechanism. GFAP

immunohistochemistry demonstrated astrogliosis in the form of

hypertrophy of astroglial process within the regions of axonal

degeneration. Astrogliosis was also more evident at 12 months compared

to 1 month post-exposure. These observations demonstrate that exposure

to HZE radiation is a potent stimulator of CNS injury and astrogliosis and

suggest a delayed and sustained mechanism. (Supported by NASA Grant

#NNJO4HD80G to AO)

[97] EFFECTS OF RADIATION ON MACROPHAGE FUNCTION. M.J.

Pecaut 1,2, F. Baqai 1,2, E. Bayeta 1 & D.S. Gridley 1,2 1Dept of Radiation

Medicine and 2Dept of Biochemistry & Microbiology, Loma Linda

University and Medical Center, Loma Linda, CA.

With current technology, there are at least four ever-present and

currently inescapable factors inherent to the spaceflight environment that

may be responsible for changes in immune status: 1) Mission-related

psychological stress, 2) Changes in inertial condition (i.e. microgravity), 3)

Low-dose/low-dose-rate radiation, and 4) Potentially dangerous levels of

microbial contamination. Of these environmental factors, combined

exposures to radiation and microbial challenge are most likely to pose a

significant threat to astronaut safety. Bacterial clearance is typically

accomplished within a few hours in healthy individuals due to pre-existing

innate or natural immune mechanisms. However, in experimental models,

as well as in humans after the use of atomic bombs at the end of World

War II, radiation-induced mortality has long been associated with enteric

bacterial infection. To date, there are few studies involving radiation

effects on survival after bacterial challenge. Susceptibility appears to be

enhanced after exposure to whole-body x-rays where mice succumb in

greater numbers to lower bacterial titers. Reports also indicated that

radiation did not lead to the selective growth of more virulent bacterial

populations and that mortality was due to circulating endotoxin.

We propose that whole-body exposure to low-dose radiation will alter

immune function and host resistance, leading to an increased vulnerability

to infection. These changes will be due to a reduction in the efficacy of

systemic macrophage function. We have already have evidence that

exposure to whole-body, proton irradiation can influence the response to a

subsequent challenge with live E. coli, in terms of clearance rates in the

peritoneal cavity and blood. Here, we show that macrophages irradiated in

vitro, with doses as low as 0.1 to 4 Gy γ-rays, have altered phagocytic and

oxidative burst capacity, suggesting that radiation can influence

macrophage function on a the level of cellular mechanisms.

(Supported by NASA Cooperative Agreement NCC9-149)



Biotechnology and Cell

Biology

Peter Scherp, Chair

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[98] DID EARTH-MARS METEORITIC EXCHANGE SPREAD

MICROBIAL LIFE? DNA-BASED LIFE DETECTION FOR MARS,

WITH HUMAN SPACEFLIGHT APPLICATIONS. C.E. Carr1, M.T. Zuber1, and G. Ruvkun2,3 1Department of Earth, Atmospheric and Planetary Sciences, Massachusetts

Institute of Technology, Cambridge, MA. 2Department of Genetics, Harvard University, Cambridge, MA. 3Department of Molecular Biology, Massachusetts General Hospital,

Boston, MA.

On Earth, the DNA-based polymerase chain reaction (PCR) provides a

simple, standard, and powerful method to detect life. A soil sample from

an extreme environment can be surveyed for the signature of life, a DNA

fragment of a gene that is universal to life on Earth, in less than 2 hours in

any standard molecular biology laboratory. Due to massive meteoritic

exchange between Earth and Mars, a reasonable case can be made for life

on Mars to be related to life on Earth. We are developing a PCR detector

for in situ analysis on other planets, most immediately, Mars. If present,

even low levels of microbial life on Mars could be detected by the

instrument, which would also be able to classify the evolutionary

relationship between any “positives” and the “tree of life” through in-situ

analysis. We are working to develop a prototype of the DNA amplification

module of this instrument that will be validated using terrestrial samples.

In addition to searching for extant life ancestrally related to life on Earth,

the instrument also has applications for planetary protection and human

spaceflight, including medical diagnosis and environmental monitoring.

