NASA Technical Memorandum 4258

1989-90 NASA Space Biology

Accomplishments

Edited by

Thora W. Halstead

NASA Office of Space Science and Applications

Washington, D.C.

National Aeronautics and

Space Administration

Office of Management

Scientific and Technical

Information Division

1991
I _ _ _
PREFACE

An individual technical summary of each research task within the NASA

Space Biology Program is presented in this publication. Each summary,

prepared by the principal investigator, consists of a description of the

research, a listing of the project's accomplishments, an explanation of the

significance of the accomplishments, and a list of the publications resulting

from the past year's research. Since spaceflight experiments, submitted in

response to the Space Biology Dear Colleague letter, have become an integral

part of the Program, reports on the activites of this related research are

integrated in the report. Accomplishments of the scientists in the NASA

Space Biology Research Associates Program, which provides opportunities

for postdoctoral scientists to conduct research in the fields of gravitational

and space biology, and the NASA Graduate Student Researchers Program,

which supports promising students pursuing advanced degrees in science

and engineering, are also included. The participants in these programs have

been outstanding and merit independent recognition.

This publication has two objectives: first, to provide the scientific

community and NASA with an annual summary of the accomplishments of

the research pursued under the auspices of the Space Biology Program, and

second, to stimulate an exchange of information and ideas among scientists

working in the fields of gravitational and space biology.

Thanks are due to the Program participants and postdoctoral and graduate

student scientists whose research and cooperative response to our requests

for information made this report possible. Editorial support provided by

Janet V. Powers, Katherine J. Dickson, and Elizabeth L. Hess is gratefully

acknowledged and appreciated, as well as the technical assistance provided

by April Commodore Roy.

Additional information about this report of the Space Biology Program can

be obtained by writing to me at the following address:

Dr. Thora W. Halstead

Code SBR

NASA Headquarters

Washington, D.C. 20546

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=
CONTENTS

Preface

Introduction - The NASA Space/Gravitational Biology Program

Accomplishment Highlights

Plant Projects

ROBERT S. BANDURSKI and MARK F. DESROSIERS

An Attempt to Localize the Gravity Sensing Mechanism of Plants:

Effect of Gravity on the Electrical Parameters of the Stele-Cortex

Junction in Corn Shoots

ROBERT CLELAND

The Role of Calcium in Gravity Sensing

DANIEL J. COSGROVE

Mechanism of Differential Growth During Stem Gravitropism

KATHRYN L. EDWARDS

The Gravitational Response of Phycomyces With Respect

to Gadolinium and Factors Affecting Calcium Function

MICHAEL L. EVANS

Role of Calcium in Signal Transduction in Root Gravitropism

LEWIS FELDMAN

Physiological, Biochemical and Molecular Processes

Associated with Gravitropism in Roots of Maize

JAMES S. FERRARO

Circadian Rhythms Persist in Space with a Decreased Frequency:

Preliminary Results from STS-32 Life Sciences Experiment

"Characterization of Neurospora Circadian Rhythm in Space"

PETER B. KAUFMAN, NADARAJAH KARUPPIAH,

CASEY LU, and DONGHERN KIM

Gravitropic Response Mechanism in Cereal Grass Shoots

ABRAHAM D. KRIKORIAN

Cells, Embryos, and Development in Space

A. CARL LEOPOLD

Perception and Transduction of Graviresponses in Plants

NORMAN G. LEWIS

Determining the Effects of Gravitational Stresses on Lignin

Formation and Structure: A New Approach

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TERRI L. LOMAX

Analysis of Components of the Gravitropic Response in Plants

Using Purified Plasma Membrane Vesicles

CARY A. MITCHELL and LAURA L. COE

Mechanical Stress Regulation of Plant Growth and Development

RANDY MOORE

Signal Transmission and the Role of the Root Cap

During Gravitropism

KENNETH L. POFF

Gravitropism in Arabidopsis thaliana: A Genetic Approach

B.W. POOVAIAH

Calcium Messenger System in Gravitropic Response in Plants

V. RAGHAVAN

Effects of Hypergravity on Spore Germination in the Sensitive

Fern Onoclea Sensibilis

DAVID L. RAYLE

Cellular and Molecular Processes of the Gravitropic Response

STANLEY J. ROUX

Assessing Potential Targets of Calcium Action in Gravitropism

FRED D. SACK

Cellular Polarity and Interactions in Plant Graviperception

FRANK B. SALISBURY

Gravitropism in Dicot Stems

ROBERT D. SLOCUM

Amide Oxidase and Peroxidase Regulation of Differential

Growth Responses in Gravistimulated Plants

Animal Projects

DANIEL D. BIKLE and BERNARD P. HALLORAN

Effect of Skeletal Unloading on Bone Formation

ROBERT H. BLANKS

Physiology of Developing Gravity Receptors and Otolith-Ocular

Reflexes in Rat

STEPHEN B. DOTY

Studies of Intercellular Communication and Intracellular Responses

by Bone Cells to Simulated Weightlessness

PAULINE JACKIE DUKE

Effect of Altered G on Chondrocyte Differentiation

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CI_SARD. FERMIN

Influencesof SynapticGenesisontheArchitectureof the

VestibularEpithelia

WILLIAM F.GANONG

NeuralMechanismsBy WhichGravitationalStimuli andStress

Affect theSecretionof ReninandOtherHormones

RAPHAEL GRUENER

Vector-FreeGravityInterfereswith SynapseFormation

JOHNM. HOROWlTZ andBARBARA A. HORWITZ

TheEffectsof GravitationalFieldsonNeuralSignaling

in theHippocampus

MILLIE HUGHES-FULFORD

Mechanismof Controlof BoneGrowthBy Prostaglandins

DONALD E.INGBER

Mechanochemical TransductionAcrossExtracellularMatrix

TIMOTHY A. JONES

VestibularElectrophysiologicalResponseThresholds:

Effectsof SpaceflightandLinearSineWaveMotion

CHRISTINE E. KASPER

Molecular Basis of Tension Development in Muscular Atrophy

THOMAS P. KERR and DENNIS G. DRESCHER

Mechanisms of Vestibular Ion Transport

WILLIAM J. LANDIS and LOUIS C. GERSTENFELD

Collagen Synthesis, Assembly, and Mineralization in

Chicken Osteoblast Cell Cultures

EDWIN R. LEWIS

Micromotional Studies of Utricular and Canal Afferents

GEORGE M. MALACINSKI and ANTON W. NEFF

Gravity as a Probe for Understanding Embryonic Pattern

Specification in the Amphibian Model System

EMILY MOREY-HOLTON

Structural Development and Gravity

NICOLA C. PARTRIDGE

Skeletal Collagen Turnover By the Osteoblast

CAREY R. PHILLIPS

Gravitational Effects on Early Development in Amphibian

Embryos

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W. EUGENEROBERTS

Cell Kinetic andHistomorphometricAnalysisof

MicrogravitationalOsteopenia

MURIEL D. ROSS

StructureandFunctionof MammalianGravityReceptors

MARTIN P.SCHREIBMAN

Development,MaturationandPhysiologyof theBrainPituitary-GonadalAxis of Fishin theCEBAS/AquarackSystem

DOROTHYB. SPANGENBERG

Effectsof WeightlessnessonAurelia Ephyrae

Differentiation and Statolith Synthesis

MARC E. TISCHLER

Skeletal Muscle Metabolism in Hypokinetic Rats

FRED H. WILT

The Role of Calcite Skeletal Matrix Proteins in

Biomineralization

DEBRA J. WOLGEMUTH

Effects of Microgravity on Mammalian Development and

Differentiation

Special Activities

SPACE BIOLOGY RESEARCH ASSOCIATES PROGRAM

X.J. Musacchia

STEVEN D. BAIN

The Interaction of Skeletal Remodeling with Systemic Disorders:

An Obstacle to Extended Spaceflight?

WALLACE D. BERRY

Effects of the Unloading Model on Dihydroxyvitamin D

and Cellular Immunity

EDWARD M. GREENFIELD

Regulation of Osteoclastic Bone Resorption by Osteoblasts

A_A LYSAKOWSKI

Synaptic Relations of Type I and Type II Hair Cells in the

Crista Ampullaris of the Chinchilla

KENNETH J. McLEOD

Regulation of Bone Remodeling Activity Through the Control

of Stress Generated Potentials

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KENNETH G. POTE

Temporal Expression and Spatial Distribution of the Major

Proteins in the Otoconia of Two Species of Vertebrate

DENNIS M. REINECKE

Plant Hormone Catabolism and Tropically Induced Asymmetric

Growth

LAWRENCE D. TALBOTT

Changes in Cell Wall Polymer Size as Indicators of Gravitropic

Response Mechanisms

DONALD B. THOMASON

Mechanisms of Decreased Actin Synthesis During Rodent

Non-weightbearing

NASA GRADUATE STUDENT RESEARCHERS PROGRAM

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ix

INTRODUCTION

THE NASA SPACE BIOLOGY PROGRAM

Thora W. Halstead

Life Sciences Division

Office of Space Science and Applications

National Aeronautics and Space Administration

Washington, D.C. 20546

Introduction

One of the major features of the physical environment of the surface of Earth is the constant

presence of the force of gravity. The phenomenon of near-weightlessness encountered on

spacecraft provides a unique biological research opportunity to study the importance of

gravity to life on Earth. Access to space provides an opportunity to manipulate gravity

from its normal value down to almost zero, effectively providing the full spectrum of

gravitational research capability for the first time. This capability, combined with the

stability and pervasiveness of gravity on Earth, its obvious impact on biological evolution,

and its continuing effect on the morphology, physiology, and behavior of living organisms, has led the Space Biology Program to concentrate its efforts and resources on

investigating the biological significance of gravity and advancing knowledge in the

biological sciences through the use of the microgravity environment of spaceflight.

Program Goals

The Space Biology Program includes both plant and animal research, and is dedicated to:

understanding the role of gravity and the effects of microgravity on biological processes;

determining the effects of the interaction of gravity and other environmental factors on

biological systems; and using the space environment as a tool to advance knowledge in the

biological sciences to improve the quality of life on Earth.

Program Scope

Space Biology includes the following research areas:

. Plant Biology. The primary areas of plant biology now demanding study

are: a) gravity perception and transduction, b) reproduction, c) growth and

development, d) transport, and e) metabolism and photosynthesis.

All tropic responses of plants to gravity begin with the perception of the

gravity vector. How gravity is perceived and transmitted to

responsive plant parts is just beginning to be understood. Research is

directed to the molecular level to determine the mechanism involved in

gravitropism and its relationship to phototropism.

Both sexual and asexual reproduction involve the mechanism of cell division

and the transmission of the genetic code through DNA-bearing chromosomes.

Aberrations in cell division and chromosomes have been observed in space,

and a careful study of the entire reproductive process of plants needs to

be conducted from germination through flowering, seed setting, and

senescence.

During growth, plants undergo a variety of qualitative changes. Their

development is controlled by hormones, which receive signals from the

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environment that influence all aspects of their metabolism. Research is

encouraged to determine the effect of microgravity on the growth and

development of plants, and to determine how the plants respond to gravity

during their life cycles.

Conduction of water, ions, and nutrients through the bodies of plants is

necessary for survival. Gravity can affect the polarization of cells and tissues

and thereby affect transport. The operations of membranes involved in

transport and their responses to gravity need to be understood.

.

Evidence from spaceflight suggests that metabolism and photosynthesis

are affected by the absence of gravity; amyloplasts and chloroplasts have been

reported to be depleted of starch in space-grown plants. Flight experiments

suggest that the structural molecules of lignin and cellulose are gravity

sensitive. These topics, as well as carbohydrate metabolism in general, are of

special interest, although total metabolic studies and investigations on the

effects of gravity and microgravity on photosynthesis are needed.

Gravi|v Sensing. Animals have developed gravity-sensing systems that

facilitate orientation and locomotion within Earth's environment. The nearweightless environmentof space=pr0videsa Unique research opportunity to

understand how gravity-sensing systems of different orgamsms have

developed, and how they process and transmit information. Specific

objectives are: __;_ :

a. To identify gravity-sensing organsand mechanisms, and to define

how they function on Earth and adapt to weightlessness;

b. To understand how gravitational information is transduced,

processed, transmitted, and integrated into a response; and

c. To understand the role of gravity in the development and evolution

of animal gravity sensing systems.

3. Cell andilDeve|0pmental Biology.

Ceil B_ology: Cells that are building blocks of systems (e.g., plant root

caps), individually functioning units of certain tissues (e.g., blood cells), and

unicellular organisms (e.g., paramecia) have been shown to be sensitive to

gravity. Research focuses on how gravitational loading influences cell

functions and the molecular mechanisms regulating them. The objective is to

determine at what level gravity affects cells and where and how they are

affected. Specific objectives are"

a. To investigate the role of gravity in maintaining normal cellular

and molecular function;

b. To determine how and where gravity affects cells;

c. To distinguish direct from indirect, extracellular, or systemic,

gravitational effects on cells;

d. To discriminate among the influences of cosmic rays, microgravity, and other spaceflight environmental factors; and

e. To assess the permanence of effects on cells exposed to microgravity.

....... DeVelopmental Bioiogyi Re'arch in this area examines the influence of

gravity and weightlessness on genetic integrity, reproduction, growth,

development, life span, senescence, and subsequent generations of animals.

Specific objectives are:

4
a.

b.

C.

To determine if organisms and multiple generations of organisms

can develop normally in microgravity;

To identify gravity-sensitive developmental stages, systems, and

mechanisms; and

To understand the effects of gravity and weightlessness on

gravity-sensitive developmental stages, systems, and mechanisms.

. Biological Adaptation. All biological species on Earth have evolved

under the influence of a gravity level of 1 g. In response to this force,

organisms have developed structures to withstand gravity loads, as well as

regulatory systems to function optimally. The objective is to understand how

gravity affects and controls the physiology, morphology, and behavior of

organisms; how gravity and other environmental stimuli and stresses interact

in this control; and the biological mechanisms by which living systems can

respond and adapt to an altered gravity environment, particularly the

environment found during spaceflight. Research also includes the use or

removal of gravity's physiological effects to explore biological problems.

Specific objectives are:

a. To understand the influence of gravity on the composition,

regulation, and function of biological support structures, with

special emphasis on biominerals;

b. To determine the role of gravity in regulating metabolism, metabolic

rate and products, fluid dynamics, and biorhythms;

c. To understand basic mechanisms of mineral and hormonal homeostasis and the role of calcium as a mediator of gravitational effects;

and

d. To identify the effects on organisms of the interaction of environmental factors (e.g., stress) with gravity, and determine the

mechanisms involved.

Focus of Program

The Program is focused on basic research that contributes to both NASA applied goals and

to improving the quality of life on Earth. Understanding how plants develop, metabolize,

and grow in space is essential for a space based Controlled Ecological Life Support System

(CELSS). Biomineralization and the mechanisms controlling the structural integrity of

bone and bone turnover are important to understanding osteoporosis on Earth and the

calcium loss and bone changes of spaceflight. The reconstruction and modeling of the

functional organization of mammalian gravity sensors are expected to lead to increased

understanding of how information is processed by biological systems.

Research Opportunities

While the research supported and encompassed by the Space Biology Program is primarily

ground-based, spaceflight experiments are an essential component of the program.

Spaceflight provides the validation for experimental hypotheses developed in ground-based

research, while gravitational experiments on Earth hone the questions, provide the

necessary baseline data, and develop spaceflight experimental protocol.

The experimental approach of the ground-based studies is to manipulate gravity on Earth

and develop weightless simulation models to: a) develop and test gravitational hypotheses,

b) identify gravity-sensitive biological systems and interacting environmental response

mechanisms, c) analyze biological systems and mechanisms known to be gravity-sensitive,

5
d) analyzeflight experimentdataanditerativelyexpandgroundresearchcapability,ande)

plananddesignfuturespaceexperiments.

Theresearchof the Space Biology Program is dependent upon several dynamic factors: the

requirements of NASA, the characteristics of flight experiment opportunities, the sensitivity

of specific biological systems to gravity, the scientific value of the research, the state of

knowledge and technology in the specific scientific areas, the interest of scientists in

studying the biological questions, and the availability of funds to support the research. Of

these factors, scientific interest in this field of biological science is paramount; for without

outspoken support from the scientific community, opportunities for space biological

research will remain static.

6
ACCOMPLISHMENT HIGHLIGHTS

SPACE BIOLOGY

ACCOMPLISHMENTS HIGHLIGHTS

PLANT

Gravitropism: Sensing

• Nature of the Receptor

The first demonstration that the hydrostatic sensing mechanism may be involved

with gravity perception was made during a study of cytoplasmic streaming in

Characean algal cells. (Leopold)

There is a severe reduction in gravitropic sensitivity in the stems of a tobacco

mutant with starch-deficient amyloplasts (starch-containing organelles thought

to be the gravity sensor). Although gravitropism is not entirely eliminated by

the lack of starch, this demonstrates that starch is necessary for complete

graviperception in tobacco and that amyloplasts function in gravitropic sensing

hypocotyls. (Sack)

Mutants of Arabidopsis that do not respond to gravistimulation have been

identified. One of the mutants is deficient in starch and one is not, thereby

providing evidence that a loss of starch does not necessarily lead to a loss of the

ability to perceive gravity. (Poff)

Phytochrome mRNA is spatially closely associated with columella cells in the

corn root cap, cells that are involved with graviperception, suggesting that the

phytochrome protein mediates some early steps in the sensing and processing of

the gravity stimulus. (Feldman)

One of the first attempts to use the technique of freeze-substitution on an entire

plant organ to avoid ice crystal damage has been successfully used to study cells

believed to be responsible for gravisensing in plant roots. (Sack)

The root cap is not necessary for the detection of mechanical and electrical

stimuli, which suggests that the cap may not be the only site for the perception

of the gravitational stimulus. (Evans)

Studies have revealed that root caps are unnecessary for root gravitropism in the

plant Tristerix aphyllus, the only advanced plant believed to have gravityperceiving cells located next to the root elongation zone instead of in the root

cap. (Moore)

• Electrical Potential

An endogenous electric voltage was measured across the stele-cortex junction of

corn shoots, which indicates that an electric potential exists across this region.

(Bandurski)

• The electric voltage between stele and cortex tissues changes rapidly following

change in shoot orientation with respect to gravity. (Bandurski)

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For seedlings turned horizontally, the lower side of the stele-cortex junction is

at a different potential than the upper side. This supports the hypothesis of a

voltage asymmetry between the upper and lower stele-cortex junctions involved

in regulation of gravitropism. (Bandurski)

Gravitropism: Transduction

The role of plant kinase proteins in transduction of the gravity stimulus has been

examined for the first time. One kinase, structurally homologous to animal and

microbial kinases involved in the transduction of many environmental stimuli,

has been identified. This kinase is located in the root cap and is not affected by

light. (Feldman)

Role of Calcium

Calcium gradients are thought to develop at the tip of roots during gravitropism

and to be essential for the gravitropic response. The first demonstration of a

calcium gradient in vivo was made with calcium microelectrodes. (Cleland)

The successful incorporation of the calcium-sensitive fluorescent dyefura-2 into

intact fern spores and germinating gametophytes will allow changes to be

measured in the level of cytosolic free calcium after phytochrome activation or

gravistimulation. This system can serve as a model for testing whether these

stimuli can induce changes in cytosolic free calcium. (Roux)

A calcium-binding calpactin-like protein from peas has been purified and is

being characterized. This protein appears to play a role in calcium-dependent

cell secretion, a process which is likely to be a major control point in plant

growth regulation. (Roux)

Enhanced levels of calmodulin mRNA and calcium-dependent protein

phosphorylation have been observed in tobacco protoplasts transformed with a

cloned plant calmodulin cDNA. (Poovaiah)

After transformation with sense constructs of calmodulin cDNA, protopiasts

from tobacco plants reveal up to a 50-fold increase in calmodulin mRNA;

however, the amount of calmoduiin protein never increases more than 2-fold.

This indicates that the amount of calmodulin in the plant cell is regulated

stringently during or after translation of mRNA. (Poovaiah) :

More calmodu!in and calmodulin-binding proteins are found in the tip than in

the base of corn roots, suggesting a coordinated regulation of calmodulin

expression and its target peptides. (Poovaiah)

• Application of gadolinium, which inhibits calcium channels, and other agents

that block the functioning of calcium inhibits gravitropism in the fungus

Phycomyces. (Edwards)

• Based on an analysis of calcium and pH in shoot ceil walls, cell wall calcium

appears to play no part in gravitropic curvature. (Cleland)

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• Role of IAA

Different concentrations of Ca 2+ applied internally or externally to isolated

zucchini plasma membrane vesicles had no significant effect on the transport of

IAA across the vesicles; this observation provides evidence against the

mechanism of coupled Ca2+/IAA flux. (Lomax)

Auxin-binding proteins were identified in isolated plasma membrane vesicles

from zucchini hypocotyls with the use of azido-IAA labeled polypeptides. The

lack of azido-IAA binding in membrane proteins of the agravitropic mutant of

tomato diageotropica (dgt) identifies an absent or altered auxin-binding protein

that is necessary for plant graviresponse. (Lomax)

cDNA clones encoding a protein believed to bind with the auxin IAA in tomato

plants are being developed. These clones will be used to study this protein and

its role in plant shoot elongation. (Rayle)

The enzyme indole-3-acetic acid oxygenase is stimulated by a lipid-soluble

cofactor which can be replaced by unsaturated fatty acids in the irreversible

oxidation of IAA. (Reinecke)

Gravitropism: Growth Response

A detailed analysis of growth patterns during the root graviresponse shows a

shifting pattern of localized, time-dependent, distinct growth rate changes,

suggesting that root curvature involves more than simply growth inhibition

along the lower side. (Evans)

Studies of cell wall autolysis in isolated walls, growing walls, and non-growing

walls suggest that cell wall autolysis may be independent of the process(es) that

cause cell wall loosening, and that it does not necessarily lead to wall

expansion. (Cosgrove)

Cell wail autolysis is qualitatively the same for epidermal (outer) and cortical

(inner) tissue walls, which is evidence against any distinctive autolytic

processes occurring in the epidermis. (Cosgrove)

Gravistimulated oat pulvini treated with gibberellic acid show significantly

higher glucan synthase activity than gravistimulated pulvini that are not given

hormone treatment. The increase in glucan synthase activity correlates well

with levels of [3-d-glucan (which is associated with cell elongation) found in the

lower halves of graviresponding oat pulvini. (Kaufman)

• A yeast invertase DNA that can be used to detect the invertase gene in oat

pulvinus tissue has been obtained. (Kaufman)

A polyclonal anti-yeast invertase antibody and four monoclonal antibody cell

lines bind soluble oat acid invertase. These antibody preparations are being

examined for specificity, and the specific probe will be used to localize invertase

at the subcellular level of oat pulvini and to probe the pathway of invertase

transport during graviresponse. (Kaufman)

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Plant growth in shootsappearsto consistof two components: a rapid acidmediatedresponseanda slowernon-acidresponse.(Cleland)

Experimentswith pH buffersandrootshavecastdoubton theideathat acidmediatedgrowthplaysarole in gravitropism.(Cleland)

Binding and blocking the activity of a 90kD wall isoperoxidase with a

monoclonalantibody inhibits thegrowth rate of coleoptile segmentsof corn

seedlings.Whetherthis indicatesthatperoxidaseis notresponsiblefor growth

rateinhibition but is neededfor normalcell wall extension,or if theantibodyantigencomplexcreatesstericproblemsthat nonspecificallyinhibit growth,is

not known. (Roux)

Certainperoxidaseisozymes(PODs)respondto short-termgravistimulationand

arebelievedto regulatetropisticbending.Two cationicPODsareexpressedon

theuppersideanda weaklyanionicPODisexpressedonthelower sideof corn

coleoptileswithin 30 minutesof gravistimulation. Secretionof this anionic

PODappearsto bedependentoncalcium. (Slocum)

In gravirespondingpeastems,cell wall polymersdecreasein sizeonthelower

sideandincreasein sizeontheuppersideof thebendingstem. Thesechanges

in polysaccharidesizeoccurprimarily in stemepidermaltissue.(Talbott)

Most of the polysaccharidesthat changesizeduring cell wall curvatureare

xyloglucanpolymers. As indicatedby electronmicroscopy,thesizeincreases

of xyloglucanaredueto theformationof a crosslinkednetwork,which maybe

dependentonion andauxin-inducedpH levels,althoughenzymaticactionis not

excluded.(Talbott)

Thereis aninitial lagperiodafter gravistimulationof plantdicot hypocotylsat

different IAA concentrationsbefore the growth rate of the bottom surface

increasessharply and upward curvature begins. As auxin concentration

increases,the lag time shortensuntil thereis nodetectablelag but a burstof

rapid growthof bothupperandlowersurfaces.(Salisbury)

Plant Metabolism

Theexactbondingpatternsof phenylpropanoidpolymersinto lignin in vascular

plantswasdetemainedwith nuclearmagneticresonance,providingthefirst

directevidencefor lignin-carbohydratebonding.(Lewis)

Environmental Factors

• Roots of corn seedlings respond not only to gravity and light, but also to

temperature. (Poff)

The rapid reduction in growth rate caused by mechanical stress in pea and

soybean seedlings suggests that the initial events are biophysical, possibly

involving reduction of turgor within the cell elongation zone. (Mitchell)

12
Circadian Rhythms

Neurospora cultures flown on STS-32 in January 1990 were examined for

changes in circadian rhythms during flight. The findings strongly support the

theory that these daily oscillations are endogenously generated -- that is, not

generated by environmental factors. (Ferraro)

Exposure to microgravity diminshed the period, phase, and amplitude of the

endogenous circadian rhythms of BND and CSP cultures of Neurospora.

(Ferraro)

Plant Cell & Developmental Biology

The somatic embryos in a carrot tissue culture system that form first multiply

initially as globular stage embryos only when the pH of the medium is allowed

to fall during the culture period. This is the first demonstration that morphology

of a higher plant system is reversibly influenced by changes in external pH.

(Krikorian)

At least six new proteins are produced by the spores of the sensitive fern

Onoclea sensibilis after the plant has been exposed to hypergravity through

centrifugation. A stronger gravity force results in a higher rate of synthesis of

these "shock" proteins. These findings suggest that specific changes in gene

expression occur in the plant spores as a response to hypergravity stress.

(Raghavan)

A cDNA library of mRNA sequences from Onoclea sensibilis spores has been

constructed. From this library cDNA clones specific for hypergravity stress are

now being selected. (Raghavan)

13
ANIMAL

Gravity Receptors and Neurophysiology

Chick embryos g,'own in space on the shuttle had elevated vestibular

thresholds when compared with Earth controls; they required stimulus

intensities nearly twice those as the controls to elicit vestibular responses. This

supports the hypothesis that gravity may play a role in determining vestibular

sensitivity. (Jones)

Studies of discharge patterns of otolith afferent in neonatal rats showed that

they have a high level of spontaneous discharge and show sensitivities to

sinusoidal and velocity steps similar to the adult. (Blanks)

In studies using the American bullfrog, the rate of change of the projection of

the gravity vector onto the utricular surface was found to be the stimulus for

the faithful representation of rotation velocity followed for small-amplitude,

low-frequency rotations about horizontal axes. (Lewis)

Three-dimensional reconstructions of terminal/receptive fields and of small

portions of the macular neural network in rats have identified four network

organizations that differ from one another in the kinds of nerve fibers

supplying the area, in terminal fields, and degree of collateralization. This

finding indicates that the intrinsic collaterals are significant in information

processing. (Ross)

Labelling of impulse initiation zones in rat vestibular nerve fibers revealed that

each vestibular nerve has but one impulse initiation zone, which is located just

distal to the heminode. (Ross)

The effect of sine wave linear motion on vestibular responses was studied in

rats using a linear sled. Linear motion was found to produce a small phasedependent modulation of vestibular response thresholds, latencies, and

amplitudes. (Jones)

The sequential development of graviceptor structures, particularly formation of

the touch-plate (part of the rhopalium which is involved in gravity reception in

the adult) was determined using Aurelia (jellyfish) ephyrae. (Spangenberg)

Studies of guinea pigs maintained on a high Na+/low K + diet and injected with

aldosterone provided evidence of a treatment capable of increasing levels of

inner-ear ATPase, and suggest that endolymphatic fluid/electrolyte balance may

be altered, at least transiently, by alterations of circulating aldosterone induced

by exposure to microgravity. (Kerr)

In chinchilla, there are differences in the afferent bouton innervation of type II

hair ceils in the central and peripheral zones of the crista. A higher

convergence ratio of hair cell synapses to afferent boutons in the central zone

of the crista implies that, anatomically the central zone appears to be specialized

for increased sensitivity. (Lysakowski)

The amino acid sequence of the 22kD otoconial protein from Xenopus laevis

was determined. (Pote)

14
Carbohydrateanalysisof theXenopus laevis 22kD otoconial protein revealed

that it is a glycoprotein, and not a glycosaminoglycan as was commonly

assumed. (Pote)

Development

Studies of synapse development in a slow clinostat focusing on the distribution

of cytoskeletal proteins that may affect acetylcholine receptor distribution reveal

that the ratio of vinculin (a cytoskeletal protein marker) to acetylcholine

receptors is larger than in control cells. This implies that cytoskeletal

organization in response to nerve contact takes place at least up to the level of

vinculin. (Gruener)

Study of fertilized amphibian eggs revealed that spawnings of eggs differ in

cytoplasmic rigidity which can be quantified. Eggs with a more rigid

cytoplasm display a higher survival rate in response to centrifugation, and a

lower rate of twinning. (Malacinski)

The neurotransmitters neuropeptide Y and dynorphin were localized in the

brain and pituitary gland of immature and mature swordtail fish, Xiphophorus

maculatus. The results suggest that these neurotransmitters play a role in

sexual maturation, and growth and differentiation. (Schreibman)

Signals which induce the amphibian ectoderm to become neural are traveling

from the blastopore lip area (the cells which initiate the cell rearrangements at

gastrulation) through the plane of the ectoderm. This differs considerably from

the old paradigm of neural induction and patterning. (Phillips)

A recombinant cDNA library was constructed of all active genes (RNA) in

amphibian cells previously shown to be sending signals for neural induction.

These will enable the study of the mechanisms for neural induction at the

molecular level. (Phillips)

Normal patterns of expression of subsets of the hsp90 and hsp70 members of

the heat shock protein gene family have unique cellular and temporal patterns

of expression in the developing mouse testis and the midgestation embryo.

(Wolgemuth)

The particular pattem of expression of the stress-inducible member of the HSP

70 family raises the possibility that cells in early stages of spermatogenesis

may be especially vulnerable to change in their environment. (Wolgemuth)

Bone and Muscle

In a study of long bones of rats flown on Cosmos 1887, vascular changes

were observed in the subperiosteal regions. This suggests that the

cardiovascular changes occurring during space flight may have a skeletal

component that significantly alters bone function. In the areas of bone where

severe vascular changes were seen, osteocytic death and significant

histochemical changes in the perivascular cells were also observed. (Doty)

15
Growthplatesofratsflown onCosmos2044showedincreasedheightandcell

numberin the proliferative zone,andreducedheight andcell numberin the

hypertrophy/calcificationzonecomparedwith unloadedcontrolgroups. There

wasevidencethat thesechangesmay bedependenton ageof animalduring

exposureto microgravity. (Duke)

Ratsflown onCosmos2044showedminimal bonegrowthduringthemission

eventhoughthebody massgainwas similar to thatof ratsflown on Cosmos

1887. Cosmos2044 rats were analyzedmorequickly postflight and were

threeweeksolderthantheCosmos1887animals.(Morey-Holton)

Minimal bonechangesoccurredin adultfemaleratsaftertwo or four weeks of

unloading, while muscle changes were similar to those reported in growing

rats. (Morey-Holton)

Rats exposed to simulated weightlessness for 3 days showed a 52% increase in

osteoblast progenitor cells and a 47% decrease in preosteoblast cells in the

tibial metaphysis. Ttiese results are similar to those seen in the periodontal

ligaments of animals exposed to both actual and simulated weightlessness.

(Roberts)

Muscle atrophy induced by hypokinesia in rats shows that fibers display "antigrouping" behavior: fiber types tend to avoid themselves and to seek

adjacencies with other fiber types, the grouping behavior seen in normal

muscular tissue. Atrophies caused by denervation show other types of fiber

grouping, suggesting that the atrophy produced by skeletal muscle unloading is

not neurologically mediated, but seems solely due to myogenic variables.

(Kasper)

Osteoblast-derived factor(s) stimulate bone resorption by maintaining the

viability of osteoclast precursors rather than by activating mature osteoclasts.

(Greenfield)

Evidence suggests that in muscle atrophy in rats due to unloading, membrane

receptor proteins are unaffected, although membrane enzymes show

differential activities. (Tischler)

Accelerated protein breakdown in unloaded muscle occurs mostly in the

cytoplasm and seems to be linked to increases in calcium-dependent

proteolysis. (Tischler)

New evidence shows that unloading results in an increased isoproterenol (betaadrenergic agonist) response to glycogen synthesis and cyclic AMP

production. (Tischler)

Neither IGF-1 nor IGF-2 (insulin-like growth factors, apparently produced by

bone through stimulation by growth hormone) prevent loss of bone mass due

to unweighting in rats. (Bikle)

In hypophysectomized rats, growth hormone sustains growth of bone, but

skeletal unweighting blocks this response. (Bikle)

In ratsl parathyroid hormone regulates collagenase gene expression, in part by

a transcriptional mechanism (Partridge).

16
A gene,p NG 3/7, thatencodesfor a spiculematrix protein in theseaurchin

S. purpuratus was isolated and begun to be characterized. A cue from the

extracellular matrix in the sea urchin's blastocoel is necessary to instruct the

primary mesenchyme cells to express this gene. (Wilt)

The rapid down regulation of protein synthesis that occurs at the onset of nonweightbearing is accompanied by a shift of polysome size toward larger

polysomes (more ribosomal subunits per mRNA). This indicates there is a

direct coupling of mechanotransduction to the control of gene expression in

muscle at the translation level in rats. (Thomason).

Micromass cultures of differentiating cartilage cells centrifuged at 2.9 g

developed cartilage 24 hrs prior to the appearance of cartilage in control

cultures. (Duke)

Cloned osteoblast cells, treated with dexamethasone to simulate the effects of

increased plasma cortisols, showed a decrease in new protein synthesis and

demonstrated that collagen type 1 accounts for approximately 97% of the total

collagen synthesis in this cell line. (Hughes-Fulford)

In addition, studies using the same cells mentioned above revealed that

glucocorticoids interfere with the cytoskeleton formation and assembly of

collagen matrix during mineralization. (Hughes-Fulford)

Results of studies of crosslink formation and collagen turnover in cultured

bone cells derived from osteoblasts of chicken calvaria indicates that the

presence of remodeling or collagenase activity appears to occur in culture,

affecting newly synthesized collagen molecules. (Landis)

Other evidence from these studies suggest that chemical crosslinks unique to

collagen are responsible for the aggregation of smaller fibrils to larger ones and

for the assembly of the extracellular collagen matrix as a whole. (Landis)

Regulatory Biology

Paraventricular lesions in rats decrease circulating angiotensinogen. For the

first time, it has been demonstrated that there is a neuroendocrine mechanism

regulating the circulating level of this component of the renin angiotensin

system. It appears that the lesions decrease secretion of the brain hormone that

stimulates the pituitary to secrete the hormone that, in turn, stimulates thyroid

hormone secretion. (Ganong)

The sympathetic nervous system plays a major role in the increase in renin

secretion produced by a low sodium diet. (Ganong)

Serotonin was effective in modulating the amplitude of one neural response in

rats exposed to hypergravity and control rats. This indicates that the decrease

in the number of receptors observed under hypergravic conditions is

insufficient to abolish a primary modulatory action of serotonin. (Horowitz)

Rats born and reared in hypergravity were able to maintain their core

temperature when cold exposed, compared with 1 g reared animals exposed to

17
cold in a2 g environment.Thisshowsthatanimalsacclimatedto hypergravic

fields haveanalteredthermo-controlsystem.(Horowitz)

Cell Biology

Demonstrated that fibronectin controls cell growth based on its ability to

support changes of cell and nuclear shape and that the effects of fibronectin on

cell form and function are mediated by specific binding interactions with cell

surface matrix receptors. These results suggest that cells may utilize a

biomechanical signal transduction system during growth control. (Ingber)

Showed that actin assembly is necessary for progression through G- 1 phase of

cell cycle. Demonstrated that fibronectin regulates actin filament assembly both

by binding to cell surface receptors and by physically resisting cell-generated

loads that are applied to those receptors. (Ingber)

18
PLANT PROJECTS

AN ATTEMPT TO LOCALIZE THE GRAVITY SENSING MECHANISM

OF PLANTS: EFFECT OF GRAVITY ON THE ELECTRICAL

PARAMETERS OF THE STELE-CORTEX JUNCTION IN CORN SHOOTS

Robert S. Bandurski

Mark F. Desrosiers

Department of Botany and Plant Pathology

Michigan State University

East Lansing, MI 48824

Description of Research

The first response of a plant to a gravity stimulus is membrane depolarization. This

response occurs within seconds and is a fundamental characteristic of a plant's response to

a stimulus, not only to gravity, but also to light. The second response is the development

of an asymmetric distribution of the plant growth hormone, indole-3-acetic acid (IAA).

The development of this chemical asymmetry appears to be the chemical transduction of the

bioelectric response. The hormone asymmetry then leads to an appropriate growth

response such that the plant grows back into its normal orientation with respect to gravity.

The objective of this research is to link the bioelectric response to gravity to the chemical

response, that is, the asymmetric distribution of growth hormone. How is a bioelectric

response transduced into a chemical asymmetry? We have attempted to explain this by

means of our potential-gating theory. Our measurements reported here of the

changes in potential difference between stele and cortex following a gravity

stimulus were predictable by the potential gating theory.

Our working hypothesis, the potential gating theory, is that the plant's internal bioelectrical

gradients serve as the sensors and transducers of the gravity response of plants. Shifts in

the plant's orientation with respect to gravity are followed by corresponding shifts in the

plant's internal bioelectric fields. The internal bioelectric fields control the voltage-sensitive

IAA-transporting channels between the plant's vascular tissue (stele) and the surrounding

cortical tissue (cortex) such that IAA accumulates on the lower side of the shoot. This

increased concentration of IAA on the lower side of the shoot leads to increased growth of

that side resulting in curvature of the shoot back to the vertical.

In previous years we tested this hypothesis by examining the effect of externally applied

potentials on growth, endogenous IAA concentration, and IAA transport from the seed

kernel to the shoot. A small, externally applied potential affected the growth rate of the

shoots in a polarity-dependent manner. The changes in potential-induced growth rate of the

shoots corresponded to changes in the endogenous IAA concentration in the plant shoot but

not to the transport of IAA from the seed kernel to shoot.

We also showed that an apoplastic transported dye would not penetrate the stele-cortex

barrier in response to the same externally applied potentials even though the electrical

resistance across the stele-cortex junction of the active growing regions was low. These

experiments suggest that the plasmodesmatal channels across the stele-cortex junction are

the controlling points for the transport of IAA from the vascular tissue to the cortical tissue

in the shoot and the controlling factor for the stele-cortex junction is the potential difference

across it.

__ .... Iq.,,_.NTIONA[E_,_ 21 PRE'CEDING PAGE BLANK NOT FILMED
Accomplishments

We have measured the endogenous potential across the stele-cortex junction at three

different regions of the shoot (the cell division region, the cell extension region, and a

mature region) as a function of the orientation of the shoot with respect to gravity.

(1) An endogenous voltage is measurable across the stele-cortex

junction.

(2) The endogenous voltage changes in response to a shift in the

orientation of the plant with respect to gravity. This is illustrated in Figure 1.

(3) For both vertical-to-horizontal and horizontal-to-vertical positional changes the

shift in the voltage across the stele-cortex barrier at the division region of the shoot was

significantly different from the other two regions of the shoot in both magnitude and

direction.

(4) For vertical-to-horizontal positional changes in the orientation of

the shoot, the voltage change across the lower stele-cortex barrier is

different from the voltage change across the upper stele-cortex barrier in

the regions of the shoot which show curvature (Figure 1).

(5) For a horizontal-to-vertical orientation change of the shoot, there was no

asymmetric potential difference between the upper and lower junctions between the stele

and the cortex (Figure 1).

(6) The electrical resistance of the stele-cortex junction is lowest in the regions of

the shoot where curvature occurs.

300

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150

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Figu,e Chang=intheelectr lpo=ntialbetweenthesteleandco.exintheme otyl of

Zea mays seedlings. Vertical lines indicate a change in orientation of the tissue.

22
Significance of the Accomplishments

Finding #1: A potential difference exists between the stele and cortex tissues.

Finding #2: The potential between the stele and cortex tissues changes within seconds

following a change in the sample's orientation with respect to gravity and continues to

change over 15 minutes. This potential change occurs throughout the length of the shoot.

Finding #3: The cell division region of the shoot behaves electrically in a manner different

from the extension and mature regions of the shoot.

Finding #4: The lower side of the stele-cortex junction is at a different potential than the

upper side of the stele-cortex junction. This supports the hypothesis of a voltage

asymmetry between the upper and lower stele-cortex junctions,when the shoot is in a

horizontal position, as being the controlling factor in regulating IAA transport out of the

vascular stele into the cortical tissues.

Finding #5: The lateral electrical asymmetry does not develop when the gravity vector is

aligned with the shoot.

Finding #6: The region of the shoot that curves in response to a lateral gravity stimulus has

a lower resistance between the stele and cortex tissues than the region of the shoot which

does not curve.

Publications

Bandurski, R.S. and Schulze, A. 1990. Preliminary report on growth hormone

concentration and distribution (GHCD) as flown on Atlantis, STS-34 (Abstract). Plant

Physiology 93(Suppl.): 80.

Bandurski, R.S., Schulze, A., Desrosiers, M., Jensen, P., Reinecke, D., and Epel, B.

1990. Voltage-gated channels as transducers of environmental stimuli. In: Inositol

Metabolism in Plants (ed. by D.J. Morr6, W.F. Boss, and F.A. Loewus). New York:

Wiley-Liss, p. 289-300.

Bandurski, R.S., Schulze, A., Jensen, P., and Desrosiers, M. 1989. Effects of

microgravity on growth hormone concentration and distribution in plants. In: Cells in

Space (ed. by J.D. Sibonga, R.C. Mains, T.N. Fast, P.X. Callahan, and C.M. Winget).

Moffett Field, CA: NASA Ames Research Center, p. 121-131. (NASA CP-10034)

Bandurski, R.S., Schulze, A., Jensen, P., Kowalczyk, S., Desrosiers, M.F., and Kesy,

J. 1989. Studies on the mechanism by which an asymmetric distribution of indole-3-acetic

acid in the maize mesocotyl is attained (Abstract). ASGSB Bulletin 3: 66.

Desrosiers, M.F. 1990. Electrical potential differences between stele and cortex in the

mesocotyl of seedlings of Zea mays (Abstract). Plant Physiology 93(Suppl.): 39.

Desrosiers, M.F. and Bandurski, R.S. 1989. Location of low resistance pathways in the

mesocotyl of corn seedlings (Abstract). ASGSB Bulletin 3: 26.

Jensen, P.J. and Bandurski, R.S. 1990. Characterization by NMR of tryptophan isolated

from seedlings of Zea mays grown on 30% deuterium oxide (Abstract). Plant Physiology

93(Suppl.): 69.

23
Kesy, J.M. andBandurski,R.S. 1990. Partialpurification andpropertiesof theenzyme

synthesizingindol-3-ylacetyl-myo-inositol(Abstract). Plant Physiology 93(Suppl.): 34.

Kowalczyk, S.W. and Bandurski, R.S. 1990. Hormone metabolizing complexing in Zea

mays endosperm (Abstract). Plant Physiology 93(Suppl): 34.

24
THE ROLE OF CALCIUM IN GRAVITY SENSING

Robert Cieland

Department of Botany

University of Washington

Seattle, WA 98195

Description of Research

The overall goal of this research is to determine how gravity is sensed in plants, and how

this information is transduced into changes in the growth rates of plant organs leading to

gravicurvature. There is convincing evidence that calcium might be required at two steps:

at the site of perception of gravity and at the site of the unequal rates of elongation growth

which actually results in the curvature. A current hypothesis states that a lateral

redistribution of calcium occurs at both sites in horizontal" stems and roots, and is essential

for the response. At the site of perception the calcium redistribution is believed to be

directly involved in the redistribution of the actual growth-controlling hormone. It has been

suggested that at the site of curvature an accumulation of calcium in the walls of the future

concave side stiffens the cell walls and inhibits acid-mediated wall extension. Direct

evidence for both of these ideas is limited. Our objective is to provide evidence as to

whether changes in extracellular calcium are directly involved in either the perception or the

reaction stages of gravitropism.

Our research during the past year has been directed along three main lines. The first is to

determine the amount and timing of the gradient of apoplastic calcium that develops at the

tip (the site of perception) of maize roots. This is followed by studies in which the gradient

has been eliminated, in order to determine whether it is needed for graviperception.

The second direction has been to determine the relationship between the wall-bound

calcium, free apoplastic calcium, wall pH and wall extensibility in soybean hypocotyl

tissues. In the first phase of this work the role of wall-bound calcium as a stiffening agent

in cell walls has been determined by measuring the effect of calcium removal on the

facilitated creep of isolated cell walls. In the second phase, the apoplastic calcium

concentration has been measured with calcium microelectrodes, and then the pH that must

have existed in the wall in order for that apoplastic calcium concentration to be in

equilibrium with the bound calcium has been determined.

The third project is a reassessment of the role of apoplastic acidity in the control of cell

elongation. Current theories, backed by considerable evidence, indicate that in the

curvature zone differential rates of auxin-induced proton excretion lead to different rates of

acid-induced wall loosening and thus growth. We have begun to reassess this concept,

with the goal of determining to what extent auxin-induced growth is acid-mediated and to

what extent a second, acid-insensitive process is involved.

Accomplishments

(1) A calcium microelectrode has been used to demonstrate that a gradient of

apoplastic calcium does develop at the tip of corn roots during graviperception. The

apoplastic calcium concentration is about 5 mM at the tips of vertical corn roots, but begins

to change within 10 min after roots are placed in a horizontal position. The calcium

concentration on the upper side decreases to one-half while on the lower side it increases

two-fold. The result is a four-fold gradient of calcium across the gravisensing region.

These data demonstrate directly, for the first time, that there is a gradient in

25
apoplastic calcium that develops across the graviperception zone of

horizontal roots.

(2) The role in graviperception of gradients of calcium, indoleacetic acid and

hydrogen ions across the root apex has been examined by fixing small lengths of capillary

tubing onto the tips and filling them with solutions. When the tubes are empty or filled

with water, gravicurvature is normal. When filled with a solution of the weak calcium

chelator dinitro-BAPTA and 1 mM Ca 2+ so as to equalize the apoplastic calcium

concentration across the tip, curvature is severely inhibited. This demonstrates that the

calcium gradient that develops across the perception zone of horizontal

roots is essential for the gravitropic response.

When the tubes are filled with 10 I.tM IAA, gravitropic curvature is also blocked. This

provides direct evidence that a gradient of auxin across the graviperception zone

is necessary for curvature. On the other hand, addition of either a pH 6.5 buffer, to

maintain a constant neutral pH, or fusicoccin, to maintain a constant acidic apoplastic pH,

has no effect on curvature. A gradient of acidity, if it occurs, is not important

in the initial steps of gravitropism.

(3) Once an asymmetrical distribution of growth regulators occurs in the

graviperception zone, it must be transmitted back to the reaction zone in roots. Evans and

co-workers have argued that this transmission occurs in the epidermal layers from

experiments where removing a girdle of epidermal cells seemed to prevent curvature

beyond the girdle. We have reinvestigated this, and have shown that even when the whole

epidermis, from the cap through the elongation zone is removed, gravicurvature still

occurs. Our data demonstrate that the epidermal cells are not required for root

gravitropism, and that the growth regulator does not have to pass through

this cell layer.

(4) Calcium microelectrodes have been used to measure the apoplastic free calcium

activity in soybean hypocotyls. The values obtained vary from hypocotyl to hypocotyl, but

the average value is about 10 I.tM. There are no comparable published values for apoplastic

free calcium from other systems.

(5) A study has been made as to the conditions of pH and free Ca 2+ which are in

equilibrium with the wall-bound calcium of soybean hypocotyl walls. It has been shown

that if the free Ca 2+ is 150 t.tM, the apoplastic solution must have a pH of about 3.5. This

pH is considerably lower than that normally accepted for the apoplastic solution, i.e., a pH

of 5.0 to 6.5. There are two possible explanations. The first is to assume that the wallbound calcium is not in equilibrium with the apoplastic free calcium. There is no reason to

believe that this could be true. The second is to assume that the uronic acids exist as part of

the Donnan Free Space (DFS), whose pH is different from that of the Water Free Space

(WFS). Theoretical studies have suggested that the pH of the DFS can be as much as 2.5

pH units lower than that of the WFS, due to protons trapped in this space to neutralize the

uronic acids.

(6) If the pH of the DFS is significantly lower than that of the WFS, addition of

exogenous Ca 2÷ would be expected to raise the pH of the DFS and lower that of the WFS,

since Ca 2+ Would substitute for H + as the ion balancing the fixed negative charges. This,

in turn, would inh_it aCid-mediated cell elongation. To provide a partial test of this idea,

the effect of exogenous calcium on the WFS pH of soybean hypocotyl sections was

determined using a newly developed assay in which the rate of uptake of a weak acid,

benzoic acid, is used to measure the pH at the external face of the plasma membrane (the

26
wvs pH).

1 pH unit.

favor.

We have shown that exogenous Ca 2+ causes a decrease in WFS pH of almost

While this does not prove the DFS concept, it provides strong evidence in its

(7) It is well established that in tissues in which growth is promoted by auxin,

auxin promotes proton excretion into the apoplast, and exogenous acid has the ability to

cause these cells to elongate in the absence of auxin. The acid-growth theory states that

auxin-induced elongation requires this apoplastic acidification, and that wall loosening only

occurs under acidic conditions. The most significant evidence against this theory is that

growth induced by exogenous acid only persists for about 2 hours while auxin-induced

growth can persist at a constant rate for many hours. Several investigators have suggested,

without direct evidence, that auxin-induced growth consists of two sequential reactions:

initially, via an acid-growth mechanism and, subsequently, via a completely different

auxin-mediated reaction. If so, it should be possible to show that auxin-induced growth

should require an acidic apoplast during the first 2 hours but then proceed equally well at

more neutral pHs thereafter. Amazingly, this simple test has never been carried out. We

have now tested this with Avena coleoptiles, and find that auxin-induced growth can best

be explained as consisting of two simultaneous processes: the acid-growth response during

the first 2 hours and a second process whose pH optimum is 5.5-6.5 and which persists

for up to 24 hours. These results are being refined and confirmed; if they are correct, the

ideas concerning the mechanism of auxin-induced growth, including gravitropism, will

have to be modified accordingly.

Significance of the Accomplishments

Three major conclusions have arisen from this year's research. The first is that gradients of

calcium do develop across the graviperception zone of the root during normal gravitropism

and are essential for the gravitropic response. It has been shown previously that applied

gradients of calcium cause curvature, but this is the first demonstration of an in vivo

calcium gradient.

The second conclusion is that calcium does not act as a major wall stiffening agent in

hypocotyl walls, and that increases or decreases in wall-bound calcium cannot be a part of

any mechanism of gravitropism. There is simply no evidence for either an in vivo

redistribution of calcium in the actual zone of curvature, or a role of extracellular calcium in

the curvature itself.

The third conclusion, which is still somewhat tentative but potentially of great importance,

is that auxin-induced acidification is only involved in the initiation of growth and that a

second, acid-insensitive process is responsible for prolonged auxin-induced growth. This

has considerable implications for gravitropism, which occurs over short time periods. Of

particular interest will be a determination as to whether the gravitropic curvature is due to

the acid-mediated growth or to the non-acid growthmechanism.

Publications

Bj6rkman, T. and Cleland, R.E. 1989. Changes in calcium activity in gravity sensing

maize roots (Abstract). ASGSB Bulletin 3: 98.

Bjrrkman, T. and Cleland, R.E. 1990. Changes in extracellular calcium activity during

gravity sensing in maize roots (Abstract). Plant Physiology 93(Suppl.): 39.

27
Bjrrkman, T. andCleland,R.E. 1990. Is theepidermisessentialfor root growthcontrol?

(Abstract) Plant Physiology 93(Suppl.): 129.

Cleland, R.E., Bagshaw, S., Taylor, D., and Virk, S.S. 1989. Calcium redistribution,

wall extensibility and gravitropism (Abstract). ASGSB Bulletin 3: 96.

Cleland, R.E. and Virk, S.S. 1989. The role of wall pH and wall calcium in the control of

cell wall extension. In: Plant Water Relations and Growth Under Stress (ed. by M.

Tazawa, M. Katsumi, Y. Masuda, and H. Okamoto). Osaka: MYU K.K., p. 283-290.

Daniel, S.G., Rayle, D.L., and Cleland, R.E. 1989. Auxin physiology of the tomato

mutant diageotropica. Plant Physiology 91: 804-807.

de Boer, A.H., Watson, B.A., and Cleland, R.E. 1989. Purification and identification of

Fusicoccin binding pro!ein from oat root plasma membrane. Plant Physiology 89: 250-

259.

Sandstrom, R.P. and Cleland, R.E. 1989. Comparison of the lipid composition of oat

root and coleoptile plasma membranes. Plant Physiology 90:1207-1213.

Sandstrom, R.P. and Cleland, R.E. 1989. Selective delipidation of the plasma membrane

by surfactants. Plant Physiology 90: 1524-1531.

Taylor, D.P. and Cleland, R.E. 1990. Inhibition of auxin- and fusicoccin-induced

elongation by calcium is transitory in soybean hypocotyls (Abstract). Plant Physiology

93(SuppL): 11.

28
MECHANISM OF DIFFERENTIAL

GRAVITROPISM

Daniel J. Cosgrove

Department of Biology

Pennsylvania State University

University Park, PA 16802

GROWTH DURING STEM

Description of Research

Gravitropism offers us a valuable opportunity to learn about the interaction of gravity with

growth processes and to gain insight into the cellular, physical, and molecular processes

that contribute to and govern plant growth. When plants are placed in a horizontal position,

their stems typically grow upright and their roots grow downwards. This gravitropic

bending occurs because of a growth redistribution on the upper and lower sides of the

organ. We are using young cucumber (Cucumis sativus L.) seedlings to investigate the

mechanism of gravitropism because they show a very vigorous response: after a lag of

about 10 min the upper stem surface ceases growth entirely whereas the lower surface

doubles or triples its expansion rate. Subsequently, this growth asymmetry reverses itself.

Since the stem diameter is only 1.5 mm, this means that ceils very close to one another

respond oppositely to gravity. Elucidation of this rapid growth response will likely tell us a

great deal about how plants integrate sensory stimuli and use this information to control

their growth.

When intracellular pressure, or turgor pressure, was measured with the pressure probe, we

found only a negligible, passive change on the two sides of the stem. This demonstrated

that cell expansion was not modulated by altering cell turgot or other related hydraulic

properties of the growing cells. By stress-relaxation analysis on growing tissues and on

isolated cell walls, we found that the wall on the upper stem was not physically stiffened

during the period when its growth was inhibited. Rather, the active process of wall

loosening was inhibited. At present the nature of this loosening process is uncertain.

We are focusing on the working hypothesis that enzymatic modification of the wall

regulates wall loosening, and that the activity of these hypothetical enzymes is regulated

during the gravitropic response, perhaps through changes in the ionic (Ca 2+, H +)

environment of the wall or through some covalent modification. In this past year we have

attempted to characterize some wall enzymes in the cucumber seedling which may be

responsible for wall relaxation. Attention was focused on enzyme activities that break

down the polysaccharides of the wall matrix.

Accomplishments

(1) Cell walls from the growing region of cucumber seedlings were found to

exhibit autolytic activities. Release of monosaccharides, oligosaccharides and uronic acids

from wall fragments was measured by anion exchange chromatography and pulsed

amperometry. A number of sugars were released in a time-dependent fashion.

(2) The pH-dependence of wall autolytic activities was found to vary

among the sugars released. The maximum rates of autolysis were found to

be at pH 5.5 or 6.5. In comparison, long-term extension of isolated

cucumber walls had a much lower pH optimum (about 4).

29
(3) The pattern of autolytically released sugars was the same from

epidermal walls as for inner cortical tissue walls.

(4) The autolytic process was relatively insensitive to most added

cations (Al +3, Cu +2, Ca +2, K "1 and others), with the exception of H + and

Hg +2. In contrast, extension of isolated walls and growing segments were

sensitive to some of these ions.

(5) Basal wall fragments also exhibited autolytic activity, but the pattern of sugar

release differed from that of the apical growing walls.

(6) A large number of proteins are associated by ionic and hydrophobic bonding to

isolated cell walls. This was demonstrated by extracting isolated walls with 3M LiCI or

with phenol/acetic acid/water, and separating the solubilized proteins by SDS gel

electrophoresis_

Significance of the Accomplishments

Finding #1 confirms and enlarges upon previous studies, showing that walls from growing

plant tissues indeed contain substantial autolytic activities. Our analytical technique has

high sensitivity (10 pmoles of glucose can be measured) and can resolve nearly all of the

wall fragments released by autolytic enzymes. Thus we have a powerful system for

examining wall autolysis and how it may be affected during gravitropic responses.

Finding #2 suggests that wall autolysis shows a distinctly different pH dependence than (a)

endogenous growth during gravitropism, and (b) extension of isolated walls. Finding #4

extends this observation to several cations. The implication is that growth and wall

extension in vitro are not rate-limited by wall autolytic reactions. It may be that wall

autolysis is independent of the process(es) causing wall loosening and relaxation. This is

reinforced by Finding #5, showing high autolytic activity in nongrowing walls. This

shows that wall autolysis does not necessarily lead to wall expansion.

