:hnical Memorandum-4"7 i I

/

1 NASA Space Biology

_lishments

NASA Technical Memorandum

1992-1993 NASA Space Biology

Accomplishments

Edited by

Thora W. Halstead

NASA Office of Life and Microgravity Sciences and Applications

Washington, D.C.

National Aeronautics

and Space Administration

Scientific and Technical

Information Office

1994

CONTENTS

Preface

Introduction -- The NASA Space Biology Program

Accomplishment Highlights

Plant Projects

M. A. BISSON and M. J. HOLLINGSWORTH

Gravity.Induced Alteration in Differentiation in Ckara

JANET BRAAM

Calmodulin-Related Proteins in Mechanostimulated Plants

Page

1

5

9

31

33

CHRISTOPHER S. BROWN

Kennedy Space Center Plant Space Biology Laboratory Flight

Experiment Support

ROBERT E. CLELAND

Plasmodesmata and the Control of Gravitropism

BOB V. CONGER

Embryogenesis of Gramineae in Microgravity

DANIEL J. COSGROVE

Mechanism of Differential Growth During Stem Gravitropism

36

4O

43

46

KATHRYN L. EDWARDS 50

Search for Structural Proteins Having Potential Function in Gravity

Sensing and for Differential Expression of Genes in Gravitropism

MICHAEL L. EVANS

Root Gravitropism in Maize and Arabidopsis

52

LEWIS FELDMAN

Physiological, Biochemical, and Molecular Processes Associated

with the Transduction of Gravity in Roots of Maize

55

SARI GALANES and PHILIP M. LINTILHAC

A New Method for Following the Gravitropic Response in Maize

Roots

57

MARCIA A. HARRISON

Interactions of Light and Ethylene in Stem Gravitropism

MARCIA A. HARRISON

Regulation of Ethylene Biosynthesis in Plants Grown in Closed

Canisters

6O

61

ifi
KARL H. HASENSTEIN 63

Probing the Gravisensing and Graviresponse Systems in Roots

PETER B. KAUFMAN, LIU.LAI WU, and DONGHERN KIM 66

Gravitropic Response Mechanism in Cereal Grass Shoots

JOHN Z. KISS 73

Gravitropism and Golgi Apparatus Function in Chara Rhizoids

TERRI L. LOMAX 76

Analysis of the lazy.2 Gravitropic Mutant of Tomato

PATRICIAN. MYERS and CARY A. MITCHELL 78

Mechanical Stress Regulation of Plant Growth and Development

BARBARA G. PICKARD 81

Transduction of the Gravitropic Stimulus

B. W. POOVAIAH 85

Calcium Messenger System in Gravitropic Response in Plants

DAVID L. RAYLE 88

Mechanism and Control of Gravitropism in Plants

STANLEY J. ROUX 90

Assessing Potential Targets of Calcium Action in Gravitropism

FRED D. SACK 93

Cellular Polarity and Interactions in Plant Graviperception

FRANK B. SALISBURY 95

Developmental Studies of Wheat in Microgravity

RANDY WAYNE 98

Gravity-Induced Polarity in Single Cells of Chara

Animal Projects

RICHARD A. BAIRD 103

Comparative Transduction Mechanisms of Vestibular Otolith Hair

Cells

DANIEL D. BIKLE and BERNARD P. HALLORAN 107

Effect of Skeletal Unloading on Bone Formation

JOHN C. CLOHISY 110

Parathyroid Hormone Stimulates c-los and c.jun mRNA Abundances

in Rat Osteoblastic Cells

STEPHEN L. COCHRAN 112

Basic Gravitational Reflexes in the Larval Frog

MICHAEL V. DANILCHIK 114

A Gravity-Sensitive Period During Frog Oocyte Maturation

iv
ROBERT S. DECKER

Mechanical Modulation of Striated Muscle Phenotype

116

J. DAVID DICKMAN

Otolith-Canai Convergence in Vestibular Nuclei Neurons

119

RANDALL L. DUNCAN

Modulation of Bone Remodelling via Mechano-Sensitive Ion

Channels

122

CI_SAR D. FERMIN

Neuronal Proteins Before, During, and After Vestibular

Synaptogenesis

124

JOHN A. FRANGOS

Microgravity In Vitro Model of Bone Cells: Flow Effects

129

WILLIAM F. GANONG 130

Neural Mechanisms by Which Gravitational Stimuli and Stress Affect

the Secretion of Renin and Other Hormones

ALFRED L. GOLDBERG 133

Muscle Protein Loss in Weightlessness Environments: Role of the

ATP-Ubiquitin-Dependent Proteolytic System

ROSALIND A. GRYMES

Gravity Influence on Signal Transfer and Differentiation

135

ERIK J. HENRIKSEN 141

Skeletal Muscle Glucose Transport During Simulated Weightlessness

EMILY MOREY.HOLTON

Structural Development and Gravity

144

JOHN M. HOROWITZ

Gravitational Fields and Neural Signaling in the Hippocampus

147

MILLIE HUGHES-FULFORD

Mechanism of Control of Bone Growth by Prostaglandins

149

DONALD E. INGBER

Mechanochemicai Transduction Across Extracellular Matrix

153

TIMOTHY A. JONES

The Effects of Gravitational Loading and Vibration on Vestibular

Ontogeny

156

CHRISTINE E. KASPER

Molecular Basis of Tension Development in Muscular Atrophy

160

JOHN O. KESSLER 164

Orientation of Cells by Gravity, Illumination, and Shear: Quantitative

Relationships
WILLIAM J. LANDIS and LOUIS C. GERSTENFELD

Collagen Synthesis, Assembly, and Mineralization in Chicken

Osteoblast Cell Cultures and other Calcifying Vertebrate Systems

169

CAREY R. PHILLIPS 173

Gravitationally Sensitive Molecular Rearrangements Involved in

Establishing Early Embryonic Axes in Amphibians

ARTHUR N. POPPER

Evolution of Gravity Receptors in the Ear

177

RAVEN REITSTETTER 179

Effects of Vector-Averaged Gravity on Single Channel Properties of

the Acetylcholine Receptor in Cultured Myocytes

MURIEL D. ROSS

Structure and Function of Mammalian Gravity Receptors

180

DAVID ROWE

Osteoblastic Response in Transgenic Mice

184

CLARENCE F. SAMS

Mechanisms of Altered Lymphocyte Activation in Simulated

Microgravity and Hypergravity

187

MARTIN P. SCHREIBMAN 190

Development, Maturation and Physiology of the Brain-PituitaryGonadal Axis of Fish m the CEBAS/Aquarack System

EDWARD SCHULTZ

Effects of Hindlimb Suspension on Skeletal Muscle Growth

193

HERMAN H. VANDENBURGH 197

Growth Factor Involvement in Tension-Induced Muscle Growth

PEDRO VERDUGO

Mechano-Transduction in Mammalian Ciliated Cells

201

FRED H. WILT 205

The Role of Calcite Skeletal Matrix Proteins in Biomineralization

DEBRA J. WOLGEMUTH

Effects of Altered Environments on Cellular Stress Genes in

Reproduction and Development

208

Space Flight Experiments

ALLAN H. BROWN

Plant Gravitational Physiology

213

MARK F. DESROSIERS and ROBERT S. BANDURSKI 215

An Attempt to Localize the Gravity Sensing Mechanism of Plants:

The Electrical Impedance Between Vascular and Cortical Tissues

vi
STEPHEN B. DOTY 219

Studies of Intercellular Communication and lntraceilular Responses

by Bone Cells to Simulated Weightlessness and Space Flight

PAULINE JACKIE DUKE 222

Altered Chondrocyte Differentiation in Response to Altered g.Level

RAPHAEL GRUENER 225

Synapse Development in Altered Gravity: A Model for the Study of

the Effects of Space Microgravity on Embryonic Development

W. C. HYMER 230

Microgravity-lnduced Effects on Pituitary Growth Hormone Cell

Function: A Mechanism for Muscle Atrophy in Manned Space Flight

W. C. HYMER 232

Electrophoretic Separation of Cells and Particles from Rat Pituitary

and Rat Spleen

A. D. KRIKORIAN

Cells, Embryos and Development in Space

234

MARIAN L. LEWIS

Mechanism of Control of Bone Growth in Microgravity

239

NORMAN G. LEWIS 241

Plant Metabolism and Cell Wall Formation in Space (Microgravity)

and on Earth

MARY E. MUSGRAVE

Developmental and Physiological Processes Influencing Seed

Production in Microgravity

245

NICOLA C. PARTRIDGE

Skeletal Collagen Turnover by the Osteoblast

248

EUGENE ROBERTS and LAWRENCE P. GARETTO 253

Cell Kinetic and Histomorphometric Analysis of Microgravitational

Osteopenia

HEIDE SCHATTEN

Microgravity Effects During Fertilization, Cell Division,

Development and Calcium Metabolism in Sea Urchins

257

GERALD SONNENFELD and EDWIN S. MILLER

STL.01 and STL.02 Experiments

Immunology

259

MARC E. TISCHLER

Skeletal Muscle Metabolism in Hypokinetic Rats

262

ARTHUR C. VAILAS

Soft Dense Fribrous Connective Tissue Response to Hindlimb

Unloading

265

vii
MICHAEL L. WIEDERHOLD 267

Early Development of a Gravity-Receptor Organ in Microgravtty

Special Activity

SPACE BIOLOGY RESEARCH ASSOCIATES PROGRAM

Gerald Sonnenfeld

273

CHRISTOPHER R. KIRBY 283

Eccentric Exercise Training as a Countermeasure to Non.Weight

Bearing Muscle Atrophy

WILLIAM K. KROEN

Competition Between Fruit Development and Root Growth in

Tomatoes Grown at High CO2

285

VINCENT LING

Characterization of a Calmodulin-Binding Protein from Plant

Roots

288

ANNA LYSAKOWSKI

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

Mammalian Crista Ampullaris

291

ED MILLER 296

Influence of Antiorthoetatlc Suspension on Resistance to Infection

MARGARET S. RICE 300

Developmental Study of Responses to Auxin in the Diagtotropica

Tomato Mutant

JOYCE L. THOMPSON

Sarcomere Lesions in Atrophied Reloaded Adductor Longus

,304

LADORA V. THOMPSON

The Role of H÷ in the Fatigability of the Soleus Following

Hindlimb Suspension

.309

LARRY E. WlMMERS

Molecular Characterization of a Pkmt Ca 2+ ATPase

312

.°°

V'LU
PREFACE

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

and NASA with a 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.

Individual technical summaries of the research conducted under the tasks

within the NASA Space Biology Program for the calendar years of 1992 and

1993 are presented in this publication. Each summary, prepared by the

principal investigator, consists of a description of the research and the

project's accomplishments, as well an explanation of the significance of the

accomplishments and a list of the publications over the last 2 years resulting

from the research. Since Small Payload Shuttle middeck space flight

experiments have become an integral part of Space Biology research, reports

on these experiments are incorporated in the document. 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 at host university laboratories,

are also included. The participants in this program have been outstanding

and merit independent recognition.

Thanks are due to the Program participants and postdoctoral scientists whose

research and cooperative response to our requests for information made this

report possible. Julianna Klejnot played a principal role in organizing and

editing the publication with the support of Mary Lou Bumell, Bruce M.

Hather, Carla J. Howard, Richard M. Poley, and Joshua Singer. Their

significant and essential contributions which made this publication possible

are gratefully acknowledged.

Thora W. Halstead, Ph.D.

INTRODUCTION

THE NASA SPACE BIOLOGY PROGRAM

Thora W. Halstead, Ph.D.

Life and Biomedical Sciences and Applications Division

Office of Life and Microgravity Sciences and Applications

National Aeronautics and Space Administration

Washington, DC 20546

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

presence of the force of gravity. We can study the importance of gravity to life on Earth by

using the unique biological research tool provided by the phenomenon of nearweightlessness encountered on spacecraft during their free-fall flight. Access to space

provides an opportunity to manipulate the force of gravity from its normal value of one

down to almost zero for prolonged periods of time. This capability has laid the foundation

for the Space Biology Program to support research using hypergravity, Earth-normal

gravity, and microgravity as environmental tools to advance fundamental knowledge about

biological systems; the role of gravity and the effects of microgravity on biological

processes; and the interaction of gravity and other environmental factors on biological

systems.

The Program emphasizes research in cell, developmental, integrative and plant biology,

structural biosystems, and gravity sensing that seeks an understanding of basic

mechanisms affected by gravity. Studies include higher and lower plants and animals as

subjects, as well as cell and tissue cultures. The scope of the research and scientific

objectives in each of these areas is described below. While the research supported by the

Program is totally ground-based, much of it must ultimately lead to flight experiments that

can confirm or refute the fidelity of ground-based models and hypotheses. The Space

Shuttle middeck Small Payload Program has been the main source of opportunities for such

flight experiments.

_. Gravity has profound influences on the development, growth, and

physiological processes of plants; yet, the basic mechanisms underlying gravity's

effects are still not well understood. The goals of plant gravitational biology are to

achieve both a fundamental scientific understanding of the effects of gravity on plants,

and to provide basic knowledge of plant development and physiology that can

contribute to the utilization of plants in space and on Earth. Research is focused in

three primary areas: understanding the basic mechanisms whereby plants perceive,

transduce, and respond to a gravitational force; elucidating the role of gravity and

microgravity on developmental and reproductilte processes in plants; and determining

the role of gravity and microgravity in metabolism, photosynthesis, and transport

processes in plants. Integral to each of these areas is how gravitational force and other

environmental stimuli are integrated biologically.

_. Cells, whether as single unicellular organisms or as the basic structural and

functional unit of multiceUular biological systems, have been shown to be sensitive to

gravity. The scientific goal of gravitational cell biology is to understand how the weak

force of gravity can act as a biological regulator that functions at the cellular level, and

thereby understand how a physical force is transduced to produce a biological effect.

To attain this goal, research is focused on identifying how cells "sense" gravity both

directly and indirectly, how this information is transduced into a biological response,

and how ceils respond to both acute and long-term variations in gravity.
Developmental Biology. This research area examines the influence of gravity and

microgravity on reproduction, genetic integrity, differentiation, growth, development,

life span, senescence, and subsequent generations of animals. The goals are to

determine whether specific animal species have evolved developmental mechanisms that

depend on a gravity force or vector for normal function and, if so, to identify the

mechanisms; to determine if altered gravity induces phenotypic or genotypic changes;

and to determine if gravity affects the capacity of animal species to reproduce and

develop normally over serial generations.

Gravity Sensinz. Animals have developed gravity-sensing systems that facilitate

orientation and locomotion within Earth's environment. The goals of this discipline are

to understand how the brain processes information by understanding the organization

and functioning of gravity-sensing organs; to test mechanisms of adaptation by

exposure to variable gravity; to elucidate the stages by which the gravity-sensing

system evolved, from invertebrates through vertebrates; and to begin to understand

where in the developmental process gravity influences genetic transcription/translation

to direct production of a specific functional architecture in gravity-sensing organs.

Structural Biosvstems. In response to the force of gravity under which all biological

species on Earth evolved, organisms have developed structures to withstand gravity

loads. The goal of this discipline is to understand the role of gravity in modulating

biological processes that regulate musculoskeletal systems. Research focuses on

identifying how gravity level and direction influence the type, pattern, and amount of

biomineralization and muscle in structural biosystems; understanding interactions

between gravity and internal (e.g., metabolic regulatory substances) and external (e.g.,

environmental) factors in these systems; and specifying the time course of

musculoskeletal adaptation to altered gravity and readaptation to normal Earth gravity.

Integrative Biolo2v. Living systems have evolved in a gravitational field and are regulated

t-o function wi-thin tightly controlled limits. Asymmetries in the environment (including

presumably gravity) result in disturbances in these regulatory mechanisms to which

living systems adapt and evolve. The goal of integrative biology is to understand how

gravity affects mechanisms regulating homeostasis, adaptation, and the ability of living

systems to respond to internal and external signals. Research focuses on understanding

the role of gravity on animal regulatory mechanisms: the generation and/or entrainment

of circadian rhythms, the internal synchronization of several circadian functions, and

one or more selected homeostatically controlled systems; e.g., the regulation of body

temperature and the associated neuroendocrine regulation of energy and water.

The use of the unique microgravity environment of spaceflight is an integral partof these

research efforts. Flight experiments in gravitational biology permit identincation,

confirmation, and understanding of effects due to microgravity. At the same time, groundbased research activities lay the groundwork for validation by flight experiments, identify

gravity-sensitive biological systems, and utilize modern research techniques and

instrumentation to target analyses at the most fundamental levels.

6
ACCOMPLISHMENT HIGHLIGHTS

SPACE BIOLOGY

ACCOMPLISHMENTS HIGHLIGHTS

PLANT

Gravitropism: Sensing

Nature of the Receptor

The Potential Gating Theory, a working hypothesis, attempts to link the internal

bioelectric fields of the plant to the transport of the growth hormone, indole-3-acetic

acid (IAA). Briefly, the theory states that the transport of IAA from the vascular stele of

the plant to the cortical cells is through plasmodesmatal connections and is regulated by

the bioelectric potential difference between the stele and cortex. (Desrosiers/Bandurski)

In order to study the role of amyloplasts in plant gravisensing, amyloplast manipulation

by forces other than gravity is desirable and achieved using a high gradient magnetic

field. The magnetic field generates a ponderomotive force that is superimposed to the

force of gravity and allows the application of various forces to all or a subset of

amyloplast in, for example, the root cap. The extent of amyloplast displacement

correlates with the subsequent growth direction (curvature) of roots. (Hasenstein)

The statolith compartment in Chara rhizoids is more complex than prior reports

indicate. In addition to containing the previously reported barium sulfate crystals, this

compartment has an organic matrix consisting of protein and carbohydrate moieties.

(Kiss)

• The response to gravity is directly correlated with the number of statoliths in Chara

rhizoids. (Kiss)

Gravitropic roots of the aquatic angiosperm, Limnobium, have sedimented amyloplasts

in their elongation zone but not in their rootcap. If amyloplast sedimentation is

responsible for gravitropic sensing, then the site of sensing in Limnobium roots is the

elongation zone and not the root cap. Comparison of several different moss genera

establishes that amyloplast sedimentation is present whenever the protonema is

gravitropic. (Sack)

Hydrostatic pressure mimics gravitational pressure. Finding that a unidirectionalapplied hydrostatic pressure mimics gravitational pressure in inducing the physiological

response. (Wayne)

Gravireceptor may be an integrin-like protein. Demonstrates that the receptors are

localized at the plasma membrane-extraceUulax matrix junction at the ends of the cell and

that the mechanoreceptor for gravitational and hydrostatic pressure is an integrin-like

protein. (Wayne)

Gravireceptor is distinct from touch-receptor in the same cell. The touch receptor is

distinct from the receptor involved in sensing gravitational and hydrostatic pressure.

(Wayne)

9
Role of Membranes

Cell-to-cell communication through plasmodesmata (PDM) is under metabolic control.

When adenosine triphosphate (ATP) levels are reduced in wheat roots, the maximum

size molecule that passes through PDM increases from about 800 to over 5,000 MW.

Under normal circumstances ATP is able to pass from cell to cell through the PDM.

(Cleland)

Cell-to-cell communication through PDM is also modulated by gravity. In oat

coleoptiles, the movement of carboxyfluorescein from subepidermal cells to cortical

cells is greatly increased when a vertical coleoptile is placed in a horizontal position.

(Cleland)

Cytoskeletal protein extracts from Phycomyces sporangiophores contain protein bands

reacting with monoclonal antibodies to g-actin, myosin, and integrin in western blots.

These proteins are being considered in a cytoskeletal framework and the anchoring of

that framework to the membrane and cell wall which could, in conjunction with the

vacuole, serve in the detection of gravity stress in this organism. (Edwards)

It has been proposed that mechanical tension is passed from cell wall to the plasma

membrane and its mechanosensory calcium channels by way of adhesion sites that are

functionally similar to those of animal ceils. To explore this concept, cell membranes

were exposed by digesting away wall polymers so that antibodies to the animal

adhesion molecules integrin, vitronectin, and fibronectin could be applied. These

antibodies were then stained with fluorescent secondary antibodies and covisualized

using total computation, optical sectioning microscopy. The antigens occur in punctate

loci, with extensive overlap. This and other evidence supports the idea that adhesion

sites in plants have many similarities to those in animals. (Pickard)

A change in membrane potential is not involved in the signal transduction chain

involved in sensing gravitational or hydrostatic pressure. (Wayne)

Role of Phytochrome

Towards understanding the role of phytochrome in regulating root gravitropism, there

is evidence for the hypothesis that a phytochrome-associated protein kinase modulates

light-regulated root gravitropism in vivo and that the protein kinase may function to

regulate receptor activity. (Feldman)

Gravitropism: Transduction

It was determined that the cells that mediate curvature induced by application of calcium

gradients near the root tip are cells of the postmitotic isodiametric growth (PIG) zone.

The PIG zone is a group of cells between the meristem and the main elongation zone.

This is the same group of cells that plays the primary role in the response of roots to

gravity and to touch. (Evans)

The calmodulin gene from Phycomyces has common homologies to both higher plant

calmodulins and calmodulins from other fungi. A 339 base pair (bp) fragment of the

gene has been obtained by polymerase chain reation (PCR) and will be used in an effort

to sequence the entire gene (Braam and Davis, 1990). (Edwards)

10
To measuresignal-inducedchangesin the level of cytosolic Ca2+concentration,

transgenicplants carrying the aequoringenefrom jellyfish were produced. Using

confocalimageanalysis,signal-inducedchangesin free Ca2÷concentrationin these

transgenicplantsweredetected.(Poovaiah)

Two distinct classesof Ca2÷channelsareinvolved in gravisensing,a third is not.

Ca2÷channelslocatedattheendsof thecellsaresensitiveto organicchannelblockers

and areinvolved in gravisensing(ClassesI andII). A third classof Ca2÷is found

throughoutthe plasmamembrane,is insensitiveto organicchannelblockers,and is

involvedin sensingelectricalstimulationandtouch(Classm). (Wayne)

Use of lanthanidesto probe the structuresof the threeclassesof Ca2÷ channels

indicatesthatClassI is anelasticperistalticchannelwith threebindingsiteswithin the

poresfor Ca2÷.Likewise,ClassHI is anelasticperistalticchannelwith threebinding

sitesfor Ca2÷;however,the binding sitesaredefinitely multidentate. By contrast,

ClassII channelshaveanexternalvestibulethatcanonly beblockedby largecations.

(Wayne)

Role of Calmodulin

To study the consequences of altered levels of calmodulin on plant growth and

development and signal transduction, transgenic potato plants were generated carrying

the calmodulin cDNA (PCM-I) in sense and antisense orientations driven by

constitutive and inducible promoters. These transgenic plants exhibit striking

differences in growth and development. (Poovaiah)

Role of Calcium/Calmodulin Regulated Kinases

Towards understanding the hypothesized role of calcium/calmodulin-regulated kinases

in root gravitropism, a cDNA encoding a homologue of mammalian CaMK II was

isolated from root caps of maize. This suggests a physiological function for a CaMK in

light-regulated root gravitropism. (Feldman)

A Ca2+/calmodulin-dependent protein kinase from corn root tip has been purified. This

protein (56 kDa) autophosphorylates in the presence of Ca2+a and calmodulin. In

addition, a Ca2+/calmodulin-dependent protein kinase has been cloned from lily.

(Poovaiah)

Role of Electrical Sign_tin_

The response of maize roots to applied electrical fields initially shows curvature toward

a negatave electrode followed by curvature toward a positive electrode. The electrical

response of maize roots to thigmostimulation was tested, and dramatic touch-induced

depolarizations of surface potential were found. (Evans)

R91¢ of H0rmon¢_

Roots inhibited by auxin show rapid, strong gravitropic curvature. This curvature is

mediated exclusively by gravistimulated enhancement of elongation on the upper side of

the PIG zone and tells us that gravi-induced rapid elongation on the upper side of the

PIG zone is not mediated by a gravi-induced shift in auxin distribution. (Evans)

11
In red-pretreatedpeaepicotyls,adecreasein ethyleneprecursor,1-aminocyclopropane1-carboxylicacid (ACC), paralleledincreasedACC-malonyl (MACC) conjugation

within 30-minutegravistimulation. Thisredlight regulationof ethylenebiosynthesis

may be responsiblefor altering cell growth in the stem after gravistimulation.

(Harrison)

Theoxidationof ethyleneprecursor,ACC,is alteredinpeaseedlingsgrowninone-liter

closedcanisterswithin a 5-dayincubationperiod. This resultwasaccompaniedby

ethylene accumulation within the canisters and indicates a reduction in oxygen during

the incubation period. (Harrison)

Phytochrome is the pigment involved in regulating the light-induced reversed

gravitropic response of stems of the lazy-2 gravitropic mutant of tomato. The reversed

gravitropic response of lazy-2 shoots results from stimulation of growth on the upper

side of gravistimulated stems. (Lomax)

Wild type and diageotropica tomato seedlings do not respond to gravity when

submerged in high concentrations of indole-3-acetic acid (IAA) or IAA transport

inhibitors N-l-naphthylphthalamic acid (NPA) and 2, 3, 5 - triiodobenzoic acid

(TIBA). This finding suggests that IAA transport plays an important role in the

gravitropic response of tomato seedlings. (Rice)

Role of Proteins

Towards understanding the hypothesized role of calcium-dependent protein kinases

(CDPKs) in root gravitropism, it has been shown that root caps contain four CDPK

peptides; two of which co-migrated with those found in other tissues, and two of which

appear to be unique to the root cap. (Feldman)

An investigation of developmentally regulated calmodulin-binding proteins in Viciafaba

(lava bean) seedlings led to the identification of glutamate decarboxylase as a calciumdependent calmodulin-stimulated enzyme in roots. The product of glutamate

decarboxylase activity, gamma-aminobutyrate (GABA), is known as a stress-induced

metabolite in plants and as an inhibitory neurotransmitter in animals and insects. The

biochemical linking of calcium-mediated signal transduction to stress physiology offers

new directions of research in plant gravitropism. (Ling)

Phytochrome, the same pi.gment that mediates the effects of light on gravitropism,

induces increased guanosme triphosphate (GTP)-binding protein activity in the

envelope of pea nuclei. There results implicate nuclear GTP-binding proteins in the

signaling pathway by which phytochrome regulates changes in plant growth and

development. (Roux)

Confocal laser microscopy of immunostained rhizoid cells of Dryopteris has revealed

that Ca2+-binding annexin proteins are concentrated at the extreme tips of these cells

during their initiation and elongation. Ca2+ is also concentrated in this region. These

results suggest that annexins may play a major role in Ca2+-mediated exocytosis events

critical for the polar growth of rhizoids, and may participate in the asymmetric growth

response of gravitropism. (Roux)

12
Use of Mutants

In general, there is a close correlation between auxin sensitivity and gravitropic

sensitivity; i.e., the greater the reduction in sensitivity to growth inhibition by auxin,

the greater the reduction in graviresponsiveness. (Evans)

Roots of the tomato (Lycopersicon escalentum, Mill.) mutant diageotropica (dgt)

exhibit an altered phenotype. We speculate that the insensitivity of dgt root growth to

auxin transport inhibitors and ethylene is an indirect result of the loss of sensitivity to

auxin in this single gene-recessive mutant. (Rayle)

A tomato mutant, diageotropica, displays a developmentally regulated response to

gravity. Young (5-20 day old) seedlings which are turned horizontally will curve

upward to vertical, although they are slower than wild type. After development of the

first intemode, the maximum attainable gravicurvature declines substantially. (Rice)

Gravitropism: Growth Response

Specific wall proteins, expansins, that mediate the acid-extension responses of isolated

walls have been identified. These novel proteins are important for the extension growth

of most plant cells and may be ubiquitous components of plant cell walls. (Cosgrove)

A new method has been developed for studying the old problem of how roots bend

down in the Earth's gravitational field. Method allows continuously following of the

amount of force the root generates as it tries to bend down. This will allow building a

biomechanical model of root performance in terms of work done and power output.

(Galanes/Lintilhac)

Auxin binding protein (ABP) in graviresponding oat (Avena) shoots has been isolated

and has had its action related to the signal transduction process and to increases in auxin

sensitivity in lower halves of leaf-sheath pulvini that are responding to gravistimulation.

Key components of the signal transduction process are: (a) Ca2+ requirement, (b) a

plasma membrane ATPase-driven proton pump, and (c) an increase in the number of

high affinity binding sites on the ABP for auxin. (Kaufman)

• Differential gene expression occurs early during the gravitropic response in cereal grass

pulvini. (Kaufman)

Antibodies against complex polysaccharides of higher plants cross-react with Chara

rhizoids in a specific manner. These antibodies will allow investigation of the vesicle

blockage hypothesis (for the response mechanism) since apical vesicles can be divided

into several categories depending on size, staining patterns, and antibody labeling.

(Kiss)

Stem segments and intact seedlings of the diageotropica tomato mutant do not elongate

in response to exogenous IAA at the time of development. However, diageotropica

seedlings can respond to gravity, which suggests that IAA-induced elongation and the

asymmetric growth produced during gravistimulation axe different. (Rice)

• A second gene encoding a putative Ca2+-ATPase has recently been identified and

cloned. (Wimmers)

13
Cvtoskeleton

Cortical cells and columella cells of graviresponding roots show significant

reorientation of cortical microtubules. Reorientation is most explicit in the outer cortex

along the lower side of the elongation zone. The upper, convex side of roots is not

affected. The specificity of the reorientation indicates that the outer cortex, not the stele

or epidermis, is the tissue most influenced by the growth inhibition that leads to

gravicurvature. (Hasenstein)

Gravity and the Plant Cell

Nuclei, as well as amyloplasts, sediment consistently and completely in the cells of the

elongation zone in roots of Limnobium. The location of cells containing nuclear

sedimentation is under tight developmental control. (Sack)

Apical cells of protonemata of the moss Ceratodon are unusual among plant cells with

sedimentation in that only some amyloplasts sediment and these do not fall completely

to the bottom of vertical cells. Application of cytoskeletal inhibitors to protonemal cells

of the moss Ceratodon suggests that microtubules restrict the sedimentation of plastids

along the length of the cell and that microtubules are load-bearing for all plastids in the

apical cell. (Sack)

Plant Metabolism

Changes in soybean cotyledon starch concentration due to altered gravity treatments

(lower in clinorotated and higher in centrifuged plants) appears to be directly related to

ADP glucose pyrophosphorylase activity. This enzyme is known to be rate limiting for

starch synthesis in many plants under other conditions. This is the first time ADP

Glucose Pyrophosphorylase has been suggested to play a role in gravity-mediated

changes in starch concentration. (Brown, C.)

By using cell cultures of Pinus taeda (Eberhardt, et al., Journal of Biological

Chemistry, 1993), it has been possible to attain a cell line capable of undergoing a

developmental-like transition from an unlignified primary wall to a lignified secondary

wall (~SI deposition). (Lewis, N.)

Plant Cell and Developmental Biology

Alterations of the orientation of isolated segments of Chara plants affect the

differentiation pattern of rhizoids which develop from these segments. While rhizoids

normally have oblique division planes which occur at some distance from the tip,

occasionally they develop a pattern of periclinal and anticlinal divisions at the tip which

are more similar to the pattern seen in shoot tissue. (Bisson/Hollingsworth)

Orchard grass (Dactylis glomerata L.) leaf segments cultured in vitro can be

preincubated at 4"C for at least 7 days without affecting somatic embryogenesis and

plant regeneration. (Conger)

A system was developed to maintain orchard grass leaf segments in contact with agar

medium in Petri dishes. Leaf segments are overlaid with 1800 V.m Teflon TM mesh.

The mesh is secured with a polypropylene ring which is slightly smaller in diameter that

the Petri dish and which fits between the lid and the mesh. (Conger)

14
Strategieshavebeendevisedfor handlingembryogenicplant cellsandtissuesprior to

theinitiationof anexperimentin spacefrom theperspectiveof havingnoor absolutely

minimumdevelopmentalprogressiononEarth. Low temperaturestrategyhavealso

beentestedto enableinvestigationswith totipotentplant cells to be setup andkept

quiescentandthusenableaninvestigatorto initiate anexperimentwith culturesby

changingor modifyinga singleparameter.Thisprovidesa simpleanddirectmeansof

initiatinganexperimentonceit isin space.(Krikorian)

The CHROMEX-4 payload had severalscientific componentsand representeda

coordinatedeffortto optimizesciencereturn. Onepartdealtwithreproductionandseed

formation;theseconddealtwith cell division, chromosomes,andgeneticstability;the

third part dealt with cell wall formation and geneexpression;the fourth with nonstructuralcarbohydratesandbiochemicalpartitioningof carbon. Only thosecellsthat

were absolutely clearly scorableas aberrationswere recorded. The number of

aberrationsfound werefarfewerthanhasbeenfound in rootsof otherspeciesgrown

in earlierspaceexperiments.Karyotypeanalysisof chromosomeswhich weremade

from root cells in metaphasein both groundcontrols and in spaceflight was also

carriedout. Nodistinctivechangesin karyotypeweredetectable.(Krikorian)

A unifyinghypothesisbasedonstressin space-grownplantcellsandsystemshasbeen

developed taking into account data gained from cytological and karyological

examinationsof spacegrownplant materials.Statedin its barestessentials,the more

developmentally advanced a system is, the less likely it is to suffer catastrophic stress

effects in the space environment, the less advanced or morphologically complex it is,

the greater the vulnerability. (Krikorian)

Data gained from nuclear examinations suggest that cells of species with large nuclei,

large chromosomes and DNA content, with variously located centromeres, show signs

of considerable perturbation in space. Although interpretation has been drawn from

only a few species, cells of polyploids with large chromosomes that are essentially

metacentric show very few disturbances. The younger the somatic embryo stage in

terms of its developmental progression, the more sensitive it is; the more advanced, the

less sensitive. The more polyploid the system, the more resistant to perturbation it

seems to be but the higher the DNA level in the nucleus the more sensitive it seems to

be. Ceils of species with small chromosomes and low DNA content show far fewer

mitotic anomalies. (Krikorian)

The smaller the responding embryogenic initial, the greater the vulnerability to

perturbation and hence more "damage" is shown in space (daylily). Embryogenic cells

dispersed in a semi-solid medium show less perturbation than those in liquid or on

semi-solid media (carrot). (Krikorian)

A growing procedure has been developed that permits the utilization of pre-flowering

plants in the Plant Growth Unit (PGU). The flexibility of the PGU has been extended

for use in all stages of the plant life cycle. (Musgrave)

Gr_vitaxis of Micro-Organisms

Methods

A direct computer-based numerical method for analyzing videotape images of

swimming cells, acquired through a microscope, was adapted (mechanically and

through the generation of dedicated software) for the task of analyzing the statistical

information required for a complete description of the distribution of swimming

15
velocities(speed,angle,spread,sensoryinput correlations).Themethodcanbeused

live or with videorecordsacquiredandstoredpreviously. It is thereforesuitablefor

analyzingmicrogravityandhypergravitydata. (Kessler)

Invention of the rotating cuvette method for analyzing (a) gravi-phototaxis without

statistical bias, (b) gyro-phototaxis, (c) shear + gravity addition as function of

centrifugal force, and (d) for analyzing sedimentation and sedimentation interactions

among small particles (applications in materials science, gas/grain studies). A

prototype apparatus was constructed and shown to operate as expected. A final version

was shown to require much improved (but available, costly) bearings. (Kessler)

Theory

Theoretical predictions were developed for the variation of angular width of the microorganisms' velocity distribution with magnitude of the effective acceleration of gravity.

This theory can be applied in the entire range of micro -> hypergravity. The theory is

ready for experimental test, using the computer/microscope instrumentation and the

NASA ARC centrifuges for initial runs at hypergravity. (Kessler)

Environmental Factors

The mechanostimulus-inducible TCH3 gene of Arabidopsis encodes a unique

calmodulin-related protein that harbors 6 potential Ca2÷-binding EF hands. The protein

is abundant in the meristematic dome and vascular tissue of 8-day-old Arabidopsis

shoots. TCH3 also accumulates at branch points, suggesting that TCH3 may be

regulated not only by externally applied mechanical stress, but also by physical strains

that occur during morphogenesis. (Braam)

The mechanostimulus-inducible TCH3 gene of Arabidopsis encodes a protein with

significant sequence to xyloglucan endotransglycosylase (XET). This is intriguing

because XET has been proposed to function in cell wall loosening and cell expansion,

processes necessary for thigmomorphogenesis and gravitropism. (Braam)

The rapid (<1 minute) initial collapse of growth rate (60%-90%) by dark-brown

soybean seedlings in response to a single episode of thigmic stress applied to the tip of

the hook was accompanied by a 20%-50% reduction in turgor in the cell elongation

zone of the hypocotyl during the first hour following treatment. Increasing calcium

from 1-5 mmol in the growth medium enhanced seedling growth rate and shortened the

time of recovery to the pre-stress elongation rate. (Myers/Mitchell)

Few studies have investigated plant responses to the low irradiances available in current

growth chambers for space experiments. Results emphasize the importance of

increasing the light in such chambers. Although plants may ap .pear relatively normal

when grown close to the light compensation point, development is greatly delayed and

yields are reduced-- although yields are somewhat higher than expected because of the

longer exposure to the low light. These adverse affects make it very difficult to

evaluate the actual effects of microgravity. High CO2 and substrate-moisture problems

compound these difficulties. (Salisbury)

Excellent equipment has been developed to monitor such environmental factors as CO2,

02, light, air and leaf temperatures, pressure, and substrate moisture in future space

experiments with plants. This will make it possible to diagnose and better understand

the observed plant responses. (Salisbury)

16
ANIMAL

Gravity Receptors and Neurophysiology

Hair cells with differing hair bundle morphologies differ in their voltage-dependent

conductances and the rate and extent of their adaptation to their bundle displacement.

Vestibular nerve afferents innervating the otolith organs may derive their low-frequency

response dynamics from the adaptation kinetics of their innervated hair cells. (Baird)

Mechanisms responsible for mediating gravitationo-ocular reflexes in the bullfrog

tadpole were investigated by utilizing the isolated head preparation. A method has been

developed to quantify spontaneously occurring events such as action potentials

recorded from extraocular motor nerves. Nose-up tilt of the isolated head results in an

increase in activity of trochlear motoneurons and a reciprocal decrease in the activity of

medial rectus motoneurons, lontophoresis of antagonists to the excitatory transmitter

glutamate into the extraocular motor nuclei silences motor nerve activity. Similarly,

excitatory post-synaptic potentials (EPSPs) evoked by electrical stimulation of afferent

pathways are reversibly abolished by these same glutamate antagonists. (Cochran)

Galvanic polarization of the labyrinth in pigeons can selectively silence the most

.irregular firing afferents while leaving the regular f'tring afferents either unaffected or

only slightly reduced. These effects have been effectively used to determine h_w

convergence of regular and irregular firing afferents upon vestibular nuclei neurons

produces responses to rotational stimulation. (Dickman)

The anatomical orientation of the semicircular canals in pigeons were determined in

order to specify the alignment between canal activation directions and afferent response

directional selectivity. (Dickman)

A correspondence between expression of S-IOOB and the appearance of mature afferent

synapses and myelin implies an association between S-100B and these processes.

(Fermin)

Afferent vestibular neurons that connect the inner ear to the brain express the calcium

binding p.rotein/neurotrophic factor S-100B in their nuclei, whereas afferent auditory

neurons m the same nerve of the same animal during the same embryonic stage,

express this protein diffusely in the cytoplasm and nuclei. (Fermin)

Gravity-related otoconia in the chick inner ear are perfect calcite crystals with an organic

matrix that in fixed material is distorted, but nevertheless has a close resemblance to a

computer-generated model derived from assembling various two-dimensional plates of

organic matrix chemically fixed and stained histochemically. (Fermin)

Objective quantification of afferent neuron-related molecules is facilitated by color

thresholding, which, contrary to gray levels of intensity densitometry, does not require

a prior knowledge of specified Irdter combinations. (Fermin)

Modulation of hippocampal cell activity is maintained in animals that have been exposed

to hypergravic fields. Thus, even though anatomical evidence shows that the number

of receptors for serotonin is decreased, there appears to be a surplus of receptors so that

neural information processing is maintained and electrical activity is not altered.

(Horowitz)

17
Thresholds fall markedly following 19days of incubation in the chick embryo,

suggestingthatnormallydevelopinggravityreceptorsbecomemoresensitiveto stimuli

asdevelopmentproceedsbeforeandafterhatching.(Jones)

The useof purelymorphologicalfeaturesto distinguishsubclassesof typeI hair cells

belonging to different afferent classes provides a significant contribution to studies of

the vestibular periphery. The general trends in regional synaptic innervation observed

in the crista of the chinchilla, as well as in the monkey, a primate model, seem to be

valid. This has implications for studies of human synaptic innervation of the vestibular

periphery. (Lysakowski)

Type I-like and type II vestibular hair cells have now been identified in two

taxonomically very distinct teleost fishes, providing strong evidence that these are

ubiquitous among at least telosts. This suggests that hair cell heterogeneity is very

important for the function of the fish ear, and supports the suggestion that multiple hair

cell types arose far earher than with the origin of arnniotes. (Popper)

Two types of vestibular hair cells, resembling the mammalian type I and type II hair

cells, are found in both the saccule and utricle of the goldfish, Carassius auratus. The

two hair cell types are in discrete epithelial regions, lending the preparation to

physiological studies of hair cell function. The morphologically different hair cell types

can be closely correlated with physiologically distinct hair cells that have been reported

in the literature for goldfish hair cells. (Popper/Lanford)

Biochemical studies strongly suggest that hair cell heterogeneity may extend to the fish

lateral line. Only one type of hair cell in the lateral line is damaged by the ototoxic drug

genamicin sulphate, and these cells also regenerate, even in the presence of the drug.

(Popper/Song)

A study of rat macular hair cell synaptic plasticity conducted on the Space Life

Sciences 1 (SLS-1) mission demonstrated that plasticity is retained into adult stage.

Synapses were increased by -55 percent in type II cells and by -41 percent in type I

cells in animals that had flown compared to ground animals. Tissues were collected

beginning 4.5 hours after landing of the Shuttle (R + 0). (Ross)

Much more transmission electron microscopical data were collected, illustrating the

origin of efferent terminals from afferent nerve fibers and calyces. These terminals end

on type !1 hair cells, other nerve fibers, and on calyces. This means that the number of

efferent terminals of extrinsic origin are fewer in number than is commonly believed.

(Ross)

There is a shiP, toward the sphere-like central body in ribbon synapses of both kinds of

hair cells in maculas of R + 0 flown rats (p = <0.0001). This may indicate a generation

of new synapses since the sphere-like ribbon is considered to be the primitive form by

developmental biologists. There is also an increase in pairs and in clusters of synapses

in type II hair cells of R + 0 flight rats compared to controls (p = <0.0001). Stress

may influence synaptic number in the macular hair cells. (Ross)

The development of the statocyst of Aplysia was studied in embryos and larvae reared

at 1, 2, 3, and 5.7 g. The statolith in embryos becomes progressively smaller as the g

force is increased. At increasing g-levels, the percentage of statocysts with no statolith

increases. Similarly, post-metamorphic specimens produce fewer and smaller

statoconia at high g-levels. (Wiederhold)
The time course of appearance and growth of the otolith organs and semicircular canals

in larvae of the Japanese red-bellied newt, Cynops pyrrhogaster, has been established.

Three morphologies of otoconia have been identified: in the adult utricle and early larval

utricle and saccule, barrel-shaped otoconia are made of calcite; at a later stage, first

fusiform and then prismatic otoconia appear in the saccule, and both of these forms are

made of aragonite. (Wiederhold)

Development

Hair cells in the bullfrog vestibular otolith organs regenerate following aminoglycoside

ototoxicity. Regenerating hair cells, recognizable by their short, well-formed hair

bundles, can be classified into distinct hair cell types by the same morphological criteria

used to classify their mature counterparts. The 5-bromo-2'-deoxyuridine (BrdU)

labeling studies suggest that hair cell recovery in the vestibular otolith organs takes

place via both mitotic and non-mitotic mechanisms. (Baird)

The frog egg develops a functional bilateral symmetry before fertilization. The source

of this axial polarity does not arise during oogenesis as previously thought but is

acquired during meiotic maturation, when reorganization of the cytoskeleton permits

gravity-driven cytoplasmic rearrangements to occur. This finding provides an

explanation for the observed wide variability in the spatial relationship between the

point of sperm entry and the process of dorsal-ventral axis specification. (Danilchikj"

Parallel experiments were performed on slow clinostat and in space flight to examine

the effects of altered gravity on the aggregation of the nicotinic acetylcholine receptors

and the structure of the cytoskeleton in cultured Xenopus embryonic muscle cells.

Concordance of results between space flight and the clinostat show the slow clinostat is

a relevant simulation paradigm. (Gruener)

Space-flown cells showed marked changes in the distribution and organization of actin

filaments and had a reduced incidence of acetylcholine receptor aggregates at the site of

contact with polystyrene beads. The sensitivity of the synaptic receptor aggregation

and cytoskeletal morphology suggests that in the microgravity of space cell behavior

may be importantly altered. (Gruener)

Gravity normally plays a role in the establishment of the dorsal ventral axis during early

development of the amphibian Xenopus laevis. A clone was isolated for an RNA

found to be dorsally enriched during the fin'st cleavage cycle in Xenopus. Sequence

analysis indicated that this clone represented the small subunit of mitochondria. In situ

hybridization analysis showed that mitochondria become localized to the presumptive

dorsal side of the embryo shortly after fertilization. The relocation of mitochondria to

the presumptive dorsal side of the embryo was found to be gravitationally sensitive.

(Phillips)

Results show that excitable cells are sensitive to alterations in the perceived gravity

field. The function of acetylcholine receptor (AChR) channels, however, is not

drastically affected, possibly due to a process of cellular adaptation. Taken together,

these results indicate that the development of the neuromuscular junction may be

affected during exposure to microgravity. (Reitstetter)

The identification of neuroregulatory peptides, neurotransmitters, and pituitary

hormones at key stages of development is critical to the fundamental understanding of

reproductive system maturation and function, the control systems that orchestrate these

processes, and how they may be affected by conditions in space. The data we are

gathering are essential to recognize and correctly interpret the influence of hypogravity

19
andotherenvironmentalconditionsof spaceflight ontheneuroendocrineregulationof

thereproductivesystem.(Schreibman)

Tobacco hornworm (Manduca sexta) pupae, which normally undergo adult

development in a horizontal position, are sensitive to a head-up vertical position as

evidenced by (1) more rapid development within 7 days of inversion, (2) elevated

hemolymph concentrations of alanine and phenylalanine, and (3) increased protein

content of the fully developed dorsolongitudinal flight muscle. (Tischler)

The hsp702 gene is testis-abundant, not testis-specific. It is expressed at lower levels

in other adult murine tissues and in predominantly extra-embryonic tissues in the midgestation conceptus. At the level of in situ hybridization, we confirmed the

developmentally regulated pattern of expression of hsp70.2 during mouse

spermatogenesis. The expression of Hsp70 and Hsp90 in the adult testis was

confirmed, at the protein level, to be most abundant in mouse germ ceils from

pachytene spermatocyte to round spermatid stages. Hsp90 protein was found to coimmunoprecipitate with a protein of 70 kDa, which appears to be a member of the

Hsp70 family. Studies on the expression of hsp70.2 have revealed the surprising

observation of transcripts in both the sense and antisense orientations from this area of

the genome. (Wolgemuth)

Bone and Muscle

Alendronate, a bisphosphonate, can prevent the bone loss subsequent to skeletal

unloading. The data suggest that alendronate or like drugs may be useful for preserving

bone during long-term space flight. However, this drug inhibited normal bone

modeling and may be detrimental to the growing skeleton. (Bikle)

Determining the mRNA and protein levels of insulin-like growth factor-1 (IGF-1),

IGF-2, and their receptors from fetal life into old age permits us to gauge changes in

these important skeletal growth factors at different ages and provides correlative

information regarding the role of these factors in skeletal development. (Bikle)

Space flight and hindlimb elevation increases IGF-1 mRNA and protein. The MRNA

levels for IGF-1 do not decrease with skeletal unloading as was expected from the fall

in bone formation; rather, they increase, suggesting a compensatory mechanism for

resistance to IGF-1 observed during skeletal unloading. (Bikle)

Hindlimb elevation results in a decreased ability of IGF-1 and growth hormone to

stimulate bone growth. Understanding the nature of this resistance could provide the

key to understanding the decline in bone formation with skeletal unloading. (Bilde)

Differences were demonstrated in stromal cell proliferation in ceils obtained from the

bones of young and old rats. If such cells remember their history of loading in vivo

(in addition to age) when evaluated in vitro, this model will permit us to perform a

number of experiments in vitro to attempt to unravel the decline in osteoblast function

during skeletal unloading. (Bikle)

Parathyroid hormone is a potent stimulator of bone resorption and could be involved in

the process of microgravity-induced bone loss. Two immediate early gene responses

which may be fundamental to PTH-altered gene expression in the osteoblast have been

identified and characterized. (Clohisy)

• Observations derived from non-beating, passively loaded, rabbit heart cells indicate that

catecholamines can maintain and even promote the fractional rate of heart cell growth

2O
andmodulatetheturnoverof contractileproteins,butadrenergicactivationis incapable

of inducingtheassemblyof thesenascentproteinsintomyofibrilsin non-beatingrabbit

cardiacmyocytes.(Deckeretal.)

Endostealosteoblastsof compactboneof thetibia from 9-daySLS-1flight showless

alkalinephosphataseactivitythantheadjacentprecursorcells. Pro-collagen-containing

secretorygranulesin osteoblastshowedanincreasein numbersduring9-dayrecovery

periodfollowing SLS-I flight. (Doty)

Preliminaryresultsfrom the 14-daySLS-2flight indicatethatthealkalinephosphatase

activity of endostealosteoblastswasreducedbyspaceflight. (Dory)

The micromass cultures of embryonic mouse limb mesenchymeflown aboard

InternationalMicrogravityLaboratory1(IML-1) showedlittle, if any,matrixproduced

during spaceflight. Until late in the mission (4-5 days) the flight cells were

abnormallysmooth,havingnomatrixassociatedwith thecells. (Duke)

Chronic, intermittent mechanicalstrain (CMS) applied to osteoblastsincreases

whole-cellconductance,mediatedthroughthemechanosensitivechannel.Thesedata

suggestthat mechanicalstrainmayprime the mechanosensitivechannelto tonically

respondto continuedstrain. Furthermore,CMS also up-regulatestheproductionof

bonematrixproteins,typeI collagen,osteopontin,andosteocalcin.Theup-regulation

of osteopontinand osteocalcinis independentof Vitamin D stimulation. These

observationssuggesthatCMS producesamoredifferentiatedosteoblastandthat the

signaling mechanismfor CMS may be transducedthrough the mechanosensitive

channel.(Duncan)

Modelingtheendosteumandperiosteumof boneasa monolayerof osteoblastsgrown

on filters showsosteoblastsrepresent a significant barrier to flux of fluid, and the

hydraulic conductivity can be regulated by calcitropic hormones, calcitonin, and

parathyroid hormone. (Frangos)

Studies have provided strong evidence that the ATP-dependent proteolytic system

involving ubiquitin and the proteasomes is the major degradative pathway and is

activated by a variety of physiological stimuli that lead to muscle wasting, including

stress (e.g., cortisol) and disuse. (Goldberg)

Results indicate that unweighting of the soleus muscle by tail-cast suspension causes a

marked increase of insulin-stirnulated glucose transport activity, with no enhancement

of the insulin-independent pathway for glucose transport. (Henriksen)

Prostaglandin E2 (PGE2) acts as bone cytokine. When prostaglandin is added to serum

deprived osteoblasts, cell growth is increased from 2-3 times the control.

Prostaglandins induce oncogene message synthesis. Prostaglandin causes a 20-fold

increase in expression of c-fos; in addition, the c-fos response of the osteoblast is

proportional to the amount of PGE2. The rise in c-fos expression is followed by an

increase in c-jun. Also, messages for 13-actin and v-actin are induced with the addition

of prostaglandin. (Hughes-Fulford)

The decrease in cytoplasm to nuclear ratio in atrophied muscle fibers could suggest that

when lower cytoplasmic levels are maintained the availability of myonuclei are no

longer a modulatory factor in controlling fiber size. (Kasper)

21
Eccentricexercisetraining(lengtheningmusclecontractions)duringnon-weightbearing

attenuated,but did not prevent, the loss of soleus muscle wet weight and noncollagenous protein by 77 and 44 percent, respectively. (Kirby)

The absence of increases in skeletal muscle cAMP during the first 4 hours after a single

10- or 60-minute bout of running by untrained rats suggests that either cAMP is not

part of the mechanochemical link between endurance exercise and increased

mitochondrial density or that the response of the other elements in the adenylate cascade

may correlate better with run duration. (Kirby)

An investigation of the temporal relationship between extracellular matrix formation,

collagen assembly, and mineral deposition in a bone cell (osteoblast) culture of

embryonic chicken calvaria showed that mineral formation is dependent on the state of

maturation or degree of assembly of collagen fibrils. (Landis)

Collagen deposition in a bone cell (osteoblast) culture of embryonic chicken calvaria

has been studied and was shown to be controlled at several post-translational stages

during fibrillogenesis, including cross-link formation, lateral fibril formation, and

increasing fibril stability. (Landis)

Studies of a bone cell (osteoblast) culture of embryonic chicken calvaria grown in the

presence of vitamin D3 [1,25(OH)2D3] have demonstrated that different initial

osteoblast cell populations yield more mature cells of a greater heterogeneous nature.

The effects appear to depend upon the embryological or developmental stage. (Landis)

The mineral crystals from bone cell (osteoblast) cultures of embryonic chicken calvaria

and normal calcified tissues have been described for the first time by high-voltage

electron microscopy and three-dimensional image reconsmaction methods. (Landis)

Alendronate dramatically increased cancellous bone mass in growing animals by

decreasing both bone and cartilage resorption; however, the drug also inhibited bone

formation. (Morey-Holton)

During a 9-day space flight, individually housed growing rats showed more dramatic

bone changes than group-housed animals. During recovery, the group-housed animals

appeared to recover more rapidly that the individually housed rats. (Morey-Holton)

Within 24 hours of return from space flight, young, growing rats showed -300 percent

increase in steady-state levels of mRNA expression of proteins associated with bone

matrix formation. (Morey-Holton)

The morphology of the rat osteosarcoma cells flown on Spacelab-Japanese was slightly

different compared to our ground controls, suggesting that microgravity causes slight

retraction of osteoblastic cells from the substratum. Rat tissue inhibitor of

metalloproteinases-2 was cloned, and regulation in osteoblastic cells was examined.

Parathyroid hormone increases transcription of this gene as a primary response through

cAMP-dependent pathway. Collagenase is expressed as a late-differentiation gene in

normal, differentiating, mineralizing rat osteoblasts. (Partridge)

A specific domain within the type I collagen promoter is utilized in bone cells. This

region is distinct from sequences required for promoter activity in other type I collagen

producing tissues. This is called the basal bone element, and it is believed that it will be

crucial in understanding the molecular control of collagen formation by activated bone

cells. (Rowe)

22
The type I collagenpromotertransgenecontaining3.5 kb of 5 ft. flanking sequence

respondsto the signal for the osteoblastto increaseboneformation in responseto

mechanicalloading.(Rowe)

Satellitecell mitotic activity of therat soleusandextensordigitorum longusmuscle

decreasesbelow weight-bearingcontrol levels within 24 hoursof the initiation of

hindlimb unweighting. Hindlimb unweightingreducesthe quality and quantity of

regenerationin rat soleusmuscles.The reductionappearsto be relatedto a slowed

developmentof fibersduringregenerationratherthana slowedformationof fibers,the

result of reduced satellite cell proliferative activity during the early phaseof

regeneration.(Schultz)

Focal contractions and hyperstretch lesions demonstratedweaker, gradually

diminishing, staining pattern; while wide-A, opaque, and missing-A regions

demonstratedabruptabsenceof staining. (Thompson,J.)

Lesionsproducedastheresultof theapplicationof eccentricloadsto atrophicmuscle

showcompromisedimmunostainingwith antimyosinantibody,indicatingalterationsin

the myosin protein conformation between lesioned and non-lesioned areas.

(Thompson,J.)

Theeffectsof hindlimb unloadingon single-fiberfunction (fiber atrophy,decreased

peaktension,andincreasedmaximumshorteningvelocity)of theslow typeI fibersof

the soleuswere confirmed. As pH was loweredfrom 7.0 to 6.2, both single-fiber

force and velocity decreasedsignificantly, suggestingthat the fibers from hindlimb

unloadinganimalsarelessresistantto H+ion. Hindlimb unloadinginducesa shift

from type I fibers with slowervelocitiesto fiberswith fastervelocities. The fibers

exhibiting the fastestvelocities showedthe greatestreductionin force as pH was

lowered. Thissuggeststhattheincreasedfatigability following hindlimb unloadingis

in part causedby an increased susceptibility of the contractile apparatus to H+ ions.

(Thompson, L.)

Juvenile rats flown on STS-48 showed a similar pattern of hindlimb muscle protein

changes (i.e., soleus atrophy, reduced growth of plantaris and gastrocnemius), a

comparable intensified effect of insulin on soleus glucose uptake, and elevated soleus

interstitial fluid volume as has been observed using the tail-cast hindlimb suspension

model. (Tischler)

Atrophy of the juvenile soleus muscle due to hindlimb unweighting is characterized by

a preferential loss of myofibrillar proteins, which show accelerated protein degradation

not evident for non-myofibrillar proteins in the same muscle. (Tischler)

Heightened effects of insulin, insulin-like growth factor, and catecholamines were

demonstrated to be physiologically significant in the soleus of unweighted hindlimbs by

injecting the muscle and conducting in situ measurements of hormone responsiveness.

(Tischler)

Previous voluntary running does not lessen the effect of unweighting on atrophy of the

soleus muscle. (Tischler)

Studies with glucocorticoid antagonist, RU 38486, showed that unweighting atrophy

of the soleus is definitely not a consequence of elevated circulating glucocorticoids or

increased muscle binding capacity for these hormones. (Tischler)
Older rats exposedto short-durationmicrogravity showedfewer changesin bone

structureand material properties than youngerrats. Therefore, animal age and

endocrinestatusare important factors in predicting the duration of microgravity

exposurethat would induce significant changesin bone structure and material

properties. The load history of the muscle plays an important role in the

compartmentalizationof collagenandits maturationduringgrowthanddevelopment.

(Vailas)

There is a residual amount of lysylpridinoline in hypertrophic cartilage of newly

hatched chicks. These findings suggest that the unusual presence of this crosslink of

non-mineralized fibrocartilage may be associated with a change in calcification.

(Vailas)

Significant interactions occur between mechanical forces and growth factors in

regulating skeletal muscle size when tissue-cultured skeletal myofiber cultures are used.

Mechanical stimulation increases both the sensitivity to, and efflux of, anabolic

autocrine/paracrine cytokines and growth factors such as prostaglandins and insulinlike growth factor-l. Stress-related glucocorticoids cause muscle atrophy, and

mechanical stimulation protects myofibers from this atrophy by generating anabolic

cytokines of the prostaglandin family. Two molecular mechanisms have been identified

by which muscle stretch increases cytokine production: (1) increased synthesis and

activity of cyclooxygenase, the enzyme which synthesizes prostaglandins; and '(2)

activation of several phospholipases, which increases the availability of arachidonic

acid from phospholipids. Both processes are dependent on stretch-activated G

proteins. (Vandenburgh)

The protein matrix of sea urchin embryo calcitic spicules is a collection of over 30

different proteins. Specific antibodies have been raised against two of them; anti-SM50

and anti-SM30. Antibody localization studies show that these proteins are located in

different portions of the spicule matrix. These same antibodies have been used to show

that adult mineralized tissues contain these same matrix proteins. Epitope tagged

versions of the mRNA for SM50 are expressed in the embryo after the mRNA is

microinjected into the zygote. (Wilt)

Regulatory Biology

Hypophysectomy blocks the xylazine-induced increase in angiotensinogen, as does

adrenalectomy, but replacement treatment with glucocorticoids in adrenalectomized rats

restores the response. Therefore, it appears that xylazine acts by an extrapituitary route

but requires the presence of glucocorticoids for the response to occur. (Ganong/Reid)

Recent studies have (a) confirmed the localization of nitric oxide synthase in the macula

densa of rabbits using the nicotinamide adenine dinucleotide phosphate (NADPH)

diaphorase histochemical technique, (b) demonstrated that inhibition of nitric oxide

synthesis with L-Ng-nitro-L-arginine methyl ester (L-NAME) in conscious rabbits

markedly suppresses the renin secretory response to activation of the macula densa

control of renin secretion by furosemide, and (c) demonstrated that inhibition of nitric

oxide synthase increases vasopressin secretion in conscious rabbits. These

observations provide new evidence that nitric oxide participates in the regulation of

renin and vasopressin secretion. (Ganong/Reid)

24
Cell Biology

Dermal fibroblasts detect mechanical force vectors and rearrange their cell bodies to

align parallel to the applied force. Corneal fibroblasts fail to re-align. Force

perception/response requires integrity of the actin cytoskeleton and may also depend on

intercellular connections and cell/matrix adhesion. (Grymes)

Mechanical force interacts with growth factor signal transduction pathways in dermal

fibroblasts of a genetic disease condition (Werner's syndrome). A normal plateletderived growth factor (PDGF) response profile is recovered during mechanical stretch

and through adaptation to a pliable culture substrate. (Grymes)

Clinostat g-force averaging inhibits contraction of collagen gels by corneal stromal

fibroblasts, while application of oscillating rotational forces enhances contraction.

Environmental forces interact with cellularly generated forces in matrix reorganization

behaviors. (Grymes/Johnson-Wint)

A magnetic twisting device was developed which allows one to apply controlled

mechanical stresses directly to specific transmembrane receptors and simultaneously

measure the cellular response in living cells. Using this device, it was demonstrated

that integrin receptors act as cell surface mechanoreceptors and transmit mechanical

signals to the cytoskeleton via a specific molecular pathway. (Ingber)

Mechanical measurements obtained with the magnetic twisting device provide

experimental data in support of the tensegrity (tensional integrity) model of cell

architecture described in previous research. New studies with three-dimensional

tensegrity models demonstrate that use of this form of architecture by cells could

explain how remodeling of the cytoskeleton occurs at the molecular level in response to

mechanical stress application. (Ingber)

The concept of "solid state" signaling is proposed, in which mechanochemical

transduction would be mediated simultaneously at multiple locations by force-induced

cytoskeletal rearrangements that result in redistribution of associated elements of the

cell's metabolic machinery. (Ingber)

In both 3T3 mouse osteoblasts (Hughes-Fulford) and Xenopus muscle cells (Gruener)

grown on small coverslips in the Materials Dispersion Apparatus flown on STS-56, the

actin cytoskeletal filaments were coalesced, disorganized, and showed significantly

altered morphology compared to ground controls. This strongly suggests that the

cytoskeleton may be involved in altered cell response to space flight and that

microgravity sensing directly involves the cytoskeleton. (Lewis/HughesFulford/Gruener)

Mono- and polyclonal antibodies were generated for the characterization of

microtubules, microtubule-organizing centers, and the centrosomes, as well as to

investigate the dynamic microtubule reorganizations necessary for the union of sperm

and egg nuclei, the formation of the mitotic apparatus and further development. The

role of calcium for these processes was investigated by interfering with

calcium-requiring events using inhibitors such as dithiothreitol. These inhibitors

interfered to varying degrees with the organization of cytoskeletal proteins, with

membrane fusions, and with the formation of spicules during later development.