(Supported by NASA grant NNG05GK27G.)

[99] DETECTION OF STRAND CLEAVAGE AND OXIDATION

DAMAGE USING MODEL DNA MOLECULES CAPTURED IN A NANOSCALE PORE. V. DeGuzman1, A. Solbrig1, B. Nogal1,2, W.

Vercoutere3; 1Education Associates Program, NSGF, Moffett Field, CA; 2Cornell University, Ithaca, NY; 3NASA ARC, Moffett Field, CA.

Human travel beyond Earth’s atmosphere and magnetosphere requires an

understanding of the biological impact of cosmic radiation. Among the

biological effects, damage to DNA can lead to some of the most

deleterious results, including cell death, tissue degeneration, mutation,

tumorigenesis, and premature aging. The excess energy of radiation can

directly and indirectly cause purine or pyrimidine oxidation, single strand

scission and double strand scission. The greater the damage to DNA, the

higher the risk is to human health. However, the relationship between

dose, radiation type and DNA damage is not clear. This may be clarified

by analysis of differences in damaged DNA at the molecular level. Our

previous work has shown that a biological nano-scale pore can be used to

detect structural and dynamic differences among individual DNA

molecules. Here we show that this detector can discriminate DNA

molecules designed to differ only by a single 8-oxo-guanine or a single

phosphodiester backbone nick, common modifications that can arise from

radiation exposure. We also show preliminary results indicating that

frequency of damage can be measured. We are developing a library of

molecular signatures attributable to specific types of DNA damage. In

future studies, the compiled library of molecular signatures may be used to

interpret the types of damage caused to DNA samples irradiated with

different doses and particle types of cosmic radiation. Assessment of

human health risk and our capacity to recover from exposure will be

improved by establishing the relationship of cosmic radiation exposure

levels to type and frequency of DNA damage. (Supported by NASA ARC

IR&D 2004-2005)

[100] HIGH-RESOLUTION GENE EXPRESSION PROFILING IN

SINGLE CELLS AND SMALL TISSUES P. Scherp and K.H.

Hasenstein, Biology Department, University of Louisiana, Lafayette

70504-2451

Gene expression profiling has become a standard method to investigate

the genetic response of animal and plant cells to internal and external

stimuli. Utilizing an mRNA extraction method with high spatial and

temporal resolution (Solid Phase Gene Extraction, SPGE) and real-time

PCR we analyzed single Drosophila eggs and roots of germinating flax

seeds for the expression of specific genes in different sites over time. In 0

– 1 h old Drosophila eggs the distribution of nanos and bicoid, two

maternally expressed genes that determine the development of the

embryo’s body-axis, showed distinct polarity. The highest abundance of

nanos mRNA occurred in the posterior of the egg while bicoid was

concentrated in the anterior.

In flax, the onset of actin gene expression during germination and early

stages of seedling growth showed discrete patterns that are essential for

general cell functions, development and mechanosensing. The primordial

and juvenile root (3-48 h after imbibition) was sampled at three different

sites (cap, meristem and elongation zone) by inserting an oligo-dT-covered

needle ca. 100 µm into the tissue. The extracted mRNA was analyzed for

two actin genes. In the early stages of imbibition (3-6 h) the actin genes in

the root cap, the root meristem and the elongation zone showed less than

45000 copies per sample (cps) for ACT1 and less than 2000 cps for ACT2.

The expression of ACT1 in the root cap peaked 9 h after imbibition and

increased 11 fold in the elongation zone between 15 h and 18 h after

imbibition, which coincides with the onset of root elongation. ACT2

expression increased between 6 h and 12 h after imbibition in the root cap

(1000 fold) and the meristem (130 fold) which precedes the onset of

mitosis at 12 h after imbibition.

These data represent the first high resolution mapping of gene expression

in single Drosophila eggs and primordial and germinated roots of plants.

In flax, the data connect the expression levels of ACT1 and ACT2 with the

onset of elongation growth and mitosis, respectively. Supported by NASA

grant NNA04CK48G.