Finding #3 shows that autolysis is qualitatively the same for epidermal and inner tissues.

This is important to know because the epidermal wall is hypothesized to be the major

restraint to stem growth and the major site of action of auxin and gravitropic growth

responses. We find no evidence for distinctive autolytic processes in the epidermis.

Finding #6 indicates that plant walls may be enz.ymatically complex mixtures. The results

also provide a basis for further investigations into possible alterations of wall enzymes

during gravitropic responses.

Publications

Cosgrove, D.J. 1989. Dynamics of relaxation in growing tissues: Examples and

interpretations. In: Plant Water Relations and Growth under Stress (ed. by M. Tazawa,

M. Katsumi, Y. Masuda, and H. Okamoto). Osaka: MYU K.K., p. 309-316.

,7

Cosgrove, D.J. 1989. Gravitropism in plants: Hydraulics and wall growth properties of

responding cells. In: Cells in Space (ed. by J.D. Sibonga, R.C. Mains, T.N. Fast, P.X.

Callahan, and C.M. Winget). Moffett Field, CA: NASA Ames Research Center, p. 153-

156. (NASA CP-10034)

30
Cosgrove,D.J. 1990. Gravitropismof cucumberhypocotyls: biophysicalmechanismof

alteredgrowth. Plant, Cell and Environment 13(3): 235-241.

Cosgrove, D.J. 1990. Rapid, bilateral changes in growth rate and curvature during

gravitropism of cucumber hypocotyls: implications for mechanism of growth control.

Plant, Cell and Environment 13(3): 227-234.

Cosgrove, D.J. 1989. Wall relaxation and water uptake in growing cells. In: Plant

Membrane Transport: The Current Position (ed. by J. Dainty, M.I. DeMichelis, E. Marre,

and F. Rasi-Caldogno). Amsterdam: Elsevier Science Publishers, p. 567-570.

Cosgrove, D.J. and Hedrich, R. 1990. Stretch-activated cation, anion and calcium

channels coexist in guard cells (Abstract). Plant Physiology 93(Suppl.): 17.

McQueen-Mason, S., Durachko, D.M., and Cosgrove, D.J. 1990. Wall-associated

protein that induces extension of qell walls (Abstract). Plant Physiology 93(Suppl.): 75.

Schmalstig, J.G., Kao, T.-H., and Cosgrove, D.J. 1990. Gibberellic acid effects on gene

expression during stimulation of stem elongation (Abstract). Plant Physiology 93(Suppl.):

98.

Spalding, E.P. and Cosgrove, D.J. 1989. Large plasma-membrane depolarization

precedes rapid blue-light-induced growth inhibition in cucumber. Planta 178:407-410.

Spalding, E.P. and Cosgrove, D.J. 1990. Mechanism of blue-light-induced membrane

depolarization (Abstract). Plant Physiology 93(Suppl.): 30.

Stahlberg, R. and Cosgrove, D.J. 1990. Rapid alterations in stem growth rate and surface

electric potentials upon excision (Abstract). Plant Physiology 93(Suppl.): 74.

Taylor, A. and Cosgrove, D.J. 1989. Gibberellic acid stimulation of cucumber hypocotyl

elongation. Plant Physiology 90:1335-1340.

31
THE GRAVITATIONAL RESPONSE OF PHYCOMYCES WITH RESPECT

TO GADOLINIUM AND FACTORS AFFECTING CALCIUM FUNCTION

Kathryn L. Edwards

Biology Department

Kenyon College

Gambier, OH 43022

Description of Research

One of the simplest systems which detects and responds to gravity is the large singlecelled, upright, reproductive sporangiophore of the terrestrial fungus, Phycomyces. As a

single cell it contains all mechanisms for the detection of gravity to the asymmetrical cell

wall growth response. This system is being considered because of the appealing

possibility that a number of molecular mechanisms for gravitropism may be evolutionarily

preserved and be common to a wide variety of cell wall-bound organisms, including the

higher plants.

Preliminary studies indicated that the lanthanide gadolinium, which has been shown to

inhibit gravitropism but not linear growth in higher plants, has a similar effect on

Phycomyces gravitropism. The focus during the last year has been to describe the uptake,

movement, and location of gadolinium in the sporangiophore to more closely describe the

effect of gadolinium on the kinetics of gravitropism, and to preliminarily examine the

effects of calcium modulators on gravitropism. Much of this work has been done in

conjunction with two Honors Research students, Bill Joiner and Tawny Stecker.

Accomplishments

(1) The Phycomyces sporangiophore has a chitinous cell wall covered with a

cuticle to reduce water loss. These structures are minimalized in the growing zone at the

apex just beneath the sporangium. Radiolabelled gadolinium applied in the agar medium on

which the fungal mycelium is grown or applied as a thin layer of liquid to the top of the

agar medium moves slowly (days) into the sporangiophore itself. Gadolinium is readily

taken up into sporangiophores when applied to them directly, either by: (a) inverting intact

sporangiophores and submerging them in solution or (b) applying the gadolinium to the

apical region by agar block.

(2) Radiolabelled gadolinium is incorporated largely into cytosolic and membrane

fractions of the sporangiophore and is tightly though not covalently bound. Gadolinium

is associated with precipitable proteins in both of these fractions and to one

class of large molecular weight proteins in the cytosol. Since gadolinium is

highly charged it is known to be generally "sticky," but we find that tightly, held gadolinium

appears to be associated with specific proteins. The nature of these proteins is not known.

Further characterization is underway.

(3) The sporangiophore displays a large number of tropistic movements of which

gravitropism is one of the 'weaker' resp.onses. It is over-ridden by phototropism with

adequate light, for example. Examinauon of sporangiophore kinetics requires careful

technique under infrared conditions, yet among individuals there is considerable variability,

which is enhanced by many physical disturbances (e.g., air currents, touch, heat). For

these reasons we initially wanted to avoid physically dipping sporangiophores into

gadolinium chloride solutions to study its effects on the kinetics of gravitropism. Instead,

vertical sporangiophores produced from 5-7 day-old mycelia grown on agar containing

32
low, 1-7I.tm,gadoliniumwereorientedhorizontally,andthekineticsof gravitropismwere

examined.Gadolinium,evenat 1 _tM,consistentlyextendedthelatentperiodprior to the

onsetof asymmetricupwardcurvatureby 2.5hr. Controlsinitiatedgravicurvatureafter30

min reorientationto gravity while gadoliniumexperimentalsbeganrespondingafter 3 hr.

The curvatureratewas reduced20-40%in thepresenceof gadolinium,aswas thelinear

growth rate. Thus, long term exposure to gadolinium affects both gravity

detection/transduction/growth and linear growth. Short-term exposure of

sporangiophores to gadolinium may, as in higher plants, similarly eliminate the effect of

linear growth rate. This possibility is being examined. However, it is clear that

gadolinium does affect gravity perception/transduction.

(4) Preliminary study of various calcium effectors on sporangiophore gravitropism

was pursued by application in small 1-3 mm agar blocks to one side of the sporangiophore

apex. Blocks containing gadolinium chloride, EGTA (external calcium

chelator), BAPTA/AM (internal calcium chelator), and Compound 48/80

(calmodulin inhibitor) induced curvature towards the block when applied to

vertical sporangiophores; they were not affected significantly by control agar blocks.

BAPTA/AM and C48/80 were the most effective agents and were able to override upward

gravitropism when applied to the lower surface of horizontal sporangiophores. We suggest

that internal calcium and calmodulin may serve in stimulating wall growth and asymmetric

curvature.

Significance of the Accomplishments

Phycomyces sporangiophores grown on medium containing gadolinium at low micromolar

concentrations show reduced ability to detect and transduce gravitational signals and have a

slower curvature rate. Gadolinium has been shown to bind to proteins in the cytosol and

membranes of the sporangiophore. These proteins may be involved in both

sensing/transduction and growth. The sensing/transduction mechanism theoretically could

entail stretch-activated ion channels or voltage-gated calcium channels which have been

shown to be inhibited by gadolinium in frog oocytes (Yang and Sachs, 1989) and hybrid

neuroblastoma-glioma cells (Docherty, 1988), respectively. Asymmetric growth could be

realized through the involvement of voltage-gated calcium channels and calmodulin, for

which gadolinium competes with calcium, resulting in inhibition (Buccigross et al., 1986).

Calmodulin is present in Phycomyces and is known to be similar to that in animals. The

results from the agar block experiments suggest involvement of calmodulin and internal

calcium, in at least the regulation of growth. We are presently undertaking the

characterization of stretch-activated and calcium ion channels in sporangiophore tonoplast

and plasma membrane vesicles to better our understanding of gravity detection and signal

transduction.

33
ROLE OF CALCIUM

GRAVITROPISM

Michael L. Evans

Department of Botany

Ohio State University

Columbus, OH 43210

IN SIGNAL TRANSDUCTION IN ROOT

Description of Research

This research is directed toward understanding the influence of gravity on plant growth --

in particular how roots become oriented with respect to gravity. (gravitropism). The

detection of gravity occurs at the tip of the root while adjustments xn growth rate occur in

the growing region about 0.5 cm behind the tip. There is evidence that calcium within the

cap plays a role in linking gravity detection to asymmetric distribution of the growthinhibiting hormone, auxin, in the growing region of the root. We are interested in the

pathway of movement of signal from the cap to the elongation zone. We are also interested

in characterizing the signal detection function of the root cap for signals other than gravity,

e.g., pressure or electrical gradients. It is likely that these detection mechanisms have

features in common, and the elucidation of these features will help in the understanding of

stimulus/response coupling in plants.

Our research has centered on the following: (1) Measurement of calcium levels in

protoplasts from cells of the elongation zone and cap of maize roots, and testing whether

treatments known to affect gravitropic sensitivity also affect cell calcium; (2) Testing the

effects of antibodies to auxin and abscisic acid (a second candidate for the hormonal

mediator Of gravitropism) on the longitudinal transmission of the gravitropic signal. In

these experiments we remove entire rings of cortical tissue from around the root at various

positions between the cap and elongation zone, fill the gap with agar or with agar

containing antibody, and then determine the effect of these treatments on gravitropism. In

related experiments, we measure the relative responsiveness of different cell layers to auxin

using longitudinally bisected roots; (3) Use of a computerized video analysis system to

determine the detailed kinetics of growth changes during gravitropic curvature; and (4)

Analysis of the effects of two different stimuli (mechanical impedance of root elongation

and imposition of an electrical field across the root tip) on root growth patterns. The

objective is to compare detection and response to these signals with the graviresponse. It is

likely that these detection/response systems have features in common.

Accomplishments

(1) We isolated protoplasts from both the cap and the elongation zone of maize

roots, loaded them with the calcium indicator dyes indo-1 and fura-2 and measured

cytoplasmic free calcium. The cytoplasmic free calcium level is much higher in protoplasts

from the elongation zone than in protoplasts from the root cap. Preliminary results indicate

that auxin has no effect on the cytoplasmic calcium content of protoplasts from root cap

cells. We also found that transient plasmolysis of the root cap prevents gravityinduced calcium redistribution across the cap but does not prevent

gravitropic curvature.

(2) Earlier, we found that when we remove a narrow (0.5 to 1 mm) ring of cortical

tissue from the root within the apical portion of the elongation zone, gravitropic curvature

occurs apical to the girdle but not behind it. The signal will pass a girdled zone filled with a

34
diffusion mediumsuchasagar. We haveobtainedantibodiesto free auxinand abscisic

acidandplanto testtheirability to blocksignalmovementhroughanagar-filledgirdle.

(3) We havemadeprogressin the reliability andaccuracyof acomputer-based

videodigitizer systemfor automatedanalysisof growthduringgravitropism.We usedthe

system to obtain a detailed characterization of localized growth rate changes in

gravirespondingmaize roots (Figure 1). The results reveal large time-dependent

changes in localized growth rates during the gravity response. In

particular, the pattern of growth rate distribution changes dramatically

during the response from an early phase with rapid growth on the top and

zero growth on the bottom to a later phase with the opposite pattern (rapid

growth on the bottom and zero growth on the top). The analysis also reveals a shift in the

growth zone toward the apex, especially along the upper side.

2° tUI t

E : ' ' '

v , topside: :

>, I ._- J--e-e'-..._ , ,.P -- -o ,

R lo _,. . _ , o :

. , , _o ,, •---"---;'-_--o.

8_ , o o\ _ - o.--,

r , , : ,\ /i.,;.o. .... - :

I .... .\ i '

0 ol ! i • • I i

d "0 u o 0..O' u

| II n • ._0 I" - 0 "._.1_7..-.--. _\ / _ I , ,

I _4: _-o_Op--_oo : _oa"-_ - 3oo 4oh

| t_ ' bottom side '

-0.4L _" ' :

Timei(min)

: l

I

, i

I

I II III IV V

0

l

u

I

f

Vl

Figure 1. Time-dependent variation of total rate of extension (velocity, mm h"l) on the upper

(convex surface of bend) and lower (concave surface of bend) side of a gravistimulated maize

root. Convex surface of bend = solid line. concave surface of bend = dashed line. The

curvature response is arbitrarily divided into six phases indicated I-VI.

(4) We have tested the ability of roots to respond to mechanical pressure and to

electrical gradients and compared these responses to the gravitropic response. Roots

respond to an electrical gradient by curving strongly toward the positive (Figure 2). They

respond to mechanical resistance by increasing their growth rate and, if the

resistance is great enough, initiating curvature resulting in growth around the

obstacle. Both the responses to mechanical resistance and to electrical fields

occur even when the cap is removed from the root. The response to gravity

requires the cap.

35
RESPONSE OF MAIZE ROOTS TO AN ELECTRICAL GRADIENT

Figure 2. Electrotropic curvature of the primary root of a maize seedling (cv. Merit).

Curvature is toward the positive electrode. Roots with the cap removed respond strongly

to electrical fields but do not respond to gravity stimulation.

Significance of the Accomplishments

Finding #1: Calcium has been proposed to mediate transduction of the gravitropic signal.

The finding that the cytoplasmic calcium level is unusually low in root cap protoplasts

suggests that these cells may be especially sensitive to calcium as a signaling ion. Our

observation that transient plasmolysis prevents gravity-induced calcium redistribution but

not curvature indicates that, although calcium is necessary for the gravitropic response, a

gradient of calcium may not be critical.

Finding #2: Last year we reported that the gravitropic signal can pass across a region of the

elongation zone from which a ring of cortex has been removed, provided the gap is filled

with a diffusion medium such as agar. This system provides a convenient system for

testing the nature of the signal transmitted from the cap to the elongation zone. The planned

experiments in which antibodies to auxin and abscisic acid will be incorporated into the

agar should provide a specific test of whether the signal moves in the form of one of these

two hormones.

Finding #3: The detailed analysis of growth patterns during the gravitropic response

shows that curvature cannot be attributed to a simple inhibition of growth along the lower

side as proposed by some models. Not only does the growth zone itself shift, but the

gradient of growth rates reverses completely at least once during the gravitropic response.

This information is critical to evaluation of models of gravitropism. Our data indicate that

multiple factors control growth rate distribution during the response. This makes it

especially important to investigate the role of changes in sensitivity to hormones (or to the

gravity stimulus itself) during the response.

Finding #4: The fact that the root is sensitive both to electrical stimulation and to

mechanical resistance provides two related stimuli that can alter root growth performance.

In both cases growth rates are affected and curvature is induced (only above a certain

threshold for mechanical impedance). It is particularly significant that both of these

responses also occur in decapped roots, i.e., the root cap is not necessary for the detection

of either of these stimuli. The fact that cells of the root other than from the cap are highly

sensitive to environmental stimuli suggests that they may also participate in gravity sensing.

36
This raisesthe possibility that,althoughthecapis necessaryfor thegravity response,it

maynotbethesolesiteof gravityperception.Characterizationof theresponseto electrical

fields andmechanicalimpedancemayprovide importantcluesconcerningthe natureof

gravitydetectionbyplants.

Publications

Bodo, K., Young, L.M., and Evans, M.L. 1989. Downward auxin transport polarity

across gravistimulated roots is cation dependent (Abstract). ASGSB Bulletin 3: 79.

Evans, M.L., Kiss, H.G., and Ishikawa, H. 1990. Interaction of calcium and auxin in

the regulation of root elongation. In: Calcium in Plant Growth and Development (ed. by

R.T. Leonard and P.K. Hepler). Rockville, MD: American Society of Plant Physiologists.

Ishikawa, H. and Evans, M. 1990. Effects of IAA and calcium on maize root elongation:

Time-dependent changes in growth patterns (Abstract). Plant Physiology 93(Suppl.): 70.

Ishikawa, H. and Evans, M. 1990. Electrotropism of maize roots: Relationship to

gravitropism (Abstract). Plant Physiology 93(Suppl.): 79.

Ishikawa, H., Yang, R.L., and Evans, M.L. 1989. Electrotropism of corn roots

(Abstract). ASGSB Bulletin 3: 126.

Kiss, H.G. and Evans, M.L. 1990. Cytoplasmic calcium measurements of root cell

protoplasts of Zea mays using Fura and Indo (Abstract). Plant Physiology 93(Suppl.): 39.

Kiss, H.G. and Evans, M.L. 1989. Implications of using Indo and Fura to measure

calcium in corn root protoplasts (Abstract). ASGSB Bulletin 3: 130.

Kiss, H.G. and Evans, M.L. 1989. Measurement of cytoplasmic free calcium in corn root

protoplasts using FURA-2 (Abstract). Plant Physiology 89(Suppl.): 94.

Lee, J.S., Hasenstein, K.-H., Mulkey, T.J., Yang, R.L., and Evans, M.L. 1990.

Effects of abscisic acid and xanthoxin on elongation and gravitropism in primary roots of

Zea mays. Plant Science 68: 17-26.

Lee, J.S., Wha-Kyung, C., and Evans, M. 1990. Effects of ethylene on curvature and

auxin redistribution in gravistimulated roots of maize (Abstract). Plant Physiology

93(Suppl.): 80.

Moore, R., Evans, M.L., and Fondren, W.M. 1990. Inducing gravitropic curvature of

primary roots of Zea mays cv. Ageotropic. Plant Physiology 92:310-315.

Yang, R. and Evans, M. 1989. The kinetics of aluminum action on the elongation of

primary roots of Zea mays (Abstract). Plant Physiology 89(Suppl.): 121.

Yang, R.L. and Evans, M. 1990. Differential effects of auxin and acid on the elongation

of cortical and epidermal tissue in maize roots (Abstract). Plant Physiology 93(Suppl.):

70.

Yang, R.L. and Evans, M. 1989. Movement of gravitropic effectors in maize roots:

Microsurgical evidence for transmission through the epidermis and/or cortex (Abstract).

ASGSB Bulletin 3: 78.

37
Yang,R.L. andEvans,M.L. 1989.Effectof localizedremovalof cortical andepidermal

cell layersonpatternsof auxinmovementthroughcornroots(Abstract). ASGSB Bulletin

3: 128.

Yang, R.L., Evans, M.L., and Moore, R. 1990. Microsurgical removal of epidermal and

cortical cells: evidence that the gravitropic signal moves through the outer cell layers in

primary roots of maize. Planta 180: 530-536.

Young, L.M., Evans, M.L., and Hertel, R. 1990. Correlations between gravitropic

curvature and auxin movement across gravistimulated roots of Zea mays. Plant Physiology

92: 792-796.

Young, L.M., Rapp, K., and Evans, M.L. 1989. The effects of root cap

plasmolysis/rehydration on gravicurvature and gravi-induced asymmetric auxin

redistribution (Abstract). ASGSB Bulletin 3:129.

38
PHYSIOLOGICAL, BIOCHEMICAL AND MOLECULAR PROCESSES

ASSOCIATED WITH GRAVITROPISM IN ROOTS OF MAIZE

Lewis Feldman

Department of Plant Biology

University of California

Berkeley, CA 94720

Description of Research

On Earth roots typically respond to gravity by growing downward. Our research focuses

on the physiological, biochemical, and molecular steps involved with converting the

stimulus of gravity into a biological response.

For this effort we would like to be able to "turn gravity off" at will and establish some base

line with regard to biochemical and physiological processes, and after having done this,

"turn gravity back on" and observe what new processes are initiated. Unfortunately, being

able to turn gravity on or off at will is not yet available to us, especially for Earth-based

materials. Hence we use systems in which the response of the tissues to gravity, even in

the presence of gravity, is incomplete unless some other requirement is also satisfied. For

our work, that other requirement is light. Roots of certain varieties of corn will not

respond completely to gravity unless the root is also provided with some light. Light

therefore initiates some biochemical processes which are necessary for complete response

of the roots to gravity. Our hope was that by learning about the processes initiated by light,

we might gain an understanding of steps involved in converting the gravity stimulus into a

growth response. Our efforts for this project have concentrated on defining the role of a

particular pigment, phytochrome, which we earlier showed was responsible for mediating

the light/gravity response in roots. Our efforts have shown that messenger RNA (mRNA)

for phytochrome (the compound coding for the phytochrome protein) is located

preferentially in the root cap where gravity is perceived and processed in roots. Moreover,

the phytochrome mRNA is highly expressed in specific cells within the root cap, the socalled columella cells, where we now believe gravity is first detected by the root. The

intimate spatial association between these cells and the phytochrome mRNA makes it likely

that the phytochrome protein mediates very early steps in the processing of the gravity

stimulus. Our best guess fight now is that probably the phytochrome protein affects

receptors involved with gravity perception. Other evidence showing how rapidly (kinetics)

phytochrome can affect a response also supports our notion of this molecule being involved

in early steps of gravity processing. The challenge now will be to determine which events

are phytochrome-mediated and, in particular, how they are associated with gravity

perception and/or processing.

The second focus of our work has been on understanding the relatively later steps in the

processing of the gravity stimulus. These steps occur after the involvement of

phytochrome and are believed to make use of particular proteins known as kinases. These

proteins function by phosphorylating (by putting a phosphate onto other proteins), thereby

changing their structure and allowing for the transduction of an environmental signal.

Based on analogies with animal and microbial systems, it is hypothesized that the

processing of the gravity signal involves a kinase. Unfortunately, little is known about

plant kinases. Indeed, none has ever been completely purified. Thus, we have taken a

molecular approach and looked for kinases which, based on their characteristics, may be

involved with processing various environmental signals, such as gravity. To date this

effort has resulted in the positive identification of a kinase, which is structurally highly

homologous to other animal kinases involved in the transduction of many environmental

39
stimuli. The kinaseis locatedin the root cap and is not affected by light. The challenge

ahead will now be to further characterize this kinase with regard to its possible role in

mediating the transduction of the gravity signal in roots.

Accomplishments

The major findings from these studies are:

(1) There is an intimate association between the phytochrome mRNA

and the cells in the root cap believed to be associated with gravity

perception.

(2) Light leads to very rapid changes in the level of the mRNA for

phytochrome and the kinetics of this light-induced change correlate very

strongly with the light-induced gravity response.

(3) A kinase homologous to those involved with environmental signal

transduction in animals and microbial systems is present in the root cap.

This kinase is not light-regulated, therefore implying that it works late in

the signal transduction chain.

Significance of the Accomplishments

Virtually nothing is known about the biochemistry involved with converting the gravity

stimulus into a developmental response in plants. Our recent work has shown an intimate

spatial association between the phytochrome mRNA and those cells involved with the

perception of gravity, thus making it likely that phytochrome mediates some early steps in

processing gravity. Knowing this, we can now concentrate on looking for those steps

which are phytochrome-mediated, and hence which may be involved with the perception of

gravity. Our work with kinases is the first to look at the role of this important group of

proteins specifically in the transduction of the gravity stimulus. We have demonstrated the

presence in the root cap of a kinase protein which is similar to proteins in animals and

microbes already shown to transduce a variety of environmental signals, thus making it

likely that the root kinase functions in a similar way (although not necessarily for gravity)

in roots of maize.

Publications

Bierman, B.J. and Feldman, L.J. 1990. Expression of a maize cDNA homologous to the

catalytic domain of second messenger-dependent protein kinases (Abstract). Plant

Physiology 93(Suppl.): 79.

Biermann, B.J. and Feldman, L.J. 1989. Maize root caps contain an mRNA species with

homology to the conserved catalytic domain of cyclic AMP-regulated protein kinases

(Abstract). ASGSB Bulletin 3: 94.

Biermann, B.J. and Feldman, L.J. 1989. Maize root tips contain an mRNA species with

homology to the conserved catalytic domain of protein kinases (Abstract). Plant

Physiology 89(Suppl.): 105.

Goliber, T.E. and Feldman, L.J' 1989. Analysis of leaf development in the

heterophyllous aquatic plant Hippuris vulgaris (Abstract). Plant Physiology 89(Suppl.):

103.

40
Goliber, T.E. and Feldman,L.J. 1990. Developmentalanalysisof leaf plasticity in the

heterophyllousaquaticplantHippuris vulgaris. American Journal of Botany 77: 399-412.

Goliber, T.E. and Feldman, L.J. 1989. Osmotic stress, endogenous abscisic acid and the

control of leaf morphology in Hippuris vulgaris L. Plant, Cell and Environment 12: 163-

171.

41
CIRCADIAN RHYTHMS PERSIST IN SPACE WITH A DECREASED

FREQUENCY: PRELIMINARY RESULTS FROM STS-32 LIFE SCIENCE

EXPERIMENT "CHARACTERIZATION OF NEUROSPORA CIRCADIAN

RHYTHMS IN SPACE"

James S. Ferraro _.

Department of Physiology

Southern Illinois University

School of Medicine

Carbondale, IL 62901

Description of Research

An experiment utilizing the fungus Neurospora crassa was conducted on NASA space

shuttle flight STS-9 in late 1983 to examine the function of circadian rhythms (i.e.,

biological oscillations with periods approximately 24 hou,'r in length) when removed from

the daily geophysical rhythms induced by the Earth's rotation. The STS-9 experiment

reexamined whether the drive of circadian oscillations is generated endogenously or

imposed exogenously via environmental influences. While it is known that environmental

oscillations such as light provide time cues which influence diurnal oscillations in the

physiology of most organisms, it is also known that many physiological rhythms persist in

the absence of these geophysical times cues. The STS-9 results did not conclusively

support the endogenous generation of biological rhythms. The results demonstrated a

significant reduction in the clarity of conidial banding (rhythm of asexual spore formation)

which has been suggested to be a reflection of circadian rhythm amplitude. This damping

of the conidiation rhythm was sufficient to obliterate the distinction of daily cycles,

resulting in apparent arrhythmicity in 25% of the cultures. The damping and arrhythmicity

in the conidiation rhythm, while unexpected, was reversed by the inflight marking

procedure. Following this procedure, in which the cultures were exposed to light (and

acceleration via handling), rhythmicity was reinstated and persisted for the remainder of the

flight, suggesting that endogenous biological clocks can function in space. Other findings

included an increase in variability of the free-running period and growth rate. This increase

in variability of the period may have obscured an apparent decreased frequency of the

rhythm in space.

Postflight studies at the University of California-Davis Acceleration Laboratories supported

a hypothesis that the hypergravity of launch (centrifugation at 3 g for 15 min) could have

been responsible for the damped circadian rhythm. Furthermore, the damping effect of

hypergravity was eliminated by a light pulse following centrifugation. These studies

suggested a gravity sensing system in the pacemaker of Neurospora.

To test the hypothesis that Neurospora possesses a circadian rhythm of conidiation that is

endogenously generated and that Neurospora has a gravity sensing system which can affect

aspects of circadian function (e.g., period, phase and amplitude), we conducted an

experiment on shuttle flight STS-32 in January of 1990. Substantial alterations in the

design of the STS-9 package were implemented to greatly increase further delineation of the

timekeeping function of organisms in space. These changes consisted of: doubling the

sample size; adding a second strain of Neurospora (CSP; which possesses a more robust

rhythm and appears less susceptible to hypergravity); subdividing the package into three

sections {one package treated similarly to the original STS-9 experiment (Blue), a second

package of tubes wrapped in a red filter which allowed the tubes to be marked without

exposure to white light (Red), and a third package left undisturbed during the first marking

procedure (White)}; marking tubes earlier inflight (Figure 1); increasing inflight marks; and

42
addinggassamplingsyringes.Oneflight package(G) andfour 25oc synchronousground

controls (Bl-B4) were compared.Two asynchronousOrbital EnvironmentalSimulator

(OES)controlsandone25oc asynchronousgroundcontrolwererunatKSCin May 1990,

to discernthe effectsof variationsin orbiterambienttemperatures,humidity, andcarbon

dioxidefrom theeffectsof microgravity.TheOESdatais currentlyunderanalysis.

ORIGINAL PAGE

BLACK AND WHITE PHOTOGRAPH

Figure 1. STS-32 mission specialist Marsha Ivins performs the first inflight marking procedure nine hours

after launch. She is holding one of the 25 "race" tubes in the "Blue" package up to the mid-deck light and

marking the growth front. Only the "Red" and "Blue" packages were marked at MET L+00/09:00. Gas

samples were also taken at thistime,

Accomplishments

While there was some damping in flight, the amplitude of the conidiation rhythm of

Neurospora flown on STS-32 was much more robust than that seen on STS-9. Only two

flight cultures became arrhythmic in space. The conidiation rhythm in these tubes was

quickly reinstated by a marking procedure late on flight day 5. Therefore, the results of the

flight experiment support the hypothesis that while influenced by environmental

perturbations in light, temperature, and gravity, circadian rhythms appear to be

endogenously generated diurnal oscillations.

43
The rhythm amplitude of the BND cultures in the "Red" flight package was significantly

more damped than in only one of the four "Red" ground control packages. The BND

cultures in the "Blue" flight package were, however, significantly more damped than three

of the four "Blue" ground control packages. There was no significant difference in rhythm

amplitude between the "White" flight or any of the '_Vhite" ground packages.

The CSP cultures in the "Red" and "Blue" flight packages had a significantly higher

percentage of tubes with a damped amplitude of the circadian rhythm of conidiation (88%

and 100%, respectively) than any of the four ground control "Red" and "Blue" packages

(80% of the "Red" and "Blue" ground cultures demonstrated no rhythm damping). Again

there was no significant difference between the CSP cultures in the "White" flight vs. the

"White" ground control packages.

28"0 t [ PcriodFlight I Period Ground

27.0"1 a

b

21.0

20.0

RED/BND BLUE/BND WHITE/BND

Package/Strain

Figure 2. Mean + SEM free-running period (in hours) of the circadian rhythm of conidiation in the BND

strain ofNeurospora crassa for each of the three flight packages ("Red", "Blue", and "White") and each of the

three packages of the mean of the four sets of ground controls. The number at the lower portion of the bar

represents the number of cultures in each mean. Bars with dissimilar superscripts are significantly different

(p,:0.01).

44
To some extent in the BND strain, but to a greater extent in the CSP strain

of Neurospora, in both the "Red" and "Blue" packages, there was a

decrease in the amplitude of the conidiation rhythm in space. The rhythm in

the "White" flight package, however, was similar in amplitude to "White" ground control

packages for both strains. While the UC-Davis studies demonstrated that the hypergravity

of launch could cause damping of the circadian conidiation in Neurospora, the flight data

suggest that microgravity can also induce a diminution of the rhythm of

conidiation. Some aspect (e.g., light exposure, acceleration) of the marking

procedure can reinstate high amplitude rhythmic conidiation.

The period length of the circadian rhythm of conidiation in the BND cultures in the "Red"

flight package (26.1+0.7 hr) was significantly longer than any of the "Red" ground

packages (21.6+0.6, 21.1+0.3, 21.3+0.1 and 20.9+0.1 hr; p<0.01) which were not

significantly different from one another. The BND cultures in the "White" flight package

had a mean period length (24.9+0.7 hr) which was also significantly longer than any of the

"White" ground control packages (20.9+0.1, 21.5+0.2, 21.8+0.6 and 21.7+0.6 hr;

p<0.01). The "White" ground packages were not significantly different from one another.

While the BND cultures in the "Blue" flight package had significantly longer free-running

periods (24+0.2 hr) than the BND cultures in the "Blue" ground packages (22.2+0.7,

22.9+0.3, 21.3_+0.3 and 20.6+0.1 hr; G vs. B2, p<0.05; G vs. B1, B3, or B4, p<0.01),

there were also smaller, yet significant differences in the average period of BND cultures

among the four ground control packages (see Figure 2). The overall trend, however, was

that spaceflight was correlated with tremendous increases in the length of

the free-running period of the circadian oscillator in the BND strain of

Neurospora.

The effect of gravity on the period length of the circadian rhythm of

conidiation was more attenuated in the CSP strain. Spaceflight, however, did

significantly increase the free-running periods of the rhythm in the "Red" and "White"

flight packages when compared to ground controls (Red G vs. Red B1, p<0.05; Red G vs.

B2, B3, or B4, p<0.01). The CSP cultures in the "Blue" flight package, however, had an

average free-running period that was not significantly different from the "Blue" B 1 ground

control package. In fact, CSP cultures in the "Blue" flight package had an average period

length that was significantly shorter than three of the four "Blue" ground control packages

(p<0.05; see Figure 3).

Significance of the Accomplishments

The findings of STS-32 Life Science Experiment CNCR-01 strongly suggest that circadian

rhythms can persist in space and support the hypothesis of endogenous generation of these

daily oscillations (Figure 4). The results also suggest that gravity, like light, is an

environmental influence that can affect the period and perhaps the phase and amplitude of

the daily rhythms generated by the circadian pacemaker. The suggestion that gravity (or its

absence) can be an environmental Zeitgeber (time cue) is umque and profound. The

circadian pacemaker has been suggested as being an endogenous time-keeping mechanism

that is very accurate over long periods of time without external input. The system has an

inertial component that resists alterations in frequency and an environmental sampling

system which corrects for temporal discrepancies between internal time and diurnal

oscillations of the environment. The environmental sampling system is restricted to a

limited number of inputs which are almost exclusively confined to highly predictable

geophysical rhythms. Environmental factors that are not valuable as reliable indicators of

diurnal and/or seasonal time are substantially ignored by the pacemaker (e.g., the

pacemaker is relatively temperature compensated; i.e., Q 10 of approximately 1); however,

45
they may induce temporary alterations in observed overt rhythms. This brief modification

in an observable rhythm is termed "masking" and usually has little, if any, impact on

subsequent circadian phase or period length. Therefore, the discovery of another

geophysical force that provides temporal information to the pacemaker is of substantial

importance. The suggestion that organisms can receive temporal information from minor

fluctuations in gravitational force will have substantial impact on the study of biological

rhythms on Earth and could have significant consequences for the health and performance

of astronauts, as well as for life science experiments using plants and animals during longterm space travel.

,mal

¢h

_laml

¢¢

28.0

27.0

26.0

25.0

24.0

23.0

21.0

20.0

b

a

RED/CSP BLUE/CSP

Package/Strain

J Period Flight Ground ]

a

WtlITE/CSP

Figure 3. Mean + SEM free-running period (in hours) of the circadian rhythm of conidiation in the CSP

strain ofNeurospora crassa for each of the three flight packages ("Red", "Blue", and "White") and each of the

three packages of the mean of the four sets of ground controls. The number at the lower portion of the bar

represents the number of cultures in each mean. Bars with dissimilar superscripts are significantly different

(p<0.01).

46
ORIGINAL PAGE

BLACK AND WHITE PHOTOGRAPH

Figure 4. Inflight photograph taken by STS-32 mission specialist Marsha Ivins of a "race" tube from the

"Blue" package during the second marking procedure at MET L+05/22:15. The preflight mark (L-00/15:50)

and the first inflight mark (MET L+00/09:00) are visible in the lower portion of the tube. It is readily

apparent that the banding pattern of the Neurospora crassa conidiation rhythm is quite regular and of high

amplitude. The gross observation suggest clock functions similar to those found normally on the ground.

Publications

Ferraro, J.S. 1990. Nocturnal illumination maintains reproductive function and simulates

the period-lengthening effect of constant light in the mature male Djungarian hamster

(P hodopus sungorus ). Journal of Interdisciplinary Cycle Research 21: 1-16.

Ferraro, J.S., Fuller, C.A., and Sulzman, F.M. 1989. The biological clock of

Neurospora in a microgravity environment. Advances in Space Research 9(11): 251-260.

Ferraro, J.S., Happe-Shelton, D.J., and Dorsett, J.A. 1989. The effects of temperature

on growth rate and circadian rhythmicity in Neurospora crassa (Abstract). ASGSB

Bulletin 3: 64.

47
Ferraro, J.S., Krum, H.N., Bartke, A., Wassmer, G.T., Chandrashekar, V., Michael,

S.D., and Sulzman, F.M. 1990. Nocturnal illumination does not necessarily stimulate the

photoperiodic response, despite mimicking the effects of constant light on the circadian

system in the male Syrian hamster. Physiology & Behavior 47: 577-588.

Ferraro, J.S. and Steger, R.W. 1990. Diurnal variations in brain serotonin are driven by

the photic cycle and are not circadian in nature. Brain Research 512: 121-124.

Ferraro, J.S. and Steger, R.W. 1989. Diurnal variation in brain serotonin is driven by the

photic cycle and is not circadian in nature (Abstract). Society for Neuroscience Abstracts

15: 727.

48
GRAVITROPIC RESPONSE MECHANISM IN CEREAL GRASS SHOOTS

Peter B. Kaufman, Nadarajah Karuppiah,

Casey Lu, and Donghern Kim

Cellular & Molecular Biology Group

Department of Biology

University of Michigan

Ann Arbor, MI 48109

Description of Research

The primary goal of our research is to determine the mechanism by which gravistimulation

causes negative gravitropic curvature (upward bending) in cereal grass shoots. The locus

of this response is the swollen leaf-sheath pulvinus located next to a node at the base of

each leaf. Gravitropic curvature in the pulvinus involves differential cell elongation, with

zero elongation at the top and maximal elongation at the bottom. In a given pulvinus, such

differential elongation has the potential to cause upward bending in the shoot portion above

the pulvinus 90 ° to an upright position.

The gravitropic response mechanism has three basic components: graviperception,

transduction, and asymmetric growth response. In our earlier work, we focused on the

graviperception component and showed unequivocally that starch statoliths (starchcontaining chloroplasts) are the gravisensor organelles in the cereal grass pulvinus system.

When they are rendered starch-free by pretreatment in the dark, no graviresponse occurs.

When starch-free statoliths resynthesize starch by sucrose feeding, the graviresponse is

restored. We have postulated that starch statoliths may act as membrane pressure probes to

open ion/hormone channels, serve as information carders (e.g., serve as a source of

hormone deconjugating enzymes or a source of Ca2+), or provide substrate (as D-glucose)

for cell wall biosynthesis that occurs during gravity-induced cell elongation.

More recently, we have probed into the nature of the transduction process, where key

effector hormones, IAA (indole-3-acetic acid) and gibberellins, become asymmetrically

distributed in the graviresponding pulvinus. Our current model of transduction portrays the

pulvinus storing inactive conjugates of IAA and GAs (gibberellic acids) when it is in an

upright, non-gravistimulated position. During the course of gravistimulation, the free IAA

and GAs become differentially released from their respective conjugates and differentially

synthesized so as to give a 1:1.5-2.0 top/bottom asymmetry of the free hormones. Such

ratios have been experimentally determined for both IAA and GAs in the pulvinus system.

Currently, we are testing the model to determine precisely how and when these hormone

asymmetries are established in relation to the kinetics for the upward bending response.

The asymmetric growth response component of gravitropic curvature in the cereal grass

pulvinus involves cell wall loosening, celt wall biosynthesis, starch degradation, changes

in 13-D-glucan composition in the cell wall, and differential enhancement of sucrose

hydrolysis.

Accomplishments

(1) Invertase Gene Expression in Oat Pulvinus Tissue: The pBR 322

plasmid containing yeast invertase gene in E. coli HB101 was obtained from Carlson et al.,

Department of Human Genetics and Development, Columbia University, NY. E. coli was

grown overnight in LB medium in our laboratory and the plasmid DNA was isolated.

49
Qualitativeagarosegel electrophoresisindicatedplasmidpurity andexpectedsize(ca.23

kb). The plasmidDNA thusobtainedwas initially probedwith biotin-labelled ATP (a

nonradioactivelabel)for SouthernBlot analysis.Total genomicDNA wasisolatedfrom oat

pulvini andthenpartially digestedwith restrictionenzyme(BAMH1). Following DNA

agarosegel electrophoresisand transferonto HyBond-N hybridization membrane,the

DNA was hybridized with the biotin-labelled probeand detectedwith a Streptavidinalkalinephosphataseconjugatesystem. Resultsobtainedwere quite satisfactory. The

experimentwas repeatedwith a radioactive32p probeto increasesensitivity. Results

indicated that the yeast invertase DNA could be used to detect the invertase

gene in oat pulvinus tissue. The next stage of the present study will be to construct

an oat recombinant plasmid library in an effort to isolate and clone the invertase gene and

sequence it. Simultaneously, employing the same probe, invertase mRNA levels will be

monitored following the effects of gravity and plant hormone treatments.

(2) Invertase Localization in Oat Pulvinus Tissue: It has been

demonstrated that acid invertase activity increases in the bottom halves of gravistimulated

oat and barley pulvini. In addition, two forms of acid invertase (soluble and cell wallbound) have been detected in this tissue system. They exhibit different Km's and pH

optima. To more accurately determine the subcellular locations of these enzymes, an

immunolocalization study has been undertaken.

We will attempt to localize invertase in oat (Avena sativa cv. 'Victory') at the cellular and

subcellular levels via light and transmission electron microscopy utilizing histo/

cytochemical and immunochemical techniques. We hope to be able to determine (from a

time-course series of studies on gravistimulated tissue) the specific tissue and cellular

location(s) where invertase production is turned on, where invertase is acting inside

graviresponding cells, ar, d the cellular transport pathway that invertase follows on its way

to being secreted into the cell wall.

Polyclonal antibodies, raised against soluble acid invertase from barley, bound both

invertase and several other barley proteins, as determined by protein A-Sepharose affinity

chromatography and gel electrophoresis. Thus, a second attempt at raising invertase

specific polyclonal antibodies (this time raised against yeast invertase) by the Kaufman

laboratory was undertaken. Also attempted was raising monoclonal antibodies against

soluble oat invertase by the University of Michigan's Hybridoma Facility, located on the

medical campus.

The polyclonal anti-yeast invertase antibody does bind soluble oat acid

invertase as determined by ELISA. Four monoclonal antibody cell lines

also bind soluble oat acid invertase, again determined by ELISA. We are

currently checking the specificity of both the polyclonal and monoclonal antibodies with

western blotting assays. Preliminary results indicate that the polyclonal and three out of

four monoclonal antibody preparations may be specifically binding oat acid invertase. We

are now carefully checking for antibody specificity before we continue with the next step of

monoclonal antibody production or begin our immunolocalization work using the

polyclonat antibody preparation. Once we have a specific antibody probe, it will be used in

conjunction with an immunogold conjugated second antibody to localize invertase at the

subcellular level, when increased invertase expression is induced by gravity and hormone

treatments.

(3): Glucan Synthase ActiVity in Graviresponding Oat Pulvini: The

leaf-sheath pulvinus is the primary gravisensor organ in cereal grass shoots. During

gravistimulation, we find asymmetric growth in an upward-bending pulvinus. There are

50
two mechanismsinvolvedin thisphenomenon:cell wall looseningandcell wall elongation.

In cell walls of cerealgrasses,a mixed-linkage(I]-1,3,_-1,4) -D-Glucanis synthesized

only whencellsexpandandturnoverof thispolymeris coincidentwith cell elongation.A

gravistimulation time-chaseexperimentwasdoneto check how 13-D-glucansynthase

activity changesin top andbottomhalvesof oatpulvini. Whenpulvinus-containingstem

segmentsaregravistimulatedin thedark at 25oc andfed with O.1M sucrosefrom the

bases,theystartto bendupwardby 3 hr. Upperandlowerhalvesof pulvini werecollected

andassayedfor glucansynthaseactivity afterbeinggravistimulatedfor 0, 3, 6, 12,and24

hr. Glucan synthaseactivity increasednoticeably after 12 hr of gravistimulation as

comparedwith thatin pulvini of upright control segments.Lower halvesof pulvini had

higher enzymeactivity thanupperhalves(Figure 1). Oat pulvini gravistimulatedfor

24 hr and treated with 30 I.IM GA 3 had significantly higher glucan synthase

activity than those pulvini without any gravistimulation and hormone

treatment.

1000

0

Upper Hal/ Lower Hall

Figure 1. Glucan synthase activity of upper and lower halves of oat pulvini after 24 hrs of

gravistimulation.

(4) Analysis of IAA/IAA-Conjugates in Gravistimulated Oat Pulvini:

Experiments were conducted to analyze the asymmetric distribution of IAA and its

conjugation products in p-1 (next-to-last) pulvini of oat shoots during gravistimulation.

P-1 segment of oats were placed horizontally and incubated in a dark growth chamber at

room temperature for gravistimulation. At 0, 3, 6, 9, 12, and 24 hr after gravistimulation,

segments were harvested and partitioned into "top" and "bottom" halves. Analysis of IAA

and its amide-linked and myo-inositol conjugates in oat p-1 pulvini have been carried out

according to the methods of Chen (1987) with a little bit of modification.

Samples were prepared for free IAA analysis. Now free IAA in these samples is being

9urified by using HPLC (with reverse phase C18 columns packed with Whatman Partosil

,3DS-3) and quantitated by using mass spectrometry (ms).

Significance of the Accomplishments

Finding #1: Invertase Gene Expression: Yeast invertase DNA will be used as a probe to

detect the invertase gene in oat pulvinus tissue. An oat recombinant plasmid will be

51
constructedin pBR322. Followingtransformationin E. coli, the colonies will be screened

with the yeast invertase DNA probe to isolate, clone, and sequence the invertase gene.

Once this is achieved, we can assess how oat pulvinus invertase gene expression is

regulated by gravity and by the two hormones, IAA and GAs, that are involved in the

transduction process. This will allow us to couple gravity transduction with one of the

early steps in the asymmetric growth response mechanism.

Finding #2: Invertase Localization: Once an antibody specific to invertase is produced, we

will have a very powerful tool capable of detecting invertase at the subcellular level. The

antibody probe will allow us to: (a) produce visual data (micrographs) corroborating the

biochemical data on the invertase asymmetry at the tissue level; (b) extend our

understanding of where invertase asymmetry occurs at the subcellular level; (c) probe the

pathway of invertase transport as the gravity response proceeds; and (d) probe the pathway

of invertase transport after hormone treatment.

Finding #3: Glucan Synthase Activity Changes Induced by Gravistimulation: Our data on

differential expression of glucan synthase activity in graviresponding pulvini indicate that

this enzyme may be a primary regulatory site involved in differential enhancement of cell

wall synthesis in our pulvinus system. The significant increase in glucan synthase activity

in lower halves of pulvini, as compared with upper halves, correlates well with enhanced

levels of 13-D-glucan found in cell walls of tissues located in the lower halves of pulvini that

are responding to gravistimulation.

Finding #4: IAA and IAA-Conjugate Analyses: By analyzing the distribution of IAA and

its conjugates in gravistimulated oat pulvini, as compared with that in upright control

pulvini, we can provide evidence for our current model of signal transduction during

gravistimulation, in which free IAA is differentially released from inactive conjugates of

IAA stored in the pulvinus, and/or is differentially synthesized during gravistimulation.

Publications

Brock, T.G., Kapan, U., Ghosheh, N.S., and Kaufman, P.B. 1989. The effect of

gravistimulation on auxin transport in oat pulvini (Abstract). ASGSB Bulletin 3: 127.

Brock, T.G. and Kaufman, P.B. 1990. Movement in grass shoots. In: The PuIvinus:

Motor Organ for Leaf Movement (ed. by R.L. Satter, H.L. Gorton, and T.C. Vogelmann).

Rockville, MD: American Society of Plant Physiologists, p. 59-71.

Brock, T.G., Lu, C.R., Ghosheh, N.S., and Kaufman, P.B. 1989. Localization and

pattern of graviresponse across the pulvinus of barley Hordeum vulgare. Plant Physiology

91:744-748.

Chang, S.-R., Ghosheh, N.S., and Kaufman, P.B. 1990. Changes in glucan synthase

activity in response to gravistimulation and hormone treatment in oat shoot leaf-sheath

pulvini (Abstract). Plant Physiology 93(Suppl.): 79.

Chang, S.-R., Karuppiah, N., and Kaufman, P.B. 1989. Changes in the activity of 13-

glucan synthase in response to gravistimulation in oat (Avena sativa) leaf-sheath pulvinus

(Abstract). ASGSB Bulletin 3: 65.

Karuppiah, N., Vadlamudi, B., and Kaufman, P.B. 1989. Purification and

characterization of soluble (cytosolic) and bound (cell wall) isoforms of invertases in barley

(Hordeum vulgare) elongating stem tissue. Plant Physiology 91: 993-998.

52
Lu, C.R.,Karuppiah,N., andKaufman,P.B. 1990. Cellular and molecularstudieson

invertasesin gravirespondingcerealgrasspulvini (Abstract). Plant Physiology 93(Suppl.):

90.

53
CELLS, EMBRYOS, AND DEVELOPMENT IN SPACE

Abraham D. Krikorian

Department of Biochemistry and

Cell Biology

State University of New York

Stony Brook, NY 11794

Description of Research

It is now generally recognized that the problems of development constitute a major part of

the objectives of modern biology. The ultimate aim of our research plan is to furnish

systems at different levels of initial organization that will enable the effect of microgravity

in the space environment to be tested on the behavior of plants in contrast or comparison to

their performance at 1 g and in ground controls. While the main focus is aimed towards the

broad effects of near zero gravity that operate on systems as they grow and develop, the

systems in being are or will be adaptable to a variety of more specific tests. For example,

any one of our test systems is capable of being used to ascertain whether there might be

differences in the normal rate, frequency, and patterning of cell division, or in the fidelity

of partitioning of the chromosomes of their cells during or after exposure to spaceflight.

The thrust of the investigations deal with: (a) the induction of active growth, cell

proliferation, and metabolism in otherwise mature quiescent cells as they exist in situ. This

is a problem that has involved and still involves the identity and mode of action of relatively

simple growth regulating substances of low molecular weight, their synergists and

cofactors; (b) the obtaining and multiplication in culture of free cells and their contrasted

development into unorganized callus masses, on the one hand, and as somatic (nonzygotic) embryos into plantlets, on the other; (c) the growth, morphogenesis, and

metabolism of intact plantlets and tissue-culture-derived propagules with their established

growing regions of shoot and root, in response to interacting factors which are both

environmental (i.e., different regimes of photoperiodicity and changing temperature) and

nutritional; (d) the development of protocols which have a high level of reliability for

establishing chromosomal characteristics and profiles for the plant species we are working

with, while at the same time seeking to extend the principles so gained to a still broader

range of species; and (e) the management of cultured systems from the perspective of being

able to use them effectively and with a minimum of human intervention in a space

environment setting.

Accomplishments

Work has focused largely on characterizing further our initial finding that somatic embryos

can be produced from two tissues of cultivated carrot On hormone-free medium. We had

learned that mericarps ("seeds"), after germination of their zygotic embryos, reliably yield

somatic embryos. Mechanically wounded zygotic embryos also yielded somatic embryos,

but were a much poorer source of somatic embryo production.

(1) Attempts to increase somatic embryo production were realized. Experiments

showed that hormone-free nutrient medium, containing 1 mM NH4 + as the sole nitrogen

source, fostered production of somatic embryos from wounded zygotic embryos to the

same high degree initially found with mericarps.

54
(2) Growth on 1mM NH4+-containingnutrient medium preventsthe initially

formed somaticembryosfrom developing into later stages,but doesnot preventcell

multiplication. The apparentinability to continue developmentresulted in cultures

consistingentirelyof preglobularstageproembryos.

(3) Thehistologicalcharacterizationof productionor initiation of somaticembryos

from woundedzygoticembryoswascardedout. Thedatasuggestedthatpreglobularstage

proembryoscouldbemaintainedassuchon 1mM NH4+-containingmediumdueto adrop

in pH.

(4) Thef'trst-formedsomaticembryoswill continuedevelopmentinto laterembryo

stages,without continuedsecondaryembryoformation,if themediumpH is maintained

above4.5 (testedatpH 4.5, 5, 5.5,and6).

(5) The establishment of cultures consisting entirely of preglobular stage

proembryosis a process,not anevent. Thefirst-formedsomaticembryosmultiply in the

beginningasglobular stageembryosonly whenthepH of themediumis allowedto fall

during theculture period. During eachsuccessiveculture period,thevolume per tissue

massmadeupof globularstageembryosdecreaseswhile thevolumeof preglobularstage

proembryosincreases;atotalof 4 to 6 transfersof theentiretissuemassaftertheinitiation

of somaticembryosisrequiredto establishacultureconsistingentirelyof preglobularstage

proembryos.

(6) Establishmentof preglobularstageproembryoscanbehastenedby repeated

mashingor woundingof thefirst formedglobularstageembryosat thetime transfersare

made.

Significance of the Accomplishments

It has long been suspected that the mineral element composition of culture media influences

virtually every aspect of in vitro culture including initiation, maintenance, and development

of somatic embryos. Most often, though, these suspicions have been ignored and

emphasis has been placed on the extent of somatic embryo formation, and only when

exogenously added growth regulators were used to initiate and maintain the cultures. Also,

special attention has been given to developing protocols that can yield somatic embryos,

which in turn develop into mature plants phenotypically identical to the plant from which

the original explant was derived. Moreover, the requirements for, or the ability to respond

to, certain mineral elements and other media components has widely been thought to be

controlled by the exogenously added growth regulator(s) and these requirements, in turn,

then direct the developmental fate of those cells. Hence, there has been little incentive to

investigate inorganic components in isolation from the more usual organic, hormonal

additives. This work shows conclusively that the so-called classic or model carrot system,

thought in the past only to be possible in a reliable way via added growth regulator(s), is

achievable with high confidence without the use of any added growth regulator.

We have, as yet, no evidence of what the physical or chemical nature of the pH effect is on

the morphogenetic fate of preglobular stage proembryos. Therefore, we have no direct

evidence of its mechanism of action. Even so, this is the first demonstration that

morphology of a higher plant system is reliably and reversibly influenced

by changes on external pH. Since pH changes probably exert control indirectly via

changes in internal pH, particular attention will now be paid to examining this hypothesis.

The ability to promote continuation of early embryogenesis by means of pH adjustment

55
providesavehiclewherebyflight experimentationcaneasilybyaccomplishedwith minimal

technologicalintervention.

Publications

Krikorian, A.D. 1989. Polarity establishment, morphogenesis and cultured plant cells in

space. In: Cells in Space (ed. by J.D. Sibonga, R.C. Mains, T.N. Fast, P.X. Callahan,

and C.M. Winget). Moffett Field, CA: NASA Ames Research Center, p. 87-95. (NASA

CP-10034)

Krikorian, A.D., Kann, R.P., and Fitter Corbin, M.S. 1989. Daylily. In: Handbook of

Plant Cell Culture Volume 5, Ornamentals (ed. by P.V. Ammirato, D.R. Evans, W.R.

Sharp, and Y.P.S. Bajaj). New York: McGraw-Hill Publishing Company, p. 375-412.

Levine, H.G., Kann, R.P., O'Connor, S.A. and Krikorian, A.D. 1989. Preliminary

results from the Chromosomes and Plant Cell Division in Space Experiment (CHROMEX)

flown aboard shuttle mission STS-29 (Abstract). ASGSB Bulletin 3: 96.

Smith, D.L., Kelly, K., and Krikorian, A.D. 1989. Ethylene-associated phase change

from juvenile to mature phenotype of daylily (Hemerocallis) in vitro. Physiologia

Plantarum 76: 466-473.

Smith, D.L. and Krikorian, A.D. 1989. A new way to study strategic control points in

plant somatic embryogenesis on Earth and in space (Abstract). ASGSB Bulletin 3:81.

Smith, D.L. and Krikorian, A.D. 1989. Release of somatic embryogenic potential from

excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormonefree medium. American Journal of Botany 76:1832-1843.

56
PERCEPTION AND TRANSDUCTION OF

PLANTS

A. Carl Leopold

Boyce Thompson Institute for Plant Research

Cornell University

Ithaca, NY 14853

GRAVIRESPONSES IN

Description of Research

The research in this project is divided into two components: a search for the gravity

perception mechanisms in plants which lack amyloplasts, and an analysis of the

transduction mechanisms which may be involved in root gravitropism, where amyloplasts

are directly involved.

In an effort to develop a simpler model of gravisensing than that found in the root of

angiosperm seedlings, we focused our studies on the polarity of cytoplasmic streaming in

internodal cells of Characean algae. Our intent is that hypotheses derived from this simple

system could then be tested using the more complex systems of higher plants.

As a continuation of our research on the gravity responses of higher plants, we are seeking

a linkage between the orthogravitropic response, the diagravitropic response, and the

phenomenon of springback, or loss of curvature upon withdrawal of the gravity stimulus.

Accomplishments

In our studies of Characean responses to gravity, we have demonstrated that hydrostatic

qualities of the external medium are a component of gravity perception. The polarity of

streaming can be altered by either altering the osmolarity of the ambient medium, or by

altering the density of the medium. As the density of the medium is increased to equal that

of the cytoplasm, or to exceed the density of the cytoplasm, polarity is diminished, and

then actually reversed. This is the first demonstration of a hydrostatic role in

gravity-sensing in plants.

From these results, we became concerned about a possible role of hydrostatic sensing in

roots. Rice roots were employed because of their tolerance of immersion; with this material

we found that increased density of the ambient solution could readily inhibit

gravitropism in rice roots.