(Schatten)

25
Developedtheoptical andelectronichardware,aswell asthesoftwareof a powerful

andinexpensivesystemto detectchangesof intracellularCa 2+ concentration with high

temporal and spatial resolution. (Verdugo)

Discovered that the free ionized fraction of Ca 2+ inside the endoplasmic reticulum of

ciliated cells does not remain constant at millimolar levels as it had been thought.

Instead it stays at submicromolar concentration, but it can reach submillimolar levels

following stimulation. (Verdugo)

Immunology

The in vitro activation of B-lymphocytes was tested on STS-56 (April 1993) with

human spleenic cells exposed during flight to lipopolysaccharide (LPS), S. aureus

cowan (SAC), anti CD3, and CD40 ligand. Post-flight analyses showed no significant

difference between flown and ground control cells. This is in agreement with other

reports that B-cells do not appear to be affected by microgravity. (Lewis/Neff)

Mouse T-lymphocytes stimulated with Staphylococcal Enterotoxin (SEB) of anti-CD3

monoclonal antibody in the presence of MHC-presenting ceils on small coverslips were

flown in the Materials Dispersion Apparatus on STS-56. The T-cells initiated DNA

synthesis indicating that T-cell receptor-mediated activation is not impaired during space

flight. This is in contrast to the consistently blunted response of T-lymphocytes flown

in the presence of Con-A. This differential activation of T-cells in rnicrogravity can

provide a tool for investigating mechanisms of abnormal or altered cellular responses to

mediators. (Lewis/Principato)

Antiorthostatic suspension by the tail enhanced the capacity of mice to resist a primary

infection with virulent Listeria monocytogenes. It also enhanced the capacity of mice to

express protective immunological memory during a secondary infection with virulent

Listeria monocytogenes. (Miller)

Thymus cells harvested from rats flown on STS-54 produced enhanced titers of

interleukin-3 (IL-3) and interleukin-6 (1L-6) compared to ground controls. Spleen cells

harvested from the same rats produced enhanced levels of IL-6 but normal levels of IL3. (Miller)

• Whole body antiorthostatic suspension did not influence the capacity of neutrophils

harvested from rats to generate the oxidative bursL (Miller)

Gentle dispersion and clinorotation had similar inhibitory effects on in vitro lymphocyte

activation by mitogenic lectins. Data suggest that dispersion of ceils and other indirect

environmental effects significantly contribute to the inhibition of lymphocyte activation

observed in clinostats and that inhibition occurs at very early steps regulating entry into

the activation sequence. (Sams)

Phorbol esters and ionophores, which bypass the surface events and directly activate

intracellular signal transduction systems, allow the initiation of early activation events in

suspended or clinorotated lymphocytes. The data suggest that alteration in surface

signaling is responsible for the inhibition of lectin-induced lymphocyte activation in

clinostats. (Sams)

• Current study suggests that space flight can inhibit the differentiation of precursor cells

to mature macrophages. This result is in accordance with previous human and animal

26
studieswhichsuggesthatthemacrophageisa keycell in space-flight-inducedimmune

dysfunction.(Sonnenfeld/Miller)

27

PLANT PROJECTS

GRAVITY-INDUCED ALTERATION IN DIFFERENTIATION IN CHARA

M.A. Bisson and M.J. Hollingsworth

Department of Biological Sciences

State University of New York

Buffalo, NY 14260

Description of Research

Conversion Structures:

Chara corallina is a freshwater alga with a complex morphology with rhizoids and shoots

comprised of multicellular nodal complexes separated by long intermodal cells. The peripheral

cells and basal branchlet cells of the node can give rise to axillary shoots and rhizoids. Shoots are

thick, with a diameter around 250 I.tm, and green. They show a characteristic pattern of anticlinal

and periclinal tip divisions, and they are negatively gravitropic. Rhizoids are thin, having a

diameter around 30 I.tm, and colorless. They display oblique division planes often far from the

apex, and are positively gravitropic. Isolated plant segments consisting of internodes with the

nodal complex on each end can be experimentally cultured so that rhizoids and axillary shoots

initiate from both nodes.

Occasionally, anomalous differentiation occurs, in which some rhizoids later develop shoot-like

properties. We call these shoot-like rhizoids "conversion structures." Two types of conversion

structures can take place: tip division and greening. Only tip division was affected by orientation

with respect to gravity (Table 1). We call this phenomenon "gravi-morphogenesis."

Table 1. Summary of normal and anomalous rhizoid development

in isolated Chara nodal complexes

[Mean + SD, summary of 4 experiments]

Condition Total Rhizoids Dividing, % Greening, %

Normal 549-__302.16 3.62+3.35 5.40+-5.07

Inverted 641+_358.79 8.34+_5.43 5.40-2-_3.68

In order to determine the time at which cellular differentiation is fixed, one-half of the nodes were

reversed in orientation after a certain number of days. Nodes that were originally normal were

switched to inverted, and those originally inverted became normal. In two experiments, one-half

of the segments were reversed in orientation on day 7, when no conversion structures were

apparent.

Accomplishments and Significance of the Accomplishments

Segments which were reversed in orientation developed as expected for the final orientation. That

is, segments whose orientation was changed from normal to inverted had a greater percentage of

rhizoids with dividing tips than segments in the normal orientation. Segments whose orientation

was changed from inverted to normal had a lower percentage than segments maintained in an

inverted orientation. This indicates that the signal resulting in enhanced tip division was not fixed

at 7 days. Inversion on day 11 or day 16 resulted in an enhanced conversion rate m segments

whose orientation was altered from normal to inverted. Segments whose orientation was altered

from inverted to normal at these later days, however, did not show a decreased number of

conversion structures. This suggests that a decision made to convert, between 7 and I 1 days, is

31
not readily reversed; but a normal rhizoid can still convert on a later day if its orientation occurs

later.

Molecular Biology:

Since changes in differentiation entail changes in gene expression, we began experiments to

characterize gene expression in Chara. We have generated cell-specific cDNA libraries from shoot

and rhizoid tissue by the Haselkorn method (Bauer et al., 1993, PNAS (USA)90: 8812), using

N6-oligomers to generate small (200 bp) cDNA from rhizoids and shoots, as well as larger cDNA

libraries by conventional methods. Whole alga DNA has also been isolated for generation of a

genomic library.

We are at present screening the cDNAs to determine whether there is differential expression of

ATPase isoforms, which axe known to be differentially expressed in higher plants both in a tissuespecific manner and in response to environmental stimuli.

Publications

Gerber, N.R. and Bisson, M.A. 1992. Control of differentiation ofceU type in Chara australis by

the gravity vector. ASGSB Bulletin 6:7 I.

Gerber, N.R., Chau, R., Dombkowski, S.E., Hollingsworth, M.J., and Bisson, M.A. 1993.

Alterations in the gravity vector modify differentiation of Chara australis rhizoids. ASGSB Bulletin

7:58.

32
CALMODULIN-RELATED PROTEINS IN MECHANOSTIMULATED PLANTS

Janet Braam

Department of Biochemistry and Cell Biology

Rice University

Houston, TX 77251-1892

Description of Research

The long-term goal of this research is to understand the role(s) of the TCH proteins of

Arabidopsis thaliana in the plant's responses to mechanical stimuli, including gravity, touch,

wind, and rain. The TCH genes were discovered due to their strong and rapid induction of

expression following stimulation of plants with mechanical stimuli. Three of the TCH genes

encode calmodulin-related proteins, strongly implicating involvement of a calcium ion signaling

system in the mechanosensory pathway of Arabidopsis. Very recently we have found that a

fourth TCH gene product is closely related to a xyloglucan-specific endotransglycosylase. This

is exciting because these enzymes may be involved in cell wall loosening and cell expansion. To

gain insight into the functions of the TCH proteins, we are overproducing them in E. coli for

direct functional assays and also for generating antibodies as specific tools for monitoring the

accumulation, stability, localization, and post-translational modifications of the proteins. As we

dissect the functions of the TCH proteins in response to varied mechanical stimulation, we hope

to shed light on the complex and undoubtedly important roles of the calcium ion as a second

messenger in plant responses to its environment. Through an understanding of the cellular

events that occur in plants following mechanical stimulation, the mechanism(s) and signal

transduction pathways that lead to mechanostimulus-induced developmental changes will be

elucidated.

Accomplishments

(1) We have isolated, mapped, and sequenced the genomic locus and nearly full-length

cDNA of the TCH3 gene. The sequence information obtained indicates that TCH3 is novel in

several respects: (a) It encodes a calmodulin-related protein (60 percent identity) that has six

potential calcium ion binding sites. (b) The conceptual protein product contains linker regions

joining pairs of calcium ion binding sites that are distinct from the linker region of calmodulin in

that they are significantly longer (17 amino acids). (c) The TCH3 gene may have evolved

relatively recently, as the 5' end of the gene (including sequence encoding of the first two

calcium ion binding sites and the first intron) is a nearly identical duplication of the region

encoding the second pair of calcium ion binding sites.

(2) We have isolated, mapped, and sequenced the genomic locus and nearly full-length

cDNA of the TCH4 gene. Although TCH4 expression is regulated similarly to the TCH1-3

genes, it is unique in that the conceptual protein sequence is unrelated to calmodulin. However,

our analysis indicates that the conceptual TCH4 protein is significantly similar to regions of

Arabidopsis Meri-5 (80 percent identical) and to a xyloglucan-specific endo 1,4 b-D glucanase

enzyme (50 percent identical) postulated to function in cell wall expansion. We have already

succeeded in generating antibodies against a portion of the TCH4 protein that is unrelated to

Meri-5. Our preliminary evidence suggests that this antibody recognizes a single protein from

Arabidopsis cells and is therefore likely specific for the TCH4 protein. This antibody will be a

valuable tool in assessing the accumulation, localization, and function of the TCH4 protein. We

are currently generating transgenic antisense TCH4 Arabidopsis plants and TCH4-gusA fusion

plants for tools to examine the in vivo functions of the TCH4 protein and the regulation of

expression of the TCH4 gene.

33
(3) We havegeneratedpolyclonal antibodiesdirectedagainstthe regionof theTCH3

protein containing the first throughfourth calcium-binding sites. This antiserumis able to

recognizetheTrpE-TCH3fusion proteinusedasantigenandthe b-galactosidase-TCH3fusion

protein(demonstratingrecognitionof theTCH3portionof theprotein). This antibodyis specific

in that it is unableto detecttheTCH1 andTCH2 fusion proteins, indicating that the antibody

does not recognize a nearly full-length calmodulin protein (TCH1) or a calmodulin-related

protein (TCH2). This is important because of the presence of multiple expressed calmodulin

genes and calmodulin-related proteins in plants. These data cannot rule out the possibility that

other calmodulin-related proteins in Arabidopsis will be recognized by the TCH3 antibody;

however, as shown by western analysis, a single band of the expected size (33-kD) is detected in

total protein from cultured Arabidopsis cells. Our immunolocalization experiments demonstrate

that the TCH3 protein is abundant in the meristematic dome and vascular tissue in 8-day-o|d

Arabidopsis shoots. TCH3 also accumulates at branch points. These studies of TCH3 are

actively continuing to determine the developmental and tissue-specific localization and

accumulation after stimulation in Arabidopsis plants.

Significance of the Accomplishments

Finding 1: Complete characterization of the TCH3 genomic locus provides us with information

concerning the primary structure of the conceptual protein product. As described above, TCH3

has several features indicating that, although it is a calmodulin-related protein in that it possesses

EF-harid structures predicted to be capable of binding calcium ions, it is significantly distinct

from calmodulin itself and therefore very likely has distinct functions. Indeed, the region of

TCH3 corresponding to the domain believed to be important for interaction with target enzymes

diverges from calmodulin. This feature suggests that, if TCH3 protein functions to regulate

target enzymes, the targets are likely distinct from those of calmodulin.

Finding 2: The molecular characterization of the TCH4 gene is also a valuable step forward in

determining the identity, functions, and control of this mechanostimulus-regulated gene.

Sequence analysis indicates that the conceptual protein product of the TCH4 gene is significantly

similar to enzymes involved in cell wall modifications and possibly cell expansion. This is

intriguing because it would be expected that thigmomorphogenesis and gravitropism

(mechanostimulus responses) would require either an alteration in the orientation of cell

expansion or asymmetric cell wall expansion, respectively.

Finding 3: Immunodetection of the TCH3 protein using the newly generated polyclonal antiserum has demonstrated that, in young Arabidopsis shoots, the protein is abundant in the meristematic dome, vascular tissue, and branch points. The significance of the localization to the

meristem is not yet clear, and it should be noted that accumulation of protein or RNA in this

region may reflect the relative abundance of small ceils rich in cytoplasm (this localization is

often seen with "housekeeping" molecules). The accumulation in branch points is especially

intriguing, as these are the regions that would be expected to be under mechanical strain during

development, thus suggesting that TCH3 may be regulated not only by externally applied

mechanical stress, such as wind or touch, but also by physical strains that occur during

morphogenesis. These studies of TCH3 are actively continuing to determine the developmental

and tissue-specific localization and accumulation after stimulation in Arabidopsis plants.

Publications

Braam, J. 1992. Regulated expression of the calmodulin-related TCH genes in cultured

Arabidopsis ceils: Induction by calcium and heat shock. Proceedings of National Academy of

Science USA 89: 3213-3216.

34
Braam,J. 1992. Regulationof expressionof calmodulin and calmodulin-relatedgenesby

environmentalstimuli in plants.Cell Calcium 13: 457-463.

Braam, J. 1992. Touch-induced regulation of expression of the calmodulin-related TCH genes

and thigmomorphogenesis in Arabidopsis. In: Plant Growth Substances (Karssen, C.M.,

VanLoon, L.C. and Vreugdenhil, D., Eds.), Dordrecht, The Netherlands, Kluwer Academic

Publishers, pp. 82-95.

35
KENNEDY SPACE CENTER PLANT SPACE BIOLOGY LABORATORY FLIGHT

EXPERIMENT SUPPORT

Christopher S. Brown

Bionetics Corporation

Mail Code BIO-3

Kennedy Space Center, FL 32899

Description of Research

Growing plants in the microgravity of space comes with a unique set of problems and

opportunities. For example, the lack of biologically significant gravitational force has been

shown to impact the growth, metabolism, and reproductive capability of plants. Additionally,

there are unusual horticultural concerns in microgravity which must be addressed, including

appropriate nutrient delivery and lighting subsystems.

The overall goal of the research effort at the Kennedy Space Center Plant Space Biology

laboratory is to provide scientific advice and testing of plant flight hardware. To this end, studies

are conducted (1) to determine if gravity influences photosynthesis and carbohydrate metabolism

in higher plants by using the ground-based techniques clinorotation, centrifugation, and/or

tropistic stimulation; (2) to determine the impact of microgravity on carbohydrate metabolism

and photosynthesis in higher plants; and (3) to investigate nutrient delivery and lighting systems

which could have application for plant culture in microgravity.

Accomplishments

(1) In 6-day-old soybean cotyledons, the activity of the starch biosynthetic enzyme ADP

glucose pyrophosphorylase was reduced in clinorotated plants and was increased in centrifuged

plants relative to the control plants (Figure 1). None of the five other starch metabolic enzyme

activities measured were affected.

60

_U

5o

0 ,

0

._ 40

_30

r =0.9965 ._..._y.. _/_N T

.1. VERT

_HORZ

I I 1 I

0 4 5 6 7 8

Starch

( mg- (I00 mg DW) "I)

Figure 1. Relationship between ADP glucose pyrophosphorylase activity and starch concentration

in soybean cotyledons exposed to altered gravity conditions. Horizontal clinorotation (HORZ),

vertical rotation (VERT) and centrifugation at 5g (CENT).

36
(2) Using in vivo protein labeling with 1-D and 2-D PAGE analysis, no changes in

soluble protein expression were found in chronically rotated Arabidopsis (21-day treatment) and

maize (6-day treatment) plants compared to the non-rotated control plants. There were slight

differences in growth.

(3) The porous tube nutrient delivery system was configured for testing during parabolic

flight aboard the KC-135. Control of tube surface moisture levels was maintained on all of the

tubes, regardless of pore size (0.3, 0.7, and 2.0 larn) at all of the gravity levels (0.01 - 1.8g).

(4) Investigations into the use of light emitting diodes (LEDs) for supporting plant

growth commenced. Reductions in growth, photosynthetic rates, and disease resistance were

noted in pepper plants grown under red (660 nm) or red+far red (660 +735 nm) compared to the

metal halide-grown control plants. The addition of just 4.2 p,mol m -2 s-1 of blue light

(1.4 percent of the total photosynthetically active radiation [PAR]) significantly reduced these

differences.

(5) Wheat seedlings grown under high-intensity red light from LEDs did not synthesize

chlorophyll in their shoots. The root-perceived inhibition of chlorophyll biosynthesis in the

leaves was caused by the impairment of the Mg-chelatase enzyme working at the origin of the

Mg-tetrapyrole pathway.

Significance of the Accomplishments

Finding 1: Changes in soybean cotyledon starch concentration due to altered gravity treatments

(lower in clinorotated and higher in centrifuged plants) appears to be directly related to ADP

glucose pyrophosphorylase activity. This enzyme is known to be rate limiting for starch

synthesis in many plants under other conditions. This is the first time ADP glucose

phosphorylase has been suggested to play a role in gravity-mediated changes in starch

concentration.

Finding 2: Although clinorotation can be a severe stressor to plant growth and metabolism, we

found that chronically clinorotated Arabidopsis and maize plants do not show physiological and

biochemical symptoms of severe stress (reduction or retardation of growth, marked changes in

protein banding patterns). Although clinorotation is not a substitute for microgravity, it may be

suitable for certain ground-based studies.

Finding 3: The porous tube nutrient delivery system, which utilizes the capillary movement of

water through ceramic tubes to supply plant roots with moisture and soluble nutrients, is under

consideration as a possible microgravity nutrient delivery technique. The KC-135 parabolic

flight results provide strong evidence that the system will contain and supply water under

conditions other than Earth normal gravity. Plans for testing the system under microgravity

conditions on the Space Shuttle are underway.

Finding 4: Because of their minimal mass, volume, and wavelength specificity, LEDs are

promising as a radiation source for intensive plant culture systems, such as space-based research

chambers or bioregenerative life support systems. For pepper plants, it appears that while red

light (660 nm) alone will support plant growth, low levels of supplemental light in other regions

of the spectrum _blue, 300-500 nm) are necessary for optimal growth.

Finding 5: The use of high-intensity LEDs has been suggested for the culture of plants in space.

However, there may be developmental problems to be overcome, such as the inhibition of

chlorophyll biosynthesis in leaves caused by high-intensity red light. Blue light appears to

mitigate this response and may be an important addition to the spectrum.

37
Publications

Berry, W.L., Goldstein, G., Dreschei, T.W., Wheeler, R.M., Sager, J.C., and Knott, W.M. 1992.

Water relations, gas exchange, and nutrient response to a long-term constant water deficit. Soil

Science 153(6): 442-451.

Brown, C.S., Cox, W.M., Dreschel, T.W., and Chetirkin, P.V. 1992. The vacuum operated

nutrient delivery system: Hydroponics for microgravity. Hortscience 27(11): 1183-1185.

Brown, C.S. and Dreschel, T.W. 1993. Sealed plant growth chamber for clinostat. NASA Tech

Briefs 17(12): 93. (KSC 11538)

Brown, C.S. and Piastuch, W.C. 1991. Carbohydrate metabolism in germinating soybeans in

altered gravity conditions. ASGSB Bulletin 5(1): 75.

Brown, C.S., Piastuch, W.C., and Knott, W.M. Soybean cotyledon starch metabolism is

sensitive to altered gravity conditions. Advances in Space Research 14(8): 107-I 10.

Brown, C.S. and Schuerger, A.C. 1992. Growth and photosynthesis of pepper plants grown

under light emitting diodes. ASGSB Bulletin 6(1): 52.

Bushong, W.E., Fox, R.C., Brown, C.S., Biro, R.R., and Dreschel, T.W. 1993. Clinostat

delivers power to plant growth cabinets. NASA Tech Briefs 17(12): 92. (KSC 11537)

Dreschel, T.W. 1992. Hydroponics. In: The McGraw-Hill Encyclopedia of Science and

Technology 7(8): 607-610.

Dreschel, T.W. 1992. Monitoring and controlling hydroponic flow. NASA Tech Briefs 16(2):

113-114.

Dreschel, T.W. 1992. Tubular membrane plant growth unit. NASA Tech Briefs 16(2): 113.

Dreschel, T.W. and Brown, C.S. 1993. Water-conserving plant growth system. NASA Tech

Briefs 17(1): 89-90. (KSC 11536)

Dreschel, T.W., Brown, C.S., Piastuch, W.C., Hinkle, C.R., Sager, J.C., Wheeler, R.M., and

Knott, W.M. 1992. A summary of porous tube plant nutrient delivery investigations from 1985-

1991. NASA Technical Memorandum 107546.

Dreschel, T.W., Brown, C.S., Piastuch, W.C., Hinkle, C.R., Wheeler, R.M., and Knott, W.M.

1991. Technologies for plant space biology investigations in the Space Shuttle middeck locker.

ASGSB Bulletin 5(1): 90.

Dreschel, T.W., Cox, W.M., Brown, C.S., and Knott, W.M. 1992. The vacuum-operated

nutrient delivery system for hydroponics in space. ASGSB Bulletin 6(1): 84.

Schuerger, A.C. and Brown, C.S. Spectral quality may be used to alter plant disease

development in a CELSS. Advances in Space Research 14.

Takahashi, H., Brown, C.S., Dreschel, T.W., and Scott, T.K. 1991. Root hydrotropism in a

porous tube water delivery system. ASGSB Bulletin 5(1): 46.

Takahashi, H., Brown, C.S., Dreschel, T.W., and Scott, T.K. 1992. Root hydrotropism in a

porous tube water delivery system. Hortscience 27(5): 430-432.

38
Tsao,D., Okos, M.R., Sager,J.C.,and Dreschel,T.W. 1992. Developmentof physicaland

mathematicalmodelsfor theporousceramictubeplant nutrificationsystem(PCTPNS). NASA

TechnicalMemorandum107551.

39
PLASMODESMATA AND THE CONTROL OF GRAVITROPISM

Robert E. Cleland

Botany Department, KB-15

University of Washington

Seattle, WA 98195

Description of Research

Gravitropic curvature occurs when there is an unequal rate of cell elongation on the two sides of

a non-vertical plant organ; e.g., in stems the lower side grows faster than the upper side with the

result that the stem curves upward. This difference in rate of cell elongation is due to an

asymmetric distribution of the growth hormone, auxin. The normal downward polar auxin

transport stream becomes diverted laterally in non-vertical stems. I have proposed a new theory

to explain how this occurs. My research is directed toward a direct test of this theory.

Polar auxin transport occurs when auxin, taken up symmetrically into cells, is exported by auxin

efflux carriers restricted to the base of these cells. This results in a directional flux of auxin from

cell to cell. However, plant cells axe interconnected by protoplasmic tubes, the plasmodesmata

(PDM), which are believed to pass any molecule smaller than the size exclusion limit (SEL) of

about 800 Da. If so, auxin should back/low into the exporting cell through the PDM and

equalize the auxin, preventing polar transport. I propose that auxin moves through cells which

perceive gravity and that in these cells the PDM are closed in the vertical direction but open in

the horizontal direction. This prevents backflow of auxin and permits polar auxin transport in a

basipetal direction. When the stem is reoriented horizontally, the lateral PDM close, because

they are now the "vertical" PDM, and polar auxin is now lateral rather than longitudinal. This

idea can be tested by microinjecting fluorescent dyes into such cells and determining the

direction of movement by epifluorescence microscopy. It is predicted that if a small amount of

dye is injected into cells of a vertical stem, it will move laterally because the PDM are open in

this direction. It will not move vertically because PDM are closed in this direction. When the

tissue is placed in a horizontal orientation, the injected dye will now move longitudinally rather

than laterally.

In order to learn this technique, I participated in a project in the laboratory of Dr. W.J. Lucas, at

the University of California, Davis. I examined the question as to whether the size exclusion

limit of PDM of wheat roots is affected by intracellular adenosine triphosphate (ATP), as there

had been a suggestion that reduction in ATP levels would cause PDM to close. I therefore

determined the effect of anaerobic conditions and azide on the SEL of these roots. It was shown

that both treatments resulted in a reduction of intracellular ATP to less than 10 percent of control

levels. In addition, I examined the possibility that ATP, itself, might be able to move from cell

to cell via the PDM.

Accomplishments

(1) I have shown that when the intracellular ATP levels are reduced by azide or by

anaerobiosis, the size exclusion limit of wheat root epidermal and subepidermal cells increases

from <1 kDa to 5-10 kDa within 30 minutes.

(2) I have shown that when a fluorescent analogue of ATP, 2'0-(trinitrophenyl)

adenosine-5'-diphosphate (TNP-ADP) (mol wt 681 ), is injected into wheat root cells, it is able to

move freely through the PDM to neighboring cells.
Significance of the Accomplishments

Finding 1: The fact that a reduction in cellular ATP causes the SEL of PDM to increase nearly

10-fold indicates that cell-to-cell communication in plants is under direct metabolic control. This

raises the possibility that constriction of PDM to prevent movement of molecules > 1 kDa is due

to phosphorylation of some specific PDM protein by a protein kinase. The idea that PDM might

be able to regulate cross-talk between cells in this way is a new one.

A second point is that these data indicate that when root cells are subjected to localized

anaerobiosis, a frequent stress for plant roots, they are able to compensate for the stress by

increasing PDM conductance. This should facilitate movement of sugars into the anaerobic

region, increasing anaerobic respiration. In addition, it should permit reduced beta-nicotinamideadenine dinucleotide (NADH) to escape the anaerobic cells and move to an aerobic environment

where it can be converted to NAD +, further increasing anaerobic ATP production.

Finding 2: The fact that TNP-ADP can move freely from cell to cell through the PDM means

that cells are interconnected in terms of their energy charge, since ATP and ADP should be able

to equilibrate from cell to cell through the PDM. The idea that plant cells have a common pool

of ATP has never been considered before. It means that cells can cooperate in production of

ATP, with cells with a high ATP demand not having to have a high rate of ATP production

themselves.

These results are going to have a major impact on the way that PDM are considered in plants and

the mechanisms of cell-to-cell communication in plants.

Publications

Bagshaw, S.L. and Cleland, R.E. 1992. Vacuolar calcium does not interfere with measurement

of wall calcium distribution during gravitropism of sunflower hypocotyls. ASGSB Bulletin 6: 70.

Bagshaw, S.L. and Cleland, R.E. 1993. Is wall-bound calcium redistributed during the

gravireaction of stems and coleoptiles? Plant Cell and Environment 16: 1081-1089.

Bagshaw, S.L. and Cleland, R.E. 1993. The effects of enhanced levels of calcium on the

gravireaction of sunflower hypocotyls. Plant Cell and Envtronment 16:1091-1097.

Cleland, R.E. 1992. A possible role for plasmodesmata in plant gravitropism. In: Abstracts,

2nd International Workshop on Basic and Applied Research in Plasmodesmatal Biology,

Oosterbeek, The Netherlands, p. 83-84.

Cleland, R.E. 1992. Auxin-induced growth of Avena coleoptiles involves two mechanisms with

different pH optima. Plant Physiology 99:1556-1561.

Cleland, R.E. 1992. Evaluation of the contribution of acid-extension to the total auxin-induced

growth of Avena coleoptiles. ASGSB Bulletin 6: 96.

Cleland, R.E. 1993. Role of acidification in cell wall expansion. The extracellular matrix of

plants: molecular, cellular, and developmental biology. Journal of Cellular Biochemistry 17A

(Suppl.): 4.

Cleland, R.E. and Lucas, W.J. 1993. ATP regulation of the plasmodesmal size exclusion limit

in wheat roots. Plant Physiology 102: $22.

41
Lavee,S. and Cleland, R.E. 1993. Responsesof intact and excisedyoung beanplantsto

fusicoccin. Plant Growth Regulation 12: 255-262.

Rayle, D.L. and Cleland, R.E. 1992. The acid growth theory of auxin-induced cell elongation is

alive and well. Plant Physiology 99:127 l- 1274.

42
EMBRYOGENESIS OF GRAMINEAE IN MICROGRAVITY

Bob V. Conger

Department of Plant and Soil Science

University of Tennessee

Knoxville, TN 37901-1071

Description of Research

The overall objective of this research is to provide information on the influence of microgravity

on embryo initiation, differentiation, development, and the ultimate reproductive capacity of

resultant plants utilizing an in vitro culture system in orchard grass (Dactylis glomerata L.) in

which the target cells remain in situ. Somatic embryos form directly from mesophyll cells in

cultured leaf segments and develop fully to a germinable stage. The basal 3 cm of the youngest

two leaves are split down the midvein. These axe cut transversely into segments approximately

3 mm square. Segments from one-half leaf are used for various treatments, and the

corresponding sister segments serve as controls. This provides a precise control for each treated

leaf segment and the opportunity to use paired statistics for the analyses of data. Observations

and data will be collected on quantity and quality of embryo formation, axis determination, and

polarity. This will involve estimation of embryo number using an already developed formula

and extensive histology. Plants will be established from somatic embryos and used for'mitotic

chromosome analyses. The same plants will be transferred to the field and used for meiotic

chromosome analyses and estimation of pollen fertility.

The findings will aid in corroborating or refuting those results previously obtained with other

plant tissue culture systems, as well as provide new information. In addition to the advantages

and uniqueness of the system already mentioned, the experiments provide the opportunity to

obtain data on a species representing the Gramineae.

Accomplishments

We have been conducting work on this project for approximately 9 months. During this time,

the research has been concentrated on developing our system into a flight experiment. Among

the most important factors is adherence of the leaf segments to the medium during vibration and

vertical orientation as might be encountered during different phases of a space flight. We have

devised a system in which the leaf segments are overlaid with either polypropylene or Teflon TM

mesh after they have been placed on agar solidified medium. To hold the mesh in contact with

the medium and leaf segments, a polypropylene ring, approximately 8 mm wide and slightly

smaller in diameter than the Petri dish containing it, is placed on the mesh inside the dish before

covering the dish with the lid. The lid holds the polypropylene ring firmly to the mesh. This

scheme is illustrated in Figure 1. Segments from one-half of each of the innermost and next leaf

outward are plated serially from the basal to the distal portion. The corresponding segments

from the sister leaf halves are plated identically except that they are not overlaid with mesh;

these, as mentioned above, serve as controls.

So far, we have conducted experiments with mesh having openings of 710 I.tm, 1800 _tm, or

2200 l.tm. The mesh was kept on top of the leaf segments for 2 weeks and then removed. This

time period was chosen because the duration of Shuttle missions is usually about 10-14 days.

Preliminary data indicate either no influence from overlaying with mesh or a slightly improved

response. Response in terms of relative growth (qualitative observations) and plant regeneration

appears to be better with mesh having larger rather than smaller openings. Segments overlaid

43
with mesh respond equal to, or better than, those not overlaid with mesh (controls). We are still

in the process of conducting experiments and collecting data.

DISTAL L

E

A

F

fourth

third

second

first

BASAL

ring

mesh

Innermost leaf Second leaf outward

ring

mesh

___ medium

S

E

G

M

Figure 1. Diagram of mesh scheme to hold orchard grass leaf segments in contact with the

medium. After the segments are plated onto the medium, a piece of mesh of exact diameter as the

inside of the Petri plate is placed on top of them. A polypropylene ring of slightly smaller

diameter is then placed on top of the mesh. The width of the ring is such that it fits exactly

between the mesh and the lid of the Petri plates.

Significance of the Accomplishments

Adherence of the experimental material, in this case, orchard grass leaf segments, to agar

medium during various stages of a space flight is extremely important to success of the

experiment. The frequently observed improved response by the overlay of our leaf segments

with mesh may be due to the mesh holding more of the leaf surface in contact with the medium.

Our system may improve agar stability and, moreover, have application, in either its present or

modified form, to other biological systems.

Publications

Bell, L.M., Trigiano, R.N., and Conger, B.V. 1993. Relationship of abscisic acid to somatic

embryogenesis in Dactylis glomerata. Environmental and Experimental Botany 33: 495-499.

44
Dutta Gupta, S., Joshi, P.A., Hovanesian,J.C., and Conger, B.V. 1992. Ultrastructural

characterizationof somaticembryosregeneratedfrom NaC1selectedand nonselectedcalli of

Dactylis glomerata. Protoplasma 170: 177-185.

Tomaszewski, Z. and Conger, B.V. 1993. In vitro response of cultured orchard grass leaf

segments to the application of mesh to hold them in contact with agar medium (Abstract).

ASGSB Bulletin 7(1): 90.

Trigiano, R.N., May, R.A., and Conger, B.V. 1992. Reduced nitrogen influences somatic

embryo quality and plant regeneration from suspension cultures of orchard grass. In Vitro

Cellular and Developmental Biology ----Plant 28P: 187-191.

45
MECHANISM OF DIFFERENTIAL GROWTH DURING STEM GRAVITROPISM

Daniel J. Cosgrove

Department of Biology

Pennsylvania State University

University Park, PA 16802

Description of Research

Plant growth is responsive to a number of internal and external stimuli, including gravity. Gravity

modulates the biophysical and biochemical processes that govern plant cell expansion, such that

plants tend to grow in a fixed orientation with respect to gravity and they revert to this orientation

following displacement. This gravity response, known as gravitropism, is mediated by a bilateral

asymmetry in growth of roots and stems. We axe investigating the cellular and biochemical

mechanisms underlying gravitropic growth asymmetries to gain insight into the fundamental

mechanisms controlling plant growth and to learn how gravity modifies these processes.

We study young cucumber seedlings (Cucumis sativus L.) because their stems display a vigorous

gravitropic bending upward after they are turned on their side. This reorientation is accomplished

by a transient cessation of cell elongation on the upper stem surface and a simultaneous increase

(twofold) of the growth rate on the lower stem surface. The growth asymmetry begins to reverse

itself well before the stem attains a vertical position. These fine modulations of cell growth occur

in a stem that is only 1.5 mm in diameter; that is, cells can be quite close to one another and still

react in opposite fashion to a gravitropic stimulus. By studying this gravitropic response, we hope

to elucidate how plants integrate sensory stimuli and use this information to control their growth

and form.

Our previous studies showed that cell turgor pressure and membrane hydraulic conductivity play a

secondary, passive role, not a controlling role, for this gravitropic response. Our studies showed

that water transport poses an insignificant limitation to cell expansion in cucumber seedlings and

that the major control point for plant growth rests in the biophysical relaxation of the wall.

We axe now studying the biochemical basis for wall relaxation, with the working hypothesis that

wall enzymes catalyze the wall modifications that give rise to wall stress relaxation. A major

puzzle in this field is the nature of these wall enzymes and the mechanisms by which their activity

may be regulated. We have developed a cell-flee system for studying the biochemistry of cell wall

expansion (see Figure 1), and we recendy reported the flu'st isolation of wall proteins responsible

for the "acid-growth" response of wall.

Accomplishments

(1) A novel class of proteins was identified with the ability to induce extension of isolated

walls. We call the proteins "expansins." They axe found in an active form in cucumber walls

obtained from the growing region of the cucumber hypocotyl. Cucumber walls contain two active

proteins of 29 kD and 30 kD nominal molecular mass (determined by sodium dodecyl sulfatepolyacrilamide gel electrophoresis [SDS-PAGE]). These proteins induce extension of wa.l!s .that

axe held in tension in an acidic buffer. They exhibit many of the peculiar biochemical sensmvmes

that characterize "acid-growth" of native walls; e.g., acidic optimum, activity enhancement with

thiol reducing reagents, activity inhibition with 1 mmol aluminum, copper and mercury ions, and

survival of brief treatment in boiling methanol.

46
cut

freeze

thaw

abrade

boil

x 1000

homogenize

collect & wash walls

extract walls w/salt

fractionate protein

J

LVDT

Figure 1. Diagram of our re,constitution system for identifying proteins that catalyze wall

extension. The growing region of etiolated cucumber seedlings are used as the biological

material because their walls display stable and sensitive wall-extension characteristics. As

substrate for the re,constitution assay, the growing hypocotyl is frozen and thawed to kill the

cells, abraded with carborundum powder to permeabilize the cuticle, boiled in water (15 seconds)

to inactivate the endogenous proteins, and clamped in a constant-force extensometer. Protein

fractions with suspected extension activity are added to the cuvette surrounding the wall sample,

and extension responses are measured with a position transducer (linear variable displacement

transducer [LVDT]) attached to the bottom clamp.

47
(2) A wail proteinantigenicallyrelatedtocucumberexpansin-29canbe extracted from oat

coleoptile walls. We purified wall extension proteins from oat coleoptile walls by high-pressure

liquid chromatography (HPLC) and related methods in combination with our wall extension

reconstitution assay (Figure 1). The single active fraction contained a single major protein band of

29 kD. In Western blots, this protein was recognized by an antibody raised against cucumber

expansin (the 29 kD form).

(3) Treatment of cucumber hypocotyls with 1 _tmol auxin leads to a change in the redox

state of the cell wall, such that wall protein sulfhydryls become more reduced. This conclusion

was established by labeling tissues (with or without auxin pretreatment) using an impermeant

fluorescent thiol reagent. Wall-bound proteins were then extracted with 1 tool NaC1. The proteinassociated fluorescence doubled in the auxin-treated samples, yet the total molar amount of protein

remained little affected.

(4) Light treatment of oat coleoptiles reduces their growth rate and also reduces their

ability to grow in response to auxin, fusicoccin, and acidic buffers. Moreover, their walls lose

responsiveness to acidic buffers and to exogenous expansins. They also reduce their rate of

gravitropic bending. In other words, it appears that the ability of the walls to respond to various

growth-stimulating agents is reduced after light treatment.

Significance of the Accomplishments

Finding 1: Identifies specific wall proteins that mediate the acid.extension

responses of isolated walls. This is the first identification of proteins with this ability.

Because acid-growth responses are nearly ubiquitous in plants with cellulosic walls, we infer that

these novel proteins are important for the extension growth of most plant ceils. Acid growth has

been implicated in the action of auxin, light, and gravity on plant cell enlargement. Therefore, it is

plausible that these stimuli affect growth via effects on expansin action.

Finding 2: Indicates that expansins are found in grass walls, and thus may be

ubiquitous components of plant cell walls. It is notable that the polymeric composition of

matrix polysaccharides in dicot walls and grass walls is very different; yet expansins are effective

on both types of walls. This implies that the biochemical mechanism for acid-induced wall

expansion is common to these distantly related plants. Both dicot and grass shoots are thought to

respond gravitropically via an auxin-induced differential acidification of the wall space; we

postulate that expansins are the wall protein factors that translate a wall pH asymmetry into a wall

expansion asymmetry. (We recognize that other mechanisms of wall expansion may additionally

be involved).

Finding 3: Is important because it shows that auxin can cause changes in the thiol

redox state of wall proteins. This fact, in combination with the observation that expansin

activity is stimulated by thiol reducing reagents, suggests that a part of auxin action and

gravitropism may involve control of wall redox state. We are now testing for asymmetries in wall

redox potential during gravitropism.

Finding 4: Indicates that changes in wall susceptibility to expansin action may

prove a mechanism for controlling plant growth. The results also provide correlative

evidence in favor of the idea that expansins play an important role in plant responses to light,

auxin, and gravity.

48
Publications

Cosgrove, D.J. 1993. How do plant cell walls extend? Plant Physiology 102: 1-6.

Cosgrove, D.J. 1993. Water uptake by growing cells: An assessment of the controlling roles of

wall relaxation, solute uptake and hydraulic conductance. International Journal of Plant Sciences

154: 10-21.

Cosgrove, D.J., Durachko, D.M., and McQueen-Mason, S. 1992. Extension of isolated plant cell

walls and its induction by endogenous wall proteins. Current Topics in Plant Biochemisto' and

Physiology 11: 74-85.

Cosgrove, D.J. and Hedrich, R. 1991. Stretch-activated chloride, potassium and calcium channels

coexisting in plasma membranes of guard cells of Viciafaba L. Planta 186: 143-153.

Cosgrove, D.J. and Li, Z.-C. 1993. Role of expansin in development and light control of growth

and wall extension in oat coleoptiles. Plant Physiology 103: 1321-1328.

Li, Z.-C., Durachko, D.M., and Cosgrove, D.J. 1993. An oat coleoptile wall protein that induces

wall extension in vitro and that is antigenically related to a similar protein from cucumber

hypocotyls. Planta 191: 349-356.

McQueen-Mason, S., Durachko, D.M., and Cosgrove, D.J. 1992. Creep of isolated wails and its

induction by wall proteins. In: Plant Cell Walls as Biopolymers with Physiological Functions

(Masuda, Y., Ed.). Yamada Conference XXXII, Osaka, Yamada Science Foundation, pp. 159-163.

McQueen-Mason, S., Durachko, D.M., and Cosgrove, D.J. 1992. Two endogenous proteins that

induce cell wall expansion in plants. The Plant Cell 4: 1425-1433.

McQueen-Mason, S., Fry, S.C., Durachko, D.M., and Cosgrove, D.J. 1993. The relationship

between xyloglucan endotransglycosylase and in vitro cell wall extension in cucumber hypocotyls.

Planta 190: 327-331.

Spalding, E.P. and Cosgrove, D.J. 1992. Mechanism of blue-light-induced plasma-membrane

depolarization in etiolated cucumber hypocotyls. Planta 188: 199-205.

Spalding, E.P. and Cosgrove, D.J. 1993. Influence of electrolytes on growth, phototropism,

nutation, and surface potential in etiolated cucumber seedlings. Plant Cell and Environment 16: 445-

452.

Stahlberg, R.S. and Cosgrove, D.J. 1992. Rapid changes in growth rate and electrical potentials

upon excision of pea epicotyls. Planta 187:523-531.

Wang, T.-W., Cosgrove, D.J., and Arteca, R.N. 1993. Brassinosteroid stimulation of hypocotyl

elongation and wall relaxation in Pak-Choi (Brassica chinensis cv. Lei-Choi). Plant Physiology

101: 965-968.

49
SEARCH FOR STRUCTURAL

IN GRAVITY SENSING AND

IN GRAVITROPISM

Kathryn L. Edwards

Biology Department

Kenyon College

Gambier, OH 43022

PROTEINS HAVING POTENTIAL FUNCTION

FOR DIFFERENTIAL EXPRESSION OF GENES

Description of Research

Gravitropism displayed in plants and fungi involves detection, transduction, growth response, and

resolution. The molecular foundations of each of these phases is arguably more complex in higher

plants, whose responsive organs are multicellular and, often, where the tissue of gravity detection

is separated from the tissue undergoing the growth response. These gravitropic mechanisms,

however, are just as likely to have some common evolutionary cores with those organisms that

have single-celled gravitropic structures, such as the sporangiophore of the terrestrial fungus,

Phycomyces, which has been the focus of our research.

Several approaches are being undertaken to understand the mechanisms for the detection of

gravity, the secondary messenger mechanisms with a particular emphasis on calcium,related

functions, and the genetic interplay in the response. Until recently we have been attempting to

understand various membrane ion channels in Phycomyces sporangiophores whose characteristics

might inherently be useful in one or more aspects of gravitropism. Our inability to satisfactorily

identify the membrane or stabilize certain channels that appeared to be stretch-activated has lead us

to reassess this approach.

Last year we (a) investigated the in situ immunolocalization of cytoskeletal proteins, the properties

of which might lend themselves to a fretwork used to detect alterations of the cell's orientation to

the gravitational field; (b) began work to sequence Phycomyces calmodulin; and (c) continued finetuning our study of the effect of gadolinium on gravitropism of Phycomyces and some of its

gravitropic mutants. Calmodulin continues to hold our interest because it has been found to be

differentially expressed in higher plants with respect to mechanical stimulation such as touch (and,

likely, gravity), and a protein partially purified from Phycomyces has shown calmodulin-like

activity (Martinez, et al., 1982).

This year we (a) are confirming the presence of cytoskeleton and related proteins of protein extracts

using immunoidentification procedures; (b) preparing to perform an mRNA subtraction or possibly

differential display in order to determine what, if any, genes are activated/expressed upon exposing

the fungal sporangiophore to gravity stress; and (c) probing for second messenger proteins likely

involved in coordinating orientation to gravity and growth response.

Accom plishments

(1)We have in situ localized polyclonal antibodies to globular (g)-actin and chicken

spectrin to a thin-lined area at the plasma membrane of the Phycomyces sporangiophore using

immunofluorescence. Though control tissues were negative, the use of polyclonal antibodies

could result in an anomalous reaction. These results are being confirmed using the more specific

monoclonal antibodies.

(2) Total protein extracts from Phycomyces sporangiophores and cytoskeletal protein

extracts using variations of an extraction procedure developed for yeasts have revealed the presence

of two 40-50 kDa bands in the range expected for g-actin in Western blots using monoclonal actin

5O
antibody. ThecytoskeletalextractionhasproducedWesternswith two 180-200kDa proteinsin

theexpectedweightrangefor myosinwith monoclonalmyosinantibodyandtwo faint bandsin the

rangeof 100-120kDa with monoclonalintegrin immunoassay.Assaysfor spectrinhavebeen

inconclusive.

(3) Wheat calmodulin cDNA (gift of R. Zielinski, University of Illinois at UrbanaChampaign)hybridizedwith two closelyassociatedbandsfrom Phycomyces genomic DNA in a

Southern blot indicates that a calmodulin gene resides in Phycomyces and bears homology to

wheat calmodulin. Although a protein with calmodulin-like activity has been described from

Phycomyces, neither the protein nor the gene has been sequenced. In order to sequence, DNA

primers were constructed from two regions homologous in the calmodulin cDNA sequences from

wheat and soybean, and three fungal species, Neurospora, Candida, and Achlya. Using these

primers, we have obtained, using PCR, a 339-base pair (bp) fragment that hybridizes to both

wheat calmodulin cDNA and genomic Phycomyces DNA in Southern blots. Thus, we believe this

fragment to be a portion of the Phycomyces calmodulin gene, and we are presently sequencing the

fragment. We will be able to extend this fragment and/or will be able to use the fragment as a

probe for the differential expression of calmodulin in gravity-stressed and unstressed cells.

Significance of the Accomplishments

Findings 1 and 2: The presence of actin and myosin is to be expected in Phycomyces. since

cytoplzismic streaming is evident in the sporangiophore. Our results support this assurfaption.

Integrins are a group of proteins involved in linking or anchoring the cytoskeleton to an

extracellular matrix in many animal cells. The presence of an integrin-like protein in Phycomyces

sporangiophores leads us to speculate on its possible role in rigidly anchoring the cytoskeleton to

the cell wall. We further speculate on the possibility of its serving to locate/activate proteins in the

cell membrane that would assist in the growth response to altered stress in the cytoskeleton due to

the orientation of the sporangiophore with respect to gravity.

Finding 3: The calmodulin gene from Phycomyces has common homologies to both higher plant

calmodulins and calmodulins from other fungi. The 339-bp fragment of the gene obtained by

PCR, after being sequenced, will be used in an effort to sequence the entire gene. The fragment

alone will be a useful probe in determining whether calmodulin is differentially expressed in

gravity-stressed sporangiophores as it is in stressed Arabidopsis stem tissue (Braam and Davis,

1990).

51
ROOT GRAVITROPISM IN MAIZE AND ARABIDOPSIS

Michael L. Evans

Department of Plant Biology

Ohio State University

Columbus, OH 43210.

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 in growth occur about 2-5 mm behind the tip.

We are interested in the nature of the signal moving from the cap to the responding zone, and we

want to determine whether gravity sensing is restricted to cells in the cap. We seek to identify the

cells showing the earliest growth changes following stimulation. This will allow us to concentrate

our efforts on these ceils in regard to the influence of gravity on factors likely to control growth

(e.g., hormone distribution, hormone sensitivity, electrical properties).

Our recent research has centered on the following: (1) Analysis of the differing effects of the

growth regulating hormone, auxin, on cells in different regions of the root; (2) Analysis of the

nature of the gravitropic response in roots pretreated with high levels of auxin, the hormone

thought to control the differential growth leading to curvature; (3) Modification of the video

digitizer system to allow analysis of the small roots of Arabidopsis seedlings, so we may utilize

auxin response mutants of this species to analyze gravitropism in plants with reduced auxin

sensitivity; and, (4) Characterizing the surface electrical potential pattern in maize roots with

emphasis on gravity-induced changes in this pattern and its spatial and temporal correlation with

growth pattern changes during gravitropism.

Accomplishments

(1) Auxin inhibits the elongation of cells in the main elongation zone but promotes the

elongation of a group of ceils close to the meristem after a lag of about 1 hour. This region of the

root has been referred to as the Postmitotic Isodiametric Growth (PIG) zone or, alternatively, as

the Distal Elongation Zone (DEZ). The promotion of elongation in the DEZ accounts for the ability

of roots to adapt to inhibiting levels of auxin. The root resumes growth because of the delayed

promotion of elongation in the DEZ. Cells in the elongation zone remain inhibited.

(2) Roots treated with a concentration of auxin sufficient to inhibit elongation 100 percent

show strong curvature upon gravistimulation. The curvature results from strong enhancement of

growth along the top of the root in the DEZ, just as occurs in untreated controls. Growth along the

bottom is nil because of the high auxin concentration.

(3) We successfully modified the video digitizer system to allow analysis of growth in

roots of Arabidopsis. The timing of the auxin response in these roots is similar to that reported in

other species. We have analyzed the auxin dose/response relationship in wild type seedlings as

well as the auxin resistant mutants axrl-3, axrl-12, auxl, and axr2. We find that there is no shift

in the dose-response curve along the concentration axis but that the mutants show enhanced

promotion by low doses of auxin, as well as reduced inhibition by high doses. The mutants show

impaired gravitropism, but the degree of gravitropic responsiveness appears to correlate more with

absolute growth rate than with auxin sensitivity per se.

(4) There is a distinct surface potential distribution along the apical region of maize roots.

The potential is rapidly but transiently reduced in a biphasic pattern upon stimulation of the root

52
surface(e.g.,touchingat a localizedpoint). Theregionof theroot mostsensitiveto electrical

perturbationuponstimulationcorrespondswith the DEZ; i.e.,it is the sameregionshowingthe

earliestgrowthresponse following gravistimulation.

Significance of the Accomplishments

Finding 1: The finding that auxin promotes elongation in the DEZ but inhibits in the elongation

zone is significant because it alters our perception of this hormone as one which can only retard the

elongation of intact roots. It is also significant because we know that there are periods during the

gravitropic response when growth is strongly enhanced in localized regions of the root. The

observation that the elongation of certain cells can be enhanced by auxin raises localized auxin

elevation as a possible regulator of growth acceleration during gravitropism.

Finding 2: The finding that roots with elongation rates reduced to zero by treatment with auxin can

exhibit gravitropic curvature with kinetics similar to that of control roots is an especially significant

observation. We have shown that the pattern of growth acceleration along the top of the root is

essentially the same in gravistimulated auxin-inhibited roots as in controls. This tells us that the

acceleration of elongation in the early stages of gravitropism is not auxin mediated and that

gravistimulation can cause strong growth enhancement in cells on the upper side of the DEZ even

in the presence of high concentrations of auxin. The conclusion is that gravity-induced growth

acceleration is auxin-independent. The major theory of root gravitropism until now holds that

differential growth is accounted for by auxin redistribution. This finding shows that this idea is

wrong, at least as applied to the growth acceleration component of gravitropism.

Finding 3: Our successful modification of the digitizer system to allow analysis of Arabidopsis

mutants opens the door for a more detailed analysis of the role of auxin in root growth and

gravitropism. This will allow us to thoroughly analyze gravitropism in a number of recently

generated mutants.

Finding 4: The finding that DEZ cells show the largest electrical perturbation in response to

localized stimulation suggests that these cells may be particularly important in root sensory

responses. Cell-to-cell electrical transmission has been suggested as a possible mode of signal

transmission during root gravitropism. Further characterization of electrical changes in the cells

which show the earliest growth response to gravistimulation should add insight regarding the role

of membrane potential changes in signal transmission or alteration of growth patterns.

Publications

Cha, M.R., Evans, M.L, and Hangarter, R.P. 1992. Localization of IAA transporting tissue in

Coleus stems and corn coleoptiles (Abstract). In Vivo 6: 566.

Cha, M.R., Evans, M.L., and Hangarter, R.P. 1993. Localization of IAA-transporting tissue in

Coleus stems and corn coleoptiles. Plant Physiology and Biochemistry 31: 263-269.

Cleland, R.E., Buckley, G., Nowbar, S., Lew, N.M., Stinemetz, C., Evans, M.L., and Rayle,

D.L. 1991. The pH profile for acid-induced elongation of coleoptile and epicotyl sections is

consistent with the acid-growth theory. Planta 186: 70-74.

Evans, M.L. 1992. What remains of the Cholodny-Went theory? (contribution to multi-author

forum). Plant, Cell and Environment 15:767-768 (entire forum = pp. 759-794).

Evans, M.L. and Ishikawa, H. 1991. Role of the postmitotic isodiametric growth zone in the

gravitropic response of maize roots (Abstract). ASGSB Bulletin 5: 44.

53
Evans,M.L., Young, L.M., andHasenstein,K.H. 1992. Therole of calciumin theregulationof

hormonetransportin gravistimulatedroots. Advances in Space Research 12:21 I-218.

Ishikawa, H. and Evans, M.L. 1992. Accelerated gravitropism in maize roots pretreated with

auxin (Abstract). ASGSB Bulletin 6: 74.

Ishikawa, H. and Evans, M.L. 1992. Calcium-induced curvature in maize roots (Abstract). Plant

Physiology 99 (Suppl.): 60.

Ishikawa, H. and Evans, M.L. 1992. Induction of curvature in maize roots by calcium or by

thigmostirnulation: Role of the postmitotic isodiametric growth zone. Plant Physiology 100: 762-

768.

Ishikawa, H. and Evans, M.L. 1992. Surface potential changes induced by thigmostimulation of

maize roots (Abstract). ASGSB Bulletin 6: 98.

Ishikawa, H. and Evans, M.L. 1993. The role of the distal elongation zone in the response of

maize roots to auxin and gravity. Plant Physiology 102: 1203-1210.

Jones, A.M., Cochran, D.S., Lamerson, P.M., Evans, M.L., and Cohen, J.D. 1991. Red-lightregulated growth. I. Changes in the abundance of indoleacetic acid and a 22-kilodalton auxinbinding protein in the maize mesocotyl. Plant Physiology 97: 352-358.

Kiss, H.G., Evans, M.L., and Johnson, J.D. 1991. Cytoplasmic calcium levels in protoplasts

from the cap and elongation zone of maize roots. Protoplasma 163: 181-188.

Stinemetz, C.L., Hasenstein, K.H., Young, L.M., and Evans, M.L. 1992. Effect of calmodulin

antagonists on the growth and graviresponsiveness of primary roots of maize. Journal of Plant

Growth Regulation 11: 419-427.

Young, L., Ishikawa, H., and Evans, M.L. 1992. Comparative auxin action in roots of wild type

and auxin response mutants of Arabidopsis (Absu'act). ASGSB Bulletin 6:71.

54
PHYSIOLOGICAL,

ASSOCIATED WITH

MAIZE

BIOCHEMICAL, AND

THE TRANSDUCTION

Lewis Feldman

Department of Plant Biology

University of California

Berkeley, CA 94720

MOLECULAR PROCESSES

OF GRAVITY IN ROOTS OF

Description of Research

Our research focuses on understanding the mechanisms used by plants to convert the physical

stimulus of gravity into a developmental response. This work is performed with roots of corn.

Normally, when roots are reoriented to the horizontal, they perceive this changed orientation and

grow downward. We know that this change in orientation is perceived and then biochemically

processed in a special region at the tip of the root known as the root cap. Within the root cap it is

hypothesized that certain biochemical steps, collectively called signal transduction steps, occur.

We are investigating the hypothesized involvement of specific proteins, known as kinases, in

signal transduction. Based upon models developed from animal and microbial systems, it is

suggested that kinases in the root cap function to convert the physical stimulus of gravity into a

developmental response. Although the mechanism of operation of kinase has been well defined

from other non-plant organisms, there is no reason to believe that the basic elements of kinase

operation differ in plants. These basic elements include a compound called a second messenger,

which regulates the activity of the kinase (i.e., does it function?), and a target (usually another

protein) which the "turned-on" kinase then affects. For this effort we are, (1) investigating the

presence of second messenger kinases in root caps, (2) investigating what proteins are regulated by

kinases, and (3) determining whether root cap kinases mediate the biochemical processing of

gravity.

Accomplishments

We undertook a molecular approach in order to determine whether second messenger kinases

operate in root caps. We constructed a cDNA library from root caps and then screened this library

with oligonucleotides fashioned after conserved regions of other second messenger kinases.

Using this approach, we have obtained several putative second messenger kinases. One, which

we have called 90.7 (Biermann and Feldman, 1992), has all the features characteristic of kinases

which fall into calcium/calmodulin, cAMP-regulated kinases. In addition, 90.7

autophosphorylates and is expressed to a high level in the root cap. In order to test effects of

possible regulators on 90.7 protein kinase activity, we developed a sensitive method for detecting

regulation of autophosphoryl labeling of protein kinases in unfractionated corn root protein

extracts. We tested a wide variety of potential second messengers, including calcium and plant

hormones. Although we were not able to show that any of these compounds increased 90.7

activity, we could demonstrate that intermediates of carbohydrate metabolism (e.g., sugar

phosphate intermediates) down-regulated the autophosophorlyating activity of the 90.7 kinase.

This indicates that the regulation of kinases in plants, specifically the 90.7 kinase, is different from

that known in animals and fungi, further suggesting operation of unique protein kinase regulatory

mechanisms in plants. Details of this work are in Biermann and Feldman (1992). In addition to

finding the 90.7 kinase, we have also recently sequenced a kinase which has all the characteristics

ofa calcium/calmodulin II kinase (CaMK II). This kinase was isolated from a root cap library, and

we are now in the process of defining how the activity of this kinase is regulated. Based upon the

finding of a CaMK II in root caps, we undertook a study of the effects of drugs which are

supposedly specific inhibitors of CaMK II. One such drug, KN-93, was applied to roots which

55
werethenreorientedwith respectto gravity. We found that this drug preventedthe root from

completingthe gravity response, curving downward, but did not inhibit root elongation. These

results are detailed in the publication by Lu, Feldman, and Hidaka (1993) and provide strong

evidence that a CaMK 1I pathway, or one involving CaMK II homologues, operates in root caps

and is involved in processing the gravity signal.

Significance of the Accomplishments

During the previous year, we have investigated the hypothesized role of protein kinases in

mediating the conversion of the physical stimulus of gravity into a developmental response. A

significant accomplishment of this work has been the demonstration of several different kinases in

root caps as well as our having shown that, when we inhibit the activity of a kinase apparently

belonging to the calcium/calmodulin subfamily of kinases, we simultaneously affect the gravitropic

response. These data thus suggest that not only are second messenger regulated kinases present in

roots but also, when the activity of one of these kinases is inhibited, we can prevent roots from

responding to gravity.

Publications

Lu, Y-T, Feldman, L.J., and Hidaka, H. 1993. Inhibitory effects of KN-93, an inhibitor of

Ca2+/calmodulin-dependent protein kinase 11, on light-regulated root gravitropism in maize. Plant

Physiology and Biochemistry 31 : 857-862.

56
A NEW METHOD FOR FOLLOWING

MAIZE ROOTS

Sari Galanes and Philip M. Lintilhac

University of Vermont

Burlington, VT 05405

THE GRAVITROPIC RESPONSE IN

Description of Research

The primary objective of this work has been to develop a continuous measure of the bending force

output of gravitropically responding roots. Our long-term goal is to develop a means of following

the mechanical parameters associated with the gravitropic bending response in maize roots in order

to build a true biomechanical picture of the process. Additionally, we hope to provide a more

representative study of gravitropic bending by providing mechanical constraints against which the

bending root must act.

Traditional methods for following the gravitropic response involve goniometric measurements of

unrestrained roots bending in humid air. Besides being a highly unnatural environment for roots,

the traditional method affords no opportunity to analyze mechanical factors in the bending

response.

Accomplishments

We have developed a method which allows us to follow the bending force output of the responding

root.

We place the root between two narrowly separated horizontal Teflon" surfaces which act as an

artificial crevice to restrain downward bending. The forces generated in the vertical direction are

measured continuously with a Vitrodyne V-200 mechanical testing frame. The load generated by

the root is measured as a restoring force necessary to maintain constant displacement between the

two Teflon TM surfaces as the root tries to force them apart by bending downward.

Initial characterization of the system shows pH, calcium, and light dependence of the response.

Decapping also eliminates all bending force production by the root. Simultaneous axial growth

measurements reveal a link between gravitropic bending and root elongation.

Characteristic curves representing bending force production over time are shown in Figures 1-3.

Figure 1 shows root bending force output in distilled water buffered to pH 7.0. Figure 2 shows a

dramatic increase in force output in 10 mmol CaC12. Figure 3 shows an equally dramatic

depression of force output when available calcium in the medium is bound by EGTA*. These

curves illustrate the dynamic nature of force production by the root and show a typically

multi-modal character. Superimposed on these plots are curves illustrating overall root extension

determined by simultaneous time-lapse photography.

* Ethylene Glycol-bis (]3-Aminoethyl Ester) N,N,N'.N.-Tetraacetic Acid.

57
11

10

9

8

7

u..

o

_ 5

4

3

2

1

II

H20-- Control -- pH 7.0

Force

Length -- -- --

o

• ._.....f

.1.o•

J

1 2 3 4 5 6 7 8 9 10 11

Time (Hrs)

11

10

9

8

7

_.--.

6 wE

4

3

2

1

-0

Figure 1. Root bending force output in distilled H20.

11

10

9

8

7

_ 5

4

3

2

1

0_

10 mM CaCI 2

Force

Length

/

/

/_.;,....

f

\

r'3 \

I.