[101] EFFECT OF GRAVITY VECTOR CHANGES ON

INTRACELLULAR CALCIUM CONTROL IN CULTURED GLIAL

CELLS. M.A. Masini1, F. Strollo2, F. Ricci3, P. Prato1, and B. Uva1 1Dipartimento di Biologia, University of Genova, Italy, 2Unita’

Endocrinologia INCRA & Università di Roma “La Sapienza”, Italy, 3ENEA C.R. Casaccia Roma Italy.

Glial cells, like any other eukariotic cells, rely on calcium ions to display

a large variety of signalling capabilities inside the cell and among

neighbouring cells. This calcium signalling is based on the existence of a

calcium gradient between the extra cellular and intracellular compartments

that is carefully regulated by the glial cells themselves by means of

extrusion channels, buffering proteins and intracellular organelles

(Endoplasmic Reticulum and Mitochondria). Gravity vector changes

causes severe alterations to the cells damaging the cytoskeleton, as

observed in lymphocytes during Space Flights (Cogoli-Greuter et al., J.

Gravit. Physiol.1994; Lewis et al., Faseb J., 1998) and in on ground

conducted experiments (Uva et al., Brain Res .2002), plasma membrane as

well as mitochondria were also damaged (Uva et al., MST, 2005).

Addition of minerals to the culture medium has proved to protect

microtubules from depolarization in cultured glial cells (Masini et al.,

Gravit. Space Biol. 2006). Aim of the present study was to verify if a

period of rest and the addition of calcium to the culture medium may

restore, after 1h 3D RPM rotation, calcium metabolism in astroglioma

cells in culture. For the purpose, glial cells (C6 cell line from rat

astroglioma) were submitted to 3D RPM clinorotation for different length

of times (1h to 24h) with or without addition of calcium ions to the culture

medium. After clinorotation, the cultures were kept for different length of

time (1h to 24h) in a rest condition. At the end of the experiment, the cells

were fixed and submitted to immunohistochemistry using the following

antibodies: Ab to α-tubulin, Ab to Na+/K+ATPase, Ab to Na+/K+/Cl-

cotransporters, Ab to Ca2+ATPase, Ab to proteins of inner mitochondria

cristae, Ab to Caspase 7 executioner, Ab to HSP. DNA fragmentation was

visualized by TUNEL method. Cytosolic calcium concentration was

visualized by FURA method. (Supported by ASI)



[102] HYPERGRAVITY INCREASES OSTEOBLASTOGENESIS AND

OSTEOCLASTOGENESIS BY BONE MARROW CELLS IN CULTURE. H. Kondo1, R.K. Globus2, C. Limoli1, E. Almeida2, D.J.

Loftus2, W. Vercoutere2, R. Mojarrab2, M. Atwal2, N.D. Searby2. 1Department of Radiation Oncology, University of California, Irvine; and 2Life Sciences Division, NASA ARC

Spaceflight results in bone loss, and it is thought that altered gravity

contributes significantly to this loss. Artificial gravity, with gravity (g)

levels greater than Earth’s 1-g, or hypergravity, is being considered as a

possible spaceflight countermeasure to add back gravity loading. We

tested if application of hypergravity to bone marrow (BM) cells regulated

osteoblastogenesis or osteoclastogenesis, since these cells are pertinent to

bone health. Bone marrow cells were collected from 6 – 9 week old

C57BL6/J male mice and grown in alpha-MEM with 15% fetal bovine

serum and factors that promote either osteoblast or osteoclast

differentiation. On day 2 (d2) or d4 of culture, cells were centrifuged at 5-

g, 10-g, and 50-g for 5 to 180 min. Osteoblastogenesis was assessed by

alkaline phosphatase (ALP) activity and production of mineralized matrix

(Alizarin red staining). BM stromal (BMS) cell numbers were assessed by

manual cell counting or by measuring DNA content (CyQUANT). BMS

cell adhesion was assessed by measuring changes in DNA content 5 min

after the hypergravity stimulus. Osteoclastogenesis was assessed by

counting Tartrate Resistant Acid Phosphatase (TRAP)-positive cells. We

found that 5 min 50-g increased ALP activity 2.3-fold on d8, and 180 min

50-g increased mineralized nodule area 1.4-fold on d21. Cell number 2d

after hypergravity increased for all lengths and magnitudes of stimuli

tested. Cell numbers immediately after 5 min 10-g increased 1.5-fold,

indicating increased cell adhesion. A 5 min 50-g stimulus increased the

number of TRAP-positive cells 1.3-fold on d8. In summary, hypergravity

increased osteoblast differentiation, number, and adhesion as well as

osteoclast differentiation, which may support the idea of using

hypergravity to improve bone health. (Supported by NASA: Grant from

solicitation # NNH04ZUU005N)