Turning to the problem of the regulation of directional responses to gravity, we have

evidence that the cessation of a gravity stimulus can cause a change in the direction of the

curvature response. In short, we are able to convert orthogravitropic roots into

diagravitropic roots by withdrawal of the gravity stimulus.

Significance of the Accomplishments

Our work with Characean cells opens an entirely new area of study of the perception of

gravity in plants. Our experiments with rice roots indicate further that the hydrostatic

sensing mechanism may be involved in the graviresponses of roots of angiospermous

seeds as well.

57
Publications

Leopold, A.C. and Wettlaufer, S.H. 1989. Springback in root gravitropism. Plant

Physiology 91: 1247-1250.

Perdue, D.O., Leopold, A.C., and Wayne, R. 1989. Comparative effects of Ca ++ on

two types of root gravitropism (Abstract). ASGSB Bulletin 3: 130.

Perdue, D.O., Wayne, R.O., and Leopold, A.C. 1989. Calcium requirements for

diagravitropism and orthogravitropism in roots (Abstract). ASGSB Bulletin 3: 132.

Staves, M., Leopold, A.C., and Wayne, R. 1989. The effect of gravity on the polarity of

cytoplasmic streaming (Abstract). ASGSB Bulletin 3" 131.

Staves, M.P., Wayne, R., and Leopold, A.C. 1990. Hydrostatic factors affect the

gravity-induced polarity of cytoplasmic streaming in Characean algae (Abstract). Plant

Physiology 93(Suppl.): 78.

Wayne, R., Staves, M.P., and Leopold, A.C. 1990. Gravity-dependent polarity of

cytoplasmic streaming in Nitellopsis. Protoplasma 155: 43-57.

Williams, R.J. and Leopold, A.C. 1989. The glassy state in corn embryos. Plant

Physiology 89(3): 977-981.

58
DETERMINING THE EFFECTS OF GRAVITATIONAL STRESSES

LIGNIN FORMATION AND STRUCTURE: A NEW APPROACH

Norman G. Lewis

Department of Wood Science/Forest Products

and Biochemistry

Virginia Polytechnic Institute

Blacksburg, VA 24061

ON

Description of Research

Lignins are complex phenlypropanoid polymers produced by terrestrial vascular plants.

One of their roles is to provide compressive strength to plant cell walls, thereby allowing

such structures to stand in upright forms. There is some tentative evidence suggesting that

the lignin content of plants can be affected by gravitational loads experienced during

growth.

The specific objectives of this research are to determine the effect of gravitational forces on:

(a) the rate of uptake and binding of specifically labelled lignin precursors; (b) the bonding

patterns of lignin in situ; (c) the function, formation, and activities of specific peroxidase

isozymes responsible for lignin formation; (d) cinnamyl alcohol dehydrogenase activity;

and (e) development of vascular tissue.

In this reporting period, we have grown plants (Leucaena leucocephala) and loblolly pine

(Pinus taeda) aseptically on hydroponic media, supplemented with various specifically

labelled (14C, 13C) lignin precursors. Lignifying P. taeda cell suspension cultures have

also been obtained in order to study initiation of the lignification process in culture. All

experiments were first carded out at 1 g in order to establish appropriate controls. A

similar sequence of experiments is currently underway using aseptic, hydroponically

grown plants on clinostats. In addition, L. leucocephala plants were grown under identical

conditions on soil for 10, 20, and 30 days on clinostats (horizontally rotating, vertically

rotating, and vertical static). This was done to establish differences in cell-wall

development (i.e., ultrastructure) and polymer composition (e.g., lignin content and

monomer ratio, and alpha-cellulose and hemicellulose content), and activities of key marker

enzymes.

Accomplishments

(1) Uptake and Binding of Isotopically Labelled Lignin Precursors

into Lignin in Vascular Plant Tissue. [U-14C] phenylalanine was demonstrated to

be intactly and efficiently incorporated (>10%) into leaf, stem and root sections of L.

leucocephala, T. aestivum, N. tabacum, and P. taeda plants, which were grown

hydroponically under aseptic conditions. [2-14C] ferulic acid, on the other hand, was only

efficiently incorporated into root tissue and was not significantly translocated further.

Following administration of [U-14C] Phe and [2-14C] ferulic acid to hydroponically

grown plants, the lignin composition (i.e., ratio of monomer degradation products, phydroxybenzaldehyde (H), vanillin (V), and syringaldehyde(S)) was determined; only data

for roots is reported here (Table 1). As can be seen, a significant perturbation of the H:V:S

ratio occurred with ferulic acid; this was not observed with administration of phenylalanine.

Similar results were obtained with stem sections.

59
Molar Ratio Soil-Grown Hydroponically Grown

With Ferulic Acid With Phenylalanine

H/V 0.14 + 0.00 0.06 + 0.00 0.14 + 0.00

S/V 0.56 + 0.00 0.48 + 0.00 0.64 + 0.01

H:V:S 8:59:33 4:65:31 8:56:36

Where H = p-hydroxybenzaldehyde; V = vanillin; S = syringaldehyde

Table 1. Lignin composition ofL. leucocephala roots, as determined by nitrobenzene oxidation

Since [U-14C] Phe was a more efficient and natural precursor, as regards maintaining both

lignin content and monomer composition, the distribution of radioactivity into L.

leucocephala roots and stems was next determined (Table 2). As can be seen,

approximately 90% of radioactivity in the stem sections was associated with wall-bound

(i.e. lignified) components.

Fraction

R-oots

Organ

Stems

Aqueous Solubles (%)

Organic Solubles (%)

Insoluble (%)

Total Recovery (%)

33.9 + 5.09 12.7 + 1.32

1.56 + 0.62 0.73 + 0.04

64.5 + 4.48 86.6 + 1.36

74.0 + 2.57 91.3 + 5.87

Table 2. Distribution of radioactivity in L. leucocephala roots and stems administered [U-14C]

phenylalanine

(2) Uptake of [1-13C], [2-13C], and [3-13C] Phenylalanine. Next,

L. leucocephala were

1 plants _grown for 28-day periods and were individually administered

[1-13C], [2- 3C], and [3-13C] phenylalanine. Solid state C-13 nuclear magnetic

resonance (NMR) spectroscopic analyses of the resulting tissues were then carded out.

Results are shown in Figure la-c; assignment of the signals due to substructural units in the

lignin polymer are included.

60
METABOLIC SPECTROSCOPIC ANALYSES OF PHENYLALANINE

ADMINISTERED INTO LIGNIN IN PLANT STEMS

R

• _H20H

_ ¢_ 0C_3 mCH2OH

";°" \ / o_,,

J :'_ "

(a) f °-)4

R

_"'°"h-_,l

L ICH--O i,

0%I

CHitOH |

c.-o-t

(c)

_O OCH3 R%• • R

0% CH_OH \ L

/ I- H_.-o_o_,,"

p-....OCH_ J

R OCH 3

HIOH

,_.-o L

R_OCH3

CIC_4 3

OCH:3

CH2OH

=l

HO CH _R

R_OC :=

°%

260 1_0 160 5'0 8 PPM

Figure l.

stems. L=lignin; C=carbohydrate

Incorporation of (a) [1-13C], (b) [2-13C], and (c) [3-13C] phenylalanine into L. leucocephala

61
ill:

(3) Design of Clinostats for Aseptic, Hydroponic Growth of Plants.

As described in the last reporting period, clinostats were developed, each of which

permitted the horizontal or vertical growth of six plants in a relatively vibration-free

environment under controlled conditions. These have now been adapted to permit growth

of aseptic, hydroponically grown plants on a horizontally rotating clinostat. The

corresponding 14C and 13 C experiments, previously conducted at 1 g, are now underway

with this configuration; appropriate vertical rotating and vertical static control experiments

will be carded out.

(4) Function of Specific Peroxidases in Lignin Formation. Cell

suspension cultures of P. taeda have been obtained which excrete two peroxidase isozymes

into the nutrient media. These have been preparatively separated, and appropriate kinetic

data (for monolignol oxidation and H202 formation with NADH) is being determined.

The formation of each isozyme with clinostat-grown plants and their correlation with active

lignification is currently under investigation.

(5) Cinnamyl Alcohol Dehydrogenase. We consider this to be an important

marker enzyme in the lignification process. Crude cinnamyl alcohol dehydrogenase was

obtained from P, taeda suspension cell cultures (250 mg) by an extraction-grinding

procedure (100 i.tM tris-HC1, pH 7.5 containing 10 l.tM DTT, 5% PEG, insoluble PVP

(100 mg) and sea-sand) in a chilled mortar. The homogenate was then centrifuged (40,000

g) and the crude enzyme desalted on a Sephadex G-25 column. Isoenzymes were

separated by PAGE (8% polyacrylamide), using a stacking gel, and desalted with

Coomassie Blue and coniferyl alcohollNADH with an appropriate azo dye. The appearance

and correlation of these isozymes in clinostat-grown, actively lignifying plants is presently

being investigated.

Significance of the Accomplishments

First, it was established that the uptake of exogenously supplied phenylalanine and its

subsequent translocation and incorporation into lignified root and stem tissue did not

adversely affect the lignification process; i.e., the lignin content and monomer ratios were

essentially identical to "normally grown" plants. This was not the case when ferulic acid

was supplied as a precursor.

Second, this methodology allowed us to probe the exact intermonomeric unit bonding

environments of specific side chain carbons in the phenylpropanoid polymer; these

represent most of the interunit linkages. The spectra following metabolism of [ 1-13C] '

[2-13C], and [2-13C] Phe into lignin are shown in Figure 1. It can be seen that the most

abundant bonding environment corresponds to the ]3-0-aryl linkage (Figure lb). In

addition, the first direct evidence for lignin-carbohydrate bonding was obtained (Figure

lc).

Third, a clinostat design for growing and maintaining plants hydroponically on aseptic

media was developed. This now permits the determination of (I) any changes in lignin

composition and structure with plants grown under "reduced" gravitational loads, and (2)

any differences in the enzymatic activities of key enzymes involved in the lignification

process.

62
Publications

Lewis, N.G., Inciong, M.E.J., Dhara, K.P., and Yamamoto, E. 1989. Highperformance liquid chromatographic separation of E and Z-monolignols and their

glucosides. Journal of Liquid Chromatography 479(2): 345-352.

Lewis, N.G. and Yamamoto, E. 1990. Lignins: Occurrence, biogenesis and

biodegradation. Annual Review of Plant Physiology and Plant Molecular Biology 41: 455-

496.

Pullockaran, A.J., Kingston, D.G.I., and Lewis, N.G. 1989. Synthesis of

stereospecifically deuterated desoxypodophyllotoxins and 1H-NMR assignment of

desoxypodophyllotoxin. Journal of National Products 52: 1290-1295.

63
ANALYSIS OF COMPONENTS OF THE GRAVITROPIC RESPONSE

PLANTS USING PURIFIED PLASMA MEMBRANE VESICLES

Terri L. Lomax

Center for Gene Research and Biotechnology

Oregon State University

Corvallis, OR 97331

IN

Description of Research

The Committee on Space Biology and Medicine (National Research Council) and the

Workshop on Plant Gravitational and Space Research (NASA) both have emphasized the

need for subcellular investigations into the mode of action and regulation of the transport of

the plant growth regulator auxin and of calcium. The goal of ourr_search is to examine

components of the plasma membrane (PM) which are involved in the gravitropic response

of plants. The technical breakthrough which made this study possible was the development

of a method to obtain large quantities of highly purified PM from plant tissues. The

method involves separation of different kinds of membrane vesicles on the basis of both

surface charge and density; it yields PM vesicles of extremely high purity (95-98%). We

adapted this technique to membrane preparations from zucchini (Cucurbita pepo)

hypocotyls which yield vesicles that will stay tightly sealed for many hours.

Accomplishments

In the past year, we have used the PM vesicle system to analyze: (1) the interaction of

Ca 2+ with auxin transport, (2) Ca2+-stimulated protein kinases, and (3) auxin-binding

proteins. We have also expanded the investigation of auxin-binding proteins to a

gravitropic mutant of tomato.

(1) Effects of Calcium on IAA Transport. Calcium and movement of the auxin

indole-3-acetic acid (IAA) are linked in the gravitropic response, but in vivo studies of the

transport and localization of IAA and Ca 2+ using either intact tissue or stem segments have

been complicated by the intra- and extracellular compartmentalization of IAA and Ca 2+.

This led us to examine the effect of Ca 2+ on the transport of IAA across the PM. We have

found that a wide range of Ca 2+ concentrations applied either internally or

externally had no significant effect upon IAA uptake into or efflux from

isolated PM vesicles. This indicates that the influence of Ca 2+ on IAA

movement may be more indirect and speaks against the mechanism of

coupled Ca2+/IAA flux. We also attempted to elucidate the mechanisms of Ca 2+

movement across the PM. Our results showed that the published method for measuring

Ca 2+ uptake into plant PM vesicles using the fluorescent chelator chlorotetracycline is

invalid and based on a biophysical artifact. We established that another commonly used

Ca 2+ indicator compound, murexide, is also unreliable for in vitro measurements.

Problems with murexide have also recently been observed by other workers. We are

currently exploring the use of 45Ca for such studies.

(2) Calcium-Stimulated Protein Kinases. Ca2+-regulated protein kinases can be

important regulators of physiological processes including several which are associated with

gravitropism. It has been suggested that Ca 2+ ma X exert its effect on IAA transport via

phosphorylation of the carrier proteins by a Ca:Z+-stimulated protein kinase. This

64
prompted us to identify and partially purify severalPM protein kinases which are

dependentupon Ca2+ for autophosphorylationactivity. We havenot yet beenable to

demonstrateany changesin the in vitro autophosphorylation of these serine/threonine

kinases in response to gravistimulation. However, our assay is limited in that it can only

detect those covalent modifications of the kinase which survive the isolation procedure. It

remains possible that less permanent modulations of activity may take place. We are

currently purifying the Ca2+-stimulated protein kinases in order to generate specific

antibodies which will allow us to study their in vivo involvement in the gravity response.

(3) Auxin-Binding Proteins Involved in the Gravity Response. Although the

molecular mechanism of auxin action is still unknown, it is likely that auxin initiates cellular

responses by binding to specific receptor proteins. One objective of our research is to

understand the role of auxin-specific plasma membrane (PM) receptors and transport

proteins in plant growth and development, especially with regard to the gravitropic

response. This information will provide the cornerstone of future studies aimed at the

molecular mechanism of both auxin action and gravitropism. We have identified auxinbinding proteins in highly-enriched PM vesicles from zucchini hypocotyls by photoaffinity

labeling of polypeptides using 3H-5N3-IAA (azido-IAA). Photolysis of azido-IAA in the

presence of membrane proteins from auxin-responsive tissues of a variety of species results

in the high specific-activity labeling of a low abundance polypeptide doublet of 40-42

apparent molecular weights (kD), which displays properties consistent with those expected

for an auxin receptor or transport protein. The polypeptides: (1) are widespread in

dicotyledonous, monocotyledonous, and coniferous species and in plant tissues that

respond to IAA, but are not detected in auxin non-responsive tissues such as leaves; (2) are

present at low abundance in the PM; (3) are saturated by increasing concentrations of IAA;

and (4) are selective for binding only those auxin analogs that are physiologically active

auxins or specific antagonists.

In an attempt to determine if the azido-IAA labeled polypeptides are indeed involved in

auxin mediation of the gravitropic response, we have examined the auxin insensitive,

agravitropic mutant of tomato (Lycopersicon esculentum, Mill.) known as diageotropica

(dgt). We have shown that membranes from roots and hypocotyls of the wild-type

progenitor of dgt, VFN8, contain the characteristic two polypeptides (40-42 kD) which are

labeled with high specific activity by azido-IAA. These proteins are also azido-labeled at

similar levels in roots of dgt. In contrast, membrane proteins from dgt hypocotyls are only

slightly labeled relative to similar preparations from VFN8 hypocotyls.

Significance of the Accomplishments

The lack of specific azido-IAA binding in dgt is an exciting finding as it

identifies an auxin-binding protein which is missing or altered solely in the

mutant line of tomato. Since the dgt mutation appears to involve auxin perception, this

provides not only confirmation of the probable receptor identity of the 40-42 kD

polypeptides, but also associates that receptor with a specific set of morphological and

physiological attributes which are known to be connected to auxin (i.e., altered gravity

response and vascular system, reduced growth, lack of lateral roots, and failure to grow or

produce ethylene in response to auxin). We believe that these results will be important in

more precisely identifying the relationship of the 40-42 kD polypeptides to the dgt lesion.

65
Publications

Hicks, G.R., Rayle, D.L., and Lomax, T.L. 1989. The diageotropica mutant of tomato

lacks high specific activity auxin binding sites (Abstract). Plant Physiology 89(4, Suppl.):

111.

Lomax, T.L. 1990. Molecular communication in higher plants. New Biologist 2: 133-

135.

66

=
MECHANICAL STRESS REGULATION OF PLANT

DEVELOPMENT

Cary A. Mitchell and Laura L. Coe

Center for Plant Environmental Stress Physiology

Department of Horticulture

Purdue University

West Lafayette, IN 47907

GROWTH AND

Description of Research

The objectives of this research are: (1) to characterize the effects of physical forces other

than gravity on plant growth and development, and (2) to determine the physiological basis

for these responses. Physical disturbances in the form of shaking, vibration, or contact

rubbing affect straight growth as well as differential growth (e.g., tropistic curvature), and

the responses can be modified by prevailing environmental conditions (e.g., light intensity,

temperature). The usual response is reduced growth of unhardened herbaceous plants

relative to that of undisturbed plants as assessed by measurements of both productivity

(photosynthetic rate, dry weight gain) and dimensional parameters (e.g., leaf area, stem

length). However, in some cases mild stimulation of plant growth has been detected in

response to mild vibration.

The long-range objectives of this research are to: (1) elucidate which physiological

mechanisms are common to and which are different between mechanically stressed and

gravistimulated plants, and (2) determine the threshold of sensitivity of a sensitive test

species to the frequencies and amplitudes of vibration found in orbiting spacecraft in the

absence of a confounding gravity vector.

We have selected dark-grown legume seedlings as model test systems for both

seismomorphic (shaking or vibrational effects) and thigmomorphic (contact rubbing

effects) stimuli. Seismic forces are less damaging overall, can lead to net stimulation of

growth, and are typical of the kinds of physical perturbations likely to be encountered on

spacecraft (e.g., thruster firing and astronaut activity, such as treadmill exercises and

acceleration sled). Thigrnic forces are a good model for the abrasive action of soil particles

encountered by germinating seedlings growing upward through soil. Since many firstgeneration Space Biology plant flight experiments will be done with tender seedlings

.growing in darkness or under low-light conditions (where plant sensitivity to seismic stress

is greatest), dark-grown seedlings are appropriate test systems, and they are treated in our

ground-based research program either with vibric or thigmic stress.

Elongating epicotyls and hypocotyls of pea and soybean, respectively, represent a model

growth system that depends upon axial enlargement of cells in a discrete zone just below

the plumule or hook of the dark-grown seedlings. Kinetic growth rate studies are being

performed to determine the likelihood of biophysical triggers for more sustained growth

rate changes following the initial. In vitro studies with cut sections, as well as intact

seedlings with chemical probes, are being used to elucidate the role of changes in growthpromoting hormones and stress hormones in controlling the sustained growth changes

caused by mechanical stress.

The principal investigator (CAM) has been collaborating with engineers and scientists at

NASA Ames Research Center (ARC) in the Centrifuge Facility Project Office for Space

Station regarding the conduct of a ground-based Plant Vibration Sensitivity Study with

dark-grown seedlings of Pisum sativum L. cv. Alaska and Zea mays L. cv. Merritt.

67
During 1989protocolswereestablishedfor growing test subjectsandexposingthemto

continuousvibrationequivalentto either10-3 or 10-2 g in thehorizontalor verticalplanes.

Growth, development, and ethylene evolution protocols were establishedfor plant

responseto thesetreatments,and will, of course,beoverlain with a continuous,static,

vertical 1g force. Theground-basedstudiesatARC will becompletedduringthesecond

half of 1990. Theresultscouldreinforcebutwill not replacetheneedfor plant vibration

studiesunderreal spaceflightconditions.

Accomplishments

Growth of intact seedlings was monitored using an an.gular position-sensing transducer

capable of measuring growth with a resolution of microns per minute. Dark-grown

soybean (Glycine max L. Merr. cv. Century 84) and pea seedlings exhibited immediate

growth reduction following a single mechanical exposure (20X symmetric contact to

hook). Peas began growth recovery within 10 min of treatment and recovered to 70% of

pre-stress rates by 30 min. However, preliminary results obtained with soy.beans suggest

that not all plants respond to mechanical stress similarly. In a recent experament, 60% of

test plants showed post-stress growth rates that were 36% of pre-stress rates, Whereas

remaining subjects showed no effect. In those plants that did show post-stress depression

of growth rate, the effect was immediate and dramatic. Moreover, the effect was sustained

for up to 7 hr post-stress.

Significance of the Accomplishments

The rapid reduction in growth rate caused by mechanical stress suggests that initial events

are biophysical, possibly involving turgor reduction within the cell elongation zone.

Following this immediate response is a sustained component resulting in long-term growth

inhibition. Subsequent hormonal changes are responsible for the slower growth rate. Less

auxin is transported through the elongation zone of thigmo-stressed plants. Production of

stress ethylene during the longer-term growth reduction interferes with basipetal auxin

transport and likely contributes to the reduction in axial growth.

Publications

Coe, L.L. and Mitchell, C.A. 1989. Ability of chemical growth promoters to negate

mechanical stress effects on dark grown pea seedlings (Abstract). ASGSB Bulletin 3: 63.

Coe, L.L. and Mitchell, C.A. 1990. Auxin and ethylene involvement in mechanical stress

responses of pea seedlings (Abstract). Plant Physiology 93(Suppl.): 79.

Jones, R.S. and Mitchell, C.A. 1989. Calcium ion involvement in growth inhibition of

mechanically stressed soybean (Glycine max) seedlings. Physiologia Plantarum 76: 598-

602. = .......

Jones, R.S. and Mitchell, C.A. 1989. Mechanical stress-induced changes in cell wall

extensibility and cell morphology of dark-grown soybean hypocotyls (Abstract). ASGSB

Bulletin 3: 65.

68
SIGNAL TRANSMISSION AND THE ROLE OF THE ROOT CAP DURING

GRAVITROPISM

Randy Moore

Department of Biological Sciences

Wright State University

Dayton, OH 45435

Description of Research

The objective of our research is to understand signal transmission and the role of the root

cap in root gravitropism. We studied this by examining (1) a newly discovered mutant of

Zea mays having deformed root caps to determine how changes in this gravity-perceiving

organ affect root graviresponsiveness, (2) the role of the apoplast and outer cell layers of

roots as potential pathways for stimulus transmission during root gravitropism, (3) roots of

the ageotropic (and mucilage-lacking) cultivar ofZea mays to determine how the absence of

mucilage affects root gravitropism (this cultivar secretes negligible amounts of mucilage

and is not responsive to gravity), and (4) the gravitropic responses of capless roots of

Tristerix aphyllus.

Accomplishments

(1) In the ageotropic mutant of corn, gravitropic effectors such as calcium (Ca)

move from the root cap to the root in mucilage (Figure 1). The gravitropic response can be

turned on and off by applying and removing exogenous mucilage. Thus, the apoplast,

including the mucilage, is a primary route for movement of gravitropic effectors

in roots.

Figure 1. Root tips of a wild-type (left) and the 'Ageotropic' (right) cuhivar of Zea mays. Tips of

wild-type (graviresponsive) roots are covered by mucilage secreted by the root cap and epidermis.

Root tips of the mutant lack mucilage. Applying mucilage to tips of these roots induces

graviresponsiveness, indicating that in this cultivar, gravitropi c effectors move in mucilage from

the root cap to the root.

69

ORIGINAL PAGE IS

OF POOR QUALITY
(2) Symplastic connections occur between the root cap and root across only about

10% of the area of the cap junction in primary roots of Zea mays. These connections are

unnecessary for root gravitropism.

(3) Primary roots of Tristerix aphyllus are graviresponsive despite lacking root

caps. Thus, root caps are unnecessary for root gravitropism in this species.

(4) Putative gravity-perceiving cells in primary roots of Tristerix

aphyllus are located immediately adjacent to the elongating zone. To our

knowledge, this is the only higher plant in which putative statocytes form in

this position.

(5) The outer cell layers, especially the epidermis, are important for signal

transmission in primary roots of Zea mays.

(6) Plants grown on a rotating clinostat have significantly different cellular

ultrastructures and metabolisms than those grown in microgravity. Plants grown in

microgravity aboard the space shuttle produce different amounts and types of

fatty acids than controls grown at 1 g or on a rotating clinostat.

Significance of the Accomplishments

Our finding that gravitropic effectors move through outer cell layers and mucilage is

significant because (1) it provides important information about signal transmission during

root gravitropism, and (2) it provides a relatively simple system for identifying the

gravitropic signa!(s). -........

Our finding that symplastic connections are unnecessary for root gravitropism is consistent

with the observation that gravitropic effectors move apoplastically. This conf'n'ms the value

of our mutant as a system for identifying the gravitropi c effector(s). =

Our studies of Tristerix indicate that (1) root caps are unnecessary for root gravitropism,

and (2) putative statocytes may be located immediately adjacent to the elongating zone of

roots. This system is unique in that putative statocytes in other higher plants (e.g., primary

roots of Zea) are located in the root cap.

Our studies of flight-grown plants indicate that microgravity strongly affects lipid

metabolism. These data are consistent with our previous observations indicating that

microgravity affects cellular structure. Our comparisons of clinostat-grown plants with

those grown in microgravity indicate that clinostats do not mimic the ultrastructural effects

of microgravity.

Publications :_ _ 2 ==_ 2 := =/2_2 . _ =£

Marcum, H. and Moore, R. 1990. Influence of electrical fields and asymmetric application:: i::i:

of mucilage on curvature of primary roots of Zea mays. American Journal of Botany 77:

446-452.

Moore, R. 1990. How effectively does a clinostat mimic the ultrastructural effects of

microgravity on plant cells? Annals of Botany 65:213-2i6.

Moore, R. 1989. Root graviresponsiveness and cellular differentiation in wild-type and a

starchless mutant of Arabidopsis thaliana. Annals of Botany 64: 271-277.

70
Moore,R., Cameron,I.L., andSmith,N.K.R. 1989. Movementof endogenouscalcium

in theelongatingzoneof gravirespondingrootsofZea mays. Annals of Botany 63: 589-

593.

Moore, R., Evans, M.L., and Fondren, W.M. 1990. Inducing gravitropic curvature of

primary roots ofZea mays cv. Ageotropic. Plant Physiology 92: 310-315.

Moore, R. and McClelen, C.E. 1989. Characterizing pathways by which gravitropic

effectors could move from the root cap to the root of primary roots of Zea mays. Annals of

Botany 64: 415-423.

Moore, R. and Miller, I. 1990. Defective secretion of mucilage is the cellular basis for

agravitropism in primary roots of Zea mays cv. Ageotropic (Abstract). Plant Physiology

93(Suppl.): 40.

Yang, R., Evans, M.L., and Moore, R. 1990. Microsurgical removal of epidermal and

cortical cells: evidence that the gravitropic signal moves through the outer cell layers in

primary roots of maize. Planta 180: 530-536.

71
GRAVITROPISM IN ARABIDOPSIS

A GENETIC APPROACH

Kenneth L. Poff

MSU-DOE Plant Research Laboratory

Michigan State University

East Lansing, MI 48824

THALIANA:

Description of Research

Our ultimate objective is to achieve an understanding at the molecular level of the

mechanism whereby a plant measures and responds to a gravitational stimulus. Toward

that end, this project is developing a collection of mutant strains of Arabidopsis with

alterations in gravitropism. These lines will permit the identification of the elements in the

transduction pathway, and will permit an analysis of the interrelations between the two

sensory responses, gravitropism and phototropism. :

The work is divided into several aspects. First, a family of mutants has been identified

with alterations in gravitropism and phototropism. Second, individual mutants are

characterized physiologically and genetically. Third, particularly interesting mutants will be

mapped with respect to morphological markers and restriction fragment length

polymorphisms in preparation for cloning the genes of interest.

Accomplishments

(1) Several mutants have been identified which exhibit no hypocotyl gravitropism,

but which exhibit normal phototropism. These include a mutant which is starch-deficient

and one which contains wild-type amounts of starch. Thus, a loss of starch is not

necessary for a loss of graviperception.

(2) A critical review of the older literature (particularly German) shows that the data

on site of graviperception in roots indicates that perception is located in the apical 2-3 ram.

This is not compatible with the assumption stated in the more recent literature without

supporting data that perception is localized to the columella cells of the root cap.

Thus,

which

(3) Roots of corn seedlings grow toward the warmer side in a thermal gradient.

thermotropism must be added to the list of environmental stimuli to

roots respond.

(4) A system has been developed with which gravitropism of seedlings can be

monitored in physiological "darkness," using infrared radiation (wavelengths greater than

800 nm) and an infrared-sensitive CCD camera. This system permits the kinetics of a

single seedling's response to gravity to be monitored in the absence of light as a

confounding factor.

Significance of the Accomplishments

An evaluation of the role of starch in graviperception has proven to be an intractable

problem. The identification of mutants in which graviperception is independent of starch

synthesis demonstrates the utility of this genetic approach.

72
Publications

Fortin, M.-C., Pierce, F.J., and Poff, K.L.

formation in curving roots of Arabidopsis

Environment 12: 337-339.

1989. The pattern of secondary root

thaliana (L.) Heynh. Plant, Cell and

Fortin, M.-C. and Poff, K.L. 1990. Thermotropism by primary roots of maize

(Abstract). Plant Physiology 93(Suppl.): 40.

Khurana, J.P., Best, T.R., and Poff, K.L. 1989. Influence of hook position on

phototropic and gravitropic curvature by etiolated hypocotyls of Arabidopsis thaliana.

Plant Physiology 90: 376-379.

Khurana, J.P. and Poff, K.L. 1989. Mutants of Arabidopsis thaliana with altered

phototropism. Planta 178: 400-406.

Khurana, J.P., Ren, Z., Steinitz, B., Parks, B., Best, T.R., and Poff, K.L. 1989.

Mutants of Arabidopsis thaliana with decreased amplitude in their phototropic response.

Plant Physiology 91: 685-689.

Orbovic, V. and Poff, K.L. 1990. An infra-red imaging system for the analysis of

tropisms in Arabidopsis thaliana seedlings (Abstract). Plant Physiology 93(Suppl.): 79.

Poff, K.L. 1989. A genetic system for the analysis of gravitropism in Arabidopsis

thaliana (Abstract). ASGSB Bulletin 3: 99.

Poff, K.L. and Martin, H.V. 1989. Site of graviperception in roots: a re-examination.

Physiologia Plantarum 76:451-455.

73
CALCIUM MESSENGER SYSTEM IN

PLANTS

B.W. Poovaiah

Laboratory of Plant Molecular Biology

and Physiology

Department of Horticulture

Washington State University

Pullman, WA 99164

GRAVITROPIC RESPONSE IN

Description of Research

Investigations during the last decade indicate that calcium and calmodulin play a central role

in gravity signal transduction in plants. Recent results suggest that key enzymes involved

in the calcium messenger system such as calcium- and calmodulin-dependent protein

kinases can be modulated in two ways: (1) alteration in the calcium flux in response to the

signal which activates calcium-calmodulin-dependent enzymes and (2) changes in the

amount of calmodulin. Thus the calcium messenger system may involve changes in the

concentrations of both the messenger (calcium) and receptor (calmodulin). It is likely that

target cells regulate the levels of these components to evoke a specific response. In our

laboratory we have taken the approach that one or both of these possibilities exist in gravity

signal transduction.

Accomplishments

We have cloned a plant calmodulin eDNA and shown signal-induced changes in the

expression of calmodulin (Proceedings of the National Academy of Sciences, USA 86:

3644-3648, 1989). To study the consequences of altered levels of calmoduIin, we

transformed tobacco protoplasts with calmodulin cDNA driven by cauliflower mosaic virus

(CaMV) 35S promoter. We observed increased levels of calmodulin mRNA and

calcium-dependent protein phosphorylation in protoplasts transformed with

calmodulin. To further study the expression of calmodulin mRNA and the level of

protein in transgenic plants, we regenerated several independent transgenic plants

containing short (mostly the coding region) or long (the entire cDNA containing 5' and 3'

untranslated regions) constructs in sense or antisense orientation driven by the CaMV 35S

promoter (Figure 1).

Characterization of these plants revealed up to 50-fold higher levels of

calmoduHn mRNA in plants containing the sense constructs, whereas plants

carrying the long construct in antisense orientation showed generally lesser

amounts of the calmoduHn message as compared to controls. In addition, the

steady-state level of the calmodulin antisense RNA was much lower compared to the

calmodulin mRNA driven by the CaMV 35S promoter. However, the amount of

calmodulin protein was, at most, two-fold higher in sense plants, and there was a slight

reduction in the level of calmodulin in plants carrying the long antisense construct as

compared to control. These results suggest that the amount of calmodulin in the plant cell

is regulated stringently at the translational and/or post-translational level.

T

To detect calmodulin'binding proteins, microsomal fractions were obtained from the tip and

base of corn roots and analyzed for calmodulin-binding proteins using a [1251] calmodulin

gel overlay assay. A distinct difference with respect to number and abundance of

calmodulin-binding proteins was observed between the microsomal membrane fractions

74
from thetip andthe baseof theroot. The root tips werefound to havemorecalmodulin

and also more calmodulin-binding proteins. These results suggest the existence of

coordinated regulation in the expression of calmodulin and its target

peptides.

Changing the orientation of corn roots from a vertical to a horizontal position increased the

phosphorylation of specific polypeptides. These changes were observed within 3 min after

changing the orientation. Likewise, depletion of Ca 2+ with EGTA reduced this

phosphorylation, and replenishment of Ca 2+ restored it.

I I" |

I I i II

R ZD P XH R

I I I I II I

t

"°, ,°"

,, o°,"

• o

• ,o °.

"°.° °,_'

11.6- DZLORCB

I

\

°°;t. )

LA

I

5.8

pGA924

pGA 926

Figure 1. Illustration of the constructs of calmodulin that were used for stable transformation.

Cloning of calmodulin cDNA from the pPCM-1 clone (Jena et al., Proceedings of the

National Academy of Sciences USA 86: 3644-3648, 1989) into pGA748 in both sense and

antisense orientations, pGA924-925 contains the short fragment (Scal-Hpal fragment) of

calmodulin cDNA, mostly the coding region, in sense and antisense orientation, respectively

(short constructs), pGA926-927 contains the intact EcoRI fragment of calmodulin cDNA

including the 5', 3' untranslated regions and the coding region (long constructs). A, Sacll; B,

Bgll; C, Clal; D, Hindl]l; H, tIpal; I, Mlul; K, Kpnl; L, Sail; M, BamHi; O, Xhol; P, Sphl;

R, EcoR]; S, Sacl; T, Pstl; V, EcoRV; X, Xbal; Z, Scal.

Significance of the Accomplishments

The seedlings of R1 and R2 generations of transgenic plants could be used to study the role

of calmodulin in gravitropism. Furthermore, our observation that there is a coordinate

75
regulation of calmodulin and its binding proteins in roots suggests that the binding proteins

might have a role in the calcium messenger system. Our studies also indicate that calciumdependent changes in protein phosphorylation are involved in gravity signal transduction.

Publications

Friedmann, M., Mannan, R.M., and Poovaiah, B.W. 1990. Calmodulin-binding proteins

and protein phosphorylation in corn root tips (Abstract). Plant Physiology 93(Suppl.): 79.

Jena, P.K., Reddy, A.S.N., and Poovaiah, B.W. 1989. Molecular cloning and

sequencing of a cDNA for plant calmodulin: Signal-induced changes in the expression of

calmodulin. Proceedings of the NationaI Academy of Sciences, USA 86: 3644-3648.

Jena, P.K., Wang, Z., Bollinger, J., and Poovaiah, B.W. 1990. Characterization of

auxin-regulated calmodulin gene expression in strawberry fruit (Abstract). Plant

Physiology 93(Suppl.): 3.

Mannan, M., Jena, P.K., and Poovaiah, B.W. 1989. Role of calmodulin and calmodulindependent multifunctional protein kinase in gravitropism (Abstract). ASGSB Bulletin 3:

131.

Reddy, A.S.N., Chengappa, S., and P_xwaiah, B.W. i990. Induction and repression of

specific genes by auxin in developing strawberry fruit (Abstract). Plant Physiology

93(Suppl.): 3.

Wang, Z., Reddy, A.S.N., An, G., and Poovaiah, B.W. 1990. Transformation of

tobacco with sense and antisense constructs of calmodulin (Abstract). Plant Physiology

93(Suppl.): 157.

76
EFFECTS OF HYPERGRAVITY ON SPORE

SENSITIVE FERN ONOCLEA SENSIBILIS

V. Raghavan

Ohio State University

Department of Botany

Columbus, OH 43210

GERMINATION IN THE

Description of Research

Previous studies have shown that germination of spores of the sensitive fern Onoclea

sensibilis is delayed when they are subjected to hypergravitational forces of low to

moderate intensity and that the delayed germination and recovery are accompanied by the

synthesis of new proteins. The long-range goal of this research is to analyze the role of

centrifugation-stress proteins during germination of spores of O. sensibitis and to use this

as a model system to study the molecular biology of centrifugation stress in plants.

Shock proteins are known to be synthesized when cells are subjected to various treatments

such as temperature or pressure. The possible role of these proteins in compensating for

gravity-induced stress during germination of spores of O. sensibilis was investigated by

pulsing spores at various times after centrifugation with _5S-methionine, followed by

SDS-PAGE and fluorography. Results of these experiments showed that at least six new

proteins are synthesized after hypergravity stress, the new bands showing up stronger in

spores stressed at 10,000 g than in spores stressed at 100 g. The bands were visible within

the first two hours after centrifugation and persisted for the next 4-8 hours, but disappeared

as spores recovered from stress. These results suggest that hypergravity stress

may be tied to specific changes in gene expression as part of the spore's

strategy to overcome the effects of the stress. From this perspective the general

thrust of our research is to construct and screen a cDNA library of mRNA sequences from

centrifuged spores and to select cDNA clones specific for centrifugation stress. By a

multifaceted approach involving Northern blot, RNA dot blot, and Southern transfer, we

intend to determine the centrifugation-stress specifically of the cDNA clones, the timing of

their transcription, their relative abundances during germination, and their copy number.

Preparatory to the proposed work on the isolation and characterization of centrifugationstress mRNA sequences, we have already made a cDNA library to polyA+RNA of dormant

spores and by differential screening have isolated several germination-specific clones.

Accomplishments

(1) Construction of cDNA library to polyA+RNA of dormant spores

by the Gubler and Hoffman method using a commercial cDNA kit.

(2) Primary screening of transformed bacterial clones with random-prime labeled

single-stranded cDNA prepared from polyA+RNA of dormant spores.

(3) Secondary screening of positive clones with single-stranded cDNA probes

prepared from 20-day old gametophytes and leaves of O. sensibilis and identification of

spore-specific recombinants.

(4) Establishment of spore-specificity of recombinants by Northern blot of RNA

from spores, gametophytes, and leaves with labeled probes of recombinants.

77
(5) Selectionof germination-specificmRNAclonesbyNorthernblotof RNA from

germinating spores and dark-imbibed non-germinating spores probed with labeled

recombinants.

Significance of the Accomplishments

We have isolated from dormant spores of O. sensibilis a series of mRNA sequences which

are used to code for proteins of germination. Since the hypergravity environment delays

spore germination, with a concomitant appearance of new proteins, we are in a position to

follow the effect of centrifugation stress on the expression of these mRNA sequences.

This information, along with our proposed studies on the isolation of cDNA clones specific

for centrifugation-shock, will provide great insight into the molecular biology of

centrifugation effects on the germination of spores of Onoclea sensibilis.

Publications

Raghavan, V. 1989. Developmental Biology of Fern Gametophytes.

Cambridge University Press, 361 p.

Cambridge:

78
CELLULAR AND MOLECULAR PROCESSES

RESPONSE

David L. Rayle

Department of Botany & Plant Pathology

Oregon State University

Corvallis, OR 97331

OF THE GRAVITROPIC

Description of Research

The mechanism by which plants transduce information about the direction of gravity into a

predictable pattern of growth is an interesting problem in developmental biology and has

important ramifications regarding our ability to grow and utilize plants in the microgravity

environment of space. When a plant shoot is placed in a horizontal position, it begins to

curve upward in a smooth arc within minutes, and it reestablishes its original vertical

orientation within several hours. For the purpose of discussion and experimentation, this

phenomenon, known as negative gravitropism, can be divided into three components: (1)

gravity perception, (2) signal transduction, and (3) asymmetric cell elongation. The longrange goal of this research is to understand the cellular and molecular processes that occur

between signal transduction and asymmetric growth.

Asymmetric shoot growth in most plant species involves a reduced rate of cell elongation

on the upper side of a gravistimulated shoot and an increased rate of cell elongation on the

lower side relative to vertical controls. It is quite likely that an asymmetric distribution of

some growth factor must precede asymmetric cell elongation. There is a large volume of

literature suggesting that, in shoots, the hormone auxin (IAA) is a growth factor that

modulates asymmetric shoot elongation. This in turn makes it likely that auxin-specific

receptors are an essential link between transduction events and the resulting response of

gravistimulated tissue. Thus, a logical starting point toward a molecular understanding of

the gravitropic response is the identification and characterization of auxin-specific

receptors.

The analysis of mutants is a powerful approach to determining the physiological function of

certain proteins. Several mutants of higher plants exist which are insensitive to the growth

hormone auxin. The tomato (Lycopersicon esculentum, Mill.) mutant known as

diageotropica (dgt) is a particularly promising candidate for an analysis of auxin-regulated

growth and development. Diageotropica is a spontaneous, single gene recessive mutant of

the parental variety VFN8. Mutant plants do not elongate in response to IAA. They also

exhibit other morphological abnormalities which suggest that these plants may have a defect

associated with a primary site of auxin perception or action. We have recently shown that

the photoaffinity auxin analogue 3H-5N3-IAA (azido-IAA) labels a polypeptide doublet at

40-42 kD at high specific activity in membrane preparations from shoots of VFNS, but the

identical procedure fails to label polypeptides from dgt shoots. These findings suggest that

this 40-42 kD polypeptide doublet is a physiologically important auxin receptor which

displays an altered pattem of expression in the mutant. Additional evidence supports the

notion that these polypeptides are receptor proteins. The polypeptides are: (1) observed

only in plant tissues that respond to IAA, (2) present at low abundance, (3) associated with

the plasma membrane, (4) saturable with increasing concentrations of IAA, and (5) specific

for binding to those auxin analogs which are also active auxins or specific antagonists.

The next step towards more precisely identifying the nature of the 40-42 kD polypeptides is

the isolation of specific cDNA clones encoding these auxin-binding polypeptides. In

79
working towardthis goal,anoligonucleotideprobecorrespondingto aninternalregionof

anauxinbindingproteinwasusedto screena tomatofruit cDNA library in )_gt 11. Out of

200,000 recombinant plaques screened, 12 apparent positive clones were selected. Of

these, four remained clearly positive upon rescreening. These clones are currently being

characterized as to insert size. Verification of the identity of the cloned cDNAs will come

from a variety of experimental approaches. These will include: direct sequence

comparison with known protein and nucleic acid sequence information; expression of the

proteins, either by direct expression of the fusion proteins or by translation of in vitro

transcripts followed by azido-labeling, and/or the complementation of the dgt phenotype by

the production of plants transgenic for wild type sequences.

Accomplishments

(1) Auxin-binding proteins were photoaffinity labeled by the addition of 3H-5N3-

IAA to membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by

subsequent PAGE and fluorography,

(2) At -196°C, high specific-activity labeling of a 40-kD and a 42-kD

polypeptide was observed in a variety of plant species.

(3) Shoots of the tomato

have reduced amounts of the 42

controls.

mutant diageotropica (dgt) were found to

kD-labeled polypeptides relative to wild-type

(4) The above findings and physiological studies of d.gt and wild-type tomato

seedlings suggest the radiolabeled polypeptides are auxm receptors.

(5) A tomato library was screened in an attempt to isolate specific cDNA clones

encoding these auxin-binding polypeptides.

(6) Preliminary information suggests we have isolated clones encoding the 40-42

kD polypeptides.

Significance of the Accomplishments

It has been shown that the azido-labeled 40-42 kD membrane proteins are (1) ubiquitous in

plant tissues that respond to IAA, (2) of low abundance, (3) saturable with increasing

concentrations of IAA, and (4) capable of binding specific analogues that are also active

auxins or specific antagonists. These data suggest that the two polypeptides are part of a

physiologically importmat auxin receptor system. It is hoped that further characterization of

these polypeptides will make it possible to dissect the mechanism of auxin action and thus

provide us with a clearer understanding of plant gravitropism. Toward that goal, cDNA

clones have been isolated whlch appear to encode the 40-42 kD polypeptides. Sequence

information from clones representing both dgt and wild-type genes should help to

distinguish both the nature of the mutation and the relatedness of these polypeptides to

other recently characterized auxin binding proteins. Such knowledge should prove useful

in efforts to manipulate the growth and yield of plants.

Publications

Daniel, S.G., Rayle, D.L., and Cleland, R.E. 1989. Auxin physiology of the tomato

mutant diageotropica. Plant Physiology 91: 804-807.

80
Daniel, S.G.,Rayle, D.L., and Cleland, R.E. 1989. Auxin physiology of the tomato

mutantdiageotropica (Abstract). Plant Physiology 89(4, Suppl.): 111.

Hicks, G.R., Rayle, D.L., and Lomax, T.L. 1989. The diageotropica mutant of tomato

lacks high specific activity auxin binding sites (Abstract). Plant Physiology 89(4, Suppl.):

111.

81
ASSESSING POTENTIAL

GRAVITROPISM

Stanley J. Roux

Department of Botany

University of Texas

Austin, TX 78713

TARGETS OF CALCIUM ACTION IN

Description of Research

Light greatly accelerates the gravitropic response of roots, coleoptiles, and stems in a wide

variety of plants. This indicates that some cellular response initiated by light affects at least

one of the gravity-induced cellular responses necessary for gravitropism. Our research

objective is to identify one or more of these responses, thus clarifying the cellular

mechanisms regulating gravitropic growth.

Several lines of evidence indicate that calcium may play an important role in transducing the

stimuli of both light and gravity into growth changes in plants. However, full confidence

in the validity of this hypothesis will require that at least two additional pieces of evidence

be obtained: (1) the identification of specific targets of calcium action that have a clear and

significant impact on growth and (2) a demonstration that the gravity and/or the light

stimulus alters the concentration of cytosolic free calcium ([Ca2+]cyt) in the responding

cells. As yet there is little information on these two critical sets of evidence. This past year

we carded out some experiments aimed at narrowing this information gap.

In most well-described cases, the immediate target of calcium action during the transduction

of an environmental stimulus is a calcium-binding protein. Such proteins tend to be highly

conserved evolutionarily, with very similar types occurring in both plants and animals.

The best known target of calcium action in plants and animals is the calcium-binding

protein calmodulin. More recently, calcium-binding kinases, including protein kinase C

and phospholipid-independent kinases, have been described in both plants and animals.

Still other calcium-binding proteins are well characterized thus far only in animals,

including, prominently, the annexin family of proteins. One sub-set of this family was

originally named the calcimedins, and two years ago we reported finding cross-reactivity

between a pea antigen and an antibody to a mammalian calcimedin. This year we

succeeded in partially purifying and characterizing an annexin-like protein from peas, and

here we describe these results.

Wall isoperoxidases have been reported to be under the control of calcium, and last year we

reported evidence that a 89 kD acidic isoperoxidase present in the cell walls of corn tissues

may help mediate the growth responses of corn coleoptiles and mesocotyls to light. These

results predicted that selectively inhibiting this isoperoxidase could alter growth rates in

corn. This year we report the results of our initial efforts to inhibit this isoperoxidase in

situ by reacting it with a monoclonal antibody that blocks its activity.

Detecting gravity-induced changes in [Ca2+]cyt i n complex multicellular tissues would be

technically difficult. This measurement, however, would be easier in organisms with only

a few cells. This past year we succeeded in measuring [Ca2+]cyt in germinating spores of

the fern Onoclea and in the rhizoid cell produced by the first cell division of the newly

germinated spore, using incorporated fura-2 as a fluorescent indicator of [Ca2+]cyt in these

cells. The rhizoid cell of a newly germinated Onoclea spore shows normal growth and

82
gravitropismevenwith fura-2presentin its cytoplasm.This findingsetsthestagefor usto

learn whether [Ca2+]cyt changesin the rhizoid cell when it is gravistimulated, i.e.,

reorientedby 90°.

Accomplishments

(1) We have purified to near homogeneity a calcium-binding

calpactin-like protein (CLP) from peas. Like animal calpactin, it has a mass of 35

kD and binds to phospholipids in a calcium-dependent fashion. It also cross-reacts with

antibodies raised to animal calpactin, a protein thought to help regulate calcium-dependent

secretion from cells.

(2) We have raised polyclonal antibodies to the pea CLP and have completed an

initial study of its localization in pea stem and root cells. We found that the CLP appears to

be most concentrated in the periphery of cells that are actively engaged in secretion, such as

root cap cells and young developing tracheary elements.

(3) Perfusing coleoptile segments of etiolated corn seedlings with a

monoclonal antibody (mAb) that binds to and blocks the activity of a 98 kD

wall isoperoxidase inhibits the growth rate of these tissues. Perfusing

coleoptiles with pre-immune serum or mAbs to phytochrome (a cytosolic protein) has no

effect on their growth.

(4) The calcium-sensitive fluorescent dye, fura-2, can be

successfully electroporated into intact fern spores and into intact newly

germinated gametophytes, such that they continue to show both normal growth and

development and normal responsiveness to light and gravity after the electroporation and

incorporation of the dye. The dye appears to be restricted to the cytoplasm, where the

[Ca2+]cyt measured is 0.1-0.2 _tM.

Significance of the Accomplishments

Findings #1 and #2: As yet there is only one other report of an annexin-like protein in

plants, and it is preliminary and incomplete. Our results provide more convincing evidence

both that calpactins are present in plants, and that they may play a role in secretion, a critical

process which is likely to be a major control point in the regulation of growth.

Finding #3: All prior reports on the use of antibodies to inhibit wall enzyme activity also

report growth inhibition by the antibodies. Because peroxidases are thought to inhibit

growth by their cross-linking activities, inhibition of these wall enzymes would

theoretically be expected to promote growth. The fact that growth is inhibited by mWP3

suggests either that peroxidase function may be needed for normal cell wall extension or

that the reaction of any antibody with any antigen in the wall can create steric problems that

nonspecifically inhibit growth. Further experiments will be needed to resolve which of

these two alternatives is the more likely explanation of the results.

Finding #4: Successful incorporation of fura-2 into intact fern cells such that they continue

to grow, develop, and respond to growth stimuli normally after the dye incorporation will

allow us to measure even very rapid kinetics of any calcium changes that result from either

gravitropic or light stimulation. Thus the germinating fern spore can serve as a valuable

model system for directly testing whether phytochrome activation or gravitropic stimuli can

induce changes in [Ca2+]cyt.

83
Publications

Clark, G., Dauwalder, M., and Roux, S.J. 1989. Partial purification and characterization

of calcium-phospholipid binding proteins in peas (Abstract). Journal of Cell Biology 109:

304a.

Kim, S.-H., Shinkle, J.R., Reichler, S., and Roux, S.J. 1989. Dynamic changes in the

immunodetectable level of a wall peroxidase precede growth changes in maize seedlings

(Abstract). ASGSB Bulletin 3: 94.

Kim, S.-H., Shinkle, J.R., and Roux, S.J. 1989. Phytochrome induces changes in the

immunodetectable level of a wall peroxidase that precede growth changes in maize

seedlings. Proceedings of the National Academy of Sciences USA 86: 9866-9870.

Li, H. and Roux, S. 1990. Partial characterization of a calcium-dependent protein kinase

from the chromatin of pea nuclei (Abstract). Plant Physiology 93(Suppl.): 63.

Roux, S.J. 1990. Calcium as a mediator of plants' directional growth response to gravity.

In: Fundamentals of Space Biology (ed. by M. Asashima and G.M. Malacinski).

Tokyo/Berlin: Japan Scientific Societies Press/Springer-Verlag, p. 57-67.

Roux, S.L 1990. Calcium-regulated metabolism in seed germination. In: Recent

Advances in the Development and Germination of Seeds (ed. by R.B. Taylorson). New

York: Plenum Press, p. 127-138.

Scheuerlein, R., Wayne, R., and Roux, S.J. 1989. Calcium requirement of

phytochrome-mediated fern-spore germination: No direct phytochrome-calcium interaction

in the phytochrome-initiated transduction chain. Planta 178: 25-30.

84
CELLULAR POLARITY

GRAVIPERCEPTION

Fred D. Sack

Department of Plant Biology

Ohio State University

1735 Neil Avenue

Columbus, OH 43210

AND INTERACTIONS IN PLANT

Description of Research

Our long term goal is to understand the mechanism(s) of gravitropic sensing in higher and

lower plants. Although it had long been thought that the movement of amyloplasts

heavy, starch-filled plastids -- probably triggers sensing, the isolation of several starchdeficient, but gravity-perceiving, mutants indicates that amyloplasts are not necessary for

sensing. However, in the last few years, we have demonstrated that starchless roots of

Arabidopsis and starch-deficient roots of tobacco are less sensitive to gravity than are wildtype roots with amyloplasts. We therefore concluded that starch (or presumably the weight

it confers) is necessary for full sensitivity and that amyloplasts function as statoliths.

Last year we provided an initial description of gravitropism in the moss Ceratodon, which

has filaments of cells (protonemata) that grow upward in the dark. We found that this cell

has a distinct plastid zonation which includes a zone that is apparently specialized for

amyloplast falling when the cell is horizontal, but not vertical. Also, using infrared

videomicroscopy to study upward curvature in living cells reoriented to the horizontal, we

found that before this cell curves up, there is a "wrong-way" (downward) curvature that

takes place within minutes after reorientation.

Accomplishments

(1) A tobacco mutant with reduced starch has very disoriented,

weakly gravitropic stems (hypocotyls). Electron microscopy confirmed that the

tobacco mutant stems contain plastids with <10% of wild-type (WT) starch in comparable

cells which have sedimented amyloplasts in the WT. Vertically grown (in the dark) mutant

hypocotyls are severely disoriented (ratio of variabilities of mutant: WT = 5.3). In

contrast, in vertically maintained tobacco roots, the corresponding ratios for light and darkgrown roots were 1.3 and 1.6, respectively. The tobacco mutant hypocotyls are somewhat

gravitropic since (a) they are oriented a mean of 47 ° from the vertical (vs. 11o for the WT)

and (b) upon a 90 ° reorientation, they curve upwards 10 ° in 72 hours (vs. 70 ° in the WT).

(2) The presumed gravity-sensing cells in roots have been visualized

in the electron microscope after entire roots were rapidly frozen using high

pressure freezing followed by freeze substitution. The cytoplasmic faces of all

membranes displayed electron dense coats. Other new details of columella cell structure

were seen such as vacuolar contents, turgid compartments, and intercisternal elements in

the Golgi apparatus.

(3) Gravitropic curvature in corn roots does not start with an asymmetrical growth

of the rootcap itself. Over the years, it has been reported that the very first growth

response of a root to gravistimulation is a differential growth of the rootcap, a differential

which later extends to the elongation zone. Growth markers were placed on the rootcap of

85
(4) Amyloplast sedimentation correlates with the recovery of

gravitropism after basipetal centrifugation of Ceratodon protonemata. When

amyloplasts were centrifuged out of the tip, growth resumed but gravitropism was absent

until amyloplasts migrated back close to the tip and sedimented. Upward curvature

sometimes took place without amyloplasts in the apical dome, but wrong-way curvature

only occurred when amyloplasts were present in the very tip (apical dome).

Significance of the Accomplishments

Finding #1: The severely reduced gravitropism of the Nicotiana mutant hypocotyls

demonstrates again that starch is necessary for full gravitropic sensitivity and that

amyloplasts function in gravitropic sensing hypocotyls. While starch-deficiency depresses

gravitropic functioning much more in hypocotyls than in roots, it does not completely

eliminate all sensing. It is hypothesized that the starch-deficient plastids that remain in the

mutant account for the residual sensing that exists in the hypocotyls. A comparison with

Arabidopsis hypocotyls (starchless mutant and WT) is in progress.

Finding #2: Development of a successful protocol for freezing an entire plant organ

without ice crystal damage means that one of the first freeze-substituted cells to be seen

below the surface of an organ is of the cells that are presumed to sense gravity in roots.

This new technique provides a means of preserving tissues that appears to be less

artifactual than conventional chemical fixation techniques. This technique should not only

provide a more accurate view of the structure of these cells, but it should also open up the

possibility of doing high-resolution immunolabelling, e.g., of labile membrane antigens in

cells that have been heretofore inaccessible.

Finding #3: This experiment shows that the rootcap is not the initial target of its own

orientational signals in corn. This was one of the most sensitive assays used to study this

question. Thus, rootcap asymmetry is not a universal feature of gravitropism, and this

method provides a way of testing other genera to determine where, if anywhere, in the

rootcap an asymmetry develops.

Finding #4: These and previous results suggest that the sedimenting amyloplasts function

in gravitropic sensing in upward curvature and that the non-sedimenting amyloplasts in the

apical dome are associated with an initial wrong-way curvature. This is the first modern

description of a cell that both perceives and responds to gravity and that appears to employ

amyloplasts as statoliths. These results reinforce the concept that the sedimentation of

amyloplasts is generally associated with sensing. But the absence of dramatic

sedimentation before the start of wrong-way curvature in Ceratodon protonemata indicates

that sedimentation is not required fbr the earliest phase of protonematal gravitropism. An all

or none interpretation regarding the importance of sedimentation appears inappropriate.