_,"

1 2 3 4 5 6 7 8 9 10 11

Time (Hrs)

11

10

9

8

7

6 _t_

4

3

2

1

-0

Figure 2. Increase in root bending force output m _at.:t2.

58
11

10

9

8

7

5

4

3

2

1

1_ _---

A,, r

1 2 3

EGTA 1.0 mM

Force

Length

m

Q

p

4 5 6 7 8 9 10 11

Time (Hrs)

11

10

9

8

7

6

5

4

3

2

1

-0

Figure 3. Depression of root bending force output with calcium in the medium bound by EGTA.

Significance of Accomplishments

The forces produced during gravitropic bending by the root axis are a reflection of differential

growth in the zone of elongation, and they serve two purposes: First, they move the root tip in

space, thereby guiding its growth; and, second, they allow the root to move small obstacles which

lie in its path and aid the root in soil penetration.

With our measurement system, we can follow small changes in the gravitropic response which

reflect the dynamic nature of the controls involved. This may allow us to break down the time

course of the response into changes in root stiffness versus overall root growth. In the future, we

hope to be able to relate the time-variable character of bending force output to local variations in

root extensibility on the upper and lower surfaces of the root.

Other measurements made possible by this system include axial growth rate measurements under

continuous controlled tip load. We hope to extend these measurements to generate work and

power output measurements for gravitropicaUy responding roots.

59
INTERACTIONS OF LIGHT AND ETHYLENE IN STEM GRAVITROPISM

Marcia A. Harrison

Department of Biological

Marshall University

Huntington, WV 25755

Sciences

Description of Research

Increased ethylene production occurs after gravistimulation in many plant stems. However, it is

generally thought that ethylene is primarily involved in modifying the later phase of curvature

rather than regulating early, upward bending.

Conflicting reports exist concerning the effect of light on ethylene biosynthesis. Studies focusing

on etiolated pea shoots have reported light-inhibited as well as light-stimulated ethylene production.

In our previous studies using etiolated pea stems, red-light-inhibited ethylene production has

altered the kinetics and locus of curvature during gravistimulation. Thus, red-light treatment of

etiolated tissue may alter gravitropism by affecting ethylene biosynthesis.

In this study, we examined the regulation of ethylene production during stem gravitropism in

etiolated (given dim green light daily) and red-pretreated, etiolated pea seedlings. We evaluated the

levels of ethylene precursor, l-aminocyclopropane-l-carboxylic acid (ACC), the malonylconjugate of ACC (MACC), and in vivo ACC oxidation.

Accomplishments

(1) In red-pretreated pea epicotyls, decreased ACC paralleled increased MACC levels

within 30 minutes of gravistimulation. Tissue levels of ACC and MACC did not significantly

change during the time course of gravistimulation in etiolated seedlings.

(2) ACC oxidation did not change in either etiolated or red-treated pea epicotyls during

gravitropic curvature.

Significance of Accomplishments

In this research, red-pretreated etiolated pea stems exhibited changes in ACC and MACC levels

during gravitropism. Ethylene biosynthesis was not significantly changed in gravistimulated

etiolated epicotyls. These results suggest light regulation of ACC malonylation and/or ACC

synthesis.

It is proposed that gravistimulation causes a decrease in ethylene biosynthesis via conjugation of

ACC. An ethylene decrease within a restricted zone of the stem's subapical region could result in

the localized gravitropic bending observed in red-pretreated plants. These findings support the

previous observation that inhibition of ACC synthesis (by aminoethoxyvinylglycine) also alters the

kinetics and locus of curvature in etiolated stems in a manner similar to red pretreamaent.

611
REGULATION OF ETHYLENE

CLOSED CANISTERS

Marcia A. Harrison

Department of Biological Sciences

Marshall University

Huntington, WV 25755

BIOSYNTHESIS IN PLANTS GROWN IN

Description of Research

Growth space is a limiting factor in NASA's Space Shuttle experiments and will be critical for the

cultivation of plants in space. Small closed environmental canisters are currently used for plant

studies aboard the Space Shuttle middeck locker facility. Ethylene accumulation in small closed

chambers may limit plant growth and affect the interpretation of results from Shuttle experiments.

Altered ethylene production can result from a change in ACC synthesis, conversion of ACC to

ethylene, or from the conjugation of ACC into MACC. The conversion from ACC to ethylene is

oxygen dependent and requires the enzyme ACC oxidase. Malonylation of ACC serves as a

mechanism to dissipate excess ACC and participates in the regulation of ethylene biosynthesis.

This in_cestigation (supported by the WV Space Grant Consortium) was conducted to determine

changes in ethylene biosynthesis in pea seedlings grown in closed, l-liter canisters, using

measurements of ethylene, ACC oxidase, ACC, and MACC.

Accomplishments

(1) ACC oxidase activity decreased in closed canisters after 5 days of incubation. This

result is most likely due to decreased atmospheric 0 2 and/or increased ethylene levels within the

chamber. Overall slowing of ethylene biosynthesis may occur in longer term experiments within

closed chambers.

(2) Tissue ethylene production and ACC levels did not change in seedlings grown in

closed canisters over the 5-day incubation period. Overall, ACC levels in roots were found to be

greater than those in stems. The accumulation of ACC in roots is consistent with previous reports

that ACC is produced in the roots and transported to the stem where it is aerobically converted to

ethylene.

(3) MACC in roots of closed canisters was greater than MACC in roots of open canisters

after 5 days of incubation. This was accompanied by a slight decrease in MACC in stems of

closed chambers. High conjugation activity in the roots may result in lowered ethylene production

by reducing the amount of substrate (ACC) for ACC oxidase.

Significance of the Accomplishments

The findings suggest that slight changes in ethylene biosynthesis (ACC oxidation and MACC

formation) begin to occur in etiolated pea seedlings grown in small enclosed canisters over the

relatively short incubation period of 5 days. Within this time, ethylene accumulation and oxygen

depletion may begin to affect the ethylene biosynthesis system as well as ethylene-sensitive

enzymes and ethylene-regulated genes involved in plant growth.

61
Publications

Steed, C. and Harrison, M.A. 1993. Regulation of ethylene biosynthesis after short-term

heat-stress in etiolated pea stems. Physiologia Plantarum 87: 103-107.

62
PROBING THE GRAVISENSING

ROOTS

Karl H. Hasenstein

Biology Department

University of Southwestern Louisiana

Lafayette, LA 70504-2451

AND GRAVIRESPONSE SYSTEMS IN

Description of Research

Gravisensing is typically associated with amyloplast displacement. However, an analysis of the

mode of action of amyloplasts is lacking. It is unknown which percentage of amyloplasts needs to

be displaced or whether the act of displacing, or a particular position inside the receptor cells,

constitutes gravisensing. Since reorientation always involves the entire plant and not just

amyloplasts, we attempt to isolate amyloplast movement from other factors.

Instead of displacing the amyloplasts by reorientation, we induce displacement by a high gradient

magnetic field (HGMF). The HGMF is generated between permanent magnets by inserting a

magnetically soft particle in a wedge-shaped form. The transition from high-field intensity in the

vicinity of the wedge to the uniform field between the permanent magnets generates a gradient that

exerts a ponderomotive force on magnetic substances. If a substance, such as amyloplast starch,

which is more diamagnetic than the cytoplasm, is positioned in sufficient proximity to the gradient,

it will be repelled from the gradient (or wedge). This technique is being used to manipulate

amyloplasts in roots and correlate curvature and growth rate with amyloplasts distribution and

forces.

Among other parameters, plant growth depends on the amount and direction of deposition of new

cell wall material. There is substantial evidence suggesting that the orientation of cellulose fibrils is

correlated with the orientation of rnicrotubules (MTs). We study the behavior and possible role of

cortical MTs in growth control in curving roots. Our studies attempt to characterize the time frame

and extent of changes in the cytoskeleton of graviresponding roots. The investigations are based

on indirect immunofluorescence and confocal microscopy.

Accomplishments

(1) The measurement of the parameter that determines the magnitude of the ponderomotive

force, the magnetic susceptibility, is not complete; but we were able to visualize the phenomenon

of a magneto-ponderomotive force acting on starch grains (see Figure I).

Figure 1. Sedimentation of starch

grains in the presence a high gradient

magnetic field (left) and in the absence

of a high gradient field (right). A

ferromagnetic cone (C) was positioned

next to a water-filled capillary

(0.5 mm diameter) between two

10 mm thick SmCo magnets (not

shown). This distorted the otherwise

uniform magnetic field such that the

starch grains moved away from the

cone leaving a space devoid of starch

grains (arrows). In the absence of a

cone, the starch grains sediment

uniformly (right).

63
We tested various combinations of generation of HGMF and the effect on root curvature and found

a high correlation between the fraction of amyloplasts that are displaced and curvature in vertically

oriented or clinostatted roots. We compared roots that were either positioned horizontally (i.e.,

control, showing natural graviresponse, no magnetic gradient applied) or clinostatted (1 rpm) in

two configurations. Vertical clinostatting is when the (horizontal) axis of rotation is perpendicular

to the root/shoot axis (i.e., gravity vector changed longitudinally) of vertically oriented seedlings.

Horizontal clinostatting indicates rotation parallel to the root/shoot axis of horizontally placed

seedlings. Table 1 summarizes the data on growth and curvature obtained in controls and

magneto-stimulated roots.

Table 1. Curvature (degrees, +SE) and growth (mm) of flax (Linum

utitatissimum) roots in the absence or presence of an HGMF.

Horizontal Orientation Vertical Clinostatting Horizontal Clinostatting

Time (no magnetic field) (HGMF) (HGMF)

Growth Growth Growth Angle

34.9+- 11.2

Angle

16.5+_7.4

Angle

1 h 0.76_-+0.5 0.59+-0.16 23.8_+11.6 0.49+-0.24

2 h 47.1+10.2 1.46+-0.59 32.7+10.4 1.25_+0.32 32.74-11.6 1.23_+0.34

The presence of the high-gradient magnetic field induces curvature, and the distribution of

amyloplasts (as measured by microscopical analysis of root caps) is highly correlated with the

extent of curvature. The results suggest that the mode of action of amyloplasts in gravisensing can

be investigated by substituting the force of gravity by a magneto-ponderomotive force. It is also

conceivable that a magnetic field can physiologically substitute the gravity signal under

microgravity conditions.

(2) The studies of cortical MTs of graviresponding corn roots (cv. Merit) revealed a

wealth of information. The most important result is that there seems to be a graviresponse-specific

reorientation of MTs in the lower cortex but not lower epidermis of roots, while MTs of the upper

cortex are not affected. MTs in cap cells also reorient after gravistirnulation. Dark-grown roots of

this variety typically do not curve and also do not exhibit MT reorientation along the lower side of

the cortex. IAA application to vertically growing roots resulted in MT reorientation in regions

ranging from 1.8 mm (10 -6 mol) to the maturation zone (10-s mol). However, IAA causes MT

reorientation in light and dark grown roots.

Significance of the Accomplishments

We were able to induce amyloplast displacement using HGMF in root caps of flax. The curvature

obtained under conditions of dynamic weightlessness (clinostatting) or vertically oriented roots

suggests that the amyloplast position, rather than directional movement, induces changes in

differential growth.

We have established that cortical microtubules behave in a highly specific manner during

gravicurvature of roots.

Publications

Blancaflor, E.B. and Hasenstein, K.H. 1992. Organization of microtubules in curving corn

roots. ASGSB Bulletin 6: 53.

Blancaflor, E.B. and Hasenstein, K.H. 1993. Kinetics of microtubule reorientation in graviresponding maize roots (Abstract). ASGSB Bulletin 7: 58.

Blancaflor, E.B. and Hasenstein, K.H. 1993. Kinetics of microtubule reorientation in graviresponding maize roots (Abstract 677). Plant Physiology 102: 120.

64
Blancaflor,E.B. andHasenstein,K.H. 1993. Organizationof microtubulesin graviresponding

cornroots.Planta 191: 231-237.

Kuznetsov, O.A. and Hasenstein, K.H. 1993. Novel approaches to investigate the gravisensing

system of plants (Abstract). ASGSB Bulletin 7: 92.

65
GRAVITROPIC RESPONSE MECHANISM IN CEREAL GRASS SHOOTS

Peter B. Kaufman, Liu-Lai Wu, and Donghern Kim

Cellular and Molecular Biology Group

Department of Biology

University of Michigan

Ann Arbor, MI 48109-1048

Description of Research

The primary goal of our NASA Space Biology Program research project is to unravel the

gravitropic response mechanism in cereal grass shoots. Our objectives are threefold: (1) to show

how gravity perception occurs, (2) to understand the signal transduction process, and (3) to

delineate the key physiological and molecular steps represented in the response mechanism

cascade. The working model we are currently using, which depicts the key steps for gravity

perception, signal transduction, and response in cereal grass shoot pulvini, is shown in Figure 1.

Portions outlined in solid-line rectangular boxes have been demonstrated by us, and others, to

occur in graviresponding cereal grass shoot pulvini. Portions outlined by dotted rectangular boxes

are assumed to occur but have not been directly characterized.

In our cereal gravitropism research, we are focusing on two major components of this model: (1)

how auxin-mediated signal transduction occurs in graviresponding pulvini, and (2) early molecular

events involving gravity/hormone-enhanced invertase gene expression that starts the response

mechanism cascade in cereal grass pulvini.

Accomplishments

(1) Gravitropic tissue elongation of oat shoot leaf-sheath pulvini at

different pHs: The effects of the buffered pH treatment on the gravitropic response and

pulvinus elongation in oat shoots has been measured. After 24 hours of the gravistimulation under

buffered pH conditions, the gravitropic curvature response and gravitr.opic tissue elongation of the

bottom flank of the pulvini were determined. Figure 2 shows the gravltropic bending response and

the gravitropic elongation growth of the bottom half of oat pulvinus segments. The following

points can be made: (a) The most acidic conditions (pH 4.5) promote the elongation of "bottom"

halves of pulvini by 52.4 percent of their original length. (b) "Bottom" halves at pH 5.5 and 6.5

elongate by 45.2 and 35.7 percent of their original lengths, respectively. (c) The highest tissue

elongation of the unbuffered control "bottom" halves (by 64 percent of the original length) is not

significantly different from pulvinus elongation at pH 4.5. (d) Under these buffered pH

conditions, gravitropic curvature responses of pulvinus segments containing "bottom" halves are

reduced to 62.9 to 55.7 percent of that of the unbuffered control pulvinus segments. However,

such variation in the reduction of gravitropic curvature is not statistically significant.

66
I

IGRAVITY PERCEPTION I _ [

/',, I

I REGULATION GENE EXPRESSIOOF |N I SIGNAL TRANSDUCTION I

Fall of starch statoliths (within 1 rain.)

Gravity perception requires 60 to 120 sec.

----4_

LOWER HALF ...................ASYMMETRIC UPWARD BENDING RESPONSE

7

Activation of mechanotransductive ion

I channels I

I Calcium ion transport into statocyte cells I

Modification of an auxin binding protein

I Asymmetric activation of proton pump I

Asymmetric distribution of free IAA and

GAs across the width of pulvinus tissue

caused by hormone transport, by release

from hormone conjugate, and/or

enhanced synthesis

I Increased efflux proton I I Increased celiwallh,drolases activities of[[ [invertase[genes Upregulation of i

',,, /

Cell wall loosening occurs

Cell elongation ]

I

Invertase mRNA increases,

starting within 1 h [

Istarting within 2 to 3 hrs I

I" I

. Water uptake & solute .

(1 increase inpulvinus _-_

Pressure

UPPER HALF

,L

Increased and/or secretion cellulose activity I

Cell walls are loosened I

and will respond to I

gravistimulation when [

pulvini are rotated 180°I

I"Ev"e'nt_'liy, [ rigidifydue cell to wall walls I[

synthesis, lignification,

Hydrolysis of sucrose and [ I and/or silicification I

release of hexoses (D-glucose

& D-fructose increase) I

L.

r

Cell walls thicken

I"-- I

Cell wall rigidities by,

I lignification and/or I

I silicitication I

I

ener_ Hexoses forprovide metabolicsubstrate pathwa_,s & ]

I Cell wall synthesis increases I

Figure 1. Working model for gravitropic response mechanism.

67
(2) Changes in sensitivity to exogenous indole.3-acetic acid (IAA) of

gravistimulated oat shoot pulvini: The elongation of oat pulvini has been measured after 24

hours of gravistimulation treatment in the presence of various concentrations of IAA. Figure 3

shows the relative growth of the top and bottom sides of the gravistimulated pulvini, as well as

vertically-held control pulvini, as compared to their original lengths. The following points can be

made: (a) The maximum growth of vertically-held pulvini occurs at 10-5 M IAA. (b) The top part

of the graviresponding pulvini shows the same pattern of dose-response tissue growth induction

by IAA as vertically-held pulvini. (c) The bottom parts of the graviresponding pulvini show

maximum growth induction by IAA as vertically-held pulvini. (d) The bottom parts of the

graviresponding pulvini show maximum growth at 10-8 tool IAA. (e) A difference in the

responsiveness to exogenous IAA between top and bottom halves of the graviresponding pulvini is

about 1000 times.

(3) Kinetic induction of invertase mRNA in graviresponding oat shoot

pulvini: Northern blot analysis was performed using the partial-length invertase cDNA (PCR

product) as a probe. Results show that a 1.90 kilobase (Kb) invertase mRNA is detected from the

pulvini of oat plants (Figure 4A). The level of this 1.90 Kb mR.NA is very low at time zero, but it

is significantly induced 1 hour after initiation of gravistimulation treatment, after which time it

decreases. In order to examine the induction pattern of the gravistimulated invertase mRNA in

more detail, mRNA was separately isolated from top and bottom halves of oat-shoot pulvini after

different times of gravistimulation. As shown in Figure 4, there is a clear pattern of kinetic

changes in invertase mRNA level during the gravitropic response of the respective halveg of oat

shoot pulvini. The 1.90 Kb mRNA was detected at a relative low level in the top halves of the

pulvini (Figure 4B). Interestingly, this mRNA was shown to occur at a very high level in the

bottom halves of the pulvini (Figure 4C). The amount of mRNA rapidly increases to a peak at

1 hour after initiation of gravistimulation, and then it gradually decreases afterwards. Using a

densitometer, the maximum level in these bottom halves of the pulvini that occurs at 1 hour

represents about a five-fold increase above that of the time zero control (vertical pulvini).

(4) Genomic invertase gene(s) estimation: Genomic DNA from oat plants was

purified and digested with seven restriction enzymes to estimate the size of invertase gene(s). Four

bands were detected using the partial-length invertase cDNA as a probe (Figure 5). One band

showed a very strong hybridization to the probe, indicating that oat contains only one locus

corresponding to the partial-length invertase cDNA. The other weakly hybridizing bands may

correspond to other members of the invertase gene family.

68
z

o

1--

(.9

Z

o

_1

iii

u.I

3

co

(/)

I--

180

160

140

120

100

GRAVtTROPIC CURVATURE

"nSSUE ELONCC_TION

I I I I

C) "O "O '_

0 -r -r -1-

Z

= ;.,I o_ tn

O

I--

50

4O _<

--I

o

30

0

20 0

lO _

-I

c

:o

0 m

Figure 2. Gravilropic tissue elongation and bending response of the bottom side of oat shoot

pulvini under different buffered pH conditions. Initial pH of the control was 6.5.

A

-r- 200

P

0

rr 160

m 120

if)

r--- 8o

LU

40

,<

--I

U.I

e¢"

--e- VERTICAL

-e-TOP

--e--BOSOM

0 I I I I I I I I

CONTROL 10 .8 10 .6 10 .4 10 .2

CONCENTRATION OF IAA (M)

Figure 3. Relative pulvinus growth induced by IAA with or without gravislimulation.

69
A -25S

-I.90 Kb

-I 8S

-I.90 Kb

C

• k

-I.90 Kb

0 1 3 6 1224h

Time

Figure 4. Northern blot analysis of oat shoot invertase mRNA isolated from gravistimulated oal

shoot pulvini. Poly(A)+RNA (2 ttg/well) was elecm3phoresed in I percent asarose gel, blotted

onto a nylon memtnane, and hybridized with a-32PdCTP-labeled invenase cDNA (PCR product) as

a probe. Figta_e 4A. mRNA isolated from intact pulvini. Figure 4B. mRNA isolated from the top

halves of the pulvini. Figure 4C. mRNA isolated from bottom halves of the pulvini. Lanes 1-6

represent mRNA isolated from oat pulvini during different times of gravisbmulafion. Lane l:

0 hour is the vertical control. Lane 2: gravistimulated for 1 hour, Lane 3:3 hours, Lane 4:6

hours, Lane 5:12 hours, Lane 6:24 hours of gravistimulazion, respectively.

7()
M

% % % '_ %

23.1m ......

9.4--

6.6-- _ -.* u_ It -,m

4.4 -- g D

0.6

Figure 5. Southern blot analysis of oat genomic DNA. DNA (25 lxg/well) was digested with restndion

enzymes and electrophoresed on a 0.8 percent agarose gel. The gel was denatured, neutralized, and DNA

transferred onto a nylon membrane filter. The filter was hybridized to digoxgenin-dUTP-labeled partiallength invertase cDNA as a probe. The bands were detected with Lumi-Phos 530 (Boehringer-Mannheim

Biochemicals) and exposed to X-ray film for 30 minutes at room temperature with an intensifying screen.

M: DNA molecular weight standard markers. Restriction enzymes used are: EcoR !, BamH !. Hind Iii,

Kpn. I, Pst !, Hae IH, and Sac i.

Significance of the Accomplishments

Finding 1: Results in accomplishment (1) indicate that the gravitropic tissue elongation of oat

pulvini is explained partly by the "Acid Growth Theory." This conclusion is based on the

observation that the tissue elongation of the bottom sides of the graviresponding pulvini is

enhanced more under acidic buffer conditions as compared to that under neutral buffer conditions.

However, the results also demonstrate that a substantial portion of the gravitropic bending

response is not affected by the environmental pH, since buffered pH treatment of oat pulvini does

not nullify the gravitropic response of the pulvini, regardless of the pH of the buffer solutions.

Finding 2: When oat pulvini are gravistimulated in the presence of IAA, tissue elongation of

bottom and top sides of the pulvini shows the different dose-dependent response to IAA. These

results demonstrate that the bottom halves of graviresponding pulvini respond to exogenously

supplied IAA with 1000 times higher sensitivity than do the top halves of the pulvini and vertically

held control pulvini. These results demonstrate that tissue sensitivity changes in oat shoot pulvini

elicited by gravistimulation treatment is a very important component of the signal transduction

process in the gravitropic response mechanism.

Finding 3: We observed an induction of invertase mRNA by gravistimulation treatment in top and

bottom halves of oat shoot pulvini. We report here that there is a very low level of invertase

mRNA before the onset of gravistimulation of oat shoot pulvini. However, the mRNA level

dramatically increases to a maximum at 1 hour following the gravistimulation treatment and

gradually decreases afterwards (Figure 4). This indicates that the induction of invertase mRNA by

gravistimulation of oat pulvini is rapid. Particularly, we observed that the level of invertase mRNA

71
in the lower halves of pulvini was much higher than that in the top halves during the

graviresponse,with aboutthree-to five-fold increasesobservedat thespecifiedtimes(Figures4B

and 4C). We here demonstratefor the first time that the expressionof invertasegenesis

up-regulatedby gravistimulationandisanearly,essentialmolecularstepin thecascadeof events

thatoccursin thegravitropicresponsemechanism.

Finding4: Southernblot analysisshowedthat onegenomicDNA fragmentfrom oatsstrongly

hybridizesto thecDNA probe,andthreefragmentsshowa weakhybridizationsignal(Figure5).

This indicatesthat theremay bean invertasegenefamily that existsin oat plantsand that the

partiallengthcDNA couldbeoneof thegenesin thefamily encodingfor a particularisoformof

oat invertase. This is, to our knowledge,the flu:sttime that aninvertasegenefamily hasbeen

shownto exist in oat plants. Eachof theseinvertasegenescould be differently regulatedby

gravistimulation.

Publications

Brock, T.G., Burg, J., Ghosheh, N.S., and Kaufman, P.B. 1992. The role of calcium in

growth induced by indole-3-acetic acid and gravity in the leaf-sheath pulvinus of oat (Avena

sativa). Journal of Plant Growth and Regulation 11: 99-103.

Brock, T.G., Kapen, E.H., Ghosheh, N.S., and P.B. Kaufman. 1991. Dynamics of auxin

movement in the gravistimulated leaf-sheath pulvinus of oat (Avena sativa). Journal o_"Plant

Physiology 138: 57-62.

Brock, T.G. and Kaufman, P.B. 1991. Elongation growth of the leaf sheath base ofAvena sativa

seedlings: Regulation by hormones and sucrose. Plant Physiology (Life Science Adv.) 10: 125-

130.

Brock, T.G. and Kaufman, P.B. 1991. Growth regulators: An account of hormones and growth

regulation. In: Plant Physiology: A. Treatise, Vol. X. Growth and Development, Chapter 5

(Bidwell, R.G.S., Ed.). New York: Academic Press, Inc., pp. 277-340.

Karuppiah, R. and Kaufman, P.B. 1992. A rapid and inexpensive micro-elution method of

nucleic acid and protein from agarose and polycrylamide gels. Biotechniques 13(3): 368.

Kim, D., Brock, T.G., Karuppiah, N., and Kaufman, P.B. 1992. Green and non-green callus

induction from excised Ace (Oryza sativa) embryos: effects of exogenous plant growth regulators.

Plant Growth Regulator Society of America Quarterly 20 (4): 189-199.

Lu, C.R., Kim, D., and Kaufman, P.B. 1992. Changes in the ultrastructure of cell walls,

cellulose synthesis, and glucan synthase from gravistimulated pulvini of oat (Avena sativa).

International Journal of Plant Science 153(2): 164-170.

Wu, L.-L., Song, I., Karuppiah, N., Kaufman, P.B. 1993. Kinetic induction of oat pulvinus

invertase mRNA by gravisfimulation and partial cDNA cloning by polymerase chain reaction. Plant

Molecular Biology 21:1175-1179.

72
GRAVITROPISM AND

RHIZOIDS

John Z. Kiss

Department of Botany

Miami University

Oxford, OH 45056

GOLGI APPARATUS FUNCTION IN CHARA

Description of Research

The purpose of this research is to characterize the gravitropism pathway in Chara rhizoids and to

determine the role of the major organelles and structures in each step of the pathway. We have

been using rhizoids of the alga Chara as a model system since they are strongly gravitropic and

since all stages (i.e., perception, transduction, and response) of gravitropism occur in a single cell.

In contrast to higher plants which appear to utilize amyloplasts as statoliths in gravity perception,

Chara rhizoids have single membrane-bound compartments located near the cell apex that function

as statoliths. These vesicles settle to the new lower cell wall within minutes following horizontal

reorientation.

Our specific goals have been (1) to reappraise the ultrastructure of Chara rhizoids with special

emphasis on organelles and other structures related to the gravitropic response by using

cryofixation and immunocytochemical techniques; (2) to analyze the structure and biosynthesis of

statoliths in order to determine their role in the development of gravitropic competence.

Accomplishments

(1) Chara rhizoids can be successfully frozen and preserved by

cryofixation. We can now routinely cryofix rhizoids by high-pressure freezing. Compared to

chemical fixation, rhizoids prepared by cryofixation showed a number of significant stractural

differences, such as improved preservation of the Golgi apparatus and cell wall and improved

preservation of antigenic determinants (see Kiss and Staehelin, 1993).

(2) Antibodies against complex polysaccharides of higher plants cross-react

with Chara rhizoids in a specific manner. Results to date show that several antibodies

against higher plant pectic polysaccharides cross-react with Chara rhizoids. These include a

polyclonal antibody to polygalacturonic acid/rhamnogalacturonan I (PGA/RG-I), a monoclonal

antibody to methyl-esterified pectin (JIM 7), a monoclonal to de-esterified pectin (JIM 5), and a

monoclonal antibody to RG-I (CCRC-M2). The binding of these antibodies is specific, as

indicated by controls including preabsorption of a particular antibody with its appropriate

carbohydrate. These antibodies bind to the Golgi apparatus, apical vesicles, and the cell wall.

(3) The statolith compartment in Chara rhizoids contains carbohydrate and

protein in addition to the previously reported barium sulfate crystals. We have used

cytochemical and immunocytochemical assays to better characterize the statolith compartment.

Coomassie Brilliant Blue cytochemistry indicates that statoliths contain protein. In addition,

CCRC-M2 (a monoclonal antibody to RG-I) labels the statoliths (Figure 1), which indicates that

these compartments contain a carbohydrate matrix.

(4) The response to gravity is correlated with the number of statoliths in

Chara rhizoids. Differential-interference-contrast microscopy demonstrated that rhizoids from

soil water (SW) medium grown plants typically contain 50-60 statoliths per cell while rhizoids

from artificial pond water (APW) grown plants contain 5-10 statoliths per cell. RhJzoids from SW

are more responsive to gravity than rhizoids from APW since (1) SW rhizoids were oriented to

73
gravity during vertical growth while APW rhizoids were relatively disoriented, and (2) curvature of

SW rhizoids was three to four times greater throughout the time course of curvature (Figure 2).

The growth rate of APW rhizoids was significantly greater than that of SW-grown rhizoids.

S

Figure 1. Ultrathin section of a Chara rhizoid that was immunolabellcd with the CCRC-M2

antibody (against the carbohydram RG-I) followed by a secondary gold label. The gold particles arc

localized in the statolith (S) compartment (arrowheads), and label also is present in vesicles. Scale

bar-- ltun.

8O

70

_ 6o

• 5o

_ 40

_ 3o

_ 2o

_ lO

0 o

o

./+/+

4 8 12 16 20 24

Time (hours)

Figure 2. Time course of curvature of Chara rhizoids from plants grown in either soil-water or

artificial pond water (APW) medium. The downward curvature of rhizoids was measured following

placement n a horizontal orientation. Data points are mean curvature :t SE (15 < N < 2A).

Significance of the accomplishments

Finding 1: In previous studies chemical fixation of rhizoids was employed, which can cause

numerous well-documented artifacts. However, our data suggest that the new methodological

approach of cryofixation will yield insights regarding the role of the Golgi apparatus and the cell

wall in the differential growth in response to gravity. Cryofixation will also allow us to accurately

determine antibody labeling patterns in the rhizoids (see below).

Finding 2: The specific binding of higher plant antibodies to Chara is consistent with the fact that

cell walls in Characean algae are biochemically similar to higher plant cell wails; i.e., they axe

composed of cellulose, hemicellulose, pectin, and protein in a similar ratio. Our battery of

antibodies will allow us to investigate the vesicle blockage hypothesis (for the response

74
mechanism)sinceapicalvesiclescanbedividedinto severalcategoriesdependingonsize,staining

patterns,andantibodylabeling.

Finding3: Previousreportshavedemonstrated that the statolith compartment contains barium

sulfate crystals. Our studies demonstrate that this compartment is more complex in that, in addition

to these crystals, carbohydrate and protein also are present. Improved knowledge about the

composition of the statolith compartment is important for understanding statolith synthesis and the

precise role of statoliths in gravitropism.

Finding 4: While there have been several reports which show a correlation between the presence

of statoliths and gravitropic sensitivity, our studies provide strong evidence for the importance of

statoliths in Chara rhizoids. In addition, this system is in many ways comparable to the studies

with higher plant starch-deficient mutants (Arabidopsis and Nicotiana) which provide support for a

statolith-based mechanism of gravity perception in plants.

Publications

Kiss, J.Z. 1992. Gravitropic competence in developing Chara rhizoids (Abstract). ASGSB

Bulletin 6: 75.

Kiss, J.Z. 1993. Changes in statolith density in Chara rhizoids (Abstract). ASGSB Bulletin

7: 94.

Kiss, J.Z. and McDonald, K. 1993. Electron microscope immunocytochemistry following

cryofixation and freeze substitution. Methods in cell biology 37:311-341.

Kiss, J.Z. and Staehelin, L.A. 1993. Structural polarity in the Chara rhizoid: A reevaluation.

American Journal of Botany 80: 273-282.

75
ANALYSIS OF THE LAZY-2 GRAVITROPIC MUTANT OF TOMATO

Terri L. Lomax

Department of Botany and Plant Pathology

Oregon State University

Corvallis, OR 97331-2902

Description of Research

We are studying a tomato mutant that appears to provide an ideal system with which to test the

theories pertaining to shoot gravitropism. The shoots and petioles of the single gene-recessive,

gravitropic mutant lazy-2 (lz-2) exhibit the striking phenotype of downward curvature in

response to gravity. That is, curvature of lz-2 shoots is in the same direction as the gravity vector

(positive gravitropism), whereas the normal response of shoots is upward curvature (negative

gravitropism). The roots of lz-2 are wild type in their response to gravity. The abnormal

downward curvature of the shoots is manifested only in the light. When lz-2 plants axe grown in

the dark, the shoots exhibit a wild-type gravitropic response, and they revert to the wild-type

phenotype if returned to the dark after illumination. Other than the gravitropic response of the

shoots and petioles, lz-2 plants appear completely normal; they are v_gorous and healthy, are

easily crossed, and produce many fruit and plenty of seed.

Since only the gravitropic response of the lz-2 shoots is affected and there are no other apparent

phenotypic abnormalities, a genetic lesion specific to some aspect of the shoot gravitropic

pathway is indicated. There are many other documented agravitropic or sluggishly gravitropic

mutants which are generally caused by starch deficiencies or are of a pleiotropic nature.

However, we know of no other mutant which can actually carry out gravttropism, but in a

direction reversed from wild type. We have shown I that lz-2 plants can perceive gravity and also

have the ability to respond with asymmetric growth but that some component of signal

transduction appears to be reversed. These studies also demonstrated that the light-induced

switch from negative to positive gravitropism in lz-2 is potentiated by red light. This indicates

the involvement of the photoreceptor phytochrome.

We have now established that phytochrome is indeed the pigment involved in regulating the lz-2

phenotype. 2 The red light induction of downward growth can be reversed by a far red light

pulse, and downward curvature reaches a maximum 16 hours after a I-hour red light pulse, after

which dark reversion is observed. Fluence response curves indicate that this response has

characteristics of both low-fluence and high-irradiance phytochrome responses.

The physiological characterization of lz-2 has allowed us to test the sufficiency of the

Cholodny-Went theory for plant gravitropic responses. The Cholodny-Went theory would

predict that in plants which have been exposed to red light, the IAA asymmetry during

gravitropism is reversed in lz-2 as compared to "wild type". This would lead to a growth

asymmetry resulting in greater cell elongation on the upper side relative to the lower side, thus

causing downward curvature. In order to test this hypothesis, we have measured the relative

growth rates on either side of both lz-2 and wild-type seedlings, either gravistimulated or not.

These studies have shown that, as compared with "wild type", red light stimulates growth on

both sides of the stem, with the highest growth rate on the upper side. 3 This results in the

downward curvature of the lz-2 stems. The increase in growth rate on both sides of lz-2 stems

after light induction indicates that the reversed curvature is not a result of increased

concentrations of IAA inhibiting growth on the lower side. Rather, it suggests that either

increased IAA is transported to the upper side or an inhibitor is removed.

76
Duringthisgrantingperiod,wealsobegan genetic studies designed to both map the lz-2 lesion to

its chromosome and to study the effect of creating double mutants with other tomato gravitropic

and phytochrome mutants. For mapping, we created an interspecific cross between lz-2

(in Lycopersicon esculentum) and a wild tomato species, Lycopersicon pennellii. The FI

progeny from this cross has been grown to maturity and allowed to self. Genomic DNA from the

resulting F2 plants will now be used with restriction fragment length polymorphism (RFLP)

probes to determine the chromosomal linkage of the lz-2 lesion and to place the lz-2 gene on the

extensive tomato RFLP map.

To study the interaction between two distinct mutations in the gravitropic mechanism, we

constructed a double mutant between lz-2 and diageotropica (dgt), a tomato mutant which

responds sluggishly to gravistimulation and which is insensitive to exogenously applied auxin.

The resulting Iz-2/dgt double mutants were both selfed and backcrossed to the respective parents

to establish that they are truly double mutants. Interestingly, the plants carrying both mutant

genes have an lz-2 type shoot gravitropic response (positively gravitropic) and a dgt-like root

response (agravitropic), slower shoot gravitropic response, and auxin insensitivity. This

indicates that the two genes do not complement each other (and are, thus, not the same) and that

one is not dominant over the other. Since dgt is affected in its auxin responsiveness, these results

indicate that auxin is involved in the reversed gravitropic response of lz-2 and that the dgt gene is

likely to lie downstream of lz-2 in the gravitropic signal transduction pathway.

References

IGaiser and Lomax, 1992.

2GaJser and Lomax, 1993.

3Kim and Lomax (in preparation).

Publications

Gaiser, J.C. and Lomax, T.L. 1992. The gravitropic tomato mutant lazy-2 is altered in signal

transduction. In: Progress in Plant Growth Regulation (Karssen, C.M., van Loon, L.C., and

Vreugdenhil, D., Eds.). Dordrecht, The Netherlands: Kluwer Academic Publishers, pp. 928-937.

Gaiser, J.C. and Lomax, T.L. 1993. The altered gravitropic response of the lazy-2 mutant of

tomato is phytochrome-regulated. Plant Physiology 102: 339-344.

Lomax, T.L., Coenen, C., Gaiser, J.C., Hopkins, R., Rayle, D.L., and Rice, M.S. 1993. Auxin

perception and the regulation of tomato growth and development. In: Molecular Biology of

Tomato: Fundamental Advances and Crop Improvement (Yoder, J., Ed.). Lancaster, PA,

Technomic Publishing Co., pp. 129-138.

Verhey, S.D. and Lomax, T.L. 1993. Signal transduction in vascular plants. Journal of Plant

Growth Regulation 12: 179-195.

77
MECHANICAL STRESS REGULATION OF PLANT GROWTH AND

DEVELOPMENT

Patricia N. Myers and Cary A. Mitchell

Center for Plant Environmental Stress Physiology

Department of Horticulture

Purdue University

West Lafayette, IN 47907-1165

Description of Research

This project has two main objectives: (1) To characterize plant responses to mechanical stress; and

(2) To determine the physiological mechanisms of the responses.

Mechanical stresses, forces, or stimuli are natural occurrences in the growth and development of

higher plants on earth. Whether these forces occur as gravity, wind, or seedling movement

through the soil, they affect the growth, development, and orientation of seedlings and mature

plants. These physical forces not only inhibit growth and development, but also strengthen plants

against subsequent physical and environmental stresses. The dark-grown soybean (Glycine max

Merr. cv. 'Century 84') seedling has been selected as a model system for characterizing and

analyzing events involved in the inhibitory response because of its rapid growth rate and extreme

sensitivity to mechanical perturbation. Thigmic (contact rubbing) stress is used as the model for

seedling movement between abrasive soil particles during emergence. A l0 second application of

thigmic stress is accomplished by 20 upward strokes with the thumb and forefinger to the apical

hook and subtending 5 mm of the hypocotyl. The typical response to a single application of

thigmic stress is a temporary inhibition of hypocotyl elongation by the dark-grown seedling.

Understanding the early events and controlling factors of the growth response will better enable us

to provide proper growth conditions in a space environment.

Accomplishments

Characterization of seedling responses to a single episode of thigmic stress has been a primary

focus. We have defined several characteristics and have determined several factors affecting the

response:

(1) The response of dark-grown seedlings to a single (10 seconds) application of thigmic

stress is very rapid (seconds) and dramatic (up to 90% inhibition of elongation). The initial

response is followed by a long (hours) recovery period which is responsible for the cumulative

effect of reducing hypocotyl length. After 24 hours, thigmo-stressed seedlings are as much as

30% shorter than unperturbed control seedlings.

(2) The cumulative response to thigmic stress by seedlings of the same starting height and

equivalent rates of elongation was affected by the time it took for seeds to germinate (Figure 1).

Seedlings were selected for planting 24, 48, or 72 hours after imbibition, when the radicle length

reached 1 era. Seedlings in the earliest germinating group (radicle length of 1 cm, 24 hours after

imbibition) had the greatest response to thigmic stress; seedlings had 30% shorter hypocotyls than

did control seedlings 24 hours after stress application. Seedlings grown from later germinating

seeds, 48 or 72 hours after imbibition, had a diminished response to thigmic stress resulting in

seedlings 25% or 15% shorter, respectively, than unperturbed control seedlings in each

germination group.

78
A

E

o 12

e-

.o

•_ 10

O1

C

O

m a

i

o 6

0

¢,j

"1" 4

N 2

E

° 0

I I I I

Planted 24 h after start of Imbibltion:

[3 Control _1_

• Thigmic jl_ /,.

Planted 48 h after imb'biti°n: //_ /.,_

Z_ Control .1_ _ /

• Thigrnic _ .__/ /

Planted 72 h after: _ /,_ ,_ /_

0 Control izl/ _/It K / _'

• Thigmic _'-_ ./S

Stress Stress

, , I = I

48 72 96 120

Time After Imbibition (h)

Figure 1. Response to a single episode of thigmic stress (arrow) by dark-grown soybean seedlings differing in age

(time after imbibition to reach a radicle length of 1 cm) at time of planting. Once emerged, all three undisturbed

control groups elongated at the same rate and were at equivalent heights when stress was applied. Bars represent +_.

the standard error of the mean of 32 to 36 seedlings.

(3) Although the initial, rapid response to thigmic stress was consistent among all

treatments, recovery time was variable and dependent on nutritional status of the seedling.

Increased Ca2+ in the growth medium from 1 to 5 mM increased hypocotyl elongation rate from <1

mm.h-I to >2 mm-h'l. Recovery time following a single episode of thigmic stress decreased

significantly from >9 hours to <3 hours by increasing Ca2+ from 1-5 mM in the growth medium.

Supplementing the medium with 5 mM K + further enhanced recovery time.

(4) After elongation rate had recovered for thigmo-stressed seedlings, the solute potential

of solution expressed from the elongation zone of the hypocotyl was significantly less than that of

unperturbed control seedlings (-0.96 vs. -0.80 MPa).

Significant of Accomplishments

Finding #1: The initial response of soybean seedlings to a sing!e episode of thigmic stress is so

rapid that we hypothesize a turgor collapse of cells in the elongauon zone of the hypocotyl may be

occurring. We currently are investigating this possibility using a pressure microprobe.

Finding #2: The rapid recovery from thigmic stress by seedlings selected for long (>48 hours)

germination time suggests that any environmental stress prior to, or during, germination which

slows the germination process, may render the seedlings more resistant to the long-term effects of

mechanical stress.

Finding #3: The variability of recovery time can be accounted for by the pre-stress physiological

condition of the seedling and the nutrient composition of the culture medium. Time for recovery to

occur can be enhanced by increasing the Ca2+ level, as well as by including 5 mM K ÷ in the

growth medium.

79
Finding #4: The decreased solute potential in the elongation zone following growth rate recovery

may indicate that the stressed seedlings were adjusting osmotically in order to regain and maintain

the pre-stress elongation rate and turgor potential.

These findings not only aid our basic understanding of stress-induced growth inhibition, but also

will help in controlling the effects of a stressful environment. Potential application to the field of

gravitational and space biology is to provide information to assist in plant culture in the space

environment and to understand the mechanisms by which plants respond to physical forces such as

gravity and mechanical stress.

Publications

Jones, R.S. and Mitchell, C.A. 1992. Effects of physical agitation on yield of greenhousegrown soybean. Crop Science 32: 404-408.

Mitchell, C.A. 1992. Modification of plant growth and development by acceleration and

vibration: concerns and opportunities for plant experimentation in orbiting spacecraft. Advances

in Space Research 12: 219-225.

Myers, P.N. and Mitchell, C.A. 1992. Factors affecting short and long term growth responses to

mechanical stress by dark-grown soybean seedlings (Abstract 162). ASGSB Bulletin 6(1): 98.

Myers, P.N. and Mitchell, C.A. 1992. Mechanical-stress inhibition of hypocotyl elongation in

dark-grown seedlings (Abstract 778). Plant Physiology 99 (1, Suppl.): 131.

Myers, P.N. and Mitchell, C.A. 1993. Modification of mechanical stress effects on elongation of

dark-grown soybean by Ca2+ and K + (Abstract In Press). HortScience.

Myers, P.N., Ackerman, J.D., and Mitchell, C.A. 1993. Growth and turgor responses of darkgrown soybean seedlings to mechanical perturbation (Abstract 892). Plant Physiology 102 (1,

Suppl.): 156.

Myers, P.N., Coe, L.L., Jones, R.S., and Mitchell, C.A. 1991. Investigations of the

biophysical basis for mechanical stress inhibition of hypocotyl elongation by dark-grown soybean

seedlings (Abstract N-18). ASGSB Bulletin 5( 1): 79.

Myers, P.N., Cutler, J.W., and Mitchell, C.A. 1993. SuperSorb-C as a growth matrix for Ca2+

nutrition studies of dark-grown seedlings (Abstract In Press). HortScience.

8O
TRANSDUCTION OF THE GRAVITROPIC STIMULUS

Barbara G. Pickard

Department of Biology

Washington University

St. Louis, M0 63130-4899

Description of Research

The short-range goals of our recent research, funded partly by the Space Biology Program and

partly by the Innovative Research Program of the Office of Space Science and Applications,

were (1) to identify modulators, promoters, and inhibitors of an ion channel that putatively serves

as a gravitropic sensor for higher plants; (2) to utilize such modulators and modifiers for

biochemical characterization of the channel; and (3) to study diverse roles of the channel.

The long-range goals of our research program, now several years old, have been (1) to

characterize the primary transducer of the gravitropic stimulus; (2) to test the hypothesis that this

mechanotransducer acts as a signal integrator in plant development and maintenance; and (3) to

work out the s_uctural, biochemical, and physiological features of the control system in which

we believe the transducer is embedded.

Accomplishments

In our last report, we described a stretch-activated ion channel and provided evidence that it is a

primary gravitropic signal transducer. Activity of this channel was shown to be strongly

modulated by transmembrane voltage and by temperature, so the channel can serve as a

multimodal signal integrator.

Acid and the regulation of growth. We have now shown that acid in the free space of the cell

wall also strongly modulates channel activity. Since one of the effects of gravitropic stimulation

is often a spatially patterned shift in wall pH, this modulation suggests the occurrence of a

variety of feedback loops in which the channel may be critically involved.

In particular, we have developed a model purporting to explain how mechanical and electrical

stimuli could regulate the rate and the vectors of cell expansion during straight growth, as well as

control the net direction of the mediational hormone auxin during the asymmetric growth

required for gravitropic curvature.

The feedbacks of this model involve many steps, as would be expected of complex cellular

regulation, and are summarized in a paper devoted to the topic (Pickard and Ding 1993) Briefly,

the model suggests that Ca2÷ passing inward through the sensory channels results, via a "signal

transduction chain," in pumping of H+ into the wall, loosening it, and allowing it to yield to cell

turgor and expand. But expanding the wall around a set of channels creates a weak zone around

them; and, therefore, mechanical stress such as is always experienced by the turgid cells will be

focused on that group of channels, leading to a positive feedback between channel opening and

wall loosening. Damping of channel activity by increasing wall acidity, however, could replace

such runaway behavior with a regulatory negative feedback and allow continual adjustment of

the local rate of wall expansion so that reasonable uniformity of the wall is maintained and so

that neighboring cells maintain their fit in the tissue.

Other responses to Ca2+ and H+ are postulated to address the vectorial control of expansion.

When Ca2+ in the cytosol adjacent to the activated channels reaches a certain threshold,

81
microtubulesarepossibly releasedfrom certainproteinsthat moorthemto thecell membrane.

At the sametime, acid at the other sideof the membranemay freethe mooringproteinsfrom

associationwith transmembraneproteinsthat normally fix their orientationwith respectto the

cell wall. Now,assumingthat themooringproteinscontainanappropriateelectricaldipole and

thattheyareclusteredto increasenetdipole strength,theyarefreeto orientin theaxial electrical

field which normally prevails in growing tissue. As is well known, the orientation of the

microtubulescontrolstheorientationof newlyforming cellulosefibrils, andthesein turn control

the direction in which thecell wall canbestretchedif loosened. Finally, asthe elevationof

mural acid reduceschannelopening, the microtubules are locked in place again, ready to

maintainallometryby participatingin thenextcycleof local expansion.Or, if therehasbeena

changein orientingconditions,themicrotubulesarereadyto shift thevectorsof growth.

As introduced in our last report, we are currently working with fluorescent tags and a

computational optical sectioning microscopein order to observenot only the individual

componentsof themodelbutalsohowtheirarrangementsandactivitiescanchangewith time.

Aluminum toxicity. Over the past year, we have shown that the channel is inhibited by low

levels of aluminum ions, suggesting that blockage of this or a closely related stretch-activated

channel might play a key role in the agriculturally and sylviculturally detrimental acidic soil

syndrome.

Figure 1. illustrates such inhibition by aluminum ions. It is a temporally compressed patch

clamp recording of an excised, outside-out, plasmalemmal patch from onion bulb scale

epidermis. It shows (see pointers) the absence of channel currents in the absence of membrane

tension, the presence of large currents when suction is applied, and the immediate inhibition of

currents when a large amount of dissolved aluminum chloride is perfused past the patch. At the

pH of the perfusate, only a little of the aluminum is in the trivalent ionic form commonly thought

to be the major toxic aluminum species. Experimental conditions are specified in the fourth

reference listed below; of particular note is the provision of K+ rather than Ca2÷ ions to support

the currents, since in the absence of Ca2+ the conductance for K÷ is higher than that for Ca2÷.

0 kPa

Y

7.1 kPa

Y

.... JL,ll J ,IL,, ,I,.. ....L,L.,..I .......

Figure 1. Inhibition by aluminum ions.

Target ofafungal toxin. A potent fungal toxin recendy described as a gravitropic inhibitor (A.

Sievers and M.B. Busch, Planta 188:619, 1992) has an immediate enhancing effect on stretch

activation of the channel, and after a lag begins to activate the channel in the absence of

externally imposed tension. It is possible that these effects are due to insertion of the molecule in

the bilayer. Regardless of how the stimulation is achieved, it suggests a possible mechanism by

which cytosolic Ca2+ might be elevated during causation of certain plant rots. We previously

predicted that the channel would be a target for a variety of pathogens, and we hope to check to

what extent the action of this and other toxins may partly account for various disease syndromes.

Meanwhile, in view of the use of the toxin by plant physiologists as a putatively specific agent to

82
interdict a ratherdifferent cell function, as discussedby SieversandBusch,the data serve

warningthatprevious"pharmacological"studiesneedto bereevaluated.

Turgor regulation. We have tested that a moderately low concentration of the channel inhibitor

gadolinium conspicuously and reversibly delays the response of protoplasts to osmotic shifts in

the surrounding medium. Evidently, the putative gravitropic receptor can also serve in

osmoregulation. There is a growing sense that turgor sensors play diverse roles in the plant,

ranging from control of syntheses to partitioning of photosynthate. Thus, extension of channel

inhibitor studies to such processes might prove highly profitable.

Significance of the Accomplishments

Finding 1: If, as we propose, the alleged gravitropic transducer is also a primary target of toxic

aluminum ions in acidic soils, it should now be possible to make faster progress in understanding

why many crop and forest plants do not thrive in these soils. In the very long range, it might be

possible to work out mechanisms by which the effects of acidic soils may be ameliorated.

Finding 2: More generally, the data on channel modulation by acid in the wall space extend the

concept presented in the preceding report that the alleged gravitropic transducer is a multimodal

sensor with a second messenger output. Because of the integrational capabilities of this channel,

and because of the accumulating evidence that it may play a role in control of turgor and of the

rate and vectors of cell growth, our statement in the preceding annual report seems even stronger:

"...systematic expansion of the gravity sensor program into the arena of molecular architecture

and function should greatly broaden understanding of the plant cell and of the developmental

processes and maintenance activities of plants." As exemplified in a publication by Pont-Lezica,

listed below and discussed in the preceding report, the new data on the multimodal sensory

channel can be combined with high-resolution optical sectioning microscopy, molecular biology,

and antibody staining to further our understanding of how responses and feedbacks of this

system help to integrate cell behavior.

Publications

Badot, P.M., Ding, J.P., and Pickard, B.G. 1992. Mechanically activated ion channels occur in

vacuoles of onion bulb scale parenchyma. Comptes Rendus Academy of Science, Paris Ser. III,

315: 437-443.

Ding, J.P., Badot, P.M., and Pickard, B.G. 1993. Aluminum and hydrogen ions inhibit a

mechanosensory calcium-selective cation channel. Australian Journal of Plant Physiology 20:

771-778.

Ding, J.P. and Pickard, B.G. 1993. Mechanosensory calcium-selective cation channels in onion

epidermis. Plant Journal 3:83-110.

Ding, J.P. and Pickard, B.G. 1993. Modulation of mechanosensitive calcium channels by

temperature. Plant Journal 3:713-720.

Pickard, B.G. and Ding, J.P. 1992. Gravity sensing by higher plants. Chapter 5 in: Advances in

Comparative and Environmental Physiology: Vol. 10: Comparative Aspects of Mechanoreceptor

Systems (F. Ito, Ed.). Berlin - Heidelberg: Springer, pp. 81-110.

Pickard, B.G. and Ding, J.P. 1993. The mechanosensory calcium-selective ion channel: Key

component of a plasmalemmal control center? Australian Journal of Plant Physiology 20: 439-

459.

83
Pont-Lezica,R.F.,McNally, J.G.,andPickard,B.G. 1993. Wall-to-membranelinkers in onion

epidermis.Plant, Cell & Environment 16:111-123.

84
CALCIUM MESSENGER SYSTEM IN GRAVITROPIC RESPONSE IN PLANTS

B.W. Poovaiah

Laboratory of Plant Molecular Biology and Physiology

Department of Horticulture

Washington State University

Pullman, WA 99164-6414

Description of Research

The primary goal of our research on gravitropism is to determine the mechanisms involved in the

plant's detection of the gravity signal and resulting differential growth of the upper and lower

halves of roots. Signal-induced change in free Ca2+ concentration in the cell has generally been

portrayed as a switch regulating various cellular activities. Plant responses to external signals are

believed to be achieved by changes in intracellular free concentration; which, in turn, modulate

the activity of Ca2+ and Ca2+/calmodulin-dependent protein kinases. Figure 1 shows a schematic

diagram that illustrates how elevated levels of cytosolic Ca2+ affect various enzymes and

proteins in the cell.

Increase in _ Regulation of

cytosolic Ca 2+ expression of CaM

Activalion of Ca 2+.

dependent enzymes

(e.g., CDPKs)

_ CaM-finding proteins

(e.g., protein kinase,

ATPases)

Regulation of

express-ton of

CaM-binding proteins

RESPONSE

Figure 1. Schematic illustration of the proposed events involving Ca 2+ in signal transduction.

Many difficulties exist in making free Ca2÷ measurements in intact plants and cells. Precise

measurement of changes in the level of cytosolic Ca2+ concentration in response to external

stimuli is crucial in establishing the role of Ca2÷ in signal transduction. Aequorin, the best

known of many Ca2+-activated photoproteins, emits a blue light with an intensity proportional to

the Ca2+ concentration. This is a photoprotein isolated from the jellyfish, Aequorea foskalea.

We are using a molecular approach to quantitate gravity-induced changes in free Ca2+

concentration by introducing aequorin gene into plants. Our ultimate goal is to study gravityinduced changes in intracellular free Ca2+ concentration and its relationship to the regulation of

Ca2+ and Ca2+/calmodulin-dependent protein kinases in plants.

85
Accomplishments

(1) During the past year, we have produced transgenic plants carrying the aequorin gene

that can report changes in cytosolic free Ca2+ concentration. To accomplish this, the

apoaequorin-coding region from complementary DNA clone was fused to the cauliflower mosaic

virus (CaMV) 35S promoter and transferred to plants using the Agrobacterium tumefaciens

binary vector system. Northern analysis revealed that we have a set of transgenic plants

expressing different levels of apoaequorin. F1 progeny from the transformant expressing the

highest levels of apoaequorin were selfed and homozygous plants were produced.

(2) Two approaches have been taken to detect and characterize Ca2+-regulated protein

kinases in plants. In the first approach, two degenerate oligonucleotide primers corresponding to

the highly conserved regions, GGELFD and DLKPEN of serine/threonine protein kinases were

used. These primers were used to amplify a DNA fragment from a corn root tip cDNA library by

polymerase chain reaction (PCR). The PCR-amplified product was used to screen the library to

obtain corresponding cDNA clones. Sequence analysis of one of the isolated cDNA clones

revealed that it represents a Ca2+-dependent protein kinase. This is a unique protein kinase

which has both a kinase domain and an adjoining calmodulin-like domain with four Ca2+-

binding EF-hand motifs in a single polypeptide. The junction domain between the kinase and the

calmodulin-like domains has a putative calmodulin-binding region. In situ hybridization studies

indicate that the expression of this kinase is higher in the root tip. The genomic Southern

revealed that there are other related protein kinases. Two other PCR products show very high

homology to Ca2+ and Ca2+/CaM-dependent protein kinases.

By screening the corn root tip cDNA expression library with 125I and 35S-labeled calmodulin as

probes, several cDNA clones were obtained. Two partial cDNA clones (CBP-1 and CBP-5) have

been characterized. Comparison of the deduced amino acid sequence of both the clones showed

100 percent conservation of the 34 amino acid stretch at their carboxy-terminal ends. The highly

conserved 34 amino acid stretch contained putative calmodulin-binding domain, a basic

amphiphilic alpha helix. Results indicate that mRNA for one of the clones (CBP-5) increases

when the seedlings are subjected to mechanical perturbations.

(3) To study the regulation of calmodulin gene expression, transgenic plants carrying a

chimeric fusion between the calmodulin promoter and chloramphenicol acetyltransferase (CAT)

reporter gene were produced. Depletion of Ca2+ by EGTA and ionomycin resulted in decreased

expression of the CAT activity. Plants carrying CaMV 35S promoter with the reporter gene

CAT were used as controls. Ca2+ depletion did not affect the CAT expression driven by the

CaMV 35S promoter. Furthermore, verapamil, a Ca2+ channel blocker, also decreased the

expression of the reporter gene driven by the calmodulin promoter. These results suggest a role

for Ca2+ in regulating the transcription of calmodulin gene.

Significance of the Accomplishments

Finding 1: Our results suggest that free Ca2+ concentration in the cell and the changes in protein

phosphorylation that are under the control of Ca2÷ and Ca2+/calmodulin-dependent protein

kinases are closely linked. A better understanding of the interrelationships between Ca2+-

regulated protein kinases and signal-induced changes in free Ca2+ concentration would be of

immense value in understanding the Ca2+ messenger system and its role in gravity signal

transduction.

86
Publications

Friedmann, M. and Poovaiah, B.W. 1991. Calcium and protein phosphorylation in the

transduction of gravity signal in corn roots. Plant and Cell Physiology 32: 299-302.

Poovaiah, B.W. and Reddy, A.S.N. 1991. Calcium and root development: Importance of

calcium in signal transduction. In: Plant Roots: The Hidden Half(Waisel, Y., Kafkafi, U., and

Eshel. A., Eds.). New York: Marcel Dekker Inc., pp. 205-227.

Poovaiah, B.W., Reddy, A.S.N., An, G., Choi, Y.J., and Wang, Z.Q. 1992. Calmodulin gene

expression and Ca2+/calmodulin-dependent protein kinase II in plants. In: Progress in Plant

Growth Regulation (Karssen, C.M., VanLoon, L.C., and Vreugdenhil, D., Eds.). Dordrecht, The

Netherlands: Kluwer Academic Publishers, pp. 691-702.

87
MECHANISM AND CONTROL OF GRAVITROPISM IN PLANTS

David L. Rayle

Department of Biology

San Diego State University

San Diego, CA 92182-0057

Description of Research

How plants respond to gravity to produce 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. Our research objective is to understand the signal

transduction events that lead from gravity perception to asymmetric cell elongation in seedling roots

and shoots. To dissect the steps in root and shoot gravitropism, we are using the tomato mutant

diageotropica (dgt).

The dgt mutant arose from a spontaneous, single gene-recessive mutation of the parental variety,

VFN8. Shoots of dgt exhibit a set of morphological and physiological abnormalities including an

altered stem gravitropic response, which suggests that these plants may have a defect associated

with some primary event in auxin action.

Other than qualitative data showing that dgt plants fail to form lateral roots and do not respond to

gravity, no information on the dgt lesion in roots and its resultant phenotype was available. Here

we report on our efforts to characterize the growth and gravitropic behavior of dgt roots.

Accomplishments

(1) The gravitropic response of dgt roots is abnormal. Although the dgt radical has some

capacity for positive gravitropism when it emerges from the seed, after reaching a length of

approximately 5 mm, the primary roots of dgt seedlings become agravitropic. When seedlings are

placed horizontally or at various angles relative to the gravity vector, they continue to grow at that

orientation, exhibiting no curvature. In contrast, the roots of VFN8, the wild-type tomato from

which dgt is derived, have a normal gravitropic response.

(2) Relative to wild-type roots, dgt roots are less sensitive to growth inhibition by

exogenously applied IAA and auxin transport inhibitors (phytotropins) and the roots exhibit a

reduction in maximal growth inhibition in response to ethylene.

(3) IAA transportthrough roots, binding of the phytotropin, tritiated naphthylphthalamic

acid ([3H]NPA) to root microsomal membranes, and NPA-sensitive IAA uptake by root segments

are similar in mutant and wild-type roots.

Significance of Accomplishments

We speculate that the insensitivity of dgt root growth to auxin transport inhibitors and ethylene is

an indirect result of the loss of sensitivity to auxin in this single gene-recessive mutant. We

conclude that dgt roots, like dgt shoots, exhibit abnormalities indicating they have a defect

associated with, or affecting, a primary site of auxin perception or action.

_8
Publications

Lomax, T.L., Coenen, C., Gaiser, J.C., Hopkins, R., Rayle, D.L., and Rice, M.S. 1993.

Auxin perception and the regulation of tomato growth and development. In: Tomato Molecular

Biology (Yoder, J., Ed.). Lancaster, PA: Technomic Publishing Co.

Rayle, D.L., and Cleland, R.E. 1992. The acid growth theory of auxin-induced cell elongation is

alive and well. Plant Physiology 99:1271-1274.

89
ASSESSINGPOTENTIAL TARGETS OF CALCIUM ACTION IN GRAVITROPISM

Stanley J. Roux

Department of Biology

University of Texas

Austin, TX 78713

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 ions may play an important role in transducing the

stimuli of both light and gravity into growth changes in plants, including the important fact that

both stimuli alter the concentration of cytosolic free calcium in the responding cells. However,

full confidence in the validity of this hypothesis will require that at least one additional piece of

information be obtained, namely, the identification of specific targets of calcium action that have

a clear and significant impact on growth.

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.