[103] GRAVITY DEPENDENCE OF LEUKOCYTE ADHESION TO A

SIMULATED BLOOD VESSEL WALL. D.F. Kucik, R.L. Rouleau,

L.W. Smith, and K.B. Gupta. Dept. of Pathology and Biomedical

Engineering, University of Alabama at Birmingham and Birmingham VA

Medical Center, Birmingham, AL.

It has been known for many years that space travel can affect the

immune system, but the nature of the effects and their molecular

mechanisms are not yet well understood. Of particular interest is the effect

of microgravity on white blood cells, which has been shown to be

independent of effects of cosmic radiation and physical stress.

One important aspect of white-cell function that has been difficult to

address experimentally is regulation of leukocyte adhesion to the blood

vessel wall. This is a vital, early step in the initiation of an immune

response, without which effective immunity is not possible.

We designed an integrated flow-chamber adhesion assay and used this

system to measure leukocyte adhesion under flow conditions in actual

microgravity on a series of parabolic flights. Flight data at 0g, 1/6

(lunar)g, 1g, and 1.8g have established a gravity dependence for leukocyte-

endothelial cell adhesion under conditions simulating those in a blood

vessel. Our results suggest that leukocyte-endothelial cell adhesion may

be severely impaired during microgravity, which could have important

consequences in the event of a serious injury or illness during prolonged

flight. Some theories predict that the presence of red blood cells has a

positive effect on white blood cell adhesion, increasing the frequency of

initial WBC contact with the blood vessel wall by hydrodynamic effects.

This would imply that maintaining a high hematocrit might be a useful

countermeasure for decreased WBC adhesion. In our experiments, RBCs

did not demonstrate this positive effect at zero g. This work has important

implications for the safety of prolonged spaceflight. Further experiments

using our system can be instrumental in developing and testing

countermeasures to spaceflight-associated impaired immunity. (Supported

by NASA: NNJ04HB26G)

[104] INFLUENCE OF 3D CLINOROTATION ON THE ACTIVATION

OF RHO INVOLVED IN ACTIN FIBER REMODELING. A.

Higashibata1,3, M. Imamizo-Sato2,3, M. Seki2,3 T. Yamazaki1 and N.

Ishioka1,3. 1Institute of Space and Astronautical Science, Japan Aerospace

Exploration Agency, Tsukuba, Japan, 2Space Station Engineering

Department, Advanced Engineering Services Co., Ltd, Tsukuba, Japan,

3Department of Space Environmental Medicine, Kagoshima University

Graduate School of Medical and Dental Science, Kagoshima, Japan.

We studied effects of 3D clinorotation on actin fiber remodeling process

in bovine brain microvascular (BBM) endothelial cells. The actin stress

fiber in clinostat-cultured cells (CCCs) was significantly disorganized

under clinorotation as observed in the previous flight experiments. Our

western blotting results, however, showed that the expression of RhoA

protein was enhanced in CCCs in spite of actin fiber disorganization.

These results indicated that Rho activation process was influenced by

clinorotation and it subsequently caused disorganization of actin fiber.

Based on the results above, we examined the effect of clinorotation on the

expression of LARG that is a Rho guanine exchange factor. The result

from mRNA fingerprinting method showed that 62 fragments showed

different gene expression in the CCCs. From these fragments, we found a

part of LARG fragment. To confirm the mRNA fingerprinting result, we

measured LARG gene expression by quantitative real time PCR. LARG

gene expression was decreased in CCCs. Our western blotting result also

showed that LARG expression was decreased under clinorotation. These

data indicated that LARG expression was downregulated under

clinorotation. Furthermore, we measured the amount of active-formed

RhoA by ELISA method. The formation of active-formed RhoA

significantly decreased in the CCCs. From these results, we conclude that

Rho activation process responds to clinorotation and it causes the

disorganization of actin fiber.