Publications

Giddings, T., Staehelin, L.A., Kiss, J., and Sack, F. 1990. Differentiation of Golgi in

high-pressure-frozen root tips (Abstract). Plant Physiology 93(Suppl.): 77.

Kiss, J. and Sack, F. 1989. Reduced gravitropic sensitivity in a starch-deficient Nicotiana

mutant (Abstract). ASGS B Bulletin 3: 123.

Kiss, J.Z., Giddings, T.H., Jr., Staehelin, L.A., and Sack, F.D. 1990. High pressure

freezing/freeze substitution of root tips (Abstract). Plant Physiology 93(Suppl.): 37.

I

=

m,

86
Kiss, J.Z. and Sack,F.D. 1989. Reduced gravitropic sensitivity in roots of a starchdeficient mutant ofNicotiana sylvestris. Planta 180: 123-130.

Kiss, J.Z., Staehelin, L.A., and Sack, F.D. 1989. Superior preservation of columella

cells by high-pressure freezing (Abstract). ASGSB Bulletin 3: 126.

Kiss, J.Z., Young, J.C., and Sack, F.D. 1990. Severely reduced gravitropism in

hypocotyls of a starch-deficient Nicotiana mutant (Abstract). Plant Physiology 93(Suppl.):

39.

Sack, F.D. and Kiss, J.Z. 1989. Plastids and gravity perception. In: Physiology,

Biochemistry, and Genetics of Nongreen Plastids (ed. by C.D. Boyer, J.C. Shannon, and

R.C. Hardison). Rockville, MD: American Society of Plant Physiologists, p. 171-181.

Schwuchow, J., Sack, F., and Hartmann, E. 1989. Gravity affects the distribution of

microtubules in gravitropic protonema of the moss Ceratodon (Abstract). ASGSB Bulletin

3: 123.

Walker, L. and Sack, F. 1989. Different classes of amyloplasts in the gravitropic tip cell

of the moss Ceratodon purpureus. In: Physiology, Biochemistry, and Genetics of

Nongreen Plastids (ed. by C.D. Boyer, J.C. Shannon, and R.C. Hardison). Rockville,

MD: American Society of Plant Physiologists, p. 258-260.

Walker, L. and Sack, F. 1989. Recovery of gravitropism after centrifugation correlates

with amyloplast sedimentation (Abstract). ASGSB Bulletin 3: 125.

Walker, L.M. and Sack, F.D. 1990. Amyloplasts as possible statoliths in gravitropic

protonemata of the moss Ceratodon purpureus. Planta 181: 71-77.

Walker, L.M. and Sack, F.D. 1990. Recovery of gravitropism after basipetal

centrifugation in Ceratodon protonema (Abstract). Plant Physiology 93(Suppl.): 39.

White, R.G. and Sack, F.D. 1990. Actin microfilaments in presumptive statocytes of root

caps and coleoptiles. American Journal of Botany 77: 17-26.

Young, J.C. and Sack, F.D. 1990. Time-lapse analysis of gravitropism in Ceratodon

protonema (Abstract). Plant Physiology 93(Suppl.): 78.

87
GRAVITROPISM IN DICOT STEMS

Frank B. Salisbury

Plants, Soils, and Biometeorology Department

Utah State University

Logan, UT 84322

Description of Research

We continue to concentrate on the role of auxin (indoleacetic acid, IAA) in the gravitropic

response of stems. The Cholodny-Went hypothesis explains gravitropic stem bending by

suggesting that auxin moves to the lower side of a horizontal stem, causing those tissues to

grow more than top tissues and thus causing the stem to bend up. We have emphasized an

alternative mechanism; namely, that tissue sensitivity to auxin changes in response to

gravistimulation whether auxin concentration changes or not_We obtain dose-response

curves for upper and lower surfaces of stem segments immersed in auxin solutions over a

wide concentration range (0, 10 -8 to 10 -2 M IAA). Sections are photographed at intervals

(usually half an hour), and negatives are projected onto a digitizer. Suitable software is

used to measure bending and growth in length of top and bottom surfaces. Maximum

growth of bottom tissues occurs in zero or very low auxin concentrations; higher

concentrations progressively inhibit growth. Growth of top tissues, on the other hand, is

minimal in low auxin concentrations, increasing to a maximum at about 10 -4 M IAA and

then decreasing in the higher concentrations. Maximum growth of top tissues never quite

equals maximum growth of bottom tissues. Because of these relationships, stem segments

bend up in low auxin solutions and down in high auxin concentrations (because top

surfaces are growing more than bottom surfaces). These data suggest that Vma x sensitivity

of top tissues is never as great as that of bottom tissues (because maximum growth is

always less), and K m sensitivity of bottom tissues greatly exceeds that of top tissues

(because much lower auxin concentrations will cause one half of maximum growth).

During the past year, we began studies of the kinetics of changing sensitivity induced by

gravistimulation to auxin in top and bottom tissues. We have investigated effects of

temperature and the initial changes in stem growth immediately after stems are turned to the

horizontal. We have also tested some species other than the sunflower and soybeans used

previously.

Accomplishments

(1) For several months after moving to a new laboratory, we did not have

temperature control in the two physiological darkrooms. Experiments were run at room

temperature (18-22oc) instead of the higher temperature (33oc) we had used previously.

Bending was reduced, and the downward bending at high auxin concentrations was often

not apparent. Eventually, temperature control was installed in both darkrooms, so we

could compare sunflower hypocotyl sections from the same batch at two different

temperatures. No new phenomena were discovered. Although bending is slower at

reduced temperatures, and sections at lower temperatures sometimes fail to bend as much

as those at higher temperatures, in some experiments bending is essentially the same at both

temperatures if one waits for segments at the lower temperature to catch up with those at the

warmer temperature. Because sections are immersed in non-aerated solutions, it may not

be surprising that bending is somewhat reduced when it is extended (i.e., by cool

temperatures) over a long interval.

88
(2) We have observedthe kinetics of bending and growth of top and bottom

surfacesfollowing gravistimulation. Changeswere small after short times, so thereis

someuncertaintyin theresults. Nevertheless,therewasclearly a lag of about 30to 60

minutesafter gravistimulationbeforecurvaturebegan;that is, beforegrowth of top and

bottom surfacesdiffered. Typically, sectionsbentdown slightly during that initial lag

period,eventhough,at all concentrations,therewasa slight burstof growth that lasted

about15minutesimmediatelyfollowing gravistimulation(i.e.,topgrewslightly morethan

bottom). Then, after about 2.5 hr in buffer with no auxin or in 10 -8 M IAA,

there was a sharp increase in growth rate of the bottom surface; growth rate

of top surfaces stayed fairly constant. As auxin concentration increased,

the lag time became shorter until, at about 10 -5 M IAA and higher, there

was no detectable lag but a large burst of rapid growth of both surfaces that

slowed after about 15 minutes.

(3) We tested the following species in our system, using hypocotyl, stem, or

coleoptile sections from seedlings: cabbage, pea, tomato, maize, and oats. Responses

were similar to those of sunflower and soybean, but there were some interesting

differences. In several experiments, pea stem sections failed to bend (although we did

observe the usual response once). Although we grew the plants at different temperatures as

well as in light or darkness, we have not resolved the discrepancies. Variability was

always high. We were interested in maize because most evidence for the Cholodny-Went

hypothesis has been obtained with monocots, including maize. In three experiments

completed so far, maize coleoptiles exhibited bending, much like sunflower and

soybean hypocotyls: rapid upward bending zn zero and low auxin

concentrations, virtually no bending or some downward bending (one

experimenO at the highest concentrations. A single analysis of top and bottom

growth of maize coleoptiles, however, produced the unexpected result that growth of

bottom surfaces continued to increase with increasing auxin, reaching a maximum at 10 -3

M IAA, the same auxin concentration that produced maximum growth of the top surface,

which was a little higher, accounting for the downward bending. Growth of the top

surface was essentially zero in buffer, increasing to its maximum at 10 -3 M IAA, but

increasing more steeply than did bottom growth.

Significance of the Accomplishments

Finding #1: Temperature experiments were somewhat disappointing. We had expected to

find qualitative differences at cool and warm temperatures (based on earlier observations),

but the differences proved to be only quantitative.

Finding #2: Studies of the lag in bending are more interesting. We wonder if this lag time

is required for sensitivity to auxin to change in response to gravistimulation.

Disappearance of the lag at high auxin concentrations is especially interesting. It is as if

auxin penetration and response to auxin were virtually instantaneous at those

concentrations. If the initial lag represents the development of auxin sensitivity, its

shortening by higher auxin concentrations suggests (as others have also suggested) that

sensitivity to auxin can develop in response to auxin itself and that higher concentrations

lead to more rapid increases in auxin sensitivity. In bottom tissues, however, sensitivity is

already so great at zero or very low external auxin levels that endogenous auxin leads to

maximum response, and increases in solution auxin push the response system over the top

of the curve to the descending or inhibitory part. The striking thing remains the marked

difference in response to auxin of top and bottom tissues.

89
Finding #3: Resultsso far suggestthat the generalizationgainedfrom sunflowerand

soybeancan probably be extendedto other species,but the observeddifferences in

responsearealsointeresting. It is especiallyimportant to extendtheobservationswith

maizeto seeif thepatternobservedsofar appearsin futureexperiments.

Publications

Bugbee, B.G. and Salisbury, F.B. 1989. Controlled environment crop production:

Hydroponic vs. lunar regolith. In: Lunar Base Agriculture: Soils for Plant Growth.

Madison, WI: ASA-CSSA-SSSA, p. 107-129.

Rorabaugh, P.A. and Salisbury, F.B. 1989. Gravitropism in higher plant shoots. VI.

Changing sensitivity to auxin in gravistimulated soybean hypocotyls. Plant Physiology 91:

1329-1338.

Salisbury, F.B. 1990. Controlled environment life support system (CELSS): A

prerequisite for long-term space studies. In: Fundamentals of Space Biology (ed. by M.

Asashima and G.M. Malacinski). Tokyo/Berlin: Japan Scientific Societies Press/SpringerVerlag, pl_i_i83, .......... _- ...... k_ .: ..... .....

Salisbury, F.B. 1989. Farming on the moon: Reaching the potential of crop productivity.

In: Proceedings of the lOth Annual Conference of the ltydroponic Society of America,

May 5-7, 1989, Tucson, Arizona, p. 47-57.

Salisbury, F.B. and Gillespie, L. 1989. Effects of gravistimulation on sensitivity to

auxin: New methods (Abstract). ASGSB Bulletin 3: 129.

Salisbury, F.B. and Gillespie, L. 1990. Studying the lag in changing sensitivity to auxin

during gravistimulation (Abstract). Plant Physiology 93(Suppl.): 39.

90
AMIDE OXIDASE AND

DIFFERENTIAL GROWTH

PLANTS

Robert D. Slocum

Biology Department

Williams College

Williamstown, MA 01267

PEROXIDASE

RESPONSES IN

REGULATION OF

GRAVISTIMULATED

Description of Research

Our long-term goal is to understand the biochemical and physiological mechanisms by

which plants respond to a perceived gravity stimulus. An elucidation of such mechanisms

will facilitate our understanding and prediction of plant growth responses in altered gravity

environments, such as the hypogravity environment of space.

Our specific goal is to determine the extent to which peroxidase-mediated coupling

mechanisms play a role in the regulation of plant growth by modulating cell wall

extensibility. In particular, we want to characterize specific peroxidase isozymes (PODs)

that are responsive to short-term gravistimulation and, therefore, may regulate differential

growth (i.e., tropistic bending). We expect that cross-linking reactions catalyzed by these

inducible PODs should be largely reversible, while other "housekeeping" PODs may carry

out lignification and other types of cross-linking reactions which are irreversible, providing

long-term structural support for the plant.

We have been examining a new model for plant growth regulation, the key components of

which are cell wall-localized: (1) amine oxidases generating H20 2 as a product of

polyamine oxidation, and (2) PODs utilizing the H202 to carry out cross-linking of

phenolic moieties of wall constituents. This cross-linking would decrease wall extensibility

and growth potential of the tissue. Differential growth accompanying tropistic curvature of

the gravistimulated organ could result from gradients in the activities of amine oxidases,

their polyamine substrates, and/or PODs.

We have shown previously, using both cytochemical and biochemical methods, that lateral

gradients in amine oxidase activities are not seen in either gravistimulated pea epicotyls or

corn coleoptiles. However, these organs do exhibit a lateral asymmetry in concentrations

of polyamines (higher on the upper, slower growing sides, consistent with the model),

which could be sufficient to produce H202 gradients and differential growth responses. In

addition, corn coleoptiles exhibit lateral asymmetries in the expression of certain POD

isozymes, some of which are induced after only 30 min gravistimulation. Expression of a

few of these PODs also appears to be induced by Ca 2+ and auxin, which are thought to

play important, often antagonistic, roles in regulating plant growth. We are presently

characterizing these PODs in relation to the gravitropic bending response in the coleoptile.

Accomplishments

(1) We have carried out kinetic analyses of changes in the activities of soluble and

ionically and covalently bound PODs in gravistimulated corn coleoptiles.

(2) We have shown that the activities of two cationic PODs are increased

on the upper, slower growing sides of gravistimulated corn coleoptiles;

induction of these isozymes occurs as early as 30 rain after presentation of

the gravity stimulus.

91
(3) We havecharacterizedchangesin theexpressionof various POD isozymes in

response to exogenous applications of Ca 2+, the Ca `'`+ ionophore A23187, and H20 2 in

non-gravistimulated corn coleoptiles.

(4) Ca 2+ inhibition of PODs in vitro has been demonstrated.

Significance of the Accomplishments

Finding #1: While the expression of specific isozymes has frequently been correlated with

growth and development in plants, expression is generally examined over a period of days

or weeks. To my knowledge, this is the first time that rapid changes in the

expression of specific POD isozymes has been demonstrated for any growth

response. Our findings suggest that it is largely ionically bound PODs that

respond to the gravity stimulus; thus they are the most IikeIy candidates for further

studies involving the role of PODs in growth regulation. Isozyme staining profiles for

soluble and covalently bound PODs are essentially unchanged during gravistimulation.

Finding #2: After only 30 min, the activities of two cationic POD isozymes are induced in

tissues on the upper, slower growing side of the gravistimulated coleoptile (Figure 1).

Induction of these PODs precedes visible bending in the coleoptile and these enzymes may

initiate cross-linking of the cell wall, setting up the differential growth response. In

addition, a weakly anionic POD from the soluble fraction is expressed in tissues on the

lower side. Interestingly, this anionic POD is responsive to Ca2+; in non-gravistimulated

coleoptiles, its activity is increased in tissues on the side opposite that side to which

exogenous Ca 2+ is applied. All three PODs are found in cell wall exudates. Ca 2+-

dependent secretion of extracellular POD has been reported by other workers and may

function in POD expression in the gravistimuiated coleoptile.

1

2

vertical

control

" 4 Cl

3

130m ni I C2

Figure 1. Kinetic analysis of cationic peroxidase isozyme (POD) expression in gravistimulated corn

coleoptile. Lane numbers on gel correspond to tissue sections from which the PODs were extracted, as

shown in diagram at left. Tissue segments #3-6 were harvested at 30 min, 1 hr, or 3 hr after the onset of

gravistimulation. Each lane represents 5 lag of total protein. Two major isozymes are seen; both are

located predominantly in apical coleoptile tissues. POD activity (isozymes CI and C2) is higher on the

upper, slower-growing section #3 side than on lower side (section #4). In more basally located tissues,

weak POD activity in isozyme C1 is initially higher on lower side (section #6) than on upper side (section

#5), but becomes uniform after 3 hr gravistimulation.

92
I
Finding #3: Unilateral application of H20 2, Ca 2+, and A23187+ Ca 2+ all produce

marked changes in the activities of several weakly anionic/cationic PODs from the soluble

and ionicaUy bound POD fractions. We have observed that application of H20 2 to vertical

coleoptiles stimulates bending toward the side of application and it now seems likely that

this growth response is due to the induction of specific PODs, several of which are

responsive to gravistimulation (see above) or Ca 2+ treatment. The most exciting discovery

to date is that an alternating lateral asymmetry in the activity of one cationic POD is

consistent with an "autotropic straightening" mechanism by which plant growth is guided

relative to the gravity vector.

Finding #4: We wanted to know whether Ca 2+ availability could regulate the activities of

POD, apart from its possible involvement in POD secretion into the cell wall, indirectly

influencing growth by regulating POD-mediated cross-linking reactions. Ca 2+ inhibits the

activity of all classes of PODs in enzyme assays, but only at high concentrations (5-10

mM). Gel staining assays suggest that it is the weakly anionic/cationic PODs that are

inhibited to the greatest extent. Ca 2+, however, is an integral part of the POD molecule

and does stimulate POD activity in preparations in which Ca 2+ has been depleted using a

chelator such as EDTA. In summary, it seems likely that Ca 2+ does not regulate POD

activity directly, but may control POD secretion into the wall.

Publications

Slocum, R.D. and Moyant, E.C. 1989. Characterization of polyamine oxidase activity in

gravistimulated corn coleoptile (Abstract). ASGSB Bulletin 3: 98.

Slocum, R.D. and Weinstein L.H. 1990. Osmotic stress-induced putrescine accumulation

as a mechanism of ammonia detoxification in oat leaves (Abstract). Plant Physiology

93(Suppl.): 24.

Slocum, R.D., Williamson, C.L., Poggenburg, C.A., and Lynes, M.A. 1990.

Immunological characterization of plant ornithine transcarbamylases. Plant Physiology 92:

1205-1210.

93
ANIMAL PROJECTS

___ IN_'_NTIONAL_'t pRE_EDLMG PAGE B_t_,Lw_ l_C}'i rtLWIED

EFFECT OF SKELETAL UNLOADING ON BONE FORMATION

Daniel D. Bikle and Bernard P. Halloran

Veterans Administration Medical Center

and

University of California

San Francisco, CA 94121

Description of Research

The long range goal of this research program is to understand the effects of gravity on

skeletal development and bone metabolism. Present objectives are to define the effects of

gravity or mechanical stress on bone formation and resorption, and to determine the

mechanisms (hormonal or paracrine) by which mechanical stress is coupled to bone cell

activity.

Bone is a dynamic, living tissue. It is continually undergoing change, or remodeling,

which involves a delicate balance between bone formation and bone resorption. This

balance is influenced by systemic hormones such as parathyroid hormone, glucocorticoid

hormones, growth hormone, and the vitamin D metabolites, as well as local factors such as

blood flow, neuromuscular activity, and mechanical stress. Recently, a number of

cytokines have been observed to stimulate or inhibit bone formation and resorption. Some

of these cytokines, such as insulin-like growth factor-1 (IGF-1), transforming growth

factors, and fibroblast growth factors, have been identified in bone. IGF-1 production by

bone appears to be regulated by growth hormone. Such cytokines may participate in

coupling mechanical stress to bone cell activity, or the activity of one type of cell to the

activity of another type of cell, for example, osteoblast activity to osteoclast activity.

The major focus of work conducted during the past year concerned the role of IGF-1 in

bone formation. IGF-1 is produced by bone and stimulates bone formation in an autocrine

or paracrine fashion. Growth hormone stimulates IGF- 1 production in a number of tissues

including bone. Since IGF-1 levels may be reduced in bone from the hindlimb-unloaded

rats (the model of skeletal unweighting used in this research), it was determined whether

either growth hormone, IGF-1, or IGF-2 (an insulin-like growth factor similar to IGF-1

produced primarily in fetal tissues which may also stimulate bone formation infusion) could

reverse the bone loss seen in the tibiae of the unloaded rats. In addition we developed the

cDNA probes to permit the examination of whether unloading leads to a reduction in the

message for IGF-1 and IGF-2.

Accomplishments

(1) Demonstrated that growth hormone sustains growth of bone in

hypophysectomized rats, but that skeletal unweighting blocks this

response.

(2) Demonstrated that IGF.1 had a greater effect on bone mass than

IGF.2, but that neither IGF-1 nor IGF-2 prevented the decrement in bone

mass due to unweighting.

(3) Demonstrated that the growth plate from rat bone contains a comparable

complement of IGF- 1 transcripts to the liver, and that hypophysectomy reduces the amount

of IGF-1 transcript in both tissues to comparable degree.

97

.... INI_NTf0NI[_, _ PRE'CEDIi_G PAGE BLAI,_K NOY t:ILMED
(4) Demonstrated that fetal bone and liver contain IGF-2 transcripts in much higher

concentrations than adult tissues.

Significance of the Accomplishments

Current efforts are directed toward determining whether a systemic hormone, 1,25(OH)2D

(the active metabolite of vitamin D), interacting with a local growth factor, IGF-1, regulates

bone formation, and if so whether it is this regulation which is disrupted by skeletal

unweighting. To date it has not been possible to reverse the inhibition of bone formation

caused by unweighting with infusions of 1,25(OH)2D, IGF-1, or growth hormone which

should increase the local production of IGF-1. Nevertheless, it is known that serum

1,25(OH)2D levels in the blood fall with skeletal unloading, and preliminary evidence

suggests that local levels of IGF- 1 do likewise. We are now in a position to test, in a more

sensitive fashion because of the availability of cDNA probes for the IGF-1 message in bone

whether skeletal unloading suppresses IGF-1 production by skeletal tissue. This will

permit the reexamination of whether iGF-1 production fails with skeletal unloading, and

the reassessment of whether growth hormone and 1,25(OH)2D fail to prevent the loss of

bone with unloading because of their failure to raise IGF-I production.

Publications

Halloran, B.P., Bikle, D.D., Cone, C.M., and Morey-Holton, E. 1989. Skeletal

unloading: Effects of hypophysectomy and growth hormone replacement (Abstract).

ASGSB Bulletin 3:112.

Patterson-Buckendahl, P., Globus, R.K., Bikle, D.D., Cann, C.E., and Morey-Holton,

E. 1989. Effects of simulated weightlessness on rat osteocalcin and bone calcium.

American Journal of Physiology 257:R1103-1109.

Sessions, N.D., Halloran, B.P., Bikle, D.D., Wronski, T.J., Cone, C.M., and MoreyHolton, E. 1989. Bone response to normal weight bearing after a period of skeletal

unloading. American Journal of Physiology 257:E606-E610.

98
PHYSIOLOGY OF DEVELOPING GRAVITY

OTOLITH-OCULAR REFLEXES IN RAT

Robert H. Blanks

Department of Anatomy and Neurobiology

and

Department of Surgery

University of California

Irvine, CA 92717

RECEPTORS AND

Description of Research

Our long-term objective is to examine the effects of microgravity on the physiology of the

developing and adult mammalian gravity receptors. This proposal examines: (1) the

physiological responses of otolith afferents in the adult rat and during postnatal

development, and (2) the otolith organ contribution to the vertical vestibulo-ocular reflexes

(VOR).

These experiments will provide important new information on the physiology of the rat

gravity receptors during development, interaction of otolith-canal information in trochlear

motoneurons and at the motor output (vertical vestibulo-ocular reflex), and will examine the

adaptive behavior of the vestibular system subsequent to otoconial demineralization, i.e.,

reduced otoconial mass, as a model for examining microgravity effects on the developing

and mature otolith system. Such information will be required to interpret the results from

future experiments with orbited rats to assess the effects of microgravity on the developing

and mature labyrinth.

Accomplishments

We have made progress in three areas:

(1) Physiology of adult otolith afferents. Studies have been conducted to

characterize the physiologic response of otolith afferents in the adult rat. In response to

ramp changes in head position, the vast majority of otolith units (57/68) show

predominantly "tonic" responses in which the discharge rate is proportional to head

position and is independent of the velocity of transition. The few remaining otolith units

(11/68) have "phasic-tonic" responses characterized by an overshoot (or undershoot)

during transition followed by a return, within 4-12 sec, to a new steady state rate. Within

the range of stimulus parameters used, tonic and phasic-tonic afferents respond in an

essentially linear manner. These data from first-order otolith afferents will permit a

quantitative comparison between peripheral and central parts of the otolith-ocular pathways

and will allow us to chart the postnatal stages in the physiological development of the

peripheral otolith system.

(2) Physiology of otolith afferents in early postnatal development. Studies have

been conducted to examine the discharge patterns of otolith afferents in neonatal rats.

Otolith afferents of postnatal day 14 rats already have a high level of

spontaneous discharge and show sensitivities to sinusoidal and velocity

steps similar to the adult. Additional experiments are underway to characterize the

other postnatal periods.

(3) Postnatal development of gravity-related postural reflexes; studies using

labyrinthectomized animals and animals with demineralized otoconia as a result of their

99
rearingon manganese-deficientdiets. Considerableprogresshasbeenmadethis yearin

characterizingthepostnataldevelopmentof gravity-relatedposturalreflexes,includingairfighting, surface-righting,swimming, negativegeotaxis,and headelevation. There is

someevidencefor otoconialdemineralizationwithevenshort-durationspaceflight(6days).

The manganese-deficient paradigm produces some variability in the

completeness of otoconial defect from animal to animal. Behavioral testing

revealed that over half of the animals showed asymmetries in three or more tests, and there

was consistent laterality of the symptoms within one animal. The most sensitive indicators

of vestibular disorders in the Mn-deficient animals were swimming, surface- and airfighting. Studies are now underway to analyze the changes in the otoconia of the animals

undergoing the behavioral trials.

Significance of the Accomplishments

Research analysis results on the characterization of the otolith afferents in the adult rat are

now being compared to the same afferents during postnatal development in an attempt to

identify critical milestones for development of the otolith system. Detailed studies in other

sensory systems (visual, auditory) show that it is during development that neural systems

are most susceptible to alterations in the stimulus environment. A knowledge of these

developmental milestones will be helpful in assessing possible effects of microgravity on

animals born and reared in space. The study of animals with demineralized otoconia will

be important for evaluating the ability of the vestibular system to adapt to changes in

otoconial demineralization possibly occurring with microgravity. Our results have shown

that the neonatal animals compensate for demineralization, and this compensation proceeds

with a time course similar to recovery from hemilabyrinthectomy in young animals. A

significant factor in compensation is the animals' ability to see, as evidenced by a correction

in postural asymmetry at a developmental time when the eyes are open.

Publications

Blanks, R.H.I., Clarke, R.J., Torigoe, Y., Pham, S., and Giolli, R.A. 1989. Projections

of the lateral terminal accessory optic nucleus (LTN) of the marmoset (Abstract). Society

for Neuroscience Abstracts 15: 458.

Blanks, R,H.I. and Torigoe, Y. 1989. Orientation of the semicircular canals in rat. Brain

Research 487: 278-287.

[

100
STUDIES OF INTERCELLULAR COMMUNICATION AND

INTRACELLULAR RESPONSES BY BONE CELLS TO SIMULATED

WEIGHTLESSNESS

Stephen B. Doty

Hospital for Special Surgery

535 E. 70th Street

New York, NY 10021

Description of Research

The hypogravity environment encountered both in space and in models of weightlessness

here on Earth results in a reduction of new bone formation. The mechanism(s) that cause

this reduction in formation are unknown. These researchers have previously shown that a

change in blood flow or vascular injury to the blood vessels in bone may be one

explanation. However, the bone forming process is complex. The bone forming cells

(osteoblasts) are dependent on a normal blood supply, but they are also sensitive to

hormonal and mechanical influences which regulate the normal physiology of the skeleton.

For example, significant changes have not been found in non-weight-bearing bones such as

the ribs or calvaria compared with the changes found in the long bones which are weightbearing and subject to high levels of mechanical stress.

Because of the variability in anatomy and physiology of different bones of the skeleton the

investigators have chosen to use histological techniques to study bone cell function.

Enzyme activity is localized with histochemical methods, cell structure is studied with

electron microscopy, and cellular components are described by cytochemical localization of

monoclonal antibodies for specific proteins and carbohydrates. Studies to date have

concentrated on the bone forming cells (osteoblasts) and the cells embedded within the

bone matrix (osteocytes), as well as the vascular association with these cells.

Accomplishments

(1) It has been established that vascular changes occur within bone as a

result of spaceflight and that these alterations are anatomically confined to

the subperiosteal region of the long bones. Results from spaceflight onboard

Cosmos 1887 have helped establish this finding.

(2) In areas of bone where severe vascular changes are seen, osteocytic death

and significant histochemical changes in the perivascular cells also occur.

(3) Techniques have been developed to embed fresh unfixed bone in plastic resin in

such a way as to preserve enzyme and structural information in the osteocyte population.

Using this technique it is now possible to visualize cytoskeletal components of the

osteocyte population and study cell-cell relationships within the bone matrix.

Significance of the Accomplishments

The vascular changes in bone are unusual in that they are confined to only subperiosteal

regions of the weight-bearing bones (Finding #1). This suggests that the cardiovascular

changes which occur during spaceflight may have a skeletal component that significantly

alters bone formation. This effect has not been found in simulated weightlessness,

although a slow down in blood flow through bone has been observed. Thus the model and

actual spaceflight are not entirely comparable with regard to the intensity of physiological

101
changesthatoccur during non-weight-bearing. It appears that the vascular changes also

initiate cellular necrosis in the osteocyte and perivascular cell population (Finding #2). It

was interesting that histochemical studies demonstrated which cells were undergoing cell

death even when morphological studies indicated no significant change.

The new techniques of working with fresh unfixed embedded bone will permit

cytochemical studies not previously possible. For example it is now possible to localize

specific proteins and cellular components with antibodies (Finding #3) as well as enzyme

activities which previously were denatured by the tissue preparation. Other studies indicate

that the osteocyte population contains some enzymes that are very responsive to mechanical

stimuli. This new methodology will permit the study of these ceils and relate their activity

to mechanical and hormonal changes that take place during studies of weightlessness.

Publications

Cone, C.M., M0rey-Holton, E., and Doty, S.B. 1989. Development of a mineralizing,

gravity-sensitive culture system for primary rat osteoblasts (Abstract). ASGSB Bulletin 3:

116.

Doty, S.B. 1989. Electron microscopic studies Of the vasculature in bone (Abstract).

ASGSB Bulletin 3:115.

Doty, S.B., Morey-Holton, E.R., Durnova, G.N., and Kaplansky, A.S. 1990. Cosmos

1887: morphology, histochemistry, and vasculature of the growing rat tibia. FASEB

Journal 4: 16-23.

Doty, S.B. and Schofield, B.H. 1990. Histochemistry and enzymology of bone-forming

cells. In: Bone: A Treatise. Volume 1. The Osteoblast and Osteocyte (ed. by B.H. Hall).

Caldwell, NJ: Telford Press, p. 71-102.

Holton, E., Beretta, D., Doty, S., Roberts, W., and Garetto, L. 1990. Experiment K-6-

03. Part I: Gravity and Skeletal Growth. In: Final Reports of the US Experiments Flown

on the Soviet Biosatellite Cosmos 1887 (ed. by J.P. Connolly, R.E. Grindeland, and

R.W. Ballard). Moffett Field, CA: NASA Ames Research Center, p. 115-121. (NASA

TM-102254)

E

102
EFFECT OF ALTERED G ON CHONDROCYTE DIFFERENTIATION

Pauline Jackie Duke

University of Texas Dental Branch

Dental Science Institute

Houston, TX 77225

Description of Research

The response of the skeletal system to microgravity is well-documented, with all

components thus far examined (bone, tendon, cartilage) showing significantly altered

differentiation, including altered matrix organization, after spaceflight.

The projects in our laboratory are directed toward the question of the effect of gravitational

changes on pre- and postnatal skeletal development, with emphasis on endochondral

ossification. Several approaches to this question are used: in vivo and in vitro

centrifugation, whole animal unloading, and spaceflight. In the last case, we are analyzing

bones from recent Cosmos spaceflight missions and are developing hardware for what will

be the first study of cultured skeletal cells in space.

Because of the complexities involved in whole animal systems, and the lack of inflight

animal sacrifice, cell culture systems to address mechanisms of gravitational responses in

the skeletal system are of importance. One such system, using differentiating cartilage cells

in high density micromass culture, will be flown in space on the Spacelab mission

International Microgravity Laboratory-1 (IML-1). Besides being the first culture of skeletal

cells in space, this experiment also has the most extensive medium changes and fixations

ever carried out in space.

Accomplishments

Centrifugation, in vitro

(1) Centrifuged (2.9 g) micromass cultures developed

hrs prior to appearance of cartilage in control cultures.

cartilage 24

Centrifugation, in vivo

(2) Analyses of skeletal muscle of mice centrifuged at 3 g for 14 weeks were

carried out in Berne, Switzerland, and demonstrated an increase in mitochondrial and lipid

total volumes. This finding is similar to changes seen in endurance training. However,

there was also an increase in muscle mass which is seen in strength rather than endurance

training.

(3) Dr. Cogoli of Zurich found a four-fold higher incorporation of

3H-thymidine in lymphocytes from the spleens of animals centrifuged for

12 months and stimulated with the mitogen Concanavalin A when compared to

controls.

Spaceflight

(4) Increased height and cell number in the proliferative zone of

growth plates of flight rats were found after exposure to microgravity for 14 days

aboard the Soviet biosatellite Cosmos 2044.

103
(5) The hypertrophycalcification zone of rat growth plates was

significantly reduced in height and cell number after spaceflight aboard

Cosmos 2044,

(6) No significant changes were found in growth plates of the

unloaded control group, in contrast to previous studies which had shown this to be a

good simulation model for spaceflight.

(7) Response of the growth plate to microgravity depends on age.

Cosmos 2044 results provided the first indication that growth plate may respond differently

to microgravity at different ages of the animal.

Hindlimb unloading

(8) Hypergravity was used as a countermeasure to the effects of hindlimb

unloading. Unweighted animals were allowed to bear weight for 1 or 2 hrs/day, at 1 g

(ground), or by centrifugation at 1.5 or 2.6 g. Height and cell number per zone of the

growth plate were determined. In all experimental groups, total plate height and total cell

number/column were significantly less than in controls. However, the results indicate

some preventive or reparative effect of reloading at 1 g for 2 hrs or 1.5 g for 1 hr/day. In

1.5 g (1 hr) animals, the reduction in proliferative and hypertrophic/calcification Zones was

less than in other centrifuged groups and not significantly different from controls. Longer

or higher g exposures produced significant reductions in cell number and height.

Significance of the Accomplishments

(1) Previous studies in this laboratory showed that precocious chondrogenesis

occurs in intact embryonic limb buds exposed to hypergravity. The current study shows

that the same result occurs when the cells of the limb are dissociated and exposed to

hypergravity in culture. With the micromass culture system, the response of precartilage

cells to a complete range of g forces, including simulated microgravity using a clinostat and

true microgravity on our shuttle flight, can be determined. With cartilage cells and

quantitative measurements, it should be possible to come to some scientific conclusions

regarding the relationship of centrifugation to spaceflight and the validity of clinostating.

(2) This study demonstrates the benefits of long range tissue sharing, and the value

placed upon operation of our animal centrifuge by other laboratories. It also adds to our

knowledge of centrifugation effects, and with the advent of centrifugation on shuttle and

space stations and the current lack of centrifuge scientists, such studies have become

extremely important.

(3) The increased activation seen in hyper-g exposed animals is in contrast to the

lower activation seen in spaceflown animals.

(4&5) These animals were exposed to earth gravity for about 10 hrs prior to

sacrifice, so these results represent both effects of microgravity exposure and initial stages

of recovery from that exposure. The results also confirm previous observations of altered

chondrocyte proliferation and differentiation in growth plates exposed to microgravity.

(6&7) The lack of response to unloading seen in these animals is likely due to age,

because these were the oldest unloaded group we have studied. However, we have not

studied plates from the particular model used by the Soviets, which does not allow

movement in 360 ° . Whichever the case, the results demonstrate the need for caution in

interpretation of unloading studies.

[

104
(8) Thispioneeringstudy,cardedout with Dr. FrankBoothof Universityof Texas

Medical School,demonstratestheadvantagesanddisadvantagesof usingg forceshigher

than1to preventeffectsseenin unloadingor spaceflight.Theadvantageis thatwith 1.5g,

shorterloadingtimesareneededthanwith 1g; thedisadvantagesarethattoo muchg force

compressestheplateandstopsthegrowth,andthattheanimalsaresomewhatstressedby

thecentfifugation. Additionalstudiesarebeingcardedoutto determinewhatcombination

of g force and time is bestto maintainbonegrowth. It is also possible that different

systems,e.g.,muscleandbone,requiredifferentthresholdsfor healthmaintenance.

Publications

Duke, P.J., Durnova, G., and Montufar-Solis, D. 1990. Histomorphometric and electron

microscopic analyses of tibial epiphyseal plates from Cosmos 1887 rats. FASEB Journal

4: 41-46.

Duke, P.J., Montufar-Solis, D., Durnova, G. 1990. Experiment K-6-06. Morphometric

and EM analyses of tibial epiphyseal plates from Cosmos 1887 rats. In: Final Reports of

the US Experiments Flown on the Soviet Biosatellite Cosmos 1887 (ed. by J.P. Connolly,

R.E. Grindeland, and R.W. Ballard). Moffett Field, CA: NASA Ames Research Center,

p. 169-182. (NASA TM-102254)

Duke, J., Moore, J., and Montufar-Solis, D. 1989. Continuing studies of "CELLS" flight

hardware (Abstract). Physiologist 32: $57-$58.

Montufar-Solis, D. and Duke, J. 1989. Altered distribution of matrix vesicles sizes in

Spacelab 3 rats (Abstract). ASGSB Bulletin 3:113.

Montufar-Solis, D. and Duke, J. 1989. Changes in rat growth plates due to strain, diet

and housing (Abstract). ASGSB Bulletin 3:114.

105
INFLUENCES OF SYNAPTIC GENESIS

THE VESTIBULAR EPITHELIA

Crsar D. Fermin

Department of Pathology

Tulane University Medical School

New Orleans, LA 70112

ON THE ARCHITECTURE OF

Description of Research

The objective of this project is to study, with the electron microscope and modern

biotechnological applications, the vestibular epithelia of the utricle and saccule in order to:

(1) delineate modifications of cells known to be influenced by synaptic genesis in other

sensory organs and (2) investigate the relationship that might exist between refinements of

the epithelia and changes in the formation of the statoconial membrane that rests above the

maculae.

This study is needed because the maculae utriculi and sacculi are _avity detectors and thus

their development should be influenced by the micr0gravity environment of spaceflight.

The modifications caused by changes in the gravity vector and force in the developing

gravity sensors could occur at the organ level, at the tissue level, and/or at the cellular level.

At each level, morphological and biochemical manifestations of the changes can be

followed during development and critical periods can be determined based on the

significance of the changes. Normal values from Earth-based experiments are needed in

order to identify changes that may occur in spaceflight.

Accomplishments

In order to establish normative Earth-based data, the saccule and utricle of chick embryos

and newly hatched birds were carefully studied with the light and electron microscopes and

with computerized video microscopy. In this report analysis of the bipolar neurons in the

vestibular nerve is described. The bipolar neurons are cable-like structures that

connect the inner ear with the brain. These neurons change in size as the

distance between the inner ear and the brain becomes larger due to

embryonic growth. It is a well known fact that an increase in the length of nerve fibers

is accompanied by an increase in the axoplasmic cross sectional area and wrapping

thickness of myelin sheaths. The ear from 11-day old embryos (El 1) processed in parallel

with newly hatched chicks (P2) was analyzed.

Statistical manipulations of the data obtained from computerized video-morphometric

measurements showed a significant increase (p<O.O0003) in the surface area of

the bipolar neurons of P2 over Ell. No significant difference was found in the

shape of the neurons from these groups. At E11, the nerve fibers of the vestibular neurons

have almost no myelin sheaths, and the perikarya of the neurons still display the

characteristic appearance of actively growing structures, i.e., heterochromatic nuclei and

large quantities of polysomes. At P2, the utricle and saccule resemble the adult, and their

appearance corroborates the precocious development of chicks, a characteristic essential for

evaluating the processes of embryonic development and its relationship to maturation.

Significance of the Accomplishments

The neurons of the vestibular nerve that connect the inner ear to the brain undergo a

significant change in size of perikarya but not in shape (Figures 1 and 2). This probably

106
means that the shape factor (circle = 1.0) of perikarya, through a given sectional plane,

tends to remain nearly the same. On the other hand, the perikaryal size increases probably

to accommodate the larger size of its axons. A constant change of perikaryal size would

occur as transition between temporary and permanent synapses takes place. Such has been

the case for the auditory portion of the inner ear of chicks.

[]

SHAPESONH

o

SHAPE SO37

I I I I I I

0.6 0.7 0.8 0.9 1.0 1.1

Figure 1. Comparison of neuronal shape between E11 (bottom) and P2 (top) vestibular

nerves. A value of one represents a perfect circle. The main body of the neurons (perikarya)

does not significantly change between the onset of myelination and time of hatching.

I I I I °° "

AREASONH

I Io"

AREA SO37

I I I I I I i i

0 100 200 300 400 500 600 700

Figure 2. Contrary to the shape of the perikarya, the cross sectional area (_un2 on the x axis)

changed significantly between E11 (bottom) and P2 (top).

Since studies in chicks by other investigators show a progressive maturation of vestibular

responses, a gradual change and correspondence between morphology and

physiology is reasonable. In fact, in many other sensory systems,

behavioral responses do have an underlying morphological basis. Future

aims of this project are to examine other aspects of the inner ear and vestibular nerve in

order to provide morphological data needed by other investigators in this field. A

corresponding alteration of the perikarya and axons of neurons should also exist, which

should be evident in statistical analysis of data obtained from various developmental

groups. In the utricle, hair cells proximal to the nerve entrance (habenula perforata) are

innervated first while those away from the entrance of the nerve are innervated last. This

pattern of innervation constitutes a gradient which has also been shown in the auditory

lagena by this laboratory.

Image processing is being used in this laboratory in conjunction with true color/real time

analysis for examining the association of structural components in the developing inner ear.

The computerized video system allows us to emphasize subtle differences barely visible in

107
unprocessed raw images. The real advantage of this technology is its capacity for true

objective quantification of differences between structures, since this system can recognize

millions of color variations and then match the color threshold with those presented for

analysis. For instance, gray levels of intensity cannot distinguish between the density of a

blue nucleus counterstained with hematoxylin and the density of a nucleus stained brown

with peroxidase precipitate for a given antibody. True color image processing, however,

can make this distinction, and thus it is a dream come true for researchers wishing for

objective quantification of immunological reactions.

Publications

Fermin, C.D. and Gerber, M.A. 1989. Morphometric and densitometric analysis with

true color/real time video processing (Abstract). ASGSB Bulletin 3: 107.

Fermin, C.D., Lovett, A.E, Igarashi, M., and Dunner, K., Jr. 1990. Immunohistochemistry and histochemistry of the inner ear gelatinous membranes and statoconia of

the chick (Gallus domesticus). Acta Anatomica 138: 75-83.

Vellinger, J., Deuser, M., Castonguay, M., Kavookjian, H., Fermin, C., Kerschmann,

R., Hester, S., Hullinger, R., Sharp, P., Hunter, S., and Holick, M. 1989. Effect of

weightlessness on bone development and mineralization in chicken embryos aboard the

space shuttle Discovery (Abstract). ASGSB Bulletin 3: 104.

108
NEURAL MECHANISMS BY WHICH GRAVITATIONAL

STRESS AFFECT THE SECRETION OF RENIN

HORMONES

William F. Ganong

Department of Physiology

University of California

San Francisco, CA 94143

STIMULI AND

AND OTHER

Description of Research

The long-term goal of this research is delineation of the brain pathways and

neurotransmitters that mediate gravitational and stress-induced changes in the secretion of

renin and other salt and water-regulating hormones. Evidence from this laboratory

indicates that stimulation of certain serotonin-secreting neurons in the dorsal raphe nucleus

of the midbrain increases renin secretion, and that these neurons project to the

hypothalamus. One goal of this research has been determination of the specific parts of the

hypothalamus that affect circulating renin and angiotensin. Another goal has been analysis

of the importance of this region in the physiologic control of renin secretion. A third goal

has been determination of the pathways from the nervous system to the renin-secreting cells

in the kidney. To study the role of the hypothalamus and other parts of the brain in the

regulation of renin secretion, stimuli have been used which increase renin secretion in

diverse ways. Emphasis has been placed on the postural stress of 45 ° head-up tilt. Other

standard tests include: (1) administration of the serotonin-releasing drug p-chloroamphetamine (PCA); (2) the psychological stress of immobilization; (3) the chronic volume

depletion stress of a low sodium diet; and (4) the acute volume depletion stress of

nonhypotensive hemorrhage. The role of the vasopressin-secreting neurons that connect

the hypothalamus to the brainstem and spinal cord in the regulation of renin secretion is

also being studied.

Accomplishments

(1) Demonstration that the sympathetic nervous system plays a major

role in the increase in renin secretion produced by a low sodium diet. The

drug propranolol blocks the receptors that mediate the effects of sympathetic nerves on

renin secretion, and its effect on various responses have been studied. We previously

showed that propranolol blocks the renin responses to immobilization, head-up tilt, and

PCA. The effect of propranolol on the response to a low sodium diet has been studied and

it has been found that renin is reduced compared to vehicle-injected controls on the same

diet. However, injection itself is a stress, and in the animals that have been handled and

injected, it is not possible to say whether propranolol is reducing the overall response to

sodium depletion or simply blocking the response to injection stress. Therefore, chronic

cannulas were placed in rats so that we could inject the animals without handling them. In

the animals on a normal sodium diet, propranolol did not reduce renin secretion when

administered in this fashion.

(2) Demonstration that vasopressin probably acts within the brain to inhibit renin

secretion. In addition to regulation of angiotensinogen secretion, the paraventricular nuclei

of the hypothalamus are involved in the regulation of the secretion of renin from the

kidneys in some situations. Most of the paraventricular neurons secrete vasopressin into

the circulation from the posterior pituitary, but others project to the cardiovascular

regulatory areas in the brainstem and spinal cord. The vasopressin secreted by the neurons

109
Brattleboro rats that (due to a geneticdefect) have no vasopressinin their brains or

circulatingblood,we foundthat 13-adrenergicblockadeproducedby propranolollowered

circulatingrenin to essentiallynormallevels. Thisindicatesthat theBrattlebororatshave

chronically increasedsympatheticdischarge. In anotherset of experiments,we used

osmotic minipumps to infuse vasopressinsubcutaneouslyin Brattleboro rats in a dose

which restoredcirculating vasopressinto normal. The increasein renin secretionwas

unaffectedeventhoughurine volume and waterintake weremarkedlyreducedand the

concentrationof the urinewasincreased.This seemsto rule out a peripheralstimulusto

sympatheticdischarge.However,sincevery little vasopressinpenetratestheblood-brain

barrier,theincreasedsympatheticdischargethatproducesthereninhypersecretioncouldbe

central in origin. If our working hypothesisis correct, then injection of vasopressin

directly into thebrainshouldinhibit reninsecretion.In experimentscardedoutduringthe

pastyear,this appearsto bethecase. Additional experimentsareunderwayto makesure

thatthiscentralinhibitionis reproducible,clearcut,anddoserelated.

(3) Confirmation of the hypothesis that paraventricular regulation of

angiotensinogen secretion is mediated via neuroendocrine control of the

thyroid gland. Several years ago, we demonstrated that lesions of the paraventricular

nuclei decreased circulating angiotensinogen. We postulated that this effect was

neuroendocrine, since the paraventricular nuclei regulate the secretion of thyroid and

adrenocortical hormones via the anterior pituitary gland, and these hormones increase

circulating angiotensinogen. We found that removal of the anterior pituitary duplicated the

effect of paraventricular lesions. This year, we completed experiments m animals with

paraventricular lesions in which thyroid hormones or adrenocorticotropic hormone (ACTH)

were replaced by injection. ACTH did not restore angiotensinogen to normal, but

replacement of thyroid hormones did. Therefore, it seems clear that paraventricular

lesions decrease circulating angiotensinogen because they decrease

secretion of the brain hormone that stimulates the pituitary to secrete the

hormone that in turn stimulates thyroid hormone secretion.

(4) Discovery of a unique effect of anesthetics on circulating angiotensinogen. In

the course of experiments described in the preceding paragraph, we made the chance

observation that 24 hours after surgical stress and some anesthetics, plasma

angiotensinogen was elevated with little if any change in plasma renin

activity. This year, we have demonstrated that this increase cannot be explained by an

increase in ACTH, thyroid hormones, luteinizing hormone, or renin. However, it is

blocked by hypothosectomy. Therefore, we are actively investigating the role of the

remaining pituitary hormones in mediating the response.

Significance of the Accomplishments

The experiments described above and experiments conducted in previous years have done

much to elucidate the role of the brain in the regulation of renin secretion. This has

appreciable significance for NASA because both postural changes and stressful stimuli

increase renin secretion, and renin not only stimulates the secreuon of the salt-retaining

hormone aldosterone, but affects water balance and maintains blood pressure. In addition,

our demonstration that brain lesions lower circulating angiotensinogen is important because

it demonstrates for the first time that there is a neuroendocrine mechanism regulating the

circulating level of this important component of the renin-angiotensin system.

h

110
Publications

Ganong, W.F. 1989. Angiotensin II in the brain and pituitary: Contrasting roles in the

regulation of adenohypophyseal secretion. Hormone Research 31 (1-2): 24-31.

Ganong, W.F. 1990. Renin secretion and 5-hydroxytryptamine. In: Cardiovascular

Pharmacology of S-hydroxytryptamine: Prospective Therapeutic Applications (ed. by P.R.

Saxena, D.I. Wallis, W. Wouters, and P. Bevan). Dordrecht: Kluwer Academic

Publishers, p. 329-337.

Ganong, W.F., Deschepper, C.F., Steele, M.K., and Intebi, A. 1989. Renin-angiotensin

system in the anterior-pituitary of the rat. American Journal of Hypertension 2: 320-322.

Golin, R., Keil, LC., and Ganong, W.F. 1989. Role of angiotensin II in the vasopressin

response to tilting in anesthetized rats (Abstract). In: Abstracts, Fourth European Meeting

on Hypertension, Milan, Italy, p. 302.

Golin, R., Keil, L.C., Shackelford, R.L., and Ganong, W.F. 1989. Role of the

sympathetic nervous system in mediating the exaggerated renin response to immobilization

in Brattleboro rats (Abstract). In: Proceedings of the XXXI International Physiological

Congress, Helsinki, p. 246.

Golin, R.M.A., Gotoh, E., Keil, L.C., Shackelford, R.L., and Ganong, W.F. 1989.

Lack of effect of vasopressin replacement on renin hypersecretion in Brattleboro rats.

American Journal of Physiology 257: R 1117-R 1122.

Gotoh, E., Ishigami, T., Ishii, M., and Ganong, W.F. 1989. Role of the sympathetic

nervous system and circulating vasopressin in the renin secretion in response to stress

(Abstract). Japanese Journal ofNephrology 31: 556. (Japanese)

Intebi, A.D., Flaxman, M.S., Ganong, W.F., and Deschepper, C.F. 1990.

Angiotensinogen production by rat astroglial cells in vitro and in vivo. Neuroscience 34:

545-554.

Murakami, K., Akana, S., Dallman, M.F., and Ganong, W.F. 1989. Correlation

between the stress-induced transient increase in corticotropin-releasing hormone content of

the median eminence of the hypothalamus and adrenocorticotropic hormone secretion.

Neuroendocrinology 49 (3): 233-241.

Williams, W.M., Ganong, W.F., Mangiapane, M.L. 1989. Lesion of area postrema (AP)

impairs control of circulating adrenocorticotropic hormone (ACTH) (Abstract). FASEB

Journal 3(4):A529.

111
VECTOR-FREE GRAVITY INTERFERES WITH SYNAPSE FORMATION

Raphael Gruener

University of Arizona

College of Medicine

Department of Physiology

Tucson, AZ 85724

Description of Research

Prolonged spaceflights and plans for the colonization of space have opened two new areas

of research in biology. The first concerns the well-being of developing biological systems

in flights of even short durations, and the second concerns the possibility of investigating,

from an evolutionary perspective, the contribution of gravity to development.

The continued objective of this research is to examine, in-depth, specific aspects of nervous

system development, under simulated microgravity, to gain a better understanding of how

gravity influences ontogenetic development of the brain while at the same time exploring

the possibility that the constancy of gravity on Earth has contributed significantly to cellular

evolution.

The investigators have already shown that exposure of neurons and myocytes to vector-free

gravity profoundly affects synapse development including abnormalities in neuronal

morphology. Over the past 12 months, attention has been focused on the distribution of

cytoskeletal proteins which may be the underpinnings of the alterations in myocyte

acetylcholine receptor distribution and the alterations in neuron morphology (appearance of

swellings along neuritic shafts). Using immunocytochemistry to label cytoskeletal

proteins, we are probing changes in the structure of the cellular scaffolding responsible for

the anchoring of proteins to specific locations in the cell subsequent to growth of cells in

vector-free gravity.

Accomplishments

Our results show that synapse development in a slow clinostat (1-10 rpm) results in

dramatic reductions of the appearance of nerve-induced acetylcholine receptors (AChR)

accumulation at the point of contact between nerve and muscle cells in culture. Figure 1

shows typical results from cells which were rotated and then stained for 43kD protein and

for vinculin, both of which are cytoskeletal protein markers. Our results show that 43kD

protein always co-distributes with AChRs. This is not surprising since 43kD is

now known to be directly, and very tightly, linked to the cholinergic receptor. Therefore,

in the absence of AChR (after rotation), 43kD protein is also expected to be absent. The

story for vinculin is more complicated mostly because the exact location of vin.culin, in

reference to AChR, is still unknown. Our data show that in rotated cells the ratio of

vtnculin to AChR is larger than in control cells. This implies that

cytoskeletal organization in response to nerve contact takes place at least up

to the level of vineulin (vinculin is present in rotated cells even when AChR numbers

are diminished, hence the increase in vinculin/AChR ratio). Thus, the loss of

anchoring of AChR may be due to cytoskeletal elements which are located

between vinculin and the receptors. We are now pursuing this lead. Possible

candidates for these elements include: talin, actin oligomers, spectrin, and/or extracelIular

matrix proteins (see Figure 2 for a model of the system).

112
DISTRIBUTION OF CYTOSKELETAL MARKERS

ORIGINAL PAGE

BLACK AND WHITE PHOTOGRAPH

IN MYOCYTES

Figure 1, Panels A-H. Preliminary data showing distribution of cytoskeletal markers in

control and rotated myocytes.

Figure 1A-D: Coincidence of acetylcholine receptors (AChRs) with 43kD protein. Panel A shows the

distribution of AChRs in a control myocyte. Panel B shows that 43kD protein codistributes with the

AChRs indicating the close relationship between the receptor and cytoskeletal proteins. Panel C shows

AChR distribution from a rotated myocyte (most myocytes do not in fact have AChR at the point of

innervation after rotation; see text). Panel D shows the corresponding distribution for 43kD, the

cytoskeletal marker. Note that although 43kD is present, its distribution appears to be reduced when

compared to the control (A, B). In cases where AChRs are absent from the nerve-muscle contact zone, after

rotation, 43kD is also absent. This indicates that the tight binding between the two proteins is not affected

by rotation. Absence of receptors, after rotation, also results in absence of 43kD. The two proteins behave

as a unit.

113

ORIGINAL PAGE IS

OF POOR qUALr/'y
DISTRIBUTION OF CYTOSKELETAL MARKERS IN MYOCYTES

Figure 1E-H: Coincidence of AChR binding sites with vinculin binding sites. Panel E shows AChR

distribution in a control myocyte, and panel F reveals that vinculin, a cytoskeletal linker protein,

codistributes with the AChRs. In contrast, panels G and H, from a rotated cell, show that vinculin is

present at the nerve-muscle contact zone despite the absence of AChR subsequent to rotation. This implies

that nerve-muscle interactions, during rotation, take place normally as far as assembly of the cytoskeleton is

concerned up to the level of vinculin, but that despite this partial assembly, the AChRs cannot be retained

at the nerve-muscle contact zone.

ORIGINAL PAGE

BLACK AND WHITE PHOTOGRAPH

ORIGINAL PAGE IS

OF POOR QUALITY

114
PROPOSED MODEL OF RECEPTOR ACCUMULATION/CYTOSKELETAL ASSEMBLY AT THE NEUROMUSCULAR SYNAPSE

t act=n ohgomer

(Adapted from Bloch & Pumplin, 1988; Am. J. Physiol. 254: c345)

Figure 2: Cartoon model of the mechanism of receptor patching and its association with the cytoskeleton

at the neuromuscular junction. This model, adapted from Bloch & Pumplin (1988), provides a hypothetical

framework through which we examine how simulated lag may affect the receptor accumulation process and

the assembly of the associated cytoskeleton. For example, the initiating signal is derived from the nerve (or

polystyrene beads as a substitute) which contains extracellular matrix ligands (ECM1) that bind to their

hypothetical receptor on the ECM of the myocyte. This initiates the assembly of the underlying

cytoskeleton (e.g., vinculin). By an as yet unknown process this signal is carried to non-patched, randomly

distributed AChR in the myocyte. This signal results in receptor accumulation at the nerve-muscle zone.

Shortly after the arrival of AChR, at the forming neuromuscular junction, the receptors are capped by

anchorage to the underlying cytoskeleton (via 43kD protein, spectrin, actin, etc.). By probing for

distribution of specific elements, we expect to gain information concerning the specific mechanism(s)

affected by clinostat rotation which may be associated with the failure of AChR to accumulate at the nervemuscle contact zones (see text for further details).