Calmodulin and calcium-dependent protein kinases are the best known targets of calcium action

in plants. More recently, a new class of calcium-binding proteins called annexins has been

discovered in both plants and animals. All animal annexins tested to date exhibit ion channel

functions in biological membranes, several of them promote the fusion of membranes in cells

and in model systems, and annexin VII shows voltage-dependent channel activity that is highly

selective for Ca2+. In our 1990-1991 Summary of Accomplishments, we reported the

purification, initial biochemical characterization, and localization of an annexin-like protein in

peas, called p35. In this year's Summary, we describe additional biochemical properties of pea

annexin and provide initial localization evidence that implicates plant annexins in the control of

secretion and growth.

Accomplishments

(1) We have localized p35 in ferns and dicots predominately in cells that are rapidly

elongating or actively engaged in secretion, lmmunolocalization reveals that p35 is highly

concentrated in rapidly elongating cells undergoing xylogenesis or differentiating into phloem

sieve tubes, in peripheral root cap cells that are actively secreting mucilage, and at the apical tips

of polarly growing fern rhizoids and tobacco pollen tubes. At the ultrastructural level, they are

found predominately in association with Golgi vesicles and the plasma membrane.

(2) Using the calcium-sensitive fluorescent dye, fura-2, we have found calcium gradients

within polarly growing rhizoids of the fern, Dryopteris, with the highest concentration at the

growing tip. This work was done in collaboration with Drs. M. Poenie (University of Texas) and

Robert Scheuerlein (University of Erlangen) to help develop this fern as a model system for

studying gravitropism in single cells (the rhizoid is positively gravitropic). Furthermore, there is

a significant literature demonstrating that red-light-activated phytochrome plays a major role in

inducing both the germination of Dryopteris rhizoids and the subsequent growth of the rhizoids,

9O
sogerminatingfern sporesshouldbe idealfor studyingbothlight- and gravity-inducedsignal

transductionin the same cell.

(3) We have sequenced five peptides generated by lys-protease digestion of p35 and

found them to be highly similar to known annexins. The peptides were purified by reverse-phase

HPLC chromatography. Their sequences were all at least 40 percent identical to one or more

known annexins with conserved substitutions at several of the non-identical positions.

(4) We have found that annexin expression is co-induced with xylogenesis in cultured

Zinnia cells. Cultured Zinnia cells can be induced to undergo xylogenesis by hormone treatment.

In collaboration with the laboratory of Dr. C. Haigler (Texas Tech University), we have found

that, co-incident with the induction of xylogenesis in these cells, there is a great increase in the

immunodetectable level of annexins.

Significance of the Accomplishments

Findings 1 and 2: Both in rapidly elongating cells undergoing xylogenesis and in polarly

growing rhizoids and pollen tubes, there is active secretion of wall materials into the newly

forming or expanding walls. Calcium is thought to play a major role in this process, but the

target of calcium action in polar growth is unknown. Our findings suggest that calcium-binding

annexin proteins, which are known to promote the fusion of secretory vesicles with the plasma

membrane in animal cells, are highly concentrated in cells active in the secretion of wall

materials and are thus major candidates for being a target of calcium action in this process. The

coincidence of calcium and annexin gradients in fern rhizoids is consistent with the hypothesis

that annexins may play a role in calcium transport in these cells. If annexins play an integral role

in delivering vesicles to expanding walls for growth, they should be key agents in the process of

differential growth leading to gravitropic curvature.

Finding 3: These structural data further support the conclusion drawn from our earlier functional

analyses that p35 is indeed a member of the annexin family. These amino acid sequences are

among only a few known for plant annexins at the present time. These data also provide a

valuable information base for verifying the identity of cDNA clones that we have selected from a

pea cDNA library by antibody screening as ones that encode annexin-like proteins.

Finding 4: These results indicate that annexins are selectively induced by hormones that

promote the elongation and differentiation of cultured cells into xylem cells. Hormones are also

thought to be key promoters of the differential growth leading to gravitropism. Therefore,

learning more about the role of annexins in hormone-induced growth should provide valuable

insights into the cellular mechanisms of gravitropism.

Publications

Clark, G., Dauwalder, M., and Roux, S.J. 1991. Characterization and localization of an annexinlike protein in plant cells. Journal of Cell Biology 115: 258a.

Clark, G., Dauwalder, M., and Roux, S.J. 1991. Purification, biochemical characterization and

localization of a calcium-phospholipid binding protein in peas. Plant Physiology 96 (Suppl.): 67.

Clark, G., Dauwalder, M., and Roux, S.J. 1992. Immunolocalization of annexin-like proteins in

a variety of plant cells. Molecular Biology of the Cell 3 (Suppl.): 18a.

Clark, G., Dauwalder, M., and Roux, S.J. 1992. Purification and immunolocalization of an

annexin-like protein in pea seedlings. Planta 187: 1-9.

91
Clark, G., Dauwalder, M., and Roux, S.J. 1992a. Characterizationand localization of an

annexin-likeproteinin plantcells. Plant Physiology 99 (Suppl.): 42.

Clark, G.B., Dauwalder, M., and Roux, S.J. 1993. Immunolocalization of annexin-like proteins

in a variety of plant cells. Plant Physiology 102 (Suppl.): 114.

Clark, G.B., Memon, A.R., Thompson, G.A., Jr., and Roux, S.J. 1993. Phytochrome regulates

GTP-binding protein activity in the envelope of pea nuclei. Molecular Biology of the Cell

(Suppl.) 4: 148a.

Clark, G.B., Memon, A.R., Tong, C.-G., Thompson, G.A., Jr., and Roux, S.J. 1993.

Phytochrome regulates GTP-binding protein activity in the envelope of the pea nuclei. Plant

Journal 4: 399-402.

Clark, G.B. and Roux, S.J. 1993. Localization evidence implicating calcium-binding annexin

proteins in mechanisms of secretion and growth control in plant cells. ASGSB Bulletin 7: 34.

Haas, C.J., Scheuerlein, R., and Roux, S.J. 1991. Phytochrome-mediated germinated and early

development in spores of Dryopterisfelix-mas: Phase-specific and non-phase-specific inhibition

by staurosporine. Journal of Plant Physiology 138: 747-751.

Li, H.and Roux, S.J. 1992. Ca 2+-stimulated protein kinases in the pea nucleus: Potential

targets of calcium action during light-stimulated gravitropism. ASGSB Bulletin 6: 97.

Roux, S.J. 1992. Calcium-regulated nuclear enzymes: Potential mediators of phytochromeinduced changes in nuclear metabolism? Photochemistry and Photobiology 56:811-815.

Roux, S.J. 1993. Casein kinase-2-type protein kinases in plants: Possible targets of polyamine

action during growth regulation? Plant Growth Regulation 12: 189-193.

Roux, S.J. 1993. What remains of the Cholodny-Went theory? Riding the transduction chain to

the answer. Plant Cell and Environment 15: 783-784.

Scheuerlein, R., Haas, C., Poenie, M., and Roux, S.J. 1991. Germinating spores of Dryopteris:

A model system for studying the role of calcium in light- and gravity-induced changes in growth

and development. ASGSB Bulletin 5: 74.

Scheuerlein, R., Schmidt, K., Poenie, M., and Roux, S.J. 1991. Determination of cytoplasmic

calcium concentration in Dryopteris spores: A developmentally non-disruptive technique for

loading of the calcium-indicator fura-2. Planta 184:166-174.

Tong, C.-G., Dauwalder, M., Clawson, G.A., Hatem, C.L., and Roux, S.J. 1993. The major

nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common

epitopes also present in nuclear lamins. Plant Physiology 101:1005-1011.

Zhang, S., Jin, C.-D., and Roux, S.J. 1993. Casein kinase H-type protein kinase from pea

cytoplasm and its inactivation by alkaline phosphatase in vitro. Plant Physiology 1-3: 955-962.

92
CELLULAR POLARITY AND INTERACTIONS IN PLANT GRAVIPERCEPTION

Fred D. Sack

Department of Plant Biology

Ohio State University

1735 Neil Avenue

Columbus, OH 43210-1293

Description of Research

Our long-term goal is to understand the mechanisms of gravitropic sensing in higher and lower

plants. This involves identifying the cells that sense gravity and determining the cellular mass that

gravity acts upon to trigger sensing. We hope to learn which events occur during the transduction

of a physical signal into a physiological signal that affects growth.

A second goal is to understand how cells have "used" gravity in orienting and organizing

themselves. A related question is what mechanisms have evolved that prevent the stratification of

cell components with respect to gravity?

One major focus of our research has been the tip-growing cells of the protonemata of the moss

Ceratodon, which grow up in the dark. We have shown that the process of upward curvature

involves a redistribution of microtubules close to the growing zone.

Accomplishments

(1) Gravitropic roots of the aquatic angiosperm, Limnobium, have sedimented

amyloplasts in their elongation zone but not in their root cap.

(2) Nuclei as well as amyloplasts sediment in the elongation zone.

(3) Plastids sediment in vertical cells of the moss Ceratodon, but sedimentation is

incomplete and the number of plastids that sediment and the location and extent of plastid

sedimentation is highly regulated in vertical cells.

(4) Microtubules restrict plastid sedimentation in vertical protonemata; i.e., microtubule

deploymerizers cause complete plastid sedimentation, whereas microfilament inhibitors have no

effect on sedimentation.

Significance of the Accomplishments

Finding 1: These data extend our previous findings from roots of the more primitive plant,

Equisetum, that amyloplast sedimentation in roots can occur outside the cap. While this does not

prove that the elongation zone is capable of sensing, it does provide structural data that warrant a

reinvestigation of the question of whether sensing is confined to the cap in all roots.

Finding 2: As far as I know, these cells in roots of Limnobium and Equisetum are the only cases

reported where nuclei sediment at 1-g. Presumably, this demonstrates that nuclei have enough

mass to sediment in other systems as well but do not do so, perhaps because of restraint by the

cytoskeleton.
Finding 3: Comparison of upright and inverted cells shows that plastids do sediment in vertical

cells, not just in horizontal cells. However, heretofore, such sedimentation has been difficult to

detect since sedimentation is incomplete; i.e., plastids are present throughout the length of vertical

cells and plastids do not fall through the length of these cells. Inversion reveals subtle but real

amyloplast sedimentation. Such restricted sedimentation argues strongly for a role for the

cytoskeleton in maintaining plastid distribution.

Finding 4: These data establish a central role for microtubules in controlling plastid sedimentation

in these moss cells. This uncovers a second gravity-related role for microtubules in these cells,

since microtubules also become redistributed prior to and during upward gravitropic curvature.

The finding that microtubules restrict sedimentation demonstrates the importance of the

cytoskeleton in maintaining organelle position and cell organization against the force of gravity.

Although it has been proposed that the cytoskeleton evolved, in part, to prevent the stratification of

cell components with respect to gravity, there are few direct proofs of this role in the literature.

Publications

Ridge, R. and Sack, F.D. 1992. Cortical and cap sedimentation in gravitropic Equisetum roots.

American Journal of Botany 79: 328-334.

Sack, F. 1993. Gravitropism in protonemata of the moss Ceratodon. Bulletin of the Torrey

Botanical Club 25:36-44 (in review).

Sack, F.D., Kim, D., and Stein, B. 1993. Organelle sedimentation in gravitropic roots of

Limnobium is restricted to the elongation zone. ASGSB 7: 91.

Sack, F.D. and Schwuchow, J. 1993. Microtubules limit plastid sedimentation in protonemata of

the moss Ceratodon purpureus (Abstract 3020). Yokohama: XV International Botanical Congress.

Schwuchow, J. and Sack, F.D. 1992. The influence of cytoskeletal inhibitors on plastid position

in inverted and vertical Ceratodon protonemata. ASGSB 6(1): 96.

Schwuchow, J. and Sack, F.D. 1993. Effects of inversion on plastid position and gravitropism

in Ceratodon protonemata. Canadian Journal of Botany 71: 1243-1248.

Walker, L.M. and Sack, F.D. 1991. Recovery of gravitropism after basipetal centrifugation in

protonemata of the moss Ceratodon purpureus. Canadian Journal of Botany 69: 1737-1744.

Walker, L.M. and Sack, F.D. 1992. Effects of inversion on plastid position and gravitropism in

Ceratodon protonemata. ASGSB 6(1): 75.

Walker, L.M. and Sack, F.D. 1992. Ultrastructural analysis of dark grown protonemata of the

moss Ceratodon. ASGSB 6(1): 38.

Young, J.C. and Sack, F.D. 1992. Time-lapse analysis of gravitropism in Ceratodon

protonemata. American Journal of Botany 79:1348-1358.

94
DEVELOPMENTAL STUDIES OF WHEAT IN MICROGRAVITY

Frank B. Salisbury

Department of Plants, Soils,

Utah State University

Logan, UT 84322-4820

and Biometeorology

Description of Research

Since 1987, we have been preparing for flight experiments with a superdwarf cultivar of wheat on

the U.S. Shuttle and the Russian Space Station Mir. During 1992, it became apparent that the

Shuttle experiments might be indefinitely delayed; but, in December of 1992, two of our

experiments carried out in cooperation with personnel at the Institute of Biomedical Problems in

Moscow were incorporated as part of the Shuttle/Mir rendezvous (called Spacelab Mir-I or

SLM-l) scheduled to take place in 1995. These exercises will include a seed-to-seed experiment

for up to about 120 days followed by a second crop for about 30 days, at which time the U.S.

Shuttle will arrive. Fixed and dried samples from the first crop and frozen samples from the

second crop will be returned for analysis. Virtually all previous long-term experiments with plants

in microgravity have resulted in plants with retarded developmental stages and greatly reduced

yields; however, these experiments have never been done with environmental conditions known to

produce normal plants on Earth. Thus, the goals of our experiments include the improvement of

growing conditions plus the diagnosis of reasons for observed growth responses.

The Russian plant growth chamber, Svet, is being provided with environmental sensors to monitor

various aspects of the plant environment, including substrate moisture levels, temperatures,

irradiances, pressure, oxygen, atmospheric humidity, and carbon dioxide during growth of both

crops. Gail Bingham in the Space Dynamics Laboratory at Utah State University is the project

engineer who is developing equipment for this monitoring, which will allow calculation of

photosynthesis, dark respiration, and transpiration. During the seed-to-seed crop, plants will be

sampled and chemically fixed at five stages: prefloral (6 days after planting), early floral ( 14 days),

boot (head surrounded by leaf), anthesis, and seed filling. All remaining plants will be harvested

at maturity, and the resulting seed will be tested for viability and ability to produce normal plants.

Samples will be studied by William Campbell (anatomy, histology) and John Carman (a spectrum

of plant hormones, other parameters), both at Utah State University; and David Bubenheim

(various biochemical parameters) at NASA Ames Research Center. Boris Yendler, also at Ames,

will examine the returned root module in cooperation with Igor Podolsky in Moscow and other

investigators in Utah. Leaf area measurements will be made on collected samples at each sampling

time.

In addition to hardware development, ground-based research emphasizes growth of plants under

conditions comparable to those that will be encountered in the space experiments. These include

relatively low light levels (125--250 mmol m "2 s-l, photosynthetic photon flux) and high CO2

levels (up to about 1.0 percent before canisters are changed). The root module appears to be an

especially challenging problem. The substrate (Balkanine, an ion-exchange material charged with

plant nutrients) may be saturated at some points and dry at others, judging by previous experience

with this system. We can only monitor this situation this time but hope to achieve better control in

future space studies.

Accomplishments

Our studies with superdwarf wheat (25--30 cm tall) have so far emphasized responses to the low

light levels available in space-rated plant growth chambers. We have grown plants in a controlled

95
environment(temperature,CO2levels)undera tiltedbankof fluorescentlamps. Plantsclosestto

the lampsreceiveabout350 I.tmolm-2sl, while plants farthestfrom the lampsreceiveabout

50I.tmol m"2 s"1. Plantsunder the lowest irradiancesare about at the compensationpoint;

photosynthesisproducingaboutthesameamountof fixed carbonasrespirationuses;hence,there

is virtually nogrowth(althougha few leavesform usingfoodstoredin theseedandasterilehead

mighteventuallyform). Plantsunderthehighestirradiancesareessentiallynormalalthoughtheir

yields arereducedcomparedwith plants undereven higherlight levels (e.g.,full sunlight=

2000_tmolm"2s'l). To summarizetheresultsof severalexperiments:All developmentalstages

(e.g.,formation of heads)aredelayedat lower irradiancescomparedwith higherlight levels,but

final yieldsrise sharplywith irradiancesabovethecompensationpoint andthenlevel off. The

levelingis becauseplantsat lowerirradiancestakemuchlongerto matureandthushavea longer

time to accumulatelight energy. In addition to our studieswith light, we havetestedvarious

substrates.Our foll same wit
0 0 TD

e hence,

compared

com9nmo nta2ao,0l2TD (a) Tj 9lpslugj 26 0 TDo
-438 -12n Tjj 59 0 TD2 altho0(point)0 TD (Tocsheir) Tj 

 TD (delahnD (2
 -12 1ltho0(point)0 TD (Toed) 

leveli5j 9lpslug 0 r aformation levelTD2

compared lo5j 9lps/SI1 -Tf

(Svet si4

-437 s./SR1 -Tf

(mockup (levelTD (under Tj 37 0 TD 

b0 TD (at.) Tj0 TD (altl33 0 TD ) Tj 27 Inst tuwigelonger3 Tj 27 BiomedicalTj D 

1 25 0 TDl33 0 TD 

altpared

altl33 0 TD 18 0 TDdirectio7 0 TD4
Although not describedin detail in this report, hardwaredevelopmenthasprogressednicely.

Excellentequipmentexiststo monitorsuchenvironmentalfactorsasCO2,02, light, air andleaf

temperatures,pressure,andsubstratemoisture.This will makeit possibleto diagnoseandbetter

understandthe observedplant responses.The newly developedinstrumentationcould be of

considerablevaluein futurespaceexperimentswith plantsby othersaswell asourselves.

Publications

Salisbury, Frank B. 1991. Gravitropism in Dicot Stems. In: NASA Space Biology

Accomplishments 1989-1990. NASA Technical Memorandum 4258, pp. 88-90.

Salisbury, Frank B. 1991. The Systeme Internationale: The use of SI units in plant physiology.

Journal of Plant Physiology 139: I-7.

Salisbury, Frank B. 1992. Building a Moon base: Life-support systems. In: 1989 Proceedings

of National Space Society's Eighth Annual International Space Development Conference (Liss,

Jeffrey G. and Chapman, Glenn H., Eds.). Chicago: Space Development Conference, Ltd., pp.

137-142.

Salisbury, Frank B. 1992. Some challenges in designing a lunar, Martian, or microgravity

CELSS. Proceedings of the 9th IAAA Man in Space Symposium, Cologne, Germany,

June 1991. Acta Astronautica 27:211-217.

Salisbury, Frank B. 1992. What remains of the Cholodny-Went theory? A potential role for

changing sensitivity to auxin. Plant, Cell & Environment 15(7): 787-788.

Salisbury, Frank B. 1993. Gravitropism: Changing Ideas. Horticultural Reviews, Volume 15

(Janick, Jules, Ed.). pp. 233-277.

Salisbury, Frank B. 1993. Lunar agriculture and growth of superdwarf wheat in microgravity.

In: X Reuni6n Nacional de la S.E.F.V. III Congreso Hispano-Portuguds de Fisiologia Vegetal.

Libro De Resumenes, Pamplona, Spain. pp. 11-17.

Salisbury, Frank B. and Ross, Cleon W. 1991. Plant Physiology, 4th Edition. Belmont, CA,

Wadsworth Publishing Company, 682 pages.

97
GRAVITY-INDUCED POLARITY IN SINGLE CELLS OF CHARA

Randy Wayne

Section of Plant Biology

Cornell University

Ithaca, NY 14853

Description of Research

The overall goal of our research program is to elucidate the gravireceptor and the biophysical

events that the activated gravireceptor initiates in order to understand how cells perceive and

respond to a gravitational stimulus. In order to accomplish our goals, we have studied the effects

of gravity on the polarity of cytoplasmic streaming in the single intemodal cells of characean

algae.

We have proposed a model for gravisensing in which the entire protoplast experiences the force

of gravity and settles within the extracellular matrix. Consequently, proteins connecting the

plasma membrane to the extracellular matrix at the ends of the cells experience a differential

compression or tension as a result of gravitational pressure. This leads to the activation of

certain classes of Ca2+ channels, localized at the ends of the ceils, and subsequently a change in

the polarity of cytoplasmic streaming.

Accomplishments

(1) Hydrostatic pressure mimics gravitationalpressure. In order to test the hypothesis

that ceils sense a differential pressure on the top and bottom of the cell induced by gravity (i.e.,

gravitational pressure), we subjected horizontal cells to a unidirectionally applied pressure. We

find that a unidirectionally applied hydrostatic pressure mimics gravitational pressure in inducing

the physiological response. Both stimuli produce physiological responses that are identical in

terms of ionic requirements, inhibitor sensitivity, etc.

(2) Gravireceptor may be an integrin.like protein. In order to characterize the

mechanoreceptor responsible for sensing gravitational and hydrostatic pressure, we treated ceils

with a variety of impermeant enzymes and peptides, including Arg-Gly-Asp. These experiments

demonstrate that the receptors are localized at the plasma membrane-extracellular matrix

junction at the ends of the cell and that the mechanoreceptor for gravitational and hydrostatic

pressure is an integrin-like protein.

(3) Gravireceptor is distinct from touch-receptor in same cell In order to test the

hypothesis that a single mechanoreceptor is involved in all responses to mechanostimulation, we

characterized the mechanoreceptor involved in the touch-induced action potential in characean

internodal cells. We find that the touch receptor is distinct from the receptor involved in sensing

gravitational and hydrostatic pressure. The two classes of receptors vary in their spatial

localization, their sensitivity to proteases and peptides, their energetics, their ability to perceive

direction, etc.

(4) A change in membrane potential is not involved in the signal transduction chain

involved in sensing gravitational or hydrostatic pressure. In order to test the hypothesis that a

change in membrane potential contributes to the signal transduction chain involved in

gravitational or hydrostatic pressure sensing, we measured the membrane potential of ceils

subjected to either gravitational or hydrostatic pressure. The resting membrane potential

measured with intracellular electrodes is approximately -0.22 V. The membrane potential at the

98
endsof the cells, measured with intracellular electrodes, varies by less than 0.001 V when cells

are subjected to hydrostatic pressure. The difference in membrane potential at the ends of the

cells, measured with extracellular electrodes, varies by less than 0.001 V when cells are

subjected to gravitational pressure. These variations in membrane potential are too small to be

involved in signaling and may be the result of changes in the partial membrane conductance.

(5) Two distinct classes of Ca2+ channels are involved in gravisensing, a third is not.

Pharmacological and localization experiments indicate that two distinct classes of Ca2+ channels

are localized at the ends of the cells, are sensitive to organic channel blockers, and are involved

in gravisensing (Classes I and II). A third class of Ca2+ channel is found throughout the plasma

membrane, is insensitive to organic channel blockers, and is involved in sensing electrical

stimulation and touch (Class III). These conclusions are supported by flux measurements using

Sr2+ as a tracer.

(6) Characterization of the structure of the Ca2+ channels. We have used the

lanthanides to probe the structures of the three classes of Ca2+ channels. We find that Class I is

an elastic peristaltic channel that has three binding sites within the pore for Ca2+. Likewise,

Class III is an elastic peristaltic channel with three bindings sites for Ca2+; however, the binding

sites are definitely multidentate. By contrast, Class II channels have an external vestibule that

can only be blocked by large cations. Lanthanide flux studies show that the lanthanides go

through at least Class I and Class III channels and kinetically inhibit channel activity. We are

currently testing whether or not they go through Class II channels or remain in the external

vestibule.

Significance of Accomplishments

Finding 1: We have established an artificial system to mimic the effect of gravity and test the

gravitational pressure hypothesis.

Finding 2: We have determined that the gravireceptor may be an integrin-like protein.

Finding 3: We have demonstrated that the mechanoreceptor involved in gravisensing is distinct

from the mechanoreceptor involved in touch sensing in the same cell, indicating that there are

multiple classes of mechanoreceptors.

Finding 4: We have found that a change in membrane potential is not involved in the signal

transduction chain involved in sensing gravitational pressure in characean ceils.

Finding 5: We have established that two distinct classes of Ca2+ channels are involved in

gravisensing, a third is not.

Finding 6: We are characterizing the structure of the Ca2+ channels in order to determine how

the structure of a channel relates to the function in which that channel is involved.

Publications

Hepler, P.K. and Wayne, R.O. 1993. Calcium and plant development. Citation Classic.

Current Contents 24: 8.

Magalhaes, B.P., Wayne, R., Humber, R.A., Shields, E.J., and Roberts, D.W. 1991. Calciumregulated appressorium formation of the entomopathogenic fungus Zoophthora radicans.

Protoplasma 160: 77-88.

99
Sawyer,A.J.,Griggs,M.H., andWayne,R. 1993. Dimensions,density,andsettlingvelocity of

entomophthoraleanconidia: Implications for aerial disseminationof spores. Journal of

Invertebrate Pathology 63: 43-55.

Staves, M.P. and Wayne, R. 1993. The touch-induced action potential in Chara: Inquiry into

the ionic basis and the mechanoreceptor. Australian Journal of Plant Physiology 20: 471-488.

Staves, M.P., Wayne, R., and Leopold, A.C. 1991. Hydrostatic factors affect the gravity

responses of algae and roots. Physiologist 34:570-571.

Staves, M.P., Wayne, R., and Leopold, A.C. 1992. Hydrostatic pressure mimics gravitational

pressure in characean cells. Protoplasma 168: 141-152.

Wayne, R. 1992. What remains of the Cholodny-Went theory? Plant, Cell and Environment

15: 791-792.

Wayne, R. 1993. Excitability in plant cells. American Scientist 81: 140-151.

Wayne, R. 1993. Plant Movements. In: 1994 McGraw-Hill Yearbook of Science and

Technology. New York, McGraw-Hill, pp. 307-308.

Wayne, R., Kadota, A., Watanabe, M., and Furuya, M. 1991. Photomovement in Dunaliella:

Fluence rate-response curves and action spectra. Planta 184:515-524.

Wayne, R. and Staves, M.P. 1991. The density of the cell sap and endoplasm of Nitellosps and

Chara. Plant and Cell Physiology 32:1137-1144.

Wayne, R., Staves, M.P., and Leopold, A.C. 1991. The effect of gravity on cytoplasmic

streaming in characean cells. Biological Bulletin 181: 508.

Wayne, R., Staves, M.P., and Leopold, A.C. 1992. The contribution of the extracellular matrix

to gravisensing in characean cells. Journal of Cell Science 10 l: 611-623.

I00
ANIMAL PROJECTS

COMPARATIVE TRANSDUCTION MECHANISMS

HAIR CELLS

Richard A. Baird

R.S. Dow Neurological Sciences Institute

Good Samaritan Hospital and Medical Center

Portland, OR 97209

OF VESTIBULAR OTOLITH

Description of Research

Using in vitro preparations, 5-7 we have previously shown that hair cells in different regions of the

bullfrog sacculus and utriculus differ markedly in hair bundle morphology and physiological

response properties. More recently, using the aminoglycoside antibiotic gentamicin sulfate, we

have demonstrated our ability to induce in vivo the degeneration and subsequent regeneration of

saccular and utricular hair cells in the sacculus and utricular striola. 1,3 Hair ceils in these organs are

differentially sensitive to gentamicin, with saccular hair cells and hair cells in the utricular swiola

being damaged at lower gentamicin concentrations than hair cells in the utricular extrastriola.

Regenerating hair ceils in these studies were easily distinguished from their mature counterparts by

their hair bundles, which were distinctly smaller and shorter than those of more mature hair cells.

These cells were also classified into a number of types using the same morphological criteria used

to identify their mature counterparts.

More recently, we have developed in vitro explant cultures of the bullfrog saccular and utricular

maculae to extend our earlier in vivo studies of hair cell regeneration in the vestibular otolith

organs. These preparations have allowed us to control the concentration and duration of gentamicin

application and to continuously follow the fate of individual regenerating hair cells. In

morphological studies, we have studied the formation and migration of individual regenerating hair

cells, documenting changes in their cellular and hair bundle morphology. Ultimately, we hope to

document changes in the physiological response properties of hair cells, correlating these changes

with the time of acquisition or changes in the amplitude or gating kinetics of specific membrane

currents.

Accomplishments

(1) In vivo studies of cellular proliferation. The results of earlier in vivo studies

suggested that hair cell regeneration in the vestibular otolith organs might involve the mitotic

production of new hair cells. To more closely examine the role of mitotic division in hair cell

recovery, we injected bullfrogs with 5-bromo-2'-deoxyuridine (BrdU), a thymidine analogue that

is incorporated into mitotic cells. We were then able, using immunocytochemical methods, to

measure ongoing and gentamicin-induced cell proliferation in the vestibular otolith organs. The

primary aim of this study was to determine if mitotic division in gentamicin-treated animals was

upregulated from the ongoing rate, if any, of mitotic division in normal animals. We also wished,

by examining the spatial and temporal distribution of BrdU-labeled cells in gentamicin-treated

animals, to compare the patterns of cell proliferation and hair cell regeneration in these organs, and

to identify the progenitor cells of new cells in the vestibular otolith organs.

BrdU labeling was seen in normal and, to a greater extent, in gentamicin-injected animals. Labeling

in the saccular macula was more extensive than in the utriculus, consistent with the greater damage

caused by gentamicin to the former organ. BrdU-labeled cells were initially seen in the peripheral

macular margins and, within the maculae, in a subset of supporting ceils immediately adjacent to

the basement membrane. These supporting cells had spherical cell bodies and, unlike typical

supporting cells, had little or no apical projections. At later times, hair ceils and supporting cells

103
were alsolocatedfurther from the basementmembrane,suggestingthat mitotic ceils andtheir

progenyhadmigratedto moreapicalpositions.Themaculardistributionof BrdU-labeledcellswas

not correlated with the pattern of gentamicin-inducedcellular damage.First, the macular

distribution of BrdU-labeledcells was similar in both normalandgentamicin-injectedanimals.

Second,proliferatingcells werenotrestrictedto only thosemacularareasdamagedbygentamicin

treatment.Proliferatingcells in thesacculus,for example,werelargelyrestrictedto theabneural

halfof thesensorymacula.In theutricularmacula,proliferatingceilswereseenin boththestriolar

andextrastriolarregions.

Cellularproliferation wasinsufficient to explainhair cell recoveryseenin thevestibularotolith

organs.Moreover,proliferatingcellshadadifferentmaculardistributionfrom thatofregenerating

hair cells.In addition,thegreatmajorityof regeneratinghairceilsin thevestibularotolith organs

were not BrdU-labeled, suggestingthat hair cell recovery in the vestibular otolith organsis

primarily determinedby non-mitotic mechanisms.In lessdamagedareas,suchasthe utricular

striola,hair cell recoveryappearedto belargelycarriedout by hairbundlerepair.In moreheavily

damagedregions,unlabeledsupportingcells with immaturehair bundlesandcellularmorphology

transitionalbetweenthatof hair cells and supporting cells were often seen, suggesting that hair cell

recovery in these regions was primarily determined by the transdifferentiation of undamaged

supporting cells into hair cells. Supporting cells have long apical projections which have extensive

surface contact with hair cells. We hypothesize that the loss of this contact due to local hair cell

death may trigger intercellular signals which initiate the transdifferentiation of supporting ceils into

hair cells.

The results of these studies have been presented in abstract form at the Annual meeting of the

Society for Neuroscience 4 and are now being written up in manuscript form. s

(2) Phalloidin labeling of filamentous actin. Phalloidin labeling was used to label

filamentous actin in the cell bodies and hair bundles of hair cells. This technique has significantly

improved the visibility of immature hair bundles. In addition, the cellular distribution of

f'damentous actin in regenerating hair cells, particularly at early developmental times, differs from

that of normal hair cells, enabling us to identify new hair cells before the appearance of their hair

bundles.

In normal animals, phalloidin strongly labeled the intercellular adherens junctions at the apical

epithelial surface and the stereocilia array of hair bundles. The distribution of filamentous actin in

intercellular adherens junctions revealed a continuous reticular lamina throughout the confines of

the former sensory surface. Little or no labeling, by contrast, was observed within hair cells or

supporting cells. Twenty-four to forty-eight hours after intraotic gentamicin sulfate injection, little

or no labeling was seen above the macular surface, confirming the rapid loss of hair bundles

throughout the saccular macula and within the utricular striola. In heavily damaged regions, the

apical surfaces of supporting cells had expanded in size to fill spaces normally occupied by hair

cells.

Hair cell recovery in the sacculus and utriculus of gentamicin-treated animals proceeded in a fixed

spatial order. In the sacculus, new hair bundles were first seen in the peripheral macular margins

adjacent to the entrance of the saccular nerve, indicating the presence of new hair cells in the

saccular macula. Similar bundles were observed in the abneural portion of the maculae only at later

times. By 7-9 days after gentamicin injection, new hair bundles were observed in both the neural

and abneural regions of the saccular macula, although hair bundles in the abneural region were

often smaller than those in the neural region. The line of reversal of hair cell polarization in the

saccular macula remained largely devoid of hair bundles. Large epithelial holes, surrounded by

supporting cells, were also observed in the abneural, but not the neural, region. In the utricular

macula, new hair bundles were seen only in the striolar region. Hair cells repopulated the striola in
a fixed manner,with new hair cells appearingfirst on the outer striolar rows and only later

appearingontheinnermostrowsof thestriolarregion.

The cellular distribution of filamentousactin in regeneratinghair cells, particularly at early

developmentaltimes, differed from that in maturehair cells. In normal animals,little or no

phalloidinlabelingwasseenwithin hair cellsor supportingcells.By contrast,newhaircellswere

diffusely labeledjust belowtheirapicalsurface,indicatingthattheir cuticularplatescontainedan

appreciableamountof filamentousactin.This labelingwasparticularly obviousin theutricular

striola andat theline of reversalof hair cell polarizationin the sacculus.In theseregions,many

cellswereobservedwith cuticularplatelabelingbutnohairbundles.Thesecellswereassumedto

representnewcellswhichwerein theprocessof creatingor repairingtheirsensoryhairbundles.

The resultsof thesestudieshavebeenpresentedin abstractform at theannualmeetingof the

Societyfor Neuroscience4 and are now being written up in manuscript form. 9

(3) Cell proliferation and hair cell regeneration in explant organ cultures.

We have created explant organ cultures by removing saccular and utricular maculae from the

membranous labyrinth in cold, oxygenated HEPES-buffered saline, trimming these organs of

excess nervous and connective tissue to improve the visibility of hair bundles and removing the

otolith membranes of these organs with proteolytic digestion and gentle mechanical agitation.

Excised organs were then incubated in Wolfe-Quimby incubation media (GIBCO) and place_l, hair

bundles upward, in lab-built culture chambers. Cultured organs were maintained for 7-14 days,

replacing half of the culture medium with fresh culture medium every 2 days.

The extent of cellular damage in normal cultures was assessed using Nomarski optics and vital

stains with light (Trypan blue) and fluorescent (Ethidium bromide) microscopy. Normal cultures

displayed little or no cellular damage and exhibited normal morphology for periods up to 14 days.

Exposing organ cultures for 6-18 hours to varying concentrations of gentamicin sulfate

(100-400 I.tmol), we have induced in vitro patterns of hair bundle and cellular damage similar to

those seen in vivo. Hair cell recovery in the saccular and utricular maculae, as measured by hair

bundle and nuclear density, was also similar to that seen in earlier in vivo studies. We have also

cultured normal and gentamicin-treated organ cultures with varying concentrations of BrdU.

Sacrificing cultures at varying times subsequent to gentamicin application, we have then been able

to monitor the number, macular location, and cellular morphology of both proliferating and

regenerating hair cells. These preliminary studies have demonstrated that our in vitro organ cultures

are a good model system for the study of cell proliferation and hair cell regeneration following

gentamicin ototoxicity.

In recent studies, we have demonstrated that hair cell regeneration takes place even in the absence

of cell proliferation. This was verified by maintaining organ cultures in aphidicolin, a blocker of

mitotic division, subsequent to gentamicin treatment. Under these conditions, little or no cell

proliferation was seen, but hair cell regeneration occurred at close to its normal rate. The results of

these preliminary studies, when complete, will be written up for pubhcation, acknowledging the

support of NASA Grant 2-651.

References

1Baird, R.A., Torres, M.A., and Schuff, N.R. 1993. Hair cell regeneration in the vestibular

otolith organs. Association of Research Otolaryngologists Abstracts 16: 103.

2Baird, R.A., Bancroft, J., and Schuff, N.R. 1993. Regional differences in lectin binding

patterns of vestibular hair cells. Hearing Research 65:151-163.

105
3Baird, R.A., Tortes, M.A., and Schuff, N.R. 1993. Hair cell regenerationin the bullfrog

vestibularotolith organs.Hearing Research 65: 164-174.

4Baird, R.A., Torres, M.A., and Schuff, N.R. 1993. Ongoing and gentamicin-induced hair cell

regeneration in the bullfrog vestibular otolith organs. Society of Neuroscience Abstracts 19: 1580.

5Baird, R.A. and Schuff, N.R. 1994. Peripheral innervation patterns of vestibular afferents in

the bullfrog utriculus. Journal of Comparative Neurology 342: 279-298.

6Baird, R.A. 1994. Comparative transduction mechanisms of utricular hair ceils. I. Responses to

inliacellular current. Journal ofNeurophysiology 71: 666-684.

7Baird, R.A. 1994. Comparative transduction mechanisms of utricular hair cells. II. Sensitivity

and response dynamics to hair bundle displacement. Journal ofNeurophysiology 71: 685-705.

106
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

Skeletal unloading, whether a result of bedrest, immobilization, or space travel, leads to a

reduction in bone mass. In a remodeling skeleton this is due both to a decrease in bone

formation and an increase in bone resorption. However, in a modeling skeleton such as in the

growing rat the relative loss of bone is thought to be due primarily to a decrease in bone

formation. We have evaluated the response of bone formation to skeletal unloading in a rat

model in which the animal's hindlimbs are unweighted by elevation of the hindquarters while the

forelimbs remain weightbearing. We observed that bone formation in the unloaded bones ceases

after 5 days of hindlimb elevation but resumes after an additional 5 days despite continued

unloading. Bone mass reaches a new and lower steady state, which is then maintained. Upon

normal weightbearing, bone formation is accelerated until bone mass is recovered.

Serum 1,25(OH)2D levels parallel the changes in bone formation during skeletal unloading;

falling during the first week of skeletal unloading, returning to control by 2 weeks, and

increasing upon normal weightbearing. However, infusion of 1,25(OH)2D to prevent its fall

during skeletal unloading did not prevent the fail in bone formation. Following this observation,

a significant question to be investigated was whether the locally produced bone growth factor

IGF-I was involved. Surprisingly, the levels of IGF-1 mRNA tended to increase during the

initial stages of unloading. Infusions of IGF-1 or growth hormone (which increases IGF-I

production by bone) increased bone formation in normally loaded bones but did not increase

bone formation in unloaded bones. These results are consistent with the hypothesis that skeletal

unloading tends to counteract the bone-growth-promoting actions of IGF-1 (and GH) at several

levels including IGF-1 production.

We are currently testing this hypothesis by evaluating the level at which skeletal unloading

blocks the ability of IGF-1 and GH to regulate events involved in bone formation. In particular,

the effect of skeletal unloading on GH and IGF-1 receptors, IGF-1 production, osteoblast

function (osteocalcin, collagen, and alkaline phosphatase), and the effect of skeletal unloading on

the ability of GH and IGF-1 to regulate these functions are being determined. We expect to be

able to determine at what level resistance to these bone regulating hormones is encountered

during skeletal unloading.

Accomplishments

(1) Demonstrated the changes in the mRNA and protein levels for IGF-I, IGF-2, and

their receptors as a function of development from 5 days before birth to 28 months.

(2) Determined the mRNA levels for IGF-I, IGF-1 receptor, alkaline phosphatase,

collagen, and osteocalcin as a function of skeletal unloading.

(3) Demonstrated that the potent bisphosphonate, alendronate, reversed the relative loss

of bone incurred during skeletal unloading.

(4) Established the in situ hybridization method for IGF- 1 mRNA using fetal bone.

107
Significance of the Accomplishments

The striking changes in mRNA levels for IGF-1, IGF-2, and their receptors during fetal through

adult development served to validate our methods and provided important insight into the

relative contributions of IGF-1 and IGF-2 in bone development. Surprisingly, the mRNA levels

for IGF-I and IGF-1R are much lower in bones from post-weaning and early adolescent rats than

in bones from the fetal or adult rats. Such observations suggest that, during this period of rapid

growth, IGF-I may not be the dominant regulator of bone growth. In light of the failure of

IGF- 1 mRNA to change during skeletal unloading, this possibility needs to be considered further.

In this regard, the collagen and osteocalcin mRNA levels fell with skeletal unloading, but the

IGF-1 and IGF-1R mRNA levels did not. Measurements of IGF-1 protein are in progress, but

these data indicate a surprising dichotomy between parameters of bone formation and the

production of a factor thought to be responsible for bone formation, i.e., IGF-1. We are

examining the localization of the mRNA for IGF-1 by in situ hybridization as affected by

skeletal unloading, since changes in the mRNA levels in cells critical for bone formation and

affected by skeletal unloading may be obscured by the IGF-1 mRNA levels in other cells not

responding to skeletal unloading, yet contributing to the total mRNA signal in whole bone

extracts.

Alendronate, a potent inhibitor of bone resorption, was evaluated for its ability to affect bone

mass during skeletal unloading as a test of the hypothesis that bone resorption contributes little to

the relative bone loss in the growing rat subjected to skeletal unloading. The results indicated

that the contribution of bone resorption was higher than expected. Alendronate resulted in

increased bone in the metaphyseal region in both controls and unloaded rats, loss of contact

between osteoclasts and the underlying bone, and inhibition of bone formation in the tibia-fibula

junction. Thus, protection against bone loss during skeletal unloading is not without some

potentially serious side effects. Nevertheless, these data indicate that bone resorption contributes

substantially to the relative loss of bone during skeletal unloading even in the growing rat and

suggests a potential means of reducing bone loss during space flight.

Publications

Bikle, D.D. 1992. What can we learn about the impact of space flight on bone metabolism? In:

Proceedings of the Conference on Space Flight and Bone Metabolism, Tokyo, Japan.

Bikle, D.D., Currier, P.A., Morey-Holton, E.R., Doty, S.B., and Halloran, B.P. 1993.

Alendronate preserved bone mass during skeletal unloading. ASGSB Bulletin 7: 36.

Bikle, D.D., Halloran, B., Fong, L., Steinbach, L., and Shellito, J. 1993. Elevated 1,25

dihydroxyvitamin D levels in patients with chronic obstructive pulmonary disease treated with

prednisone. Journal of Clinical Endocrinology and Metabolism 76:456-461.

Bikle, D.D., Halloran, B.P., Harris, J., Cone, C., and Morey-Holton, E. 1991.

unloading results in increased mRNA for IGF-1 but decreased mRNA for collagen.

Bulletin 5: 36.

Skeletal

ASGSB

Bikle, D.D., Halloran, B.P., Harris, J., and Morey-Holton, E. 1993. Skeletal unloading induces

resistance to IGF-1. Journal of Bone and Mineral Research 8: S155.

Bikle, D.D., Halloran, B.P., Harris, S.T., and Portale, A.A. 1992. Progestin antagonism of

estrogen stimulated 1,25 dihydroxyvitamin D levels. Journal of Clinical Endocrinology and

Metabolism 75:519-523.

108
Halloran,B.P.,Bikle, D.D., Hams, J., Cone, C., Foskett, H.C., and Morey-Holton, E. 1992.

Changes in 1,25-dihydroxyvitamin D kinetics and the plasma concentrations of calcium,

phosphorus and l:rlT-t during skeletal unloading. ASGSB Bulletin 6: 92.

Halloran, B.P., Bikle, D.D., Harris, J., Daly, R., Thai, P., Cone, C.M., and Morey-Holton, E.

1991. Production and metabolic clearance of 1,25(OH)2D during skeletal unloading. ASGSB

Bulletin 5: 64.

Halloran, B.P., Bikle, D.D., Harris, J., Foskett, H.C., and Morey-Holton, E. 1993. Skeletal

unloading decreases production of 1,25 dihydroxyvitamin D. American Journal of Physiology

264: E712- E716.

Orwoll, E., Ware, M., Stribrska, L., Bikle, D.D., Sanchez, T., Andon, M., and Li, H. 1992. The

effect of dietary protein deficiency on mineral metabolism and bone mineral density. American

Journal of Clinical Nutrition 56:314-319.

Tanner, S.F., Doty, S.B., Morey-Holton, E.R., Bikle, D.D., and Halloran, B.P. 1993. The effect

of bisphosphonate on weighted and unweighted cancellous bone. ASGSB Bulletin 7: 80.
PARATHYROID HORMONE STIMULATES

ABUNDANCES IN RAT OSTEOBLASTIC CELLS

John C. Clohisy

Saint Louis University Medical Center

Department of Orthopedic Surgery

3635 Vista Avenue at Grand Boulevard

St. Louis, MO 63110

c-fos AND c-jun mRNA

Description of Research

Exposure to microgravity results in hypercalciuria and loss of skeletal mass. The cellular and

intracellular events which mediate these physiologic changes remain unclear. We hypothesize that

in a weightless environment bone cells, specifically the osteoblasts, may have an altered sensitivity

to certain resorptive factors like parathyroid hormone (PTH). The objective of my

NASA-supported research was to define further the intracellular mechanisms which mediate

PTH-altered gene expression in the osteoblast. We were specifically interested in analyzing the

immediate-early genes, c-fos and c-jun. This work was designed to establish a model which could

serve as a foundation for future experiments conducted at zero gravity.

We utiiized the rat osteoblastic osteosarcorna cell line, UMR 106-01, which is commonly u_ed as a

model of the osteoblast. The effects of PTH and other agents of interest were measured at the

mRNA (Northern blot analysis) and transcriptional (nuclear run-on assay) levels. We studied the

immediate-early genes c-los and c-jun which are proto-oncogenes that respond to, and mediate, the

effects of many extracellular stimuli. These genes encode the protein productsfos and jun which

are major components of the activator protein- 1 transcription complex.

Accomplishments

Our work clearly demonstrated that in the UMR 106-01 osteoblastic cell line PTH stimulated a

marked, transient increase in c-los (50 fold) and c-jun (5 fold) mRNA levels. Both transcripts

were maximally elevated approximately 30 minutes after hormone treatment. Nuclear run-on

assays demonstrated that PTH also stimulated a transient increase in the rate of c-los and c-jun

transcription which was maximal 20 minutes after hormone exposure. Additional experiments

were performed to further define the signal transduction pathways involved in these responses.

We demonstrated that the protein synthesis inhibitor cylcloheximide had no effect on the

PTH-induced increase in c-los and c-jun rnRNAs. Second messenger analog experiments showed

that PTH stimulates c-los and c-jun mRNA abundance via a mechanism which is partially

mimicked by protein kinase A activation and is independent of PMA-sensitive protein kinase C

pathways.

Significance of Accomplishments

Parathyroid hormone is a potent stimulator of bone resorption and could be involved in the process

of microgravity-induced bone loss. Nevertheless, little is known about the intraceUular events

which mediate PTH-altered gene expression. The osteoblast is the PTH target cell in bone; and,

therefore, we analyzed the effect of PTH on immediate-early gene (c-los and c-jun) expression in

this cell type. Our work identified and characterized two immediate-early gene responses which

may be fundamental to PTH-altered gene expression in the osteoblast. These data have enhanced

our understanding of the intraceUular events which mediate the action of PTH. By further

understanding the "normal" cellular response to PTH, we can formulate experiments to determine

110
whether these responsesare altered at zero gravity and whether they play a role in

microgravity-inducedboneloss.

Publications

Clohisy, J., Scott, D., Brankenhoff, K., Quinn, C., and Partridge, N. 1992. Parathyroid

hormone induces c-fos and c-jun mRNA abundances in rat osteoblastic cells. Molecular

Endocrinology 6:1834-1842.

Scott, D., Quinn, C., Brankenhoff, K., Clohisy, J., and Partridge, N. Parathyroid hormone

induces transcription of collagenase in rat osteoblastic cells by a mechanism utilizing cAMP and

requiring protein synthesis. Molecular Endocrinology 6:2153-2159.

Scott, D., Weaver, W., Clohisy, J., Brankenhoff, K., Kahn, A., and Partridge, N. 1992.

Regulation of an H-ras-related transcript by parathyroid hormone in rat osteosarcoma cells.

Molecular Endocrinology 6: 1425-1432.

111
BASIC GRAVITATIONAL REFLEXES IN THE LARVAL FROG

Stephen L. Cochran

Department of Otolaryngology

University of Texas Medical Branch at Galveston

Galveston, TX 77555

Description of Research

Little is known about how vertebrates are able to sense gravity and how they process this

information to generate appropriate motor responses. This investigation is designed to determine

how a primitive vertebrate, the bullfrog tadpole, is able to sense and process gravitational stimuli.

Because of the phytogenetic similarities of the vestibular systems in all vertebrates, the

understanding of the gravitational reflexes in this relatively simple vertebrate should elucidate a

skeletal framework, on an elementary level, upon which the more elaborate reflexes of higher

vertebrates may be constructed.

The tadpole manifests a powerful counter-rolling response of the eyes to static tilt of the head. In

addition, the fact that amphibians are cold-blooded means that their cellular metabolism is much

lower than that of mammals. Consequently, the entire head can be maintained in vitro with the

brain, sensory structures, and eye muscles exposed. The strong gravitational reflexes are still

evident under these conditions and persist for several days. Such conditions permit detailed and

reproducible electrophysiological and anatomical investigations of this reflexive behavior.

The purpose of this study is to understand how the nervous system of the larval amphibian

processes gravitational information. This study involves predominantly electrophysiological

investigations of the isolated, alert (forebrain removed), bullfrog tadpole head. The focus of these

experiments is threefold: (1) to understand from whole extraocular nerve recordings the signals

sent to the eye following static gravitational tilt of the head; (2) to localize neuronal centers

responsible for generating these signals through reversible pharmacological ablation of these

centers; and (3) to record intraceUularly from neurons within these centers in order to determine the

single neuron's role in the overall processing of the center. This study will provide information on

the mechanisms by which a primitive vertebrate processes gravitational reflexes.

Accomplishments

(1) Quantification of the behavior. A software event detection technique, which has

been developed over the last 15 years, has been finalized and published (Cochran, 1993). This

technique allows for analysis of spontaneously occurring voltage deflections over time.

Electrophysiological recordings digitized (at 50 kHz) over many minutes can be rapidly and

automatically analyzed. The publication illustrates this technique for spontaneously occurring

synaptic potentials recorded intracellularly from VllIth nerve afferents that are consequent from

convergent hair cell innervation of the afferents. This technique allows for tens of thousands of

excitatory post-synaptic potentials (EPSPs) to be analyzed during this period. With respect to the

experiments in this study that involve whole nerve recordings from the trochlear and medial rectus

nerves, this same technique can be applied to detect and quantify the extracellularly recorded nerve

action potentials. Since these whole nerve recordings are more stable than the intracellular

recordings of EPSPs, digitized periods are often over an hour in duration, allowing for continuous

quantification of hundreds of thousands of spike potentials that occur during the recording period.

Since the amplitude (as well as other parameters) of each spike potential is quantified, whole

nerve-recorded spike potentials can be parceled according to size and their size can then be related

to the time of occurrence during a tilt stimulus.

(2) Responses to static tilt of the head. Trochlear motoneurons increase their

frequency of firing with nose-up tilts and decrease it with nose-down tilts. Motoneurons

112
innervatingthemedialrectusmusclerespondreciprocally;i.e., theyincreasetheir activity with

nose-downtilts anddecreasetheir activity with nose-uptilts. For boththetrochlearandmedial

rectusmotoneurons,thelargersizedunitsarephasicandadaptmorerapidly thanthesmallersized

units,whichappearto havealargertoniccomponentandadaptlessrapidly. Typically largerunits

fire only during a movement(relatingthemmoreto velocity), while smallerunits appearto be

modulatedbothby thetable movementaswell asby theabsolutepositionof thetable(relating

thembothto tablevelocityandposition).

(3) Pharmacological studies. Iontophoresis of the kynurenic acid, an antagonist of

the excitatory transmitter glutamate, into the extraocular motor nuclei reversibly abolishes spike

activity in the extraocular motor nerves. Intracellular recordings from oculomotor neurons show

complex mixtures of EPSPs and inhibitory post-synaptic potentials (IPSPs) following electrical

stimulation of the vestibular nerves and afferent brainstem pathways to this of nucleus. Bath

application of kynurenic acid reversibly blocks over 95 percent of the EPSPs. Both of these

findings are consistent with the hypothesis that the principal excitatory transmitter to the

oculomotor nucleus is glutamate or a related compound.

Significance of the Accomplishments

In general, the above accomplishments provide a step toward understanding how these primitive

vertebrates sense gravitational stimuli and how they process these stimuli and integrate them into

reflex control.

The development of the quantification technique is an important step in acquiring the ability to

investigate how gravity can influence eye movements. Moreover, this technique is generalizable to

any spontaneously occurring events that can be represented as voltage deflections over time,

indicating that it has a potential use other than its strict application in this study.

The similarity of the responses from the trochlear versus medial rectus motoneurons (although their

receptive field properties are different) suggests that different inner ear end organ signals converge

upon these motoneurons but that the quality of the processing of these signals is similar. The

contribution of individual end organs and specific vestibular nuclear complex neurons in

channeling the static and dynamic signals remains to be determined.

The finding that glutamate is the main excitatory transmitter to the oculomotor nucleus indicates that

glutamate is the principal excitatory transmitter of the entire vestibulo-ocular reflex. Glutamate (or

a related compound) is the hair cell transmitter, it is the transmitter of VIIIth nerve afferents, it is

the transmitter of excitatory commissural vestibular neurons, and it is the transmitter of vestibulooculomotor neurons.

These findings provide basic information as to the organization of gravitational-ocular reflexes in a

relatively simple lower vertebrate. These studies can provide a basic framework for understanding

how these reflexes are organized and elaborated upon in the more complex vestibulo-ocular

behaviors exhibited by higher vertebrates.

Publications

Cochran, S.L. 1992. Responses of medial rectus motoneurons to static tilt of the isolated tadpole

head. ASGSB Bulletin 6: 93.

Cochran, S.L. 1992. Synaptic input to oculomotor neurons of the frog. Society for Neuroscience

Abstracts 18: 28.

Cochran, S.L. 1993. Algorithms for detection and measurement of spontaneous events. Journal

of Neuroscience Methods 50: 105-121.

113
A GRAVITY-SENSITIVE PERIOD DURING FROG OOCYTE MATURATION

Michael V. Danilchik

Department of Biological Structure and Function

Oregon Health Sciences University

Portland, OR 97201-3097

Description of Research

The unfertilized amphibian egg appears to be radially symmetric about its animal-vegetal axis.

Establishment of the embryo's bilaterally symmetric body plan, referred to as dorsal-ventral (DV)

axis specification, requires a 30 ° rotation of the vegetal yolk mass relative to the egg surface during

the first cell cycle. The direction of this rotation reliably determines the DV axis orientation and

usually reflects the sperm entry position (SEP). Thus, the SEP is often used to predict the plane of

bilateral symmetry. However, the spatial relationship between the SEP and the DV axis is actually

quite variable, suggesting that cues other than the SEP also bias the rotation mechanism. One wellknown external influence is gravity; i.e., when fertilized eggs are tilted from their usual orientation,

gravity-driven internal rearrangements result in rotation directions different than that specified by

the SEP. Endogenous cues to direct rotation are also present in the egg prior to fertilization; for

example, activated eggs exhibit a normal, directed rotation, even though they have not been

fertilized. We observed that, in eggs of the frog Xenopus laevis tilted 90 ° off axis during in vitro

maturation, the maturation spot appears about 15 ° from the center of the animal hemisphere closest

to the point of the equator that was positioned upward. When such eggs are activated, the vegetal

yolk mass rotates toward this point. The fact that normal spawned eggs behave similarly suggests

that off-axis influences also occur under in vivo conditions. As in oocytes matured in vitro, the

yolk mass of spawned eggs rotates toward the maturation spot, confirming the presence of an

endogenous gravity-sensitive cue in the egg. The goal of this project is to identify the gravitysensitive mechanism by which eggs direct the assembly of the parallel microtubule bundles that

constitute the cortical rotation apparatus.

Accomplishments

We demonstrated a strong functional correlation between maturation spot position and yolk mass

rotation direction in activated eggs. Rotation direction was recorded by time-lapse confocal

fluorescence microscopy of fluorescently labeled subcortical mitochondria, a method that gives an

accurate overall view of cortical/cytoplasmic rearrangements occurring over the entire vegetal

surface. Both spawned eggs and eggs that had been experimentally manipulated to produce an

eccentric maturation spot showed a strong bias to rotate toward the maturation spot meridian,

supporting the hypothesis that the orientation of oocytes undergoing maturation affects the polarity

of the cortical rotation machinery. Significantly, in fertilized eggs, the direction of yolk mass

rotation is detectably influenced by this gravity-based cue, acting in cooperation with the sperm

aster. Because the rotation direction ultimately establishes the orientation of the dorsal-ventral axis,

the wide variability in the relationship between the SEP and the dorsal side probably stems from

the random orientations experienced by individual oocytes during maturation. These results

suggest two possible mechanisms by which gravity could cue the rotation direction: (a) the cortical

cytoskeleton of the maturing egg becomes entrained in a way that promotes directed microtubule

assembly in the vegetal cortex; or (b) a microtubule organizer, perhaps one associated with the

female pronucleus, becomes displaced from a concentric axial location.

Significance of the Accomplishments

We have established that obliquely applied gravitational force experienced by Xenopus eggs during

meiotic maturation affects the rotation-determining cortical cytoskeletal machinery. It is well

114
knownfrom work in variouslaboratories,includingourown,thatthevegetalyolk massrotation

directioncan be experimentally redirected by simply tipping the egg for a few minutes at the

beginning of the rotation phase of the first cell cycle. This sensitivity to gravity implies a positive

feedback relationship between rotation itself and the directed growth of cortical microtubules in the

shear zone that develops between the cell surface and the deep cytoplasm. Recently, it was

revealed that many, if not most, cortical microtubules are continuous with radial microtubules

emanating from the cell center.

A plausible scenario for the sequence of events leading to directed rotation would begin with the

growing microtubule tips arriving at the egg surface early in the first cell cycle. Once at the

surface, they could easily respond to shear-generated alignment that would result from any relative

movement between cortex and inner cytoplasm. Their subsequent parallel growth would, in turn,

reinforce continued motion in the same direction by enabling (+) end-directed transport of vesicular

components of the yolk mass, for example, the endoplasmic reticulum (ER). Thus, a preexisting

rotation-fixing bias might take the form of asymmetries induced in the yolk mass during

maturation.

Alternatively, non-microtubular components of the vegetal cortex might have become aligned by

shear during meiotic maturation. One candidate for such alignment would be the network of

intermediate filaments (cytokeratin) in the cortex. It is known that cytokeratin filaments undergo

disassembly and reorganization during maturation. However, whether the organization of the

reassembled network reflects directional shear experienced during maturation has not been

investigated.

Finally, there is the possibility that the microtubule organizer at the cell center becomes displaced

from its original location toward one side during maturation. Following parthenogenetic

activation, microtubules emanating from this eccentric cell center would arrive at the egg cortex on

one side more quickly than elsewhere. This initial asymmetry might then be sufficient to promote a

limited yolk mass movement relative to the cortex that would then entrain the growth of

microtubules arriving at the surface in other regions.

We are presently carrying out experiments to address these various possibilities.

Publications

Brown, E.E., Denegre, J.M., and Danilchik, M.V. 1993. Deep cytoplasmic rearrangements in

ventralized Xenopus embryos. Developmental Biology 1(60): 148-156. (NAGW 3798).

Danilchik, M.V., Brown E.E., and Margelot, K.M. 1993. Cues for dorsalventral axis formation

in unfertilized Xenopus laevis eggs (Abstract 102). ASGSB Bulletin 7(1): 76 (NAGW 3798).

Denegre, J.M. and Danilchik, M.V. 1993. Deep cytoplasmic rearrangements in axis-respecified

Xenopus embryos. Developmental Biology 1(60): 157-164. (NAGW 3798).

Phillips, C.R. and Danilchik, M. 1993. Gravitational influence of mitochondrial localization

during the first cleavage cycle in Xenopus embryos (Abstract 150). ASGSB Bulletin 7(1): 96

(NAGW 3798).

115
MECHANICAL MODULATION OF STRIATED MUSCLE PHENOTYPE

Robert S. Decker

Department of Medicine/Cardiology

Northwestern University

Medical School

Chicago, IL 60611

Description of Research

Mechanical load has been documented to regulate the mass of the heart and skeletal muscles. This

investigation employs cultured adult cardiac myocytes and skeletal muscle myotubes to evaluate

how mechanical load controls the expression, turnover, and assembly of contractile proteins and to

determine whether specific cytoskeletal proteins are crucial to the maintenance of the striated

muscle phenotype and its contractile properties. The hypothesis being presently tested is that

microgravity alters the forces that the heart and skeletal muscles must overcome to conduct

meaningful work and that a reduction in mechanical workload alters cytoskeletal-myofibrillar

interactions in these tissues. Experiments described briefly in the progress report reveal that the

cytoskeletal-associated proteins, a-actinin and vinculin, appear to have a pivotal role in

mainte.nance of myofibril structure. The a-actinin appears to be important in the early phases of

myofibril assembly, whereas vinculin redistribution may be associated with the ability of

myofibrils to withstand force generation. Regulation of cytoskeletal protein expression, turnover,

and incorporation into myofibrils will provide new information on the subcellular mechanisms that

mediate mechanical load-dependent myofibril assembly and potential signal pathways that may be

modulated in a microgravity environment, where gravitational forces are markedly reduced and

mechanical work is believed to be diminished proportionately.

The principal objective of year 1 was to determine how activation of beating altered the turnover of

contractile cytoskeletal proteins in cultured adult cardiac myocytes and whether such changes

modulated growth or atrophy of the heart cell. The specific aims of year 1 were (1) to monitor the

redistribution of the cytoskeletal-associated proteins a-actinin and vinculin in beating heart cell

preparations by immunofluorescence microscopy, (2) to determine the turnover rates of

contractile/cytoskeletal proteins, and (3) to explore the potential pathways of proteolysis and their

contribution in regulating contractile/cytoskeletal protein composition.