[105] TWO SPECIAL INSTRUMENTS FOR THE STUDY OF SIMULATED ALTERED GRAVITY. M.A. Benjaminson, S. Lehrer,

J.A. Gilchriest, L. Schonbrun and M.B. Madigan NSR-Touro Applied

Bioscience Research Consortium, Bay Shore, NY 11706.

We designed and fabricated a clinostat and a centrifuge able to

accommodate specimens in petri dishes. We established quality control

protocols which improved the accuracy of our measurements of simulated

altered gravity effects on small biological systems (Dictyostelium). The

D1003 clinostat provides a calculated value of 0.0058 g. The D1006

centrifuge provides values of g from 1.5-10. Cocultures of D. discoideum

and E. aerogenes (prey) were segregated at 1g or clinorotated to provide

0.0058 g. The ratios of mature/reproductive individuals to immature forms

was 20:1 at 1g and 5.4:1 at 0.0058 g. This indicates that clinorotation

slows the process of development, paralleling results with other clinostats.

The centrifuge was tested with identical cocultures at 10g. All cultures

survived at this level of hyper g. Preliminary data indicate that

centrifugation at 10 g slowed development, showing ratios of 1:1.3 at 1g

and 11:1 at 10g. The high survival rate of the test biota and excellent

performance of the two instruments speak well for their future

performance in gravitational biology research (Supported by NASA:

NAG8-1809)

Gravitational and Space Biology 20(1) November 2006 A-1

Au

thor

Ind

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AUTHOR INDEX – 2006 ANNUAL MEETING ISSUE

A-2 Gravitational and Space Biology 20(1) November 2006

Abstract numbers follow each author’s name. Abstract numbers in bold-faced italics indicate the author

is the presenter. Non-bold-faced numbers indicate co-authorship of an abstract. The abstracts are in

numerical order in both the detailed program (pages ix – xxiv) and the abstract section (pages 1-52).