ORIGINAL PAGE IS

OF POOR QUALITY

115
Publications

Coggan, J.S., Kreulen, D.L., Matsumoto, S.G., and Gruener, R. 1989.

responses from celiac ganglion neurons in primary culture (Abstract).

Neuroscience Abstracts 15:184.

Muscarinic

Society for

Gruenerl Riand Hoeger, G. i990_ Vector-free gravity disrupts synapse formation in cell

culture. American Journal of Physiology 258: C489-C494.

Hoeger, G. and Gruener, R. 1989. Synapse formation in vector-free gravity:

cytoskeleton sensitive to altered gravity? (Abstract) ASGSB Bulletin 3: 105.

Is the

116
THE EFFECTS OF GRAVITATIONAL FIELDS ON

IN THE HIPPOCAMPUS

John M. Horowitz and Barbara A. Horwitz

Department of Animal Physiology

University of California

Davis, CA 95616

NEURAL SIGNALING

Description of Research

Our research has as an ongoing, long-term objective the elucidation of the effects of altered

gravitational fields on modulatory and regulatory neural networks. Recently our

experiments have dealt with the effects of serotonin, a neuromodulator, on neurons in the

hippocampus. We studied the hippocampus because rats flown on Spacelab-3 showed an

increased number of serotonergic receptors in this region of the central nervous system

after seven days in space. In addition, a more recent study has shown that rats exposed to

a hypergravic field of 2.0 g for seven days show a decreased number of receptors.

Serotonin is a well-studied neuromodulator involved in many types of neural activity,

including sleep and temperature regulation. In addition, in the hippocampus serotonin may

modulate long-term potentiation, a proposed cellular mechanism for memory and learning.

Our immediate goal is to determine how serotonergic effects on hippocampal cells are

altered by gravitational fields.

An additional motive for studying the effect of serotonergic activity in the hippocampus is

that other types of neural activity are known to be modified by exposure of animals to

hypergravic fields. Temperature regulation of animals in a microgravity environment has

been reported to be impaired. This year we also completed studies on thermoregulation in

hypergravic-acclimated rats (briefly described below), and found that when raised from

birth in a 2.1 g field rats were better able to cope with cold exposure in hypergravic

environments than were 1 g controls. While there are many studies by several investigators

showing that thermoregulatory systems are altered by changes in gravitational fields, there

are relatively few studies on the effects of gravitational fields on serotonergic mechanisms,

and more studies are needed to more fully delineate altered serotonergic activity.

Accomplishments

(1) One set of experiments focused on selected aspects of serotonergic modulation

of neural activity in rats exposed for seven days to 2 g fields. Experiments showed that

serotonin was effective in modulating the amplitude of one neural response

(the population spike) in both the rats exposed to 2 g for seven days and

the 1 g controls. More specifically, even though there was a 26.9% decrease in

receptor number in rats exposed for seven days to a 2 g field, this was not sufficient to

markedly alter the action of serotonin at 5-HT 1A receptors. (Additional experiments are in

progress to determine if there is a subtle change in serotonin's effectiveness in decreasing

population spike amplitude.)

(2) In extending the experiments to the action of serotonin acting at other receptor

types on hippocampal cells, experiments were performed on rats and hamsters. In

experiments at 1 g, population spikes had a clear biphasic response, with a depression in

neural activity averaging 52.4 + 10.1% less than control levels followed by a rebound of

neural excitability which averaged 33.7 + 9.3% greater than control levels. In rats at 1 g

117
the depressionwasalsoclearly present,averaging76.4 + 5.5% less than control levels;

however, the rebound was often small or masked in background activity, averaging only

7.4 +__4.8% of controls. The rebound effect was not blocked by 5-HT1A blockers, and

thus was the result of the activation of cellular mechanisms by serotonin acting on a second

type of hippocampal serotonergic receptor.

(3) When rats were exposed to cold (9°C), concurrently with a hypergravic field of

2.1 g, the core temperature of rats raised at 1 g fell markedly (by 6°C), while that of rats

born and raised at 2.1 g remained relatively constant (falling only l°C). Measurements of

0 2 consumption showed that the rats reared at 1 g and then cold exposed at 2.1 g failed to

activate thermogenic mechanisms.

Significance of the Accomplishments

Finding #1: Depression of the amplitude Of population spikes during serotonin perfusion

in rats exposed to hypergravic fields showed that one modulatory effect of serotonin is not

abolished in animals exposed to hypergravic fields. Thus the decreased number of

receptors observed under the same experimental conditions (exposure to a hypergravic field

using the same small animal centrifuge) is insufficient to abolish a primary modulatory

action of serotonin.

Finding #2: The rebound following serotonin administration was larger in the hamster than

in the rat. Thus to study the characteristics of rebound (an enhancement elicited by a

different serotonergic receptor than the 5-HT1A receptor type) under hypergravic

conditions, the hamster is the preferred model.

Finding #3: While a hypergravic field of 2.1 g is a novel environment for animals raised at

earth gravity (1 g ), it is not novel for the animals reared from birth in a 2.1 g field. The

rats acclimated to 2.1 g were able to maintain their core temperature when cold exposed in

the 2.1 g environment whereas the 1 g acclimated rats could not. These experiments show

that an animal acclimated to hypergravic fields has an altered neural

thermocontrol system and can more effectively regulate core temperature in

a hypergravity field than can an animal acclimated to 1 g.

Publications

Hamill, N.J., McGinn, M.D., and Horowitz, J.M. 1989. Auditory brainstem responses in

ground squirrels arousing from hibernation. Journal of Comparative Physiology B 159:

167-172.

Hamill, N.J., McGinn, M.D., and Horowitz, J.M. 1989. Characteristics of auditory

brainstem responses in ground squirrels. Journal of Comparative Physiology B 159: 159-

165.

Horowitz, J.M. and Horrigan, D.J. 1989. Thermal dependence of serotonergic effects on

neural activity in the hamster hippocampus (Abstract). FASEB Journal 3(4): A396.

Horrigan, D.J. and Horowitz, J.M. 1989. Modulatory effects of serotonin on neural

networks after exposure to 2.0 g gravitational fields (Abstract). ASGSB Bulletin 3: 35.

118
Horrigan, D.J. and Horowitz, J.M. 1989. Serotonergicmodulation in the hamster

hippocampal slice is different than in the rat hippocampus(Abstract). Society for

Neuroscience Abstracts 15: 534.

Krelstein, M.S., Thomas, M.P., and Horowitz, J.M. 1989. Thermal dependence of

long-term potentiation in hamster hippocampal neurons (Abstract). FASEB Journal 3(4):

A397.

Monson, C.B., Patterson, S.L., Horowitz, J.M., and Oyama, J. 1989. Thermoregulation

in hypergravity-acclimated rats. Journal of Applied Physiology 67: 383-389.

119
MECHANISM OF CONTROL OF BONE

PROSTAGLANDINS

Millie Hughes-Fulford

Veterans Administration Medical Center

University of California Medical Center

San Francisco, CA 94121

GROWTH BY

151F

Description of Research

Biomedical studies of manned spaceflight have consistently shown a loss of weight-bearing

bone. Various lines of evidence from human and animal studies suggest that this loss is

due to the lack of bone formation in the absence of gravity. The mechanism by which bone

growth is regulated is not known; however, clinical observations have demonstrated that

increases of endogenous cortisol as seen in Cushings syndrome are associated with bone

loss and osteoporosis. Treatment of patients with asthma and rheumatoid arthritis has also

demonstrated the deleterious effects of glucocorticoids on bone formation. The bone loss

associated with glucocorticoids involves trabecular bone and examination of patients treated

with synthetic glucocorticoid prednisone shows a reduction in bone formation which is

probably due to a direct inhibition of osteoblastic function.

These data suggest that the glucocorticoids could also play a role in the loss of bone that

occurs in spaceflight. In the Skylab missions urinary cortisols of the nine crew members

increased almost two fold. The direct cause of glucocorticoid-induced bone loss is not

known and an understanding of the relationship between glucocorticoids and

prostaglandins in bone cell growth and differentiation may increase our ability to

understand the underlying mechanism of bone formation in microgravity. The

glucocorticoids and prostaglandins may play a role in the physiological control of bone cell

growth and mineralization at the cellular and molecular level. We are using an in vitro

osteoblast model treated with comparable amounts of glucocorticoid as seen in spaceflight

to determine if: (a) glucocorticoids affect prostaglandin synthesis and cell growth; (b) if

glucocorticoids affect bone mineralization; and (c) if these glucocorticoid-induced changes

are related to changes in protein synthesis or assembly.

Accomplishments

Our bone model is the MC3T3-E1 cell, a cloned 0steoblast line that retains synthetic

functions of normal bone tissue, including production of alkaline phosphatase,

prostaglandin E 2 and mineralized matrix containing hydroxyapatite. In these studies, we

have used 100 laM dexamethasone treatment on the osteoblast MC3T3-E1 cells to simulate

the effect of increased plasma cortisols on bone ceils seen in spaceflight over a 13-day

period of growth and differentiation.

(1) The levels of collagen synthesis in the growing osteoblast are barely detectable.

However once the cells are confluent and differentiated, collagen is the major protein

synthesized by the osteoblast. Using western blot analysis and affinity purified collagen

antibody, we have found that collagen type I accounts for approximately 97% of

the total collagen synthesis in this cell line.

(2) Dexamethasone treatment of the mineralized osteoblast causes a decrease in

14C proline incorporation into new protein synthesis. Using western blot analysis with

radioautography and video densitrometry, this observation has been expanded to

120
demonstratethat collagentype I synthesisis specifically inhibited by glucocorticoids.

Procollagenal(I) anda2(I) synthesisis decreasedby approximately50% while collagen

al(I) anda2(I) is decreasedup to 70%whencomparedto control after 13daysof growth

andmineralization.

(3) The glucocorticoidtreatedcellswereexaminedby microscopyfor changesin

cell structure. Using phasemicroscopy,the shapeof the treatedcells was found to be

irregular whencomparedto controls. Using specificprobesfor f-actin cytoskeletonwe

found that the irregularity seen in overall cell shape is reflected by the actin

cytoskeleton.

(4) The distribution of the collagen matrix in the treated cells differed from the

control osteoblasts. In general, the collagen matrix formation in the treated cell was not as

uniform as the control and appeared to be discontinuous in portions of the matrix.

Significance of the Accomplishments

The importance of these findings is that the temporal regulation of collagen synthesis in this

cultured osteoblast corresponds to published observations made in vtvo. Collagen

synthesis is of minor importance during the first stages of osteoblast growth, but becomes

increasingly important during mineralization. Finding #2 suggests that collagen synthesis

is inhibited by the glucocorticoids at concentrations approximating the levels of cortisol

seen in man during spaceflight. In addition to decreased collagen synthesis, there are

changes in the f-actin cytoskeleton assembly and collagen matrix formation in the

mineralizing cultures. These findings suggest that not only do the glucocorticoids decrease

the ability of the bone cell to synthesize collagen matrix needed for mineralization, but also

interfere with the cytoskeleton formation and assembly of the collagen matrix during

mineralization. This, along with other findings, suggests that the glucocorticoids may

inhibit bone formation, both by interfering with the growth of the osteocyte and also by

inhibiting synthesis and uniform assembly of the collagen matrix during mineralization.

Publications

Hughes-Fulford, M. and Scheld, H.W. 1989. Thin film bioreactors in space. Advances

in Space Research 9(11): 111-117.

Hughes-Fulford, M., Schmidt, J., Kumegawa, M., and Appel, R. 1989. Glucocorticoids

cause a decrease in collagen matrix formation in cloned osteoblast cells (Abstract). ASGSB

Bulletin 3:117.

Hughes-Fulford, M., Schmidt, J., Kumegawa, M., and Appel, R. 1989. Glucocorticoids

modulate collagen synthesis in MC3T3 osteoblast cells (Abstract). Journal of Cell Biology

109: 43a.

Pliam, N.B., Smith, R.L., Kumegawa, M., and Hughes-Fulford, M. 1989. Effects of

FGF b, TGF b, IGF-1, PDGF, and PGE 2 on osteoblast growth and morphology

(Abstract). ASGSB Bulletin 3:118.

121
MECHANOCHEMICAL TRANSDUCTION

MATRIX

Donald E. Ingber

Surgical Research Laboratory

Department of Pathology

Harvard Medical School

300 Longwood Avenue

Boston, MA 02115

ACROSS EXTRACELLULAR

Description of Research

The general goal of this project is to characterize the molecular mechanism by which

mechanical signals, such as those that result from the action of gravitational forces, are

transduced into intracellular metabolic alterations. The more specific aim is to analyze the

process by which mechanical forces are conveyed by extracellular matrix molecules,

transmitted across the cell surface, and transduced into changes of actin polymerization

inside the cell. This approach is based on the concept that cell shape and, thus, the form of

the cytoskeleton, depends on a dynamic equilibrium between tensile forces that are

generated within contractile actin-containing microfilaments and resisted both by internal

structural dements and by extracellular matrix attachment sites on the surface of the cell. If

this type of tensional integrity or "tensegrity" mechanism is used by cells, then externally

applied mechanical loads may affect complementary force interactions, change local

thermodynamic parameters, and thereby alter cytoskeletal filament assembly. Changes of

cytoskeletal organization, in turn, can alter the distribution and hence function of much of

the cell metabolic machinery. Characterization of this process could have much greater

implications for understanding the effects of gravity on a wide variety of metabolic

functions.

We have focused on the biomechanical mechanism by which actin filament assembly is

regulated inside the cell. Ceils normally generate tension within their contractile

microfilaments and apply these forces on their points of attachment on extracellular matrix

molecules. Thus, one specific objective of this research is to study the process by which

transmembrane matrix receptors resist cell-generated mechanical forces and transduce them

into alterations of microfilament assembly in the cell. Our long term goal is to determine

whether externally applied forces utilize a similar matrix-based mechanism for

transmembrane mechanochemical coupling.

Accomplishments

(1) Demonstration of the Importance of Cell Shape in Growth

Control: We have developed a simplified in vitro system for regulation of capillary

endothelial cell growth responsiveness to soluble growth factors, such as basic fibroblast

growth factor. In this system, cell shape and DNA synthesis are controlled by varying the

coating density of a single type of extracellular matrix molecule, such as fibronectin. Using

this system in combination with chemically defined medium, we have been able to clearly

demonstrate that fibronectin (FN) controls cell growth based on its ability to

support changes of cell and nuclear shape. We have also demonstrated that the

effects of fibronectin on cell form and function are mediated by specific

binding interactions with cell surface matrix receptors, specifically the alpha 5,

beta 1 integrin receptor.

122
(2) Micro filament Assembly Mediates the Effects of Fibronectin on

Growth: Immunofluorescence studies using antibodies directed against G-actin or using

rhodaminated-phalloidin demonstrate an increase in the number and length of actin bundles

as fibronectin coating densities are raised. However, no increase in actin filament staining

is seen in suspended cells cultured in the presence of soluble fibronectin or fibronectincoated microbeads which do not support cell spreading. Furthermore, use of cytochalasinD, an inhibitor of microfilament assembly, revealed that actin polymerization is required for

both cell spreading and entry in S-phase. Importantly, cytochalasin had no effect when

added at later times after cells have entered the synthetic phase of the cell cycle. These

results suggest that actin assembly is necessary for progression through the

G-1 phase of the cell cycle.

(3) Quantitation of Actin Polymerization within Cells Spreading on

Fibronectin: We have used quantitative immunoblotting techniques in combination with

anti-actin antibodies and a NBD-phallicidin binding assay to measure actin polymerization

during the initial phases of cell attachment and spreading on fibronectin-coated dishes.

Both of these techniques showed similar results and demonstrated that monomeric actin

shifts into a polymerized F-actin form and becomes associated with the cytoskeleton

beginning approximately 10 minutes following cell attachment to fibronectin-coated dishes.

The effects of FN on actin assembly nicely parallel its early effects on cell spreading.

(4) Application of Mechanical Loads to Fibronectin Receptors: We

have just begun to develop a system in which cells are plated on silastic membranes to

which controlled mechanical loads can be applied. These membranes are non-adhesive for

endothelial cells under the culture conditions we utilize. We can therefore vary the number

of structural interconnections between the cell and the substratum (and thus the amount of

force experience by the cell) by varying the density of fibronectin coated on these elastic

substrata. Experiments measuring the effect of externally applied mechanical loads on actin

polymerization are just beginning to get underway.

Significance of the Accomplishments

Finding #1: The finding that cell proliferation can be controlled by varying the density of

fibronectin attachment sites on the surface of the cell suggests that extracellular matrix may

serve as a basic regulator of cell growth responsiveness in the local tissue

microenvironment. These studies also suggest that matrix molecules regulate cell

proliferation based on their ability to provide a physical anchoring

substrata that can resist cell-generated tensile forces and thereby promote

cell extension. The finding that fibronectin regulates cell growth by modulating cell

shape also suggests that cells may utilize a biomechanical signal transduction

system during growth control

Finding #2: The demonstration of the effects of fibronectin on actin assembly as well as

inhibition of cell growth by the addition of inhibitors of actin polymerization strongly

suggests that the effects of fibronectin on growth are mediated via its effects

on the cytoskeleton.

Finding #3: The finding that fibronectin promotes cell spreading and actin polymerization

in parallel, whereas soluble FN does neither, is consistent with our hypothesis that

fibronectin regulates actin filament assembly both by binding to cell surface

receptors and by physically resisting cell-generated loads that are applied to

those receptors.

123
Finding#4: We now haveanin vitro system which can be used to analyze the molecular

path of force transmission across the cell surface as well as the mechanism of

mechanochemical transduction.

Publications

Ingber, D.E. 1990. Fibronectin controls capillary endothelial cell growth by modulating

cell shape. Proceedings of the National Academy of Sciences U.S.A. 87: 3579-3583.

Ingber, D.E. and Folkman, J. 1989. How does extracellular matrix control capillary

morphogenesis? Cell 58: 803-805.

Ingber, D.E. and Folkman, J. 1989. Mechanochemical switching between growth and

differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: Role of

extracellular matrix. Journal of Cell Biology 109: 317-330.

124
VESTIBULAR ELECTROPHYSIOLOGICAL RESPONSE THRESHOLDS:

EFFECTS OF SPACEFLIGHT AND LINEAR SINE WAVE MOTION

Timothy A. Jones

University of Nebraska Medical Center

College of Dentistry

Department of Oral Biology

Lincoln, NE 68583

Description of Research

The principal aim of this research is to examine the role played by gravity in controlling or

influencing the ontogeny of peripheral and central vestibular function. Ultimately an indepth comparison will be made of how vestibular function develops and matures under the

influence of gravitational fields having strengths less than (< 1.0 g, hypodynamic) equal to

(1.0 g) and more than (>1.0 g, hyperdynamic) the natural gravitational field strength of the

Earth. Studies have been undertaken to examine how the gravitational vector strength and

direction may modulate or influence normal response to transient stimuli. Efforts have

been directed to investigate how vestibular function may adapt to changes in gravitational

fields and to evaluate the vestibular system's ability to adapt as a function of the organism's

age. Through these kinds of studies we may begin to appreciate the limits to and

determinants of physiological adaptation in the vestibular system under a variety of

gravitational environments.

This research program has led to the recording of short latency vestibular responses to

pulsed linear cranial acceleration for the first time. Salient features of these responses

include short latencies (4 to 7 peaks occurring within 8 msec following the stimulus) and

resistance to intense auditory masking (96 to 100 dB SPL). Moreover, the responses are

abolished upon complete bilateral destruction of the labyrinth but are little affected by

selective bilateral destruction of the cochlea and lagena. This research has demonstrated

that responses to pulsed linear acceleration in anesthetized birds are vestibular in origin and

likely represent compound action potential of the vestibular nerve and central relays.

Although the precise generators are not yet known, our results suggest that the lagena

contributes little to response components.

Concurrent studies in rats demonstrated that similar responses could be obtained using

pulsed linear acceleration stimuli, These responses occurred within 8 msec following the

stimulus, had amplitudes less than 10 and were resistant to high intensity auditory masking

(up to 105 dB SPL). As in birds, response latencies and amplitude were linearly dependent

upon stimulus intensity. Moreover, response thresholds decreased as a function of the rate

of change in acceleration (i.e., da/dt) in both species. The effects of selective bilateral

destruction of the cochlea and labyrinth in rats are currently being studied.

New research described in the current report sought to: (a) determine the effects of

spaceflight on vestibular ontogeny by measuring vestibular response thresholds in birds

flown as embryos on the shuttle Discovery (March 1989) and (b) complete pilot research

evaluating the effects of linear sine wave motion on vestibular responses to pulsed linear

acceleration.

125
Accomplishments

(1) Does linear sine wave motion affect vestibular responses?

Hypothetically, vestibular responses to pulsed linear acceleration are generated, at least in

part, by neurons innervating otolith structures of the labyrinth. It is well known that linear

sine wave motion can be used to dynamically stimulate otolith sensory structures while

producing little or no effect on canal sensors. Evidence suggesting that linear motion can

modify vestibular responses to pulsed acceleration would support the hypothesis that these

responses depend on activity in otolith organs.

Pilot research was undertaken in the Vestibular Research Facility at the NASA-Ames

Research Center, Moffett Field, CA. In a series of experiments a linear sled was used to

examine the effects of sine wave linear motion on vestibular responses. Linear motion

was found to produce a small phase.dependent modulation of vestibular

response thresholds, latencies and amplitudes. These preliminary findings

provide some support for the 'otolith' hypothesis. They demonstrate that such

measurements can be made and serve as a preface to indepth studies exploring the precise

vestibular origins of the responses.

(2) Vestibular responses in unanesthetized animals: An innocuous

method of recording vestibular responses was developed to enable measurements of

vestibular function in unanesthetized birds. Responses to pulsed linear acceleration

in unanesthetized animals were highly resistant to intense auditory masking

(104 dB SPL), were little affected by the removal of both cochleae and

lagenae, but were virtually eliminated with bilateral destruction of the

labyrinth. Therefore, responses to pulsed linear acceleration in unanesthetized animals,

like those in anesthetized animals, are of vestibular origin and represent compound action

potentials of the vestibular nerve and central relays.

(3) A role for gravity in shaping, embryonic vestibular development?

Eight birds (n-8) incubated for 7 days tn space as embryos (approximately

embryonic days E9-E16 during the March 1989 flight of the space shuttle Discovery) had

elevated vestibular thresholds when compared to 8 sibling Earth controls

raised at 1.0 g. Indeed, to elicit vestibular responses, the animals exposed to

weightlessness required stimulus intensities nearly twice those of their Earth counterparts

(mean threshold + sd: space birds = 0.053 gm + 0.018, Earth = 0.033 g + 0.026;

Mann/Whitney U:p < 0.028). Moreover, the effect of spaceflight was not transient since

the measurements of threshold were made approximately four weeks after the animals

returned to Earth. These results are provocative and support the hypothesis that gravity

may play a role in determining vestibular sensitivity.

Significance of the Accomplishments

The results support the hypothesis that, during ontogeny, gravity may help shape the

ultimate functional attributes of the vestibular system. The research leads directly to studies

designed to evaluate the effects of hyper- and hypodynamic environments on the ontogeny

of vestibular function. Responses to pulsed linear acceleration appear to depend on

neurons that are more sensitive to the rate of change in acceleration than acceleration itself.

Hypothetically, the responses therefore may reflect the activity of the so-called 'irregular'

type of vestibular primarily afferents that innervate the utricles and saccules. Moreover, the

sensitivity of these neurons may be modified during ontogeny as a result of exposure to

weightlessness. These findings may serve to improve our understanding of how

organisms develop normally on Earth and be of value in forming the strategies used in

planning for space exploration.

126
Publications

Jones, T.A. 1989. Volume conducted short latency vestibular responses to pulsed linear

acceleration. Abstracts of the Annual Meeting of the Association for Research in

Otolaryngology #299: 248.

Jones, T.A. and Pedersen, T. 1989. Short latency vestibular responses to pulsed linear

acceleration. American Journal of Otolaryngology 10: 327-335.

Lange, M.E. and Jones, T.A. 1989. Mammalian short latency electrophysiological

responses to pulsed linear acceleration (Abstract). ASGSB Bulletin 3:31.

Lange, M.E. and Jones, T.A. 1990. Short latency electrophysiological responses to

pulsed linear acceleration in the mammal (Abstract). Abstracts of the Annual Meeting of the

Association for Research in Otolaryngology #390: 343.

Roll, R., Jones, T.A., and Morley, B.J. 1989. A model to study the effects of chronically

infused neuropharmacological agents on developing embryos (Abstract). Society for

Neuroscience Abstracts 15: 1023.

Weisleder, P., Jones, T.A., and Rubel, E.W. 1989. Potentiale evocados vestibulares en

Gallus domesticus (Vestibular evoked potentials in Gallus domesticus). Archivos de

Neurobiologia 52: 307-313. (Spanish)

Weisleder, P., Jones, T.A., and Rubel, E.W. 1990. Vestibular evoked potentials (VsEP)

to pulsed linear acceleration in the chick (Abstract). Abstracts of the Annual Meeting of the

Association for Research in Otolaryngology #389: 342.

127
MOLECULAR BASIS OF

ATROPHY

Christine E. Kasper

School of Nursing

University of California

Los Angeles, CA 90024

TENSION DEVELOPMENT IN MUSCULAR

Description of Research

The long-range goal of our research has been to assess the effects of immobility and

hypokinesia on skeletal muscle function -- in particular, the effect of exercise on skeletal

muscle following prolonged atrophy. It is the aim of this projectto _describe the mechanism

by which calcium ions and calcium-mediated physiologic mechanisms act in conjunction

with gravity during hypokinesia.

Our work has focused on the atrophy-associated cellular changes which might predispose

skeletal muscle to injury during re-use and recovery. Preliminary studies in this laboratory

have shown that exercising atrophied skeletal muscle following hypokinesia produces

myofibrillar damage and degeneration. A major finding was the appearance of Type IIC or

transitional fibers and extensive fiber damage coincident with training during exercised

recovery from hypodynamia. The damage appeared in the form of necrotic fibers, central

nuclei, phagocytosis, and fiber debris in the intrafasicular and intrafibrillar spaces of the

soleus.

Initial studies have sought to: (1) characterize the occurrence and distribution patterns of

control, transitional, and damaged fibers which occur in atrophied soleus during the course

of recovery; (2) determine the contribution of the cytoskeletal element Titin in the

production of skeletal muscle damage during the recovery of skeletal muscle from atrophy;

and (3) determine the role nuclear control domains play in skeletal muscle.

Accomplishments

(1) Spatial Analysis of Control and Atrophied Skeletal Muscle: It is

well known that fiber type grouping can occur in response to denervation; indeed, "type

grouping" is often taken to be the pathognomonic sign of neurogenic atrophies. Therefore,

it is of interest to determine whether fiber types are distributed randomly in the normal

muscle and also to determine whether they are distributed randomly in non-neurogenic

pathologies, for example, in disuse atrophy.

We sought to determine: (1) whether there were any nonrandom spatial patterns in soleus

muscle of control adult rats, and (2) the spatial patterns of fiber types of unloaded soleus

muscle relative to controls. Specifically, our purpose was to determine whether fibers of

any type showed a preference to be adjacent to fibers of the same or any other type, and

whether any fiber type showed a preference for being non-randomly found in association

with fascicle boundaries. The presence of spatial patterns in control groups has to be

determined in order to serve as a standard for all other groups. In addition, the presence of

nonrandom patterns in hypokinetic groups may offer clues to the nature of the processes

that produce atrophy in a manner analogous to the fact that fiber type grouping is generally

held to be a pathognomonic sign of neurogenic atrophy.

Our principal finding is that the normal muscle fiber type pattern of actively

interspersed fibers persists throughout the hypokinetic paradigm. In contrast

128
to thefibertypegroupingthatis commonlyseenin neurogenicatrophies,ourfinding is that

both normal and hypokinetic atrophic soleus muscle show "anti-grouping";

that is, a tendency for fiber types to avoid themselves and to seek adjacencies with other

fiber types.

(2) Titin Banding Patterns in Skeletal Muscle: Using single fibers from

soleus and plantaris muscles dissected in a relaxing solution (pCa 10.0; EGTA 0.05), we

have been able to stain fibers with monoclonal anti-Titin 9D10 IgG (M. Greaser, Univ.

Wisc.) which were incubated with 1:20 diluted FITC conjugated goat anti-mouse IgG. We

measured the stained Titin doublets and sarcomere lengths. A ratio of Titin

spacing/sarcomere length revealed that there was a significant difference

(p _< 0.001) between slow soleus and fast plantaris muscles (Figure 1). Soleus

muscle was 0.380 + .004 SEM, while plantaris ratios were 0.290 + .004 SEM.

Furthermore, when compared by fiber type, irrespective of muscle of origin, there was a

significant difference (p < 0.001) between fast and slow fibers. Results are consistent with

morphometric data from previous ultrastructural studies that have shown that the width of

Z bands vary between fiber type with slow (type I) fibers the widest at 1445 ,_, and fast

(type II) fibers narrower at 611-881 ,/_. These findings are consistent with others that

show ultrasmactural differences between fibers of different types.

ORIGINAL PAGE

BLACK AND WHITE PHOTOGRAPH

Figure 1. Myofibrils from a single skeletal muscle fiber labeled with monoclonal anti-Titin 9D10 IgG

which were incubated with 1:20 diluted FITC conjugated goat anti-mouse IgG (magnification X 1000).

129
(3) Nuclear Control Domains in Skeletal Muscle: Using the intact single

skeletal muscle fiber technique, fiber segments from soleus and plantaris muscles of adult

female rats were mechanically dissected and analyzed for nuclear control domains by fiber

type. Pieces from the same cell were classified according to fiber type and the third fiber

piece was stained with Hematoxilyn and Eosin for determination of the number of nuclei,

estimated fiber volume, and sarcomere spacing. In order to count only the subsarcolemmal nuclei, fiber segments with extraneous fiber debris or disrupted sarcolemma

were excluded from this study. Cytoplasmic volume (lam3)/nucleus differences were

found to be significant (p _<0.01) between the fast and slow fibers (139,116 + 13,541

SEM vs. 71,843 + 5776 SEM, respectively). Furthermore, significant (p _< 0.01) fiber

type differences were found in the number of sarcomere lengths per nucleus (5.98 + 0.38

SEM in fast versus 3.40 + 0.21 SEM in slow fibers.) Results of volume/nucleus and

sarcomere lengths/nucleus may be interpreted to be theoretical nuclear control domains and

are consistent with data from previous biochemical studies that have shown that slow

muscles have higher DNA concentrations than fast muscles. Since lower threshold motor

units with slow skeletal muscle fibers are recruited tonically, we interpret our results to

reflect a higher protein turnover rate and smaller nuclear control domain necessary for cell

maintenance in the slow versus the fast fibers.

Significance of the Accomplishments

Finding #1" Spatial Analysis: Our findings indicate that the atrophy produced by the

unloading of skeletal muscle does not appear to be neurologically mediated but seems

solely due to myogenic variables.

Finding #2: Titin: Titin (Mr, 3 x 106) is a long, flexible filamentous muscle that links

thick myosin filaments to Z lines in the sarcomere and is responsible for the maintenance of

the structural and mechanical stability of skeletal muscle. It has been proposed that Titin is

a major component of the "third" filament system of the myofibril, and has been

immunologically detected to be among "gap" filaments that are ultrastructurally visible in

overstretched muscle. Direct evidence of this elastic role was described by using ionizing

radiation to ablate preferentially Titin and nebulin from isolated myofilaments. Removal of

these filaments causes the myofibril to become much less elastic and become axially

misaligned. The significant difference in Titin doublet spacing between fiber types may

relate to the differential ability of slow and fast muscle types to withstand increased loading

following atrophy.

Finding #3: Nuclear Control Domains: A Nuclear Control Domain is, or a DNA unit

is defined as, the volume of cytoplasm controlled by a single nucleus. In skeletal muscle,

the cumulative nuclear output gives rise to the genetic products that comprise all metabolic,

membrane, and structural muscle proteins. Accurate enumeration of the numbers and

appearance of nuclei between different fiber types has not previously been accomplished.

The unique linear array of nuclei along the length of slow fibers differs greatly from that of

fast (type II) fibers which appear to be randomly distributed. The reason for this distinct

nuclear arrangement is not readily apparent.

If the area that each nucleus controls is so significantly different between fiber types, then

one may speculate that transitions between fiber types may be the result of alterations in the

domain of the nucleus. It is known that cardiac nuclei change shape during contraction.

Perhaps the nuclei is able to sense changes in the use of a muscle, as in the case of

decreased activity during weightlessness and hypokinesia, and consequently decrease its

rate of protein synthesis. Further study of this area may give rise to the initial activity

sensing mechanism that produces skeletal muscle atrophy. Further studies are planned in

130
experimental animals that have undergonefunctional adaptation,e.g., hypertrophy,

atrophy,denervation,andexercisetraining.

Publications

Kasper,C.E., St. Pierre,B.A., Page,G.G.,Garfinkel,A., andWoo, M. 1989. Spatial

analysisof fiber type distributionsin rat soleusmuscle(Abstract). ASGSB Bulletin 3:

119.

Kasper, C.E. and Townsley, J.R. 1990. Titin spacing in mechanically peeled single

fibers with respect to fiber type (Abstract). Biophysical Journal 57(2): 538a.

Kasper, C.E., White, T.P., and Maxwell, L.C. 1990. Running during early recovery

from hindlimb suspension induces transient muscle injury. Journal of Applied Physiology

68(2): 533-539.

Thompson, J.L., Kasper, C.E., and Garfinkel, A. 1989. Targetoid muscle fibers in

reloaded hypokinetic rat soleus (Abstract). ASGSB Bulletin 3:119.

Tseng, B.S., Kasper, C.E., and Edgerton, V.R. 1990. Nuclear density in isolated single

rat skeletal muscle fibers with respect to fiber type (Abstract). Biophysical Journal 57(2):

551a.

131
MECHANISMS OF VESTIBULAR ION TRANSPORT

Thomas P. Kerr and Dennis G. Drescher

Department of Otolaryngology

Wayne State University

School of Medicine

Detroit, MI 48201

Description of Research

The two classes of vestibular sensory organs, the maculae and ampullae, differ by virtue of

the accessory structures which mediate their respective mechanical sensitivities to linear or

angular acceleration. In contrast, the mechanoreceptive hair cells associated with each type

of sensory organ show many similarities of organization and function within the sensory

epithelia. The hairs of the receptor cells, in each type of structure, extend toward the lumen

of a fluid compartment filled with endolymph. This specialized extracellular fluid is

distinguished by its unique low-sodium/high potassium ionic composition. Vestibular

transduction is thought to commence when mechanical displacement of the sensory hairs

results in the opening of "transduction channels" at the apical cell surface, with augmented

flow of ionic current from endolymph into the hair cell. This "transduction current" --

which is carded, in mammalian hair cells, almost entirely by endolymphatic potassium ion

-- exits the hair cell by way of ion channels situated in the basolateral membrane.

Given the importance of potassium movements for vestibular transduction, and the

documented losses of body fluid and electrolytes which occur upon exposure to

microgravity, the long-term objective of this project is the characterization of molecular

mechanisms mediating active and passive ion flux in vestibular endolymph and sensory

epithelia. As an additional goal, we seek to determine whether these mechanisms

participate in possible adaptive responses to changes in fluid and electrolyte balance, and to

altered patterns of vestibular stimulation experienced in microgravity.

The only mechanism yet demonstrated for active transepithelial transport of K + into the

endolymphatic compartment is Na +, K+-ATPase, a transmembrane enzyme which

"pumps" Na + and K + in opposite directions across cell membranes and across epithelia.

Much physiological evidence implicating Na +, K+-ATPase in endolymphatic K + transport

has been obtained by local application of ouabain, a specific inhibitor which binds to an

extracellular site on the enzyme. Exposure of inner-ear tissues to ouabain in vivo is

associated with a decrease of endolymphatic [K+], and an increase of [Na+].

Virtually nothing is known of possible mechanisms which may modulate levels of inner-ear

ATPase. However, this enzyme also plays a major role in the regulation of whole-body

fluid and electrolyte balance, primarily in ion-transporting epithelia of exocrine organs such

as the kidney. In many of these tissues, cation transport is subject to regulation by the

steroid hormone aldosterone Caldo"), which promotes resorption of Na + and secretion of

K +. One known mechanism by which the hormone exerts these effects involves an

increased population of Na +, K+-ATPase enzyme sites in the target tissues. Since aldo

levels are significantly altered in human subjects upon exposure to microgravity,

demonstration of an aldo effect on inner-ear ATPase levels would raise the possibility that

alterations in endolymphatic fluid and electrolyte balance occur during vestibular adaptation

to the microgravity environment.

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Studiescompletedin thepastyearwereintendedto evaluatethehypothesisthataldosterone

maymodulatethepopulationof inner-earATPasesites. To estimateenzymesitedensities,

guineapig tissueswereisolatedby microdissectionandincubatedwith 3H-ouabainin vitro;

specific binding was normalized to tissue dry weight. Parallel studies of blood chemistry

were conducted.

Accomplishments

(1) Scatchard analysis of cochlear lateral wall from normal guinea pigs produced a

linear function, with kD of 2.82 [tM. This value represents the ouabain concentration

required for half-maximal saturation of binding sites. Cochlear lateral wall incorporates the

ion-transporting epithelium responsible for maintenance of cationic concentrations in

cochlear endolymph, and is more readily isolated than the corresponding vestibular iontransporting epithelia.

(2) Pairs of male guinea pigs, weighing 200-250 gm, were maintained on a high

Na+/low K + diet for 4-5 days (to suppress endogenous secretion of aldosterone). Twentytwo hours prior to sacrifice, the experimental animal received an injection of aldosterone

(10 lagm/100 gm body weight), while the paired control was sham-injected. Tissues were

incubated with 3H-ouabain at a concentration of 5 _tM; in each experiment, ouabain binding

was assayed in lateral wall of the basal cochlear turn and in the three ampullae of either ear.

The duplicate determinations of ouabain-binding in lateral wall and ampullae from each

animal were then averaged. In every experiment, ouabain binding in the aldo-injected

animal was higher than that in the control, and these differences attained statistical

significance, both in the lateral wall and ampullae (Table 1).

Tissue n(1) A, (2) p(3)

(Aldo-Control)

Lateral wall (4) + 2.35 + 1.64 <0.05

(basal cochlear turn)

Ampulla (5) + 2.33 + 1.70 <0.02

(1) Means of duplicate determinations

(2) Mean difference, aldo-control: picomoles 3H-ouabain bound per mg dry tissue,

+ standard deviation; the concentration of 3H-ouabain in the incubation medium

was 51.tM

(3) p-values determined by one-tailed t-test for paired scores

Table 1. Changes in 3H-ouabain binding: Aldosterone injection vs. sham-injected control. Pairs of

male guinea pigs were maintained on a high Na+/low K + diet, as described in Finding #2. Twenty-two

hours prior to sacrifice, one animal received an injection of aldosterone (10 lagm/100 gm body weight),

while the paired control was sham-injected. Inner-ear tissues were isolated by microdissection, and

incubated with 3H-ouabain. Tissue-bound ouabain was assayed by liquid scintillation spectrometry.

(3) Recognizing that differences in ouabain binding might arise from indirect

effects of the experimental treatment upon whole-body fluid/electrolyte balance, we

analyzed blood sera obtained by exsanguination at the time of sacrifice. In both the

133
aldosteroneandcontrol animalsmaintainedon ahigh Na+/low K+ diet, [Na+] and[C1-]

wereelevated,and[K+] wasreduced,relativetonormalvalues.

(4) Maintenanceof animalsona high Na+/lowK+ diet resulted in a statistically

significant decrease of aldosterone levels relative to normal values. Twenty-two hours

following injection, aldo levels in aldo-injected animals were significantly higher than those

in sham-injected animals, but still did not reach normal values.

(5) There were no significant differences in serum electrolyte values or serum

osmolarity between aldo-injected and control animals maintained on the altered diet.

Significance of the Accomplishments

Finding #1: Linearity of the Scatchard plot is indicative of a single class of saturable

binding sites, presumably associated with Na +, K+-ATPase. Non-specific binding, in

contrast, is not saturable. Determination of the kD value permits the selection of an ouabain

concentration which should produce nearly complete saturation of specific ouabain-binding

sites in the experiments described under Finding #2.

Finding #2: A statistically significant increase in ouabain binding was observed in both

cochlear and vestibular tissues of aldo-injected animals. This result is consistent with the

hypothesis that aldosterone promotes an increase in the population of innerear Na +, K+.ATPase sites.

Findings #3 and #4: These results confirm the anticipated effects of the experimental

manipulations. Maintenance of subjects on a high Na+/low K + diet resulted

in an increase of serum [Na+l, decrease of [K+1, and decrease of

aldosterone. Administration of exogenous aldosterone resulted in a

significant elevation of serum aldo, but not beyond physiologic levels.

Finding #5: The absence of significant differences, in serum electrolyte values and

osmolarity, between aldo-injected and sham-injected subjects maintained on a high

Na+/low K + diet argues against the possibility that observed differences in ouabain binding

resulted from an indirect effect on inner-ear ATPase. Such an effect could be mediated, for

example, by changes in extracellular electrolyte concentrations. The present observations,

however, are consistent with the hypothesis that inner-ear ion-transporting epithelia

represent target organs subject to a direct influence of aldosterone, and that

the hormone's action includes modulation of inner-ear ATPase levels.

Findings #1-#5: Taken together, these results provide first evidence of a treatment

capable of increasing levels of inner.ear ATPase, and suggest that

endolymphatic fluidelectrolyte balance may be altered, at least transiently,

by alterations of circulating aldosterone induced by exposure to

microgravity.

Publications

Drescher, D.G., Khan, K.M., Arden, R.L., Drescher, M.J., Kerr, T.P., and Hatfield,

J.S. 1989. Protein associated with the sensory cell layer of the rainbow trout saccular

macula. Brain Research 485: 225-235.

134
Kerr, T.P., Bass,J.T., and Drescher,D.G. 1989. Autoradiographicdemonstrationof

presumptivemuscarinic acetylcholinereceptor sites in mammalianvestibular organs

(Abstract). Abstracts of the Annual Meeting of the Association for Research in

Otolaryngology, p. 326.

Kerr, T.P., Pitovski, D.Z., and Drescher, D.G. 1989. Aldosterone mediates an increase

of inner-ear ouabain binding sites (Abstract). Society for Neuroscience Abstracts 15: 209.

Pitovski, D.Z., Drescher, D.G., and Kerr, T.P. 1989. Aldosterone promotes increased

binding of ouabain to inner-ear tissues (Abstract). ASGSB Bulletin 3:31.

Pitovski, D.Z., Drescher, D.G., Kerr, K.L., and Kerr, T.P. 1990. Effects of aldosterone

on the binding of ouabain to inner-ear tissues (Abstract). Abstracts of the Annual Meeting

of the Association for Research in Otolaryngology, p. 303.

135
COLLAGEN SYNTHESIS, ASSEMBLY, AND

CHICKEN OSTEOBLAST CELL CULTURES

William J. Landis and Louis C. Gerstenfeld

Laboratory for the Study of Skeletal

Disorders and Rehabilitation

Harvard Medical School and Children's Hospital

Boston, MA 02115

MINERALIZATION IN

Description of Research

The purpose of this research is to describe the basic molecular biology and the

biochemistry, morphology, and ultrastructure of a bone cell culture system to be utilized at

a later date for spaceflight experiments under microgravity conditions. At the current time,

we are characterizing the culture system in the laboratory under normal gravity. We are

interested in understanding the growth and development of cultured bone cells derived in

our case from osteoblasts of chicken caivaria and in studying the onset and progression of

mineralization in the culture extracellular matrix. We are concerned with elaborating (1) the

mechanisms of collagen gene expression, (2) the temporal relationship between collagen

cross-links and collagen ultrastructure, (3) the assembly of collagen fibrils into orthogonal

arrays, (4) the precise interaction between collagen fibrils and mineral deposition in the

cultures, and (5) the effects of non-collagenous proteins on mineralization in the system.

In our previous experiments, we have found that the cultured chick osteoblasts

reproducibly mineralize in a temporal sequence of differentiation following the osteoblast

phenotype. Over a 30-day culture period, parameters of both cell and extracellular matrix

development have been examined, including determinations by biochemical and electron

microscopic means of collagen gene expression; collagen synthesis, processing, and

accumulation; cellular growth; presence and function of non-collagenous proteins; and

extraceltular matrix composition, assembly, and mineralization. Of major importance, our

earlier work with our bone ceil cultures has shown that they closely approximate chemical,

physical, and biological events described for bone in vivo; that extracellular matrix

formation is dependent on post-translational modification affecting collagen accumulation

and not collagen synthesis; that collagen accumulation is not dependent on the rate of

synthesis but may also be dependent on the efficiency of collagen fibril formation; and that

mineralization occurs only if a preliminary matrix is present in the system.

Accomplishments

Continuing studies with the culture system have now established the following results:

(1) In cooperative research with Dr. David Eyre (University of Washington,

Seattle), we have found that lip and LP forms of non-reducible pyridinium

crosslinks, specific to collagen, increase over the 30-day time course of

bone culture development, the HP:LP ratios and molar values are identical

to those of bone collagen controls, and the kinetics of such crosslink

formation are found to correlate directly with increases in collagen fibril

diameters.

(2) By assaying crosslink formation and collagen turnover, through collaboration

with Drs. Lila Graham and Paul Gallop (Children's Hospital and Harvard School of Dental

Medicine, Boston), it was found that the presence of remodeling or

136
collagenase activity appears to occur in culture, affecting newly

synthesized collagen molecules.

(3) Treatment of bone cell cultures with o_-aminopropionitrile, a

potent crosslink inhibitor, results in a 50% decrease of collagen

accumulation over the 30 day culture period, the appearance of collagen

fibrils unusual in their diameter sizes and size distribution as a function of

culture time, the absence of extensive orthogonal arrangements of collagen

fibril layers, and the loss of bulk mineral. Alkaline phosphatase and osteocalcin

levels remain unchanged in the presence of the inhibitor.

(4) A 66 kD phosphoprotein, which we previously found by biochemical and

immunocytochemical methods to be expressed concurrently in culture with alkaline

phosphatase and osteocalcin and localized in association with initial extracellular mineral

foci in the system, contains an arginine-glycine-asparitic acid sequence among its amino

acids. This result is suggestive of a role in cell attachment for the

phosphoprotein, but its accumulation at mineralization sites distant from

cell surfaces implies alternative function(s) which are also being investigated.

(5) Mineralization of the culture system occurs in association with

collagen, and the interaction, size, shape, and distribution of mineral crystals within

fibrils were demonstrated with topographic imaging, a microscopic technique we have

developed and applied to both tissue in situ and the bone culture system. The method

provides a unique and direct three-dimensional view of ultrastructure. The mineral

crystals are thin platelets, rather than rodshaped crystals, apparently

arranged in stacks within collagen hole zones (the putative nucleation sites for

mineral within the fibrils). Platelet lengths and widths exceed the size of the

hole zones, a result with significant implications into the basic pattern of collagen fibril

aggregation and mineral growth.

(6) Initial experiments in collaboration with Dr. Marc Grynpas (Mt. Sinai Hospital

and the University of Toronto, Ontario, Canada) utilizing inductively coupled plasma

ashing measurements show that strontium in low concentrations substitutes in a

proportional manner for calcium in the extracellular matrix of the bone ceil

cultures.

Significance of the Accomplishments

The findings are very important in terms of describing some of the events of biological

mineralization in an in vitro system closely resembling the mineral deposition of normal

calcified vertebrate tissues. The results are providing insights into details of calcification

still incompletely understood and otherwise unattainable with other models. The results

cited in findings # 1, 2, and 3 above suggest that chemical crosslinks unique to collagen are

responsible for the aggregation of smaller fibrils to larger ones and for the assembly of the

extracellular collagen matrix as a whole. Changes in the characteristics of collagen fibrils

accompanying crosslink inhibition noted in finding #2 indicate the culture osteoblasts may

produce collagenase as a means for remodeling the system. The observation in finding #4

that the 66 kD phosphoprotein is localized at culture sites distant from the osteoblast implies

that the cell binding capacity of the molecule may be only transient in nature and that this

culture constituent has additional functions. From finding #5, collagen-mineral interaction

seen by microscopic topographic imaging, a newly applied technique in its own right for

direct three-dimensional viewing of ultrastructure, shows the manner by which the crystals

are normally accommodated by the fibrils, perhaps as oriented stacks of thin platelets which

137
follow gapsor channelswithin specificregionsof largecollagenassemblies.Finding #6

suggeststhatstrontiummaybeusedasareliablemarkerfor determiningtherateof mineral

accumulationin collagenin theculturematrices.

In total,thesedatacharacterizemoreextensivelythedevelopmentandgrowthof bonecells,

theassemblyof theirextracellularmatrix, and thedepositionof_ineral in culture. The

information from the bonecell culturesis extremelyvaluablefor comparisonwith that

obtainedfrom calcifyingtissuesthemselvesandfor its contributionstowardexplainingthe

mechanism(s)of vertebratemineralization. Suchground-basedculturesalsoprovidethe

controlmodelagainstwhichmineralizationeventsmaybeanalyzedfrom thesamecultures

subjectedto themicrogravityconditionsof laterinflight experiments.Fromthoseresults,it

maybepossibleto understandthebasisof oneof themostseriousproblemsof spaceflight

sufferedby humansandexperimentalanimals,themasslossof theskeletonandits related

effects.

Publications

Gerstenfeld, L.C., Lee, D.D., Landis, W.J., and Eyre, D.R. 1990. Deposition of a bone

specific collagenous matrix by chick osteoblasts in vitro (Abstract). Transactions of the

Annual Meeting, Orthopaedic Research Society 15: 23.

Gerstenfeld, L.C., Lian, J.B., Gotoh, Y., Lee, D.D., Landis, W.J., McKee, M.D.,

Nanci, A., and Glimcher, M.J. 1989. Use of cultured embryonic chicken osteoblasts as a

model of cellular differentiation and bone mineralization. Connective Tissue Research 21:

215-225.

Landis, W.J. 1990. Mineral crystal structure and organization in calcifying tissues studies

by novel microscopic techniques (Abstract). Ultramicroscopy 32(2): 197.

Landis, W.J. and Hodgens, K.J. 1990. Visualization of sulfur-containing components

associated with proliferating chondrocytes from rat epiphyseal growth plate cartilage:

Possible proteoglycan and collagen comigration. Anatomical Record 226(2): 153-167.

Landis, W.J., Moradian-Oldak, J., and Weiner, S. 1990. Mineral-collagen structure and

organization studied by electron microscopic topographic imaging (Abstract). Transactions

of the Annual Meeting, Orthopaedic Research Society 15: 422.

McKee, M.D., Nanci, A., Landis, W.J., Gerstenfeld, L.C., Gotoh, Y., and Glimcher,

M.L 1989. Ultrastructural immunolocalization of a major phosphoprotein in embryonic

chick bone. Connective Tissue Research 21: 21-29.

138
MICROMOTIONAL STUDIES

AFFERENTS

Edwin R. Lewis

Department of Electrical Engineering

and Computer Sciences

University of California

Berkeley, CA 94720

OF UTRICULAR AND CANAL

Description of Research

The long-range goal of this research is to refine our understanding of the sensitivity of the

vestibular components of the ear to very-low-amplitude motion, and especially of the role

of gravity in this sensitivity.

We focused on the American bullfrog, a common animal subject for vestibular sensory

research. Our principal experimental method was to apply precise, sinusoidal

microrotational stimuli to an anesthetized American bullfrog, to record the resulting

responses in an individual vestibular nerve fiber from the intact ear, and to use intracellular

dye to trace the fiber and thus identify the vestibular sensor that gave rise to it. In this way,

we were able to identify specific micromotional sensitivities and to associate those

sensitivities definitively with specific sensors. Furthermore, by recording from nerve

fibers after they leave the intact inner-ear cavity, we were able to achieve these

identifications without interrupting the delicate micromechanics of the inner ear. We were

concerned especially with the relative roles of the utricle and the anterior semicircular canal

in the sensing of microrotational motion of the head about horizontal axes, and with the role

of gravity in mediating that sensing process in the utricle. The functional characterization

of individual nerve fibers was accomplished with a conventional analytical tool, the cycle

histogram, in which the nerve impulse rate was plotted against the phase of the sinusoidal

stimulus.

Accomplishments

Using intracellular dye to trace functionally characterized vestibular axons to their

peripheral origins, we separated anterior vertical canal axons from utricular axons and

demonstrated conclusively that there is a sizable population of utricular striolar axons that

mimic the rotational sensitivity usually attributed to anterior vertical canal axons. For

small-amplitude, low-frequency rotations about horizontal axes, the firing rate in a subset

of utricular axons follows rotational velocity more faithfully (in both phase and amplitude)

than does that in any of the vertical canal axons we studied. We have demonstrated

definitively that the stimulus for this faithful representation of rotational

velocity is the rate of change of the projection of the gravity vector onto the

utricular surface and not centripetal acceleration of the otoconial mass of

the utricle. This rotational velocity response spans a very large part of the normal

motional range of the head, from approximately 0.1 Hz to more than 4.0 Hz.

Scanning electron microscopy studies have shown that the sensory surface (crista) of the

anterior semicircular canal is divided into five regions, characterized by different

distributions of sensory hair bundles (the transduction apparatus). The hair bundles have

been classified as types A (those with the longest sterocilia equal in length to the

kinocilium) and B (those with the longest stereocilia being less than 2/3 as long as the

kinocilium). The two peripheral (planar) regions have only type B bundles, which are

densely distributed. Between the two planar regions is the ridge. The central region of the

139
ridge hasonly type A bundles,alsodenselydistributed. The regionsof the ridge lying

betweenthis centralregionandtheplanarregionshavesparselydistributedtypesA andB

bundles.

Individual axonstendto innervateeithertheridgeregionsor a planar region, but not both.

Axons from the planar regions consistently have conspicuously lower gain and more

periodic (less random) spontaneous firing than those from the ridge regions. On the other

hand, no differences in gain or regularity of firing was found between the axons

innervating the central region of the ridge and those innervating the other two ridge regions.

Thus, gain and regularity of firing do not appear to be determined by hair

bundle type.

In principle, if the spontaneous firing of a nerve fiber were perfectly regular, then even the

smallest stimulus-induced variation in firing pattern would be detectable. Irregulari.ty of

firing makes detection more difficult, and can be interpreted as being equivalent to no_se m

the channel. We found that the stimulus amplitude required to exceed the nerve-fiber's

noise level was independent of where the fiber arose. Those from the ridge exhibited

higher gain, but also higher noise; those from the planar regions exhibited lower gain, but

also lower noise. In terms of signal detectability, both types of fibers were approximately

equivalent.

Significance of the Accomplishments

It is well known that the human vestibulo-ocular reflex operates very well for minute head

rotations. Traditionally, the sensors responsible for this reflex have been assumed to be the

semicircular canals. The otoconial organs of mammals have been considered sensors of

lineal motion and of head orientation relative to gravity. Our observations of gravitymediated rotational velocity sensitivity in the frog casts serious doubt on this traditional

view. Furthermore, our results suggest that serious deficits in rotational motion sensitivity

could occur in microgravity environments.

Publications

Lewis, E.R. and Henry, K.R. 1989. Responses of the mammalian cochlea to waveform

singularities (Abstract). Society for Neuroscience Abstracts 15: 209.

140
GRAVITY AS A PROBE FOR UNDERSTANDING EMBRYONIC

PATTERN SPECIFICATION IN THE AMPHIBIAN MODEL SYSTEM

George Malacinski and Anton W. Neff

Department of Biology and Medical

Sciences Program

Indiana University

Bloomington, IN 47405

Description of Research

The fertile amphibian egg shows a clear gravitational response. Fertile eggs naturally orient

with respect to the gravity vector, and the polarity of the future embryo can be influenced

by manipulation of the force environment ranging from hypergravity to microgravity. The

large size of the egg, the polar distribution of its cytoplasmic components, and the ease of

experimental manipulation make the amphibian egg a unique model system to study how a

single cell senses gravity and how this information is transduced into changes in pattern

specification.

We are using gravity as an experimental tool to investigate the developmental biology of

primary pattern specification. Our focus has been directed along two main lines: (a) the

biological explanation for the considerable variation in the response of individual amphibian

eggs to given gravitational perturbation and (b) the extent to which the reorganization of the

egg cytoplasm in response to gravity drives embryonic pattern specification.

Accomplishments

(1) Individual spawnings of eggs differ in cytoplasmic rigidity. The

apparent cytoplasmic viscosity of individual inverted eggs can be quantified. Previous

reports from our laboratory utilized cytoplasmic mobility (CM) as the scoring system. We

have changed the designation to cytoplasmic immobility (CIM) to more accurately reflect

what is being measured. Individual spawnings of eggs show a wide range in mean CIM

values (52 l.tm-192 [tm/20 spawnings), and each individual spawning shows a unique

spread of individual CIM values around its mean.

(2) Eggs with a more rigid cytoplasm display a higher survival rate in

response to centrifugation. The survival of fertile eggs subjected to a centrifugation

regimen that induced twinning was monitored. Eggs with high CIM values have a higher

survival rate.

(3) Individual spawnings of eggs show considerable variation in twinning. Each

spawning had a distinctive percentage of eggs that showed a double axis (% twinning=%T)

(Figure la) and a distinctive severity of twinning (twinning index=TI) in response to

centrifugation. In agreement with data reported by others, the variation in %T was

substantial (the range at 15 g was 0.0 to 100%; at 30 g it was 0.0 to 87%).

(4) Variation in cytoplasm rigidity (CIM) can account for variation in

gravity sensitivity measured by twinning frequency. The relationship between

CIM and twinning was analyzed. There was a significant negative correlation between

CIM and TI (e.g., r=0.80 at 15 g) and CIM and %T (Figure lb). Low CIM eggs produce

more twins.