Accomplishments and Significance of the Accomplishments

The experiments conducted in the past year were designed to distinguish the direct actions of

catecholamines from beating (i.e., mechanical loading) on the turnover and reorganization of

contractile proteins and the maintenance of myofibrillar structure. Observations derived from nonbeating, passively loaded rabbit heart cells indicate that catecholamines can maintain and even

promote the fractional rate of heart cell growth and modulate the turnover of contractile proteins,

but adrenergic activation is incapable of inducing the assembly of these nascent proteins into

myofibrils in nonbeating rabbit cardiac myocytes (Decker et al., 1993a). Norepinephrine enhanced

contractile/cytoskeletal protein synthesis to a greater degree than either the 13-agonist,

isoproterenol, or the ot-agonist, phenylephrine. However, these catecholamines provoked

myofibrillar disruption, suggesting that a- and/or 13-adrenergic stimulation, in the absence of

contractile activity, may enhance protein turnover but fail to promote myofibrillogenesis in these

heart cells.

In cultured adult feline myocytes however, adrenergic agonists with [31 activity induced contractile

activity. The 131-agonist, isoproterenol, stimulated the synthesis of cytoskeletal proteins, and

116
myofibrillar reorganizationwasapparentwithin24hoursaftertheinductionof beating(Simpsonet

al., 1993). If beating was blocked by membrane depolarization with KC1, then myofibril

reassembly was prevented even though changes in contractile protein synthesis were not altered by

potassium. The results of these experiments support the hypothesis that catecholamines stimulate

cytoskeletal protein synthesis, but changes in mechanical loading (i.e., beating) are required to

assemble these proteins into functional myofibrils (Decker et al., 1993c).

To further explore the role of mechanical load in regulating the turnover and assembly of contractile

proteins in the absence of adrenergic stimulation, feline myocytes were either passively stretched

10 percent of their rest length or subjected to electrical field stimulation at 1 Hz (60 beats per

minute). Both experimental paradigms enhanced the rate of contractile protein synthesis, although

field stimulation elevated the fractional rate of protein synthesis about 55 percent within 4 hours of

electrical activation, whereas passive stretch induced an increase of only 35 percent over the same

interval (Decker et al., 1993b). Both passive stretch and field stimulation induce myofibril

reassembly, which appears to be preceded by the appearance of primitive ct-actinin/vinculin

positive focal contacts. New myofibrils are probably assembled at these sites, but future

experiments will be required to verify this assumption (Decker et al., 1993e).

The extent to which heart and skeletal muscles maintain the mass of their contractile proteins must

reflect the difference between the amount of protein synthesized and the amount degraded.

Experiments designed to explore proteolytic mechanisms have uncovered two pools of protein in

cardiac myocytes. The f'trst is a small pool accounting for 10-15 percent of total myocyte [9rotein

(Simpson et al., 1993). This pool has a half-life of about 22 hours and is composed of nascent

contractile proteins that may represent precursors for myofibrillogenesis. The second pool consists

of predominantly contractile proteins whose half-lives approach 2 weeks. Isoproterenol

significantly slows the degradation rate in the small, rapid-turnover pool. Since myofibrillar

proteins (e.g., myosin, actin, tropomyosin, etc.) can be found in both pools, changes in pool size

and the turnover rate within these pools may provide some insight into how mechanical load

regulates the composition of the contractile apparatus in cardiac and skeletal muscle.

Publications

Clark, W.A., Rudnick, S.J., Simpson, D.G., La Pres, J.J., and Decker, R.S. 1993. Isolated

adult cardiac myocytes maintain the protein synthetic capacity of adult heart during prolonged

culture. American Journal of Physiology (Heart and Circulation) 264: 573-582.

Decker, M.L., Behnke-Barclay, M., Janes, D.M., Clark, W.A., and Decker, R.S. 1993b.

Electrical field stimulation and passive stretch enhance contractile protein synthesis in cultured adult

heart cells. Journal of Molecular and Cellular Cardiology 25(3, Suppl.): $28.

Decker, M.L., and Decker, R.S. 1993e. Regulation of lysosomal biogenesis and protein

degradation in cultured rabbit cardiac myocytes. Molecular Biology of the Cell 4: 446a.

Decker, R.S., Cook, M., Behnke-Barclay, M., Decker, M.L., Lesch, M., and Samarel, A.M.

1993a. Catecholamines modulate protein turnover in cultured quiescent rabbit cardiac myocytes.

American Journal of Physiology (Heart and Circulation) 265: H329-H339.

Decker, R.S., Cook, M.G., Behnke-Barclay, M., Decker, M.L., and Clark W.A. 1993. A new

approach to study adrenergic regulation of cellular hypertrophy in adult cardiac myocytes. AHA

Conference, "Cellular and Molecular Regulation of Cardiac Hypertrophy," Asilomar, CA.

Decker, R.S, Decker, M.L., Behnke-Barclay, M., and Clark, W.A. 1993c. Beating and cell-cell

contact regulate turnover and distribution of cytoskeletal proteins in cultured adult heart cells.

Journal of Molecular and Cellular Cardiology 25(3, Suppl.): $82.

117
Decker,R.S.,Simpson,D.G., Behnke-Barclay, M., Decker, M.L., Terracio, L., and Borg, T.K.

1993d. Early aspects of myofibril reassembly in cultured adult cardiac myocytes. Molecular

Biology of the Cell 4: 388a.

Simpson, D.G., Decker, M.L., Clark, W.A., and Decker, R.S. 1993. Contractile activity and

cell-cell contact regulate myofibrillar organization in cultured adult heart ceils. Journal of Cell

Biology 123: 323-336.

Simpson, D.G., and Decker, R.S. 1993. The cardiac cytoskeleton: Functional integration of the

contractile apparatus. Heart Failure 6: 239-254.

118
OTOLITH-CANAL CONVERGENCE IN VESTIBULAR NUCLEI

J. David Dickman

Department of Surgery (Otolaryngology)

The University of Mississippi Medical Center

2500 North State Street

Jackson, Mississippi 39216

NEURONS

Description of Research

The primary objective of the current investigation is to determine how information regarding head

movements and head position relative to gravity is received and processed by central vestibular

nuclei neurons in the brain stem. Specialized receptors in the vestibular labyrinths of the inner ear

function to detect angular and linear accelerations of the head, with receptors located in the

semicircular canals transducing rotational head movements and receptors located in the otolith

organs transducing changes in head position relative to gravity or linear accelerations of the head.

The information from these different receptors is then transmitted to central vestibular nuclei

neurons, which process the input signals and then project the appropriate output information to the

eye, head, and body musculature motor neurons to control compensatory reflexes. Although a

number of studies have reported on the responsiveness of vestibular nuclei neurons, it has not yet

been possible to determine precisely how these ceils combine the information from the different

angular and linear acceleration receptors into a correct neural output signal. However, by utilizing

a mechanical stimulation technique for the semicircular canals, along with electrical polarization of

afferent fibers and traditional rotational and linear acceleration stimuli, the current project will be

able to discretely activate the different receptor systems and thereby directly examine these

questions.

Accomplishments

Vestibular afferent and nuclei neuron responses elicited by electrical stimulation.

Before the synthesis of information from the different linear and angular acceleration receptors by

the central vestibular neurons was examined, it was first necessary to determine if selective types

of afferents could be differentially activated by their thresholds to electrical stimulation.

Previously, it had been demonstrated in monkeys that small electrical currents applied to the

labyrinth can selectively either silence or excite afferent fibers that have irregular discharge rates,

while afferents with more regular fixing patterns are little affected. It is currently believed that these

regular and irregular firing afferents converge upon vestibular nuclei neurons in unique patterns to

control different types of muscle movements. For example, there is evidence to suggest in

monkeys that (a) regular afferents are more involved in the control of eye movements, while (b)

irregular afferents primarily are involved in the control of head and/or postural movements. As an

added tool in the current investigation of synthesis of converging linear and angular acceleration

inputs onto vestibular nuclei neurons, electrical stimulation of the labyrinth was used to examine

the effectiveness of selectively stimulating the regular and irregular afferent fibers in pigeons.

Electrical stimulating electrodes were implanted into the perilymphatic space of the labyrinth, while

extracellular electrophysiological recordings were made from the ipsilateral semicircular canal and

otolith afferent fibers. Both anodal and cathodal electrical direct currents were utilized, with

current magnitudes ranging between -100 and +100 I.tA. In addition, for many neurons,

sinusoidal rotational (semicircular canal afferents) or off-vertical axis rotational (OVAR or otolith

afferent) stimulation was applied with and without simultaneous electrical stimulation, in order to

determine if the responsiveness of the fiber changed during the electrical stimulation. To date. 67

afferents have been studied using electrical stimulation, with effects observed in both semicircular

canal and otolith afferent fibers.

119
Similar to the results reported in monkeys, pigeon afferent fibers can be selectively affected based

upon their discharge regularity. All afferent fibers were inhibited by cathodal (positive) currents

and were excited by anodal (negative) currents. Irregular firing afferent fibers had substantially

lower thresholds to electrical currents applied to the labyrinth than did regular afferent fibers. In

fact, irregular afferent fibers can be selectively silenced with low amplitude (5-25 I.tA) anodal

currents. Regular fh-ing afferents, however, could never be silenced even when administering

large amplitude (100 }.tA) currents. The afferents' response magnitudes were correlated with the

electrical stimulus magnitude, with the sensitivity of the afferents to electrical stimulation being

proportionally related to the discharge regularity. When electrical stimulation was applied to the

afferent fiber during either rotational or OVAR stimulation, the discharge rate and the sensitivity of

the neuron to the acceleration stimulus was altered for irregular firing afferents but not for regular

afferents with low amplitude (25 l.tA) electrical currents, as shown in Figure 1. Irregular f'tring

semicircular canal fibers could be ablated (silenced) with electrical stimulation, even during

sinusoidal rotations at 20" or 30" per second peak velocity. Similarly, irregular fLring otolith

afferent fibers could also be ablated during off-vertical axis rotations of 36" per second constant

velocity. However, with regular fu-'ing afferents, either no effect or only a slight change was

observed in the sensitivity of the fibers to OVAR or angular rotation.

2o0o ,-,o_ A

200.01 +sOv'L B

i°°

1 _ ooK AAAAAAAA

• IVVVVVVVVV rr -40.0 ....

0.0 20.0 40.0 80.0 80.0

Seconds

Figure 1. Neural response

histograms of vestibular nuclei

neurons to sinusoidal rotational and,

simultaneous electrical current

stimulation. A and B = responses

from a vestibular nuclei neuron that

received probable monosynaptic

input from the ipsilateral ear (1.3

msec latency) and was classified as

a horizontal canal Type I neuron.

The neuron did not respond to

rotations in the vertical canal

planes. C = responses from a

second horizontal canal related

vestibular nuclei neuron that

received polysynaptic input from

the ipsilateral ear (4.0 msec

latency). Rotational stimuli

(bottom trace) were applied in the

horizontal plane at a frequency of

0.1 Hz and a peak velocity of

20o per second. Electrical

stimulation (dark bar) consisted of

30 sec +25 I.tA of current applied

bilaterally.

Now that the response characteristics and sensitivity of the afferent fibers to electrical stimulation

have been established, studies of the irregular and regular afferent fiber inputs to the vestibular

nuclei neurons can be conducted. Recordings are currently being obtained from these central

neurons while electrical ablation of the irregular afferent inputs is utilized. The responses of the

central neurons to both rotational and linear accelerations are obtained, first, without electrical

stimulation to determine the response sensitivity, and then, with electrical stimulation while

simultaneously stimulating the labyrinth with either rotational or linear accelerations. Differences

in the response sensitivity can then be correlated with the types of afferent input (regular or

irregular) to the neuron, as well as the amount of convergence between semicircular canal and

otolith organ afferents.

120
Significance of the Accomplishments

During manned space flight, acute vestibular disturbances often occur, leading to physical stress

and a loss of performance. The purpose of the current project is to determine how vestibular nuclei

neurons synthesize information from the different linear and angular acceleration receptors in the

labyrinths into an integrated output signal. However, it was first necessary to delineate the

different types of afferent fibers, since it is thought that regular and irregular firing afferents differ

in their input patterns onto vestibular nuclei (VN) neurons and their subsequent control over eye,

head, and postural musculature. Using electrical polarization of the labyrinth while obtaining

extraceUular recordings from afferent fibers, it was found that both semicircular canal and otolith

fibers could be selectively affected based upon the cell's discharge regularity. Irregular-firing

afferent fibers axe much more sensitive to the electrical stimulation and can be reversibly ablated

with low-amplitude stimulation. Thus, while delivering electrical stimulation to ablate the irregular

firing afferents, simultaneous rotational and linear acceleration stimuli are now being applied to

study the effect of convergence of these different types of inputs onto vestibular nuclei neurons. It

is possible that information regarding linear and angular accelerations of the head axe encoded

differently in the central neurons, since muscle groups concerned with compensatory motor

reflexes produce differing responses. Investigations during the next year will determine how the

central vestibular nuclei neurons synthesize the different irregular and regular firing afferent inputs

along with their varied linear and angular acceleration signals. In this way, we hope to discover

how the vestibular system is affected by the changes in linear acceleration that occur during

manned space flight.

Publications

Dickman, J.D. 1993. Spatial orientation vectors of semicircular canal afferent fibers in pigeons

(Abstract). ASGSB Bulletin 7: 78.

Dickman, J.D. and Angelaki, D.E. 1993. Changes in responses of canal-related vestibular

neurons via galvanic ablation of irregular afferent fibers in pigeons. Washington, DC, Society of

Neuroscience Abstracts 19:139.

Dickman, J.D. and Coreia, M.J. 1993. Communications between contralateral vestibular

labyrinths in pigeons. Neuroscience 57:1097-1108.

121
MODULATION OF BONE REMODELLING

CHANNELS

Randall L. Duncan

Renal Division

Jewish Hospital of St.

Washington University

216 S. Kingshighway

St. Louis, MO 63110

Louis

School

Blvd.

of Medicine

VIA MECHANO-SENSITIVE ION

Description of Research

One of the most negative physiologic effects resulting from extended periods of weightlessness is a

rapid loss of bone mass as well as total body calcium. This condition is similar to a pathology

which results from prolonged immobilization, termed disuse osteoporosis. Conversely, chemical

observations noted during in vivo studies suggest that physical exercise or applied mechanical load

has a positive effect on bone formation. Continuous or intermittent compressive force applied to

bone or bone cells has been shown to increase a number of parameters which are associated with

increased osteoblast activity. While mechanical force has been shown to modulate the second

messenger levels in the osteoblast, such as cyclic AMP, inositol phosphate, and intracellular

calcium, the signal transduction mechanism by which physical force is converted to chemical

message is still unclear. We have characterized a mechanically sensitive cation selective channel in

the osteoblast-like osteosarcoma cell line UMR-106.01, which we hypothesize may be the signal

transducer for mechanically induced osteogenesis. The goal of this research is to determine the

effects of mechano-sensitive ion channel activity in osteoblasts and to f'md the effects of these

channels on osteoblast function. In addition, we will characterize the effects of hormonal factors

on these channels and whether hormonal factors will potentiate the response of the osteoblast via

these mechano-sensitive channels. To attain these goals, we will combine the patch-clamp

technique to study ion channel activity with methods from cell biology. We will characterize the

effects of stretch on this mechanically sensitive channel and determine their role in formation and

secretion of bone matrix proteins, such as collagen, osteopontin, and osteocalcin. We will

determine if second messengers such as cyclic AMP, intracellular calcium, diacylglycerol, or

inositol phosphate are involved in the translation of biophysical stimuli in the biochemical event.

We will also determine if hormones such as the parathyroid hormone (PTH) and glucocorticoids

have an effect on this channel activity and whether these hormones have an additive effect on

stretch or mechanical stimulation in bone. During this reporting period, the focus of this research

centered on the modulation of these channels in response to PTH and the second messenger system

by which PTH modulates this channel.

Accomplishments

(1) Patch-clamp analysis of UMR-106.01 cells demonstrated that PTH modulates the

mechanically sensitive cation channel in two distinct ways. PTH produced a two to fourfold

increase in channel activity during stretch and also increased the conductance of the channel.

(2) Application of membrane-permeable second messengers to the UMR cell indicated that

cyclic AMP and an increase in intracellular calcium could mimic only one of the effects of PTH,

which was an increase in single-channel conductance.

(3) Patch-clamp studies demonstrated that this mechanically sensitive channel was

dependent on attachment to the cytoskeleton of the cell. When cytochalasin D, which cleaves actin

filaments, was added to the cell, the mechanically sensitive ion channels increased their channel

activity in the same way in which PTH modulated the stretch-activated cation channel. These data

122
would indicate that PTH modulates osteoblast behavior possibly through affecting the cytoskeleton.

Significance of the Accomplishments

PTH is a calciotropic hormone which is released in response to low plasma calcium. PTH,

however, has paradoxical effects on the osteoblast. Acutely, PTH raises serum calcium by

stimulating the osteoblast to release signaling messengers to stimulate osteoclast activity. However,

long-term PTH stimulation appears to induce the osteoblast to increase bone formation. Since

these channels are a likely candidate for the signal transduction of mechanical stimulation, the

stimulation of this channel by PTH would suggest this channel is an important site of convergence

of two distinct osteoblast activator pathways.

The significance of the observations described in Accomplishments (2) as well as (3) would

indicate that PTH modulates this mechanically sensitive cation channel through two distinct

mechanisms. One is mediated through the classical second messengers of PTH, which are

increases in cyclic AMP in the cell as well as increases in the level of cytosolic calcium. These

second messengers stimulate this channel by increasing single-channel conductance. By increasing

single-channel conductance, the channel is kinetically changed so that more ions can traverse the

channel in the same open times. PTH has also been shown to effect the shape of the osteoblast,

causing a shrinkage of the osteoblast into a stellate shape. This shrinkage would appear to be

mediated through the actin cytoskeleton, as evidenced by the effects of cytochalasin D, which

produces similar morphologic changes. Alteration of the cytoskeleton also affects the stretchactivated cation channel, but in a different way. Data acquired by holding the channel open for a

longer period of time for each channel opening indicate that effects of PTH being modulated

through two different mechanisms would increase the amount of ions going through the channel,

resulting in a much more rapid signal for the PTH response. Since we hypothesize that these

channels are responsible for the signal transduction of the mechanical strain into chemical events in

the cell, these findings would suggest that these channels may be responsible for the osteogenic

activity of PTH.

Publications

Duncan, R.L., Hatter, L.V., and Levin, D.W. 1992. Dual activation of mechanicallystimulated

cation channels in osteoblasts by parathyroid hormone (Abstract). ASGSB Bulletin 6: 92.

Duncan, R.L., and Hruska, K.A. 1992. Parathyroid hormone exhibits dual actions on the

stretch-activated cation channel of the osteoblast (Abstract). Journal of Bone and Mineral Research

7(1, Suppl.): $242.

Duncan, R.L. and Hruska, K.A. 1992. Regulation of stretch-activated cation channel activity via

the cytoskeleton is similar to hormonal modulation (Abstract). Molecular Biology of the Cell 3:

38a.

Duncan, R.L., Hruska, K.A., and Misler, S. 1992. Parathyroid hormone activation of stretchactivated cation channels in osteosarcoma cells (UMR- 106.01). FEBS Letters 307:219-223.

123
NEURONAL PROTEINS

SYNAPTOGENESIS

C6sar D. Fermin

Department of Pathology

Tulane University Medical

New Orleans, LA 70112

BEFORE, DURING, AND AFTER

School

VESTIBULAR

Description of Research

The long-term objective of our investigation is to determine, at the light and the electron

microscopy level, the anatomical changes that cells and tissues of the inner ear undergo during

embryonic development. The expression of neuronal proteins in the axons and neurons is

quantitated with color thresholding. In previous work we showed that ultrastructural modifications

of cells in gravity detecting organs of the chick are very similar to those modifications reported for

mammals, reinforcing the usefulness of the chick embryo (Gallus domesdcus) model in space

experimentation. Previous analyses of the otoconia and cells of the gravity detecting organs at

various stages of development emphasized the importance of understanding the modifications

occurring at lg before we can properly evaluate any change occurring in microgravity. The

neurons under study carry sensory information about the body position from the inner ear to the

brain. In this report the 8th cranial nerve is emphasized. The peripheral neuron body with nuclei

are called perikarya and their grouping form a ganglion. Projections from the perikarya toward the

hair cells of gravity detecting organs, and toward the brain stem form the vestibular nerve which

carry information from the inner ear to the brain (see diagram below).

. Components of balance & hearing

_k_ Target Perikaria

[ I hair cell or ganglion .... "'. _ . -- _ _i.

I. 1 _ cellbipolar _ _';" ;;" _'.i_i_..

Iwl \\ .eorons

Afferent Afferent ...... _ ' i " .' _ i i i _ '

fibers GANGLION fibers _ i i " _ _ i i i _ • • .

pro x=mal ' distal a_smJ ............ ......... .. r_caw=m

toear toear ' [ [ [ . [ i i _ [ " " [ "

Figure A. The peripheral neuron's body with nuclei are called perikarya, and their grouping forms a ganglion.

Projections from the perikarya toward the hair cells of the gravity-detecting organs and toward the brain stem

form the vestibular nerve, which carries information from the inner ear to the brain.

124
Modified gravity induces changes in gravity sensorsof the inner ear during embryonic

development.Suchmodificationsrangefrom alterationsof vestibularbrain stemresponsesto

ultrastructuralmodificationsof cells and tissuesincludingthe branchingof afferentfibers that

connecttheinnerearto thebrain.At eachlevel,morphologicalandbiochemicalmanifestationsof

thechangesatcritical periodsof developmentcanbestudiedandthesignificanceof thechanges characterized.

We statedin last year's report that,in addition to neurofilamentsand microtubule associate

proteins, which are both expresseddifferentially during the development of neurons,the

expressionof S-100proteinalsoincreasedin theinnereargravity-detectingorgansof thechick.

Wemappedthedistributionof thisproteinin neuronsof the8thcranialnervethatconnecttheinner

earto thebrainandshowedthatS-10013is foundin thenucleiof theneuronalbody(perikarya)in

additionto theaxonsandSchwanncells. Thereactionproductis restrictedto perikaryain different

partsof the vestibularganglion. Until our work, S-100 was found mainly in supportcells of

peripheraland control origin. A few reports indicated that S-I0013may have neurotrophic

propertiesneededfor guidingaxonsto find haircells.

Accomplishments

We investigatedthereactivityof S-100toandS-100Bin the8th cranialnerveof thedeveloping

chick. In thevestibularportionof thenerve,afferentbipolar neuronsandtheir nervef'tbersare

covered by myelin of Schwanncells. Schwanncells of the peripheral8th nerve serve the

equivalentfunctionof cerebralglial cells.S-100aa1313 wasoriginallyisolatedfrombrainglial cells,

and it was later shownto react with Schwanncells and other ectodermicderivedceils. We

determined(a) that expressionof S-10013in the chick inner ear vestibular afferentsis time

dependent,and(b) thatexpressionof S-10013in thevestibular,afferent,nerveterminalscontacting

hair cellschangesbeforeandaftersynaptogenesis,indicatingthat theperiodbetweenembryonic

day9 (E9)andE13of incubationmaycorrespondto acritical stageof developmentduringwhich

S-10013expressionis upregulatedconcomitantlywith myelinationof axons.

A color-thresholdingcomputerizedanalysisof theperoxidaseandthealkalinephosphatasereaction

productsshowedincreasedconcentrationof S-10013in theafferentaxonsfrom E9to hatchingE21.

Theincreasedstainof afferentaxonswasmostpronouncedin thechalicesaroundhaircellstypeI,

where the nerveendingshaveno myelin. Thus, expressionof S-10013aroundhair cells was

probablynotcontributedby Schwanncellsinsidetheepithelia.

Theexpressionof S-10013in theneuronsof thevestibularganglion(VG) appearedsimultaneously

(Figs. 1and2, arrows)in bothVG andauditoryor statoacousticganglion(SAG)of the8thnerve

at E9. However,more perikaryanuclei expressedS-100Bearlier in the VG than the SAG,

indicatingthattheexpressionof S-10013follows thewell-knownaccelerateddevelopmentof the

vestibularorganswhencomparedto theauditoryorgans.By El3 whenmyelinationis underway,

expressionof S-100Bincreasedin thenucleiof perikaryain bothganglia(Figs. 3-4,arrow), but

thenumberof positiveperikaryavariedin eachganglion. Somenucleiremainednegative(Fig. 3,

arrowhead),indicatingthatthereis adifferentialexpressionof thisproteinin theperikaryaof the

vestibularand auditory ganglion. Table 1 showsthe percentageof stainedneuronsin each

ganglionatdifferentstages.Notethattheexpressionof S-100isnot thesamein eachganglionof

thesameage. By El6, aftermyelinationis morethanhalf completedandsynapsesareestablished,

S-IO0 is distinctly presentin the axons and Schwanncells (arrowheads),whereasnuclear

expressiondecreased(Figs. 5-6, arrows), suggestingthat increasedexpressionof S-10013is

probablyrelatedto myelinationandsynapseformation.

125
Expressionof S-IOOBin lateralcristaisabsentfromtheterminalendingsaroundhaircells(H) and

minimal(arrows)of fibersatEl3 (Fig.7), but isprominentat hatchingin thechalices(arrows)of

hair cells (H) at P5 (Fig. 8), suggestinganassociationbetweenthe expressionof S-IOOBand

maturation of afferent neurons. Specifically, the antibody reaction was demonstratedby

preabsorbingtheanti-S-IOOBwith pureS-100Bprotein.Bothneurons(Fig. 9)andhaircells(Fig.

10)did notreact.

9 lOpm

1.o_P_m 10

Figures BI-10. The expression of S-100B, in the neurons of the VG appeared simultaneously (Figs 1-2, arrows) in both VG

and SAG of the 8th nerve at E9. However, more perikarya nuclei expressed S-1008 earlier than in the SAG, indicating that

the expression of S-100B follows the well known accelerated development of the vestibular organs when compared to the

auditory organs. By El3 when myelination is under way, expression of S-100B increased in the nuclei of perikarya in both

ganglions (Figs. 3-4. arrows), but the number of positive perilcarya varied in each ganglion. Some nuclei remained

negative (Fig. 3, arrowhead) indicating that there is a differential expression of this protein in the perilcarya of the

vestibular ganglion. By El6 after myelination is more than half completed and synapses are established, S-100B is

distinctly present in the axons and Scbwann cells (arrowheads), whereas nuclear expression decreased (Figs. 5-6, arrows),

suggesting that increased expression of S-100B may be related to the process of myelination and synapse formation.

Expression of S-100B in lateral crista is absent from the terminal endings around hair cells (H) and minimal (arrows) in

fibers at E13 (Fig. 7). but is prominent at hatching in the chalices (arrows) of hair cells (H) at P5 (Fig. 8), suggesting an

association between the expression of S-100B and the maturation of synaptic contacts. Specifically the antibody reaction

was demonstrated by preabsorbing the anti-S-100B with pure S-100B protein. Both neurons (Fig. 9) and hair cells (Fig 10)

did not react.

126
Table1.

DAY VG SAG

E9 74 59

Ell 29 31

E13 39 45

E16 9 13

E21 5 15

Percentages of S-100 Positive Perikarya in the 8th Nerve

Significance of the Accomplishments

The growth cones (growing tips) of nerve fibers use different cues for searching and contacting

their targets, and S-10013 may be one of several factors expressed during synaptogenesis and

myelination of afferent terminals onto hair cells. S- 10013 may also assist in the maintenance of

neuronal function, consistent with its expression in the 8th nerve fibers and terminals contacting

hair cell type I after hatching. Another possibility is that S-10013 acts as a cofactor needed for the

function of cytoskeletal proteins required for neurite outgrowth; this is because of the parallel

expression of S- 10013 with microtubule associate protein and neurof'dament protein observed by us

(1990-1991 progress report) and by other investigators.

Inner ear structures (e.g., utricle, cochlea, etc.) develop according to gradients that determine

hierarchy of end organ maturation. The hierarchy ensures multiple levels of response in the mature

organism and, most importantly, gradual maturation of specific areas of those organs. There are

five vestibular end organs and an auditory organ in the chick inner ear, where each organ receives

afferent nerve terminals from the 8th nerve. Since each end organ is located at different distances

from the ganglion, nerve endings probably arrive to those organs at different times. In the cochlear

or SAG fibers, more soma stained far away from the vestibule (distal end of basilar papilla) than

close to the gravity organs, suggesting that neurons at the distal end may contact younger hair

cells.

A transient expression of S-10013 in portions of the ganglion (Table 1) suggests that S-10013

expression is related to a gradient of maturation of afferent neurons. While further proof of this

is required, S-10013 could serve as a selective marker for assessing the relative maturation of

certain neurons.

Publications

Fermin, C.D. 1993. High resolution and image processing of otoconial matrix. Microscopy

Research and Technology 25: 297-303.

Fermin, C.D. and Garry, R.F. 1992. Cytopathic effects linked to early interactions of human

immunodeficiency virus with the cell surface. Virology 191: 941-946.

Fermin, C.D., Gerber, M.A., and Torre-Bueno, J.R. 1992. Color thresholding and objective

quantification in bioimaging. Journal of Microscopy 167( 1): 85-96.

Fermin, C.D., Gerber, M.A., and Torre-Bueno, J.R. 1992. Quantifying immunohistochemistry

objectively. The molecular biology of hearing and deafness. University of California at San Diego,

May 1-4.

127
Fermin,C.D. andMartin, D.S. 1992.Neurofilament(NF200)andmicrotubuleassociatedprotein

(MAP5) in thechick (Gallusdomesticus) embryo inner ear. The molecular biology of hearing and

deqfness. University of California at San Diego, May 1-4.

Fermin, C.D. and Martin, D.S. 1993. MAP5 and NF200 in developing cranial nerve 8th. New

Orleans, LA, FASEB Experimental Biology Meeting 4: A624.

Fermin, C.D., Martin, D.S., Vellinger, J., Deuser, M., Jones, T., Hollinger, R., and Hester, P.

1992. Morphometric inner ear findings in shuttle STS-29 space flown chicks. 15th Midwinter

Meeting of the Association for Research Otolaryngologists 16: 85.

Gallahar, W., Fermin, C.D., Henderson, L., et al. 1992. Membrane interactions of HIV.

Implications for pathogenesis and therapy in AIDS: Attachment, fusion, and cytopathology. In:

Advances in Membrane Fluidity (Aloia, Roland C., Ed.). New York: Wiley-Liss, Inc.,Vol. 6,

pp. 113-142.

Guth, P.S., Fermin, C.D., Pantoja, M., Edwards, R., and Norris, C.H. 1993. Three different

hair cell shapes and their distribution along the crista ampullaris of the frog. 16th Midwinter

Meeting of the Association for Research Otolaryngologists 16:112.

Guth, P., Norris, C., Fermin, C.D., and Pantoja, M. 1993. The correlated blanching of synaptic

bodies and reduction in afferent firing rates caused by transmitter-depleting agents in the frog

semicircular canal. Hearing Research 66: 143-149.

Hester, P.Y., Mcginnis, M.E., Vellinger, J.C., Deuser, M.S., and Fermin, C.D. 1993. Avian

embryogenesis in microgravity aboard shuttle STS-29: Effect on shell mineral content and

post-hatch performance. Acta Veterinaria BRNO 62(6, Suppl.): $43-$47.

Jones, T.A., Fermin, C.D., Hester, P.Y., and Vellinger, J. 1993. Effect of microgravity

onvestibular ontogeny: Direct physiological and anatomical measurements following space

flight(STS-29). Acta Veterinaria BRNO 62(6, Suppl.): $35-$42.

Lee, D.H., Martin, D.S., and Fermin, C.D. 1992. Post-embedding EM signal-to-noise ratio of

S- lOOfL ASGSB Annual Meeting.

Tenenbaum, S.A., Martin, D.S., Garry, R.F., and Fermin, C.D. 1992. Heat shock protein

during vestibular injury. ASGSB Annual Meeting.

Tenenbaum, S.A., Martin, D.S., Garry, R.F., Jones, T., and Fermin, C.D. 1993. Peripheral

vestibular injury induces heat shock and c-fos protein production in brain stem tissues. 15th

Midwinter Meeting of the Association for Research Otolaryngologists 16:112.

Yantsos, V., Lee, D., Martin, D.S., and Fermin, C.D. 1992. S-1001] protein in the chick (Gallus

domesticus) embryo inner ear. Symposium: The molecular biology of hearing and deafness.

University of California at San Diego, May 1-4.

Yantsos, V., Martin D.S., and Fermin, C.D. 1992. Expression of S 10013 in the vestibule of chick

embryos. ASGSB Annual Meeting.

Yoshihara, T., Kaname, H., Narita, N., Ishii, T., Igarashi, M., and Fermin, C.D. 1992.

Development of the endolymphatic sac in chick embryos, with reference to the degradation of

otoconia. Oto-Rhino-Laryngology 54(5): 235-240.

128
MICROGRAVITY IN VITRO MODEL OF BONE CELLS: FLOW EFFECTS

John A. Frangos

Department of Chemical Engineering

Pennsylvania State University

University Park, PA 16802-4400

Description of Research

It is well established that vascularization is required for effective bone healing. This implies that

blood flow and interstitial fluid (ISF) flow are required for healing and maintenance of bone.

ISF flow in bone results from transcortical pressure gradients produced by vascular and

hydrostatic pressure and mechanical loading. Conditions observed to alter flow rates include

increases in venous pressure in hypertension, fluid shifts occurring in bed rest and microgravity,

increases in vascularization during the injury-healing response, and mechanical compression and

bending of bone during exercise.

It is the objective of our research to characterize mechanisms that regulate ISF flow and the effect

of ISF flow on bone cell physiology.

Accomplishments and Significance of Accomplishments

(I) Endosteal hydraulic conductivity. By modeling the endosteum and periosteum

of bone as a monolayer of osteoblasts grown on filters, we have been studying their ability to act

as a regulated barrier to the lransosteal flux of solvent. Our findings are: (a) Osteoblasts represent

a significant barrier to flux of fluid. (b) The hydraulic conductivity can be regulated by calcitropic

hormones, calcitonin, and parathyroid hormone. Changes in cellular morphology in response to

hormonal action has been documented by morphological analysis on a scanning laser cytometer

(Meridien).

(2) Osteoblast differentiation in response to flow. Osteoblasts grown on

macrocarrier beads (Verax) were maintained in a fluidized bed and on a slowly rotating shaker. By

assaying for mineralization, as determined by a method based on von Kossa staining, we

demonstrated that within 10 days there was significant mineralization by the osteoblasts on the

bioreactor beads as opposed to the slowly shaken beads. This suggests that fluid flow can increase

differentiation.

(3) Nitric oxide production by sheared osteoblasts. Osteoblasts subjected to

shear stress for 12 hours or longer produce significant amounts of nitric oxide. Nitric oxide

synthase is not present in cultured primary rat osteoblasts. There is apparently up-regulation of the

enzyme with fluid flow.

129
NEURAL MECHANISMS BY WHICH GRAVITATIONAL STIMULI AND STRESS

AFFECT THE SECRETION OF RENIN AND OTHER HORMONES

William F. Ganong

Department of Physiology

University of California

San Francisco, CA 94143-0444

Description of Research

The long-term goal of this research, which has been supported by NASA since August 1, 1983, is

delineation of the brain pathways and neurotransmitters that mediate gravitational and stressinduced changes in the secretion of renin and other salt- and water-regulating hormones. One of

our goals has been determination of the specific parts of the hypothalamus and related areas that

affect circulating renin and angiotensin. A second of our goals has been determination of the

pathways from this region to the renin-secreting cells in the kidney. A third goal has been

analysis of the importance of this region in the physiologic control of renin secretion. To study

the role of the brain in the regulation of renin secretion, we have used stimuli which increased

renin secretion in diverse ways. Emphasis has been placed on the gravitational stress of 45 ° and

60 ° head-up tilt in rats. In past research, we have demonstrated that discharge of serotonergic

neurons in the dorsal raphe nucleus increased renin secretion, so we have also tested the e._fect of

the serotonin-releasing drug p-chloroamphetamine (PCA). Additional standardized stimuli have

included the psychological stress of immobilization, the chronic volume depletion stress of a low

sodium diet, and the acute volume depletion stress of nonhypotensive hemorrhage. We have

added to this the acute volume depletion produced by extravascular administration of

polyethylene glycol, a hypertonic substance which draws fluid from the vascular system in a

reproducible way. We are also studying the role of vasopressin-secreting neurons that connect

the hypothalamus to the brain stem and spinal cord. A cumulative list of publications that report

research supported by the grant is attached.

Accomplishments

(1) Further research on the role of brain stem vasopressin and oxytocin in the

regulation ofrenin secretion. In a previous study (Golin, et al, 1989), we demonstrated that in

homozygous Brattleboro rats, which have no vasopressin in their brains or circulating blood,

renin secretion is chronically increased and there is a chronic increase in sympathetic discharge.

The increased sympathetic discharge does not appear to be peripheral in origin, since chronic

subcutaneous infusion of vasopressin failed to restore renin secretion to normal. Vasopressin

does not cross the blood-brain barrier, so it seemed likely that the increased sympathetic

discharge was due to vasopressin deficiency in the brain. During the year, repeated injections of

vasopressin directly into the brain in Brattleboro rats failed to inhibit renin secretion. However,

one is never sure with such injections that the dose and timing are right. Therefore, we have

embarked on other approaches to the same question. One of these, in collaboration with Dr.

Lanny Keil of the NASA Ames Research Center, is measurement of oxytocin and vasopressin in

the brain stem under conditions in which it is known that there is increased sympathetic

discharge producing renin secretion. An experiment on the effects of a low salt diet has been

completed, but results are not yet available.

(2) Further study of the unique effect of stress and certain anesthetics on circulating

angiotensinogen. Some years ago, we made the chance observation that 24 hours after surgical

stress, plasma angiotensinogen was elevated with little, if any, change in plasma renin activity.

Since this effect is blocked by hypophysectomy or paraventricular lesions, we explored the

possibility that it was due to increased secretion of a hormone from the anterior pituitary, which

130
was controlled by the paraventricular nuclei. No increase in ACTH, thyroid hormones,

luteinizing hormone, or prolactin has been observed in animals in which angiotensinogen is

increased. Xylazine, one of the anesthetics which produces this effect, increases plasma oxytocin

and vasopressin, but injection of these hormones or blocking their effects does not produce

relevant alterations in plasma angiotensinogen. Current evidence indicates that permissive levels

of glucocorticoids are needed for the response, and this is why hypophysectomy blocks it.

(3) Further analysis of the role of the ventromedial nuclei in the regulation of renin

secretion. In previous experiments, we demonstrated that bilateral destruction of the

ventromedial nuclei does not lower circulating angiotensinogen but prevents the increase in renin

secretion produced by PCA, head-up tilt, immobilization, and a low sodium diet (Gotoh, et al.,

1988). We then tested the effect of electrical stimulation of the ventromedial nuclei and found

that this increased renin secretion. However, microinjection of the excitatory amino acid dlhomocysteic acid failed to increase renin secretion. Since this amino acid stimulates cell bodies

but fails to stimulate fibers of passage, it appears that the ventromedial nuclei are not integrating

centers per se, but part of a descending pathway. Destruction of cell bodies with excitatory

amino acids conFtrmed this hypothesis. To look for the pathways feeding into the ventromedial

nuclei, we electrically stimulated areas of the hypothalamus and forebrain rostral to the

ventromedial nuclei and found that stimulation in the so called AV3V area and a descending

ventral pathway from it increased renin secretion. We also investigated the pathways caudal to

the ventromedial nuclei and found that bilateral lesions just behind these nuclei inhibited the

renin response to immobilization.

(4) Use of immunocytochemistry for c-fos products to determine the parts of the brain

activated by stimuli to renin secretion. We recently set up an immunocytochemical method for

visualizing the products of the c-fos immediate-early gene, with the goal of using it to identify

the cells in the central nervous system that are "turned on" by stimuli which increase renin

secretion. The method was used in a pilot experiment employing the stress of injection of

bacterial lipopolysaccharide and a paper by Drs. Tkacs and Strack on the results is now under

editorial review for publication in the Journal of Neuroscience.

Significance of the Accomplishments

The experiments described above and the grant-supported experiments conducted in previous

years have done much to elucidate the role of the brain in the regulation of renin secretion. This

has important significance for NASA because both postural changes and stressful stimuli

increase renin secretion, and renin via angiotensin II stimulates the secretion of the salt-retaining

hormone aldosterone, affects water balance, and maintains blood pressure. In addition, our

demonstration that brain lesions lower circulating angiotensinogen via a neuroendocrine

mechanism involving thyroid hormone secretion is important because it demonstrates another

way the brain can influence the amount of circulating angiotensin II and, consequently, its

effects.

Publications

Ganong, W.F. 1991. Neural mechanisms by which gravitational stimuli and stress affect the

secretion of renin and other hormones. NASA Technical Memorandum 4258, pp. 109-111.

Ganong, W.F. 1992. Renin as a stress hormone (Abstract). International Journal of

Neuroscience 63: 47-48.

Ganong, W.F. 1993. The blood, pituitary, and brain renin-angiotensin systems and regulation of

the secretion of the anterior pituitary gland. Frontiers in Neuroendocrinology 14: 233-249.

131
Ganong, W.F., Tkacs, N.C., Gryler, E.C. 1992. Renin as a stress hormone. In: Stress:

Neurochemical and Molecular approaches (Kvetnansky, R., McCarty, R., and Axelrod, J., Eds.).

Gordon and Breach, pp. 409-416.

Golin, R.M.A., Keil, L.C., and Ganong, W.F. 1991. The effect of head-up tilt on vasopressin

secretion and arterial pressure in anesthetized rats. Neuroendocrinology 54: 42-48.

Gryler, E.C., Tkacs, N.C., and Ganong, W.F. 1991. Role of the ventromedial nuclei of the

hypothalamus in the regulation of renin secretion (Abstract). ASGSB Bulletin 5: 54.

Kjos, T., Gotoh, E., Tkacs, N., Shackelford, R., and Ganong, W.F. 1991. Neuroendocrine

regulation of plasma angiotensinogen. Endocrinology 129:901-906.

Milenkovic, L., Ganong, W.F., and Weiner, R.I. 1993. Angiotensin II stimulates GnRH release

from GT1 GnRH neuronal cells (Abstract). Endocrinology 132: 503A.

Reid, I.A. and Chou, L. 1993. Role of nitric oxide in macula densa stimulation of renin

secretion. Hypertension 22: 444.

132
MUSCLE PROTEIN LOSS IN WEIGHTLESSNESS ENVIRONMENTS:

THE ATP.UBIQUITIN.DEPENDENT PROTEOLYTIC SYSTEM

Alfred L. Goldberg

Department of Cellular and

Harvard Medical School

Boston, MA 02115

Molecular Physiology

ROLE OF

Description of Research

The goal of this research proposal is to learn more about the cellular mechanisms responsible for

the marked loss of muscle mass in weightless environments. In particular, we hope to delineate

the roles of different proteolytic systems in the rapid loss of muscle protein. Recent studies

indicate that the most important pathway for protein degradation in skeletal muscle wasting is the

cytosolic process involving ATP, ubiquitin, and the proteasome particle. The biochemical details of

this pathway have been elucidated only recently. We have developed methods for analyzing this

pathway in incubated rat muscles and are studying its regulation in different models of muscle

atrophy that appear relevant to the wasting seen in weightless environments. Our recent studies

indicate that the activation of the ATP-ubiquitin-dependent pathway for protein breakdown is

primarily responsible for the muscle atrophy and loss of myofibrillar components induced by a

large variety of pathological stimuli (disuse, stress, cortisol infections, hyperthyroidism, fastin.g).

Therefore, we are trying to clarify the physiological factors that lead to its activation or tts

suppression. One goal of this research program is to find simple approaches that may suppress

overall protein breakdown in muscle by dietary or perhaps pharmacological interventions. Such

treatments might represent a novel method for combating muscle wasting in space.

Accomplishments

During the past year, under grant support from the National Aeronautics and Space Administration,

my laboratory pursued a variety of studies along the lines proposed earlier. A number of

interesting and important findings were made that provide a basis for the work we proposed to

carry out in the coming year. As proposed earlier, the main goal of these studies is to understand

the mechanisms underlying the excessive protein breakdown that leads to negative nitrogen balance

and muscle wasting in a variety of catabolic studies, such as in individuals exposed to the space

flight environment. Particular emphasis has been placed on further clarifying the mechanisms by

which glucocorticoids, the major adrenal stress hormones, may be important in enhancing muscle

proteolysis. These studies and our earlier ones had suggested that activation of the ATP-ubiquitindependent proteolytic pathway was responsible for muscle wasting under a variety of conditions.

This past year, particular emphasis has been placed on obtaining stronger evidence that this

pathway is the primary cause of the excessive proteolysis in atrophying rat muscles. We have

shown that adrenal steroids not only suppress muscle protein synthesis, but also enhance the level

of ubiquitin gene expression and ubiquitin conjugation to proteins. Moreover, adrenalectomy can

block the enhancement of muscle that is seen in food deprivation. We and our collaborators have

also obtained evidence that this ATP-ubiquitin-dependent system is activated in a large variety of

conditions where muscle atrophy is seen, including acidosis, denervation, disuse (hindlimb

suspension), infection, fasting, and hyperthyroidism. Thus, there appears to be a final common

cellular mechanism which is induced by diverse stimuli and causes muscle wasting.

In addition, we have been pursuing promising studies to understand the physiological mechanisms

that can suppress intracellular protein breakdown and that might possibly be of benefit for

treatment or prevention of muscle wasting. We have investigated the metabolic and biochemical

effects of dietary protein deficiency, which suppress protein breakdown and preserve muscle mass

(unlike fasting, infections, glucocorticoids, and denervation). We have found that decreased

133
proteinintake(with adequatecalories)leadsto a reversiblereductionin boththe lysosomaland

ATP-ubiquitin-dependentproteolyticpathwaysanda fall in the muscle'scontentof the critical

enzymes(i.e., lysosomalproteasesand the proteasomeparticle). We havealso definedthe

mechanismsof thevery rapid buildupof musclemass("catch-upgrowth") duringrestorationof

normalproteinintake.

Significance of the Accomplishments

These studies have provided strong evidence that the ATP-dependent proteolytic system involving

ubiquitin and the proteasomes is the major degradative pathway and is activated by a variety of

physiological stimuli that lead to muscle wasting, including stress (e.g., cortisol) and disuse. We

have also discovered a dietary regimen (reduced protein intake) that suppresses this degradative

process and the lysosomal degradative system in skeletal muscle, and that may thus be useful for

the maintenance of muscle mass in various stressful states.

Publications

Medina, R., Wing, S.S., Kettelhut, I. and Goldberg, A.L. 1992. Regulation of different

proteolytic systems in muscle by insulin and food intake. In: Protein Metabolism in Diabetes

Mellitus, Chapter 7 (Nair, K.S., Ed.). U.K.: Smith-Gordon, pp. 97-109.

Tawa, N.E. and Goldberg, A.L. 1992. Suppression of muscle protein turnover and amino acid

degradation by dietary protein deficiency. American Journal of Physiology E317-E325.

Tawa, N.E. and Goldberg, A.L. Protein and amino acid metabolism in muscle. In: Myology,

Second Edition (Engel, A.G. and Franzini-Armstrong C., Eds.), McGraw-Hill.

Tawa, N.E., Kettelhut, I.C., and Goldberg, A.L. 1992. Dietary protein deficiency reduces

lysosomal and nonlysosomal ATP-dependent proteolysis in muscle. American Journal of

Physiology E326-E334.

Wing, S.S. and Goldberg, A.L. 1993. Glucocorticoids activate the ATP-ubiquitin-dependent

proteolytic system in skeletal muscle during fasting. American Journal of Physiology E668-E676.

134
GRAVITY INFLUENCE ON SIGNAL TRANSFER AND DIFFERENTIATION

Rosalind A. Grymes

NASA Ames Research Center

Moffett Field, CA 94035

Description of Research

Goal: The goal of the Cellular and Molecular Biology Laboratory at NASA Ames Research

Center is to understand the effects of microgravity as part of the larger dynamic of physical stress

response in mammalian cells. Our research seeks to integrate the study of physical force

interplay, extracellular matrix interactions, and gene expression events.

Objectives: The architectures of living systems perceive both load-bearing and

tension/contraction stresses. Gravity has played a fundamental role in the ontogeny of these

systems and acts as a constant input modifier. In the body, many cell types experience either

constant or periodic tension and compression. Dermal (skin) fibroblasts undergo both stretch

and flexure, endothelial (blood vessel lining) cells are exposed to strong blood flow pulses,

osteoblasts (bone cells) sense both structural and load-bearing forces, and muscle cells provide

both contraction and support strength. One of our objectives is defining the role of external

forces on cell behavior.

Cell populations are organized with extracellular matrix molecules to form functional tissue. The

matrix _s the glycoprotein mortar that binds the cellular bricks together. Cells may be embedded

in the matrix or form layered sheets on a lining of matrix. The integral attachments of cells to

matrix molecules is part of the mechanism that allows cells to transduce and interpolate physical

force information from the environment. Accordingly, our research is directed toward defining

the role of matrix materials in modifying the stress responses of cells.

Specific membrane receptors for circulating protein growth factors deliver (when occupied by

their ligands) signals to the intracellular response machinery. Matrix links contribute to the total

informational content of these signals. One of the observed effects of microgravity is a failure of

signal transfer and appropriate response in exposed cells. This is particularly striking in studies

of immune effector ceils, but is also seen in other cell types. The defect in response is selective,

affecting some intracellular pathways and not others. Our laboratory is engaged in teasing apart

relevant signal transfer processes in the response of cells to platelet-derived growth factor

(PDGF). We study several aspects of this growth factor response, in a range of dermal cell

strains that differ by degree of senescence. Senescent cells are very similar in the reactions to

growth factor treatment, as are cells exposed to microgravity.

The communication pathways initiated by growth factor binding at the cell membrane consist of

two related networks: the chemical mediator cascade and the cytoskeleton. The cytoskeleton

provides three-dimensional structure to the cell, distributing forces through tensional elements.

An architecture of highly ordered protein polymers, it is both a structure of morphology and a

transducer of information. An important objective of our work centers on examining the

cytoskeletal changes induced by external stress, or absence of stress (microgravity).

Accomplishments

We have taken two routes to the evaluation of cellular behavior under conditions of varying

physical force.

135
First, in collaboration with NASA life sciencesengineers,we have developeda prototype

clinostat. This instrumentsupportsthe cultivation of our cells underconditionsof vectorially

changinggravity. Thereisa constant(small)forceperpendicularto thedirectionofrotation,and

the vectorof the 1-ggravitationalforceis constantlyshifting. Thecells aregrown adherento

one side of a flask in a monolayer. We have adaptedthecells to growth in mediathat are

independentof constantCO2exchangefor buffering, because the rotating culture vessels must be

sealed. Figures l and 2 show cells grown in this medium in the traditional orientation and kept

for l week on the clinostat (respectively). The gravity vector-shifted cells show a marked

alteration in intercellular contacts and colony morphology. We have successfully maintained

these cultures for 8 weeks on the clinostat. Improvements and modifications to our prototype

unit have led to design changes, and two new units incorporating these changes (and providing a

rotation-only control) will be available this summer.

Figure I. Young fibroblasts grown in C02 independent medium with 10% fetal bovine

serum, under normal 1g conditions

Figure 2. The same cells and medium described in the legend to Figure 1, but grown

under conditions of continuously vectoriaUy shifted gravity. Color differences are due

to the greater volume of media in the sealed rotated flasks.

136
E

CI.

¢J

G

rE

t--

o_

Our second series of experiments in force variation have utilized the Flexercell Strain Unit. This

instrument transmits a two-dimensional force over a 24 mm flexible membrane. The amplitude

and frequency of the stress can be controlled precisely. Monolayers of cells are grown on the

flexible membrane support and subjected to predetermined flex regimens. Figures 3 through 7

reflect the behavior of senescent dermal fibroblasts to selected regimens. Cellular response was

measured by incorporation of tritiated thymidine (which indicates DNA synthesis) and by direct

cell counts (not shown).

14O000 -

120000 -

1 000O0.

80000 •

60000 •

40000-

20000

0

142000

122000

E

D.

U 102000

G

C

82000

E 62000

e42000

-r

22000

2000

0

rl

%

Time (days)

4

Aged Cells exposed to 26.5 kPa stretch

with cycling 1" pull, 1" release

Figure 3.

r-I Flexercell

Control

!

[]

Time (days)

Aged Cells exposed to 4.1 kPa stretch

with cycling 6" pull, 3" release

[] Flexercetl

• Control

Figure 5

E

r,l

u

"o

E

t-

"v140000

120000

1O00OO

80000

60000-

40000 -

20000

B

Time (days)

Aged Cells exposed to 12.S kPa stretch

with cycling 6" pull, 3" release

Figure 4.

[] Flexercet!

• Control

E

¢:L

(J

¢D

r=

10

E

r140000

120000

100000

80000

60000

40000

20000

• |

[]

Time (days)

Aged Cells exposed to 1 2.5 kPa stretch

with cycling 20' pull, 1 0' release

[] Flexercell

• Control

Figure 6.

E

Q.

U

Q,I

el:::

"r"

140000

120000

100000-

80000'

60000"

40000 •

20000

0

B

Time (days)

Aged Cells exposed to

12.5 kPa constant stretch

Figure 7.

[] Flexet cell

• Control

137
These cells, and other dermal cells we have tested, show that many levels of stress can be

accepted. Notice that the curves representing the flexed, or stressed, cells parallel those of the

controls but show generally lower values. We interpret these lower values as indicating that

responses other than proliferation are occurring in response to the applied stress. We are

currently assessing the responses of flexed cells in terms of induced cell injury (induction of

stress proteins, release of lactate dehydrogenase) and apoptosis, or programmed cell death (DNA

fragmentation).

Our experiments over this time period have utilized a trio of dermal fibroblast cell strains. These

strains were selected to represent a range of proliferative capacities. They could also be

described as providing reference points along a timeline that starts with fetal growth and ends

with senescence. They are (1) young cell strain (AG4431), (2) chronologically aged cell strain

(WU77220), and (3) Werner's syndrome cell strain (AG780). Werner's syndrome (WS) is a

genetically determined advanced aging disorder. Many of its clinical symptoms involve the skin

and connective tissue. In culture, dermal cells from WS patients fail to respond to growth factors

such as platelet-derived growth factor (PDGF), indicating a fundamental defect of signal transfer.

These cells also overexpress the enzyme collagenase I, a gene product whose transcriptional

regulation is well studied. All dermal fibroblasts produce this enzyme, which plays a central role

in remodeling and removing collagen type I, the major constituent of both mineralized and nonmineralized matrix. The induction defect in WS is a conveniently assayable end point for

effective signal transfer to the nucleus. The WS cells also demonstrate the mitogenic anomalies

characteristic of senescence. Similarities between the generalized response failures of senescent

cells and of those exposed to microgravity are the basis for our choice of this "age range" model.

Using the flexible membrane Flexcell plates, we have analyzed the behavior of AG4.431 (fetal)

cells on untreated or collagen type I coated membranes. While both strains of old cells grow

reliably on the untreated membranes, the young cells form only tenuous attachments to the

supporting material in the absence of a collagen coating. This is particularly evident when force

is exerted on the membrane, and is irreparably damaging to the cell monolayer if the continuity

of the cell sheet is disrupted before the application of stress. We see an altered program of signal

transduction events in those young cells grown on collagen and an interaction between the

applied stress and PDGF responses. Maximal stimulation of stressed cells is achieved by a

1 unit/ml PDGF, and does not show a dose dependence relationship. Control cells demonstrate

this dose dependence of response (Figure 8). In combination with the results observed in the

+ stress, + PDGF studies, we see evidence of cooperative but separate pathways for the

transduction of physical force and growth factor response. Other evidence (now shown)

indicates that these fetal ceils are producing and responding to their own growth factors. To

clarify the experimental picture, we will be adding neonatal and adult dermal fibroblasts to our

studies this year.

60000'

SO000

u 40000

"o 30000

E

_20000

10000-

• Flexed

• Control

0.

o 1 2 4 8

PDGF dose, half-maximal units/ml

Young cells exposed to PDGF and 12.5 kPa

stretch with cycling 6" pull, 3" release

138
Previously,weevaluatedthe regulatoryregionof thecollagenasetype I genein WS cell strain

AG 780 anddemonstratedthat its sequencewasnormal. The transcriptionalregulatoryprotein

complexAP-1interactswith thisDNA region. TheFOSproteinis oneof the "immediateearly"

responsefactors. Encodedby the c-los gene, its message transcript is transiently induced

following stimuli such as PDGF binding. We analyzed c-fos and collagenase type I transcripts

+ stress and + PDGF addition. Both the young and the WS cell strains produced similar overall

amounts of c-fos. Induction of c-fos transcripts by stretch alone was observed in the fetal cells at

the 30-rrunute time point, and the response was sustained at 24 hours. In control, unflexed fetal

cells, c-fos transcripts were induced by PDGF only transiently. WS cells experiencing stretch in

the Flexercell Strain Unit unexpectedly induced c-los transcripts in the presence of PDGF. The

WS cells produced copious amounts of collagenase type I transcripts under all conditions tested.

We have also examined the production of transcripts of the c-jun gene. Both the product of

c-jun, the JUN protein, and FOS are required for the induction of coUagenase type I. Thus far,

we have been unable to detect c-jun transcripts in the senescent or WS cells using an

oligonucleotide probe. We postulate that this may indicate these aged cells are producing

another member of the jun family of related genes. We are pursuing this finding.

We have used photomicroscopy to examine our samples. We acquired a phase-contrast inverted

microscope with camera attachment for this work, and have documented the observations on

growth characteristics and healing of an experimentally produced simulated wound. Preliminary

samples are being analyzed by NASA-Joint Venture (JOVE) fellow, Prof. M.V. Brown" using

fluorescent-antibody stains for the localization of cytoskeletal components. In addition to this

work, she is examining the arrangement of the intermediate filaments and tubulin compartments

of the cytoskeleton using transmission electron microscopy and immunogold labeling. This year

Christine Sawyer, the laboratory's Research Associate, completed an intensive training course in

immunocytochemistry and photomicroscopy. We will continue to expand our capabilities in this

area.

Significance of the Accomplishments

The importance of physiologic tensional stresses has been demonstrated both in vivo and in vitro.

The cytoskeleton receives and transmits environmental stimuli, including growth factor

responses and matrix interactions. It is both a structure of morphology and a transducer of

information. As a component of structural integrity, this architectural complex is ideally suited

to sense the physical changes associated with altered gravities. At the same time, there are

clearly other pathways that contribute significantly to the transmission of growth factor signals

such as that delivered by PDGF. These intracellular chemical second messenger cascading

pathways are probably the most important determiners of cellular responses to growth factors,

with cytoskeletal/matrix interactions acting as modifiers or educators. We are linking these three

fields (tensional force integration/matrix biology/growth factor response) to more fully

understand gravity modulation of cellular response. The similarities between aged and

microgravity-exposed mammalian cells can be exploited in productive experimentation geared

towards a fuller understanding of the basis of cellular "unresponsiveness" in spaceflight.

Perhaps our most significant accomplishment lies in technology and hardware development.

Determining the specifications and response parameters for the continuous vectorially shifted

clinostat apparatus and performing preliminary experiments on cells cultivated in the prototype

unit, are essential to our pursuit of further gravitational biology studies. In addition, we have

tested the capabilities and nuances of the Flexercell Strain Unit and Flexcell plates. These permit

us to alter the dynamics of the physical forces that compose the ceils' environment.

139
Living organismsdevelopandgrow in anenvironmentwhereexternalstimuli areunboundedand

unpredictable.Understandingthecell/environmentinterface,in termsof informationacquisition

and cellular response,is a broad goal of cellular and molecular biology. Cytoskeletal

architectures are fundamental to this communication. Having evolved under the influence of

gravity, they are clearly designed as tension/compression systems. The integration of perceived

force is a summation of the gravitational vector, with constant magnitude and direction (unless

manipulated in a clinostat) and applied environmental force of varying magnitude and direction.

We believe that these force components are integrated at the level of the cytoskeleton and that

the integration is impacted in part by connections to the matrix which educate the cell as to its

local environment and homeostatic function. We hope that the intracellular conversation

between force transduction and signal transduction can be revealed by our methods.

140
SKELETAL MUSCLE GLUCOSE

WEIGHTLESSNESS

Erik J. Henriksen

Department of Exercise and Sport Sciences

University of Arizona

Tucson, AZ 85721

TRANSPORT DURING SIMULATED

Description of Research

The long-term goal of this research project is to characterize on a cellular and molecular level the

responses of the skeletal muscle glucose transport system to alterations in muscle activity state.

The focus of the current research is to identify how the regulation of the skeletal muscle glucose

transport system is altered when the muscle undergoes a period of reduced weight bearing (also

known as unweighting). Skeletal muscle glucose transport is a major regulator of glucose

removal from the blood and, therefore, will be a primary factor in blood glucose homeostasis.

Glucose transport into skeletal muscle is stimulated by two independent pathways, one sensitive

to insulin and insulin-like factors, and the other sensitive to muscle contractile activity and other

interventions which activate Ca 2+ release from the sarcoplasmic reticulum. Numerous studies

have demonstrated that the maximal effects of both pathw_tys on the stimulation of in vitro

glucose transport are completely additive, indicating that the stimulatory effects of insulin and

contractions are mediated by different mechanisms and, as such, may respond differently to

alterations in muscle activity.

To study the regulation of glucose transport in skeletal muscle independently of the influence of

muscle blood flow, the isolated soleus strip preparation has been developed. Soleus strips of

equal size are incubated in vitro and can be stimulated by a variety of factors and manipulations

under highly controlled conditions. Glucose transport activity is then assessed by measuring the

uptake into the muscle cells of a non-metabolizable glucose analog, 2-deoxyglucose (2DG).

Reduced weight bearing of the soleus, a lower leg muscle used in locomotion, is induced by the

tail-cast suspension model and results in rapid (within 3 days) and significant atrophy of this

muscle. This is a widely used model of simulated weightlessness and mimics many aspects of

actual microgravity. A major finding of previous research by this lab and others is that

unweighting of the soleus leads to an enhanced action of insulin on glucose transport activity,

likely a result of increased insulin binding capacity and enhanced intracellular levels of glucose

transporter protein. How unweighting of the soleus can affect the response of the glucose

transport system to insulin-like factors such as insulin-like growth factor I (IGF-I), vanadate, or

phospholipase C (PLC) and to contractile activity, such as occurs during exercise, was not

previously known. This line of research can provide important information on the adaptative

response of skeletal muscle in a microgravity environment and also during recovery from an

unweighting period. In addition, this line of research can be used to better understand how the

skeletal muscle glucose transport system functions under normal conditions and in such

pathophysiological conditions as diabetes and denervation.