A

Ahmed, F.N., 8

Alberts, J.R., 52

Allen, C.A., 35

Allen, J., 62

Allen, P.L., 48, 70

Almeida, E.A., 18, 102

Alwood, J.S., 18

Atwal, M., 102

Auxenne, C., 78

B

Baer, L.A., 92

Bain, J.L.W., 73

Bandstra, E.R., 34, 95

Baqai, F., 97

Bascon, J., 30

Bateman, T.A., 34, 70, 95

Bayeta, E., 97

Beckingham, K.M., 32, 90

Belton, A.S., 27, 51

Benjaminson, M.A., 105

Berkowitz, D.E., 42

Bhattacharya, S., 65

Bingham, G.E., 74

Birmele, M.N., 76

Bjorum, S., 90

Blancaflor, E.B., 57, 81

Blasiak, J., 62

Bohnert, H.J., 44

Boss, W., 55

Brown, C.S., 11, 31, 55, 80,

82

Brown, M.L., 58

Bryant, S.J., 36

Bucklin, R.A., 14

Bugbee, B., 74

Bulkowski, J., 65

Bushart, T.J., 79

C

Carr, C.E., 25, 98

Cassanova, R.A., 68

Chang, S.Y.C., 70

Chang, V.Y., 26

Chapman, K.D., 57

Cirelli, D., 40

Clément, G., 88

Conley, C.A., 75

Connor, D., 50

Correll, M.J., 14, 23

Cummings, P., 96

D

Dangi, M.D., 93

Darnell, R., 62

Davis, E., 82

Day, B.H., 65

Dean, J., 50

DeCarlo, A.R., 16, 69

DeGuzman, V., 99

Dickman, J.D., 72

Dover, H., 6

Duke, J., 78

E

Edelmann, R.E., 28

Elland, C.S., 65, 66

Eubanks, L.M., 77

Evans, M.L., 59, 60

F

Ferguson, V., 33

Fernandez, J., 65

Fitts, R.H., 73

Flowers-Aime, R., 27, 51

Franklin, J.C., 13

Freeman, J., 48

Fujishima, T., 71

Fukui, K., 71

Fukushima, G., 71

Fulford-Jones, T.R.F., 5, 13

Fuller, C.A., 87

G

Geitman, A., 3

Gerzer, R., 86

Gibbs, K., 50

Gilchriest, J.A., 105

Globus, R.K., 18, 102

Gohil, H.L., 14

Goins, T.L., 53

Goldberg, A.L., 45

Granzella, N.P., 15

Gridley, D.S., 95, 97

Grosse, E.B., 13

Guerra, E.C., 5

Gupta, K.B., 103

H

Hammond, R.L., 11, 82

Hammond, T.G., 48, 70

Hanson, A., 33

Harrison, B.R., 56

Harrison, M.A., 58

Hasenstein, K.H., 43, 100

Heafitz, A.M., 5, 13

Heer, M., 86

Heffron, D., 96

Heisler, W., 47

Higashibata, A., 104

Hoffman, J.A., 5, 13

Hogan, J., 63

Horn, E.R., 85

Howard, H.N., 8

Hung, C.Y., 8, 55

Huss, D., 72

I

Imamizo-Sato, M., 104

Irazoqui, P.P., 16

Ishikawa, H., 59, 60

Ishioka, N., 104

Ivanova, K., 86

Iversen, T.H., 83

J

Jansen, J.A., 20

Jennings, D.E., 68

Jo, H., 37

Johannes, A., 83

Johnson, C.J., 32

Johnson-Wint, B.P., 47

Jones, S.B., 74

Judex, S., 34


Gravitational and Space Biology 20(1) November 2006 A- 3

Juergensmeyer, E.A., 49

Juergensmeyer, M.A., 49

K

Kajiume, T., 17, 19, 38, 39

Kataoka, K., 19, 39

Kawahara, Y., 17, 19, 38, 39

Khodakovskaya, M., 31, 55,

80

Kimbrough, J.M., 80

Kimoto, M., 71

Kiss, J.Z., 7, 23, 28, 29, 40,

41, 54

Kittang, A.I., 83

Klaus, D.M., 8, 12, 67

Klein-Nulend, J., 4

Klement, B.J., 27, 36, 51

Kondo, H., 102

Konduri, V., 90

Koskal, E.S., 5

Kostenuik, P.J., 70

Kovo, Y., 66

Kuang, A., 62

Kucik, D.F., 103

Kumar, N.S., 28, 41

Kumar, P., 29, 54

Kumar, R., 93

Kumei, Y., 71

Kuntz, A.L., 7

Kurisu, K., 38

L

Lacey, D., 70

Lehrer, S., 105

Levinskih, M.A., 74

Limoli, C., 102

Liu, W., 37

Loesberg, W.A., 20

Loftus, D.J., 102

Lomax, A., 9

Lysakowski, A., 72

M

Madigan, M.B., 105

Magaki, T., 38

Mandell, J.W., 96

Marsh, M., 78

Marshall, C.M., 51

Martinson, V.G., 53

Masini, M.A., 101

Massa, G.D., 84

Masson, P.H., 56