141
TWINNING AND CYTOPLASMIC IMMOBILITY IN TADPOLES

60

== 4o

_r = - 0.77; p _< 0.01

0 100 200

ClM

Figure 1. Relationship between twinning and cytoplasmic immobility

(CIM). (a) Representative surviving tadpoles from a spawning with high

% twinning (%T) and a low CIM. (b) Correlation between % twinning

(%T) and CIM. Simple linear regression line correlation coefficient (r),

and p value for two-tailed significance test is shown.

MICROTUBULES IN AN INVERTED FERTILE FROG EGG

ORIGINAL PAGE IS

OF POOR QUALITY _

Figure 2. Confocal scanning microscope image of the parallel

microtubule array in the original vegetal hemisphere of an inverted fertile

egg at 75% of the time between fertilization and first cleavage.

Microtubules are visualized by immunofluorescence. Control normal

orientation eggs showed identically oriented arrays, and no arrays were

detectable in the animal hemispheres of either control or inverted eggs.

142
(5) Subcortical parallel microtubule arrays remain in the original vegetal

hemisphere in eggs inverted with respect to the gravity vector. An extensive array of

parallel microtubules appears transiently in the subcortical vegetal hemisphere in normally

oriented eggs during the time the egg cortex rotates in normal fertile eggs. Inverted eggs

show considerable reversal of pattern formation such as a cleavage furrow initiation site

and blastomere size. The location of the parallel microtubule array in inverted eggs was

investigated using antibodies to 13tubulin and immunocytology (Figure 2). The parallel

microtubule arrays did not shift in inverted eggs.

Significance of the Accomplishments

Eggs from individual spawnings show considerable variation in cytoplasmic rig.idity. This

variation can be quantified (CIM) and used to uniquely classify each spawning of eggs

(Finding #1). Amphibian eggs vary considerably in their response to a given gravitational

challenge such as directed centrifugation (Finding #3). This degree of variation, which can

range from 0 to 100%, has made gravitational experiments difficult to interpret. The

current study has provided an important new discovery. For the first time, a clear

correlation between a property of the cytoplasm (CIM) and the response to an amphibian

egg to a directed gravitational force (survival and twinning) has been documented (Findings

#2 and #4). Future interpretation of gravitational studies on amphibian eggs will require an

understanding of the physical and/or biochemical basis for CIM.

Publications

Asashima, M. and Malacinski, G.M. (Eds.) 1990. Fundamentals of Space Biology.

Tokyo/Berlin: Japan Scientific Societies Press/Springer-Verlag, 203 p.

Malacinski, G.M. 1990. Reproduction, development and growth of animals in space. In:

Fundamentals of Space Biology (ed. by M. Asashima and G.M. Malacinski).

Tokyo/Berlin: Japan Scientific Societies Press/Springer-Verlag, p. 123-138.

Malacinski, G.M. and Neff, A.W. 1989. The amphibian egg as a model system for

analyzing gravity effects. Advances in Space Research 9(11): 169-176.

Malacinski, G.M. and Neff, A.W. 1989. An essay on redundancy within developmental

processes. In: Cytoplasmic Organization Systems (ed. by G.M. Malacinski). New York:

McGraw-Hill, p. 123-153.

Malacinski, G.M. and Neff, A.W. 1990. Introduction to space biology. In:

Fundamentals of Space Biology (ed. by M. Asashima and G.M. Malacinski).

Tokyo/Bedim Japan Scientific Societies Press/Springer-Verlag, p. 1-7.

Malacinski, G.M., Neff, A.W., Alberts, J.R., and Souza, K.A. 1989. Developmental

biology in outer space: Spaceflight provides the opportunity for new studies. BioScience

39: 314-320.

Neff, A.W., Ritzenthaler, J.D., and Rosenbaum, J.F. 1989. Subcellular components of

the amphibian egg: Insights provided by gravitational studies. Advances in Space Research

9(11): 117-186.

Neff, A.W., Wakahara, M., and Brustis, J.J. 1989. Gravity sensing in amphibian eggs:

Cytoplasmic compartmentalization (Abstract). ASGSB Bulletin 3: 108.

143
Radice,G.P. andMalacinski, G.M. 1989. Design challenges associated with middeck

aquatic animal breeding facility (Abstract). ASGSB Bulletin 3: 104.

Radice, G.P. and Malacinski, G.M. 1989. Expression of myosin heavy chain transcripts

during Xenopus laevis development. Developmental Biology 133(2): 562-568.

144
STRUCTURAL DEVELOPMENT AND GRAVITY

Emily Morey-Holton

NASA Ames Research Center

Moffett Field, CA 94035

Description of Research

The goal of this research is to understand the role of gravity in skeletal growth and

development. To achieve this goal, we must first learn if gravity turns bone cells on or off,

if/how these cells communicate with each other and with their environment, if/how

secretory products are altered by different gravity levels, how alterations in organic matrix

affect mineralization and material properties, and the role of local and systemic factors

(including endocrine, blood flow, and fluid shifts) in gravitational responses. To

accomplish these studies, both ground-based and spaceflight experiments are essential.

Gravity is a major factor determining the amount of structural support required by Earth

organisms. The hypotheses of this research effort are: (1) skeletal support structures will

change during spaceflight and/or unloading, (2) the magnitude of change will be dependent

upon the modeling or remodeling activity in each bone and the length of exposure,

(3) changes will be manifested primarily through altered matrix formation and/or

mineralization, (4) changes in both quantity and quality of bone will occur, and (5)

systemic and local factors will be involved in the changes. Most ground-based research

involves growing rats exposed to simulated spaceflight; three flight experiments have been

approved and will allow gathering of more information to support or negate the

hypotheses. During this report period, ground-based studies focused on innervation and

the response to skeletal unloading, correlation between bone mechanical properties and

bone biochemistry during one to four weeks of unloading, response of adult female rats to

skeletal unloading, and further definition of a bone cell culture system. Spaceflight

experiments included analysis of bones from rats flown on Cosmos 2044, and preparation

for experiments on the first and second dedicated Spacelab Life Sciences missions and the

Japanese Spacelab mission.

Accomplishments

(1) Analysis of Cosmos 2044 rat tissues showed that minimal bone

growth occurred during the mission even though the body mass gain was

similar to the rats flown on Cosmos 1887.

(2) Neonatal guanethidine-induced sympathectomy or neonatal capsaicin-induced

denervation (destruction of unmyelinated and fine myelinated sensory nerves)

did not dramatically alter the hindlimb response to unloading, However,

mechanical and chemical parameters were different in the weight-bearing

humerus of the guanethidine-treated unloaded animals as compared with the

humerus of the guanethidine-treated controls (collaborative study with Dr. Esther

Hill).

(3) Preliminary observations of rat bone cell cultures suggest that bone cells

grown on collagen-coated beads appear morphologically more like authentic

osteoblasts than control growth without beads and mineralization without

addition of organic phosphate (collaborative study with Charlotte Cone and Dr. Steve

Doty).

145
(4) Minimal bone changes occurred in adult female rats after two or

four weeks of unloading while muscle changes were similar to those

reported in growing rats.

Significance of the Accomplishments

Finding #1 suggests Cosmos 2044 rats may not be directly comparable with Cosmos 1887

animaIs. Cosmos 1887 rats were euthanized about two days after recovery of the

spacecraft while Cosmos 2044 rats were euthanized within hours of recovery, but Cosmos

2044 rats were approximately three weeks older at the beginning of the experiment due to

launch delays. Although weight gain was similar in both experiments, Cosmos 2044 rats,

if anything, had less bone than basal controls while Cosmos 1887 rats displayed evidence

of bone growth.

Finding #2 indicates that the skeletal response to unloading is not altered by the selective

destruction of unmyelinated and fine myelinated sensory nerves. However, sympathetic

innervation may be important in a systemic and/or hormonal response to unloading.

Finding #3 describes a unique bone cell culture system using primary rat osteoblast-like

cells grown on colIagen coated beads; these cells have an eccentric nucleus surrounded by

abundant cytoplasm containing well developed endoplasmic reticulum and Golgi systems,

produce alkaline phosphatase receptors, and appear to mineralize without the addition of

organic phosphate. These cells may develop a more comprehensive, osteoblast-like

phenotype due to mechanical forces or pressures not present in monolayer cultures. This

cell culture system will be flown in the Japanese Spacelab mission.

Finding #4 suggests that detection of bone changes in the unloaded long bones of adult rats

might require more sensitive measurement techniques, longer unloading periods, or

different sampling sites than were used in this experiment whereas muscle changes appear

to be age-independent.

Publications

Arnaud, S.B. and Morey-Holton, E. 1990. Gravity, calcium, and bone: Update, 1989.

Physiologist 33 (1, Suppl.): $65-$68.

Ballard, R.W., Gilbreath, W.P., Halstead, T., Richardson, M.L., and Morey-Holton, E.

1989. The reusable reentry satellite a new capability for NASA: A vehicle for international

cooperation. Paper presented at the 40th Congress of the International Astronautical

Federation, October 7-12, 1989, Malaga, Spain, 11 p. (IAF-89-015)

Cone, C.M., Morey-Holton, E., and Doty, S.B. 1989. Development of a mineralizing,

gravity-sensitive culture system for primary rat osteoblasts (Abstract). ASGSB Bulletin 3:

1i6.

Doty, S.B., Morey-Holton, E.R., Durnova, G.N., and Kaplansky, A.S. 1990.

1887: Morphology, histochemistry, and vasculature of the growing rat tibia.

Journal 4: 16-23.

Cosmos

FASEB

Garetto, L.P., Cho, H.-J., Gonsalves, M.R., Morey, E.R., and Roberts, W.E. 1989.

Nuclear volume analysis of rat mandibular condyle following 3 days of simulated

weightlessness (Abstract). ASGSB Bulletin 3:114.

146
Garetto, L.P., Gonsalves,M.R., Morey, E.R.,Durnova,G., and Roberts,W.E. 1990.

Preosteoblastproduction55hoursaftera 12.5day spaceflightonCosmos1887. FASEB

Journal 4: 24-28.

Halloran, B.P., Bikle, D.D., Cone, C.M., and Morey-Holton, E. 1989. Skeletal

unloading: Effects of hypophysectomy and growth hormone replacement (Abstract).

ASGSB Bulletin 3:112.

Holton, E., Beretta, D., Doty, S., Roberts, W., and Garetto, L. 1990. Experiment K-6-

03. Part I: Gravity and Skeletal Growth. In: Final Reports of the US Experiments Flown

on the Soviet Biosatellite Cosmos 1887 (ed. by J.P. Connolly, R.E. Grindeland, and

R.W. Ballard). Moffett Field, CA: NASA Ames Research Center, p. 115-121. (NASA

TM-102254)

Morey, E.R., Cone, C.M., Harris, J.D., Iufer, E.J., Vasques, M., and Grindeland, R.E.

1989. Response of nonweightbearing hindlimbs of growing rats to null magnetic fields

(Abstract). ASGSB Bulletin 3: 73.

Morey-Holton, E.R. and Cone, C.M. 1990. Bone as a model system for organ/tissue

responses to microgravity. In: Fundamentals of Space Biology (ed. by M. Asashima and

G.M. Malacinski). Tokyo/Berlin: Japan Scientific Societies Press/Springer-Verlag, p.

113-122.

Parazynski, S.E., Morey-Holton, E., Cone, C., and Martin, B. 1989. Musculoskeletal

unloading in skeletally mature female Sprague-Dawley rats (Abstract). ASGSB Bulletin 3:

112.

Patterson-Buckendahl, P., Globus, R.K., Bikle, D.D., Cann, C.E., and Morey-Holton,

E. 1989. The effects of simulated weightlessness on rat osteocalcin and bone calcium.

American Journal of Physiology 257:R1103-R1109.

Roberts, W.E., Seeauer, S.J., Garetto, L.P., Gonsalves, M.R., and Morey, E.R. 1989.

Osteoblast histogenesis in the periodontal ligament following 3 days of simulatedweightlessness: Potential effects of diurnal rhythm (Abstract). ASGSB Bulletin 3:117.

Sessions, N.D., Halloran, B.P., Bikle, D.D., Wronski, T.J., Cone, C.M. and MoreyHolton, E. 1989. Bone response to normal weight bearing after a period of skeletal

unloading. American Journal of Physiology 257: E606-E610.

147
SKELETAL COLLAGEN TURNOVER BY THE OSTEOBLAST

Nicola C. Partridge

St. Louis University School of Medicine

and Pediatric Research Institute

1402 S. Grand

St. Louis, MO 63104

Description of Research

Among the most overt negative changes experienced by man and experimental animals

under conditions of weightlessness are the loss of skeletal mass and attendant

hypercalciuria. These clearly result from some disruption in the balance between bone

formation and bone resorption (i.e., remodelling) which appears to be due to a decrease in

the formative functions of the osteoblast. In the present study, the clonal osteoblastic cell

line, UMR 106-01, and sections of bone have been used to investigate the regulation of

several proteins whose expression is central to both bone formation and bone resorption;

these are Type I collagen (the major organic constituent of bone), collagenase and tissue

inhibitors of metalloproteases. Expression has been monitored at the protein level using a

combination of techniques including the radiolabelling of the proteins, gel electrophoresis,

ELISA assays and immunohistochemical staining, and subsequently at the level of RNA,

by Northern blot analysis and nuclear run-on assays. This project will shed some light on

the comprehensive role of the osteoblast in the remodelling process, and, in so doing,

provide some insight into how the process might be disrupted under conditions of

microgravity.

Regulation of Collagenase Synthesis and Turnover. We have shown that collagenase is

secreted by UMR ceils and then rapidly removed from the medium by a ceil-mediated

process. Using radioiodinated collagenase, we have now been able to demonstrate the

presence of specific receptors for collagenase on UMR cells. Binding, conducted at 4°C,

was shown to be maximal at 2 hr. Scatchard analysis revealed 1800 receptors/cell, with a

single class of high affinity sites (kD~10 -11M). Specificity of the receptor was further

demonstrated by the lack of competition for binding by either human or bacterial

collagenases. This has led us to formulate the hypothesis that the collagenase receptor on

osteoblastic cells is involved in regulation of extracellular bone collagenase, and may be

responsible for either activation and/or endocytosis of the secreted enzyme.

UMR 106-01 Collagenase Clones and Regulation of Collagenase mRNA. We have

isolated two clones for rat collagenases from a UMR 106-01 cDNA library. These clones

are identical except one (UMRCase54) is 245 nucleotides longer at the 5' end. These

clones contain the entire coding sequence of the proenzyme; missing are approximately 2

amino acids of the signal sequence and 100-200 nucleotides of the untranslated 5' region of

the transcript (estimates are based on comparison to the human collagenase cDNA).

Nuclear run-on studies, together with Northern blot analyses, indicated a 4-8 fold increase

in the rate of synthesis of collagenase mRNA resulting in a 100-fold elevation in

accumulated mRNA, 4 hr after treatment with parathyroid hormone (PTH). These studies

demonstrate that the regulation of collagenase gene expression in UMR 106-01 cells is, in

part, transcriptional.

Our next goals are to isolate the regulatory regions of the rat collagenase gene. To do this,

we have constructed a UMR genomic library in lambda DASH II. Using a sequence at the

148
5'-mostendof UMRCase54we haveisolateda rat collagenasegenomicclone. We are

currentlycharacterizingthisclonewhichwill beusedto decipherthemechanisminvolved

in thetranscriptionalregulationof theUMR collagenasegene.

ImmunohistochemicalDetectionof Collagenasein Sectionsof Bonefrom Cosmos2044

Flight Animals. Frozensectionsnearthesagittalsuture(anareaof high turnover)of 64

dayold rat calvariaewerestainedfor collagenaseby immunohistochemistry.Theresults

indicatedthepresenceof collagenasein thesamerestrictedendocranialmatrix-associated

regionthatwasseenin developing14dayold ratsalthoughamountsof stainingwerelower

in theolderagegroup. Theresultsalsodemonstratedthattheenzymecouldbedetectedin

fixed, decalcifiedtissue. [Note: Only smallamountsof collagenasehavebeendetectedin

weightbearingbones(tibiae) and mostly associatedwith the primary spongiosa. These

observationshave led us to hypothesizethat collagenaseis more important in nonweightbearingskeletaltissuesandthat weightlessnessandthe relatedchangesin blood

distributionmayaffecttheamountandlocationof collagenasein calvariae.]

Prior to receiving thetissuesfrom the Cosmosflight, we conductedcontrol experiments

with similarpiecesof calvarialtissuefrom90dayold ratstoensurethevalidity of theflight

experiments.This includedtestingthetimeof fixation andtypeof fixative sincethetime

betweendissectionof theratsandarrival in Moscow wasgreaterthanusual. We found

that fixation in paraformaldehydefor 72 hr did not affect the immunohistochemistryof

collagenasestaining.

WereceivedtheCosmossamplesinvery goodconditionandclearlylabelled.We havehad

frozen sectionscut from animalsfrom the basal,flight and vivarium groupsand have

cardedout experimentsto assessour techniqueswith thesematerials. We havestained

sections from these groups both with Hematoxylin and Eosin and have used

immunohistochemistryfor collagenase.The vivarium sectionsareexcellent,but when

usingtheothergroupsweneedto cut deeperinto theblocksor at adifferentorientationto

obtainbettermaterials.With thelatter,therehavebeenproblemswith thetissueadhering

to theslides.Alternativetechniquesto attachthesectionsweretestedandwefinally found

that theuseof chrome-gelhelped. We arenow readyto conductacompletecomparative

assessment of thetreatmentgroupsfrom theflight experiment.

Accomplishments

(1) Demonstrated that the neutral metalloprotease, collagenase, is not only secreted

by osteoblastic cells but is also removed from the media by a specific receptor.

(2) Showed that PTH regulates collagenase gene expression, in part, by

a transcriptional mechanism.

(3) Isolated a genomic clone for rat collagenase from a UMR 106.01

genomic library.

(4) Observed collagenase staining in adult rat calvariae in a restricted endocranial

matrix-associated region.

Significance of the Accomplishments

Finding #1: Treatment with PTH appears to change the phenotype of the osteoblast from a

matrix-synthesizing cell to one actively involved in the resorption process. Collagen

synthesis declines while production of enzymes associated with matrix removal increases.

This situation may resemble that seen in situations of weightlessness where bone formation

149
is perturbed. Nevertheless,the cell appearsto exert tight control over the amountof

collagenasein theextracellularmediumbyrapidly re-extracting it via a specific cell-surface

receptor.

Finding #2: The demonstration that rat bone collagenase is regulated at the transcriptional

level and the availability of the cDNA clone will lead us into a new realm of

experimentation which will help us to dissect the signal transduction pathways involved in

PTH action on the osteoblast. We will also use the cDNA to assess the effects of

weightlessness on transcript abundance in bones from animals subjected to microgravity.

Finding #3: Isolation of the gene for rat collagenase and its regulatory elements will enable

us to construct materials which can be used to monitor PTH action as well as the effects of

other mediators. One use of these reagents will be to transfect osteoblasts and send them

into space to determine the effects of microgravity on hormonal responsiveness of bone

cells.

Finding #4: The detection of collagenase in adult calvariae forms the basis of studies which

are currently underway on calvarial tissues from rats flown on Cosmos 2044.

Publications

Civitelli, R., Hruska, K.A., Jeffrey, J.J., Kahn, A.J., Avioli, L.V., and Partridge, N.C.

1989. Second messenger signaling in the regulation of collagenase production by

osteogenic sarcoma cells. Endocrinology 124: 2928-2934.

Gershan, L.A., Jeffrey, J.J., and Partridge, N.C. 1989. Developmental changes in

collagenase and tissue plasminogen activator in neonatal rat calvariae (Abstract). Journal of

Bone and Mineral Research 4(Suppl. 1): $206.

Gershan, L.A., Noonan, J.E., Jeffrey, J.J., and Partridge, N.C. 1989. Developmental

changes in collagenase (C'ase) and tissue plasminogen activator (TPA) in neonatal rat

calvariae (Abstract). Pediatric Research 25(4,2): 51A.

Partridge, N.C., Scott, D.K., Kopp, K., Jeffrey, J.J., and Quinn, C.O. 1989. Hormonal

induction of collagenase mRNA in rat osteoblastic cells (Abstract). ASGSB Bulletin 3: 43.

Quinn, C.O., Scott, D.K., Kopp, K., Jeffrey, J.J., and Partridge, N.C. 1989.

Transcriptional induction of collagenase mRNA by parathyroid hormone in rat

osteosarcoma cells (Abstract). Journal of Bone and Mineral Research 4(Suppl. 1): S413.

Scott, D.K., Clifton, D.K., Kopp, K., Kahn, A.J., and Partridge, N.C. 1989. H-rasrelated gene expression and regulation in rat osteosarcoma cells (Abstract). Journal of

Bone and Mineral Research 4(Suppl. 1): $256.

Strege, D.W., Kahn, A.J., Jeffrey, J.J., and Partridge, N.C. 1990. Stimulation of

collagenase production by rat osteosarcoma cells can occur in a subpopulation of cells.

Journal of Bone and Mineral Research 5(9): 963-971.

150
GRAVITATIONAL EFFECTS

AMPHIBIAN EMBRYOS

Carey R. Phillips

Department of Biology

Bowdoin College

Brunswick, ME 04011

ON EARLY DEVELOPMENT IN

Description of Research

The long range goals of this project fall into two parts: (1) to understand how the neural

axis is determined and patterned in amphibians, and (2) to study how this pattern is altered

when embryos are fertilized and grown in an extraterrestrial environment. The more

immediate goals are to understand how the neural axis is patterned on Earth. We have

made extensive studies of the tissue interactions involved in establishing the dorsal axis and

more specifically the neural tissue. In order to understand how morphogenetic signals

pattern the neural system at the molecular level, we needed to construct molecular probes.

Therefore, another of our immediate goals has been to construct the appropriate molecular

probes so that we can study the signals which program the neural patterns and how the

presumptive neural cells respond to these signals at the molecular level.

Accomplishments

We have made considerable progress in two areas of the problem of neural pattern

formation. In the first area, we have shown that the old paradigm of neural induction and

patterning resulting from interactions from the underlying dorsal mesoderm is not entirely

correct. We have shown that signals which induce the ectoderm to become

neural are traveling from the blastopore lip area (the cells which initiate the

cell rearrangements at gastrulation) through the plane of the ectoderm as well

as possible signals from the underlying dorsal mesoderm. This finding has allowed us to

re-examine several classical questions and provide more meaningful assays to understand

the mechanisms involved in neural induction.

The second accomplishment during 1990 is that we have constructed a recombinant

DNA library of all the active genes (RNA) in the cells which we have

shown previously to be sending the signals for neural induction. We have

screened this recombinant library and have isolated clones which represent RNAs which

reside in only the cells sending the signals or those responding to the signals and becoming

neural tissue. These clones fall into two categories. One class of clones represent RNAs

which were made during oogenesis (when the egg was made). These RNAs must become

physically localized to the dorsal side of the embryo where the signaling cells will

eventually reside. The localization process of this class of RNA will be sensitive to any

gravitational perturbations which lead to an abnormal dorsal axis or a repositioned dorsal

axis. Therefore, these clones will provide excellent probes for looking at how changes in

the gravitational environment affect the distribution of specific molecules that are

implemented in the dorsal patterning process. The second class of clones represent the

earliest biosynthetic response of presumptive neural cells to the signals from the organizing

region.

Significance of the Accomplishments

Understanding the tissue interactions which lead to neural induction and pattern formation

of the brain region are extremely important if we are to understand the mechanisms which

151
control this process. There has been a major quest for finding the signals and

understandingthemechanismscontrollingneuralinductionsince1924. In 1933scientists

becameconvincedthat all of thesignalscomefrom theunderlyingdorsalmesodermand

haveapproachedtheproblemaccordingly. However,we nowknow that thereareseveral

signaling eventsoccurring prior to the time the ectodermhascontact with the dorsal

mesodermand that someof theseevents appearto be necessarybefore the dorsal

mesodermcanhaveaneffectwithin theamountof timebeforeneuraltissueis determined.

Therefore, thesefindings shouldenableus to reapproachthis very old problem with

completelydifferentassaysandexpectations.

The cloneswhich wehaveisolatedfrom therecombinantcDNA library will enableusto

studythe mechanismsfor neuralinduction at themolecularlevel, SeveralStudieshave

beendonewhich showthat thedorsalaxis canberepositionedby alteringthe embryo's

position relative to Earth's gravity. Recentstudiesindicate that amphibian embryos

fertilizedin spacewill experiencedevelopmentalabnormalities.A verylikely candidatefor

thedevelopmentalabnormalitiesis that thecytoplasmicrearrangementswhich normally

occurbetweenfertilizationandthefirst cleavagedivisiondonotoccurcorrectly. We have

clonesfor RNAs which arenormally distributedon only thedorsal sideof the embryo,

preciselywheretheorganizeror signalingcells will reside. Therefore,we now havethe

capabilityto studythemechanismsthatlocalizeaspecificmoleculewithin thesinglecelled

embryoand to studythislocalizationprocessin embryosthathavebeenfertilized and/or

raised in a spaceenvironment. The secondclass of clones correspondsto RNAs

synthesizedvery earlyin theprocessof establishingadorsalaxisandneuraltissue. These

cloneswill beimportantin helpingto identify thesignalsresponsiblefor thedorsalization

andneural inductionprocesses.We canusetheseclonesin assaysto isolatethe signals

andbeginto understandhow thefirst classof cloneswhich havebeenphysicallylocalized

to the dorsal sideof the embryoduring the first cell cycle areinitiating the processof

patternformation.

It appearsthatatleastsomeof thesignalsinvolvedin theinductionof dorsalstructuresand

the resultant patternformation arehighly conservedbetweenspecies. Therefore,it is

highly likely thatmanyof theclonedprobeswhichwehaveisolatedwill alsobeusefulin

thestudyof howotherorganismsmightrespondto a spaceenvironment.

Publications

Phillips, C.R. 1989. Gravitational effects on neural development of amphibians

(Abstract). ASGSB Bulletin 3: 40.

Phillips, C.R. 1990. Review of Experiments in Animal Development." Student Manual

(ed. by R.W. Merriam). Quarterly Review of Biology 65(3): 357-358.

152
CELL KINETIC AND HISTOMORPHOMETRIC

MICROGRAVITATIONAL OSTEOPENIA

W. Eugene Roberts

Bone Research Laboratory

Indiana University School of Dentistry

1121 W. Michigan Street

Indianapolis, IN 46202

ANALYSIS OF

Description of Research

Previous studies of cell differentiation supporting bone formation have shown that

osteoblast (bone forming cell) histogenesis is inhibited by decreased skeletal loading and

microgravity. The present goals and objectives for research in this area are: (1) to define

the cellular regulation of osteoblast production, (2) to determine how this process is

suppressed in microgravity, and (3) to develop in situ hybridization methodology enabling

further characterization and verification of previously described osteoblast precursors.

Presently, DNA labeling (3H-thymidine uptake), mitotic activity and nuclear size are used

as indices of the proliferation and differentiation aspects of osteoblast production.

Using our established techniques, kinetically and/or morphometrically distinguishable cell

compartments in the osteoblast histogenesis sequence have been described: (1) selfperpetuating, less differentiated precursor cells (A type), (2) committed osteoprogenitor

cells (A' type), (3) nonosteogenic B cells, and (4) preosteoblasts (C+D cells). The

osteoblast (Ob) histogenesis sequence is A ---> A' ---> C --_ D --->Ob (Figure 1). An

increasing nuclear volume (A --> C) is a morphological manifestation of change in genomic

expression (differentiation) and is necessary for the production of the immediate precursor

cells to osteoblasts. The nuclear volume assay is applicable to all skeletal sites tested, i.e.

periodontal ligament (PDL), tibial metaphysis, mandibular condyle and mandibular

periosteum. The rate-limiting osteogenic induction step (A' ---> C) is stimulated by

mechanical (orthodontic) force and inhibited following exposure to microgravity. The

mechanical sensitivity of the A'---> C shift appears to involve the action of prostaglandins

since administration of the prostaglandin synthesis inhibitor, indomethacin, results in a

block of osteoblast histogenesis downstream from the microgravity block.

Research conducted in the past year focused on (1) further definition of osteoblast

production in rats exposed to a ground-based simulated weightlessness model, (2) preflight

Experiment Verification Test (EVT) specimen processing and evaluation for the upcoming

SLS-1 (Spacelab Life Sciences-l) shuttle mission, and (3) development of in situ

hybridization as an advanced method for studying specific messenger RNA production

within osteoblast precursor cells.

153
OSTEOGENESIS HISTOGENESIS PATHWAY

Gi Preosteoblast G2 Preosteoblast

Committed

Osteoprogenitor

Precursor Cell

Figure 1. Schematic of cell kinetic model describing the proliferation and differentiation of cells associated

with osteoblast histogenesis. Cellular compartments are classified as A+A °, B, C or D according to the

following nuclear volume categories: 40-79, 80-119, 120-169 and >170 Ixm3, respectively. Microgravity

blocks the induction of osteogenesis (i.e., A'_ C shift).

Accomplishments

• Flight Preparation

(1) Mandibular condyles, maxillae, lumbar vertebrae and tibiae from EVT animals

showed acceptable 3H'proline labeling of bone matrix formation (Figures 2 and 3). This

test was very important since the animals also received other radioisotopes that might have

interfered with specific detection of the 3H-proline bone matrix label.

• Simulated Weightlessness (SW) Data - Unloaded Rat MGdel

(2) Unloading (3 days) resulted in a 52% increase (p < 0.05) in

osteoblast progenitor (A+A') cells and a 47% decrease (p < 0.05) in

preosteoblast (C+D) cells in the tibial metaphysis of animals euthanized in the

evening (9 PM). AM-euthanized animals showed a similar pattern, but only the decrease

(39%) in the C+D cell compartment was significantly different from control.

(3) No significant differences between the AM and PM time groups were noted in

the fractional distribution of tibial metaphyseal cell types.

154
ORIGINAL PAGE

BLACK AND WHITE PHOTOGRAPH

Figure 2. Autoradiography 3H-proline labeling of bone and PDL adjacent to maxillary first molar from

EVT rat. Concentrated band of autoradiographic gains depicts bone matrix formation adjacent to PDL.

More diffuse labeling within PDL is indicative of ongoing collagen synthesis.

Figure 3. Autoradiographic 3H-proline labeling of trabecular bone within a lumbar vertebra from EVT rat.

The concentrated band of autoradiographic grains depicts bone matrix formation adjacent to marrow space.

155 ORIGINAL PAGE IS

OF POOR QUALITY
(4) 3H-thymidine labeling of cells undergoing DNA synthesis tended to follow

shifts in the fractional distribution of cell types.

(5) Unloading (3 days) did not result in significant differences in mandibular

condyles with regard to fractional distribution of osteogenic cells, incidence of cells

undergoing DNA synthesis, width of condylar cartilage zones, or volume percent ratio of

. cartilage to bone.

• In Situ Hybridization Methods Development

(6) Initial studies have focused on the methodological problems involved in

performing this advanced technique in paraffin and methylmethacrylate embedded tissues

as well as in fresh frozen specimens, We prefer to use methylmethacrylate-embedded

tissue for the in situ hybridization because this embedding media is most amenable to the

nuclear volume cell kinetic assay. The use of methylmethacrylate-embedded tissues alone

will allow adjacent serial sections to be processed for nuclear volume cell kinetic assays or

in situ hybridization, greatly improving correlations between the two techniques.

Significance of the Accomplishments

Finding #1: Evaluation of EVT specimens indicates that the 3H-p.roline labeling methods to

be used in SLS-1 rats will adequately label bone matrix formation following spaceflight,

despite concomitant dosing of the animals with other radioactive substances (used for nonbone SLS-1 experiments).

Finding #2: These data indicate that the block of tibial metaphyseal osteoblast histogenesis

in hindlimb unloaded rats is similar to that seen in the periodontal ligament (PDL) of

animals exposed to both actual and simulated weightlessness.

Finding #3: However, in contrast to PDL cells, these data indicate that the transient

suppression of preosteoblast formation within 72 hours after initiation of

SW is not associated with disturbance of circadian rhythm of tibial

metaphyseal osteogenic cells. Alternatively, the tibial metaphyseal osteogenic cells

may have a different rhythm period.

Finding #4: Despite the size of the precursor cell (A+A') and preosteoblast (C+D)

compartments, the number of proliferating cells remained relatively constant. This

indicates a relatively constant control of cell turnover which is independent of

the total number of a particular cell type. These data are similar to that seen in

PDL.

Finding #5: The unloading model for simulated weightlessness does not mimic the effects

of spaceflight on the mandibular condyle.

Finding #6: Development of an in situ hybridization technique will allow visualization of

specific messenger RNAs as a means of further defining the state of differentiation of

osteoblast precursor cells previously described by nuclear volume assay. The maintenance

of section adhesion to the slides has proven to be a major problem that must be overcome if

methacrylate-embedded tissues are to be utilized.

156

r
Publications

Garetto, L.P., Barkett, G.D., Morey, E.R., and Roberts, W.E. 1990. Nuclear volume

analysis of osteoblast histogenesis in the rat tibial metaphysis during simulated

weightlessness. Abstracts of the Third International Conference on Bone Growth and

Development, UCLA.

Garetto, L.P., Cho, H.-J., Gonsalves, M.R., Morey, E.R., and Roberts, W.E. 1989.

Nuclear volume analysis of rat mandibular condyle following 3 days of simulated

weightlessness (Abstract). ASGSB Bulletin 3:114.

Garetto, L.P., Gonsalves, M.R., Morey, E.R., Durnova, G., and Roberts, W.E. 1990.

Preosteoblast production 55 hours after a 12.5-day spaceflight on Cosmos 1887. FASEB

Journal 4: 24-28.

Garetto, L.P., Gonsalves, M.R., Morey, E.R., and Roberts, W.E. 1989. Recovery of

osteoblast histogenesis in rat molar PDL following 7-18.5 days of spaceflight: Results

from Cosmos 1129, Spacelab-3 and Cosmos 1887 (Abstract). Calcified Tissues

International 44(Suppl.): S-96.

Garetto, L.P., Roberts, W.E., and Gonsalves, M.R. 1990. Experiment K-6-03. Part III:

Nuclear volume analysis of osteoblast histogenesis in periodontal ligament cells of Cosmos

1887 rats. In: Final Reports of the U.S. Experiments Flown on the Soviet Biosatellite

Cosmos 1887 (ed. by J.P. Connolly, R.E. Grindeland, and R.W. Ballard). Moffett

Field, CA: NASA, Ames Research Center, p. 139-146. (NASA TM-102254).

Holton, E., Beretta, D., Doty, S., Roberts, W., and Garetto, L. 1990. Experiment K-6-

03. Part I: Gravity and Skeletal Growth. In: Final Reports of the US Experiments Flown

on the Soviet Biosatellite Cosmos 1887 (ed. by J.P. Connolly, R.E. Grindeland, and

R.W. Ballard). Moffett Field, CA: NASA Ames Research Center, p. 115-121. (NASA

TM-102254)

Roberts, W.E., Garetto, L.P., and DeCastro, R.A. 1989. Remodeling of devitalized bone

threatens periosteal margin integrity of endosseous titanium implants with threaded or

smooth surfaces: Indications for provisional loading and axially directed occlusion.

Journal of Indiana Dental Association 68: 19-24.

Roberts, W.E., Garetto, L.P., and Jee, W.S.S. 1989. Differential glucocorticoid

influence on osteoblast histogenesis during mechanically induced osteogenesis (Abstract).

Calcified Tissues International 44(Suppl.): S-98.

Roberts, W.E., Seeauer, S.J., Garetto, L.P., Gonsalves, M.R., and Morey, E.R. 1989.

Osteoblast histogenesis in the periodontal ligament following 3 days of simulatedweightlessness: Potential effects of diurnal rhythm (Abstract). ASGSB Bulletin 3:117.

Rosenoer, L.M.L., Garetto, L.P., Gonsalves, M.R., and Roberts, W.E. 1989.

Indomethacin inhibits differentiation of committed osteoprogenitor cells to preosteoblasts

(Abstract). Calcified Tissues International 44(Suppl.): S-98.

157
STRUCTURE AND

RECEPTORS

FUNCTION

Muriei D. Ross

NASA Ames Research Center

Moffett Field, CA 94035

OF MAMMALIAN GRAVITY

Description of Research

The long-term goal of this research is to understand how information is processed in

gravity receptors on Earth and in space, with special reference to understanding adaptive

responses to altered gravitational environments. To help achieve this goal, more than 25

three-dimensional (3-D) reconstructions of terminal/receptive fields and of small portions of

the macular neural network of the Sprague-Dawley rat have been made, and the

connectivities of about 400 cells have been mapped. This ground-based work will serve as

a basis for interpretation of any observed changes in microstructure that occur in maculas of

rats to be flown in April 1991 on shuttle mission SLS-1 (Spacelab Life Sciences-l) and

later, on SLS-2.

For our most recent reconstructions we have cut a new series of 175 serial sections at 20

nm. This is twice as thick as is usual for transmission electron microscopy. However, this

thickness is easily handled by the Zeiss 902 transmission electron microscope (TEM)

available to us, and has many benefits in our work. Doubling the thickness has meant that

fewer sections need to be handled to cover the same distance in the macula, and has

permitted better visualization of small-diameter collaterals from calyces and nerve fibers.

Currently, we photograph alternate sections in their entirety but have also focused on a

small area for more intense study through the use of every section.

The method of reconstruction remains the same as reported previously. Following

photography at 3000X in the TEM, the micrographs are printed and assembled into

montages of the sections. Objects of interest are traced onto acetate from the sections and

are digitized into a PC where the slices are placed in register and portrayed as filled slices.

These data are then transferred to a Silicon Graphics IRIS high performance workstation

where the objects are reconstructed as shaded solid images. Animated films of the objects

turning around, or being cut into and reassembled, are made using equipment at the

Numerical Aerodynamic Simulation Facility.

Accomplishments

The accomplishments fall naturally under two headings: Results, and Theoretical

Interpretations.

Results: This study continues to provide evidence that maculas are organized as

weighted neural networks for parallel distributed processing of information. We have

mapped the distribution of over 300 ribbon and spherule synapses in type I and type II hair

cells as part of the study of weighting of information flow in the macular neural network.

No two macular terminal/receptive fields are identical in details, so that macular wiring

appears to be accomplished through constrained randomness. Moreover, various macular

areas appear to be morphologically organized for differing analyses of the incoming

acceleratory signal. This is called segregation, and means that the various parts of the

macula are likely analyzing the input for different components and, possibly, for centrally

different purposes. Thus far, we have identified four network organizations that

differ from one another in kinds of nerve fibers supplying the area, in

158
terminal fields, and degree of collateralization. With respect to collaterals, our

findings continue to indicate that the intrinsic collaterals are significant in information

processing. We still have not identified an extrinsic source for efferent terminals on

macular neural elements, but this finding must be interpreted cautiously because of the

difficulty in tracing efferents over distance within the macula. The nerve fibers are often

only 0.2 _m in diameter between boutons.

In related work, we carried out cupric ion-ferricyanide labelling of impulse initiation zones

in rat vestibular nerve fibers. The zones had not been labelled previously except in fish.

Our results showed that, whether branched or not, each vestibular nerve has but one

impulse initiation zone. It is located just distal to the heminode.

Coincidentally, it was learned that a substance in subsurface cisterns of type I hair cells is

reactive to the procedure employed, but type II hair cell cisterns react little or not at all.

This indicates that there is a fundamental difference in cisternal material in the two kinds of

hair cells.

Theoretical Interpretations: The observation that no two macular

terminal/receptive fields are identical prompted an attempt to reproduce the fields by

computer, using the Monte Carlo method of simulation. Data concerning branch lengths,

number of branches, and numbers of hair cells communicating with individual calyces were

obtained from the reconstructions and montages. These provided constraints, so that

nerves could terminate, branch or grow within specific distances and could have hair cells

within a certain range. The constraints were coupled with a random number series in a

software program to generate symbolic representations of the terminal/receptive fields. The

outcome of this study is that the fields actually observed were reproduced as were others,

within the range of possibility but not seen. The simulations lend support to the idea that

an element of randomness is introduced developmentally when the connectivities of the

maculas are being established.

A second interpretation concerns the fundamental organization of maculas, and whether it

corresponds to that in other neural tissue such as retina and other parts of the brain. The

results of the 3-D studies are being interpreted to indicate that there are channeled and

distributed modifier circuits in the maculas, as in all other neural tissue. In the mammalian

macula, type I hair cells provide channeled input to the calyx (Figure 1, left) but type II hair

cells and the intrinsic collateral system comprise the distributed circuitry that modifies

calyceal and vestibular nerve output (Figure 1, right). This interpretation brings maculas

into line organizationally with other, more complex neural tissue.

A corollary concept is that the distributed modifier microcircuits provide the system with its

greatest potential for adaptability to new environments, and have the most influence on

mechanisms underlying memory and learning. This concept will be tested in the

microgravity of space.

In collaborative work with a mathematician trained in signal processing, we have shown

that the stereocilia of the hair cell tufts have nearly optimal hexagonal organization for

detection of signals. The stereociliary tufts are compared to phased array detectors.

Significance of the Accomplishments

Our research has identified the site and number of impulse initiation zones in mammalian

vestibular nerve fibers. This information is vital to any interpretation of macular

functioning. Likewise, the research on stereocilia should provide new insights into their

functioning. While the hexagonal arrangement of the stereocilia has been known since they

were first studied using electron microscopy, the functional significance of this

159
organization has eluded discovery. These and other of our findings have been placed into a

global interpretation of macular functioning that will be helpful when flight results are

obtained. That is, we shall be looking not only for changes in number and size of ribbon

and spherule junctions, but also for alterations in subsurface and subsynaptic cisterns in the

two kinds of hair cells. The total effect is that we have a solid base of knowledge on which

to build our future research, and a solid basis for comparison of results obtained in

microgravity with those of Earth-bound studies. Our theoretical interpretation of the

fundamental organization of macular and other neural tissue will be sharpened by

experiments but, if proven to be basically correct, it provides a new conceptual framework

for studying adaptation to altered gravitational environments and for investigating the

possible causes of macular disorders wherever they occur.

INPUT oF TYPE I AND TYPE II HAIR CELLS

IN THE MACULAR CIRCUITRY

nput

- J,

I

Bipolar

cell ou,0 , IHF_ cell _ /_i ] _!

CHANNELED DISTRIBUTED MODIFIER

Figure 1. The type I (I) cell channels its output to its calyx (left). Type II cells (II) and the collateral

system comprise the distributed modifier circuit (right) that shapes calyceal output. Solid arrows indicate

input and output of hair cells; arrowheads show direction of synapses on type II cells; double headed arrows

are reciprocal synapses; dashed arrows represent modified output of bipolar cells.

160
Publications

Mugler, D.H and Ross, M.D. 1990. Vestibular receptor cells and signal detection:

Bioaccelerometers and the hexagonal sampling of two-dimensional signals. Mathematical

Computer Modelling 13: 85-92.

Ross, M.D. 1989. Meniere's disease in the context of vestibular neural networks.

In: Second International Symposium on Meniere's Disease (ed. by J. Nadol, Jr.).

Amsterdam: Kugler, p. 323-330.

Ross, M.D., Cutler, L., and Meyer, G. 1989. Biological neural networks: Beyond the

PDP model. In: Proceedings of the IEEE/International Journal of Computer Neural

Networks, p. 705-710.

Ross, M.D., Cutler, L., Meyer, G., and Dayhoff, J. 1989. Computer-assisted

visualization, animation and modeling of vestibular maculas (Abstract). Society for

Neuroscience Abstracts 15: 502.

Ross, M.D., Cutler, L., Meyer, G., Lam, T., and Vaziri, P. 1990. 3-D components of a

biological neural network visualized in computer generated imagery. I. Macular receptive

field organization. Acta Otolaryngology (Stockholm) 109: 83-92.

Ross, M.D., Meyer, G., Cutler, L., and Mugler, D. 1989. Computer visualization

techniques applied to vestibular research. In: Proceedings of the IEEE/Engineering in

Medicine and Biology Society, p. 1922-1923.

Ross, M.D., Meyer, G., Lam, T., Cutler, L., and Vaziri, P. 1990. 3-D components of a

biological neural network visualized in computer generated imagery. II. Macular neural

network organization. Acta Otolaryngo!ogy (Stockholm) 109: 235-244.

161
DEVELOPMENT, MATURATION AND PHYSIOLOGY OF THE BRAINPITUITARY-GONADAL AXIS OF FISH IN THE CEBAS/AQUARACK

SYSTEM

Martin P. Schreibman

Department of Biology

Brooklyn College, City University of New York

Brooklyn, NY 11210

Description of Research

The long-range goal of this project is to study the effects of near zero gravity on the

development and function of the brain-pituitary-gonadal (BPG) axis in the swordtail fish,

Xiphophorus helleri. The first phase of the work is to collect fundamental, relevant data in

the Closed Equilibrated Biological Aquatic System (CEBAS)/Aquarack (C/A), a project

developed by the German Space Agency (DARA), at ground level conditions in order to

establish a database that will serve as a comparison for results obtained from future space

experiments. The specific aims of this project are to: (1) Determine the effects of the C/A

system on the structure and function of the brain, pituitary gland and gonads in sexually

mature animals. This includes an analysis of the effects of the C/A system on fertilization

and gestation; and (2) Determine the effects of the C/A system on the development (birth to

maturity) of the neuroendocrine axis concerned with reproduction.

The biology of the reproductive system will be analyzed by evaluating the changes in the

synthesis, storage and release of neurotransmitters, neurohormones and pituitary and

gonadal hormones in mature and neonatal animals placed into the C/A system, and in

young immature fish born in, and permitted to reach puberty in, the system.

Immunocytochemical and morphometric methods will be the principal tools of analysis.

Accomplishments

In the initial 17 months of this program, we have processed for histological study and

immunocytochemical analysis brains, pituitary glands and gonads from 39 mature and

immature, male and female swordtails ranging in weight from 0.23 to 1.85 grams. Each

animal was photographed in Bochum before they were forwarded to Brooklyn College for

study. We have tested in pilot studies antisera to five different gonadotropins,

gonadotropin releasing hormone (GnRH), and four neurotransmitters (serotonin,

dopamine, neuropeptide Y [NPY], and dynorphin [DYN]).

Of special significance is our recent localization by immunocytochemical methods

of NPY and DYN in the brain and pituitary gland of immature and mature

Xiphophorus maculatus (the platyfish). Immunoreactive (ir-) -NPY was found in

perikarya and nerve tracts of the nucleus olfactoretinalis, telencephalon, ventral tegmentum,

the neurohypophysis and in specific cells of the adenohypophysis. Ir-DYN was found in

nerve tracts in the olfactory bulb and in association with the cells of the pars intermedia and

caudal pars distalis of the pituitary gland. The association of NPY and DYN with

brain structures and pituitary gland regions previously found to be involved

with sexual maturation, growth and differentiation, suggests that these

neuropeptides play a role in these events.

162
Significance of the Accomplishments

The data that we have already gathered on the distribution of brain neuropeptides and

neurotransmitters and pituitary hormones in Xiphophorus are enabling us to construct a

"map" that will permit us to assess brain-pituitary-gonadal axis activity under experimental

conditions. These data will enable us to study the effects of space travel on reproductive

system structure, development, and function under the conditions of the CEBAS/Aquarack

system.

Publications

Cepriano, L., Schreibman, M.P., and Holtzman, S. 1989. Serotonin and dopamine in the

pituitary gland of platyfish during development (Abstract). American Zoologist 29:18A.

Schreibman, M.P., Holtzman, S., Cepriano, L., and Swanson, P. 1989. Gonadotropins

and gonadotropes during development in platyfish (Abstract). American Zoologist 29:

18A.

163
EFFECTS OF WEIGHTLESSNESS

DIFFERENTIATION AND STATOLITH

Dorothy B. Spangenberg

Department of Pathology

Eastern Virginia Medical School

Norfolk, VA 23501

ON A URELIA

SYNTHESIS

EPHYRAE

Description of Research

The long-range goals of this research are: (1) to discover the role(s) of gravity on the

behavior and development of Aurelia ephyrae (tiny jellyfish) and on their graviceptor

structures (rhopalia) and (2) to discover the effects of microgravity on ephyrae and

graviceptor development after short-term (9-day space shuttle flight) and long-term (space

station and biosatellite) exposure to a microgravity environment.

Specific objectives are: (1) to determine whether the microgravity of space will modify the

development of ephyrae from polyps, the development of the graviceptors of ephyrae, the

mineralization and/or demineralization of statoliths of rhopalia, or the swimming/pulsing

behavior of ephyrae; (2) to discover, by comparing the features listed above in ephyrae that

develop in space with those of ephyrae that develop on Earth, the roles that gravity plays in

the development of ephyrae, their graviceptors, and their behavior on Earth; (3) to develop

a method for maintaining rhopalia in nutrient media for 1-3 months in order to study

development of more mature rhopalia (than those found in ephyrae) on Earth and in space;

and (4) to compare behavior of ephyrae with and without rhopalia.

Accomplishments

(1) Touch-plate Development in Graviceptors: For the first time, touchplate development was studied in the graviceptors (rhopalia) of living ephyrae using the

light microscope. The touch-plate is a part of the rhopalium which is involved in

graviception in medusae. A series of rhopalia were examined at 24 hr intervals following

induction of ephyra formation with iodine. At 96 hr, ephyrae which were attached to the

strobila were excised from the strobila. Rhopalia from these ephyrae and from freeswimming ephyrae at later time periods were excised and put into a wet film for

microscopic examination. The results are seen in Figure 1. Touch-plates are not found

until 144 hr and contain only a small ocellus and a few straight vibrating cilia (these may

represent the formation of a new type of mechanosensory cell not found earlier in the

rhopalia). Another observation made on these jellyfish was the formation of the first ocelli

by 96 hr while the ephyrae are still attached to the strobila. These ephyrae pulse slowly and

weakly at this time. Statolith numbers increase from the 96 hr to the 144 hr time period.

Earlier studies (see 1988-89 NASA SpaceGravitational Biology Accomplishments report)

revealed that rhopalia of ephyrae given nutrient media after release from the strobila

continued growing in size and had increased numbers of statoliths. The vibrating cilia,

thus far, have been found only in the touch-plate area of rhopalia, and they increase in

number as the touch-plate grows and the ephyrae mature into medusae.

164
TOUCH-PLATE DEVELOPMENT IN EPHYRAE GRAVICEPTORS

0

r

a

I

s

i

d

e

a

b

96 hr 120_hr

e

_14_4 hr 168 hr

Figure 1. Touch-plate formation in graviceptors of ephyrae developing after metamorphosis induction with

iodine. Side view of rhopalia: a. statoliths; b. ist oceilus; c. vibrating cilia; d. 2nd ocellus; e. touch plate.

(2) FMRF-amide-like Immunoreactivity in Graviceptor Nerves (in

collaboration with Dr. Millie Hughes-Fulford, University of California and the VA Medical

Center, San Francisco, CA): The neurons of graviceptors of Aurelia ephyrae were

examined for the presence of FMRF-amide-like (FMRF-a-I) neuropeptide using both the

immunofluorescence and peroxidase-labelled antibody methods. Microscopic examination

of the ephyrae treated with antibody against FMRF-amide revealed immunoreactive

neurons and neurites throughout the ephyrae, especially in the diffuse nerve net.

Graviceptors were immunoreactive using both methods but the peroxidase-labelled

antibody revealed individual neurons in the graviceptors. A cluster of neurons was seen at

the bases and tips of the rhopalia. Immunoreactive neurites were very closely associated

with the statoliths and the 1st ocellus. Longer neurites extended along the sides of the

rhopalia connecting the two concentrations of nerves and extending to the radial muscles.

Significance of the Accomplishments

Our research is directed toward learning as much about Aurelia development and behavior

as possible in the time interval before the Jellyfish Experiment is flown on the SLS-1

(Spacelab Life Sciences-l) shuttle flight (currently scheduled for August 1991). In

addition, efforts are being made to culture and study the jellyfish in nutrient media so that

they can be flown on a long-term unmanned flight in the future.

Finding #1: The sequential development of graviceptor structures was

determined using normal jellyfish. It was most exciting to find that touch-plate

formation proceeds in unfed newly released ephyrae and is visible within

24 hr after release from the strobila. The recent change of the SLS-1 flight from 7

to 9 days will permit a study of the formation of one group of ephyrae to the stage when the

touch-plates would form on Earth. This means, therefore, that we will have the

opportunity to learn whether microgravity interferes with the initiation of touch-plate

development, including the formation of the 2nd ocellus. In addition, of course, the

determination of this sequence of development is important from the standpoint of

understanding more about the developmental processes in differentiating and growing

ephyrae. This information will be incorporated into studies in progress, including those in

which developing ephyrae are given nutrient media.

165
Finding#2: It is veryimportantthatnervesin theephyraecanbepositivelyidentifiedusing

theFMRF-a-I immunoreactivitymethod.Thepresenceof anaturalneuropeptidewhich is

relatedto FMRF-amide in Aurelia graviceptors and in the diffuse nerve net which has

sensory function suggests that this substance plays an important role in the regulation of

pulsing and may also be involved in the orientation of the organisms while swimming. We

will be able to determine whether this neuropeptide is present in ephyrae which had

developed in space, especially in organisms which may return to Earth with

swimming/pulsing abnormalities after a spaceflight.

Publications

Spangenberg, D.B. and Hughes-Fulford, M. 1989. FMRF-amide-like immunoreactivity

in neurons ofAurelia ephyra graviceptors (Abstract). ASGSB Bulletin 3: 27.

166
SKELETAL MUSCLE METABOLISM IN HYPOKINETIC RATS

Marc E. Tischler

Department of Biochemistry

University of Arizona

Tucson, AZ 85724

Description of Research

This work concerns the mechanisms of atrophy and metabolic alterations associated with

the lack of load-bearing (unweighting), which may occur with prolonged bedrest or

weightlessness. To further understand this muscle wasting, comparisons are made with

measurements in muscles whose nerve supply is interrupted. Our work this year has dealt

primarily with carbohydrate and protein metabolism.

For carbohydrate metabolism we analyzed further the effects c ¢ isoproterenol (betaadrenergic agonist) on muscle. Measurements included glucose phosphorylation, glycogen

synthesis and production of cyclic AMP, the hormone second messenger. In relation to

our interest in the sparing of membrane proteins, we assayed several enzyme activities in

plasma membranes of unweighted muscle and compared with denervated muscles. In

terms of protein metabolism, the focus was on the mechanisms of accelerated protein

breakdown in unweighted and denervated muscle. Of particular interest were the roles of

lysosomal versus cytosolic protein breakdown.

Accomplishments

(1) Carbohydrate Metabolism

(a) Unweighting results in an increased isoproterenol (betaadrenergic agonist) response of glycogen synthesis and cyclic AMP

production. Previous studies of glycogen synthesis suggested no difference in response

but the more recent work showed that elevated glycogen in unweighted muscle can mask

isoproterenol responses. For cyclic AMP production, non-receptor-mediated effects do not

differ.

(b) Greater insulin sensitivity antagonizes the effects of isoproterenol in

unweighted muscle.

(c) Insulin control of 3-0-methylglucose uptake shows increased sensitivity in

unweighted muscle just as for 2-deoxyglucose.

(d) In preparation for our spaceflight study concerning insulin response of

glucose transport, we have found that a flight as short as 2.5 days with animal availability

by 4 hours or longer flight (5 days) with as much as a 12-hour delay in receipt of animals

will likely yield satisfactory data.

(2) Membrane Proteins

While membrane receptor proteins likely are spared during unweighting atrophy,

membrane enzyme activities show differential responses. Likely this difference reflects

varying metabolic effects on enzymes which are not evident for receptors.

(3) Protein Metabolism

(a) Protein breakdown is accelerated more with denervation than

unweighting atrophy.

(b) Accelerated protein breakdown with unweighting is primarily a

cytosolic event.

167
(c) Proteolysis in unweighted soleus is more sensitive to

alterations of intracellular calcium.

(d) In vivo proteolysis in unweighted soleus muscle can be lowered by

intramuscular injection of mersalyl, a thiol protease inhibitor, leading to termination of

atrophy for 24 hours.

(e) Accelerated protein breakdown with denervation atrophy is associated with

a role for the lysosome in accord with greater lysosomal permeability (fragility).

(f) Intramuscular injection of chloroquine, a iysosomotropic agent reduces

proteolysis and terminates atrophy of denervated muscle but not unweighted muscle.

Significance of the Accomplishments

Finding #1 : Parts la, b, c support further our observations of the sparing of membrane

receptor proteins in general (i.e., insulin and beta-adrenergic receptor). Part d results have

helped to define the potential limitations of our spaceflight experiment on shuttle flight

STS-43 in 1991.

Finding #2: Membrane enzymes do not Seem to behave identically as membrane receptor

proteins in terms of protein sparing. Likely these results indicate some metabolic alteration

instead of these activities since denervated muscle behaves in a similar manner. In contrast,

the response of receptors is different between unweighted and denervated soleus muscle.

Finding #3" The most significant aspect of these results is the finding that accelerated

protein breakdown in unweighted muscle occurs mostly in the cytoplasm. It seems to be

linked to increases in calcium-dependent proteolysis (3c) and may in particular involve the

calcium-activated protease, which is mersalyl-sensitive (3d). In contrast, denervation may

lead to increased lysosomal proteolysis (3e). If so, then an intact nerve supply may tend to

attenuate this process while with denervation the absence of some factor(s) may lead to

fragility of this organelle (3d). Of particular note was our success in differentially

terminating atrophy for 24 hours of unweighted or denervated soleus muscles.

Publications

Fagan, J.M. and Tischler, M.E. 1989. Effects of oxygen deprivation on incubated rat

soleus muscle. Life Sciences 44: 677-681.