Accomplishments

(1) Whereas soleus unweighting leads to an enhanced response to insulin for maximal

stimulation of glucose transport activity, this intervention does not alter the contractiondependent pathway for activation of this process. The hindlimbs of juvenile (~100 g) male

Wistar rats were suspended by a tail-cast system for 3 or 6 days. Glucose transport activity was

then assessed in isolated soleus strips (-18 mg) using 2DG uptake. Insulin (2 mU/ml) had a

progressively enhanced effect on 2DG uptake after 3 and 6 days of unweighting (+44 and +72

141
percentvs. control, both p<0.001),in agreementwith previousfindings. In contrast,at these

sametime points, therewas no differencebetweengroupsfor activationof 2DG uptakeby

maximally effective treatmentswith in vitro contractions (10 tetani). The response of glucose

transport activated by stimuli that work through the contraction-pathway but do not induce

sarcolemmal membrane depolarization, namely hypoxia (60 min) and caffeine (5 mM), also was

not significantly different from the control. Collectively, these results indicate that the enhanced

capacity for stimulation of glucose transport following soleus unweighting is restricted to the

insulin pathway, with no apparent enhancement of the contraction-dependent independent

pathway.

(2) Soleus unweighting leads to an enhancement of the stimulatory effects of IGF-I,

vanadate, and PLC on glucose transport activity. This study used a novel approach to further

evaluate the potential role of post-receptor binding mechanisms in this enhanced insulin effect

following unweighting. IGF-I, vanadate, and PLC were used to stimulate glucose transport

activity independently of insulin receptor binding. Soleus glucose transport activity was

evaluated in vitro using soleus strips (-18 mg). Progressively increased responses to maximally

effective doses of insulin or IGF-I were observed after 3 and 6 days of unweighting compared to

weight-matched controls. Enhanced maximal responses to vanadate (6 days only) and PLC (3

and 6 days) for 2DG uptake were also observed. The results of this study (1) provide evidence

that post-insulin receptor binding mechanisms also play a role in the enhanced response of the

insulin-dependent pathway for stimulation of glucose transport in unweighted skeletal muscle,

and (2) indicate that IGF-I action on glucose transport is included in this enhanced response in

unweighted muscle.

Significance of the Accomplishments

Finding 1: These results indicate that unweighting of the soleus muscle by tail-cast suspension

causes a marked increase in insulin-stimulated glucose transport activity, with no enhancement

of the insulin-independent pathway for glucose transport. These findings support the hypothesis

that in muscle there are two separate intracellular pools of glucose transporters, one recruited in

response to insulin (or insulin-like factors) and another recruited in response to contractions or

hypoxia. The present results would be compatible with the idea that unweighting causes an

increase in only the insulin-recruitable pool of GLUT-4 protein, with no expansion of the

contraction- or hypoxia-recruitable pool. This model of altered muscle usage, with its discordant

adaptive responses for insulin-dependent and insulin-independent glucose transport activity,

could be effectively utilized in future research endeavors to individually examine the cellular

mechanisms regulating these two pathways for stimulation of glucose transport.

Finding 2: These results provide evidence that soleus unweighting by hindlimb suspension

induces an enhancement not only in the action of insulin, but also in the action of IGF-I on the

glucose transport system. Furthermore, the enhanced effects of insulin-mimickers in unweighted

muscle suggest that multiple sites of adaptation that include post-receptor mechanisms, must be

involved in the increased effect of these hormones on glucose transport activity. Therefore, this

model of altered muscle use may be used in the future to help justify the intracellular factors that

are important in the action of insulin and insulin-like factors on the skeletal muscle glucose

transport system.

Publications

Hardy, R., Ladenson, J.H., Henriksen, E.J., Holloszy, J.O., and McDonald, J.M. 1991. Palmitate

stimulates glucose transport in rat adipocytes by a mechanism involving translocation of the

insulin-sensitive glucose transporter (GLUT4). Biochemical Biophysical Research

Communications 177: 343-349.

142
Henriksen,E.J. 1991. Effectsof phenylarsineoxideoninsulin-stimulatedsystemA aminoacid

uptakein skeletalmuscle. American Journal of Physiology 261 (Cell Physiology 30): C608-

C613.

Henriksen, E.J., and Holloszy, J.O. 1991. Effect of diffusion distance on measurement of

glucose transport in rat skeletal muscles in vitro. Acta Physiologica Scandinavica 143:381-386.

Henriksen, E.J., Louters, L.L., Stump, C.S., and Tipton, C.M. 1992. Effects of prior exercise on

the action of insulin-like growth factor I in skeletal muscle. American Journal of Physiology 263

(Endocrinology and Metabolism 26): E340-E344.

Henriksen, E.J., Rodnick, K.J., Mondon, C.E., James, D.E., and Holloszy, J.O. 1991. Effect of

denervation or unweighting on GLUT-4 protein in rat soleus muscle. Journal of Applied

Physiology 70: 2322-2327.

Rodnick, K.J., Henriksen, E.J., James, D.E., and Holloszy, J.O. 1992. Exercise training, glucose

transporters, and glucose transport in rat skeletal muscles. American Journal of Physiology 262

(Cell Physiology 31): C9-C 14.

Slentz, C.A., Gulve, E.A., Rodnick, K.J., Henriksen, E.J., Youn, J.H., and Holloszy, J.O. 1992.

Glucose transporters and maximal transport are increased in endurance-trained rat soleus.

Journal of Applied Physiology 73: 486-492.

143
STRUCTURAL DEVELOPMENT

Emily Morey-Holton

NASA Ames Research Center

Moffett Field, CA 94035

AND GRAVITY

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 (a) if gravity turns bone cells on or off, (b) if/how these

cells communicate with each other and with their environment, (c) if/how secretory products are

altered by different gravity levels, (d) how alterations in organic matrix affect mineralization and

material properties, and (e) 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

flight 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. (5) Systemic and local factors will be involved in the

changes. Most ground-based research involves growing rats exposed to simulated space flight,

and flight experiments in whole animals or primary bone cell cultures will allow gathering of more

information to support or negate the hypotheses. During this report period, ground-based studies

focused on the response to skeletal unloadin.g, with emphasis on the role of bone resorption in the

response. Flight activities included preparataon for experiments on the first and second dedicated

Spacelab Life Sciences missions and middeck rat and cell culture experiments.

Accomplishments

(1) Alendronate (AHBP), a bisphosphonate inhibitor of bone resorption, was tested in

growing rats to determine if it could inhibit the defect in mineralization during skeletal unloading.

Young male rats were injected subcutaneously with the drug (experimental group given 0.1 mg

AHBP/kg or 0.4 mg AHBP/kg), or with saline (control group) on each of the 2 days preceding

skeletal unloading. The hindquarters of half the animals in each group (n---6) were unloaded by

tail-traction. Bone markers were given before and during the experiment. The unloading lasted 9

days. The average weight gain during the experiment was approximately 33 g in each group.

Tibia mass was significantly different in all groups (basal=209-1:4 mg, unweighted vehicle

= 229-!:16 mg, weighted vehicle = 261"t-9 mg, unweighted AHBP = 286+8 rag, and weighted

AHBP = 303+7 mg) with the major difference being in the cancellous (proximal tibia) rather than

cortical (tibia diaphysis) bone. Calcium concentration in the proximal tibia increased significantly

in the AHBP treated rats compared to basal and vehicle treated groups while unloading did not

change the concentration of calcium at this bone site. The bisphosphonate caused a striking

increase in the number of osteoclasts in cancellous bone in all animals compared to saline controls.

However, the osteoclasts were apparently inactive, as the cells did not appear to be firrnly, attached

to the bone surface and they had minimal ruffled borders; also, the amount of bone was mcreased

significantly. The decrease in osteoclastic activity may be the reason for the significant increase in

cancellous bone in the proximal tibia of the bisphosphonate treated rats. Interestingly, cortical

bone formation at the tibiofibular junction was still decreased at the periosteal surface in all

unloaded rats compared with their appropriate loaded control; and, furthermore, AHBP controls

showed a significant suppression of bone formation at this site compared to vehicle treated

controls.

144
(2) Data from rats flown on STS-40(SpacelabLife Sciences1) suggestthat housing

(individual vs. group) influencesthe responseto flight with fewer changesand a morerapid

recoveryfrom flight notedin thegroup-housedanimalsascomparedto individually housedrats.

Also, thebonechangesvariednot only from boneto bonebut atdifferent siteswithin thesame

bone.

(3) A mineralizingprimaryfetalrat osteoblastsystemwastestedfor suitabilityaspartof

theSpaceTissue Loss (Walter Reed Army Institute of Research) hardware flown on STS-45 and

STS-53. Cells grown on collagen-coated beads were evaluated after incubation in the newly

developed tissue culture hardware which uses hollow-core fibers and continuous-flow medium.

Under normal temperature (37 ° C) and CO2 (5 percent) conditions, the osteoblasts thrived during

exposure to a 10-day flight after 10 days of preincubation on STS-45. Although mineralization

was not evident in the STS-45 flown cells, no ground controls were available for comparison.

During the STS-53 flight, the ground control unit malfunctioned.

Significance of the Accomplishments

Finding 1: suggests that this bisphosphonate selectively protects the filamtentous cancellous bone

found at the end of long bones and in vertebrae during unloading in growing rats but does so by

adversely changing bone chemistry, bone growth, and bone remodeling.

Finding 2: suggests that housing (group vs. individual) impacts the bone response, possibly

through differences in activity, and that the apparent bone response to space flight depends on the

bone site studied.

Finding 3: provided valuable information on flight cell culture hardware and its ability to support

primary bone cells during space flight.

Publications

Arnaud, S.B., Fung, P., Popova, I.A., Morey-Holton, E., and Grindeland, R.E. 1992.

Circulating parathyroid hormone and calcitonin in rats after space flight. Journal of Applied

Physiology 73: 166S-168S.

Bikle, D.D., Currier, P.A., Morey-Holton, E.R., Doty, S.B., and Halloran, B.P. 1993.

Alendronate preserves bone mass during skeletal unloading. ASGSB Bulletin 7: 36.

Bikle, D.D., Halloran, B.P., Harris, J., Cone, C., and Morey-Holton, E. 1991. Skeletal

unloading results in increased mRNA for IGF-1 but decreased mRNA for collagen. ASGSB

Bulletin 5(1): 36.

Cone, C.M., Morey-Holton, E.R., and Doty, S.B. 1992. Effect of space flight on primary rat

osteoblast cultures. ASGSB Bulletin 6(1): 58.

Currier, P. and Morey-Holton, E. 1992. Growth of the femur and vertebra in rats during Space

Life Sciences 1 (SLS1). ASGSB Bulletin 6(1): 50.

Doty, S.B., Morey-Holton, E.R., Durnova, G.N., and Kaplansky, A.S. 1992. COSMOS 2044:

Morphological studies of bone and tendon. Journal of Applied Physiology 73: 10S-13S.

Garetto, L.P., Morey, E.R., Durnova, G.N., Kaplansky, A.S., and Roberts, W.E. 1992.

Preosteoblast production in Cosmos 2044 rats: Short term recovery of osteogenic potential.

Journal of Applied Physiology 73: 14S-18S.

145
Halloran,B.P.,Bikle, D.D., Currier, P.A., and Morey-Holton,E.R. 1993. High dosegrowth

hormoneadministrationmay protectagainstbonelossby skeletalunloadingin thegrowingrat.

ASGSB Bulletin 7:36.

Halloran, B.P., Bikle, D.D., Harris, J., Cone, C., Roskett, H.C., and Morey-Holton, E. 1992.

Changes in 1,25-dihydroxyvitamin D kinetics and the plasma concentrations of calcium,

phosphorus, and PTH during skeletal unloading. ASGSB Bulletin 6(1): 92.

Halloran, B.P., Bikle, D.D., Hams, J., Daly, R., Thai, P., Cone, C.M., and Morey-Holton., E.

1991. Production and metabolic clearance of 1,25(OH)2 during skeletal unloading. ASGSB

Bulletin 5(1): 64.

Halloran, B.P., Bikle, D.D., Harris, J., Foskett, H.C., and Morey-Holton, E. 1993. Skeletal

unloading decreases the production of 1,25-dihydroxyvitamin D. Journal of Physiology 264:

E712-E716.

Hill, E.L., Martin, R.B., Gunther, E., Morey-Holton, E., and Holets, V.R. 1993. Changes in

bone in a model of spinal cord injury. Journal of Orthopaedic Research 11: 537-547.

Morey-Holton, E.R. 1992. Spaceflight Workshop: Preface. ASGSB Bulletin 6(1): 106.

Morey-Holton, E.R. and Arnaud, S.B.

TM-03890, pp. 34.

1991. Skeletal responses to spaceflight. ,'NASA

Morey-Holton, E.R. and Arnaud, S.B. 1991. Skeletal responses to spaceflight. In: Advances in

Space Biology, Vol. I (Bonting, S. L., Ed.), Connecticut, JAI Press Inc., pp. 37-69.

Morey-Holton, E., Cone, C., Doty, S., and Vailas, A. 1992. Biomineralization and spaceflight.

ASGSB Bulletin 6 (1): 99.

Morey-Holton, E.R., Cone, C.M., and Martin, R.B. 1991. Changes in bone metabolism and

biomechanics after 1, 2, 3, or 4 weeks of skeletal unloading. ASGSB Bulletin 5(1): 34.

Morey-Holton, E.R., Tanner, S.J., Bikle, D.D., Doty, S.B., and Halloran, B.P. 1993.

Alendronate inhibits cortical bone formation and does not protect cortical bone during skeletal

unloading. ASGSB Bulletin 7: 48.

Tanner, S. and Morey-Holton, E. 1992. Changes in the proximal femur during spaceflight.

ASGSB Bulletin 6(1): 88.

Tanner, S.J., Doty, S.B., Morey-Holton, E.R., Bikle, D.D., and Halloran, B.P. 1993. The

effect of the bisphosphonate on weighted and unweighted cancellous bone. ASGSB Bulletin 7: 80.

Tischler, M.E. and Morey-Holton, E. 1992. Research on Organs and Tissues, AIAA Space

Programs and Technologies Conference and Exhibit, Huntsville, AL, p. 8.

146
GRAVITATIONAL FIELDS AND NEURAL SIGNALING IN THE HIPPOCAMPUS

John M. Horowitz

Section of Animal Physiology

Division of Biological Sciences

University of California

Davis, CA 95616

Description of Research

Our research has an ongoing, long-term objective of elucidating the effects of altered

gravitational fields on neural networks. This past year our experiments dealt with the effects of

serotonin (5-HT), a neuromodulator, on hippocampal neurons. The hippocampus is an area of

the central nervous system associated with several types of neural activity including sleep,

temperature regulation, and, perhaps most importantly, memory and learning. Our experiments

were based on the observation that rats flown on Spacelab-3 showed a 48.6 percent increase in

hippocampal 5-HTIA receptor number after 7 days exposure to the microgravity environment,

whereas rats at 2-g for 7 days showed a 27 percent decrease in receptor number.

In our experiments, we first exposed animals to a 2-g hypergravity field for 7 days. Previous

studies showed that during this time the number of serotonergic receptors in the hippodampus

decreased, a down regulation in receptor number. Within 1 hour after the animals were removed

from the hypergravity field, the tissue was prepared for recording electrical activity. Because it

takes days for receptor numbers to change appreciably, the electrical activity reflected neural

activity of a down-regulated system. We measured electrical activity in the dentate and CA1

pyramidal cell layer in the hippocampal slice preparation before, during, and after perfusion with

serotonin and 8-OH-DPAT. Serotonin binds with several receptor types whereas 8-OH-DPAT is

a specific agonist that preferentially binds to one serotonin receptor type, the 5-HT1A receptor.

We interpreted data in terms of altered electrical activity over a trisynaptic hippocampal neural

network.

Figure l. Sketch showing synaptic transmission. (Prepared by CJ. Howard).

147
Accomplishments

(1) The hippocampus is a laminar neural structure containing a trisynaptic neural circuit,

and we examined two key locations (areas receiving major serotonergic inputs) on this pathway.

Due to the sequential nature of information transfer through the hippocampus, if serotonin affects

any one part of the pathway, it affects the output of the entire neural system. We found that two

sites along the neural pathway (the dentate and the CAI pyramidal cell layer) retain a robust

serotonergic response in animals exposed to 2-g for 7 days.

(2) We focused on characterizing the effectiveness of a particular class of serotonergic

receptors on dentate and CA1 pyramidal cells, 5-HT1A receptors, by using 8-OH-DPAT. This

set of experiments confirmed and extended observation (l) above; namely, that cells at two

critical sites along the pathway retain responsiveness to a 5-HT agonist in animals exposed to 2-g

fields. Thus, signals from the serotonergic Raphe nuclei in the brain stem, which anatomically

have been shown to project out over the dentate and CA 1 pyramidal cell layer, can modulate the

flow of information over the trisynaptic network.

(3) Based on these observations (together with receptor number studies), we are

developing a model for serotonergic modulation of information transfer over the trisynaptic

pathway in animals exposed to 2-g fields. An essential feature of the model is that modulatory

effectiyeness is maintained as shown by measurements of electrical activity in animals exposed

to 2-g conditions even though there is a down regulation of overall receptor number.

Significance of the Accomplishments

We are developing a model that accounts for a wide variety of experiments we have completed

on the effects of altered gravitational fields on the effectiveness of serotonin to modulate neural

activity. At this stage our most significant finding is that modulation of hippocampal cell

activity is maintained in animals that have been exposed to hypergravic fields. Thus, even

though anatomical evidence shows that the number of receptors for serotonin is decreased, there

appears to be a surplus of receptors so that neural information processing is maintained and

electrical activity is not altered.

The overall significance of this project is that information processing over a trisynaptic network

as modified by a neuromodulator has now been more fully elucidated. The circuit compensates

for the loss in the number of receptors so that information processing appears unchanged. The

2-g experiments were conducted on animals that were exposed to a hypergravic field for exactly

as long as animals were on Spacelab-3 so that, in addition to defining, effects in hypergravity,

information is acquired that will provide baseline data for future experiments in space. Parallel

experiments can relatively easily be carded out to test changes in a microgravity environment,

since hippocampal 5-HT receptor numbers increase in a microgravity environment.

Publications

Horrigan, D.J. and Horowitz, J.M. 1992. Effects of serotonin on signal transmission in

hibernating and cold-acclimated hamster hippocampal slices (Abstract). Society for Neuroscience

Abstracts, Vol. 18.

Horrigan, D.J. and Horowitz, J.M. 1992. Modulation of evoked responses in the dentate gyrus

by 8-OH-DPAT in rats exposed to 2g fields. ASGSB Bulletin.

Lui, A-H. and Horowitz, J.M. 1992. Modulation of paired-pulse facilitation by temperature,

calcium, magnesium, and picrotoxin in the hamster hippocampus. The FASEB Journal, Vol. 6,

A1199.

148
MECHANISM OF CONTROL OF BONE GROWTH BY PROSTAGLANDINS

Millie Hughes-Fulford

Veterans Administration Medical Center 151F

University of California Medical Center

San Francisco, CA 94121

Description of Research

The growth and repair of bone is a complex and poorly understood process. Over the past three

decades, it has been noted that astronauts lose large amounts of calcium and bone when exposed to

the microgravity of space flight. The mechanism by which bone growth is regulated is not known;

however, clinical observations have demonstrated that increases of endogenous cortisol as seen in

Cushing's syndrome are associated with bone loss and osteoporosis. Treatment of asthma and

rheumatoid arthritis with glucocorticoids is also associated with poor bone formation in these

patients. The bone loss associated with glucocorticoids involves the trabecular bone, and

examination of patients treated with the synthetic glucocorticoid prednisone shows a reduction in

bone formation, which is probably due to a direct inhibition of osteoblast function during space

flight. In the Skylab missions, urinary cortisol of the nine crewmembers increased almost

two-fold. These data suggest that the glucocorticoids as well as microgravity play a role in the loss

of bone during space flight.

This laboratory has previously shown in osteoblast cultures that glucocorticoids cause a decrease in

endogenous prostaglandin E2 synthesis accompanied by a decrease in osteoblast growth and

mineralization. Taken together, these observations suggest that glucocorticoids may regulate bone

growth by inhibiting endogenous PGE2 synthesis. The direct cause of glucocorticoid-induced

bone loss is not known, and an understanding of the relationship between bone growth and the

eicosanoid synthetic pathway may help elucidate the regulation of bone growth. Using a cloned

osteoblast cell line as a tool to study the cellular changes in growth, we have tested the hypothesis

that endogenous prostaglandin E2 directly plays a role in regulation of cell growth by perturbing

endogenous osteoblast synthesis of prostaglandins and by testing for alterations in growth and

morphology. This year we have found that the prostaglandins are mediating early oncogene

signals much like the polypeptide growth factors.

Accomplishments

Ground based experiments: Our bone model is the MC3T3-E1 cell, a cloned osteoblast line

that retains synthetic functions of normal bone tissue, including production of alkaline

phosphatase, prostaglandin E2, and mineralized matrix-containing collagen type I and

hydroxyapatite. In these studies, we have serum deprived cloned osteoblasts and added back

PGE2 alone to test the ability of prostaglandin per se to stimulate osteoblast growth.

Prostaglandin E2 acts as bone cytokine.

(1) Prostaglandins act as autocrine cytokines to stimulate osteoblast growth, normal

growth is inhibited when prostaglandin synthesis is blocked by glucocorticords or nonsteroidal

anti-inflammatory drugs (NSAIDS), and this inhibition of growth is reversed by addition of

exogenous prostaglandin. When prostaglandin synthesis is slowed by serum depletion, growth is

also inhibited. When prostaglandin is added to the serum deprived osteoblasts, cell growth, as

measured by DNA synthesis, was increased from 2-3 times the control. These data show that

prostaglandin synthesis plays a pivotal role in bone growth.

149
(2) Prostaglandins induced oncogene message synthesis. Within 30 minutes,

prostaglandincausesa20-foldincreasein expressionof c-fos; addition of cyclohexamide caused a

super induction of c-fos suggesting that this up-regulation does not require new protein synthesis.

In addition, the c-fos response of the osteoblast is proportional to the amount of PGE2. The rise in

c-fos expression is followed a few hours later by a 4-fold increase in c-jun. Messages for 6-actin

and 7-actin were also induced within 4 hours of the addition of prostaglandin.

(3) In addition, we found that PGE2 causes a rise in intracellular calcium within 2 minutes

of addition of PGE2. When the L-channel blocker nifedipine (10 I.tmol) was added to the

osteoblast cultures, the signal was abolished; and removal of the blocker restored the PGE2

mediated calcium signal, suggesting that the prostaglandins regulate calcium flux through L-type

calcium channels.

(4) Using epi-fluorescent microscopy, we noted changes in the F-actin cytoskeleton of the

control and prostaglandin treated osteoblasts, with the control osteoblast having little stress fibers

in their actin architecture. Addition of prostaglandin not only stimulated actin message, but also

caused striking increases in F-actin stress fibers that were associated with increased growth of the

prostaglandin treated cells.

(5) Since PGE2 is the major eicosanoid synthesized by the osteoblast, we asked the

question if it was also an effective mitogen by testing cell growth stimulation by various

eicosanoids. We found that PGE2 is by far the most effective growth stimulatory eicosanoid we

have tested.

Space flight Experiments: Using a cloned osteoblast cell line as a tool to study the cellular

changes in growth, we have tested a second hypothesis that space flight plays a role in growth

regulation by directly affecting prostaglandin synthesis, growth, and morphology of bone in space

flight. My collaborator, Dr. Marian Lewis, University of Alabama, Huntsville, and I have

selected the MC3T3-E 1 cell, a cloned osteoblast line that retains synthetic functions of normal bone

tissue, including production of alkaline phosphatase, prostaglandin E2, and mineralized

matrix-containing collagen type I and hydroxyapatite as our bone model. Using the CMIX

apparatus, we are able to add sera while in flight, thereby activating the osteoblasts in microgravity

and then fixing them while still in microgravity, thus enabling us to study the real-time effects of

space flight on osteoblasts. In these studies, we started with serum-deprived, cloned osteoblasts

and then added serum during space flight to test the ability of serum to stimulate osteoblast growth

in microgravity.

(6) Dr. Lewis and I have developed and perfected the technique for bone cell growth on

coverslips in microgravity on Consort 5 and STS-52.

(7) We have flown the MC3T3-E1 cells on STS-56 and found that mammalian osteoblasts

have a significantly reduced glucose utilization in space. These results agree with the work

completed on Skylab with the Hayflick WI-38 cells in 1973.

(8) Using microscopic analysis, we have also found reduced osteoblast cell growth in

microgravity conditions.

(9) In addition, the actin cytoskeleton of the flown osteoblasts has a reduced number of

actin stress fibers and drastically changed cell morphology.

(10) We are currendy measuring prostaglandin synthetic capacity of the flown osteoblast as

compared to its flight profile controls.

150
(11) These observations suggest reduced growth and DNA synthesis in bone osteoblasts

grown in microgravity. We spent over a half-year in 1993 perfecting new molecular biology

methods to allow us to quantitate specific messenger RNA content in osteoblasts grown in

microgravity conditions. Quantitative reverse transcriptase polymerase chain reaction (RTPCR)

methods are now in place to allow us to assay very small quantities of material flown in space.

(12) Looking at liver samples from rats (flown and ground controls), Dr. Tom Dietz and I

have measured several genes, including heat-shock, c-fos and others, to establish what, if any,

differences in gene expression are found in flown animals. These findings will be ready in abstract

form for the 1994 ASGSB meetings in San Francisco.

Significance of the Accomplishments

Biomedical studies of space flight crews have consistently indicated a loss of weight-bearing

skeletal bone. Various lines of evidence from humans and animals suggest that this loss is due to a

lack of bone formation in the absence of gravity. Biomechanical properties of both cortical and

trabecular bones show strength deficits that appear to represent failure of normal increases in

growth rather than an accelerated loss of bone. Joint U.S./U.S.S.R. biosatellite flights of small

animals have shown clear morphological evidence that bone formation is reduced during flight.

The cause of this loss of bone has been postulated to be related to the lack of biomechanical

stimulation during flight, to increases in systemic hormones associated with space flight,, or to a

direct effect of microgravity on the cell. We have begun studies in microgravity, testing the effect

of weightlessness on the growth response of the osteoblast. Even in the absence of elevated

glucocorticoids, microgravity reduced the growth of the osteoblasts, and caused changes in

morphology and actin stress fiber expression. We are currently testing the prostaglandin synthetic

capacity of cells in microgravity. These studies suggest a primary role of microgravity in growth

regulation of osteoblast cells in space flight.

Urinary cortisols of crewmembers during the Skylab missions increased from a preflight value of

54+4 I.tg/day to 94_+5 I.tg/day during flight, with individual crewmembers increasing

1.2- to 2.8-fold during flight. Osteoporosis seen in Cushing's syndrome patients is associated

with elevated cortisols (2- to 5-fold control values), which suggests that the elevated cortisols seen

in space flight may contribute to decreased bone mass. Since cortisol and the other glucocorticoids

like dexamethasone cause a decrease in endogenous prostaglandin synthesis, prostaglandins have

been implicated in the regulation of bone loss both in Cushing's syndrome and in long-term space

flight. We have recently reported our observation of the glucocorticoids on osteoblast growth and

prostaglandin synthesis.

In order to test further our hypothesis that prostaglandins play a direct role in the regulation of bone

growth, we have used a serum-deprived osteoblast model to test the mitogenic signaling of the

prostaglandin E2. In this publication, we report that eicosanoids increase osteoblast growth, DNA

synthesis, and c-fos oncogene message. Our results agree with studies by Web Jee and others,

who have demonstrated that prostaglandin E2 causes significant increases in bone cell growth in

vivo. Also, our epifluorescent microscopic studies have demonstrated an alteration of actin

cytoskeleton resulting in changes in cell morphology when prostaglandins axe added to osteoblasts.

These data add to the growing evidence that prostaglandin E2 is a natural regulator of osteoblast

growth and that PGE2 directly plays a pivotal role in bone cell growth and formation.

Publications

Hughes-Fulford, M. 1993. Growth and repair of mineralized osteoblasts. In: Spinal lnstabili_

(Holtzman, R., McCormick, P., et al., Eds.). New York: Springer-Verlag, pp. 3-14.

151
Hughes-Fulford,M. 1993. Reviewof the biological effectsof weightlessnesson the human

endocrinesystem.Receptor 3(3): 145-154.

Hughes-Fulford, M. and Bishopric, N.H. Prostaglandin E2 up regulation of c-fos, c-jun and actin

expression in MC3T3-E-1 cells. Journal of Bone and Mineral Research.

Hughes-Fulford, M. and Fukushima, J. 1993. Control of cell cycle by metabolites of

prostaglandin D2 through a non c-AMP mediated mechanism. Life Science Advances--

Experimental and Clinical Endocrinology 12: 57-64.

Hughes-Fulford, M., Khalifa, R., and Snowdowne, K. 1992. Prostaglandin E2 stimulation of

calcium influx in the cloned MC3T3 osteoblast cell (Abstract). ASGSB Bulletin.

Hughes-Fulford, M., McGrath, M., Hanks, D., Erickson, S., and Pullium, L. 1992. The effect

of dimethyl prostaglandin A1 on Herpes Simplex and Human Immunodeficiency Virus replication.

Antimicrobial Agents and Chemotherapy (V) 36: 2253-2258.

Hughes-Fulford, M., Renaissance, Appel, Kumegawa, M., and Schmidt, J. 1992. Effect of

dexamethasone on proliferating osteoblasts: Inhibition of prostaglandin E2 synthesis, DNA

synthesis, and alterations in actin cytoskeleton. Experimental Cell Research 203:150-156.

Lin, H-S., Hughes-Fulford, M., Kumegawa, M. and Snowdowne, K. 1993. Cytoplasmic pH

influences cytoplasmic calcium in MC3T3-E 1 osteoblast cells. Journal of Bone and Mineral

Research 8: 725-732.

152
MECHANOCHEMICAL TRANSDUCTION ACROSS EXTRACELLULAR MATRIX

Donald E. Ingber

Departments of Surgery and Pathology

Children's Hospital and Harvard Medical School

300 Longwood Ave.

Boston, MA 02115

Description of Research

The general goal of this project is to characterize the molecular mechanism by which mechanical

signals, such as those due to gravity, are transduced into changes in cell form and function. The

more specific objective is to analyze the process by which mechanical forces are conveyed by

extracellular matrix (ECM) molecules, transmitted across the cell surface, and transduced into a

cytoskeletal response. This approach is based on the concept that cell shape and, thus, the form

of the cytoskeleton depend on a dynamic balance between tensile forces that are generated within

contractile microfilaments and resisted by both internal cytoskeletal struts and ECM adhesion

sites at the cell periphery. If this type of tensional integrity or "tensegrity" mechanism is used

by cells, then externally applied mechanical loads may alter the cellular force balance and change

cytoskeletal and nuclear structure by inducing internal cytoskeletal rearrangements as well as

changes in cytoskeletal filament polymerization inside the cell. These changes in cytoskeletal

organization would then alter the distribution and, hence, the function of associated elements of

the cell's metabolic machinery. Cells normally generate tension within their contractile

microfilaments and apply these forces on their points of attachment to ECM. Thus, one specific

aim of this proposal is to determine whether transmembrane ECM receptors, which are known as

integrins, mediate mechanochemical transduction. For the purpose of this grant, we initially

focused on the biomechanical mechanism by which actin microfilament assembly is regulated

inside the cell. However, during the past year, we have also measured the effects of varying cell

ECM binding on microtubule polymerization as well as nuclear structure.

Accomplishments

Demonstration that Cell Shape Depends on a Mechanical Force Balance. A system was

developed in which cell shape control could be studied in membrane-permeabilized cells.

Endothelial cells adherent to fibronectin-coated dishes were permeabilized in saponin using a

buffer that retains the functional integrity of the actin cytoskeleton. Addition of ATP was shown

to induce retraction and rounding of permeabilized cells over a period of approximately 10-30

minutes. ATP-induced rounding was also accompanied by nuclear retraction as well as

redistribution of actin microfilaments. All ATP-driven changes in cell and nuclear structure

could be inhibited using a synthetic myosin peptide that prevents mechanical tension generation

within contractile microfilaments. In addition, both the rate and degree of cell and nuclear

retraction could be augmented by using soluble arginine-glycine-aspartate (RGD)-containing

peptides to dislodge immobilized fibronectin from cell surface integrin receptors at the same time

that internal cytoskeletal tension is raised using ATP. In contrast, the same peptides had little

effect in the absence of ATP (i.e., in the absence of cytoskeletal tension).

Altering the Cellular Force Balance Controls Tubulin Monomer Levels. Changes in tubulin

monomer and polymer mass were measured in hepatocytes that were cultured on different

densities of immobilized ECM ligands that differ in their ability to resist cell tractional forces.

Increasing the laminin coating density from low to high (I to 1000 ng/cm) promoted cell

spreading and resulted in formation of greatly extended microtubule networks, as determined by

immunofluorescence microscopy. Nevertheless, the steady-state mass of tubulin polymer was

similar at 48 hr, regardless of cell shape or ECM density. In contrast, round hepatocytes on

153
low-densitylaminin containedathree-foldhighermassof tubulinmonomerwhencomparedwith

spreadcells onhigh laminin. Ongoingexpenmentsaredesignedto analyzehow thesechanges

in tubulin monomerlevels arecontrolled in cells that likely exhibit autoregulationof tubulin

proteinsynthesis.

Probing the Molecular Basis of Force Transfer across the Cell Surface. Studies were carded

out using a prototype magnetic twisting device to apply controlled mechanical stresses directly to

cell surface receptors in the absence of a large-scale cell shape change. In this system, adherent

cells are allowed to bind to spherical ferromagnetic microbeads (5.5 I.tm) that are coated with

specific cell surface receptor ligands. The beads are magnetized in the horizontal direction, and

then magnetic stress (torque) is exerted on the adherent beads by applying a twisting magnetic

field in the vertical direction. A sensitive in-line magnetometer allows us to measure changes in

the rate and degree of bead rotation in real time and, thus, to quantitate bead deformation

(angular strain) in response to a defined applied stress. Preliminary results suggest that the ECM

receptor, integrin, can support a force-dependent stiffening response, whereas nonadhesion

receptors (e.g., acetylated LDL receptors) cannot. Studies are currently underway to further

dissect the molecular basis of transmembrane force transfer and to confirm that the cellular

response to stress is mediated by the cytoskeleton.

Significance of the Accomplishments

Our studies with membrane-permeabilized ceils clearly demonstrate that large-scale changes in

cell, cytoskeletal, and nuclear shape result from the action of mechanical tension that is generated

within the cytoskeleton via an actomyosin filament-sliding mechanism transmitted across

integrin receptors and physically resisted by immobilized adhesion sites within the ECM. Rapid

and coordinated changes of cell, cytoskeletal, and nuclear form result when this cellular force

balance is altered. In other words, ECM molecules do not control cell and nuclear form

exclusively via chemical mechanisms; rather, they largely function as mechanical anchors which

resist cell tractional forces, as suggested by the tensegrity paradigm. Thermodynamic analysis

of tubulin polymerization predicts that increasing the mechanical load on a microtubule will

result in microtubule disassembly if the tubulin monomer set point is held constant. This would

occur because compressing a microtubule increases the critical concentration of tubulin that is

required to maintain tubulin in a polymerized form. Results from our experiments on

microtubule metabolism are consistent with this type of mechanochemical regulatory mechanism

in which changes in ECM contact formation alter local thermodynamic parameters inside the

cell. Apparently, cells have evolved the ability to maintain a constant mass of microtubule

polymer in the face of these thermodynamic alterations by producing coordinate changes in the

total mass of tubulin monomer, as the critical concentration for tubulin is mechanically altered.

These results are, therefore, consistent with the mechanotransduction mechanism, in which

cytoskeletal filament assembly is modulated by changes in the balance of mechanical stresses

that are distributed across transmembrane ECM receptors and through the cytoskeleton. Our

preliminary results suggest that we are now in a position in which we can analyze the molecular

path of force transmission across the cell surface as well as the molecular basis of

mechanochemical transduction. These initial findings are consistent with our hypothesis that

integrins provide a molecular path for transmembrane force transfer and, hence, act as cell

surface mechanoreceptors. The ability to measure stress/strain relationships in living cells

should allow us to begin to identify molecular elements (e.g., different cytoskeletal filament

systems) that are critical for mechanotransduction and thus, to test the tensegnty hypothesis more

directly. If the specificity of force transfer can be confirmed using the magnetic twisting device,

then it may also provide a novel method for mechanically stimulating cells as well as for

measuring a cellular response.

154
Publications

Hansen, L.K., and Ingber, D.E. 1992. Regulation of nucleocytoplasmic transport by mechanical

forces transmitted through the cytoskeleton. In: Nuclear Trafficking (Feldherr, C., Ed.). San

Diego, CA: Academic Press, pp. 71-86.

Ingber, D.E. 1990. Fibronectin controls capillary endothelial cell growth by modulating cell.

Proceedings of the National Academy of Sciences of the United States of America.

Ingber, D.E. 1991. Control of capillary growth and differentiation by extracellular matrix: Use

of a tensegrity (tensional integrity) mechanism for signal processing. Chest 99: 34S-40S.

Ingber, D.E. 1991. Integrins as mechanochemical transducers. Current Opinion in Cell Biology

3: 841-848.

Ingber, D.E. 1993. Cellular Tensegrity: Defining new rules of biological design that govern the

cytoskeleton. Journal of Cell Science 104:613-627.

Ingber, D.E. 1993. The riddle of morphogenesis: A question of solution chemistry or molecular

cell engineering? Cell 75: 1249-1252.

Ingber, D.E., Karp, S., Plopper, G., Hansen, L., and Mooney, D. 1993. Mechanochemical

transduction across extracellular matrix and through the cytoskeleton. In: Physical Forces and

the Mammalian Cell (Frangos, J.A. and Ives, C.L., Eds.). San Diego, CA: Academic Press, pp.

61-79.

Mooney, D.J., Langer, R., and Ingber, D.E. 1991. Intracellular tubulin monomer levels

controlled by varying cell extracellular matrix contacts (Abstract). Journal of Cell Biology 115:

38a.

Plopper, G. and Ingber, D.E. 1993. Rapid induction and isolation of focal adhesion complexes.

Biochemical and Biophysical Research Communications 193:571-578.

Sims, J., Karp, S., and Ingber, D.E. 1992. Altering the cellular mechanical force balance results

in integrated changes in cell, cytoskeletal, and nuclear shape. Journal of Cell Science 103:1215-

1222.

Wang, N., Butler, J., and Ingber, D.E. 1992. Integrin 1 transmits mechanical forces across the

cell surface and to the cytoskeleton (Abstract). Molecular and Cellular Biology 3: 232a.

Wang, N., Butler, J.P., and Ingber, D.E. 1993. Mechanotransduction across the cell surface and

through the cytoskeleton. Science 260:1124-1127.

155
THE EFFECTS OF GRAVITATIONAL

VESTIBULAR ONTOGENY

Timothy A. Jones

Department of Surgery

School of Medicine-Columbia

Columbia, MO 65212

LOADING AND VIBRATION ON

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 in-depth

comparison will be made of how vestibular function develops and matures under the influence of

gravitational fields having strengths less than l-g (hypodynamic); i.e., equal to l-g, and greater

than l-g (hyperdynamic); i.e., the natural gravitational field strength of Earth. Studies will be

undertaken to examine how the gravitational vector strength and direction may modulate or

influence normal responses to transient stimuli. Efforts will also be 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 age of the organism. 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.

The research program has led to the development of a new vestibular functional test for gravity

receptors. The test consists of recording short latency vestibular responses to linear cranial

acceleration. 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 104

dBSPL). 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. The

research has demonstrated that responses to pulsed linear acceleration in birds are vestibular in

origin and represent compound action potentials of the vestibular nerve and central relays. Similar

responses have been recorded in rats and have been shown to be resistant to intense auditory

masking and depend on the eighth nerve bilaterally. The effects of selective cochlear destruction

are currently under study in rats. Among other things, the responses allow us to measure the

sensitivity of gravity receptors (i.e., vestibular thresholds) and whether or not sensory information

is processed at normal rates through vestibular relays. These functional attributes of gravity

receptors can be tested and characterized throughout development.

New research summarized in the current report sought to (a) begin the evaluation of normal

vestibular development and factors that may alter development including microgravity and drugs;

(b) evaluate the potential for functional recovery following vestibular injury (c) estimate the

statistical power of vestibular response testing, if threshold shifts are very near the step size for

stimulus intensity; and (d) better define the adequate stimulus for vestibular responses.

Accomplishments

Normal Development. Vestibular thresholds to linear acceleration were studied for ages

ranging from El9 (embryos at 19 days of incubation) to 4 weeks post-hatch. Thresholds fall

markedly during the first 10 days of this period and undergo much less change thereafter.

Similarly, latencies shorten and amplitudes tend to increase as the vestibular system matures.

These findings suggest that the developing gravity receptors systematically become more sensitive

to acceleration stimuli as development proceeds on Earth. Moreover, the neural circuits

transmitting vestibular impulses operate progressively faster and are better able to follow repetitive

stimulation as they mature.

156
Effects of the microgravity environment. Vestibular responses were measured in chicks

exposed late in development to 5 days of space flight (E9-E14, STS-29). Vestibular response

latencies and amplitudes were the same in all groups studied. However, thresholds were

significantly higher in animals incubated in space compared to synchronous, but not vivarium,

controls. These mixed results were due to grossly abnormal responses found only in a fraction of

the flight animals (38 percent).

In this fraction of flight animals, did the sensitivi_ of vestibular neurons fail to increase normally

during maturation ? The findings leave open the question of what caused the abnormal responses.

We cannot rule out space flight effects; and, therefore, the results underscore the importance of

doing additional space flight studies using bird embryos.

Effects of drug exposure. One advantage of using birds in developmental studies is that

embryos may be studied without concerns about a potentially confounding maternal response to the

experimental conditions. This is especially useful when testing drug effects on the developing

embryo. Our experiments evaluated the effects of nicotine on embryonic vestibular development.

The study revealed that despite our expectations of a regulatory response there was no change in

receptor densities or in vestibular function as a result of 9 days of nicotine exposure. These results

suggest that embryonic regulatory mechanisms serving nicotinic receptor populations may operate

differently than their mature counterparts.

Can gravi_ receptors recover from serious injury? Until recently it was thought that destruction of

gravity receptor hair cells of warm-blooded vertebrates would produce permanent functional loss.

It is now apparent that vestibular hair ceils may be capable of regeneration. It was not known,

however, whether or not regenerated hair cells would actually work. To better understand the

recuperative abilities of gravity receptors, we measured vestibular responses before and after

producing severe drug-induced lesions. Streptomycin (an antibiotic that can destroy gravity

receptor hair cells, 600 mg/kg/day) virtually eliminated vestibular responses over a period of 7

days in chicks. Within 2 weeks following drug treatment, thresholds recovered to normal levels.

Response latencies and amplitudes returned to normal slowly within 70 days following cessation

of drug treatment. These results clearly demonstrated that a virtually complete recovery of function

can occur following severe hair cell destruction. These findings, and those of others, prompt new

hope for the discovery of ways to restore hearing and balance senses in patients suffering from hair

cell lesions.

What is the statistical power of vestibular tests when threshold changes are very near the step size

of stimulus intensity? The vestibular response threshold is a valuable quantitative means of

identifying changes in the sensitivity of gravity receptors. It is important to determine the resolving

power of this vestibular test and, therefore, estimate the smallest threshold shift that can be detected

using vestibular responses. Given a normally distributed sample, a sample size of 5, an acceptable

statistical power of 0.8, a traditional a-level of 0.05 and the mean and variance of 27 randomly

chosen control animals from our laboratory, the minimum detectable threshold shift would be

6.6 dB when 6 dB intensity steps are used. These theoretical exercises suggest that even with

small samples we can expect to discern threshold changes having magnitudes near the test

measurement step with some confidence. The smallest intensity step used currently is 3.0 dB.

Are embryonic responses really vestibular? To demonstrate that responses to pulsed linear

acceleration were in fact vestibular in the embryo, new studies were completed. Bilateral

destruction of the labyrinth eliminated the response, but bilateral destruction of the cochlea did not.

These results serve as def'mitive evidence that responses cannot depend on the cochlea and thus are

vestibular in the embryo.

Is the adequate stimulus for vestibular responses linear acceleration (g) or linear jerk (time

derivative of acceleration dg/dt, g/msec)? A new stimulus paradigm was tested in detail. Results

157
showthatresponsethresholds,latenciesandamplitudesmaybedependentuponjerk magnitudes

(unitsof g/msec)andnot simplyaccelerationamplitudes.Theseresultsprovidenewinsightinto

theneuralgeneratorsof thevestibularresponsesaswell asthecharacteristicsof naturalstimuli for

gravity sensorsA detailedstudyof theeffectsof stimulusrisetime onvestibularresponseswas

carriedout. Theresultsprovidesupportfor thehypothesisthat vestibularresponsesareelicited

principallybylinear jerk rather than linear acceleration.

Significance of Accomplishments

The results presented here suggest that the gravity receptors of developing birds are dynamic in that

they exhibit a sharp increase in sensitivity in late embryos and early hatchlings. There may be as

yet undetermined factors that can alter maturational changes. One such factor could be related to

space flight. Ongoing studies are evaluating the influence of substrate vibration and hypergravity

fields on receptor function. Gravity receptors in maturing chicks are resilient and are capable of

complete recovery following severe injury. The discovery of mechanisms controlling recovery

could lead to new clinical strategies for the deaf and dizzy patient. Vestibular responses to pulsed

linear acceleration likely reflect a subset of gravity receptor neurons, in particular those signaling

linear jerk. This knowledge further improves our understanding of vestibular responses and our

ability to characterize the developing vestibular system. We continue to study changes in vestibular

function during ontogeny using a new physiological test. It is critical that we clearly define the

nature of the test and establish that it is in fact a vestibular test for all ages studied. The results

summarized here add significantly to our understanding of the origins and nature of vestibular

responses and, ultimately, to our understanding of vestibular ontogeny. Moreover, these studies

provide important insights that may lead to the successful application of the new vestibular test in

the diagnosis of the dizzy human patient.

Publications

Burkard, R., DeVries, S., and Jones, T. 1993. Conventional and cross-correlation BAERs in the

White Leghorn chick: Rate manipulations (Abstract). Association for Research in Otolaryngology

244: 61.

DeVries, S.M. and Jones, T.A. 1992. Neural generators of the chick far-field auditory response

evidenced by near-field recordings (Abstract). Association for Research in Otolaryngology 406:

137.

Fermin, C.D., Martin, D.S., Vellinger, J., Deuser, M., and Jones, T. 1992. Morphometric inner

ear findings in shuttle STS-29 space flown chicks (Abstract). Association for Research in

Otolaryngology 253: 85.

Jones, T.A. 1991. Vestibular compound action potentials recorded from the skull surface in

unanesthetized animals (Abstract). Association for Research in Otolaryngology 131:40.

Jones, T.A. 1992. Gravity and the ontogeny of animals. The Physiologist 35(1): $77-$79.

Jones, T.A. 1992. Vestibular short latency responses to pulsed linear acceleration in

unanesthetized animals. Electroencephalography and Clinical Neurophysiology 82(5): 377-386.

Jones, T.A. 1993.

1990-1991 NASA

pp. 160-161.

Short latency vestibular responses and the ontogeny of vestibular function. In:

Space Biology Accomplishments, NASA Technical Memorandum 4501,

Jones, T.A., DeVries, S.M., DuBois, L.M., and Nelson, R.C. 1993. Vestibular ontogeny:

Measuring the influence of the dynamic environment. The Physiologist 36(1): $46-$49.

158
Jones,T.A., Fermin, C., Hester,P.Y., and Vellinger, J. 1993. Effects of microgravity on

vestibularontogeny: Direct anatomicalandphysiologicalmeasurementsfollowing spaceflight

(STS-29).Acta Veterinaria Brno 62(Suppl. 6): $35-$42.

Jones, S.M. and Jones, T.A. 1993. The effects of cochlear ablation on short latency responses to

linear acceleration in the embryo. ASGSB Bulletin 6(1): 60.

Jones, T.A. and Nelson, R.C. 1992. Functional recovery of the peripheral vestibular system

following severe streptomycin injury (Abstract). Association for Research in Otolaryngology 487:

161.

Jones, T.A. and Nelson, R.C. 1992. Recovery of vestibular function following hair cell

destruction by streptomycin. Hearing Research 62: 181-186.

Jones, T.A., Nelson, R.C., and DeVires, S.M. 1992. Stimulus rise time and vestibular evoked

potentials. ASGSB Bulletin 6(1): 6.

Jones, T.A., Vellinger, J.P., Hester, Y., and Fermin, C. 1991. Weightlessness and the ontogeny

of vestibular function: Evidence for persistent vestibular threshold shifts in chicks incubated in

space. The Physiologist 34(1): S143-S144.

Nazareth, A.M. and Jones, T.A. 1991. Central and peripheral generators of short latency

vestibular responses (Abstract). Neuroscience 17(1): 29

Nazareth, A.M. and Jones, T.A. 1991. Vestibular nerve components of responses to pulsed

linear acceleration. ASGSB Bulletin 5(1): 40.

Roll, R.L., Jones, T.A., Benowitz, N.L., and Morley. B.J. 1993. The chronic infusion of

nicotine into the developing chick embryo does not alter the density of (-)-[3H] nicotine binding

sites or vestibular function. Brain Research 604: 260-265.

Tenenbaum, S.A., Martin, D.S., Garry, R.F., Jones, T., and Fermin, C.D. 1993. Peripheral

vestibular injury induces heat shock and c-fos protein production in brain stem tissues (Abstract).

Association for Research in Otolaryngology 448:112.

159
MOLECULAR BASIS OF TENSION DEVELOPMENT IN MUSCULAR ATROPHY

Christine E. Kasper

School of Nursing

University of California, Los Angeles

Los Angeles, CA 90024-6918

Description of Research

The long-range goal of our research has been to assess the effects of immobility and skeletal

muscle unloading on skeletal muscle function--in particular, the effects of exercise on skeletal

muscle following prolonged atrophy. It is the aim of this project to describe the role calcium

ions and calcium-mediated physiologic mechanisms play in conjunction with gravity during

decreased weight bearing.

Our work has focused on the atrophy-associated cellular changes that might predispose skeletal

muscle to injury during reuse and recovery. Preliminary studies in this laboratory have shown

that exercising atrophied skeletal muscle following unloading 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 exercise recovery from atrophv. The

damage appeared in the form of necrotic fibers, central nuclei, and fiber debris"_n the

intrafascicular and intrafibrillar spaces of the soleus.

Initial studies have sought to (1) characterize the occurrence and distribution patterns of control,

transitional, and damaged fibers that occur in atrophied soleus muscle during the course of

recovery, (2) determine the contribution that the cytoskeletal element titin makes in the

production of skeletal muscle damage during the recovery of skeletal muscle from atrophy, and

(3) determine the role that nuclear control domains play in skeletal muscle.

Accomplishments

(1) Titin isoform adaptation in atrophied skeletal muscle. Initial studies by this

laboratory have quantitatively compared the location and relative amount of the cytoskeletal

protein titin in single fibers of a known myosin type from control, atrophied soleus, and plantaris

muscles. Limited data have also been obtained from tibialis anterior and gastrocnemius muscles

exposed to 5.3 days of microgravity [STS-48, Physiological and Anatomical Rodent Experiment

1 (PARE 01)]. Our preliminary findings have been: (a) There are differences in titin

electrophoretic mobility and antibody location between muscles, but not in the relative amount of

titin associated with myosin type. (b) There is a decrease in the relative amount of titin, as well

as changes in titin electrophoretic mobility and antibody location in atrophied skeletal muscle.

(c) Atrophied single muscle fibers normalized for fiber size are significantly more fragile than

control fibers at resting lengths.

Titin in Control and Atrophied Muscle. We examined titin antibody localization in single

muscle fibers of adult rat soleus and plantaris using immunofluorescent microscopy and sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The first fiber segment was

used for demonstration of myosin type: either fast or slow as demonstrated by SDS-PAGE or

typed with slow myosin heavy chain (MHC) monoclonal antibody ($58). Fiber segments used

for gel electrophoresis were also analyzed for relative amounts of titin protein and titin

electrophoretic mobility. The second fiber segment was processed for titin antibody staining

with 9D 10 (M. Greaser, University of Wisconsin) specific for titin under fluorescent microscopy.

The distance of titin antibody localization to the Z-band and sarcomere spacing were determined.

We found that fibers from control soleus muscle demonstrated a significantly different antibody

160
to Z-disc distance (Ab-Z) from control plantaris fibers (Table 1), irrespective of myosin type.

This difference was maintained when the Ab-Z was normalized by sarcomere length. However,

the relative amount of titin and myosin was not significantly different between muscles.

The ability of titin to adapt in response to altered muscle function has not been previously

demonstrated. Therefore, a study was performed to examine the effects of 28 days of hindlimb

suspension (HK) on titin (Table 1). Atrophied single fibers demonstrated a significant loss of

relative titin weight between muscles and between groups within the same muscle.

Table 1. Pooled data from all fibers irrespective of myosin type (N = 207) + SEM

Fiber

N

Ab-Z/SL

Titin Wt.

Myosin Wt.

Control

34

0.459-20.017

0.173_+0.005

0.040-L,-0.003

0.160-M).011

Soleus

HK

37

0.402_+0.013"

0.164!'0.004

0.029"M).003"

0.116:1.'0.015"

Control

60

0.406:t0.009t

0.159i-0.003t

0.048:k0.003

0.185_+0.014

Plantaris

HK

76

0.427-t-0.007"+

0.178_+0.003"t

0.043_+0.003

0.179-&0.012

* = Significantly different from control, p < 0.05

t = Significantly different from soleus, p<_0.05

SL : Sarcomerelength

Wt. : Relative weight of protein

Titin in Slow and Fast Single Fibers from Soleus and Plantaris. Additional fibers from control

and atrophied muscles were analyzed for myosin type and Ab-Z distance in order to determine if

a portion of the data variability could be accounted for by myosin type. We found that Ab-Z

distance is characteristic of myosin type. However, while fast fibers in control and atrophied

fibers are similar irrespective of muscle of origin, type I fibers are different between muscles. In

atrophied type I fibers from soleus, titin Ab-Z distances decrease by 17 percent compared to

control values, whereas distances increase by 8 percent in soleus type II fibers compared to

controls. These Ab-Z distances are not significantly different from control values of the opposite

type. These data would indicate that atrophy may drive an adaptation in titin isoform to

accommodate an altered resting tension matched to fiber size and force-generating capacity. The

dramatic decrease in the relative amount of titin in soleus fibers during hindlimb unloading may

account for the selective degeneration of type I fibers during reloading of atrophied soleus.

Effect of short term microgravity on titin. Samples of muscle obtained from the STS-48 mission

(PARE.01, Sept. 1991) demonstrated that removal of weight-bearing by weightlessness was

sufficient to cause significant changes in titin Ab-Z distances and a significant loss of relative

titin weight in the gastrocnemius. As both the tibialis anterior (99 percent type II) and the mixed

gastrocnemius (93 percent type 1I) are "fast muscles", changes in titin were not anticipated in this

time frame. It is possible that the titin molecule is very sensitive to gravitational forces.

(2) Effect of short.term microgravity on myonuclei. The purpose of this investigation

was to examine the adaptation of myonuclei, and cytoplasm-to-myonucleus ratios, to short-term

microgravity. The fast-twitch tibialis anterior (TA) and gastrocnemius (G) muscles from young

SPF (S/D) female rats (N=8/group) were exposed to 5.3 days of microgravity (STS-48 mission,

PARE.01, M. Tischler, PI). The flight group (S) was compared to ground-based controls (C) and

hindlimb unloaded controls (HK). Single skeletal muscle fiber segments were dissected from

TA and G muscle and stained with acridine orange (AO) to enable counts of myonuclei. Fibers

were viewed with a Leica laser confocal scanning microscope, and fiber volume and myonuclear

161
number were calculated. Fibers were also analyzed with gradient SDS-PAGEfor MHC

isozymesand measuredwith laserdensitometry. Mean+ SD cytoplasmic volume/myonucleus

ratio was significantly decreased in the S groups ( 1.95:1-'0.07 TA; 2.13_+0.09 G) when compared

to control (2.25_+0.19 TA; 2.56_+0.13 G) and HK (2.31_+0.19T A; 2.34_+0.15 G) groups,

respectively. C and HK groups were not significantly different. The number of sarcomeres per

myonucleus was also significantly less than both C and HK groups; while, myonuclei/mm in

these group was significantly greater than C and HK groups. The increase in myonuclei and shift

in fast to slow MHC prior to an increase in fiber volume is consistent with an early adaptation of

fiber type. These results demonstrate that 5.3 days of microgravity resulted in (1) a significant

decrease in single fiber volume; (2) a significant increase in myonuclear density when compared

to either ground-based controls or HK groups; (3) an increase in myonuclear density coincided

with an early shift in MHC expression; and (4) no change in the spatial arrangement of

myonuclei.

Significance of the Accomplishments

Finding 1: Titin is the largest protein in skeletal muscle and forms a third f'tlament system for the

myofibril, which assists in sarcomeric alignment and elastic recoil. These data correlate with

altered cell size and a shift in titin isoform expression in unloaded muscles and may reflect the

ability of the muscle to modulate elasticity and compliance by the regulation of titin size

isoforms. Given that titin "holds" the contractile machinery in alignment during contraction, its

ability to adapt during microgravity and periods of atrophy could .pred.ispose skeletal muscle to

damage during reuse during periods of recovery. Further inveslagataons could determine the

optimal time frame to conduct rehabilitation and training on patients recovering from bed rest

and atrophy.

Finding 2: The decrease in the cytoplasm-to-nuclear ratio in atrophied muscle fibers could

suggest that, when lower cytoplasmic levels are maintained, the availability of myonuclei are no

longer a modulatory factor in controlling fiber size.

Publications

Kasper, C.E., McNulty, A.L., Otto, A.J., and Thomas., D.P. 1993. Alterations in skeletal muscle

related to impaired physical mobility: An empirical model. Research in Nursing and Health 16:

265-273.

Kasper, C.E. and Xun, L. 1992. Adaptation of titin to short-term microgravity. ASGSB Bulletin

6(1): 87.

Kasper, C.E. and Xun, L. 1993. Adaptation of titin to hindlimb unloading. Medicine and

Science in Sports and Exercise 25(5, Suppl.): $97.

Kasper, C.E., Xun, L., Monke, S., and Garfinkel, A. 1992. Adaptation of myonuclei to shortterm microgravity. ASGSB Bulletin 6(1): 57.

Maloni, J.A. and Kasper, C.E. 1991. Physical and psychosocial effects of antepartum hospital

bedrest: A review of the literature. Image: Journal of Nursing Scholarship 23(3): 187-192.

McNulty, A., Otto, A., Kasper, C.E., and Thomas, D.P. 1992. Effect of recovery mode

following hindlimb suspension on soleus muscle composition in the rat. International Journal

of Sports Medicine 13: 6-14.

St. Pierre, B., Kasper, C.E., and Lindsey, A.M. 1992. Fatigue mechanisms in cancer patients:

TNF and exercise effects on skeletal muscle factors. Oncology Nursing Forum 19(3): 419-425.

162
Tseng,B.S.,Allen, D.L., Sung,C.K., Kasper,C.E.,andEdgerton,V.R. 1991. Mechano-linkage

of myonuclei with sarcomerelength in isolated rat skeletal muscle fibers. Society of

Neuroscience Abstracts 17(Part 1): 650.

163
ORIENTATION OF CELLS BY GRAVITY,

QUANTITATIVE RELATIONSHIPS

John O. Kessler

Department of Physics

University of Arizona

Tucson, AZ 85721

ILLUMINATION, AND SHEAR:

Description of Research

Introduction. All life began with microorganisms, and they axe still today the majority of living

beings. Their existence depends on, and is organized by, gravity, light, their fluid habitat, and by

interactions with one another. These influences are somehow integrated by the cells into an overall

behavior pattern. In the case of swimming algal and bacterial ceils, this complex and nonlinear

integration of environmental factors is expressed as directional swimming. The probability that a

given cell swims in a certain direction and at a certain speed is thus a function of these influences.

It is one major overall objective of this research to measure the probability distribution of cell

swimming velocities with particular attention to the role of gravity and the way in which this

mechanical influence combines with fluid shear and light or oxygen, which affect cell metabolism.

It is another overall objective to analyze how the individual celrs motile behavior combines into

gravitationally mediated population dynamics.

ObjectivesGoals.

(1) To develop quantitative computer-assisted microscopy for measuring the probability

distribution of swimming velocities of microorganisms subjected to gravitational and other guiding

influences.

(2) To develop apparatus and associated methods by which swimming cells can be

exposed to fluid velocity gradients (shear) plus additional orienting influences, the resulting cell

trajectories being measurable, as in objective (1) above. The additional influences are gravity,

acceleration, molecular concentration gradients, illumination (variable incident direction,

wavelength, intensity).

(3) To develop a rotating cuvette system for (a) supplying uniform shear, and (b) for use

as an unbiased statistical sampler of motile populations.

(4) To develop quantitative methods for investigating bacterial bioconvection patterns and

their significance for gravity-mediated microbial ecology.

Accomplishments

(1) A "Motion Analysis" computer system was purchased. This system

processes in parallel many simultaneously acquired cell swimming trajectories. It calculates their

direction and speed and provides sophisticated statistical analysis of the results. The original data

can consist of hundreds of tracks, acquired during 1 or more seconds at frame rates up to 60-Is.

Typical statistical analysis takes a few minutes per run. This system is cenlral to the research

method. It provides sufficient statistics rapidly enough to guide experiments and to yield results.

However, extensive programming and trouble shooting have been required. Because of its

quantitative output, the system has delivered important cautionary information concerning

systematic sampling errors, which has resulted in the development of Objective (3).(b) cited above.

This motion analysis apparatus has been used to determine the gravity-oriented swimming

164
distributionfunction of Pleurochrysis carterae and other cells. It has been used to investigate the

effect of shear on swimming cells. P. carterae is particularly relevant to gravitational biology

because of the CaCO3 coccoliths which it produces.

(2) Two operationally distinct shear-producing apparatuses were designed

and built during the previous grant period. As of the date of this report, one of these has

been tested with swimming algal cells and the motion analysis system. With it, shear and gravity

can be parallel or orthogonal. The second apparatus allows only orthogonal gravity and vorticity

vectors, but it is more flexible than the first with respect to the possible directions of illumination.

It has been found that cells in the shear apparatus are unharmed by the moving components of the

mechanism.