McLamb, W.T., 69

Meck, J.V., 42

Melendez, D., 50

Mick, M.E., 84

Mitchell, C.A., 84

Mobley, E.M., 49

Mojarrab, R., 102

Molas, M.L., 7, 23

Montgomery, C.E., 29

Moorman, S.J., 89

Morony, S., 70

Morrow, R.C., 64

Mortenson, T.E., 76

Mulkey, T.J., 61

Musgrave, M.E., 62

Myburg, H., 11, 82

N

Naminohara, K., 17, 19

Natori, E., 59, 60

Navarro, B.J., 65, 66

Nelson, G.A., 95, 96

Nickerson, C.A., 48

Niesel, D.W., 35, 50

Nogal, B., 99

O

Obenhaus, A., 96

Ogasawara, M., 71

Okazaki, T., 38

Ormsby, S., 50

P

Paloski, W.H., 94

Pandya, U., 50

Patel, M.J., 37

Patel, O.V., 6

Paulsen, D.F., 27, 51

Pecaut, M.J., 95, 97

Perera, I., 9, 11, 31, 55, 82

Perkins, K.E., 50

Plaut, K., 6

Podolski, I.G., 74

Pohorille, A., 70

Porter, J.E., 21

Porterfield, D.M., 16, 69, 79

Prato, P., 101

Prendergast, P.J., 4

Price, S., 72

Pyle, B.H., 53

R

Rakvaag, G., 83

Ramirez-Perez, J.C., 63

Rask, J.C., 66

Rector, C., 77

Reed, D.W., 76

Ricci, F., 101

Riley, D.A., 73

Risin, D., 37

Risin, S.A., 37

Roberts, D.R., 10

Roberts, M.S., 76

Rogney, A.M., 64

Rokkam, M., 16, 69

Romatowski, J.G., 73

Romer, L., 2

Ronca, A.E., 91, 92

Rouleau, R.L., 103

Roux, S.J., 16, 69, 79

Ruvkun, G., 25, 98

S

Salinas-Mondragón, R., 80,

82

Salmi, M., 69

Sasaki, A., 17, 19, 38, 39

Sato, K., 50, 65

Scherp, P., 100

Schonbrun, L., 105

Schweighofer, K., 70

Scott, M.A., 13

Searby, N.D., 18, 102

Seki, M., 104

Selch, F., 75

Shimada, N., 89

Shoukas, A.A., 42

Shrestha, R., 57

Silvestrov, N.A., 27

Simonette, R., 32

Smith, L.W., 103

Solbrig, A., 99

Solheim, B.G., 83

Spence, A.K., 28

Stanczyk, A.R., 12

Stanga, J., 56

Stimpson, A.J., 14

Stodieck, L., 33, 48, 70


A-4 Gravitational and Space Biology 20(1) November 2006

Strollo, F., 101

Strom, P.F., 63

Suzuki, H., 71

Svare, H., 83

Sword, C., 31

Sychev, V.S., 74

Sykes, M.C., 37

Szewczyk, N.J., 75

T

Tabakov, V.Yu, 53

Takeda, M., 38

Tash, J.S., 24, 92

Teal, H.E., 50

Texada, M.T., 32

Thomas, D.J., 77

Timmerberg, B.D., 24, 92

Toda, K., 71

Todd, P., 77

Torres, A.G., 35

Trappe, S.W., 73

Travis, N.D., 95

Tripathi, K.K., 93

Truett, J.R., 14

Tuday, E.C., 42

Tuominen, L.K., 64

Turner, R.E., 68

U

ul Haque, A., 16, 69, 79

Umeda, C., 17, 19, 38, 39

Uva, B., 101

V

Vahora, N.M., 25

van Klompenberg, M.K., 6

van Loon, J.J.W.A., 1, 20

Vazquez, M.E., 34

Vercoutere, W.A., 66, 99,

102

Villanueva, I., 36

Voeikova, T.A., 53

von Deutsch, A.W., 27, 51

von Deutsch, D.A., 27, 36, 51

W

Wagers-Hughes, R., 62

Wagner, E.B., 5, 15, 26

Walboomers, X.F., 20

Wang, Y.S., 57

Ward, N.E., 37

Warren, L.E., 94

Warrington, J., 77

Wereley, S.T., 16, 69

West, T.E., 21

Willey, J.S., 95

Williams, C., 27

Williams, N., 50

Winter Sederoff, H., 11, 31,

55, 80, 82

Withers, J.C., 22

Wolfe, S., 24, 92

Wright, J., 27, 51

Wu, S.L., 17, 19, 38, 39

Wyatt, S.E., 10, 22, 30

Y

Yamaoka, K., 39

Yamazaki, T., 104

Yang, P.L., 5

Yoo, C.M., 81

Yoshimoto, R., 17, 19, 38, 39

Young, L.R., 94

Young, M., 33

Yuge, L., 17, 19, 38, 39

Z

Zenisek, J., 24

Zeredo, J., 71

Zuber, M.T., 25, 98

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