Henriksen, E.J., Kirby, C.R., and Tischler, M.E. 1989. Glycogen supercompensation in

rat soleus muscle during recovery from nonweightbearing. Journal of Applied Physiology

66: 2782-2787.

Jaspers, S.R., Henriksen, E.J., Satarug, S., and Tischler, M.E. 1989. Effects of

stretching and disuse on amino acids in muscles of rat hind limbs. Metabolism 38: 303-

310.

Kirby, C.R. and Tischler, M.E. 1989. Evidence for sparing of membrane proteins during

atrophy of unweighted rat soleus (Abstract). ASGSB Bulletin 3: 87.

Tischler, M.E., Cook, P., Hodsden, S., McCready, S., and Wu, M. 1989. In vivo

protein metabolism of developing flight muscle of tobacco hornworm Manduca sexta

(Abstract). FASEB Journal 3(3): A264.

Tischler, M.E., Henriksen, E.J., Jaspers, S.R., Jacob, S., and Kirby, C. 1989. Changes

in muscle accompanying non-weight-bearing and weightlessness. In: Advances in

Myochemistry Volume 2 (ed. by G. Benzi). London: John Libbey Eurotext, p. 325-338.

168
Tischler, M.E., Kirby, C.R., Rosenberg,S.B., and Chase,P. 1989. In vivo evidence

for different sitesof acceleratedproteolysisin unweightedsoleus(US) or denervated

soleus(DS) (Abstract). ASGSB Bulletin 3: 72.

Tischler, M.E., Satarug, S., and Henriksen, E.J. 1989. Comparison of atrophy

mechanisms in unloaded and denervated soleus muscle. In: lntracellular Proteolysis.

Mechanisms and Regulation (ed. by N. Katunuma and E. Komanami). Tokyo: Japan

Scientific Societies Press, p. 483-484.

Toth, A., Tischler, M.E.', and Johnson, P.C. 1989. In vivo quantitative determination of

NADH level (Abstract). FASEB Journal 3(4): A1403.

169
THE ROLE OF CALCITE SKELETAL

BIOMINERALIZATION

Fred H. Wilt

Department of Molecular and Cell Biology

Division of Cell and Developmental Biology

University of California

Berkeley, CA 94720

MATRIX PROTEINS IN

Description of Research

The ultimate goal of our work is to learn how calcified tissue matrix proteins preside over

the process of biomineralization resulting in the formation of hard tissues. Disturbances in

bone and calcium metabolism occur in humans and in other vertebrates during extended

spaceflight. The basic molecular and biochemical basis of how mineralized skeletal

elements are formed is poorly understood. Therefore, basic information about

biomineralization will be required to better understand the complex events that occur in

microgravity environments. We believe that the analysis of simpler model systems to study

basic aspects of biomineralization will be of substantial benefit to the space biology

program.

With this in mind, our studies have focused on the formation of the calcite spicules of the

developing larva of the California purple sea urchin, Stronglycentrotus purpuratus. The

calcite spicules of the S. purpuratus pluteus larva have an integral organic matrix consisting

of at least ten glycoproteins. Our studies have centered on learning more about these matrix

proteins. These studies have entailed isolating cDNA clones encoding spicule matrix

proteins from expression libraries by using antibodies generated against the total spicule

matrix proteins. After we isolate cDNA clones that react with the anti-total spicule matrix

antibody, we then further characterize the cDNA clone to be sure that it encodes a genuine

spicule matrix protein. Once we have isolated the appropriate cDNAs, we will not only

know the derived amino acid sequence of the various spicule matrix proteins, but we will

then be able to generate immunological and molecular probes as well as recombinant

spicule matrix proteins to use as tools to address the structure and function of these

proteins. We already have detailed knowledge of one of the spicule proteins, an

approximately 50 kilodalton protein called SM50. So, for the past year, we have been

attempting to isolate cDNA clones that encode the several other spicule matrix proteins.

Accomplishments

(1) We have isolated a cDNA clone, pNG3/7, that encodes another of the spicule

matrix proteins.

(2) We have begun to characterize pNG3]7 and its expression at a molecular level.

This analysis entails sequencing the cDNA clone as well as determining when and where

this gene is expressed.

(3) We have begun to characterize pNG3/7 expression at a cellular level. It is

apparent from our studies that a cue from the extracellular matrix in the sea

urchin's blastocoel instructs the primary mesenchyme cells to express this

gene.

(4) We have isolated a number of other cDNA clones that may encode other spicule

matrix proteins but which have not yet been well characterized.

170
(5) We have isolated and sequenced a cDNA clone from the sea

urchin Lytechinus pictus that encodes a protein homologous to the SM50

protein of the Strongylocentrotus purpuratus embryo.

Significance of the Accomplishments

The isolation and characterization of the pNG3/7 cDNA clone as well as the isolation of a

number of other possible spicule matrix cDNA clones are significant first steps towards

approaching the problem of how spicule matrix proteins preside over biomineralization.

After several screens of several cDNA libraries we have isolated a number of expression

cDNA clones that reacted with the anti-total spicule matrix antiserum. One of these clones,

pNG3/7, encodes a 30 kilodalton spicule matrix protein whose amino acid composition is

similar to that of other invertebrate matrix proteins. The derived amino acid sequence of

this clone and that of SM50 provides us with invaluable information about the structure of

these matrix proteins. While both proteins are acidic, the pNG3/7 sequence is very

different from SM50. Most notable among other differences between the two p.roteins is

that pNG3/7 does not have a repeating amino acid motif as does the SM50 protein. While

we still do not know the structure and function relationship of these proteins, these protein

sequences will be informative in comparing them to the other spicule matrix proteins.

The molecular and cellular characterizations of when, where, and how the pNG3/7 gene is

expressed are invaluable pieces of information for determining if in fact the pNG3/7 clone

encodes a spicule matrix protein. In addition, this information provides indirect clues to

how the sea urchin embryo might use this gene product to construct a spicule. For

example, experiments looking at when the RNA transcripts encoded by pNG3H and SMS0

appear in the sea urchin embryo show that they are expressed at slightly different times.

SMS0 mRNA is first expressed early by the primary mesenchyme cells (the cells that

construct the spicules), many hours in advance of the first appearance of spicules.

Alternatively, pNG3/7 transcript is first expressed relatively later by the primary

mesenchyme cells, just prior to the initial formation of spicules. These findings suggest

that there may be different functions for these proteins.

Our finding that a cue from the extracellular matrix is needed for expression of pNG3H is

significant because it is different from that of SM50. SM50 RNA transcripts will begin to

accumulate in the absence of any cues from the extracellular matrix or from other cells.

Here again these differences may be reflecting different functions for these proteins. It will

be interesting to compare the expression of other spicule matrix protein genes to these two

genes.

Our isolating and sequencing of a cDNA clone homologous to SMS0 from another species

of sea urchin (Lytechinus pictus) provides us with valuable information about important

structural features of the SMS0 protein that has been conserved over the 60 million years

since the two species of sea urchins diverged. It is apparent that the repeating amino acid

motif as well as a proline rich region of the SM50 protein are highly conserved in the L.

pictus homologue. We are currently trying to isolate a cDNA clone from L. pictus that is

homologous to pNG3/7.

While we have a number of other possible clones that encode spicule matrix proteins, these

clones have not yet been well characterized. However, we are hopeful we have isolated

cDNAs that encode other spicule matrix proteins.

171
Publications

Killian, C.E., George, N.C., and Wilt, F.H. 1989. The isolation of cDNA clones

encoding the integral acidic matrix proteins of the sea urchin embryo's calcareous spicules

(Abstract). ASGSB Bulletin 3: 71.

Livingston, B.T., Shaw, R., and Wilt, F.H. 1989. Cloning and characterization of a

cDNA encoding a Lytechinus pictus spicule matrix protein (Abstract). Journal of Cell

Biology 109(4, Part 2): 155a.

172
EFFECTS OF MICROGRAVITY ON MAMMALIAN DEVELOPMENT

DIFFERENTIATION

Debra J. Wolgemuth

Department of Genetics and Development

The Center for Reproductive Sciences

College of Physicians and Surgeons

Columbia University

New York, NY 10032

AND

Description of Research

The primary objective of our research is to evaluate the affects of altered gravitational

environments on mammalian germ cell development and early embryogenesis at the

molecular level.

We previously showed that mouse female germ cells rotated on a clinostat exhibit

anomalies of the meiotic maturation process. No marked alterations in the efficiency of

fertilization or gross abnormalities in very early embryo development were noted. We have

extended these studies to include examination of the effects of altered gravity environments

on the male reproductive system. Our studies include examination of the effects of

exposure to simulated hypogravity (using a hindlimb unweighting model) and to actual

microgravity (from spaceflight) on testicular development and spermatogenesis in rats. We

are using sensitive molecular markers of normal development in order to examine the

response of reproductive tissues to alterations in the gravity environment and to identify

stages of germ cell and embryonic development that might be most susceptible to altered

environments such as microgravity. The particular parameter that we are evaluating in

flight and unloaded testicular tissue is the expression of genes known to exhibit specific

patterns of activation during normal testicular development. Our study has focused initially

on characterization of the expression of cellular stress protein genes, also known as heat

shock protein (hsp) genes, in the rodent testis and embryo. Special attention is also

focused on examining the pattern of expression of various genes which are known to be

normally expressed in developing bone and muscle, such as certain homeobox-containing

genes. The experimental methods involve molecular cloning of these assorted genes and

both hybridization analysis of the mRNAs isolated from tissues exposed to altered gravity

and their normal counterparts and immunohistochemical and electrophoretic analysis of

their proteins.

Accomplishments

(1) Characterization of normal heat shock protein gene expression is essential in

order to evaluate alterations at the molecular level in tissues from animals in ground-based

and flight studies. To determine if the pattern of expression of heat shock genes are

modified by altered gravitational environments, we have demonstrated that the normal

patterns of expression of subsets of the hspgO and hsp70 members of the

heat shock protein gene family have unique cellular and temporal patterns

of expression in the developing mouse testis and the midgestation embryo.

Certain members of both the HSP 70 and HSP 90 families were shown to exhibit

spermatogenic stage-specific patterns of expression in the absence of exogenous stress.

Two different hsp90 transcripts were detected in the mouse testis. The testicular transcripts

exhibited cellular and developmental stage specificity of expression. The larger and more

abundant transcript was expressed at high levels in the germinal compartment of the testis,

particularly in germ cells in meiotic prophase. The smaller hsp90 transcript was expressed

173
predominantlyin the somatic compartmentof the testis. Expressionof the two hsp90

transcripts was seen in the testis of other species. In addition, expression of both hsp90

transcripts was detected in the embryonic and extra-embryonic compartment of

midgestation embryos. A stress-inducible member of the HSP 70 family was shown to be

induced by elevated temperatures in certain stages of spermatogenesis, namely the stem cell

stages, and/or in the somatic compartment of the testis, but not in later stages of germ cell

development.

(2) In collaboration with Dr. Emily Morey-Holton at NASA Ames Research

Center, we received testicular tissue from rodents used in ground-based studies on the

effects of altered gravity. We are investigating the effects of hypogravity on testicular

development and spermatogenesis at the molecular level in animals subjected to the

hindlimb unloading model system. The model was designed to simulate and test the effects

of microgravity by removing the weight-beating function of the muscle and bone of the

hindtimbs of an animal_ The mechanism used allows the-animais to move freely and

enables an ad libitum access to food and water. Dramatic decreases in testis weight have

been observed in unweighted animals, but virtually no information exists as to the nature of

this pathology. We have isolated RNA from testicuiar tissue of unloaded rats for 2, 5, 7,

and 11 days, respectively, plus their normally weighted controls. RNA isolated from testis

of unloaded rats is being examined with probes for cellular stress protein genes.

(3) An important aspect of our experiments is comparison of the observations

obtained in our studies using simulated altered gravity environments with changes observed

during spaceflight. We have received testicular tissues from rodents flown on a Soviet

Cosmos biosatellite. RNA was isolated from testes of spaceflight exposed rats and is being

examined with probes for cellular stress protein genes. RNA was isolated from the

testicular tissues of 5 rats that were flown for two weeks on Cosmos 2044, 5 rats that were

Soviet unloaded ground controls, 5 rats that were synchronous simulated-flight ground

controls, and 5 rats that were vivarium normal controls. Total testicular RNA from each of

the Cosmos-flown rats plus their controls and the unloaded rats at Ames plus their controls

was fractionated by electrophoresis and transferred to Nytran membranes. DNA probes to

Hox-1.4 (a homeobox containing gene), and hsp90 and hsp70 (subsets of the heat shock

protein gene family) were labeled with radioactive markers. Transfer and immobilization of

the RNA to the Nytran membranes facilitated retention of the RNA, thus allowing the

samples to be hybridized sequentially with multiple probes. We are currently using

densitometric scanning to semiquantitatively compare the relative sizes and levels of

transcripts from different genes within each testicular sample. Immunohistochemical

analysis will be used to extend this evaluation of gene expression by examining variation at

the level of the actual protein products.

Significance of the Accomplishments

Finding #1: Hsp genes are expressed in distinctive patterns during both normal

development and cellular stress; they are therefore useful molecular markers of cellular

perturbation. The unique cellular and temporal patterns of expression of members of the

hsp90 and hsp70 families lend support to the possibility of an important role for these

cellular stress protein genes in mammalian testicular function. The particular pattern of

expression of the stress-inducible member of the HSP 70 family raises the possibility that

cells in early stages of spermatogenesis may be especially vulnerable to changes in their

environment. This information will be valuable in predicting stages of development likely

to be sensitive to spaceflight and will be useful in designing the best experiments for actual

flight.

174
Finding #2: Northern blot hybridization permits detection of very subtle relative

differencesin theexpressionof differentgenesin tissuesamplesfrom spaceflown animals

and their ground-controlcounterparts.Suchmolecularanalysesenablethe useof small

samplesof thevery preciousflight tissuesto examinethepossibleeffectsof alteredgravity

or otherbiologicalstresses.

Publications

Tennyson, V.M., Sherman, D.L., Behringer, R.R., Brinster, R.L., Palmiter, R.D.,

Tasch, J., Crotty, D., Wolgemuth, D.J., and Gershon, M.D. 1989. Abnormalities of

enteric neuronal development in transgenic mice overexpressing the homeobox-containing

gene Hox- 1.4 (Abstract). Society for Neuroscience Abstracts 15:1121.

Wolgemuth, D.J., Gruppi, C.M., Zakeri, Z.F., and Grills, G.S. 1989. Cellular stress

protein gene expression in normal gametogenesis and embryogenesis and in tissues

subjected to altered environments (Abstract). ASGSB Bulletin 3: 107.

175

SPECIAL ACTIVITIES

PRECF-..DING PAGE BLANK NOT FILMED

SPACE BIOLOGY RESEARCH ASSOCIATES PROGRAM

X.J. Musacchia

Graduate Programs and Research

University of Louisville

Louisville, KY 40292

The Space Biology Research Associates Program provides a unique opportunity to train

individuals to conduct biological research in areas relevant to NASA's interest. To

maximize the potential for Space Biology as an emerging discipline, there is a need to

develop a cadre of scientists interested in working in this area. This grant was developed to

train biologists by offering Research Associate Awards to young scientists. These awards

provide opportunities for individuals to work on projects directly related to Space Biology

and in laboratories that provide the necessary facilities and a relevant research environment.

It is anticipated that these scientists will develop research careers in the evolving discipline

of gravitational biology, a focused area of Space Biology. The field of gravitational

biology is rapidly growing and its future will reflect the quality and training of its scientific

personnel.

The program began on June 1, 1980 with funding to support several Research Associates

each year. As of April 30, 1990, 76 annual awards had been made to 46 awardees, of

whom 30 have received a second year of funding. Table I illustrates the variety of projects,

laboratories and institutions to which the 46 Research Associates have been assigned.

These scientists represent different disciplines including: zoology, developmental biology,

botany, and physiology (animal and plant). In June 1980 there were 19 laboratories

participating. Presently there are 61 laboratories in the program. Table II illustrates the

number of awards given out each year to new recipients and to renewal candidates.

Many of the Research Associates have been asked to participate in NASA panels, national

workshops and national meetings. There have been 112 publications in refereed journals

and as many abstracts of papers presented at national and international meetings. Each year

in the fall, Research Associates attend the annual meeting of the American Society for

Gravitational and Space Biology (ASGSB). The Research Associates are active

participants in these meetings, presenting papers and posters along with their senior

colleagues. All of the current Research Associates and many of the former Research

Associates are members of ASGSB. Research Associates are encouraged to participate in

other national meetings in their own disciplines.

The scientists who have completed this program have accepted positions in colleges and

universities, with research laboratories and with NASA. An individual listing of the 46

Research Associates is provided in Table I. A more detailed description of the awardees

follows Tables I and II.

179

t[_"_...L-L_ I_r[N_0N_tt? _ PRECEDING PAGE BLANK NOT FILMED
Table I. SPACE BIOLOGY RESEARCH ASSOCIATES

S. BAIN

W. BERRY

M. BINDER

S. BLACK

H. BLAIR

J. BUCKEY

G. BURROWS

D. CLOHISY

M. COOPER

D. DICKMAN

S. GLOTZBACH

C. GOULD

M. GRAY

E. GREENFIELD

T. JONES

T. KERR

D. KLIGMAN

A. LYSAKOWSKI

K. MCLEOD

D. MEYERS

L. MINOR

D. MURAKAMI

S. PERKINS

K. POTE

G. RADICE

F. ROBINSON

J. STEFFEN

J. SZILAGYI

D. THOMASON

Y. TORIGOE

SUNY, Stony Brook

U. Louisville

Dartmouth Col.

U. Cal., Berkeley

Washington U.

U: Tex,-as, Dallas

N.I,H_

Washington U,

U. Cal.. Berkeley

U. Texas. Galveston

Slanfor/tu.

U. Louisville

Tufts U.

Washington U:

U. Cal., Davis

Wayne State U.

N.I.M.H.

U. Chicago

SUNY, Stony Brook

U. Pennsylvania

U. Chicago

U. Cal., Davis

Washington U.

U. Virginia

Indiana U.

U. Pittsburgh

U. Louisville

Cleveland Clinic

U. Texas, Houston

U. Cal., Irvine

ANIMAL PROJECTS

SKELETAL BO_ REMODELING

ROLE OF VITAMIN D/IMMUNE FUNCTIONS

CARDIAC PATHOLOGY

AMPHIBIAN DEVELOPMENTAL ORIENTATION

CELLULAR/BONE ATROPHY

CARDIOVASCULAR RESPONSES

SYNAI_E_S I S-PNE_OPATHOLOGY

OSTE_ASTOGENES iS/BONE ATROPHY

OSTEOPOROSIS/BONE ATROPHY

SEMICIRCULAR MUSCLE CANALS

NEUROPHYS IOLOCIY/C_CADIAN RHYTHM

IMMUNOLOGY/INTERFERON FUNCTIONS

MECHANICAL ENVIRONMENT IN BONE ARCHITECTURE

OS_OBLA_T RDL_gTO_I'_O_LA ST A C_V_:

NEUROPHYS IOLOGY/BRAINSTEM POTENTIALS

MAMMALIAN VESTIBULAR SYSTEM

NEURITE EXTENSION FACTOR RESPONSES

VESTIBULAR HAIR CELLS/SYNAPTIC RELATIONS

ELECTRICAL FIELDS IN BONE REMODELING

GRAVITY PERCEPTION/MICROCRUSTACEAN (FW)

RESPONSES OF SECONDARY VESTIBULAR NEURONS

HYPERDYNAMIA/VISUAL SYSTEMS

VITAMIN D/OSTEOCLAST DIFFERENTIATION

OTOCONIA Ca BINDING PROTEIN

GRAVITY-SENSORS/AMPHIBIAN EMBRYOLOGY

SENSORY MOTOR PROPERTIES IN UVULA

GLUCOCORTICOID RECEPTORS & MUSCLE RESPONSES

HYPODYNAMIC RESPONSES/ANIMAL MODEL

DECREASED ACTIN SYNTHESIS/MUSCLE ATROPHY

NEUROPHYSIOLOGY OF GUT

S. BARSEL

T. BJORKMAN

T. BROCK

M. DESROSIERS

J. GARAVELLI

J. GAYNOR

M. HARRISON

G. JAHNS

K. KUZMANOFF

M. MATILSKY

M. MUSGRAVE

D. REINECKE

B. SERLIN

R. SLOCUM

J. SLONE

L. TALBOT/"

Michigan St. U.

U. Washington

U. Michigan

Michigan St. U.

Texas A&M U.

Yale U.

Washington U.

U. Houston

Stanford U.

Princeton U.

Duke U.

Michigan St. U.

U. Texas, Austin

Yale U.

Washington U.

Washington U.

PLANT PROJECTS

PLANT CELL PHYSIOLOGY

ELECTRICAL RESPONSES/GRAVITATIONAL SENSITIVITY

AUXIN & PROTEIN SYNTHESIS IN GRAVITROPISM

ELECTRICAL POTENTIAL IN HORMONE TRANSPORT

PLANT/ALGAE CELL CHEMISTRY

AMYLOPLAST/GRAVITATIONAL SENSITIVITY

ENVIRONMENTAL ETHYLENE/GRAVITROPISM

LIGNIN BIOSYNTHESIS IN PLANT DEVELOPMENT

ENZYME REGULATORS IN PLANT CELL WALL

GRAVITY PERCEPTION/COENOCYTE

PLANT RESPIRATORY METABOLISM/SPACEFLIGHT

IAA DISTRIBUTION/PLANT GEOSENSING

CELL WALL GROWTH/CORN ROOTS

ROLE OF CALCIUM/GRAVISTIMULATION

AUXIN TRANSPORT/GRAVITROPISM

STEM GRAVICURVATURE/SPECIFIC POLYMERS

180
14

Table II. NASA Space Biology Research Associate Awards

12

_ 10 O

.<

< 8

O

a: 6

I.tJ

rn

4

Z

2

0

CE_

NEW AppoJntmenfs

RENEWED Appotntmenfs

TOTAL Awards

'81 '82 '83 '84 '85 '86 '87 '88 '89

YEAR

The cumulative fofals

from Jan.l,1981

to Apr.30,1990 are

76 awards fo

46 research associafes,

30 of whom received

a renewal award

fora second year

of funding.

RESEARCH ASSOCIATE AWARDEES

The 46 awardees are listed alphabetically, including their award term in parentheses, their

host laboratory, and current location.

DR. STEVEN BAIN (6/1/88 - 5/30/90) is working on "The Interaction of Skeletal

Remodeling With Systemic Disorders: An Obstacle to Extended Space Flight?" in Dr.

Clinton Rubin's laboratory at SUNY, Stony Brook, New York.

DR. SARA-ELLEN BARSEL (6/1/87 - 5/30/88) worked on "Molecular and Genetic

Phototropism in Arabidopsis thaliana" in Dr. Kenneth Poffs laboratory at Michigan State

University, East Lansing, Michigan. She is now working for Chemical Abstracts in

Columbus, Ohio.

DR. WALLACE BERRY (7/1/88 - 6/30/90) is working on "Lymphokine Producing

Capacity of Antiorthostatically Suspended Rats: Relationship to 1, 25-dihydroxyvitamin

D3" in Dr. Gerald Sonnenfeld's laboratory at the University of Louisville, Louisville,

Kentucky.

DR. MICHAEL BINDER (1/1/83 - 12/30/83) worked on "Congenital Heart

Malformations and Situs Inversus" in Dr. W.M. Layton, Jr.'s laboratory at Dartmouth

Medical School. He is now on a research fellowship in the Pathology Department at

Brown University, Providence, Rhode Island.

181
DR, THOMAS BJORKMAN (10/1/86 - 9/30/88) worked on "The Mechanismof

Gravity Sensing in Plants" in Dr. Robert Cleland's laboratory at The University of

Washington, Seattle, Washington. He is continuing to work in Dr. Cleland's laboratory at

the University of Washington, Seattle, Washington.

DR. STEVEN BLACK (7/1/82 - 6/30/84) worked on "Determination by Gravitational

and Centrifugal Force of the Amphibian Dorsal-ventral Axis" in Dr. Raymond Keller's

laboratory at the University of California, Berkeley. He is continuing research with Dr.

Keller and is also working with Dr. Kenneth Souza at NASA-Ames Research Center,

Moffett Field, California.

: +:

_ ]

DR. HARRY BLAIR (7/1/84 - 6/30/86) worked on "Cellular Mechanisms of Bone

Degradation" in Dr. Steven Teitelbaum's laboratory at The Jewish Hospital/Washington

University Medical Center, St. Louis, Missouri. He is continuing to work in Dr.

Teitelbaum's laboratory funded by a NIH Physician Scientist Training Grant.

DR. THOMAS BROCK (8/1/86 - 7/30/88) worked on "Comparison of Changes in

Protein Synthesis Induced by Gravity and Auxin Treatment in Pulvini and Coleoptiles of

Oat (Avena sativa L.)" in Dr. Peter Kaufman's laboratory at The University of Michigan,

Ann Arbor, Michigan. He is continuing to work with Dr. Kaufman at the University of

Michigan.

DR. JAY BUCKEY, JR. (7/1/82 - 6/30/84) worked on "2-D Echocardiography as an

Accurate Mean for Measuring Left Ventricular Volume and Central Venous Pressure

During Zero-gravity" in Dr. C. Gunnar Blomqvist's laboratory at the University of Texas

Health Sciences Center, Dallas. At the present time he is the project manager for the

cardiovascular experiment scheduled on Spacelab Life Sciences- 1 and a Research Assistant

Professor/Instructor in Clinical Medicine at the University of Texas Health Sciences

Center, Dallas, Texas.

DR. GEORGE H. BURROWS (7/1/81 - 6/30/83) worked on "Studies of

Synaptogenesis" in Dr. Marshall Nirenberg's laboratory at NIH, Bethesda, Maryland. He

is now on the staff of the National Heart, Lung, and Blood Institute, Bethesda, Maryland.

DR. DENIS CLOHISY (7/1/86 - 6/30/87) worked on "Mechanisms of Osteoclast

Precursor Differentiation" in Dr. Steven Teitelbaum's laboratory at The Jewish

Hospital/Washington University Medical Center, St. Louis, Missouri. He is now

completing his clinical training in Orthopaedic Surgery at the University of Minnesota, St.

Paul, Minnesota.

DR. MARK COOPER (1/1/85 - 12/30/86) worked on "Osteoporosis of Weightlessness

and the Electrophysiology of Bone" in Dr. John Miller's laboratory at The University of

California at Berkeley, California. He is now a Research Associate in the Department of

Molecular Neurobiology at Yale Medical School, New Haven, Connecticut.

DR. MARK DESROSIERS (7/1/86 - 6/30/88) worked on "A Search for Voltage-gating

of Plant Hormone Transport Channels" in Di'. Robert Bandurski's laboratory at Michigan

State University, East Lansing, Michigan. He is continuing to work in Dr. Bandurski's

laboratory at Michigan State University.

DR. J. DAVID DICKMAN (6/1/87 - 5/30/90) worked on "High Frequency Response

Properties of Semicircular Canal Fibers" in Dr. Manning Correia's laboratory at the

University of Texas, Galveston, Texas. He is contining to work with Dr. Correia at the

University of Texas.

182

r
DR. JOHN S. GARAVELLI (1/1/82 - 4/30/82) worked on "Chemical Characterization

of Volatile Products of Algal Cell Cultures" in Dr. Franklin Fong's laboratory at Texas

A&M University. He is now working for the Extraterrestrial Research Division at NASAAmes Research Center, Moffett Field, California.

DR. JOHN GAYNOR (1/1/81 - 12/30/82) worked on "Purification and Characterization

of Amyloplasts from Pisum sativum" in Dr. Arthur Galston's laboratory at Yale

University. He is now an Assistant Professor and Henry Rutgers Scholar in The Botany

Department at Rutgers University, Newark, New Jersey.

DR. STEVEN GLOTZBACH (1/1/84 - 12/30/84) worked on "Neurophysiological

Studies of Circadian Rhythm Control Mechanisms" with Dr. H. Craig Heller at Stanford

University and Dr. Charles A. Fuller at the University of California, Riverside. He is

continuing to work in Dr. Heller's laboratory funded by a NIH-NIRA grant, Palo Alto,

California.

DR. CHERYL GOULD (7/1/84 - 8/30/85) worked on "Effect of Weightlessness on

Various Immunological Functions Using a Murine Simulated Space Flight Model" in Dr.

Gerald Sonnenfeld's laboratory at The University of Louisville, Louisville, Kentucky.

She is now an Assistant Professor at the University of Kentucky, Lexington, Kentucky.

DR. MARTHA GRAY (7/1/86 - 6/30/87) worked on "The Correlation of Applied Strain

Distributions to the Location of New Bone Formation: A Rigorous Mechanical Analysis of

an in vivo Bone Preparation" in Dr. Clinton Rubin's laboratory at Tufts University School

of Veterinary Medicine, North Grafton, Massachusetts. She is now an Assistant Professor

at the Massachusetts Institute of Technology, Boston, Massachusetts.

DR. EDWARD GREENFIELD (7/1/88 - 6/30/90) is working on "Regulations of

Osteoclastic Bone Resorption by Osteoblasts" in Dr. Steven Teitelbaum's laboratory at

Washington University, St. Louis, Missouri.

DR. MARCIA HARRISON (7/1/83 - 8/30/85) worked on "Participation of Ethylene in

Two Modes of Gravitropism of Shoots" with Dr. Barbara Pickard at Washington

University, St. Louis, Missouri. She is now an Assistant Professor in the Biology

Department at Marshall University in Huntington, West Virginia.

DR. GARY JAHNS (1/1/83 - 4/30/84) worked on "Interactions of Light and Gravity on

the Growth, Orientation, and Lignin Biosynthesis in Mung Beans" in Dr. Joe Cowles'

laboratory at the University of Houston. He is now working at NASA-Ames Research

Center, Moffett Field, California.

DR. TIMOTHY JONES (1/1/81 - 12/30/82) worked on "The Effects of Hypergravic

Fields on Brainstem Auditory-evoked Potentials" in Dr. John Horowitz' laboratory at the

University of California, Davis. He is now an Associate Professor at the University of

Nebraska, Lincoln, Nebraska.

DR. THOMAS KERR (1/1/83 - 12/30/84) worked on "Cellular Localization of Na +,

K+-ATPase in the Mammalian Vestibular System"; the first year in Dr. Muriel Ross's

laboratory at the University of Michigan and the second year in Dr. Dennis Drescher's

laboratory at Wayne State University. He is now an Assistant Professor at Wayne State

University, Detroit, Michigan.

183
DR. DOUGLAS KLIGMAN (7/1/82 - 6/30/84) worked on "The Role of Neurite

Extension Factor Nerve and Muscle Tissue Response to Stress or Injury" in Dr. David

Jacobowitz' laboratory at the National Institute of Mental Health, Bethesda, Maryland. He

is now on the staff at NIMH, Bethesda, Maryland.

DR. KONRAD KUZMANOFF (7/1/83 - 7/30/85) worked on "Isolation and

Identification of B-glucan Synthetase: A Potential Biochemical Regulator of

Gravistimulated Differential Cell Wail Loosening" in Dr. Peter Ray's laboratory at Stanford

University. He is now a Research Associate working with Dr. Craig Beattie at the

University of Illinois at Chicago, Illinois.

.... _:__ ..... -U _ _ ....

DR. ANNA LYSAKOWSKI (7/1/89 - 6/30/90) is working on "Synaptic Relations of

Type I and Type II Vestibular Hair Cells" in Dr. Jay Gould's laboratory at the University

of Chicago, Chicago, Illin-ois.

DR. MICHAEL MATILSKY (1/1/81 - 12/30/82) worked on "Gravity Perception in the

Algal Coenocyte Caulerpa prolifera " in Dr. William Jacobs' laboratory at Princeton

University. He is now a Senior Research Scientist with Plant Biotech Industries in Ashrat,

Israel.

DR. KENNETH MCLEOD (11/1/87 - 10/30/89) worked on "In-vivo Measurement of

Strain Generated Potentials in Bone During Controlled Mechanical Loading" in Dr. Clinton

Rubin's laboratory at the State University of New York, Stony Brook, New York. He is

continuing to work in Dr. Rubin's laboratory at SUNY.

DR. DEWEY MEYERS (7/1/81 - 6/30/83) worked on "Response, Adaptation and

Gravitational Perception in a Parthenogenic Freshwater Microcrustacean, Daphnia galeata

mendotae" in Dr. Allan Brown's laboratory at the University of Pennsylvania. He was the

Science and Curriculum Coordinator in the Space Life Sciences Training Program at

Kennedy Space Center, Florida. Recently he became an Adjunct Associate Professor at

West Virginia School of Osteopathic Medicine, Lewisburg, West Virginia.

DR. LLOYD MINOR (7/1/87 - 6/30/88) worked on "Primary Vestibular Afferent Inputs

to Central Pathways Mediating the Vestibulo-ocular Reflex" in Dr. Jay Goldberg's

laboratory at the University of Chicago, Chicago, Illinois, He is now finishing his clinical

training at the University of Chicago Medical Center, Chicago, Illinois.

DR. DEAN MURAKAMI (1/1/85- 12/30/86)worked on "Influences of the

Hyperdynamic Environment on the Development of the Visual System in the Rat" in Dr.

Charles Fuller's laboratory at the University of California at Davis. He is continuing to

work with Dr. Fuller at the University of California, Davis.

DR. MARY MUSGRAVE (6/1/86 - 10/30/88) worked on "Studies of Respiratory

Metabolism" in Dr. Boyd Strain's laboratory at Duke University, Durham, North Carolina.

She is now an Assistant Professor at Louisiana State University, Baton Rouge, Louisiana.

DR. SHERRIE LYNN PERKINS (7/1/88 - 6/30/89) worked on "Vitamin D Effect on

Osteoclast Precursor Differentiation" in Dr. Steven Teitelbaum's laboratory at Washington

University, St. Louis, Missouri. She is now an instructor in the Department of Anatomic

Pathology at Jewish Hospital, Washington University.

DR. KENNETH POTE (6/1/88 - 5/30/90) is working on "An Otoconial Calcium

Binding Protein; Its Temporal Expression and Tissue Distribution" in Dr. Robert

Kretsinger's laboratory at the University of Virginia, Charlottesville, Virginia.

184
DR. GARY RADICE (7/1/81 - 6/30/83) worked on "Control of Gravity-sensing

Mechanism in Amphibian Eggs" in Dr. George Malacinski's laboratory at Indiana

University. Heis continuingto workwith Dr. Malacinski,Bloomington,Indiana.

DR. DENNIS REINECKE (11/1/88 - 9/30/89) worked on "Does Indole-3-Acetic Acid

Turnover Correlate with Topically-induced Asymetric Growth?" in Dr. Robert Bandurski's

laboratory at Michigan State University, East Lansing, Michigan. He is now an Assistant

Professor in the Department of Horticulture at the University of Minnesota, St. Paul,

Minnesota.

DR. FARREL R. ROBINSON, JR. (7/1/84 - 6/30/86) worked on "Sensory Motor

Properties of the Uvula and Nodulus" in Dr. David Tomko's laboratory at the University of

Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. He is now working as a

Research Associate with Dr. Albert Fuchs in the Physiology Department of the University

of Washington School of Medicine in Seattle, Washington.

DR. BRUCE SERLIN (7/1/84 - 6/30/85) worked on "Differential Wall Growth in

Gravistimulated Corn Roots: Its Timing and Regulation" in Dr. Stanley Roux's laboratory

at the University of Texas at Austin. He is now an Assistant Professor at DePauw

University, Greencastle, Indiana.

DR. ROBERT SLOCUM (1/1/81 - 12/30/83) worked on "Studies on the Localization

and Functional Role of Calcium in Gravistimulated Plant Organs;" the first year in Dr.

Stanley Roux's laboratory at the University of Texas at Austin and the second year in Dr.

Arthur Galston's laboratory at Yale University. He is now an Assistant Professor at

Williams College, Williamstown, Massachusetts.

DR. J. HENRY SLONE (7/1/85 - 6/30/87) worked on "Characterization of the Protein

Responsible for the Lateral Transport of Auxin During Gravitropism of Pea Shoots and

Determination Whether Phosphorylation Participates in Gravitropic Activation" in Dr.

Barbara Pickard's laboratory at Washington University in St. Louis, Missouri. He is now

working in the Plant Photobiology Laboratory at USDA, ARS, Beltsville, Maryland.

DR. JOSEPH STEFFEN (7/1/81 - 6/30/83) worked on "Glucocorticoid Receptor

Levels in Hindlimb Skeletal Muscles and Diaphragm During Prolonged (2 Week)

Antiorthostatic Hypokinesia and Recovery" in Dr. X.J. Musacchia's laboratory at the

University of Louisville. He is now an Assistant Professor at the University of Louisville,

Louisville, Kentucky.

DR. JULIANNA SZILAGYI (7/1/81 12/30/81) worked on "Progressive

Hemodynamic Changes in Simulated Weightlessness" in Dr. Carlos Ferrario's laboratory

at the Cleveland Clinic. She is now an Assistant Professor at the University of Houston,

Houston, Texas.

DR. LAWRENCE TALBOTT (7/1/89 - 12/30/89) worked on "Actuation of

Gravicurvatur in Pea Stems by Alteration of Specific Wall Polymers" in Dr. Barbara

Pickard's laboratory at Washington University, St. Louis, Missouri. He is now in a

Postdoctoral Fellowship at UCLA, Los Angeles, California.

DR. DONALD THOMASON (11/1/89 - 10/30/90) is working on "Mechanisms of

Decreased Actin Synthesis During Rodent Hindlimb Unweighting" in Dr. Frank Booth's

laboratory at The University of Texas Medical School, Houston, Texas.

185
DR. YASUHIRO TORIGOE (1/1/84 - 12/30/85) worked on "Anatomical Correlated

Underlying Vestibulo-autonomic Outflow to the Gut" with Dr. Robert H.I. Blanks at the

University of California, Irvine. He is continuing to work with Dr. Blanks at the

University of California.

186
THE INTERACTION OF SKELETAL REMODELING WITH SYSTEMIC

DISORDERS: AN OBSTACLE TO EXTENDED SPACEFLIGHT?

Steven D. Bain

Musculo-Skeletal Research Laboratory

Department of Orthopaedics

State University of New York

Stony Brook, NY 11794

Description of Research

The objectives and long-term goals of this research are intended to determine how an

organism's metabolic status interacts with bone tissue to modulate the skeleton's ability to

adaptively remodel in response to changes in its mechanical environment. As participation

in space exploration expands to include individuals of increasingly diverse metabolic status,

the question of how microgravity will interact with each individual's physiological milieu

becomes critical. For example, considering the differences in endocrine-driven remodeling

between gender, the skeletal changes in female astronauts may differ significantly, and be

potentially far more deleterious, than those stimulated in their male counterparts.

Therefore, we have proposed that the systemic state of an organism will not simply

influence, but will in fact control, the nature and extent of skeletal remodeling in response

to microgravity conditions. Ultimately, a means to predict the potential skeletal risk of each

astronaut candidate could play a pivotal role in the future selection of potential space

travelers by identifying those in greatest danger of skeletal dislress.

Determining the potential impact of a "distressed" metabolic state on the skeleton's

response to disuse requires the capacity to enhance or exclude certain components of the

bone's mechanical environment, as well as the ability to control specific aspects of the

animal's systemic milieu. These experimental criteria can be satisfied by utilizing an animal

model of disuse osteoporosis, the functionally isolated turkey ulna. In this model, while

the bone is completely deprived of any mechanical stimuli, its vasculature and innervation

are preserved. In addition, through changes in diet (calcium deficiency), hormonal balance

(endocrinopathy), or age, the skeletal response to the superimposed effects of systemic

factors can be quantified.

In our research, we are using the ulna model to address how the bone responds to the

absence of mechanical loading in each of the following systemic populations:

(1) Adult normal (healthy males, 1 yr. old, fused physes);

(2) Nutritionally deficient (calcium poor diet);

(3) Hormonally imbalanced (castrated adult males);

(4) Growing normal males (5 months of age); and

(5) Old normal males (3 years of age).

By compiling a detailed morphologic, cellular, and physical profile of the skeleton's

response to disuse in healthy adult, endocrinopathized, nutritionally deficient, growing,

and aged populations we are developing a framework with which to evaluate the impact of

metabolic status on the bone's ability to adapt to an altered mechanical environment.

187
Accomplishments

To date, we have obtained preliminary results from 4 of the 5 systemic populations; adult

normal, hormonally imbalanced, growing normal, and old animals. Results are

summarized as follows:

(1) Eight weeks of functional isolation of the ulna in normaiaduii males results in a

15% decrease of bone cross-sectional area. This bone loss is a result of cortical thinning

by the expansion of the marrow cavity with minimal intracortical porosity (Figure la). In

castrated males, the same 8 week period of functional isolation generates a decrease in

cross-sectional area equivalent to that observed in the disuse ulnae of normal males.

However, the bone is removed intracortically, not by expansion of the marrow cavity

(Figure 1b). Thus, the ability of the bone cells to perceive and/or respond to changes in the

mechanical environment are modulated by the animal's systemic state.

Figure 1. Microradiographs of 100 pm thick midshaft sections from the functionally isolated ulnae from

normal (a) and castrated (b) male turkeys. Compared to their respective, intact control ulnae, disuse in the

normal male is dominated by cortico-endosteal resorption. This contrasts markedly with the intracortical

remodeling triggered by disuse in the ulnae of castrated males.

(2) Comparisons of the intact ulnae of castrated and normal males shows an

increase in porot_c area with no real increase in remodeling events. This indicates the

presence of a remodeling imbalance in the castrated animals prior to functional isolation.

Therefore, when disuse is superimposed on this imbalance, increases in the number of

cortical remodeling events exacerbates this imbalance, leading to the development of

intracortical osteopenia.

(3) In animals systemically primed for growth (i.e., 5 month old males), exposure

of the ulnae to disuse does not trigger a decrease in bone mass. In fact, functional isolation

actually appears to accelerate bone formation rates. Compared to intact control bones,

periosteal bone apposition was increased 64% in the disuse ulnae of growing animals.

These results suggest that in the young, rapidly growing skeleton, functional stimuli exert a

strong controlling influence on bone forming and resorbing activities. Once this control is

188

ORIGINAL PAGE IS

OF POOR QUALITY
removed, the active cells escape the regulatory influence of function, and in this case,

increase their bone forming and resorbing actions.

(4) In normal adult males, total bone cross-sectional area increased 30.2% in

response to an artificial, externally applied load (3,000 microstrain, 300 cycles/day). In the

three year old males, total cross-sectional area was unaffected by the same loading

protocol. The reluctance of the bone to produce new tissue suggests that the cells in the

aged animals are "looking" for a different, perhaps more intense, signal.

Significance of the Accomplishments

It is apparent from our preliminary results that the morphologic and cellular aspects of

remodeling are dominated by the animal's systemic state. It is clear, from the endocrine

dependent site-specific resorption in the castrated animals, accelerated bone formation in the

growing animals, and the inability of the aging skeleton to respond to osteogenic stimuli,

that the systemic state must be considered in evaluating the skeletal response to disuse. By

evaluating the impact of hormonal imbalance, growth, aging and nutritional deficiency, as

regulatory factors in the skeleton's response to disuse, a profile of the remodeling

interaction potentiated by each metabolic condition can be assembled. Indeed, if there are

distinct mechanisms by which each systemic state interacts with the normal adaptive

process, then we should expect a unique morphologic response for each metabolic

population.

Spaceflight is becoming accessible to an increasingly diverse population. This will no

doubt include scientists chosen not for their physical fortitude, but for their expertise across

a wide spectra of disciplines. Certainly, the metabolic profiles of these payload specialists

will be equally diverse. To minimize the potential trauma to the skeletal system,

investigations must be designed to evaluate the influence of age, nutrition, and endocrine

related factors on the bone's response to microgravity.

Publications

Bain, S. and Rubin, C. 1989. Altered bone modeling in response to disuse: Comparisons

to adult remodeling bone (Abstract). ASGSB Bulletin 3:111.

Bain, S.D., Impeduglia, T.M., and Rubin, C.T. 1990. Cement line staining in

undecalcified thin sections of cortical bone. Stain Technology 65(3): 1-5.

Rubin, C.T., Bain, S.D., and McLeod, K.J. 1990. The inability of the aging skeleton to

respond to osteogenic stimuli: The origins of Type II osteopenia? (Abstract). Transactions

of the Annual Meeting, Orthopaedic Research Society 15: 75.

189
EFFECTS OF THE UNLOADING MODEL ON

AND CELLULAR IMMUNITY

Wallace D. Berry

Department of Microbiology and Immunology

University of Louisville School of Medicine

Louisville, KY 40292

DIHYDROXYVITAMIN D

Description of Research

The long range objective of this research is to define the mechanisms responsible for

immune system changes during exposure to the microgravity environment of spaceflight.

Our present goal is to determine how changes in the concentration of circulating 1,25-

dihydroxyvitamin D affect components of cellular immunity using the unloading model to

simulate the physical effects of microgravity_ _ =

Functional changes in components of cellular immunity accompany exposure to the

spaceflight environment. Lymphocytes from humans and animals that have flown in space

show reduced proliferative responses to mitogenic stimulation and may have reduced ability

to produce cytokines. It is presumed that these phenomena are at least partly due to the lack

of inertial loading referred to as "weightlessness" or "microgravity". The mechanisms

responsible for the immunological effects of spaceflight are unknown. However, it is

probable that physiological changes brought about by unloading interact with the immune

system during spaceflight (Figure 1).

It has been discovered that the mineral homeostatic hormone, 1,25-dihydroxyvitamin D, is

also an immunoregulatory hormone. This suggests that bone metabolism and mineral

homeostasis, which are altered during spaceflight, may interact with the immune system

through dihydroxyvitamin D. The main points of our hypothesis are: (1) altered mineral

homeostasis during spaceflight causes changes in the plasma concentration of

dihydroxyvitamin D and (2) changes in plasma concentrations of dihydroxyvitamin D

affect immune cell activity and production of cytokines.

We have employed rat whole body unloading, with and without head down flit to model the

musculoskeletal unloading and headward fluid shift experienced by humans during

spaceflight. Adult rats were unloaded with or without head down tilt for periods of one to

fourteen days. Rats were also placed in the unloading apparatus with no head down tilt and

full loadbearing on all four limbs to assess the effects of restraint. Persistency of unloading

effects were assessed by allowing unloaded rats to recover in normal caging for two or

seven days. Plasma levels of dihydroxyvitamin D were measured following each

unloading period.

Parameters of cellular immunity were quantified in the unloaded and recovered rats. Lung

macrophages were tested for changes in their ability to phagocytize foreign particles (sheep

erythrocytes). Spleen cells (monocytes and lymphocytes) were assayed for their ability to

produce the immune cytokines interleukin-1 (IL-1), interleukin-2 0L-2), interferon-gamma

(IFN-gamma), and interferon-alpha/beta (IFN-alpha/beta).

Additional experiments have been conducted to determine the role of dihydroxyvitamin D in

the immunological effects of unloading. To prevent unloading-induced changes in

dihydroxyvitamin D, the hormone was administered to unloaded rats through implanted

osmotic pumps.

190
MODEL OF PHYSIOLOGICAL AND IMMUNE SYSTEM

INTERACTIONS DURING SPACEFLIGHT

I ( SPACEFLIGHT STRESSORS I " MICROORAVITY STRESS RESP0mS_I

l ,i, I s,,=, ,+.,,.qio- / I |

,5" Cabin Atmosphere. _ : -" ._.."_" I /

* V'_ Fluid -- Muscle Veconditioning _ _, biota

EPO 4-- Redistribution Altered Bone Metabollsmq------_

J. ' _.Z." _ _tcT. I

Restxm PT][ "_ G'H _ Ceteeholmmiseml

-.'_ote_in J _ Prole©tin GLUCOCORTICOIDS

+i_c_ "_ DIHYDROXY V I'I'AMIN D]4"/'-'/ / /

-Ac,,oP,, _..,_'_ ,...o..l

, :++: + 1 ''m'+t'

+ +.)I/

_- "X . _ l+-'mtq+ib,om..t, Ln,+i.,,+

,.'-..". I'="'

T-r-u's [+il _ _,,1_,,_°11.s _" /

'.o. Ii!1, ++,,.;,----,' ,'.I,',., ;

MAUOV _ IF]i-GAMMA beta

Figure 1. Possible interactions between physiological systems and the immune system during spaceflight.

Abbreviations: PTH=parathyroid hormone, GH=growth hormone, ACTH=adrenocorticotrophic hormone,

IL-l=interleukin-1, IL-2=interleukin-2, IFN-gamma=interferon-gamma, IFN-alpha/beta=interferonalpha/beta, EPO=erythropoietin.

191
Accomplishments

(1) Plasma dihydroxyvitamin D levels were significantly reduced in unloaded rats

regardless of the type of unloading used. Circulating 1,25(OH)2 D was reduced by more

than 60% following seven days of head down unloading and by more than 70% following

fourteen days of head down unloading as compared to pair fed controls. Unloading

without head down tilt or with full loadbearing produced results essentially identical to

those for head down unloading. Plasma dihydroxyvitamin D levels returned to normal in

all unloaded rats following seven days of recovery in normal caging. Administration of

exogenous dihydroxyvitamin D prevented the decrease in plasma levels of the hormone

during unloading.

z

(2) Unloading with or without head down tilt for seven or fourteen

days or restraint in the harness system with load bearing tended to reduce

macrophage phagocytosis. Seven days of recovery were sufficient to normalize

macrophage phagocytosis in unloaded rats. Phagocytic function was normal or enhanced

in unloaded animals given exogenous dihydroxyvitamin D.

(3) Spleen cell production of IL-1 was reduced by seven or fourteen days of

unloading with or without head down tilt and also by harness restraint. Production of IL-1

was further suppressed following two days of recovery but had returned to normal after

seven days of recovery. Unloaded animals receiving exogenous dihydroxyvitamin D had

significantly increased IL-1 production compared to unloaded and unloaded controls.

(4) Spleen cell production of IL-2 was either unaffected or enhanced following

seven or fourteen days of unloading with head down tilt. Unloading without head down

tilt or with loadbearing tended to suppress IL-2 production. Dihydroxyvitamin D

administration either had no effect or suppressed IL-2 production in unloaded and control

animals.

(5) Early reports that unloading for seven or fourteen days suppressed the IFNgamma response of spleen cells have not been confirmed. In further experiments,

unloading with or without head down tilt actually appeared to enhance IFN-gamma

production. In contrast, unloading restraint with loadbearing tended to suppress IFNgamma production as did two days of recovery following unloading. Exogenous

dihydroxyvitamin D suppressed the IFN-gamma response in unloaded and control animals.

(6) The unloading model had variable effects on production of IFN-alpha/beta.

Some experiments have shown as much as 70% suppression of IFN-alpha/beta production

following unloading while other experiments have shown enhancement of production.

Production of IFN-alpha/beta was lower in rats receiving exogenous dihydroxyvitamin D.

Significance of the Accomplishments

Finding #1. Experimental and clinical evidence indicates that dihydroxyvitamin D

participates in the normal function of the immune and erythropoietic systems, brain,

vestibular system, pancreas, and bone. Therefore, changes in vitamin D metabolism would

be expected to affect these systems. These studies confirm and expand on previous

findings that the unloading model does affect vitamin D metabolism. The direct causes and

mechanisms for this phenomenon are unknown. However, because weightbearing

restraint in the unloading apparatus decreased plasma dihydroxyvitamin D levels to the

same extent as other forms of unloading, it appears that musculoskeletal unloading and/or

head-down tilt are not major contributors to the decrease in dihydroxyvitamin D observed

during unloading. Glucocorticoid hormones, which are released as part of the

192
physiologicalstressresponse,areknownto inhibit thekeyenzymein dihydroxyvitaminD

synthesis. Glucocorticoidselicited in responseto the stressof unloadingrestraintmay

haveinhibitedproductionof dihydroxyvitaminD duringunloading.

Findings #2-6 indicate that the unloading model had selective effects on the

immune system rather than an overall suppression. Differences in the

immunological effects of different unloading types indicate that unloading, head down tilt,

and restraint stress each contributed to the immunological effects of unloading modeling.

The recovery studies indicate that the observed immunological effects of unloading were

reversible within one week.

Findings #2-3. Phagocytosis of pathogens and IL-1 production by macrophages are

functions central to the initiation and regulation of the immune response. Impairment of

these functions during unloading or spaceflight would be expected to result in a suppressed

overall immune response. Enhancement of the IL- 1 response in unloaded rats receiving the

exogenous dihydroxyvitamin D suggests that changes in vitamin D metabolism may be

immunologically relevant to spaceflight.

Finding #4. Changes in IL-2 production could impair lymphocyte differentiation and

function. As with IL- 1, this could lead to suppressed or inappropriate immune responses.

Enhanced IL-2 production during unloading when dihydroxyvitamin D levels were low

was consistent with the known effects of dihydroxyvitamin D on IL-2. However,

because these effects were not consistent with respect to unloading without head down tilt

or with loadbearing, other factors such as stress may have been responsible for changes in

IL-2.

Findings #5-6. Interferons are important antiinfection, antitumor, and immune regulatory

compounds produced by lymphocytes and other cells. Interferon-gamma regulates the

expression of proteins that allow coordinated interaction with other immune cells.

Interferon-gamma also stimulates macrophage activity and IL-1 production. Interferongamma is synergistic with dihydroxyvitamin D in promoting macrophage phagocytosis and

killing of microorganisms. Interferon-alpha/beta functions significantly in immune cell

differentiation and as an antiviral agent. Abnormally increased or reduced interferon

responses could seriously disrupt immune function. As was observed for IL-2, enhanced

IFN-gamma production during unloading was consistent with reduced plasma

dihydroxyvitamin D. Suppression of IFN-gamma production in unloaded rats given

exogenous dihydroxyvitamin D was also consistent with known dihydroxyvitamin D

effects on lymphocytes. Interestingly, cell cultures flown in space have shown an

increased interferon response that could not be due to vitamin D effects.

In summary, our studies indicate that the unloading model reduces plasma

dihydroxyvitamin D levels by a mechanism not dependent on unloading or head down tilt.

The glucocorticoid mediated stress response to restraint may be responsible. Changes in

macrophage phagocytosis and IL-1 production showed the strongest correlation with

plasma dihydroxyvitamin D levels indicating that changes in vitamin D metabolism may be

immunologically relevant in unloading and spaceflight. The results obtained for the other

cytokines suggest that other factors such as stress are contributing to the immunological

effects of unloading. The recovery studies indicate that both the dihydroxyvitamin D

changes and the immunological effects are reversible.

193
Publications

Berry, W.D., Murphy, J.D., Taylor, G.R., and Sonnenfeld, G. 1989. Calcitriol,

macrophage function, and cytokine production in a suspension model of microgravity

(Abstract). Federation Journal 3: 6629.

Berry, W.D., Smith, B.A., and Sonnenfeld, G. 1989. Dihydroxyvitamin D and cellular

immunity during suspension modeling (Abstract). ASGSB Bulletin 3: 45.

Mandel, A.D., Sonnenfeld, G., Berry, W.D., Taylor, G.R., Wellhausen, S.R.,

Konstantinova, I.V., Lesnyak, A.T., and Fuchs, B.B. 1990. Experiment K-6-23: Effect

of spaceflight on levels and function of immune cells. In: Final Reports of the U.S.

Experiments Flown on the Soviet Biosatellite Cosmos 1887 (ed. by J.P. Connolly, R.E.

Grindeland, and R.W. Ballard). Moffett Field, CA: NASA Ames Research Center, p.

471-480. (NASA TM- 102254)

194
REGULATION OF

OSTEOBLASTS

OSTEOCLASTIC

Edward M. Greenfield

Department of Pathology

Jewish Hospital at Washington University

Medical Center

St. Louis, MO 63110

BONE RESORPTION BY

Description of Research

The long-term objective of this research is to understand the regulation of bone resorption

during periods of net bone loss, such as those experienced in spaceflight. Conditions that

increase bone resorption by osteoclasts, including weightlessness, are thought to function

indirectly, that is, by inducing osteoblasts, the ceils that form bone, to produce factors that

stimulate osteoclast numbers or activity. Accordingly, this project has focused on

osteoblast derived factor(s) that stimulate bone resorption by osteoclast-like cells that form

in culture from isolated osteoclast precursors.

Accomplishments

(1) Osteoblast conditioned media stimulate resorption primarily by

increasing osteoclast formation rather than by activating mature osteoclast.

like cells.

(2) Osteoblast conditioned media increase osteoclast formation by

maintaining the viability of osteoclast precursors.

Significance of the Accomplishments

Osteoblast derived factor(s) stimulate bone resorption by maintaining the viability of

osteoclast precursors rather than by activating mature osteoclasts. This finding is

reminiscent of the action of a variety of hematopoietic factors, including colony stimulating

factor- 1, which acts on cells of the monocyte lineage. Since osteoclasts derive from this

lineage, similar factors might be expected to maintain the viability of osteoclast precursors.

Prevention of apoptosis, programmed cell death, has recently been shown to be the

mechanism whereby a number of these factors maintain the viability of their target cells.

The possibility that the osteoblast derived factor(s) also inhibit the specific pattern of DNA

fragmentation that is characteristic of apoptosis is currently being investigated.

These studies have begun to describe the mechanisms that regulate bone resorption. More

complete understanding of this process may allow prevention of bone loss during

conditions such as weightlessness.

Publications

Greenfield, E.M., Teitelbaum, S.L., Alvarez, J.I., Jeffrey, J.J., McLaurine, E.A., and

Blair, H.C. 1989. Osteoblasts secrete an ~10,000 molecular weight factor that directly

stimulates resorption by isolated osteoclasts (Abstract). Journal of Bone and Mineral

Research 4(Suppl. 1): $281.