(3) A rotating cuvette (called "Clinovar") was constructed and tested. Since

the cell sample in the cuvette is always returned to the field of view, rotation eliminates the

disappearance of fast and accurately directed ceils that swim toward one end of the measuring

cuvette. Uniform rigid rotation also introduces a spatially constant vorticity equal to twice the

rotation rate, and so provides shear. The amount of shear thus varies with rotation but not with

location in the cuvette. When rotation by 180" is intermittent, it produces unbiased, bimodal

sampling but no extra shear. Programming the motion analysis system for conversion between

rotating and stationary reference frames was accomplished. It was then found that continuous

rotation requires better bearings because of the mechanical jitter, which becomes apparentat high

magnification. The theory of particle sedimentation within the rotating cuvette system was

developed, and experiments were performed to show its accuracy, even when the rotation rate is

varied.

One important result of this experimental/theoretical development is the very serious doubt that it

casts on clinostat experiments: It was shown conclusively that free organelles within fluid

comparmaents of cells move, and, presumably, tend to disrupt the cytoplasm.

(4) Gravity-mediated functional self-organization of bacterial cell

populations. The swimming direction of individual algal and bacterial cells is partly random and

partly guided by environmental factors. The environmental influences include gravity, chemical

concentration gradients, and illumination. For algae, gravity is often the major influence that

orients the swimming path of single cells. For the case of the bacteria studied during this research,

Bacillus subtilis, we have so far found no evidence that gravity has any direct influence on the

swimming of individual organisms. However, ceils do swim in the direction of increasing oxygen

concentration. In large bacterial populations (e.g., 109 cells/cm3), gravity and oxygen

consumption cause regular convective motion of large, spatially recognizable subgroups of the

population (Figures 1 and 2). The entire dynamical system, which is powered by directional cell

swimming, functions as an autonomous entity that supplies, transports, mixes, and consumes

oxygen. This spontaneous development of macroscopic ordering of a vast population of

independent undifferentiated organisms that do not interact directly with one another results from

(1) their consumption of oxygen; (2) gravitational forces that place the air/fluid interface so that

oxygen is supplied directionally, from above, thereby inducing upward swimming trajectories; (3)

gravitational forces, once more which produce convection in anisotropic fluids; and (4) the laws of

fluid mechanics, which provide dynamic long-range coherence.

165
Figure 1 (a) Top view of bacterial convection patterns. A 2-mm deep fluid layer contains -10 9 ceUs/cm3 of Bacillus

subtilis. The petri dish is 8 mm in diameter. Illumination is "dark field"; i.e., regions containing many cells are

bright, and, conversely, bacterial respiration depletes oxygen. Diffusion from the air interface above the fluid

replenishes the oxygen. Consumption and supply in the -g direction generate a gradient up, in which the cells swim.

The upward accumulation of cells generates a dense upper layer of fluid (i.e., mean fluid density is proportional to

local cell concentration). This situation is unstable. Convection rolls, and columns appear and supply oxygen to

those lower regions of bacterial population which are otherwise beyond the reach of a sufficiently rapid diffusive

supply from the surface. The cell-swimming-driven convection, of course, also mixes both the oxygen and the

bacterial ceils. The entire living fluid mechanical system functions concurrently as a transporter and consumer of

oxygen. It should be remembered that these remarkable dynamical structures are shaped by consumption of oxygen,

directed by oxygen gradients, swimming, and the force of gravity. There is no direct communication among the

cells. Figure l(b). The central portion of Figure l(a), slightly later, enlarged and copied from a time-lapse video

film. The entire structure is in slow aperiodic motion.

166
Figure2. General descriptionisthesameasforFigure 1, except that the fluid layer is slightly shallower and data

were acquired with a different cell culture.

Gravity is basic for the generation and stable development of this macroscopic ordering, which

functions to improve survival by augmenting diffusive transport of a dissolved metabolite with

convective supply and mixing. The ordering also creates diverse microenvironments,

characterized by various levels of oxygen concentration and various cell residence times.

The development of life forms clearly proceeded through stages, starting from molecular

mechanisms. Very little is known about the coalescence of individual microorganisms into

associated complex specialized groups and structures. The observations and insights gained

during the grant period suggest that one route for the development of multicellular organisms, from

an original sea of simple cells, may well have included the sort of catalysis by gravitational forces

and symmetries that is modeled by these experiments.

Significance of the Accomplishments

The integration by motile cells of environmental influences is a totally unsolved problem which

forms the core of gravitational biology/physiology for free cells. Cells which are normally part of

large organisms may obey similar laws. Some aspects of the larger biota which comprise many

cells are analogous to the patterns into which dense populations of survivors organize themselves.

This research quantitatively investigates problems of gravitational biology that relate to individual

and collective behavior. Accurate, statistically significant methods of measuring complex

individual cell behaviors are being developed. Results that concern single cells can then be

confidently integrated into theories of population structure. Applications will include insights into

the dynamics of large populations of microorganisms. For macroapplications, there will be new

information concerning individual and group behavior of phytoplankton. On a smaller scale, this

research presents the growth and self-maintenance of concentrated populations of bacteria in an

entirely new light. Areas of application include bacterial invasions of small liquid pools as in air

conditioners, sick room humidifiers, and in enriched open environments, such as waste disposal

sites. The central role of gravity in the generation of bioconvection also implies that the dynamics

of waste recycling systems may differ markedly, relative to expectations, when they are placed in a

microgravity situation.

167
Experimental Observations and Implication

Swimming cells of Pleurochrysis carterae, an alga that is a prime example for biomineralization

(CaCO3), are gravitactic. During the first 4 days after inoculating into new culture medium, the

population is bimodally gravitactic: one population segment swims up, the other down. After 7

days or more, the entire population swims upward. There is no significant change in the

swimming speeds.

These experiments suggest the new research area of Motility-Based Taxonomy. Are the observed

bimodality and its disappearance associated with explicitly observable morphological changes? Are

such hypothetical changes involved with passive versus active gravity sensing?

Gravity-Mediated Biological Self-Organization

Bacterial concentration/convection patterns that are organized by the swimming p.opulation of

Bacillus subtilis resident in dense cultures were discovered by the PI and associates. These

dynamic patterns transport and mix oxygen. They are driven by the force of gravity and by

swimming asymmetry deriving from oxygen consumption by cells. To understand these systems

deeply, one requires the velocity probability distribution function of the bacteria swimming in an

oxygen gradient. The first usable measurements of this oxygen taxis of B. subtilis were performed

during-this grant period using the computerized microscope system.

Publications

Kessler, J.O. 1992. The internal dynamics of slowly rotating biological systems. ASGSB

Bulletin 5:11-21.

Kessler, J.O. 1992. Theory and experimental results on gravitational effects on monocellular

algae. Advances in Space Research 12: 33-42.

Kessler, J.O., Doud, A.C., and Hill, N.A. 1992. Upswimming (negative geotaxis) of

Pleurochrysis carterae (Abstract). ASGSB Bulletin.

Kessler, J.O., Hill, N.A., and Haeder, D.P. 1992 Orientation of swimming flagellates by

simultaneously acting external factors. Journal of Phycology 28:816-822.

Kessler, J.O. and Tam, W.Y. 1992. The Clinovar (Abstract). ASGSB Bulletin.

Kessler, J.O and Wojciechowski, M.W. 1993. Self-organized convective transport and mixing

by swimming bacteria. Bulletin of the American Physiological Society 38: No. 12, 2306.

Pedley, T.J. and Kessler, J.O. 1992. Bioconvection. Science Progress 76: 105-123.

Pedley, T.J. and Kessler, J.O. 1992. Hydrodynamic phenomena in suspensions of swimming

organisms. Annual Review of Fluid Mechanics 24:313 - 358.

Vincent, R.V., Hill, N.A., and Kessler, J.O. 1993. Diffusion in a suspension of particles subject

to external torques. Bulletin of the American Physiological Society 38: No. 12, 2267.

168
COLLAGEN SYNTHESIS, ASSEMBLY, AND MINERALIZATION IN CHICKEN

OSTEOBLAST CELL CULTURES AND OTHER CALCIFYING VERTEBRATE

SYSTEMS

William J. Landis and Louis C. Gerstenfeid

Laboratory for the Study of Skeletal Disorders and Rehabilitation

Department of Orthopedic Surgery

Harvard Medical School and Children's Hospital

Boston, MA 02115

Description of Research

The research of this laboratory concerns describing the basic molecular biology and biochemistry,

morphology, and ultrastructure of a bone cell culture system to be utilized for future flight

experiments under zero gravity conditions. Results are correlated with investigation in vivo of

other calcifying vertebrate systems including bone, cartilage, and tendon. The bone culture system

is currently characterized under normal gravity. Cultures are derived from osteoblasts of chicken

calvaria, and the growth and development of the bone cells and the onset and progression of

mineralization in the culture extracellular matrix are studied. We are interested in defining (1) the

mechanisms of collagen gene expression, (2) the temporal relationship between collagen crosslinks _md 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. Data from the culture system

are compared with results obtained with bone, cartilage, and tendon particularly with respect to

collagen-mineral interaction deduced from electron microscopy and novel three-dimensional image

reconstruction techniques.

Our most recent previous report had shown that the mineral from our bone cell cultures is similar in

its size, shape, surface texture, crystallographic properties, and relation with collagen as that

determined from bone tissue in vivo; and that strontium may be used as a reliable and critical

marker for the determination of mineral accumulation associated with collagen in the cultures. In

addition, the interaction of collagen and mineral crystals in both tissue in vivo and the bone culture

system had been examined in initial studies utilizing high-voltage electron microscopy and threedimensional computer graphic image reconstruction techniques. Further, we had developed a cell

culture system of chondrocytes as an extrapolation of our work with bone cells, and we had

documented that this culture mineralizes as does its counterpart, normal growth plate cartilage. The

chondrocyte system then would be useful as a model describing growth plate developmental

events.

Accomplishments

The studies we have undertaken in the previous year and a half have continued with further

characterization of our bone culture system in terms of extraceUular matrix formation and regulation

and with the elaboration of the interaction between collagen and mineral during calcification of

model vertebrate systems in vivo. We have also initiated experiments to determine the atomic

surface structural features of mineral species important in biological calcification. A summary of

the respective results follows:

(1) We have studied the pre- and post-translational mechanisms controlling extracellular

matrix formation in our cultured chicken embryonic bone cells. Gene expression of specific

proteins of bone (type I collagen, osteopontin, and osteocalcin) was measured in one series of

experiments, and collagen cross-link analysis was correlated with microscopic structure in another

series of investigations. The results of the work showed two aspects of the regulation of

169
extracellular matrix formation by one cell. The first, that osteoblastgeneresponseto 1,25

(OH)2D3appearsto bedependentontheembryologicalor developmentalstageof theosteoblast;

andthesecond,thatextracellularmatrix assemblyis post-translationallycontrolled,mediatedby

polarizedsecretionof collagenandby collagencross-linkstabilization.

(2) We have determined a number of structural featuresand events related to the

developmentandmineralizationof ourbonecellcultures,aswell asa modelcalcifyingvertebrate

tissues. The work has beenaccomplishedthrough the novel use of high-voltage electron

microscopyandthree-dimensionalcomputergraphicimaging. We havefound in theosteoblast

cultures,in thenormallycalcifyingleg tendonsof turkeys,andin thelongbonesof chicksthatthe

respectiveextracellularmatricesaresimilarly composedof collagenfibrils strictly organizedand

assembledso that their holeandoverlapzonesarein contiguousregistration. This assemblage

createschannelsfor mineralaccommodationanddepositionat independentsitesthroughoutthe

collagenarrays. Crystalnucleationmayoccuralongthesurfaceandwithin thechannelsof the

fibrils, and a fusion processappearsto beresponsiblefor theformation of largercrystalsfrom

smallerones. Ultimately, the growth anddevelopmentof crystalsin associationwith collagen

forms parallel sheetsof mineral in theextracellularmatrices. Theseresultsareimportantfor

elaboratingthebasicmeansby whichmineralizationoccursamongvertebratetissues.

(3) Wehavestudieda66-kDaphosphoproteinwhichcomprisesa majornon-collagenous

proteinin our cultured chickenembryo osteoblasts.We havecharacterizedits expressionand

tissuedistributionduringosteoblastdifferentiationandculturedevelopment.A cDNA clonefor the

proteinwasisolatedfrom anexpressionlibrary madefrom embryonicchickenbonemRNA anda

completeprimary proteinsequenceof 264 aminoacidswasdetermined.Sequencecomparison,

hybridization analysis,and geneproduct induction by phorbol estersugg.esthat the 66 kDa

phosphoproteinis theavianhomologueto themammalianprotein,osteoponun.Theregulationof

avianosteopontinappearsto beundertranscriptionalcontrolduringosteoblastdevelopment.

(4) As a new approachto characterizingthe mineral depositionin vertebratetissues,

surfacestructuresof hydroxyapatiteandbrushite,two crystalsimportantin mineralization,were

determinedfor thefirst timeby atomicforcemicroscopy.A methodof suspendingthemineralsas

powdersin ethanolwasusedto isolatesmallcrystalclustersonglass.Measuredatomicspacings

correspondedto the (001) and (110) planesfor hydroxyapatiteandthe (110) crystal planefor

brushite. Theseinitial surfacestructuraldataare importantfor further work defining precise

interactionbetweencrystalsandorganicmatrixcomponentssuchascollagen,fundamentalin the

calcificationof vertebratetissues.

Significance of the Accomplishments

The current results are significant in describing a number of physical (structural), chemical, and

biological events occurring during the mineralization of a bone cell culture system which very

closely resembles the mineral deposition of normal calcified vertebrate tissues, some of which have

been independently studied in correlative investigations. Our data provide details of calcification

which are new in some instances and which cannot be obtained with other methods or model

systems. Our most recent results (1) demonstrate differences in hormonal response to

1,25 (OH)2D3 by embryonic osteoblasts isolated at different developmental ages; (2) indicate that

extracellular matrix assembly is controlled at a post-translational level through polarized secretion

of collagen and collagen cross-linking; (3) provide fundamental details of the ultrastructural

interaction between mineral and collagen: (4) isolate and characterize the first non-mammalian form

of osteopontin; and (5) report the first atomic surface structural features for vertebrate mineral

crystals. These data help expand information which is fundamentally important in understanding

bone cell growth and development, extracellular matrix organization and assembly, and

mineralization. They provide the comparison for data obtained from experiments in other systems,

170
including futurebonecell culturesflown in zero gravity. From such results, the basis of space

flight skeletal mass loss and related effects may be more readily understood.

Publications

Arena, J., McEwen, B.F., Song, M.J., and Landis, W.J. 1992. A study of the mineralization of

bone by high voltage electron microscopic tomography. In: Proceedings of the 50th Annual

Meeting of the Electron Microscopy Society of America (Bailey, W., Ed.). San Francisco: San

Francisco Press, pp. 582-583.

Bruder, S.P., Caplan, A.I., Gotoh, Y., Gerstenfeld, L.C., and Glimcher, M.J. 1991.

Immunohistochemical localization of a 66kD glycosylated phosphoprotein during development of

the embryonic chick tibia. Calcified Tissue International 48: 429-437.

Christoffersen, J.C., and Landis, W.J. 1991. A contribution with review to the description of

mineralization of bone and other calcified tissues in vivo. Anatomical Record 230: 435-450.

Gerstenfeld, L.C., Broess, M., Bruder, S., Caplan, A., and Landis, W.J. 1992. Regulation of

osteoblast extracellular matrix formation: Post-translational regulation of extracellular matrix

deposition between embryonic development and hormone response. In: The Chemistry and

Biology of Mineralized Tissues (Slavkin, H. and Price, P., Eds.). New York, Elsevier Science

Publishers, pp. 287-295.

Gerstenfeld, L.C. and Landis, W.J. 1993. Gene expression and formation of extracellular matrix

in a primary osteoblast culture system. ASGSB Bulletin 7: 30.

Gerstenfeld, L.C., Landis, W., and Eyre, D. 1992. Regulation of extracellular matrix formation

in osteoblast cultures. Connective Tissue Research 27: 136.

Gerstenfeld, L., Riva, A., Landis, W.J., and Eyre, D.R. 1991. Post-translational control of

collagen fibrillogenesis in a mineralizing chick osteoblast culture system. Proceedings of the

American Society for Bone and Mineral Research 6:$231.

Gerstenfeld, L.C., Riva, A., Hodgens, K., Eyre, D., and Landis, W.J. 1993. Post-translational

control of collagen fibrillogenesis in a mineralizing chick osteoblast culture system. Journal of

Bone and Mineral Research 8:1031-1043.

Landis, W.J., Grynpas, M.D., and Gerstenfeld, L.C. 1991. Strontium substitution for calcium

in mineralizing bone cell cultures. ASGSB Bulletin 5: 82.

Landis, W.J., Grynpas, M.D., and Gerstenfeld, L.C. 1991. Utilization of strontium as a marker

for calcium in mineralizing osteoblast cell cultures. Combined Meeting of the Orthopaedic

Research Societies of USA, Japan, and Canada 1: 293.

Landis, W.J., Hodgens, K.J., McKee, M.D., Nanci, A., Song, M.J., Kiyonaga, S., Arena, J.,

and McEwen, B. 1992. Extracellular vesicles of calcifying turkey leg tendon characterized by

immunocytochemistry and high voltage electron microscopic tomography and 3-D graphic image

reconstruction. Bone andMineral 17: 237-241.

Landis, W.J., Hodgens, D., Song, M.J., Arena, J., Kiyonaga, S., and McEwen, B. 1991.

Mineralization of collagen in tendon utilizing high voltage electron microscopic tomography and 3-

D reconstruction. Journal of CeU Biology 115: 105a.

171
Landis, W.J., Hodgens,K., Song, M.J., Arena, J., Kiyonaga, S., and McEwen, B. 1992.

Mineralandcollageninteractionduringcalcification. Connective Tissue Research 27: 107.

Landis, W.J., Hodgens, K., Song, M.J., Arena, J., Kiyonaga, S., and McEwen, B. 1992.

Mineral and collagen interaction during calcification. In: The Chemistry and Biology of

Mineralized Tissues (Slavkin, H. and Price, P., Eds.). New York, Elsevier Science Publishers,

pp. 211-219.

Landis, W.J. and Siperko, L. 1992. Imaging of hydroxyapatite crystals by atomic force

microscopy. Connective Tissue Research 27: 125.

Landis, W.J. and Song, M.J. 1991. Initial mineral deposition in calcifying tendon characterized

by high voltage electron microscopy and three-dimensional graphic imaging. Journal of Structural

Biology 107:116-127.

Landis, W.J., Song, M.J., Leith, A., and McEwen, L. 1993. Mineral and organic matrix

interaction in normally calcifying tendon visualized in three dimensions by high voltage electron

microscopic tomography and graphic image reconstruction. Journal of Structural Biology 110: 39-

54.

Landis, W.J., Song, M.J., Leith, A., McEwen, L., and McEwen, B. 1991. Mineral and organic

matrix interaction in normally calcifying tendon visualized in three dimensions by high voltage

electron microscopic tomography and graphic image reconstruction Combined Meeting of the

Orthopaedic Research Societies of USA, Japan, and Canada 1: 66.

McEwen, B.F., Landis, W.J., and Song, M.J. 1991. Electron tomographic quantitation of

mineral distribution in different collagen zones of calcifying tendon. In: Proceedings of the 49th

Annual Meeting of the Electron Microscopy Society of America (Bailey, W., Ed.). San Francisco:

San Francisco Press, pp. 190-191.

McEwen, B.F., Song, M.J., and Landis, W.J. 1992. Quantitative determination of the mineral

distribution in different collagen zones of calcifying tendon using high voltage electron microscopic

tomography. Journal of Computer-Assisted Microscopy 3:201-210.

McKee, M.D., Nanci, A., Landis, W.J., Gotoh, Y., Gerstenfeld, L.C., and Glimcher, M.J.

1991. Effects of fixation and demineralization on the retention of bone phosphoprotein and other

matrix components as evaluated by biochemical analyses and quantitative immunocytochemistry.

Journal of Bone and Mineral Research 6: 937-945.

Moore, M.A., Gotoh, Y., Rafidi, K., and Gerstenfeld, L.C. 1991. Characterization of a cDNA

for chicken osteopontin: Expression during bone development, osteoblast differentiation and

tissue distribution. Biochemistry 30:2501-2508.

Siperko, L.M. and Landis, W.J. 1992. Atomic scale imaging of hydroxyapatite and brushite in

air by force microscopy. Applied Physics Letters 61: 2610-2612.

172
GRAVITATIONALLY SENSITIVE MOLECULAR

INVOLVED IN ESTABLISHING EARLY EMBRYONIC

Carey R. Phillips

Department of Biology

Bowdoin College

Brunswick, ME 04011

REARRANGEMENTS

AXES IN AMPHIBIANS

Description of Research

Shortly after fertilization, amphibian embryos rotate so that the presumptive posterior, or vegetal

portion, is down relative to gravity and the presumptive anterior, or animal portion, is up relative to

gravity. The orientation of the anterior-posterior axis is established during oogenesis and is

reflected by a gradient distribution of yolk platelets. The dorsal-ventral axis of amphibians is

normally determined by the sperm entry position (SEP); the dorsal axis forms on the side of the

embryo opposite the SEP and the ventral side of the embryo normally forms on the same side.

However, tipping the animal-vegetal axis obliquely relative to the gravitational force vector results

in repositioning the dorsal-ventral axis so that the dorsal side forms on the gravitationally "up" side

of the-embryo, irrespective of where the sperm enters. These results suggest that some

cytoplasmic components are becoming rearranged within the single-cell embryo such that

information leading to the formation of the dorsal axial structures become localized, or activated,

on only one side of the embryo. This hypothesis is further corroborated by centrifugation studies

showing that the dorsal axial structures will form on the centripetal side of the embryo (Black and

Gerhart, 1985).

Our long-term goal is to identify the cytoplasmic components involved in dorsal axial formation

and those which become rearranged during the first cell cycle. The second portion of this study is

to understand the mechanisms responsible for the rearrangement and to understand better how

gravitational forces influence these mechanisms.

Accomplishments

We have addressed two issues relative to dorsal-ventral axis formation, both as it forms normally

here on Earth and the role gravity plays in establishing axial information. All experiments

addressed here were performed on embryos of Xenopus laevis. The first series of experiments

addressed the issue of when the presumptive dorsal animal and presumptive ventral animal cells

acquire, or are able to maintain, different identities. Using a monoclonal antibody directed against

non-neural epithelium (Epi 1), we were able to show that isolated and cultured dorsal animal

blastomeres from the eight-cell stage will not express the Epi-1 antigen, whereas the cultured

ventral animal blastomeres from the eight-celled stage will express the Epi- 1 antigen at the correct

developmental time (London, Akers, and Phillips, 1988). These results suggest that dorsal and

ventral blastomeres have received significantly different instructions by at least the third cleavage.

We went on to show that the portion of the embryo which inherits the "determinants" for

establishing dorsal axial structures is responsible for sending molecular signals which modify and

control the final pattern of Epi-1 expression (Savage and Phillips, 1989). We have subsequently

shown that the signal(s) responsible for controlling spatial patterns of gene expression travel(s)

through the plane of the ectoderm (Savage and Phillips, 1989) and that they affect the spatial

patterns of expression for several other neural specific molecules as well (Doniach, Phillips, and

Gerhart, 1992).

173
Thesedata suggested that determinants for several developmental events relating to eventual axis

formation might become localized during the first cell cycle following fertilization. We also have

preliminary evidence that the localization of many such determinants is gravitationally sensitive

movement. Therefore, we have constructed and screened cDNA libraries of maternal RNA to

isolate clones for spatially localized RNA. The first dorsally enriched maternal RNA isolated from

these libraries coded for the small ribosomal subunit of mitochondria (clone 49). In situ

hybridization probes were prepared from this clone and we have shown that mitochondria become

heavily enriched on the presumptive dorsal side of the embryo during the first cleavage cycle

(Yost, Phillips, and Danilchik, 1993). We have also demonstrated that the mechanism for the

localization of mitochondria is gravitationally sensitive. Embryos, whose animal-vegetal axis is

obliquely oriented relative to the gravitational vector, will co-localize both the enriched

concentration of mitochondria and the position of the dorsal axis (Phillips and Danilchik, 1993).

The polymerase chain reaction (PCR) display method for comparing various related populations of

RNA has been applied to the problem of isolating clones for localized RNAs in the early animal

blastomeres. In this technique, either dorsal or ventral animal blastomeres are isolated at the end of

the eight-cell stage. RNA was extracted either immediately or after the blastomeres were cultured

to the equivalent of early gastrula stages. A series of three prime primers were used in the PCR

reaction with each type of RNA. The PCR reactants are separated on a sequencing gel, and bands

of interest are cut from the gel, reamplified, and cloned into a suitable vector (Figure 1). We have

identified a number of RNAs which become localized to either the dorsal or ventral animal

blastomeres during the first cell cycle, or isolated clones for RNAs are regionally synthesized in

response to some localized components. To date, we have isolated eight clones for ventral

maternal RNAs and four clones for dorsal maternal RNAs (Table 1). In addition, we have begun

to isolate clones for regionally expressed RNAs and have obtained clones for four ventral and five

dorsal zygotic RNAs. Of particular interest is the number of clones which appear to be localized to

the ventral animal blastomeres. This finding suggests that ventral cells may be more highly

specialized than previously believed. These clones are now being sequenced and used to prepare

probes for in situ hybridization analysis. Preliminary data suggest that there are several possible

mechanisms of localization. Clones representing localized RNA are being used to study a number

of issues relating to both the mechanisms of localization and the effects of the localized RNA on the

development of the dorsal axial smictures.

174
A B C D E F

Figure 1. Dorsal and ventral animal blastomeres were removed at the eight-ceU stage. Lanes E

and F represent PCR products from RNA isolated immediately after dissection. The arrows mark

the bands which were cloned fi_m this section of the gel. Lanes C and D show the PeR products

when RNA was extracted from cells cultured to the equivalent of stage 11 (mid-gasmlla). Arrows

mark the bands which have been cloned. Lanes A and B indicate the PCR products from cells

incubated to the equivalent of stage 13 (early neurula).

175
Venwal Maternal

vrnccl I

vmsa3

vmsa4

vmc$100

vmc 8101

vmc$1

vmcal

vmc$1

Venwal Zysotic

v-a:al

Dorsal_m_

dmc$1

dm$c6

Dorsal Zy$otic

dzp3

dz_a2

Table 1. PCR products of RNA isolated from different portions of the early Xenopus

embryos were displayed on a sequencing gel (Figure 1). The bands of interest were excised

from the gel, reamplified and cloned into a Bluescript vector. Each clone was used to prepare

probes for a Northern analysis against RNA isolated from the dorsal and ventral regions of

the embryo. The clones listed above represent the selected clones which met all the criteria

for representing localized transcripts. The nomenclature indicates the three prime primers

used to obtain the clones. Each three prime primer consists of 11 Ts and two bases (ga, ca,

cg, etc.) listed within the clone designation. The clones are separated relative to the time of

their synthesis, either as stored maternal RNA or newly synthesized zygotic transcripts.

PUBLICATIONS

Black, S., and Gerhaxt, J. 1985. Developmental Biology 108:310-324.

London, C., Akers, R., and Phillips, C. 1988. Developmental Biology 129: 380-389.

Savage, R. and Phillips, C. 1989. Developmental Biology 133: 157-168.

Doniach, T., Phillips, C., and Gerhart, J. 1992. Science 257: 542-545.

Yost, Phillips, and Danilchik (manuscript submitted).

Phillips, C. and Danilchik, M. 1993. ASGSB Bulletin 7(1) p. 96.

176
EVOLUTION OF GRAVITY RECEPTORS IN THE EAR

Arthur N. Popper

Department of Zoology

University of Maryland

College Park, MD 20742

Description of Research

The vestibular system of the inner ear includes mechanosensory cells that respond to external

signals such as position and sound. Such cells in amniotes (reptiles, birds, mammals), have been

divided into two types; type I and type II. These differ in terms of ultrastructure, innervation, and

(presumably) physiology. The locations of type I and type II cells differ from one another in each

of the otolithic end organs of the ear. It has been suggested that the two types of hair cells have

functional differences.

Although type II hair cells are considered to be ubiquitous among all vertebrates, it has been argued

that type I hair cells are found only in amniotes and not in anamniotes (jawless fish, cartilaginous

fish, bony fish, amphibians). Our recent work, however, shows that sensory hair cells very much

like the type I hair cell are present in the otolithic end organs of the ears of a fish. These findings

suggest to us that there is a complex organization to the otolithic receptors of the fish ear and that

this organization may have significant similarities to the organization of mammalian otolithic end

organs. Thus, it may be possible to use the otolithic end organs of the ears of fishes to gain better

insight into the sensory cells of mammals and other amniotes.

The thrust of our research is to investigate the types of sensory hair cells in the ears of anamniotes

and to test the hypothesis that multiple hair cell types evolved very early in the origin of the

octavolateralis system, rather than at the time of the evolution of amniotes. The work also involves

determining the structural diversity of hair cells in various anamniotes, as well as determining

whether different otolithic end organs vary in their hair cell composition.

Accomplishments

(1) We have used ultrastructural techniques to analyze the structure of

sensory hair cells in the saccular end organ of the goldfish. We found that two types

of hair cells are found in the teleost species Carassius auratus at very distinct locations on the

sensory epithelium. These results correlate closely with physiological studies of hair cells done in

other laboratories and provide a potential correlation between hair cell structure and physiological

properties. Moreover, this finding suggests that the different hair cell types are potentially involved

with different responses of the ear. While we do not yet fully understand the function of the

saccule in the goldfish, there is evidence that it is an auditory receptor and that the regions of the

saccule containing different types of hair cells have different frequency response characteristics.

Thus, it is possible that the ultrastructurally and physiologically distinct hair cells of the saccule

may have evolved to respond to different frequency characteristics of sound stimulation.

(2) Multiple Hair Cell Types in the Lateral Line. The lateral line is located on

the body surface of fish and amphibians and provides for detection of hydrodynamic signals

impinging upon an animal. The lateral line system contains sensory hair cells that axe very similar

to those found in the ear of fish. Moreover, there is reason to believe that the ear and lateral line

share some common ancestor structure in the early evolution of vertebrates. Thus, if the lateral line

has two hair cell types, as does the ear, the implication is that the presence of multiple hair cell

types and multiplicity of function arose far earlier in the evolution of vertebrates than even indicated

by their presence in the ears of fishes.

177
In orderto testthis hypothesis,we havestartedto examinethelateralline systemof the oscar,

Astronotus ocellatus. In the first part of this study, we treated the lateral line using gentamicin

sulphate, an ototoxic drug that destroys type l-like hair cells but that does not appear to damage

type II hair cells. Fish were treated with very low doses of gentamicin and the hair cells of the

lateral line were examined from 1 to 12 days later using scanning electron microscopy (SEM). Our

results demonstrated that the hair cells found in neuromasts of lateral line canal organs were totally

destroyed within 1 day of treatment, while the hair cells in free neuromasts were undamaged after

12 days of treatment. The results strongly imply that there are differences in the hair cells of the

canal and free neuromast parts of the lateral line.

Significance of the Accomplishments

Finding 1: The results with goldfish, when combined with our earlier investigations of the oscar

(Astronoms ocellatus) demonstrate the possible ubiquitousness of multiple hair cell types in fish.

These two species are taxonomically very distinct from one another. Consequendy, the finding

that both species have basically similar multiple hair cell types leads us to hypothesize that multiple

hair cell types may be an important component of otolithic end organs in most fishes.

Finding 2: Our discovery of there possibly being multiple hair cell types in the lateral line is the

first demonstration of differences in hair cells of this end organ. Our hypothesis is that the

gentamicin-sensitive cells of the canal neuromasts are type-I-like and those of the free neuromasts

are type II. If this proves to be the case, it would mean that the two hair cell types evolved very

early in the origin of vertebrates. Such results would also have profound implications on questions

pertaining to lateral line function, since it would mean that different lateral line regions have

evolved to respond to signals of a very different nature.

Publications

Lanford, P.J. and Popper, A.N. 1993. Ultrastructural correlates to physiologically distinct hair

cell types in the goldfish saccule. Society for Neuroscience Abstracts 19:1580.

178
EFFECTS OF VECTOR-AVERAGED GRAVITY ON SINGLE CHANNEL

PROPERTIES OF THE ACETYLCHOLINE RECEPTOR IN CULTURED MYOCYTES

Raven Reitstetter

University of Arizona

College of Medicine

Department of Physiology

Tucson, AZ 85724

Description of Research

The objective for the investigation reported herein was to measure single acetylcholine receptor

(AChR) channel amplitudes and kinetics after clinostat rotation at different rotation paradigms by

means of the cell-attached patch clamp technique.

Our laboratory has previously reported that co-cultures of nerve and muscle cells from Xenopus

embryos show specific sensitivity to altered gravity conditions as generated by rotation in a

slow-rotating clinostat. The alterations in muscle cells included changes in cell shape and

nucleus size, as well as a reduced accumulation of AChRs at the junction between the nerve and

the muscle cell. This led us to test if clinostat rotation also alters the properties of single

acetylcholine channel molecules. During a recent space flight experiment, we were able to verify

qualitatively the reduced incidence of AChR aggregates. In addition, we found marked changes

in the distribution and organization of actin filaments in space-flown myocytes, which were in

accordance with clinostat experiments performed in parallel.

Accomplishments

AChR single-channel activity is expressed as sequential o .pening and closing events which yield

information on how much current is passed through the iomc pathway and the kinetics of channel

transitions between the closed and open state. We found that the conductance and channel mean

open time were not statistically different from controls after clinostat rotation. However, the

values for junctional AChR channel open time showed a consistent trend toward large values.

This observation may suggest an event of cellular adaptation to clinostat rotation.

Significance of Accomplishments

Our results show that excitable cells are sensitive to alterations in the perceived gravity field.

The function of AChR channels, however, is not drastically affected, possibly due to a process of

cellular adaptation. Taken together, these results indicate that the development of the

neuromuscular junction may be affected during exposure to microgravity. This conjecture needs

further investigation because of the little knowledge and small data base presently available

regarding the effects of microgravity on the function and development of excitable tissues at a

cellular level.

179
STRUCTURE AND FUNCTION OF MAMMALIAN GRAVITY RECEPTORS

Muriel D. Ross

NASA Ames Research Center

Moffett Field, CA 94035

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, my laboratory carries out

ultrastructural studies of gravity sensor organization in ground control, space flown, and

centrifuged rats. Since the basic organization of all mammalian gravity sensors is similar, the rat

can serve as a model species. The ultrastructural research is carried over to computer

reconstruction work so that, eventually, nerve terminal/receptive fields of maculas from both

ground control and space flown rats can be compared for possible differences in size and number

of neuronal processes. For the past year, effort has concentrated on a study of synapses in

utricular maculas of ground control and flight rats of the SLS-1 mission, flown in 1991.

180
Accomplishments

The accomplishments continue to fall naturally under two headings: Results and Theoretical

Interpretations.

Results.

It is necessary to introduce this section by considering a theoretical interpretation of macular

organization that will be taken up again in the following section. This concept is that vestibular

maculas consist of two main circuits, highly channeled and distributed modifying. Type I cells

feed into the highly channeled circuit while type II cells axe part of the distributed modifying

circuit. On the basis of this organization, it was predicted that type II cells of the distributed

modifying circuit would exhibit more synaptic plasticity in an altered gravitational environment

than would type I cells of the highly channeled circuit.

The primary findings of the space flight experiment, analyzed over the past year, are: (1) the

adult mammalian gravity sensor retains the potential for neural plasticity; (2) synaptic changes

are greater in the distributed modifying circuit than in the highly channeled circuit; (3)

microgravity reveals synaptic plasticity in type I cells not observed otherwise; (4) most of the

new synapses are located at new sites; and (5) stress may also cause changes in synaptic number.

These results were obtained by statistical analysis of more than 6,800 synapses in more than

1,000 hair cells. Synapses were counted in blocks of 50 section series obtained from the right

utricular maculas of 3 rats euthanized on launch day (LD), 3 ground control and 3 flight rats

euthanized on the day of Shuttle landing (R + 0), and 3 ground control and 3 flight rats

euthanized 9 days after landing (R + 9). Nine days are allowed for a recovery period (equivalent

to mission length). All animals were shared and, therefore, were subjected to handling and

manipulations unrelated to the vestibular experiment. This caused variabilities in responses that

were interesting but not necessarily related to flight, as will be discussed briefly below.

LD rats exhibited a slight but statistically insignificant rise in the mean number of synapses in

type II hair cells. This was attributed to the modest stress of injections, blood withdrawals, and

weighings conducted before Shuttle launch. R + 0 rats experienced 9 days free of experimental

handling, with the chief difference between flight and control rats being the exposure to space

flight conditions, including Shuttle ascent and descent. At R + 0, synapses were increased by -55

percent in type 11 cells and by ~41percent in type I cells in maculas of flight rats. There was a

shift in the kind of ribbon synapse from the rod-like to the sphere-like form in both types of hair

cells. Additionally, pairs of synapses nearly doubled and there was a twelve-fold increase in

groups of 3 to 6 synapses in type !1 cells. All these changes were statistically significant at the

p > 0.0001 level. Further analysis of the number of synapses distributed as groups in type I1 cells

of flight rats indicated that only a small fraction (9-12 percent) of them could be accounted for in

this way. Thus, most of the new synapses were located at new sites. The findings collectively

indicate that space flight induced a re-weighing of synapses in hair cells of utricular gravity

sensors, although the effects of a possible initiation of readaptation to Earth's 1-g cannot be

discounted at this time.

Surprisingly, at R + 9, synapse counts in type I and type II hair cells were scarcely diminished

from those at R + 0. Additionally, counts in type 1I cells of controls rose to nearly match those of

the flown rats, although counts in type I cells remained essentially unchanged. These findings are

interpreted to indicate that the stress of the many manipulations conducted postflight (injections,

blood withdrawals, weighings) resulted in the synaptic changes observed. While it is of interest

to note that stress may affect synaptic counts in vestibular maculas (another sign of neural

plasticity in an adult mammal), the findings negate any conclusions regarding macular

readaptation to Earth's 1-g following space flight.

181
The questionsraisedby thepresentresultsare:(1) What is the influenceof stresson macular

synapses?(2) Are rats already readaptingto Earth's 1-g by the time they are euthanized

postflight? Theseimportantquestionsshouldbeansweredtotally or in partfollowing theflight

of SLS-2whenrat tissuewill becollectedin spaceaswell aspostflight andwhennoprocedures

will becardedoutontheratsto beusedfor thevestibularstudy.

Theoretical Interpretations.

The findings continue to support the theory that vestibular maculas are organized similarly to

other regions of the nervous system such as the retina, olfactory bulb, and cortex. Our most

recent publications have stressed the point that the vestibular maculas seem to represent a

prototypic neural organization. That is, the maculas have a simple, highly channeled/distributed

modifying circuitry that becomes more complex in the retina and other parts of the brain as more

layers are added. In effect, there is much to learn about more complex neural tissue organization

and functioning by research on vestibular maculas. In this effort, space and centrifugation

studies will continue to play important roles.

Significance of the Accomplishments

Finding 1: Vestibular macular organization is now shown to be comparable to the fundamental

architecture of other more complex neural tissue, such as the retina, olfactory bulb, and

neocortex. This means that maculas are worthy of study as more simple examples of neural

parallel processors of information. The results of my space experiment are the first results to

demonstrate that neural plasticity is a property of the adult mammalian peripheral macula. So far

as I am aware, they are also the f'trst examples of increased neural plasticity in the distributed

modifying circuitry compared to that in the highly channeled circuit. This f'mding may later find

applications in the understanding of memory and learning, particularly in more complex neural

systems. This is because the distributed modifying circuits, also referred to as "local" or

"intrinsic," are coming to the forefront in neuroscientific research because they help to shape the

output of the system. Finally, according to a recent Deafness Research Foundation Report, it is

estimated that more than 90 million Americans over the age of 17 experience some form of

dizziness or balance disorders. Problems with balance are responsible for more than one-half of

the accidental deaths in the elderly (because of failing). Additionally, it is estimated that, in the

United States alone, more than 500,000 individuals suffer from Mtni&e's disease, a disorder of

the inner ear that affects the balance organs as well as hearing. The number of children

experiencing some form of motion sickness or inner ear distress that impairs travel and physical

activity is doubtless large but was not included in these estimates. The results of my space

research could eventually help explain some of the causality of these serious afflictions,

especially if further research should indicate that stress and changes in mobility are factors

influencing synaptic efficacy in the peripheral organs of balance.

Publications

Chimento, T.C., Doshay, D., and Ross, M.D. 1993. Compartmental modeling of macular

primary neuron branch processes. In: Analysis and Modeling of Neural Systems 2 (Eeckman, F.,

Ed.). Norwell, MA, Kluwer Academic Publishers, pp. 355-363.

Montgomery, K. and Ross, M.D. 1993. A method for semiautomated serial-section

reconstruction and visualization of neural tissue from TEM images. In: Biomedical Image

Processing and Biomedical Visualization (Ascharya, R.J. and Goldgof, D.B., Eds. ). Salem, MA,

Proceedings SPIE, pp. 114-120.

182
Ross,M.D. 1992. Synapticchangesin gravity sensorsof space-flownanimals.Proceedings of

the 14th Annual International Conference of the IEEE Engineering in Medicine and Biology

Society 4: 1304-1305.

Ross, M.D. 1993. Morphological changes in the rat vestibular system following weightlessness.

Vestibular Research 3(3): 241-25 I.

Ross, M.D. 1993. Neuroanatomy of neural networks of the vestibular system: Vestibular

maculas as prototypic neuronal networks. Vol. II, Proceedings 1993 IEEE International

Conference on Neural Networks, pp. 642-647.

Ross, M.D., Chimento, T., Doshay, D., and Cheng, R. 1992. Computer-assisted reconstruction

and simulations of vestibular macular neural connectivities. In: Sensing and Controlling Motion

(Cohen, B., Tomko, D., and Guedry, F., Eds.). New York, Annals of the New York Academy of

Sciences, pp. 75-91.

Ross, M.D., Cutler, L., Cheng, R., Doshay, D., Naddaf, A., and Chimento, T.C. 1992. The Role

of 3-D Reconstruction in the Analysis and Modeling of Neural Systems. In: Analysis and

Modeling of Neural Systems (Eeckman, F.H., Ed.). Kluwer Academic Publishers, pp. 93-99.

Ross, M.D. and Doshay, D.G. 1993. Vestibular macular circuitry: A prototypic neural

architecture. World Congress on Neural Networks. Hillsdale, NJ, Lawrence Erlbaum Associates,

Inc., and INNS Press, pp. I/-598----II-601.

183
OSTEOBLASTIC RESPONSE IN TRANSGENIC MICE

David Rowe

Department of Pediatrics

University of Connecticut

Framington, CT 06030

Description of Research

Although it is intuitive that the skeleton has evolved mechanisms to sense changes in mechanical

stress and to transduce these physical signals into a biological response that alters the structural

properties of bone, this complex process has evaded analysis at the molecular level. The effect of

mechanical strain on bone has been resistant to molecular analysis because it can be assessed only

in intact animals. This grant will develop an in vivo model to delineate molecular mechanism for

the transduction of mechanical signals in bone to a transcriptional event in osteoblasts. It will

explore the feasibility of using transgenic mice harboring DNA constructs that reflect the activity of

the a 1(I) collagen (COL 1A 1) promoter in bone as a method to probe molecular mechanisms that

alter type I collagen production in response to varying degrees of mechanical stress. The rationale

is based on the increased realization that molecular techniques of promoter analysis in a setting of a

highly differentiated cell, such as an osteoblast under mechanical stress, is not accurately mirrored

by cells cultured from intact tissue. Instead, the molecular tools that have been used in'cultured

cells must be modified to approach questions that can be asked only with respect to the intact

animal.

In our first series of experiments, we wanted to demonstrate that osteoblasts carrying the transgene

would reflect increased activity in an animal experiencing increased weight. Because the models

for bone loading in normal mice required development in our laboratory, we first turned our

attention to murine model of the human disease, osteogenesis imperfecta mouse (OIM). The OIM

produces a weakened bone matrix that does not provide adequate resistance to mechanical loading.

In this context, the OIM would exemplify chronic weighting of bone. The collagen

chloramphenicol acetyl transferase (CAT) transgene was bred into the OIM. Much to our surprise,

mice of 8 days of age did not show increased CAT activity relative to normal wild type litter mates.

At one month of age, the same observation persisted. However, by 3 months of age when the

rapid phase of somatic growth had ceased, CAT activity was 2-3 times higher in both calvarial and

humeral bone extracts relative to the normal mice. The relative increase in CAT activity was

paralleled by the content ofCOL1A1 mRNA extracted from the tissue. This work was reported in

a plenary session at the Space Biology meetings this year and is being assembled for publication.

Accomplishments and Significance of the Accomplishments

The analysis of the OIM made a number of useful points. First, as predicted by Frost, factors that

control bone formation during rapid growth are probably different from those which control bone

remodeling due to mechanical loading. I believe that the osteoblasts are maximally stimulated

during the rapid phase of somatic growth and that further activity due to mechanical loading is

obscured. Because of these studies, all our subsequent work examining the effect of mechanical

loading will be carried out in mice that are over 3 months of age.

Analysis of bone taken from normal transgertic animals loaded by sciatic or pateilar tendon scission

failed to show a significant difference in CAT activity between the weighted and unweighted side,

even after the animals were examined two weeks after the manipulation. Reports by others have

shown that in rats it is the cortical bone cells which respond to mechanical loading. Based on that

observation, we have started to examine enzymatic digest of bone for CAT activity. This has

184
showndramaticdifferencein CAT activity between the weighted and unweighted side (ten-fold)

when cortical cells are examined. Differences in the residual bone or cells of the trabecular

compartment have not shown such dramatic changes. At this point, we do not know if the

increased activity is due to more osteoblastic cells or increased CAT activity in the same number of

osteoblastic cells.

An important aspect to this project, particularly as related to the population of bone cells that are

responding to mechanical loading, is the histomorphometric analysis using CAT

immunofluoresence. The initial histological work that formed the body of the project was carded

out in fetal rat calvaria which had not yet undergone significant calcification. When applying the

immunostaining to adult bone, significant background problems were encountered. This appears

to preclude the ability to do quantitative assessment of CAT activity. However, recendy this

problem appears to be successfully resolved by Dr. Gronowicz, when she changed to a newly

developed fluorochrome.

Important progress has been made by members of our bone research group in other aspects of the

collagen promoter analysis that is relevant to the success of the current proposal. Finer deletions of

the collagen CAT construct have now delineated a region of about 40 bases that is required for

activity of the transgene in osteoblastic cells. This and other experiments have lead us to believe

that a bone enhancer element exists that is distinct from elements in other type I collagen producing

cells (e.g., tendon and periosteal fibroblasts and vascular smooth muscle cells) and is active only

whenthe osteoblast has assumed its most differentiated state in vivo. Even more exciting taas been

the identification of a nuclear factor that binds to this bone element. The factor is present only

when obtained from freshly isolated, highly differentiated bone cells. It is not present in cultured

bone cells. If this new line of work continues to bare positive results, it will eventually provide a

marker for a highly differentiated bone cell and be a likely candidate for transducing mechanical

loading to an osteoblastic response.

Publications

Bedalov, A., Breault, D.T., Sokolov, B.P., Lichtler, A.C., Bedalov, C., Clark, S.H., Mack, K.,

Khillan, J.S., Woody, C.O., Kream, B.E. and Rowe, D.W. 1993. Regulation of the al(I)

collagen promoter in vascular smooth muscle cells: Comparison with other a l(I) collagen

producing cells in transgenic animals and cultured cells. American Heart Association National

Meeting, Adanta, GA.

Bedalov, A., Salvatori, R., Kronenberg, M., Kapural B., Mack, K., Clark, S., Woody, C.,

Kream, B., Rowe, D., and Lichtler, A. 1993. Tissue specific expression of the rat COL1A1

promoter in transgenic mice: Identification of a 50 bp region required for transgene expression in

bone (Abstract). Journal of Bone and Mineral Research 8: S 119.

Bogdanovic, Z., Harrison, J.R., LaFrancis, D., Bedalov, A., Woody, C.O., Lichder, A.C.,

Rowe, D.W., and Kream, B.E. 1993. Parathyroid hormone regulation of the _1(I) collagen

promoter and NF-IL6 expression in transgenic mouse calvariae (Abstract). Journal of Bone and

Mineral Research 8: S 190.

Breault, D.T., Bedalov A., Harrison, J., Fall, P., Rumboldt, Z., Mack, K., Clark, S., Lichtler,

A., and Rowe, D. 1993. A new generation of CAT expression vectors harboring the al(I)

collagen promoter shows increased activity in collagen producing tissues from transgenic mice and

in MC3T3E1 and PY1A cells (Abstract). Journal of Bone andMineral Research 8: $316.

185
Chipman, S.D., Sweet, H.O., McBride, D.J., Davisson, M., Marks, S.C., Shuldiner, A.R.,

Wenstrup, R.J., Rowe, D.W., and Shapiro, J.R. 1993. Defective proa2(I) collagen synthesis in

a recessive mutation in mice: A potential model of human osteogenesis imperfecta. Proceedings of

the National Academy of Sciences of the United States of America 90: 1701-1705.

Kream, B.E., LaFrancis, D., Petersen, D.N., Woody, C., Clark, S., Rowe, D.W., and Lichtler,

A. 1993. Parathyroid hormone represses _tl(I) collagen promoter activity in cultured calvariae

from neonatal transgenic mice. Molecular Endocrinology 7: 399-408.

Krebsbach, P.H., Harrison, J.R., Lichtler, A.C., Woody, C.O., Rowe, D.W., and Kream, B.E.

1993. Transgenic expression of COL1A1-CAT fusion genes in bone: differential utilization of

promoter elements in vivo and in cultured cells. Molecular and Cellular Biology 13: 5168-5174.

Terzic, J., Mack, K., Rumboldt, Z., McBride, D., Shapiro, J., Rowe, D., and Clark, S. 1993.

Collagen transgene expression in an osteogenesis imperfecta mouse. Annual Meeting of the

American Society for Gravitational and Space Biology, Reston, VA.

186
MECHANISMS OF ALTERED LYMPHOCYTE

MICROGRAVITY AND HYPERGRAVITY

Clarence F. Sams

NASA Johnson Space Center

Mail Code SD4

Houston, TX 77058

ACTIVATION IN SIMULATED

Description of Research

Lymphocytes have been repeatedly studied during space flig.ht and have consistently exhibited an

apparent gravitational influence on in vitro activation by mltogenic lectins. An unresolved issue

concerning the interpretation of the currently available flight data is whether the observed effects

are a result of the direct effect of gravity on cellular systems or indirect effects, such as changes in

nutrient transfer or cell contact. Direct inhibition of the proliferative response may occur at any one

of the many events regulating the progression through the activation cascade. In addition, in vitro

lymphocyte activation is sensitive to the specifics of the cell culture environment including such

aspects as the effective cell concentration and biochemical transfer. Some of these characteristics

may be altered during culture in non-unit gravity. The objective of this investigation is to examine

these alternatives and determine the mechanisms responsible for the gravity sensitivity of in vitro

lymphocyte activation.

We hypothesize that gravity influences lymphocyte activation through one or both of the following

general mechanisms: (1) alteration of the cell culture environment (e.g., cell-cell and cell-substrate

contact, mass transfer), or (2) changes in the specific elements of the activation cascade (e.g.,

signal transduction, transcriptional regulation). To test this hypothesis, lymphocyte activation will

be characterized as a function of gravity and specific aspects of the cell culture environment.

Lymphocytes will be activated under static unit gravity, hypergravity (in a cell culture centrifuge),

hypogravity (in a clinostat), and unit gravity suspended culture (gentle stirring). Response to each

altered gravity condition will be assessed as a function of cell density, cell aggregation and mitogen

type (surface or transmembrane active). The progression of activation events will be determined as

a function of gravity and culture condition. The activation sequence will be characterized by

assessing the production of second messengers, transcription of cellular oncogenes, production of

cytokines, and expression of specific cell surface receptors.

Accomplishments

(1) A major concern in the interpretation of space flight experiments with cultured cells is

the influence of indirect environmental effects on the alterattons observed. Factors such as changes

in the effective cell concentration and an altered degree of cell contact can have major effects on the

proliferative response at specific cell concentrations. Exposure to altered gravity levels will cause

similar effects in that microgravity will support the continued dispersion of cells, while

hypergravity will promote cell and surface contact and an increase in the apparent cell

concentration. These factors may contribute significantly to the observed gravity sensitivity of in

vitro lymphocyte function.

(2) Previous reports have documented a significant decrease in mitogen activation of

lymphocytes during culture in clinostats. These results have been attributed to a reduction of the

apparent gravity on the cells to approximately 10 -2 g or less by the clinorotation at appropriate

speeds. An alternative explanation is that clinostats evenly disperse the cells and interfere with the

cell-cell and cell-surface interactions required for mitogen-induced activation. To test whether

simple dispersion was sufficient to inhibit activation, we treated lymphocytes with a mitogen while

187
cells wereeitherstatic,in a clinostat,or maintained in suspension on a hematology rocker. The

hematology rocker provided gentle suspension without the microgravity simulation of

clinorotation. Activation was monitored by the appearance of the CD69 activation marker which

appears within a few hours after stimulation with mitogens and is not expressed on resting

peripheral blood lymphocytes. Suspending the ceils caused a 60-80 percent decrease in the

percentage of T cells expressing the CD69 marker 24 hours after addition of mitogen (Figure 1).

Tritiated thymidine uptake at 72 hours was also decreased by 65-80 percent compared to the static

control (Figure 2). No decrease in the viability of the suspended sample was observed as

determined by propidium iodide staining and flow cytometry. These results suggest that

suspension/dispersion of the cells is a significant element in the decreased responsiveness observed

in clinostats. In addition, these data indicate that early events in the activation sequence are affected

by changes in culture environment when surface-active mitogens are used. Overall, these data are

consistent with the observation that very few lymphoblasts (enlarged cells which have progressed

to the G l phase of the cell cycle) are observed after stimulation with mitogens during space flight.

IO0

Figure 1

Slatlc Rocker CIInostet

Sample

Figure 1. Effects of suspension on activation of peripheral lymphocytes. Lymplx)cytes were

incubated with a mitogen (PHA) for 24 hours in static culture, suspended in a hematology rocker, or

in a clinostat. The black bars represent the activated samples and the open bats the control (without

mitogen). Data are represented as the percentage of T cells expressing the CD69 activation marker.

120

Figure 2

Control Rocker C|Inoste!

Sample

Figure 2. Effects of suspension in lymphocyte proliferation. Lymphocytes were treated with

mitogen for 72 hours and pulsed with radio-labeled thymidine. The data for the rocket and clinostat

are relxesented as a percentage of control (static) incorporation.

188
(3) Phorbolesters function as direct activators of protein kinase C and, in conjunction with

appropriate accessory signals, are effective activators of peripheral lymphocytes. These

compounds bypass the initial cell surface-mediated signaling events and provide an alternate means

for activating the cells. Our preliminary studies have shown that treatment of lymphocytes with

phorbol myristate acetate (PMA) induced similar expression of CD69 on the T cells, whether static

or suspended. The lack of sensitivity to the degree of cell dispersion observed with PMA indicates

that bypassing the receptor-mediated signaling mechanisms allows the initiation of early activation

events in the suspended state. These results further support the hypothesis that the early surfacemediated events may be critical determinants in the apparent gravity sensitivity of lymphocyte

activation. Mitogens which bypass these events are equally effective at inducing specific early

activation markers whether in suspended or static culture. Therefore, these mitogens will be useful

tools for dissecting the gravity sensitive steps in activation sequence. These data appear to contrast

with space flight experiments which indicated that PMA was ineffective at inducing ILl and IL2

production in monocyte and T cell lines during microgravity culture. This disparity may be due to

(1) differences between peripheral lymphocytes and the cell lines, (2) specific inhibition points

distal to those regulating CD69 expression but prior to interleukin production, or (3) additional

direct effects in actual microgravity. Further experimentation will be necessary to resolve these

differences.

Significance of the Accomplishments

From the above results, it is apparent that environmental or indirect effects have substantial impact

on mitogen activation and may account for a significant portion of the observed effects in clinostats

or microgravity. These effects can be observed at very early points during the sequence of

activation events and are different for surface-active mitogens and transmembrane-active mitogens.

Overall, the preliminary data confirm the validity of examining the activation sequence with

different mitogens and Culture conditions to determine specific cellular mechanisms which are

potentially responsive to direct or indirect effects of altered gravity.

This investigation will improve the understanding of environmental and gravitational influences on

cells in culture. In addition, it will provide basic data to permit the isolation of indirect gravity

effects from those cellular responses which are directly altered or influenced by gravity. This

information will be generally applicable to other cell systems and will provide basic information

concerning the effects of gravity on living organisms. Results obtained from these studies will

provide a firm scientific basis for the design and interpretation of future flight experiments and the

development of more detailed hypotheses concerning gravitational biology.

189
DEVELOPMENT, MATURATION

PITUITARY-GONADAL AXIS OF

Martin P. Schreibman

Department of Biology

Brooklyn College, City University

Brooklyn, New York 11210

AND PHYSIOLOGY OF THE BRAINFISH IN THE CEBAS/AQUARACK SYSTEM

of New York

Description of Research

Our project identifies with NASA's Space and Gravitational Biology Research Program goal to

determine the effect of long-term space flight and exposure to reduced gravity on the regulatory

mechanisms controlling and affecting the structure and function of endocrine systems. It is an

integral part of the German space program's Closed Equilibrated Biological Ao#atic System

(CEBAS)/Aquarack (C/A) Scientific Frame Program. C/A is a long-term, multigeneration

experimental system for aquatic organisms which is used in a frame program that involves German

and United States Universities and the DLR Institute of Aerospace Medicine.

The long-range goal of our project is to study

the effects of space flight conditions,

including microgravity and space radiation,

on the development, function, and regulation

of the reproductive system, specifically the

brain-pituitary-gonad (BPG) axis in aquatic

vertebrates of different age and gender in the

frame of the C/A project. The BPG axis is

the primary internal vertebrate control system

for reproduction. The biology of the

reproductive system will be analyzed by

evaluating changes in the synthesis, storage,

and release of neurotransmitters,

neurohormones, and pituitary hormones in

late embryonic, neonatal, immature, and adult

animals using immunocytochemistry, in situ

hybridization, and morphometry.

Additionally, specific sensory organs will be

studied because they are the first-line contacts

between the organism and its environment.

They can monitor the environmental

conditions of space and transmit information

back to centers in the brain which regulate the

BPG axis. This relationship is depicted in

Figure I. The swordtail (Xiphophorus

helleri), a freshwater teleost, has been

designated as the experimental vertebrate

model in the C/A Scientific Frame Program.

eye p_neal ollacto_/ cutaneous

i PNS)

Z

0

•I" o

2° Targets Gametes

Figure 1. This figure depicts the structure and functional relationship between sensory organs, the monitors of

environmental signals, and the neuroendocrine system that regulates reproductive organ development and function.

190
Thisprojectis partof a broad,joint internationalprogramof zoologicalresearchdedicatedto the

studyof reproduction,amongothertopics.Our studiescollectessential,fundamentalinformation

onthereproductivebiology of Xiphophorus so as to establish a comprehensive data base that will

permit the correlation of physiological and morphological parameters for proper evaluation of

future space flight and space station experiments.

Accomplishments

(1) We have made considerable progress in preparing a data bank for the times of

appearance and sites of localization of a variety of neuropeptides and neurotransmitters in two

closely related species of Xiphophorus, X. helleri (swordtail) and X. maculatus (platyfish). We

have reported previously the localization of some two dozen neuroregulatory peptides and

neurotransmitters in the brain and pituitary gland of Xiphophorus. More recently, we have

selected five of these neurohumors (dynorphin, FMRF-amide, neurotensin, neuropeptide Y,

galanin) for detailed analysis during the developmental period that spans late embryonic

development to sexual maturity. The data derived suggest that specific neurotransmitters and

neuroregulatory peptides are involved in the maturation of the reproductive system and that they

may possibly play important roles in communicating information between peripheral sensory

organs and the control systems for reproduction. This is suggested by their appearances during

key developmental periods in important anatomical sites in the brain and pituitary gland.

(2) We have also localized, by immunocytochemical methods, various forms of

gonadotropin releasing hormone (GnRH) [mammalian (m), chicken II (chlI), salmon (s) and

lamprey (1)] in the brain and pituitary gland of Xiphophorus at various developmental stages from

birth to sexual maturity. GnRH is responsible for controlling the synthesis and release of

gonadotropin (GTH) from the pituitary gland, a hormone that regulates gonadal development and

function. There are now two recognized forms of GTH, Beta I and Beta II. We have found that

GTH I and sGnRH are present together early in Xiphophorus development and that the onset of

sexual maturation is marked by the appearance of GTH I1 and the chlI form of GnRH. Our results

demonstrate that variant forms of GnRH and GTH are present at defined stages of development in

specific regions of the brain and pituitary gland and suggest that different forms of GnRN and

GTH regulate different aspects of reproductive system development and physiology.

Significance of the Accomplishments

The identification of neuroregulatory peptides, neurotransmitters, and pituitary hormones at key

stages of development is critical to our fundamental understanding of reproductive system

maturation and function, the control systems that orchestrate these processes, and how they may be

affected by conditions in space.

The data we are gathering are absolutely essential if we are to recognize and correctly interpret the

influence of hypogravity and other environmental conditions of space flight on the neuroendocrine

regulation of the reproductive system. This data base of morphological events will enable us to

evaluate and understand the effects of microgravity and other conditions of space flight.

Publications

Bluem, V., Breukmann, A., Schreibman, M.P., and Timmers, R. 1992. C.E.B.A.S.-Aquarack

scientific frame program: In vitro steroidogenesis in female Xiphophorus helleri brain and pituitary

tissue. ASGSB Bulletin 6: 64.

Bluem, V.B., Bungart, S., Schreibman, M.P., and Timmers, R. 1991. C.E.B.A.S.-Aquarack

scientific frame program: in vitro studies on the steroidogenic potency of Xiphophorus helleri

testes (Abstract). ASGSB Bulletin 5: 65.

191
Cepriano,L., andSchreibman,M.P. 1991. Immunoreactive-neurotensinand -galanin in the

pituitaryglandandbrainof two species of Xiphophorus (Abstract). Society for Neuroscience 17:

419.

Magliulo-Cepriano, L. and Schreibman, M.P. 1991. Neurotensin (NT), Galanin (GAL), and

FMRF-amide in the brain and pituitary gland of Xiphophorus (Abstract). American Zoologist 31:

19A.

Magliulo-Cepriano, L. and Schreibman, M.P. 1992. Localization of androgen receptors in the

brain and pituitary gland ofXiphophorus maculatus (Abstract). Society for Neuroscience 18: 97.