195
Greenfield, E.M., Teitelbaum, S.L., Alvarez, J.I., Jeffrey, J.J., McLaurine, E.A., and

Blair, H.C. 1989. Partial purification of an osteoblast-derived cytokine that stimulates

bone resorption (Abstract). ASGSB Bulletin 3:116.

Miyauchi, A., Teti, A., Greenfield, E., Carano, A., Blair, H., Barattolo, R., ZambonineZallone, A., Teitelbaum, S., and Hruska, K.A. 1989. Ca 2+ stimulates specific

mechanisms regulating osteoclast cytoskeletal rearrangement and podosome expression

during bone resorption (Abstract). Journal of Bone and Mineral Research 4(Suppl. 1):

S199.

Miyauchi, A., Teti, A., Greenfield, E., Zambonine-Zallone, A., Teitelbaum, S., and

Hruska, K. 1989. Regulation of osteoclast (OC) activity by Ca2+-induced Ca 2+ release

(Abstract). Journal of Cell Biology 109: 304a.

Teti, A., Miyauchi, A., Greenfield, E., Colucci, S., Grano, M., Teitelbaum, S.,

Zambonin Zallone, A., and Hruska, K. 1990. Cytosolic calcium controls osteoclast

activity (Abstract). Calcified Tissue International 46(Suppl.): A27.

196
SYNAPTIC RELATIONS OF TYPE I AND TYPE II HAIR CELLS IN THE

CRISTA AMPULLARIS OF THE CHINCHILLA

Anna Lysakowski

Department of Pharmacological and

Physiological Sciences

The University of Chicago

Chicago, IL 60637

Description of Research

The long-range goal of this research is to understand the structural and functional

organization of the sensory epithelium in the vestibular end-organs. These end-organs

include the crista ampullaris, which detects three-dimensional motion, and the utriculus,

which detects gravity. Our study to date has concentrated primarily on the crista

ampullaris, but preliminary data from the utriculus indicate that its regional synaptic

organization is similar to that of the crista ampullaris.

Previous work on the light microscopic level has characterized three types of afferent nerve

fibers and the two types of receptor cells (type I and type II hair cells) they innervate. The

three types of nerve fibers are: (1) calyx (or chalice) fibers, which terminate in a cup-like

ending around type I hair cells; (2) bouton fibers, which terminate as synaptic boutons at

the base of type II hair cells; and (3) dimorphic fibers, which terminate in both calyx and

bouton endings. In the adult pigmented chinchilla, type I and type II hair cells are present

in equal proportion throughout the sensory epithelium, while the three types of nerve fibers

show an unequal distribution. Calyx fibers (10% of the total) are found solely in the

central zone. Bouton fibers (20% of the total) are found solely in the peripheral zone.

Dimorphic fibers (70% of the total) are found throughout the epithelium. These three types

of fibers also have different physiological properties. Simply stated, calyx fibers have

irregular firing patterns, bouton fibers have regular firing patterns, and dimorphic fibers

have a broad range of firing patterns, which depends on their location in the sensory

epithelium. They are irregular in the central zone and regular in the peripheral zone. In

addition to these differences in discharge regularity, there are differences among the three

fiber types in galvanic sensitivity and in response dynamics. From these previous studies,

it was concluded that the physiology of an afferent is more closely related to its location in

the sensory epithelium than to its branching pattern or to the types and number of hair cells

it contacts.

The present study was undertaken to determine whether the physiological differences

described above could be related to regional differences in synaptic innervation. We have

examined, using the electron microscope, serially sectioned material from several chinchilla

end-organs. Our initial data were gathered from a series of 300 sections taken through the

entire cross-section of one superior canal crista so that all three regions (central,

intermediate and peripheral) were represented in each section. We have since switched to

an approach utilizing stereological (quantitative sampling) methods to be able to examine

tissue from several animals and to verify the general applicability of our results. Both

approaches have yielded similar results. In each case, low-power photomontages were

made of the entire sensory epithelium, using every fourth or fifth section in the series.

High power examination of every section in the series was used to determine the number

and location of all synaptic elements. These data were notated on the photomontages and

compiled using a computer spreadsheet program. In the past year, over 25,000 hair cell

profiles have been examined. In addition, we are reconstructing individual hair cells from

197
each region, using higher power serial photomicrographs and a PC-based 3-D

reconstructionprogram.

Accomplishments

(1) Peripheral type H hair cells are innervated by 30 or more boutons,

each making a single synaptic contact (Figure 1A). Central type H hair cells

have few (2.6) afferent boutons, each of which receives several synaptic

contacts (Figure 1B).

(2) Many type H hair cells in the central zone make multiple synaptic

contacts with the outer face of one or more calyx endings. In contrast,

outer-face synapses are rare in the peripheral zone.

(3) Ribbon synapses between type I hair cells and calyx endings are

plentiful in both central and peripheral zones (Figures 1C and 1D).

(4) The invaginations made by calyx endings into type I hair cells are

also plentiful, especially in the central zone.

(5) The efferent innervation of type II hair cells and of calyx endings is similar

throughout the cristae.

Significance of the Accomplishments

Finding #1: There axe differences in the afferent bouton innervation of type II hair cells in

the central and peripheral zones of the crista. A higher convergence ratio of hair cell

synapses to afferent boutons in the central zone of the crista implies that, anatomically, the

central zone appears to be specialized for increased sensitivity. In thirty-five years of

electron microscopic study of the peripheral vestibular apparatus, these regional differences

have never been reported.

Finding #2: The importance of this observation is that most so-called calyx units can be

functionally dimorphic.

Finding #3: This observation, along with the presence of outer-face calyx synapses,

should lay to rest the notion that calyx endings are not important sites of chemical

transmission.

Finding #4: Invaginating junctions are, as far as we know, not seen in other hair cellafferent synapses and so may provide a key to the peculiar function of the calyx ending.

Finding #5: This suggests that the larger efferent responses seen in central (irregular)

afferents may reflect differences in the neurotransmitter or in the density and type of

postsynaptic receptors present on calyx endings and on type II hair cells.

An understanding of the synaptic relations of hair cells with their afferent and efferent

endings is fundamental to understanding alterations during spaceflight, as well as during

development of the vertebrate vestibular apparatus in extended spaceflights. These changes

will probably be subtle, at best, and so quantitative baseline data from normal adult tissue,

such as that shown in the present study, are needed for comparison to spaceflight

experiments.

198
TYPE II HAIR CELLS OF THE SUPERIOR CANAL

OF ADULT CHINCHILLA

ORIGINAL PAGE

BLACK AND WHITE PHOTOGRAPH

CRISTA

Figure 1, Panels A-D (pages 199 and 200). Electron micrographs taken from a superior

canal crista in a normal adult pigmented chinchilla.

Panel A: Two type II hair cells (48, 49) in the peripheral zone that were serially reconstructed. The several

boutons contacting each of them were numbered consecutively based upon their first appearance in the set of

sections. Here and in the other panels, long arrows point to synaptic ribbons seen in the section, short

arrows point to synaptic ribbons seen in nearby sections. Most peripheral boutons make only single

ribbon synapses with their type II hair ceils. Panel B: A serially reconstructed type II hair cell in the

central zone. Bouton 1 makes 11 ribbon synapses, seven with cell 23 and four with cell 24, while bouton

2 receives 10 synapses from cell 23. Most of the ribbons appear in adjacent sections; the two seen in this

section (long arrows) are shown at higher magnification in the inset. An efferent bouton (E2) synapses on

cell 23 and is associated with a subsynaptic cistern (arrowhead).

199
TYPE I HAIR CELLS OF THE SUPERIOR CANAL

OF ADULT CHINCHILLA

ORIGINAL PAGE

BLACK AND WHITE PHOTOGRAPH

CRISTA

Panel C: A serially reconstructed type I hair cell (3) in the peripheral zone has eight synaptic ribbons.

Most of these ribbons appear to be associated with calyceal invaginations; the asterisk marks a case where

both structures are seen in the same section. An efferent bouton (El) contacts the calyx ending at its base.

Close appositions between type II hair cells and the outer surface of calyces are not common, but when they

do occur, there are usually thin processes of supporting cells (arrowheads) interdigitating between them.

Panel D: A type I hair cell in the central zone (21) has 14 synaptic ribbons. The calyx ending is also

contacted by a type II hair cell (22) which contains six ribbons forming synapses with the outer face of the

ending (arrows); the two synapses seen in this section of cell 22 (long arrows) are shown at higher power in

the inset.

200
Publications

Goldberg, J.M., Lysakowski, A., and Femfindez, C. 1990. Morphophysiological and

ultrastructural studies in the mammalian cristae ampullares. Hearing Research (in press).

Goldberg, J.M., Fermindez, C., and Lysakowski, A. 1990. Morphophysiological studies

of the chinchilla utricular macula (Abstract). Proceedings of the Barany Society (in press).

Lysakowski, A. and Goldberg, J.M. 1989. Regional variations in synaptic organization

in the chinchilla cristae (Abstract). ASGSB Bulletin 3: 34.

Lysakowski, A. and Goldberg, J.M. 1990. Regional variations in the synaptic

organization of the chinchilla cristae (Abstract). In: Abstracts, 13th Midwinter Research

Meeting of the Association for Research in Otolaryngology, p. 291.

Lysakowski, A. and Goldberg, J.M. 1989. Regional variations in the synaptic

organization of the chinchilla cristae (Abstract). Society for Neuroscience Abstracts 15:

502.

201
REGULATION OF BONE REMODELING ACTIVITY

CONTROL OF STRESS GENERATED POTENTIALS

Kenneth J. McLeod

Musculo-Skeletal Research Laboratory

Department of Orthopaedics

State University of New York

Stony Brook, NY 11794

THROUGH THE

Description of Research

The long-term objective of this research is to improve our understanding of the manner in

which bone is able to sense the demands of its functional environment. The basis for this

work lies in the electrophysiological properties of bone tissue. During normal functional

activity, electrical currents are endogenously induced within bone. It is believed that

skeletal tissue ceils are sensitive to these curi'ents-, because Confiective tissue cells, of which

bone cells are a derivative, are known to respond to electric currents similar in magnitude

and frequency to those induced within bone. Moreover, electric fields exogenously

induced into bone have been shown to modulate bone remodeling activity in the absence of

normal functional loading. These observations have led us to suggest that an efficient

mechanical loading protocol for the minimization or prevention of microgravity-induced

bone loss might be developed by optimizing the endogenously induced electric currents.

The specific objective of this investigation was to define a loading paradigm for the

maintenance of bone mass in the absence of normal functional loading. Our hypothesis

was that a loading paradigm which ensures the endogenous generation of osteogenic

electric fields will also maintain bone mass with a minimal mechanical work input. The

investigation therefore required the isolation of the most osteogenic electric field component

of functional strain, the establishment of the relationship between strain magnitude and

induced field intensity, and the subsequent testing of the predicted mechanical loading

paradigm.

The experimental work was based on the isolated avian ulna model of disuse osteopenia.

In this model, the ulna of adult male turkeys is isolated by proximal and distal osteotomies

such that the animal cannot apply functional loading to its ulna. Through the use of

external magnetic coils, electrical fields mimicking those produced endogenously can be

induced in the absence of functional loading. This permits the isolation of the fields which

are most osteogenic. In addition, this preparation permits the recording of induced field

intensities during controlled mechanical loading. Finally, the field characteristics and

mechano-electric transduction measurements can be used to predict an appropriate

mechanical loading protocol which can be subsequently tested in the isolated ulna

preparation.

Accomplishments

(1) Initial work in this investigation was directed toward isolating the maximally

osteogenic electric field characteristics. Through the application of broad spectrum

electromagnetic fields, with subsequent theoretical analysis, we have isolated the regime of

maximum electric field efficacy to the frequency range below 75 Hz. Using sinusoidal

stimulation, we have shown that within this low frequency range, electric fields near 15 Hz

demonstrate the greatest osteogenic effect. Calculations of the induced field intensity

within the bone preparation show that fields as low as 10 microvolts/cm or less are

sufficient to maintain bone mass at 15 Hz when applied for at least one hour per day.

202
(2) In order to associate these electric field parameters with mechanical strain, in

vitro stress generated potential recordings were made in the ulna preparation under

conditions of controlled mechanical loading. These recordings indicate that average field

intensities on the order of 10 microvolts/cm can be induced in cortical bone with peak bone

strains on the order of 10-100 microstrain.

(3) The prediction that 15 Hz mechanical strains, applied to bone for one hour per

day, should be sufficient to maintain bone mass in the absence of any functional loading

was tested in the isolated ulna preparation. As our mechanical loading apparatus cannot be

regulated at strain levels below approximately 100 microstrain, we elected to test the

predicted loading paradigm with a 250 microstrain load at 15 Hz for 5 minutes per day.

(4) Preliminary tests results suggest that very low level loading can indeed prevent

the loss of bone mass due to disuse, if the loading is at an appropriately high frequency.

While 1 Hz loading at 250 microstrain can be shown to result in up to a

11% loss of bone mass in eight weeks in the turkey, 15 Hz loading both

prevented this loss, and initiated new bone formation.

Significance of the Accomplishments

This work appears to indicate that bone senses and adapts to the relatively low amplitude

but high frequency strains presented within its environment rather that the much larger

amplitude but lower frequency components of bone strain. As the high frequency

components arise predominantly from the dynamics of muscle activity, and not through the

reaction loads associated with locomotion, these results suggest that microgravity-induced

bone loss may be preventable without strenuous activity. However, to fully understand the

normal maintenance of bone mass it will be necessary to specifically identify the origin of

high frequency mechanical strains, the specific cell population which responds to this

mechanical stimulus, and the effect of very long term controlled mechanical loading.

In the future, we will be pursuing each of these questions. Through in vivo strain gage,

accelerometer, and electromyographic recordings we will be attempting to isolate the

component within the muscular control system which gives rise to the high frequency strain

within bone during normal functional activity. In in vitro studies we will be addressing the

issue of whether the osteocyte population of bone is capable of responding directly to either

the mechanical or electrical environment. We will also be continuing with the isolated ulna

preparation in an effort to identify the minimum levels of strain necessary to maintain bone

mass when loading durations are extended into the range of several hours per day. Finally,

we will continue our use of theoretical models to address the more complex questions of

the manner in which a mechanical strain signal is utilized in the adaptation of the

morphology of bones.

Publications

McLeod, K. and Rubin, C. 1989. Strain frequency dependence of bone remodeling

activity (Abstract). ASGSB Bulletin 3:111.

McLeod, K.L, Bain, S.D., and Rubin, C.T. 1990.

frequency of induced dynamic strains (Abstract).

Orthopaedic Research Society 15: 103.

Dependence of bone adaptation on the

Transactions of the Annual Meeting,

203
McLeod, K.J. and Rubin, C.T. 1989. Dosedependentresponseof boneremodelingto

themagnitudeof inducedelectricfields (Abstract). Transactions of the Annual Meeting,

Orthopaedic Research Society 14: 309.

McLeod, K.J. and Rubin, C.T. 1990. Frequency specific modulation of bone adaptation

by induced electric fields. Journal of Theoretical Biology (in press).

McLeod, K.J. and Rubin, C.T. 1989. Prediction of osteogenic mechanical loading

paradigms from electrical response data (Abstract). Bioelectrical Repair and Growth

Society 9: 20.

McLeod, K.J. and Rubin, C.T. 1989. Skeletal resonances: An alternative mechanism for

tissue regulation (Abstract). American Zoologist 29: 36A.

McLeod, K.J. and Rubin C.T. 1989. Theoretical determination of the appropriate

frequency content of strain for the optimal influence on bone adaptation (Abstract). 13th

Annual Meeting of the American Society of Biomechanics 13: 32.

Rubin, C.T., McLeod, K.J., and Bain, S.D. 1989. Functional strains and cortical bone

adaptation (Abstract). Abstracts of the XII International Congress of Biomechanics 12: 6.

Rubin, C.T., Vasu, R., Tashman, J., Seeherman, H., and McLeod, K.J. 1989. The

nonuniform distribution of shear strains in functionally loaded bone: Implications for a

complex mechanical milieu (Abstract). Transactions of the Annual Meeting, Orthopaedic

Research Society 14: 313.

Tashman, J.S., McLeod, K.J., and Rubin, C.T. 1989. Low amplitude, high frequency

bone strain: Does it occur in the non-weight bearing skeleton? (Abstract) American

Zoologist 29: 35A.

204
TEMPORAL EXPRESSION AND SPATIAL

MAJOR PROTEINS IN THE OTOCONIA

VERTEBRATE

Kenneth Gene Pote

Department of Biology

Gilmer Hall

University of Virginia

Charlottesville, VA 22901

DISTRIBUTION OF THE

OF TWO SPECIES OF

Description of Research

This research is aimed at increasing our understanding of the function of the peripheral

portion of the vestibular system found in the inner ear of vertebrates. Specifically, this

research is interested in the evolution, chemistry, and function of a biomineralized structure

found within the inner ear. The vestibular system has evolved to detect linear acceleration

and gravity through sensory end organs, known as the maculae, through the interaction of

extracellular mineralized structures with the underlying neuroepithelium. These

biominerals, known as otoconia (literally "ear dust"), are embedded in a gelatinous mass

known as the otoconial membrane. The otoconia are thought to increase the mass of the

otoconial membrane thereby increasing the deflection of the apical projections on the

underlying sensory cell, the hair cell, during accelerations.

Otoconia are composites of protein and mineral phases. The mineral phase varies within

the vertebrates in a phylogenetic trend. Otoconia from some primitive fishes are

mineralized by calcium carbonate deposited as vaterite. Reptiles and amphibians

predominantly have otoconia mineralized by calcium carbonate in the crystal form of

aragonite. The otoconia from birds and mammals contains calcium carbonate in the form of

calcite. Each mineral type has a protein unique to that type of otoconia. This research has

focused on the proteins contained within the otoconia of the rat (calcitic) and the African

clawed frog, Xenopus laevis (aragonitic). Little is known about the nature of these

proteins, save their molecular weights. The calcitic rat otoconial protein is Mr=90k and the

aragonitic is Mr=22k. The ceils of origin, developmental expression, or turnover of the

proteins are all unknown. In addition nothing is known about interactions of these proteins

with their mineral component. Understanding the structure of these proteins will enhance

our knowledge of the development and evolution of the mammalian vestibular system, and

will serve as a model system for the field of biomineralization.

I have begun to investigate the otoconial proteins using both immunological/histochemical

and molecular biological approaches. My plan is to isolate the proteins from the otoconia

of the rat and the clawed frog, raise antibodies against them, and sequence the proteins.

The antibodies will be used for immunohistochemistry and to screen cDNA libraries

constructed from inner ear tissues. In addition, the amino acid sequence will be used to

synthesize oligonucleotide probes for screening the libraries. The clones coding for the

proteins will be used to generate in situ hybridization probes and to construct expression

clones for the production of the proteins in large amounts. Both the cDNA probes and the

antibodies are to be used to study the development and evolution of otoconia. These

approaches will enable me to answer the basic questions of the temporal and spatial

expression of these proteins. In addition, they will allow the further characterization of

these proteins and their interactions with the mineral phase using in vitro approaches.

205
Accomplishments

We have determined essentially the entire amino acid sequence of the 22 kD

otoconial protein from Xenopus laevis using a combination of mass spectrometry

and classical Edman degradation to analyze purified peptide fragments of the protein (see

Figure 1). In addition, a homology search against the NBRF protein data bank has shown

the N-terminal fragment of the otoconial protein has great sequence homology with the

phospholipase A2. This has given us insight into the manner in which the protein should

be treated prior to proteolytic digestion for sequence determination. Since the

phospholipases have seven disulfide crosslinks, we have increased the amount of

dithiothreitol used for reduction. This greatly enhanced the trypsin, CNBr, Asp-N, and

Lys-C cleavages and allowed for determination of nearly the entire amino acid sequence

(Figure 1). The entire sequence aligns with the phospholipase A2 (for example see Figure

2). We are currently mapping the disulfide linkages in this protein, to see if the linkages

are similar to those of the phospholipase A2.

N_2- T P A Q F D E M I K V T T I I Y G L A $ F S D Y G C H C G L N N Q G M P V D D I-

\ EDMAN--WHOLE--PHOTEIN- /

\---CNBr_890--/ \-----CNBr--

\--Kc--i179--/ \ ................... Kc-HIGH-MASS ...........................

\--T--I179--/ \ .................... T-HIGH-MASS ...........................

\---T-Dn--HIGH-MASS---/ \--T--Dn--1932,2160 /--/

D W C C H 0 Q D C C ? N...D Q ? Y N K A E M S G C N P v T Q T Y R F Y V E g Q K-

---CNBr-EDMAN . .................. / \ ............ CNBr-2905 .............

\------CNBr-T-I 331--/ \CNBr--T-942--/

.......... Kc-HIGH-MASS .............. / \ Kc--2584 -/

........... T-HIGH-MASS .............. / \ T--1661 -/ \--T-942--/

\--Dn-EDMAN HIGH-MASS ......................

Q VE CM KM I C EC DE KAA NC F R KE LED YN I Y F R $ F S Y LGAC R-

........ / \ CNB_ -- EDMAN-- @ -8 -Kd-S DS--PAGE

\--Kc--842--/ \--KC--II80--/ \--Kc--914--/ \ ........... Kc-HIGH-MASS .............

\--Kc-T-I 36 I--/ \Kc-T-HIGH-MASS-/

\--T--842--/ X--T--1180--/ \--T--786--/ \--\--T--1361,1489--/ k--T-HIGH-MASS--/

.................... /

G P R P F C -COOH.

/

.......... /

\Kc--T--7 8 i--/

k--T-78 i--/

Kc = Endoproteinase Lys-C.

T = Trypsin. CNBr = Cyanogen bromide.

Dn = Endoproteinase Asp-N.

-- = Sequence information confirmed by either Mass

Spectrometry or Edman.

.... Sequence information determined by a combination of HPLC and sub-digestion data.

0 = Blank cycle on the Edman sequnator.

? = Non-standard peak on the Edman sequnator.

$ = Site of sugar attachment.

.... = Unknown.

Figure 1. The amino acid sequence of the 22 kD aragonitic otoconial protein from Xenopus laevis aligned

with the sequence of a phospholipase A2 (ammodytoxin A). The single letter code for the amino acids is

used. Identical residues are identified by a colon, while conservative replacements are marked by periods.

The asterisks are residues which coordinate calcium ions within the proteins of the phospholipase A2

family. These residues are within the calcium binding loop which have the invarient residues marked by I.

The question marks have been identified as sites of glycosylation. There is 38% identity between these

sequences.

206
Xl 22k

A26535

Xl 22k

A26535

i0 20 30 40 50 60

TPAQFDEMI KVT T I IYGLA$ F SDYGCHCGLNNQGMPVDD IDWCCHXQDCCXNXDQXYNKA

: :,.. :. .. :. .. :. : : :. :: .. ::.: :.. : : : .. : : : :. :

SL IQFE TL IMKVAKKS GMFWYSNYGCYCGWGGQGRP QDATDRCCFVHDCC ...... YGKi0 20 IIIIII II I40 I50

70 80 90 100 ii0

EMSGCNPVTQTYRFYVEEQKQV ..... ECMKMICECDEKAANCFRKE LEDY-N IYFR$ F S

..... . .... o _ o •

,. : :. : .. :. : :. . : ........... . .......... :.

-VTGCDPKMDVYSF SEENGD IVCGGDDPCKKE ICECDRAAAI CFRDNLT LYNDKKYWAFG

60 70 80 90 100 110

120

Xl 22k YLGACRGPRPFC

A26535 AKNCPQEESEPC

120

Figure 2. An alignment of the major protein from Xenopus laevis aragonitic otoconia with phospholipase

A2 from viper toxin. The single letter amino acid abbreviations are used for the sequences. The colons

indicate identical residues, while the periods are conservative replacements. The I's indicate invariant amino

acids in the calcium binding domain of the phospholipases A2; Y27, G29, G31, and D48 (indicated by

asterisks) are involved in the calcium coordination. Loss of D48 results in loss of activity of

phospholipase A2. The dollar signs are glycosylation sites in X1 22 kD. The phospholipases A2 are not

glycosylated. Dashed lines are insertions made by the alignment program. The sequences have 38%

sequence identity.

The crystal structures for several phospholipases A2 have been solved. A model of the X.

laevis otoconial protein was built using the published coordinates for the carbon backbone

of the western diamondback rattlesnake (Crotalus atrox) venom phospholipase A2 by

altering the amino acid side chains to those of the otoconial protein using the University of

California, San Diego, Molecular Modeling System software. The resulting structure was

put through 160 cycles of minimization using the X-plor program. The resulting structure

was then compared to the otoconial protein before minimization and compared to the C.

atrox phospholipase A2. All of the changes in amino acid composition could be

accommodated by the backbone structure of the phospholipase A2.

We have started the carbohydrate analyses of the Xenopus laevis 22 kD otoconial protein.

We have HPLC purified the peptides containing the glycosidic modifications to analyze the

carbohydrates present in each side chain. The amount of sugar present in the protein and

each glycopeptide is shown in Figure 3. We estimate that this protein may contain as much

as 7000 molecular weight, or 34% carbohydrate.

207
Whole Protein Suqar Analysis

Suuar R_w Data _ IDcrementai_M_W. _weiaht in Protein

Fuc 1.9 2 147 294

GalNc 2.6 3 204 612

GlcNc 13.1 13 204 2652

Gal 8.7 9 163 1467

Man 6.8 7 163 1141

NeuAc 2.8 3 291 873

7039

Percentage of sugar in protein = 7,039/21,000 = 34%

GLYCOPEPTIDE ANALYSIS

COOH end #i COOH end #2 NH2 end Total

Fucose 1 / 1 / 1 1 / 1 / 1 1 / 1 / 1 2

GalNac i? i? i? 2?

GlcNac 3.1/4.6/ 4 3.8/5.6/ 4 4 / 8 / 6 i0

Galactose 2.4/2.6/ 3 2.6/2.9/ 3 3.1/5.2/ 4 7

Mannose 2.2/ 3 / 3 2.7/ 3 / 3 4.5/7.9/ 6 9

Figure 3. Summary of carbohydrate analyses of the intact Xenopus laevis otoconial protein (above) and the

I-1PLC purified glycopeptides. The glycopeptide near the carboxy-terminus was purified twice for analysis.

The glycopeptide near the nitroxy-terminus was purified once. All samples were run in duplicates. The

three numbers for the glycopeptide analyses are the first run, the second run and their whole number

average. N-acetyl-neuraminic acid (NeuAc) was not determined for the glycopeptides. Abbreviations are

Fuc=fucose, GalNc=N-acetyl-galactosamine, GlcNc=N-acetyl-glucose, gal=galactose, man=mannose. There

is general agreement in the total sugar present and that present in each glycopeptide.

Significance of the Accomplishments

Having determined that the entire amino acid sequence is significant because it allows use

of the polymerase chain reaction for amplification of the cDNA coding for the protein, the

homology search to the phospholipase A2, based on this sequence, has given insight into

the methods required to solve the problems of limited hydrolysis by the proteolytic

methods. This homology may also give insight into the evolutionary origins of the

otoconial proteins. Evidence that this otoconial protein is not a phospholipase A2 per se,

are the glycosylation (the phospholipases A2 are not glycosylated) and the modification of

the calcium binding loop. The residues invariant in the calcium binding loop of the

phospholipase A2 are shown in Figure 2. Four amino acids, tyrosine 27, glycines 29 and

31, and aspartic acid 48 coordinate the calcium ion. The first three residues are different in

the otoconial protein. This suggests that the interaction of this protein with the calcium

carbonate mineral phase in intact otoconia is not through calcium binding, per se. The

model construction is a first step in determination of the interactions between the mineral

and organic phases of otoconia.

This is the first demonstration of the type of glycosylation of an otoconial protein. It is

often assumed that proteins of the otoconial matrices are glycosaminoglycans. The

208

h
carbohydrate analyses show that this is not true. The carbohydrates present show

this protein is a glycoprotein. The sequence within the glycopeptide, X-Phe Ser,

indicates it is likely a N-linked oligosaccharide. More important for our proposed use of

the expressed protein for determination of the structure and interaction with the mineral

phases, this shows we must use an approach which will glycosylate the expressed protein.

Publications

Pote, K.G.and Kretsinger, R.H. 1990. The otoconial protein of frogs is a homolog of

phospholipase A2. In: Calcium Transport and lntracellular Calcium Homeostasis (ed. by

D. Pansu and F. Bronner). Berlin: Springer-Verlag (in press).

209
PLANT HORMONE CATABOLISM

ASYMMETRIC GROWTH

Dennis M. Reinecke

Department of Plant Biology

University of Minnesota

St. Paul, MN 55108

AND TROPICALLY INDUCED

Description of Research

Plant hormones play important roles in terrestrial plant growth and development including

roles in cell division, cell elongation, and differentiation of cell tissues. The plant hormone

indole-3-acetic acid (IAA) is rapidly redistributed to the lower faster-growing side of a

shoot following a gravity stimulus, The long-range goal of this research is tounderstand

how gravity induces a hormone/IAA asymmetry, and how microgravity may alter hormone

metabolism and action and thus affect plant growth and development.

This research has examined IAA catabolism, an important regulatory control point in IAA

metabolism which may contribute to the rapid hormone asymmetry. In corn, Zea mays L.,

I have discovered a new pathway for IAA catabolism with the oxidation of IAA to

oxindole-3-acetic acid. This pathway is an irreversible oxidation of IAA resulting in loss of

IAA/hormone activity. The regulation of this pathway may help maintain IAA growthlimiting in actively growing tissues by destroying the hormone following growth induction.

The current research has examined the mechanism and developmental occurrence of the

oxindole-3-acetic acid pathway.

Accomplishments

Accomplishments include the further characterization of the IAA to oxindole-3-acetic

pathway: (1) indole-3-acetic acid oxygenase can be separated from peroxidase/IAA oxidase

and lipoxygenase, (2) this pathway occurs in all corn tissues and developmental stages

examined. The in vitro oxygenase assay depends on a lipid soluble cofactor which can be

substituted by fatty acids and corn oil. The present study provides evidence that IAA

oxidation to oxindole-3-acetic acid is assisted by a novel enzyme present in

etiolated and light-grown tissues.

Significance of the Accomplishments

This is the first in vitro study of this pathway. The enzyme indole-3-acetic acid oxygenase

has been shown to be a novel enzyme which can be separated from peroxidase and

lipoxygenase enzymes by ion exchange chromatography and gel filtration chromatography.

The enzyme is stimulated by a heat-stable lipid-soluble cofactor which can be replaced by

unsaturated fatty acids in the oxidation of IAA. In the regulation of the IAA oxygenase

during gravitropism (as well as during vertical growth), the activity of the enzyme may be

regulated by the availability of the cofactor. The natural cofactor needs to be

unambiguously identified and quantified to determine how it affects IAA oxygenase

activity. Unrefined corn oil has been shown to stimulate IAA oxidation and may be used in

the isolation and characterization of the natural lipid-soluble factor. The mechanism of the

oxidation reaction can be examined with the purified enzyme and the following questions

asked: Is the cofactor co-oxidized during IAA oxidation? If so, does the oxidized lipid

cofactor have biological activity? Measuring the turnover rate of IAA to oxindole-3-acetic

acid, measuring IAA oxygenase activity, and measuring cofactor availability will clarify

how catabolism down-regulates IAA activity during growth. The separation of interfering

210
enzymesfrom the IAA oxygenaseand identification of thenatural cofactormakesthis

researchfeasible.

Until recently,IAA levelswerethoughtto beregulatedby decarboxylationby peroxidase

(IAA oxidase).This workwith corn(andwork byotherswith broadbean,scotspine,and

poplar) hasdemonstratedthat IAA canbe catabolizedwithout decarboxylation. This

researchhasconfirmed that IAA oxidation to oxindole-3-aceticacid occurswithout the

involvement of peroxidase,a nonspecificoxidase. Furthermore,the enzymehasbeen

shownto occurin younggreenseedlingsaswell asin etiolatedseedlings.The pathway

occursat two distinctdevelopmentalstages,supportingour hypothesisthat catabolismis

importantin planthormoneregulation.

The basic understandingof how gravity induces a hormone asymmetry and how

microgravityaffectsnormalplantdevelopmentby alterin.ghormonemetabolismandaction

will likely havepracticalbenefitsinourattemptstooptimizeplantgrowthin microgravity.

Publications

Bandurski, R.S., Schulze, A., Desrosiers, M.F., Jensen, P., Epel, B., and Reinecke,

D.M. 1990. Relationship between stimuli, IAA, and growth. In: Plant Growth

Substances 1988 (ed. by R. Pharis and S. Rood). Heidelberg: Springer-Verlag (in press).

Bandurski, R.S., Schulze, A., Desrosiers, M., Jensen, P., Reinecke, D., and Epel, B.

1990. Voltage-gated channels as transducers of environmental stimuli. In: Inositol

Metabolism in Plants (ed. by D.L Morr6, W.F. Boss, and F.A. Loewus). New York:

Wiley-Liss, p. 289-300.

Reinecke, D.M. 1990. The oxindole-3-acetic acid pathway in Zea mays. In: Plant

Growth Substances 1988 (ed. by R. Pharis and S. Rood). Heidelberg: Springer-Verlag,

p. 367-373 (in press).

211
CHANGES IN CELL WALL POLYMER SIZE AS

GRAVITROPIC RESPONSE MECHANISMS

Lawrence D. Talbott

Laboratory of Biomedical and Environmental Sciences

University of California

Los Angeles, CA 90024

INDICATORS OF

Description of Research

We intend to use structural changes in the cell walls of gravitropically active tissue to

understand the sensory and signal transduction mechanisms by which plants respond to

gravity. The structural properties of the plant cell wall are the ultimate determinant of the

rate of elongation. The length and/or degree of association of the carbohydrate polymers

composing the plant cell wall are likely to be a critical factor determining these properties.

Substantial progress has been made in understanding the changes in wall polymers during

growth responses, although the system is quite complex and we are far from a complete

understanding of the complexities and interactions of the system.

Our approach is to determine the extent and location of growth changes along a

gravitropically responding stem. By then isolating and analyzing wall components from

this stem at various times during curvature, we can correlate molecular size or

compositional changes in cell wall structure with the growth response. By analogy to

known effects and through experimentation, we will determine the likely biochemical

mechanisms controlling these structural changes. Knowledge of the final biochemical

action(s) resulting in growth control will provide a tremendous advantage for investigating

the signal transduction chain of gravitropism, to say the least. Ultimately it may be

possible to trace the chain of events from final biochemical action all the way back to the

initial sensory event.

Accomplishments

(1) We developed an HPLC system for the molecular size analysis of soluble cell

wall polymers and coupled it to an extremely sensitive electrochemical carbohydrate

detector.

(2) In pea stem, the maximum gravitropic curvature takes place between 20 and 60

minutes after horizontal placement and involves reduction of elongation on the lower side

and stimulation of elongation on the upper side. Polymers of the hemicellulose wall

fraction undergo a reduction in size on the lower side and an increase in

size on the upper side of the stem correlated with changes in elongation

rates.

(3) Monosaccharide composition analysis indicates that xyloglucan polymers

account for most of the size changes seen in the hemicellulose fraction.

(4) Polysaccharide size changes occur primarily in the epidermal

layer of the stem.

(5) Electron microscopy of fractionated polymers indicates that size

increases of xyloglucan are due to formation of a crosslinked network.

Preliminary studies indicate that formation of this network may be pH and

ion dependent.

212
Significance of the Accomplishments

Finding #1. Until now, polymer size analysis has been done on low pressure

chromatography columns requiring as long as 24 hr per separation. Recent advances in

HPLC column technology now permit similar size separations in 1 hr with better

resolution. When coupled with new electrochemical carbohydrate detectors, this system

permits practical analysis of polymer size distributions in small amounts of tissue, such as

individual stem segments or collections of epidermal peels.

Finding #2. Previously, decreases in hemicellulose polymer size have been correlated with

hormonal (IAA) stimulation of growth and with pH reductions such as those stimulated by

IAA. Increases in hemicellulose size are found in situations where cell wall extensibility

should decrease and occur as a response to sudden turgor pressure increases. Both types

of changes occur during gravitropic bending and may indicate the operation of at least two

regulatory mechanisms in the gravity response.

Finding #3. Metabolic and turnover studies indicate that xyloglucan is the most active of

the dicot hemicellulose polymers during growth. The auxin and turgor responses

mentioned above affect primarily the size of xyloglucan polymers. We now confirm that

polymer size changes in gravitropic growth regulation again involve primarily xyloglucan

polymers.

Finding #4. Much indirect evidence has focused attention on the extensibility of the

epidermis as the controlling factor governing overall extensibility of the plant stem. Our

work confirms that the majority of observed wall size changes occur in the outer few cell

layers of the gravitropically responding stem. These results focus the search for gravitropic

mechanisms to actions involving primarily the epidermal tissue.

Finding #5. The existence of pH and ion dependent molecular size changes in wall

polymers tends to support the emerging picture of the cell wall as a non-covalently linked

network as opposed to a covalent structure requiring enzymatic action for alteration.

Visualization of this network should permit direct in vitro studies of the biochemical actions

responsible for changes in wall structure. Although enzymatic action is not excluded, our

results encourage investigation of the role of ion movements and auxin-induced pH effects

in gravitropism (Figure 1).

Publications

Talbott, L.D. and Pickard, B.G. 1989. Actuation of pea stem gravicurvature by

alterations of specific wall polymers: Results with a new technology (Abstract). ASGSB

Bulletin 3: 99.

213
POSSIBLE ROLE OF ION MOVEMENTS DURING

GRAVITROPIC BENDING

Gravi_Ol_c_/lyStimula_d

(D

0 o

===================================

Figure 1. Possible mechanisms for controlling wall extensibility during gravitropic bending. Wall

polymers in the initial state form an intermediately crosslinked network with filled • and unfilled o ionic

binding sites. Gravitropic stimulation causes release of protons and/or enzymes on the bottom side which

reduce the extent of crosslinking. Ions may be transferred to the upper side, causing increased crosslinking

and slowing growth.

214 __
MECHANISMS OF DECREASED ACTIN SYNTHESIS DURING RODENT

NON-WEIGHTBEARING

Donald B. Thomason

Department of Physiology and Cell Biology

University of Texas Medical School

Houston, TX 77225

Description of Research

The purpose of this research is to define the mechanisms of skeletal muscle atrophy in a

non-weightbearing environment. The ground-based rodent hindlimb non-weightbearing

protocol has been used as a model for the atrophy of human skeletal muscle that occurs

during spaceflight. This protocol has a profound effect on the weightbearing muscles of

the hindlimb, causing rapid atrophy of the soleus muscle. Previous work by me has

shown that one of the most rapid responses of the soleus muscle is a large decrease in

contractile protein synthesis within a few hours of the onset of non-weightbearing; in these

few hours there is no change in the availability of components with which the muscle can

synthesize protein.

The hypothesis of this work is that soleus muscle actin protein synthesis rate rapidly

decreases during hindlimb non-weightbearing as a result of alterations in nascent protein

elongation and/or initiation of protein synthesis. To test this hypothesis, I proposed

experiments to measure the relative concentration of ot-actin mRNA in the polysome pool,

and the electrophoretic size profile of the polysomes containing the c_-actin mRNA.

Accomplishments

(1) A novel analytical technique was developed to evaluate skeletal muscle

polysome profiles. This technique allows simultaneous evaluation of nascent polypeptide

elongation and protein synthesis initiation.

(2) Using this technique, the rapid downregulation of protein

synthesis that occurs at the onset of non-weightbearing is accompanied by a

shift of polysome size toward larger polysomes (more ribosomal subunits per

mRNA).

(3) Using cDNA probes for ¢x-actin mRNA, the mRNA for this

protein is associated with the larger polysomes. The overall amount of the

mRNA associated with the polysomes does not change significantly.

Significance of the Accomplishments

(1) Development of this analytical technique provides a sensitive means for

evaluating the earliest changes that occur during muscle atrophy. This will be essential for

a timely and cost-effective evaluation of countermeasures to the effects of weightlessness.

(2) The observation of larger polysomes associated with the rapid downregulation

of protein synthesis in non-weightbearing skeletal muscle was surprising. The significance

of this finding is that there is a direct coupling of mechanotransduction to the

control of gene expression in muscle at the level of translation.

215
(3) Theassociationof oc-actinmRNA with thelargerpolysomesindicatesthatthis

mechanismis active(andmayevenbeunique)for thecontractileproteins. The increased

sizeof thepolysomesis consistentwith aslowing of nascentpolypeptideelongationrate

throughsome,asyet,undefinedmechanism.Thelackof asignificantchangein contentof

o_-actinmRNA on thepolysomesindicatesinitiation of proteinsynthesisis not a site of

regulationin therapiddecreasein soleusmuscleproteinsynthesis.

Publication

Booth, F.W., Morrison, P.R., Thomason, D.B., Oganov, V.S. 1990. Experiment K-6-

11. Actin mRNA and cytochrome c mRNA concentrations in the triceps brachia muscle of

rats. In: Final Reports of the US Experiments Flown on the Sovi_et Biosat¢l!ite Cosmos

1887 (ed. by J.P5 Connoliy, R.E. Gi'indel-and, and=R.W._BaHard). Moffett Field, CA:

NASA Ames Research Center, p. 275-278. (NASA TM-102254)

Graham, S.C., Roy, R.R., West, S.P., Thomason, D., and Baldwin, K.M. 1989.

Exercise effects on the size and metabolic properties of soleus fibers in hindlimb-suspended

rats. Aviation, Space, and Environmental Medicine 60: 226-234.

Thomason, D.B., Biggs, R.B., and Booth, F.W. 1989. Protein metabolism and _-

myosin heavy chain mRNA in unweighted soleus muscle. American Journal of

Physiology 257: R300-R305.

Thomason, D.B. and Booth, F.W. 1990. Atrophy of the soleus muscle by hindlimb

unweighting. Journal of Applied Physiology 68: 1-12.

Thomason, D.B. and Booth, F.W. 1989. Gene-transfer into adult rat skeletal muscle in

vivo: Retroviral-mediated, stable expression of a bacterial gene (Abstract). Journal of Cell

Biology 109: 263a.

Thomason, D.B. and Booth, F.W. 1989. Influence of performance on gene expression in

skeletal muscle: Effects of forced inactivity. In: Advances in Myochemistry 2 (ed. by G.

Benzi). London: John Libbey Eurotext, Ltd., p. 79-82.

Thomason, D.B. and Booth, F.W. 1990. Rapid decrease in nascent protein elongation

during skeletal muscle unweighting (Abstract). FASEB Journal 4: A542.

Thomason, D.B. and Booth, F.W. 1989. Soleus muscle slow myosin heavy chain

mRNA expression during hindlimb unweighting: Implications for translational control

mechanisms (Abstract). FASEB Journal 3: A698.

Thomason, D.B. and Booth, F.W. 1990. Stable incorporation of a bacterial gene into

adult rat skeletal muscle in vivo. American Journal of Physiology 258:C578-C581.

216
NASA GRADUATE STUDENT RESEARCHERS PROGRAM

In 1980, NASA initiated the Graduate Student Researchers Program (GSRP) in order to

cultivate additional research ties to the academic community and to support promising

students pursuing advanced degrees in science and engineering. Since then, approximately

800 students have completed the program's requirements while making significant

contributions to the nation's aerospace efforts. Universities have also benefitted through

strengthening their research capabilities.

Each year, NASA selects approximately 80 new students for the opportunity to receive

stipends and to work at the unique national laboratories of the NASA facilities or at their

home universities. Awardees are selected based on competitive evaluation of their

academic qualifications, their proposed research plan, and their planned use of NASA

research facilities. Fellowships are awarded for one year and are renewable, based on

satisfactory progress, for up to three years.

The Graduate Student Researchers Program is managed at NASA Headquarters by the

University Programs Branch, Educational Affairs Division. Forty of the 80 new awards

each year are sponsored by the NASA Headquarters Office of Space Science and

Applications (OSSA) in the fields of life sciences, astrophysics, earth sciences, solar

system exploration, and microgravity science and applications. OSSA fellows carry out

their research or a plan of study at their home universities. Each year they attend a two- or

three-day annual symposium at NASA Headquarters in Washington, D.C. The

symposium provides an opportunity for GSRP fellows to exchange ideas, discuss

progress, and learn more about space science and applications at NASA.

The remaining 40 new awards are distributed throughout the NASA field centers. Fellows

selected by centers must spend some time in residence at the center, taking advantage of the

unique research facilities of the installation and working with center personnel.

Of the awards currently sponsored by the Headquarters Office of Space Science and

Applications, thirteen are in the space biology and biomedical research areas. These

awardees' abstracts, which were compiled from the May 1990 annual symposium in

Washington, D.C., are included in the following pages.

Further information about the OSSA Graduate Student Researchers Program may be

obtained from: Mr. Joseph K. Alexander, Assistant Associate Administrator for Space

Science and Applications, Code E, NASA Headquarters, Washington, D.C. 20546. Mr.

Gary Gans or Mr. John Lynch, University Programs, Educational Affairs Division, Code

XEU, NASA Headquarters, Washington, D.C. 20546, may be contacted for further

information about the 40 GSRP fellowships to conduct research at NASA facilities and

centers.

217
THE DEVELOPMENT OF A GENETIC SYSTEM FOR THE DISSECTION

OF THE GRAVITROPISM PATHWAY IN ARABIDOPSIS THALIANA

Bertha L. Bullen

(Advisor: Kenneth L. Poff)

MSU-DOE Plant Research Laboratory

Michigan State University

East Lansing, MI 48823

Gravitropism was described by Charles Darwin over 100 years ago; yet, the mechanisms

of the response in higher plants remain unknown. In an effort to elucidate some of these

mechanisms, a direct screening procedure for the isolation of gravitropism mutants in

Arabidopsis thaliana was developed. Sixty mutant lines in four phenotype categories were

isolated. Some of these mutant lines exhibited different gravitropism phenotypes in the

root and the hypocotyl, supporting the hypothesis that some elements of the gravitropism

pathway are organ-specific. Additional screening of the gravitropism mutants permits the

examination of specific models for gravitropism, and the examination of the interaction of

gravitropism with other sensory pathways. Screening with iodine-potassium iodide (IKI)

showed that three of the 60 lines were also altered in starch content, allowing examination

of the role of starch in gravitropism. Tests for phototropism revealed that some of the

mutant lines were altered in their phototropic response, supporting the hypothesis that

phototropism and gravitropism share some elements of signal transduction. Since the

direct screening procedure avoids the bias inherent in procedures based on particular

models for gravitropism, it is expected that these 60 mutants represent lesions at various

steps throughout the entire gravitropism pathway.

218
EXPRESSION OF CELLULAR STRESS

EMBRYO

Carol M. Gruppi

(Advisor: Debra Wolgemuth)

College of Physicians and Surgeons

Columbia University

New York, NY 10032

PROTEINS IN TESTIS AND

The long-term goal of this project is to assess the effects of spaceflight on mammalian germ

cell development and early embryogenesis at the molecular level. We are examining the

expression of cellular stress protein genes, also known as heat shock protein genes (hsp).

These genes serve as molecular markers of both normal mammalian cellular differentiation

and the cellular response to stress. Our study has focused on the characterization of the

hsp90 gene family expression in the murine testis and embryo. Two different hsp90

transcripts were detected in the mouse testis using two human hsp90 cDNA probes. The

testicular transcripts were approximately 3.2. kb and 2.9 kb in size and exhibited cellular

and developmental stage specificity of expression. The larger, more abundant transcript

was expressed at high levels in the germinal compartment of the testis, particularly in germ

cells in meiotic prophase. The smaller hsp90 transcript is expressed p.redominantly in the

somatic compartment of the testis. Expression of two hsp90 transcripts was seen in the

testis of other species, further supporting an important role for hsp90 in mammalian

testicular function. In addition, expression of both hsp90 transcripts was detected in the

embryonic and extra-embryonic compartment of midgestation embryos. These

observations indicate that the hsp90 gene family is under developmental regulation in the

mouse testis and midgestation embryo and that it will be useful as a sensitive molecular

indicator of cellular stress.

219
EFFECTS OF LOADED, ISOTONIC MUSCLE CONTRACTIONS ON

SKELETAL MUSCLE MASS AND BLOOD FLOW IN THE CONSCIOUS,

UNLOADED RAT

Kenneth A. Mook

(Advisor: Ronald D. Fell)

University of Louisville

Louisville, KY 40292

Cardiovascular and musculoskeletal systems are adversely affected when the body is

removed from gravitational forces, as during spaceflight, in order to prevent muscle

atrophy and cardi0vascular deconditioning, both of which would hinder long-term

spaceflight, adequate animal models need to be investigated and used to evaluate a variety

of countermeasures to these adverse effects of spaceflight. In Comparing unloaded

isotonic, isometric, and loaded isotonic muscle contractions, a series of studies has

determined high resistance, isotonic muscle exercise provides the greatest benefit in

preventing muscle atrophy induced by hindlimb unloading. The purpose of current

investigati0fiS is to study the effects Of mu-scie-Substrate delivery arid rhetaboiic waste

removal on atrophy and atrophy prevention on individual skeletal muscles. Initially, these

studies have evaluated muscle blood flow (MBF) in the conscious animal. Untrained

(UT), hindlimb unloaded control soleus (SOL) muscles significantly atrophied 33%, while

plantaris (PLT) and gastrocnemius (GAST) muscles atrophied 9% and 8%, respectively.

Loaded, isotonic training (T) resulted in full prevention of PLT and GAST muscle atrophy

but only reduced SOL muscle atrophy by 10%. Absolute blood flow (ml/min) in atrophied

muscles declines to a greater degree than would be predicted by the degree of muscle

atrophy. SOL blood flow declined 68% following disuse while PLT and GAST flows

decreased by 24% and 53%, respectively. Prevention of muscle mass appears to reduce

alterations in MBF to these individual muscles. PLT and GAST blood flow declines in T

hindlimbs were reduced by 88% and 67% from UT values, respectively. Relative muscle

blood flow (RBF) (ml/min/100g tissue) demonstrated that not only muscle fiber type but

muscle function determines the distribution of blood flow in the hindlimb. Muscles are

composed of three fiber types: (1) slow contracting, oxidative, and highly fatigue resistant

(SO); (2) fast twitch oxidative glycolytic (FOG) fibers which are fatigue resistant; and (3)

fast glycolytic (FG) fibers with low fatigue resistance. Rodent SOL muscles, composed

mostly of SO fibers, had significantly lowered RBF in both UT (-43%) and T (-44%)

animals. PLT muscles composed of FG (41%) and FOG (59%) fibers, had increased RBF

in UT (22%) and T (29%) hindlimbs. GAST muscles, composed of both FG (38%) and

FOG (58%) twitch fibers, had decreased RBF in UT (-31%) while increasing 8% in T

animal hindlimbs. These two muscles (PLT and GAST) are very similar in fiber

composition, but dramatically different in blood flow distribution in both T and UT

muscles. In summary, this study demonstrated the beneficial effects of highly resistant

isotonic muscle exercise on atrophy prevention, and provided insight into differences in

blood flow distribution between unloaded and loaded hindlimb muscles. This altered

distribution may be a mechanism by which substrate delivery or metabolic waste removal

may influence muscle mass during prolonged periods of disuse. The time course of

distribution changes is important and would combine a potential mechanism if blood flow

decreases prior to mass changes. These studies are presently underway.

220
EFFECT OF PHOTOPERIOD AND LIGHT INTENSITY ON BODY

TEMPERATURE AND ACTIVITY RHYTHMS IN RHESUS MONKEYS

Edward L. Robinson

(Advisor: Charles A. Fuller)

Department of Animal Physiology and

California Primate Research Center

University of California

Davis, CA 95616

Circadian rhythms of diverse physiological, behavioral and biochemical parameters are

synchronized with environmental cues. Temporal changes in the environment often result

in physiological and behavioral alterations, but these effects are not fully understood. This

study examined the effects of light on the circadian rhythms of body temperature and

physical activity. Four adult male Rhesus monkeys were studied at two light intensities,

200 and 1500 lux, in three different 24 hour light/dark cycles: LD12:12, LDI4:10,

LD16:08. Deep body temperature and activity were recorded via telemetry. At 1500 lux

the mean body temperature was higher, the rhythm amplitude was larger, and the phase

delayed compared with 200 lux. At both light intensities in longer photoperiods, mean

temperature was elevated, the temperature rhythm amplitude increased and the phase

delayed. Responses of physical activity were more variable than body temperature. Mean

activity and rhythm amplitude were lower, and the phase delayed with increasing light

intensity and photoperiod. We conclude that the circadian timekeeping system of the

Rhesus monkey is sensitive to changes in light intensity and photoperiod.

221
A METHOD FOR STUDYING TRACHEARY ELEMENT

DIFFERENTIATION AT SINGLE CELL RESOLUTION

James N. Weinstein

(Advisor: Lewis J. Feldman)

Department of Botany

University of California

Berkeley, CA 94720

The differentiation of tile unique plant cell type, the tracheary element, has long been

considered a model system for the study of cellular differentiation in plants. In order to

study cytoplasmic events occurring in the course of tracheary element differentiation,

culture techniques were developed which permit continuous and high resolution

observations of differentiating cells. Isolated mesophyll cells of Zinnia elegans, induced to

differentiate as tracheary elements, were attached to glass cover slips and subsequently

cultured in a Sykes-Moore chamber. Differentiating cells were observed with an inverted

microscope equipped for video enhanced and differential interference contrast microscopy.

Cells treated in this way completed differentiation within 96 hours.

222
EXERCISE INDUCED GLYCOGEN DEPLETION

HINDLIMB MUSCLES AFTER TWO WEEKS

UNLOADING

Craig Stump

(Advisor: Charles M.

University of Arizona

Tucson, AZ 85724

Tipton)

IN SELECT RAT

OF HINDLIMB

This study was designed to measure the degree of glycogen depletion of different rat

hindlimb muscles of various fiber type compositions with intense exercise after exposure to

hindlimb unloading simulated weightlessness (SW). Adult male Sprague-Dawley rats

(250-320g), familiarized with treadmill running were assigned to SW, hindlimb unloading

with exercise (SW-E), cage control (CC) or cage control with exercise (CC-E) groups.

The Overton-Tipton head-down unloading (45 °) model in which hindlimbs are non-weight

bearing was utilized. After 14 days of SW or control conditions, the SW-E and CC-E

groups were exposed to five 10-minute bouts of treadmill exercise at 85-90% of maximal

oxygen consumption with 2-3 minute rest periods between bouts. Results for muscle

glycogen concentration (mg/g) immediately after exercise or without exercise were as

follows (means + SE; n = 9-10; *indicates a value significantly different from the no

exercise condition, tindicates a value significantly different from the corresponding control

condition, p < 0.05):

CC CC-E _

Soleus (SOL)

Plantaris (PL)

White Gastrocnemius (GW)

Extensor Digitorum

Longus (EDLP)

3.73 + 0.42 1.81 + 0.42* 6.15 + 0.66t 2.75 + 0.35*

4.91 + 0.48 2.30 + 0.43* 7.67 + 1.01t 1.43 + 0.31"

5.07 + 0.28 3.32 + 0.50* 5.44 + 0.49 1.09 + 0.32"t

5.21 + 0.53 2.44 + 0.47* 6.58 + 0.69 1.75 + 0.30*

Hindlimb unloading resulted in the atrophy of the SOL (14%) and PL (12%) muscles, but

not the EDL. Glucose-6-phosphate (G-6-P) concentration was measured for the SOL and

GW; but, the only significant difference observed was an increase in SOL G-6-P in SW-E

rats 30 minutes after exercise when compared to SW. These results indicate that resting

glycogen concentrations were significantly elevated in hindlimb muscles that exhibited

atrophy during SW. Furthermore, exercise induced glycogen depletion per mg of muscle is

significantly greater after SW in the PL which consists predominantly of fast oxidativeglycolytic and fast glycolytic fibers, and the fast glycolytic GW and EDL muscles, but not in

the slow oxidative SOL.

223


Report Documentation Page

"_l,_Qv'_W _ 'w',t,,,N"*, ,trot

I. Rel>on No.

NASA _,1-4258

2. Government Accession No,

4. Title and Subtitle

1989-90 NASA Space Biology Accomplishments

7, Author(s)

Thora W. Halstead, Editor

9. Pedorming Organization Name and Addre_

Space Biology Program, Life Sciences Division, NASA

Office of Space Science and Applications and The

George Washington University, Science Communication

Studies, Washington, DC 20006

12. Spon_ring Agency Name and Addre_

National Aeronautics and

Washington, DC 20546

Space Administration

3. RecJpient's Catalog No.

5. Report Date

February 1991

6. Performing Organization Code

SBR

8. Performmg Organization Report No.

10. Work Unit No.

11. Contract or Grant No.

NASW-4324

13. Type of Report and Period Covered

Technical Memorandum

14. Sponsoring Agency Code

15. Supplementary Notes

For previous edition, see NASA TM-4160

16. Abstract

This report consists of individual technical summaries of research _rojects

of NASA's Space Biology Program, for research conducted during the period

May 1989 to April 1990. This program is concerned with using the unique

characteristics of the space environment, particularly microgravity, as a tool

to advance know_edge in the biological sciences; understanding how gravity has

shaped and affected life on Earth; and understanding how the space environment

affects both plant and animal species. The summaries for each project include

a description of the research, a list of the accomplishments, an explanation of

the significance of the accomplishments, and a list of publications.

17. Key Words (Suggested by Author(S))

gravity simulated weightlessness

weightlessness life sciences

space biology .hypogravity

animal microgravity

plant hypergravity

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Unclassified Unclassified

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18. Distribution Statement

Unclassified - Unlimited

Subject Category 51

21. No. of pages I _" Price

236 1 All

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