Magliulo-Cepriano, L. and Schreibman, M.P. 1993. The distribution neuropeptide Y and

dynorphin immunoreactivity in the brain and pituitary gland of Xiphophorus maculatus, from birth

to sexual maturity. Cell and Tissue Research 271: 8 7-92.

Magliulo-Cepriano, L., Schreibman, M.P., and Bluem, V. 1993. Neurotensin, galanin and

FMRF-amide during development in Xiphophorus. General and Comparative Endocrinology 92:

269-280.

Schreibman, M.P., Cepriano, L.M., and Bluem, V. 1992. CEBAS-Aquarack scientific frame

program: Localization of androgen receptors in the brain and pituitary gland of Xiphophorus.

ASGSB Bulletin 6:114.

Schreibman, M.P., Magliulo-Cepriano, L. and Bluem, V.B. 1991. CEBAS-Aquaxack scientific

frame program: Neuropeptides in Xiphophorus; Neurotensin, galanin and FMRF-amide in the

brain and pituitary gland (Abstract). ASGSB Bulletin 5: 65.

192
EFFECTS OF HINDLIMB SUSPENSION ON SKELETAL MUSCLE GROWTH

Edward Schultz

Department of Anatomy

University of Wisconsin

Madison, WI 53706

Description of Research

Studies are focused on how the weightless environment, induced by hindlimb suspension (HS),

alters satellite cell behavior in immature growing animals. Satellite cells are very important

during skeletal muscle growth because they are cells associated with myofibers that are

responsible for the increase in myonuclei that accompanies myofiber growth. This myonuclear

increase is accomplished by the continued mitotic activity of satellite cells during the postnatal

growth period and the subsequent fusion of the cells with the enlarging myofibers. A second

important function of the cells is to provide a source of myoblasts during a regeneration response

following injury. In this sense they are regarded as the stem cells of muscle regeneration. Each

of the described functions of satellite cells in normal or injured muscle is dependent upon mitotic

divisions of the cells to provide progeny for growth or regeneration. The long-term goal of our

studies is to understand the manner in which the proliferative beh_ivior of satellite cells in

immature muscles, either in the intact growing system or during muscle regeneration, is altered

by the weightless environment.

Our previous studies indicated that when young animals are placed in HS, muscle growth is

markedly retarded. A prominent concurrent feature is a severe reduction in the proliferative

activity of satellite cells, which is most pronounced in the soleus muscle. The reduced mitotic

activity of satellite cells is sustained and appears to parallel the relative atrophy or reduced

growth exhibited by the soleus muscle and the less severely affected extensor digitorum longus

(EDL) muscle. The reduction in satellite cell mitotic activity occurs quickly after initiation of

HS, suggesting the cells in both the soleus and EDL muscles respond to the HS environment

before any morphological atrophic changes are evident in the muscles. The goal of our most

recent studies is to examine the early response of satellite cells, focusing on the period of HS

when single injections of bromodeoxyuridine (BrdU) indicate that no mitotic divisions are taking

place. A second goal is to initiate studies to examine the behavior of satellite cells during a

regeneration response in the HS environment.

Accomplishments

Immature growing rats were placed in HS for a total period of 5 days. After 3 days, the animals

were anesthetized and removed from HS. An incision was made just proximal to the base of the

tail to place an Alzet miniosmotic pump filled with BrdU (25 mg/ml) subcutaneously, parallel to

the vertebral column. The pump had been previously incubated in saline at 37 * C in order to

initiate pumping. The animals were returned to HS. At the end of the fifth day, the animals were

removed from HS and euthanized. The soleus and EDL muscles were removed and

immunostained for the presence of BrdU-labeled nuclei. The continuous infusion paradigm was

chosen because it gives a cumulative account of all mitotic activity over the duration of the

experimental period. Inspection of transverse paraffin sections for BrdU-labeled nuclei

demonstrated that control soleus muscles had abundant labeled nuclei (i.e., 86.4+18.5/unit area),

whereas HS soleus muscles had significantly fewer, i.e., 7.3:l:l.1/unit area (p< 0.01). Virtually

all labeled cells present in the HS muscles were found in the perimysium while few were found

immediately adjacent to muscle fiber peripheries. Labeling in the HS-EDL was also greatly

reduced compared to weightbearing control but not to the extent seen in the soleus. These results

add further evidence that the unweighted environment has a profound effect on the development

193
of immature muscles, producing a complete cessation of growth (in terms of nuclear accretion)

that starts within hours of initiation of HS. The results of the continuous infusion experiment

also shed light on the reduction in the number of satellite cells that was observed in EM studies.

After HS is initiated, radial growth of the soleus fibers is reduced, whereas growth in length

continues, leading to a small increase in the volume of the fibers. Presumably, the supply of

satellite daughter cells is smaller than the demand for myonuclei in the slowly growing muscles.

Continued generation of myonuclei from fusion competent satellite cells in the absence of

additional mitotic divisions would serve to deplete the population. In the EDL, there is a

reduction in the number of satellite cells at 3 days, but the reduction is never as severe as in the

soleus because division of satellite cells is never completely stopped in this muscle.

Studies of muscle regeneration have shown that the HS environment also reduces the quality of

the regeneration response. A regeneration response was induced by free-grafting the muscle or

by injecting the myotoxic snake venom Notexin. The muscles (soleus or EDL) of suspended

and weightbearing rats were removed and studied after 10-days. During the 10-day experimental

period, the rats were given BrdU injections every 12 hours. The results of these studies

demonstrated that muscle weights of the regenerated soleus muscle were significantly less than

weightbeadng controls at the end of the experimental period. The weights of the EDL muscles

of suspended animals were not significantly different than weightbearing controls. The muscle

weight results were mirrored by fiber diameters of the muscles and their respective controls; the

regenerated soleus myofibers were significantly smaller than the regenerated fibers in

weight-bearing animals. We are examining if a change in the proliferative behavior occurs

during the early phase of the regeneration response and contributes to the reduction in muscle

mass seen at 10 days. An examination of the quantity of labeled nuclei in the regenerated

muscles after multiple injections of BrdU is currently being carded out by counting the number

of immunostained BrdU positive nuclei in paraffin sections or by measuring total fluorescence.

Preliminary results suggest that there is no difference in the number of labeled nuclei in the

regenerated muscles of suspended and weightbearing muscles. The tentative conclusion is that

although satellite cell divisions are significantly reduced in intact muscles, particularly the

soleus, mitoses of satellite cells are not influenced by the weightless environment m regenerating

muscles. We speculate that it is not until myofibers are formed and start to grow that differences

in the suspended and weightbearing muscles are produced. Thus, any differences seen in

regenerating muscles are not related to the early proliferative phase but more likely occur after

fiber formation and innervation. Confirmation of this awaits further analysis.

Finally, over the past year, we have developed and described a simple apparatus for suspending

rats. The importance of the construction is that all the materials are readily available at any local

hardware store, which means the cages can be inexpensively constructed without the need for

custom work. The cages can be easily dismantled for cleaning and storage. The cages utilize an

apparatus that allows the animal to reach all areas of the cage, yet prevents them from resting

their hindlimbs against the sides or end panels.

Significance of the Accomplishments

Our results suggest that unloading a growing muscle has a profound and rapid influence on

satellite cell proliferative and fusion behavior within hours of the initiation of HS. How placing

a myofiber in an unweighted environment induces such rapid changes in satellite cell behavior is

unknown. There are no junctional complexes between satellite cells and myofibers that would

permit rapid signaling between them. In addition, behavioral changes of satellite cells in both

damaged and HS muscles are so rapid that the mechanism of activation cannot be easily

attributed to diffusion-related changes in the levels of regulatory factors from their myofibers.

One possibility is that cells in undamaged portions of injured muscles or throughout HS muscles

respond to changes in electrical activity of myofibers. Electrical activity of soleus muscles in HS

animals undergoes a significant and sudden decrease (-91percent) at the initiation of HS and

194
exhibits a slow recoverythroughday7 of HS, whenit achieves approximately 80 percent of

control values. The proliferative behavior of satellite cells parallels the electrical changes in

soleus muscles, but whether this alteration in electromyographic (EMG) activity of the soleus has

an influence on the behavior of dividing satellite cells or the changes are coincidental and

independent of one another remains unknown. However, because muscles of the anterior

compartment, such as the tibialis anterior (and probably the EDL), do not exhibit a similar

decrement in EMG activity but show parallel changes in satellite cell mitotic activity argues

against behavioral changes being induced by altered electrical activity patterns.

In summary, changes in the proliferative behavior in satellite cells of intact muscles that occur

within hours of the initiation of HS are a good indicator of future atrophic changes that will occur

in the muscles. Satellite cell proliferative activity is most severely affected in soleus muscles

where all mitoses are stopped between 3 and 5 days, and overall proliferative activity is

suppressed for as long as HS is maintained. These results suggest that the ability of growing

muscles to recover from even short periods of unweighting may be seriously compromised.

The situation is slightly different in regenerating muscles. The results of our studies suggest the

following scenario. When a myofiber is damaged, the ability of satellite cells to initiate a repair

response to replace that fiber appears not to be compromised by the unweighted environment.

That may be because the levels of mitogens that are thought to activate the cells into a

prolife.rative phase are not reduced in unweighted muscles. The proliferative response of the

cells is sufficient to form the appropriate number of myofibers. It is not until the myofibers are

formed (with their population of satellite cells) that the unweighted environment influences the

course of the regeneration response. Control of further satellite cell proliferative behavior is

regulated by the fibers. However, as is seen in intact muscle, because the myofibers are not

growing, satellite cells are not maintained in a proliferating mode. The net result is that

regeneration is reduced.

In summary, the effect of unweighting on dividing satellite cells in intact and regenerating

muscles is not the same. The variance in the response of satellite cells may be related to the

manner in which the proliferative behavior of the cells is regulated during these processes.

Publications

Danko, I., Fritz, J.D., Latendresse, J.S., Herweijer, H., Schultz, E., and Wolff, L.A. 1993.

Dystrophin expression improves myofiber survival in mdx muscle following intramuscular

plasmid DNA injection. Human Molecular Genetics 2: 2055-2061.

Jiao, S., Schultz, E., and Wolff, J.A. 1992. Intracerebral transplants of primary muscle cells: A

potential "platform" for transgene expression in the brain. Brain Research 575: 143-147.

Jiao, S., Williams, P., Safda, N., Schultz, E., and Wolff, J.A. 1993. Co-transplantation of

plasmid-transfected myoblasts and myotubes into rat brains enables high levels of gene

expression long-term. Cell Transplantation 2: 185-192.

Krabbenhoft, K.M., Lyons, G.E., and Schultz, R. 1993. Gene expression during muscle

regeneration and the effect of denervation (Abstract). Molecular and Cell Biology 4: 385a.

McCormick, K.M. and Schultz, E. 1992. Mechanisms of nascent fiber formation during avian

skeletal muscle hypertrophy. Developmental Biology 150:319-334.

Mozdziak, P.E., Schultz, E., and Cassens, R.G. 1993. Mechanism of turkey post-hatch muscle

growth (Abstract). Journal of Animal Science 71: 128.

195
Mozdziak,EE., Schultz,E., andCassens,R.G. 1993. Use of a double-labeling technique and

image analysis to quantify satellite cell proliferative activity (Abstract). Poultry Science 72:110.

Park, E. and Schultz, E. 1993. A simple hindlimb suspension apparatus. Aviation Space and

Environmental Medicine 64:401-404.

Schultz, E. 1992. Satellite cell properties which determine skeletal muscle regeneration

potential (Abstract). European Journal of Cell Biology 57: 72.

Schultz, E. 1992. Satellite cells: Basis for skeletal muscle growth and regeneration (Abstract).

FASEB Journal.

196
GROWTH FACTOR INVOLVEMENT IN TENSION-INDUCED

GROWTH

Herman H. Vandenburgh

Department of Pathology

Brown University Program

The Miriam Hospital

Providence, RI 02906

in Medicine

MUSCLE

Description of Research

Long-term manned space travel will require a better understanding of skeletal and cardiac muscle

adaptations which result from microgravity. Astronaut strength and dexterity must be maintained

for normal mission operations and for emergency situations. Although exercise in space slows the

rate of muscle loss, it does not prevent it. A biochemical understanding of how gravity, tension,

and exercise help to maintain muscle size by altering protein synthesis and/or degradation rate

should ul_nately allow pharmacological intervention to prevent muscle atrophy in microgravity.

The overall objective of this research project is to examine some of the basic biochemical processes

involved in tension-induced muscle growth. Differentiated avian skeletal myofibers and rat cardiac

myocytes can be "exercised" in tissue culture using dynamic mechanical cell stimulator devices

which simulate different muscle activity patterns. We have found patterns of mechanical activity

which significantly stimulate muscle growth, and organogenesis and alter myofiber metabolic

characteristics. With this experimental in vitro system, we are examining the interaction of

exogenous and endogenous muscle growth factors in mechanically stimulated muscle growth.

Exogenous growth factors found in serum, such as insulin, insulin-like growth factors (IGF), and

glucocorticoids modulate muscle protein turnover rates in mechanically induced muscle growth

in vitro. Endogenous growth factors, such as the prostaglandins, are synthesized and released into

the culture medium when muscle ceils are mechanically stimulated. This lipid-related family of

endogenous factors helps to regulate the rates of protein turnover in the muscle ceils.

Studies on the mechanism by which muscle mechanical activity interacts with both exogenous and

endogenous growth factors to regulate muscle protein turnover rates in these model systems will be

continued.

Accomplishments

(1) Organogenesis of single skeletal muscle cells into functional muscle organs can be

mechanically induced in tissue culture (Figure 1).

(2) The glucocorticoid dexamethasone induces atrophy of skeletal myofibers, and

mechanical stimulation reduces this atrophic response. Mechanical activity reduces muscle atrophy

by stimulating the production of the anabolic growth factor prostaglandin F2a. Prostaglandin

production is increased by stretch activation of phospholipases, which provide arachidonic acid for

prostaglandin synthesis and by stretch activation of prostaglandin synthase, the enzyme which

converts arachidonic acid into prostaglandin (Figure 2).

(3) Neonatal and adult rat cardiac cells can be mechanically stimulated in our tissue culture

model systems in a similar fashion to skeletal muscle ceils.

197
MECHANOGENESIS OF SKELETAL MUSCLE ORGANS

Figure 1. Computer-aided mechanical generation of artificial skeletal muscle organs have in vivo-like characteristics

including parallel network of long unbranched myofibers (upper left), well-organized sarcomeres (upper right),

primary and secondary-like myofibers (lower left), and concentration of the tendon-associated protein tenascin at the

end of the organ (lower right). Reprinted from the cover of FASE8 Journal 5, 1991.

GLUCOCORTICOIDS

EXERCISE/UUSCLESTRETCH

INCRF..ASED!CTIVITYOF

PHOSPHOUPASESPROSTAGLANDIN SYNTHASE

INCREASEDPROSTAGLANDIS > [ I

MUSCULARATROPHY

Figure 2. Proposed mechanism by which stretch/exercise attenuates glucocorticoid induced skeletal muscle atrophy.

198
Significance of Accomplishments

(1) Mechanical/tension/gravity effects on the organogenesis of tissues can be studied with

our mechanical cell stimulator devices.

(2) The skeletal muscle atrophying effects of the stress-related glucocorticoids are reduced

by exercise/mechanical stimulation, and this occurs by stretch-activation of anabolic prostaglandin

production.

(3) Stress/pressure/mechanical regulation of cardiac growth or atrophy can be studied in

our mechanical cell stimulators.

Publications

Chromiak, J.A. and Vandenburgh, H.H. 1991. Mechanical stimulation of cultured skeletal

muscle reduces glucocorticoid-induced decreases in myosin heavy chain and fibronectin synthesis

rates (Abstract). Journal of Cell Biology 115: 179a.

Chromiak, J.A. and Vandenburgh, H.H. 1992. Glucocorticoid-induced skeletal muscle atrophy

in vitro is attenuated by mechanical stimulation. American Journal of Physiology 262: C 1471-

C 1477.

Chromiak, J.A., Vandenburgh, H.H., Shansky, J., and Solerssi, R. 1992. Repetitive

mechanical stimulation of tissue cultured skeletal muscle mitigates glucocorticoid-induced

decreases in PGF2a synthesis and prostaglandin H synthase activity (Abstract). Physiologist 35:

213.

Samuel, J.L., Dubus, I., and Vandenburgh, H.H. 1991. Mechanically induced orientation of

adult rat cardiac myocytes (Abstract). Journal of Molecular and Cellular Cardiology

22(Suppl. IV): $55.

Vandenburgh, H.H. 1992. Mechanical forces and and their second messengers in stimulating cell

growth in vitro. American Journal of Physiology 262: R350-R355.

Vandenburgh, H.H., Chromiak, J.A., Shansky, J., Solerssi, R.L., Twiss, C., Fiore, G., and

Kim, E. 1991. Improvements in a tissue culture mechanical cell stimulator for studies on skeletal

muscle growth and atrophy (Abstract). ASGSB Bulletin 5: 35.

Vandenburgh, H.H., Haffaludy, S., Karlisch, P., and Shansky, J. 1992. Mechanically induced

alterations in cultured skeletal muscle growth. Journal of Biomechanics 24: 91-101.

Vandenburgh, H.H., Karlisch, P., and Solerssi, R.L. 1991. Insulin and insulin-like growth

factor-1 stimulation of myofiber growth in vitro is enhanced by mechanical activity (Abstract).

Journal of Cell Biology 115: 221a.

Vandenburgh, H.H., Shansky, J., Karlisch, P., and Solerssi, R.L. 1991. Mechanogenic second

messengers in stretch-induced skeletal muscle growth (Abstract). 8th International Biochemistry.

Exercise Conference 1: 47.

Vandenburgh, H.H., Shansky, J., Karlisch, P., and Solerssi, R. 1993. Mechanical stimulation

of skeletal muscle generates lipid-related second messengers by phospholipase activation. Journal

of Cellular Physiology 155: 63-71.

199
Vandenburgh,H.H., Shansky,J., and Solerssi, R. 1992. Mechanical stimulation of skeletal

muscle in vitro increases prostaglandin F2a synthesis and cyclooxygenase activity by a pertussis

toxin-sensitive mechanism (Abstract). Molecular Biology of the Cell 3: 244a.

Vandenburgh, H.H., Solerssi, R., and Fenwick-Smith, D. 1992. Insulin-like growth factor-1 as

an anabolic regulator of tension-induced skeletal muscle growth (Abstract). ASGSB Bulletin 6:

93a.

Vandenburgh, H.H., Solerssi, R., Henderson, S., and Adams, J. 1993. Repetitive stretch of

neonatal rat cardiomyocytes in vitro stimulates their rate of binucleation (Abstract). Conference on

Molecular Biology of Normal, Hypertrophied, and Failing Heart 38a.

Vandenburgh, H.H., Swasdison, S., and Karlisch, P. 1991. Computer aided mechanogenesis of

skeletal muscle organs from single cells in vitro. FASEB Journal _5: 2860-2867.

20O
MECHANO-TRANSDUCTION IN MAMMALIAN CILIATED CELLS

Pedro Verdugo

Center for Bioengineering

University of Washington WD-12

Seattle, WA 98195

Description of Research

In a broad range of species, detection of gravitational force is based on a motile/sensing organ in

which cilia operate as both a sensing transducer and a steering actuator. However, the

mechanical response of ciliated cells to changes in hydraulic load is still poorly understood. Our

studies have been aimed at investigating the role of Ca 2÷ ions in mechanical stimulus-response

coupling in ciliated cells. To accomplish this objective, it was necessary (1) to develop an assay

to characterize the response of ciliated cells to mechanical stimulation, and (2) to design an

optical system to monitor on-line fluctuations of intracellular [Ca2+]i as reported by fluorescent

probes.

Accomplishments

(1) Epitheliary ciliated cells in culture can adjust their mechanical output when

challenged by increased viscous load. This response takes place by lengthening the effective

stroke of the cilium, at the expense of shortening the recovery stroke, while keeping ciliary

frequency and amplitude relatively constant. Three forms of mechanical stimulation of ciliated

cells were tested in our laboratory: (a) local touching with a piezoelectrically driven

microneedle; (b) electromagnetically driven motion of magnetizable microspheres suspended on

top of the ciliated cells by a viscous fluid; and (c) exposing ciliated cells to increased viscous

load. Ciliated cells were found to respond to all these forms of stimulation. Although these three

methods can be precisely calibrated, the responses of ciliated cells to both electromagnetically

driven micromotion and poking with a microneedle were inconsistent, making it very difficult to

interpret results. Conversely, viscous loading was found to elicit reproducible responses of both

beat frequency and [Ca2+]i. Viscous loading also better resembles the mechanical stimulus to

which epitheliary ciliated cells are normally exposed, and it is easy to quantify and to deliver.

Additional studies to further characterize the beat cycle dynamics of rabbit respiratory ciliated

cells under conditions of increased viscous load (5-180 cp) were conducted using high-speed

cinematography (500 frames/sec). They indicate that the autoregulatory response of epitheliary

ciliated cells takes place by lengthening the effective stroke of the cilium at the expense of

shortening the recovery stroke, while keeping ciliary frequency and amplitude relatively

constant. 1

(2) Development of a system to measure changes of [Ca2+]i at high sampling rates and

with high spatial resolution. A large number of methods to detect fluctuations intracellular

[Ca2+]i have been developed in recent years. Fluorescent techniques offer the best approach to

monitor intracellular [Ca2+]i. However, with the exception of confocal microscopy, most of

these methods do not allow one to distinguish precisely where, inside the cell, these changes

occur. Also, high-speed confocal microscopy has an extremely high price tag. Other low-speed

scanning confocal systems have a rather low sampling rate, which can severely alias the Ca 2+

signals, particularly in cells where [Ca2+]i undergoes quick changes.

201
We havedevelopedthe optical andelectronichardware,aswell asthesoftwareof a powerful

andinexpensivesystemto detectchangesof [Ca2÷]iwith high temporalandspatialresolution.

This systemis basedon anepi-illumination invertedmicroscope(Nikon Diaphot),a low-cost

digital Peltier-cooledCCD videocamera(SpectraSource,M-220,CA) originally developedfor

astronomicalobservations,anda486 IBM-clone microcomputer.Thesoftwareincludesa setof

high-speedassemblylanguageinstructionsto control the communicationsbetweenthe camera

and the central processor,to commandshutters,and to read/write routines. Another set of

instructionsimplementsa programthatremovesout-of-focusoptical noise, which is the main

limitation for high-resolutionimaging in fluorescencemicroscopy.2 The strategyconsistsof

removingall theopticalinformationoutsidethedepthof field of themicroscopeobjective. Thin

optical sectionsinsidethecell canthenbeobtainedby usingashallow depth-of-fieldobjective

( 160-200nm).3,4

(3) Our preliminary results indicate that, following ciliostimulation, [Ca2+]i increases

first in the endoplasmic reticulum and then in the cytosol of cUiated cells. Calcium has a

critical role in stimulus-response coupling in a broad variety of cells. In most cells, [Ca2÷]i

remains at micromolar concentrations but can transiently increase following stimulation. These

fluctuations of [Ca2÷]i play a central role in triggering the intracellular cascade that couples

specific responses of the cell to correspondingly specific stimulus. Calcium ions can reach the

cytosolic compartment from either, or both, the extracellular space via Ca-channels of the plasma

membrane, or from intracellular sequestering sites via Ca-channels of intracellular membranous

compartments. It has been assumed that sequestered calcium found inside mitochondria or in the

endoplasmic reticulum (ER), or in the sarcoplasmic reticulum (SR) of myocytes, is in two pools:

(a) an ionized pool which is in equilibrium with (b) a bound pool associated to the chelating

proteins calsequestrin or calreticulin, which are normally found inside the SR and ER,

respectively. It has been assumed that the concentration of the ionized fraction of Ca 2+ inside

the storage compartments remains constant at concentrations much higher than the cytosol. 5

When the cell is stimulated, Ca-channels open allowing the entrance of extracellular Ca 2+ or its

release from the sequestering compartment to the cytosol. In either case, Ca 2÷ flow into the

cytosol is driven by a large diffusional gradient, raising the cytosolic [Ca2+]i. Calcium ions then

function as a second messenger, coupling the activation of a variety of cellular processes,

including phosphorylation of effector proteins via type II Ca-calmodulin-dependent protein

kinase (CaM-PKII).

A direct implication of this notion is that the fluctuations of [Ca2÷]i reported by fluorescent

probes take place only in the cytosol of the cell. Free Ca 2+ inside the storage compartments is

assumed to be at high concentrations (>btmol). Thus, Ca-probes inside the ER would be

saturated with Ca 2÷, contributing to a large fluorescence signal, but it would not vary in time. 6

This assumption has remained untested because the characteristic fuzziness of fluorescent images

cannot resolve the detailed spatial distribution and, hence, the kinetics of [Ca2÷]i at a subcellular

level.

We have found that submicromolar changes of [Ca2÷]i play an important function in the coupling

of adrenergic, purinergic, and mechanical stimulation in ciliated cells. 7 As in other cells, Ca 2÷

can enter the cytosol of ciliated cells from the extracellular space via Ca-channels, or it can be

released from intracellular membranous subcompartments, particularly the ER.

At present, our work in ciliated cells is focused on the detailed analysis of

subcompartmentalization of Ca 2÷ in the cytosol and the ER of ciliated cells undergoing viscous

loading. Using the high temporal and spatial resolution of our digital video-microscopy system,

combined with fluorescent double labeling, we have preliminarily documented that, following

202
viscous loading at 10 cp, the concentrationof [Ca2+]i (as reportedby fluorescent probes),

increasesin boththeERandthecytosolof ciliatedcells. In theseexperiments,wehaveusedthe

potentiometricfluorescentprobeDiOC6asatracerto identify theERof ciliatedcells,s We have

found that thepermeant(AM) fluorescentCa-probesaccumulatein thesamecompartmentsas

DiOC6, suggestingthat the ER of ciliated cells must havethe esterasesto turn theseprobes

impermeant.This featureis consistentamongdifferentAM Ca-probes,includingFura2,Rhod2,

andFluo3 (MolecularProbes,Inc.). The increasein cytosolic [Ca2÷]cis slightly delayedwith

respectto the Ca2+ increaseinside the ER [Ca2÷]er,and both of theseincreasesprecedethe

changesin beatfrequency. Thin optical sections(160 nm) of Rhod2fluorescently labeled

ciliated cells, takenevery 250 msec,revealthat Ca2÷seemsto be releasedfrom the ER, not

ubiquitouslyacrossthecell butin veryspecificplacesin thecytosoliccompartment.Also, when

released,theCa2÷doesnotseemto follow a typical diffusional profile butratheracooperative

patternresemblinga Ca-induced-Ca-releasemechanism. Theseobservationssuggesta new,

previouslyunrecognized,level of complexityin themechanismsthatcontrolCa2÷releaseto the

cytosolfollowing mechanicalstimulation.9

(4) We have also found that mucins, which are the principal polymer chains of the

polymer matrix of the mucusgel, canspontaneouslyself-assemble,forming liquid crystalline

domains.

References and Footnotes

1Our early observations have been published by Johnson, et al., 1991 (American Review of

Respiratory Disease. 144: 2680-85). The more recent results discussed herein were presented at

the 8th Annual Meeting of the American Society of Gravitational Biology.

2This approach was originally developed by K.R. Castelman for optical slicing of the heavens in

astronomical imaging (Digital Image Processing, Prentice Hall, 1979). It was later adapted and

validated for microscopic applications by Monk, et al., 1992 (JCB 116: 745), using a SunStation-Unix platform; and was further reprogrammed for a low-cost IBM-DOS platform by Mr.

Thien Nguyen in our laboratory (Mr. Nguyen is one of our M.D./Ph.D. students, and he will

continue with this project).

3The total cost of this system is less than $15,000, and it can provide optical sections of up to

160 nm. The maximum sampling rate is 6 images/sec using a standard 60-W mercury burnerexcitation source and up to 320 images/sec using a submillisecond chopped 2-W Argon-ion laser

excitation source.

4This material was recently presented by T. Nguyen, et al., at the 38th Annual Meeting of the

Biophysical Society (Biophysical Journal 66: A275, 1994), and it is now being prepared for

publication.

5For review see E.M. Milner, et. al., 1992 (Molecular and Cellular Biochemistry 112: 1-13).

6See J.P.Y. Kao, 1994 (Methods in Cell Biology 40: 155-181).

7See the following:

P. Verdugo, 1980 (Nature 283, 164, 765).

P. Verdugo, et al., 1980a (Fertility and Sterility 33: 193, 176).

P. Verdugo, et al., 1980b (Journal of Applied Physiology 48:868-871).

P. Verdugo, et al., 1983 (Journal of Submicroscopic Cytology and Pathology 15: 95-97). M.

203
Villalon andP. Verdugo,1982(Cell Motility and the Cytoskeleton 1: 59-65).

M. Villalon, et al. 1989. (Biophysical Journal 56: 1255-1259).

M. Villalon and P. Verdugo, 1990. In: Transduction in Biological Systems. (J. Vergara, E.

Jaimovich, J. Bacigalupo, and C. Hidalgo, Eds.). New York: Pergamon Press, pp. 221-230.

8M. Tarasaki and T.S. Reese, 1992 (Journal of Cell Science 101: 315-322).

9These results were presented at the 9th Annual Meeting of the American Society of

Gravitational Biology.

Publications

Johnson, et al. 1991. American Review of Respiratory Disease 144: 2680-2685.

Monk, M.J., et al. 1992. Journal of Cellular Biology 116: 745.

Nguyen, T. and Verdugo, P. 1993. Dynamics of intracellular Ca subcompartmentalization in

ciliated cells. ASGSB Bulletin 7: A45.

Tarasaki, M. and Reese, T.S. 1992. Journal of Cell Science 101: 313-322.

Verdugo, P. 1993. Polymer gel phase transition in condensation-decondensation of'secretory

products. Advances in Polymer Science 110: 146-156.

Verdugo, P., Deyrup-Olsen, I., Martin, A.W., and Luchtel, D.L. 1992. Polymer gel phase

transition: The molecular mechanism of product release in mucin secretion. In: Swelling of

Polymer Networks, NATO ASI Series H (Karalis, E., Ed.). Heidelberg: Springer-Verlag, 64:

671-681.

Verdugo, P., Villalon, M., and Hard, R. 1992. Beat cycle dynamics in viscous loaded ciliated

cells. ASGSB Bulletin 6: A42.

Viney, C., Huber, A., and Verdugo, P. Processing Biological polymers in the liquid crystaline

state. In: Biodegradable Polymers and Packaging (Ching, C., Kaplan, D.L., and Thomas, E.L.,

Eds.). Lancaster, UK: Technomic, pp 209-224.

Viney, C., Huber, A.E., and Verdugo, P. 1993. Liquid crystalline order in mucus.

Macromolecules 26: 852-257.

2(kl
THE ROLE OF CALCITE

BIOMINERALIZATION

SKELETAL

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 other vertebrates during extended space flight. The basic

molecular and biochemical basis of formation of mineralized skeletal elements is poorly

understood. Hence, basic information about biomineralization will be required to understand better

the complex events that occur in microgravity environments. It is hoped that 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 sea urchin, S. purpuratus. The calcite spicules of the pluteus

larva have an integral protein matrix. Our studies center on learning more about the proteins

comprising the spicule matrix. Several approaches have been used to study these proteins and their

function. First, we have directly isolated the matrix proteins and studied their properties by

biochemical methods; these ongoing studies have shown that there are over 3 dozen matrix

proteins, all but 4 of which are acidic. There axe probably 6 of them that are not glycosylated.

Second, we have isolated and cloned the genes of 2 of these proteins, naming them SM30 and

SM50. The cloned genes have been used to make specific antibodies against these two

well-characterized spicule matrix proteins, and the antibodies have been used to study the function

of the proteins in biomineralization. The cells that form the spicule matrix and spicules, the

primary mesenchyme cells, have been cultured, and biomineralization has been shown to require

an intact extracellular matrix. Finally, a powerful new approach to studying the function, in vivo,

of the SM30 and SM50 proteins has begun using genetic engineering techniques. Genes that

encode counterfeit SM50 protein have been made, and these genes and their products are being

introduced into the living embryo in order to discover, by means of the anomalies produced, what

the function of SM50 is during mineralization.

Accomplishments

(1) Significant portions of spicule matrix genes have been cloned from DNA of distantly

related species. The SM30 cDNA has been cloned from L. pictus, a species distantly related to S.

purpuratus. The promoter region of SM 50 has also been cloned from L. pictus. In each instance,

both the promoter and coding region of the two species are similar in nucleotide sequence.

(2) Cloned SM30 cDNAs have been used to study the details of expression of the gene by

RNAse protection assays. The SM30 gene is activated slightly earlier than was formerly known

because of the increased sensitivity of the assay.

(3) Both SM30 and SM50 genes have been cloned into bacteria which express these

foreign genes as proteins. The bacterially synthesized proteins have been used to produce

monospecific antibodies against the two proteins. Exposure of spicule-producing cells to these

antibodies does not interfere with biomineralization.

205
(4) Culturesof primarymesenchymecellshavebeentreatedwith agentsthatinterferewith

formation of the extracellularmatrix. Whenthe matrix is perturbed,SM 30 and SM50gene

expressionsarereduced,andthespiculedoesnot form. This resultis in contrastto treatmentof

wholeembryoswith suchagents,whereonlySM30expressionis inhibited.

(5) TheSM50geneof L. pictus has been engineered to contain the 10 amino acid epitope

of c-myc, a vertebrate gene, and the epitope tag has been inserted into 3 different places in the

SM50 protein. This allows one to locate the tagged protein, in vivo, by using antibodies against cmyc. The presence of the epitope tag, at least in one of its locations in SM50, should compromise

the function of SM50 and help one understand SMS0 function, in vivo. Such tests are underway.

Significance of the Accomplishments

Finding 1: We had previously shown that SM50 coding regions in the two sea urchin species were

closely conserved. We can now extend that generalization to the SM30 protein and to the SM50

gene regulatory region upstream of SM50. This lends strong support to the idea that important

functions of the protein are embedded in the protein structure and that, by modifying this structure,

we can learn how these proteins function in biomineralization.

Finding 2: A continued study of the SM30 genes in S. purpuratus revealed slight differences in the

mRNAs. A gene-specific probe has been designed that allows detection of only one of the three

SM30 genes; and this sensitive detection method, called RNAse protection assay, shows that this

form of SM30 is expressed at very low levels somewhat earlier than was heretofore known.

Further, this gene may be expressed only in adult spines and not in tube feet. Hence, for the first

time, there is a clear indication that the multiple genes of SM30 may subserve slightly different

functions in different situations.

Finding 3: Since both SM30 and 50 were subcloned into bacterial expression systems during the

past year, substantial amounts of pure SM30 and SMS0 can be obtained, something not formerly

possible. The pure proteins have been used to make monospecific antibodies against the two

different proteins. When such antibodies are added to cultures of embryonic mesenchyme ceils

that make spicules, there is no effect on spiculogenesis. This is consistent with our hypothesis that

the function of these two proteins is intracellular, not extracellular.

Finding 4: Perturbation of the extracellular matrix disturbs biomineralization. We had formerly

known that treatment of whole embryos with lathyrogenic agents disturbed spicule formation and

reduced SM30 expression. This result has now been reproduced in mesenchyme cell cultures,

where the phenomenon is easier to study closely. In contrast to whole embryos, SM50 expression

is also affected in cultures. Hence, the proposal that extracellular matrix function is generally

important in biomineralization receives strong support.

Finding 5: We have been able to use molecular biology techniques to produce mRNAs that encode

epitope tagged versions of SM50 and, furthermore, have engineered the mRNAs in such a way

that their function, in vivo, should be compromised. This paves the way to examination of SM50

function, in vivo, by the "dominant negative" approach, which is proving to be so powerful in

other systems.

Publications

Benson, S.C. and Wilt, F.H. 1991. Calcification of the spicule of the sea urchin embryo. In:

Calcification in Biological systems (Bonnuci, E., Ed.). CRC Press, pp. 157-178.

206
George,N.C., Killian, C.E., andWilt, F.H. 1991. Characterizationandexpressionof a gene

encoding a 30.6 kD Strongylocentrotus purpuratus spicule matrix protein. Developmental Biology

147: 334-342.

Livingston, B.T., Shaw, R., Bailey, A., and Wilt, F. 1991. Characterization of a cDNA

encoding a protein involved in formation of the skeleton during development of the sea urchin,

Lytechinus pictus. Developmental Biology 148: 473-480.

Stebbins, B., George, N.C., and Wilt, F. 1992. Disruption of extracellular matrix inhibits gene

expression of the sea urchin spicule matrix gene SM30 (Abstract). Molecular Biology of the Cell

3:316a.

Wilt, F.H., George, N.C., and Khaner, O. 1991. Sea urchin micromeres, mesenchyme and

morphogenesis. In: Gastrulation: Movements, Patterns and Molecules (Keller, R., Clark, W.,

and Griffin, F., Eds.). New York: Plenum Press, pp. 281-288.

Wilt, F.H., Killian, C.E., and Livingston. B.T. 1992. The integral matrix proteins of the

spicules of sea urchin embryos. In: Fourth International Symposium on Chemistry and Biology of

Mineralized Tissues (Slavkin, H. and Price, P., Eds.). Amsterdam: Elsevier Science Publishers,

pp. 5-92.

207
EFFECTS OF ALTERED ENVIRONMENTS ON CELLULAR STRESS

REPRODUCTION AND DEVELOPMENT

Debra J. Wolgemuth

Department of Genetics and Development

The Center for Reproductive Sciences

Columbia University

New York, NY 10032

GENES IN

Description of Research

The long-range goal of our research program is to examine the effects of the space flight

environment on mammalian development and cellular differentiation. The main aim of this project

is to characterize the response of mammalian embryos and germ cells to altered environmental

conditions and to determine the stages that are most sensitive to stress during embryogenesis and

gametogenesis. While the ideal setting for examining these effects is space flight itself, the

opportunities for such experiments are severely limited. Therefore, ground-based studies are

crucial as a means of evaluating both the aspects of development most likely to be affected in space

as well as providing background information as to potential molecular markers of perturbed

development. Our studies focus on one set of molecular markers--the cellular stress proteins. The

cellular stress protein or heat shock protein (hsp) is synthesized in response to diverse conditions

of physiological stress and environmental assaults. Our studies have focused on examining the

pattern of expression of these gene families initially at the level of RNA and, subsequently, of

proteins in mid-gestation mouse embryos and during spermatogenesis in mice.

Accomplishments

(1) One series of experiments focused on characterizing the expression of the hsp70.2

gene at the mRNA level in different mouse tissues, including adult brain, reproductive organs, and

mid-gestation mouse embryos. Hsp70.2 had been previously isolated and characterized in our lab

as a testis-specific gene of Hsp70 gene family with precise patterns of expression during

spermatogenesis. The results of the present studies now reveal that this gene, while expressed at

high levels in a specific subpopulation of spermatogenic cells, is also expressed in the adult brain

and in mid-gestation conceptuses.

The analysis utilized Northern blot hybridization with RNAs isolated from the various tissues

using a probe for the 5' end of the hspTO.2 gene, which did not cross-hybridize with other Hsp70

genes. Low levels of expression of hsp70.2 were detected in adult ovaries and prostate, and in

placenta at stage 10.5 days of gestation. Higher levels of expression were also found in the adult

brain and epididymis, in addition to the very high levels seen in the adult testis. A detailed analysis

of hspTO.2 gene expression in dissected brain structures revealed the abundance of this gene in

hippocampus, as compared to other brain regions. Other regions of the brain where hspTO.2 was

expressed included the hypothalamus, thalamus, and cortex.

(2) We also initiated studies to examine the expression of the hsp70.2 gene in mouse

tissues employing the method of in situ hybridization. This technique gives the opportunity to

observe actual expression of the gene at the cellular level. In the testis, the expression of hsp70.2

was determined to be restricted to germ cell compartment. The pattern of its expression was

shown to be developmentally regulated with the most pronounced signal in pachytene

spermatocytes, consistent with previous studies; although some expression was seen in later stages

of germ cell development (through to the round spermatid stages, again, consistent with previous

observations). The preliminary experiments also showed the expression of hsp70.2 gene in the

208
epididymisandfurtherrevealedaregionallocalizationof its expression,mostlyrestrictedto caput

region.

(3) Thedevelopmentalregulationof membersof theHsp70andHsp90genefamilieswas

examinedat the protein level using methodsof immunocytochemistry.We have begunthis

analysisusingrat monoclonalantibodies(clone7.10),which recognizedall membersof Hsp70

family, andrat monoclonalantibodies(16F1clone)which recognizedmouseHsp86andHsp84

proteins(membersof theHsp90family of proteins). Theexpressionof Hsp70andHsp90was

found in histological sectionsof testesfrom adult normal mice. The immunoreactivity was

restrictedto germcells from pachytenespermatocytesto elongatedspermatids.Since,in our

studiesat themRNAlevels,wehavedetectedexpressionof theHsp70andHsp90genefamiliesin

mid-gestationmouseembryosandplacentastages10.5-13.5daysof pregnancy,welookedat the

level of protein aswell. Whetherthe immunoreactivity,which wasdistributedthroughoutthe

tissuesections,representscross-reactingproteinsand/orubiquitousdistribution remainsto be

determined.Westernblot analysisof solubilizedmousetissuesandplacentarevealedexpression

of Hsp70proteinsin adulttestis,brain,andliver, aswell asin placentaatembryonicstages,10.5-

17.5days. Therefore,ourpreliminaryresultsshowthepresenceof Hsp70proteinsin different

adult tissuesaswell asin conceptuses,notablyin extraembryoniccompartments.Theresultsof

immunocytochemistryindicatedthat,at theproteinlevel,thebothHsp70andHsp90in thetestis

are most abundantin the germcell compartment. The expressionof theseproteins during

embryonicdevelopmentwasmostpronouncedat 10.5-13.5days.

(4) A final seriesof studieswasundertakento extendto the proteinlevel our previous

observationswhichhadestablishedthestageandcellularspecificity of expressionof Hsp86and

Hsp84in themurinetestis,in theabsenceof exogenousstressusingimmunoblotanalysis.Hsp86

proteinwasshownto bepresentthroughouttesticulardevelopment,andits levelswereshownto

increasewith theappearanceof differentiatinggermcells. Hsp86wasmostabundantin thegerm

cell populationandwaspresentat significantlylower levelsin thesomaticcells, h_contrast,the

Hsp84proteinwasdetectedin thesomaticcellsof thetestisratherthanin germcells. Thesteady

state levels of Hsp86and Hsp84paralleled the patternof the expressionof their respective

mRNAs, suggestingthat regulation at the level of translation was not a major mechanism

controllingHsp90 gene expression in testicular cells. Immunoprecipitation analysis revealed that a

70 kDa protein coprecipitated with the Hsp86/Hsp84 proteins in testicular homogenates. This

protein was identified as an Hsp70 family member by immunoblot analysis, suggesting that HspT0

and Hsp90 family members interact in testicular cells.

Significance of the Accomplishments

Finding 1: The hsp70.2 gene is testis-abundant, not testis-specific, as originally thought. It is

expressed at lower levels in other adult murine tissues, most notably in the brain, epididymis,

prostate, seminal vesicles, ovaries, and uterus, and in predominantly extraembryonic tissues in the

mid-gestation conceptus. These observations raise the question of whether its expression in these

tissues will be altered during exposure to environmental stress and changes in hormonal levels.

Finding 2: At the level of in situ hybridization, we confirmed the developmentally regulated pattern

of expression of hsp70.2 during mouse spermatogenesis. The strongest signal was observed over

meioticaUy active, pachytene spermatocytes. Furthermore, no other sites of expression in the adult

testis were observed. In the brain, in situ hybridization revealed that the highest levels of

expression were in the hippocampus.

Finding 3: The expression of Hsp70 and Hsp90 in the adult testis was confirmed, at the protein

level, to be most abundant in mouse germ cells from pachytene spermatocyte to round spermatid

stages.

21)9
Finding4:Hsp90 proteinwas found to co-immunoprecipitatewith a proteinof 70 kDa, which

appearsto beamemberof theHsp70family. It will beinterestingto observeif thisassociationis

alteredunderconditionsof alteredenvironmentalstress.

Finding 5: Studieson the expressionof hsp70.2 have revealed the surprising observation of

transcripts in both the sense and antisense orientations from this area of the genome. In the adult

mouse brain, the expression of the antisense mRNAs was highest in the brain stem, cortex,

thalamus, and the cerebellum in particular. The prostate also expressed significant levels of this

antisense transcript. These observations bring to attention new levels of complexity with regard to

the regulation arid function of the hsp70.2 gene during normal development and, ultimately, in

response to environmental changes including those encountered during space flight.

Publications

Amann, R.P., Deaver, D.R., Zirkin, B.R., Grills, G.S., Sapp, W.J., Veeramachanemi, D.N.R.,

Clemens, J.W., Banerjee, S.D., Folmer, J., Gruppi, C.M., Wolgemuth, D.J., and Williams,

C.S. 1992. Effects of microgravity or simulated launch on testicular function in rats. Journal of

Applied Physics 73(Suppl.): 174S- 185S.

Gruppi, C.M. and Wolgemuth, D.J., 1993. Hsp90 proteins are expressed in parallel with their

mRNAs and co-precipitate with Hsp70 in murine testicular cells. Developmental Genetics, 14: 199-

126.

Murashov, A. and Wolgemuth, D.J. 1993. Restricted expression of a member of the Hsp70

cellular stress protein family in the absence of exogenous stress (Abstract). ASGSB Bulletin, Vol.

7, p. 98.

Wolgemuth, D.J., Montanez, C., Murashov, A., and Gruppi, C.M. 1992. Cellular stress gene

expression in reproductive tissues and embryos (Abstract). ASGSB Bulletin, Vol. 6, p. 61.

210
SPACE FLIGHT EXPERIMENTS

PLANT GRAVITATIONAL PHYSIOLOGY

Allan H. Brown

Gravitational Plant PhysiologyLaboratory

University ScienceCenter

3401Science Center Boulevard

Philadelphia, PA 19104-3323

Description of Research

Goals: Generally stated, the long-range goal of our research is to understand the ways in which

gravity is important to plants and, by extension, to the use of plants on Earth and potentially in

extraterrestrial situations. Specifically, we want to understand the fundamental mechanisms

which enable plants to detect, measure, and respond to gravitational influences. Consequently, it

is axiomatic that in our experiments we must be able to vary the gravitational information that

test plants receive from their environment. These experimental conditions have been achieved

by increasing the G-force by centrifugation or decreasing it by performing experiments in nearEarth orbit.

All of our recent research objectives have pertained directly or indirectly to the support of space

experiments. Support from the NASA Space Biology Program enabled us to make ground-based

studies which led to the proposal of two space experiments that were flown on IML-1 in 1992.

Those experiments, even though only partially successful due to technical difficulties, provided

quantitative answers to five different scientific questions involving the kinematics of how plants

detect and respond to G-forces. Also obtained were certain serendipitous findings that set the

stage for asking new experimental questions. IML-1 research results are now being written for

publication. Within the confines of limited space, four of those questions/answers can be

described briefly.

Accomplishments and Significance of the Accomplishments

Question 1: How well does clinorotation (a simulation of weightlessness) mimic the effects

observed in space flight? If scientists could count on the validity of some kind of simulation of

weightlessness, we would be able to get answers to some scientific questions by performing

ground-based experiments without doing experiments in space. This would increase our

scientific productivity by more than an order of magnitude and decrease the cost by at least 3 or 4

orders of magnitude.

On IML-1 we were able to make nine different kinds of quantitative comparisons between the

results we obtained in space and those we obtained on Earth using the same apparatus. For five

of the nine kinds of comparison, our results showed no significant differences between space vs.

clinorotation experiments. For the other comparisons, the differences were great. Our results

only reinforced the cautious judgment that clinorotation experiments on Earth may serve as a

guide for what may be the plants' behavior in weightlessness, but we cannot describe the

clinostat as a dependable substitute for tests in space.

Question 2: Does the plant obey the "Reciprocity Rule"? The rule predicts that a plant's

responses will be the same for any stimulus (G) times duration of its application (t) even though

G and t are varied reciprocally, thus maintaining the same G x t product. Superficially, that may

sound reasonable, but there is no compelling theoretical reason why the rule must apply.

For technical reasons the data for testing the rule are less ambiguous when responses are

measured in space. The results of our IML- 1 experiment provided a NO answer; the Reciprocity

213
Rule wasnot obeyed. Thatresultis importantbecauseto plan andto interpretresultsof many

ground-basedexperimentsover the past severaldecadesthe experimentershave taken the

ReciprocityRule for granted. Now weknow that theReciprocityRuleis not valid in all cases;

and,therefore,sucha generalizationis not dependable.As a resultof our IML-1 experiment

results,wehavebeenmotivatedto developanexplanationfor thefailure of theReciprocityRule.

This is beingpreparedfor publication.

Question3: Whatis thethresholdG-stimulusfor plantsrespondingin microgravityto a rangeof

tropistic stimulations? "Threshold" meansthe weakeststimulation that elicits a detectable

response. By knowing the thresholdwe canset limits on any kind of mechanismthat could

possiblyexplainhow theplantdetects(andmeasures)gravityor otheracceleratoryforce.

Measuredin two differentwayson Earth,experimentershavepublishedthresholddatathatdiffer

by more than an order of magnitude--far more than what could be attributed merely to

experimentalerror. The supportingdataweregood, andneithermethodcanbediscreditedon

theoreticalgrounds.

We madethesamekind of measurementin spacebut bya differentmethod.Onewasin principle

unambiguous,becauseno assumptionsweremadewith regardto thevalidity of clinorotation.

Ourresultsagreedvery well with onesetof ground-basedresultsbutnotwith theother.

Question4: Will oatseedlingcoleoptilescircumnutatein weightlessness?Circumnutationis a

growthoscillationdisplayedby elongatingplantorgans.It is absolutelygrowthdependent.Like

anyotherwidespreadandpossiblyuniversalphenomena,it hasattractedexperimentalattention

andhasbeenstudiedusingseveraldifferentmethodsfor well overacentury.

Circumnutation had beenattributed to an effect of gravity; therefore,it should not occur in

weightlessness. We testedthat in the SLS-I mission and observedthat sunflower shoots

circumnutatedin space--aresultthat wasthedriver for thedevelopmentof a newtheorywhich

canexplaintheplant'scircumnutationalmechanismindependentof gravityandalsocanaccount

for its consistentoccurrence.Muchof thework thatled upto thedevelopmentof thattheorywas

supportedby SpaceBiology Programfunding.

Duringthe IML- 1missionwe hadtheopportunityto observegrowthbehaviorof wheatandoat

seedlings. Onespecies(wheat)circumnutated;the other(oat) did not. Obviously, two out of

three fall short of proof of ubiquity. Observationson more species, flown by various

investigatorsin future spaceexperiments,mayestablisha statisticallymoreadequatebasisfor

generalization.

Publications

Brown, A.H. 1991. Gravity perception and circumnutation in plants. In: Advances in Space

Biology and Medicine (Bonting, S.L., Ed.). Greenwich, CT: JAI Press, pp. 129-153.

Brown, A.H. 1992. Centrifuges---evolution of their uses in plant gravitational biology and new

directions for research on the ground and in space flight. ASGSB Bulletin 5: 43-57.

Brown, A.H. 1993. Circumnutations: From Darwin to Space Flights. Plant Physiology

101: 345-348.

Eidesmo, T., Brown, A.H., Chapman, D.K., and Johnsson, A. 1991. Tropistic responses of

Avena Seedlings in simulated hypogravity. Micrograviry Science and Technology

IV(3): 199-206.

214
AN ATTEMPT TO LOCALIZE THE GRAVITY SENSING MECHANISM OF PLANTS:

THE ELECTRICAL IMPEDANCE BETWEEN VASCULAR AND CORTICAL TISSUES

Mark F. Desrosiers and Robert S. Bandurski

Department of Botany and Plant Pathology

Michigan State University

East Lansing, MI 48824-1312

Description of Research

A plant stem when placed horizontally responds to gravity by growing back into a vertical

orientation. For the stem to grow more on the lower than the upper side, more ions, hormones,

and solutes required for growth must be transported out of the central vascular stele into the

lower side of the horizontal stem (Figure 1). It was the objective of this research to determine

how the gravitational stimulus is transduced into a selective leakage of solutes from the central

stele into the surrounding cortical tissues. The working hypothesis developed by this laboratory

is called the Potential Gating Theory and attempts to link the internal bioelectric fields of the

plant to the transport of the growth hormone, indole-3-acetic acid (IAA). Briefly the theory

states that:

(1) The growth hormone is in the vascular stele of the plant and to promote growth it

must reach target cells in the cortex and epidermis.

(2) The transport of IAA from the vascular stele to the cortical cells is through

plasmodesmatal connections and is regulated by the bioelectric potential difference between the

stele and cortex.

ENDOSPERM

I

Cortex SCUTELLUM I

Cortex I

I

ROOT KERNEL MESOCOTYL NODE COLEOPTILE

Figure 1. Diagram of IAA transport from the kernel to the growing mesocotyl in young corn seedlings as proposed

by the Potential Gating Theory. The size of the arrows represent relative magnitudes of IAA flow.

Gravity, as a stimulus, affects the system by inducing a lateral electrical gradient across a

horizontal shoot. We postulate it is this gravity-induced electrical gradient that opens the

plasmodesmatal connections between the stele and the cortex on the lower side. Consequently,

in a horizontal shoot, more IAA would flow from the stele into the lower cortex and induce

growth such that the plant would grow back to its normal upright position. (See Figure 1. The

arrows represent the direction and magnitude of IAA transport.)

215
Previouslywehaveshownthat thepotentialdifferences(seeFigure3) betweensteleandcortex

areresponsiveto gravity andstatedthat the gravity-inducedchangein the potentialdifference

could function as a regulator of IAA efflux from the stele becausea) it is rapid, b) it is

sufficientlylargein magnitude,andc) it hasthepropergeometryto produceIAA flow from stele

to cortex. The next step in testing the PotentialGating Theory was to measurephysical

responsesof the plantto gravistimulation,whichmight bea triggeringmechanismfor alteration

of thesymmetricbioelectricgradients.We reportthishasbeenaccomplished.

Accomplishments

Gravistimulation should induce a change in the distribution of charges in the cells. During

redistribution of charges, the mobility of these charged structures will change. This can be

detected by impedance spectroscopy. We found that the mobility of charges as measured by

impedance spectroscopy (mobility is inversely proportional to impedance) undergoes an

immediate transient response following gravistimulation. Figure 2 shows a plot of impedance as

a function of time and frequency.

te ra4s _ "_

-_

• I I i I

-S 0 $ I0 15

Time (minutes)

Figure 2. Impedance as a function of time and frequency in a sample at 4 nun below the mesocotyl node.

Significance of the Accomplishments

An early event in gravity detection in plants was observed. The mobility of the charges present

in a cell changed immediately following changes in orientation. This is significant because 1)

the mobility can change due entirely to physical mechanisms and hence be prior to any

biochemical responses, and 2) the fact that the gravity-induced impedance change is of limited

duration emphasizes the short-lived nature of this early event. Any gravity-induced charge

redistribution should return to equilibrium quickly.

The second step of the Potential Gating Theory states that the charge redistribution induces the

biochemical cascade. If the gravity-induced impedance change is due to physical mechanisms,

then it will occur prior to the biochemical response and can serve as the trigger for the

biochemical cascade which results in and maintains the bioelectric potentials observed following

gravistimulation.

216
Figure3 summarizesthechemical,biological,andelectricalchangesoccurringin themesocotyl

of a cornshootfollowing agravity stimulus. Theinitial event,following thegravity stimulus,is

the changein AC impedanceandDC conductancebetweenthe two tissues. This is followed

rapidly by the changein electricalchargeswhich occursbeforethe IAA asymmetry,andthat

maybethecausativefactorthatleadsto theincreasedamountsof IAA on thelower sideof the

shoot. Finally thereis a shootgrowthon thelower sideof a horizontallyplacedshootwith the

resultthattheplantgrowsbackintoa verticalposition.

¢_1.5 ' 50

?

..9. _-so

_to e

{ 5

o

0.0 - -250 -

-S

60 0.4 " 2S

o.:2; P+ 24

o.o 

30 21 _"

-0.8

O .

• 20 -,-I.0 20

0 $ I0 IS

Time Horizontal (rain)

Figure 3. A summary of the known changes in the mesocotyl of Zea mays seedlings following a shift from a vertical

to a horizontal orientation. The parameters measured are the IAA concentration in the lower side of the mesocotyl,

the mesocotyl curvature and the endogenous voltage, and DC conductance and impedance between stele and cortex

tissues.

Publications

Bandurski, R.S., Desrosiers, M.F., Jensen, P., Pawlak, M., and Schulze, A. 1991.Genetics,

chemistry, and biochemical physiology in the study of hormonal homeostasis. In: Plant Growth

Substances (Karssen, C.M., Ed.). Amsterdam: Kluwer Academic Publishing.

Bandurski, R.S., Schulze, A., Jensen, P., and Desrosiers, M.F. 1991. Results of the mid-deck

shuttle experiment, growth hormone concentration and distribution. ASGSB Bulletin 5: 28.

Bandurski, R.S., Slovin, J., and Cohen, D. 1992. The Auxins In: Plant Physiology, A Text in

Spanish (Talon, M., Ed.). Madrid, Spain: McGraw Hill.

Desrosiers, M.F., and Bandurski, R.S. 1991. Specific conductance between stele and cortex as

modified by the gravity stimulus. ASGSB Bulletin 5: 47.

Desrosiers, M.F., and Bandurski, R.S. 1992. Specific conductance between stele and cortex in

corn mesocotyls as modified by the gravity stimulus. Plant Physiology 99: 371.

Jensen, P.J., Momonoki, Y., and Bandurski, R.S. 1992. Indole-3-acetic acid biosynthesis in

maize endosperm. Plant Physiology 99: 130-133.

217
Momonoki, Y.S. 1992. Occurrenceof acetylcholine-hydrolyzingactivity at the stele-cortex

interface. Plant Physiology 100: 692-698.

Rekoslavskaya, N., Jensen, P.J., and Bandurski, R.S. 1992. IAA biosynthesis in maturing corn

endosperm. Plant Physiology 99: 101.

Schulze, A., Jensen, P.J., Desrosiers, M., and Bandurski, R.S. 1992. Studies on the growth,

indole-3-acetic acid, and abscisic acid content of Zea mays seedlings grown in microgravity.

Plant Physiology 100: 692-698.

218
STUDIES OF INTERCELLULAR COMMUNICATION AND INTRACELLULAR

RESPONSES BY BONE CELLS TO SIMULATED WEIGHTLESSNESS AND SPACE

FLIGHT

Stephen B. Doty

Hospital for Special Surgery

535 E. 70th Street

New York, NY 10021

Description of Research

Non-weight bearing and space flight cause a reduction in new bone formation, especially in the

weight bearing portion of the skeleton. The cellular mechanisms responsible for this reduction

are unknown although the bone-forming cells appear to be the cells most affected. Our present

hypothesis suggests that the cytoskeleton of these cells could be the cellular element most

sensitive to changes in gravity or microgravity. The cytoskeleton is responsible for cell shape,

cell movement and attachment, and cell secretory activity. For cells involved in collagen

synthesis, the secretory granules which contain pro-collagen are moved from the Golgi region of

the cell to the cell membrane and then released to the extracellular space. This activity requires

an intact cytoskeletal system. It has been shown that, in bone-forming cells, when the

cytoskeleton is disrupted, collagen synthesis and secretion are reduced or inhibited. Previous

studies have shown that the collagen-containing granules in these cells following space flight are

reduced in number.

We are studying the bone-forming activity of isolated cells in order to simplify and manipulate

bone formation and mineralization during space flight. This method involves growing isolated

rat bone-forming cells in a three-dimensional culture system. These cells produce significant

collagen matrix which will mineralize in culture. The mechanism of cell attachment, the

cytoskeletal components, and the collagen secretion mechanisms are much easier to study and

manipulate in this system compared to the whole animal studies discussed in the previous

paragraph.

We are also continually developing new methods to localize and quantitate specific enzyme

activities associated with bone-cell activity. We are quantitating the ability of bone cells to form

new matrix and making these measurements at specific sites within bone samples. The technique

involves our standard histochemical procedures coupled with a new imaging system and video

enhancement of the sample.

Accomplishments

The most recent whole animal studies involved analysis of long bones from Space Life

Sciences-1, a 9-day flight onboard Space Shuttle Columbia. The f'mdings from these analyses

indicated the following: (a) Stromal cells (pre-bone-forming cells) showed significantly more

alkaline phosphatase activity in the flight animals than in their controls. This was the first time

for this finding and was the reverse of what was found in the differentiated cell population. (b)

Secretory granules in the bone-forming cells were significantly increased when the flown

animals were given 9 days to recover from their flight. This suggested a reduction in secretory

granule formation during the flight itself. (c) The vascular supply to the long bones did not show

the extreme degenerative changes we found previously in the Cosmos series of flights.

The bone-forming cells grown in three-dimensional culture will form mineralized bone in the

laboratory. We have recovered cells from three Shuttle flights (STS-45, STS-53, and STS-55)

and found that they produce a collagenous matrix but have not yet found any mineralization.

219
This resultmaybepartiallyexplainedby thedifficulty of maintainingoptimalcultureconditions

duringflight. However,if theabsenceof mineralization,whichis alsowhatwefoundto occurin

the whole animal studies,is a consistentfinding, thenwe havethe basisfor future studiesof

cellularmechanisms.

By combining our histochemical methods with the video image analysis system, we are

quantitating alkaline phosphatase activity (a measure of bone formation) on a site specific basis

in each bone obtained from space flight or non-weight-bearing experiments. The results indicate

that the total alkaline phosphatase activity in trabecular bone is greater than in compact bone;

however, the individual cells in compact bone show greater activity than those same cells along

trabeculae. Thus, site specificity becomes important in the understanding of how bone responds

to space flight and non-weight bearing.

Figure 1. A scanning electron micrograph of a

single bead (B) partially covered with bone cells.

The cells have two general morphologies; a

rounded plump cell (arrowhead) or flattened cells

with numerous cytoplasmic projections (arrows).

Magnification: 2,400X.

Figure 2. This scanning electron micrograph shows

how the cells cover the beads (B) and how the beads

and cells mixed together form a mat-like structure in

the background. Magnification: 670X.

Figure 3. This scanning micrograph shows collagen

strands (arrows) stretching between beads as well as

attached to them. Magnification: 590X.

_.l)
Significance of Accomplishments

The whole-animal studies validate our assumption that the bone-forming cells and their

cytoskeletal elements should be the major focus of our continuing studies. Quantitation of the

secretory granules and analysis of the cytoskeleton components will continue to be related to

other data collected from biochemical and biomechanical studies.

The ability to manipulate and study in detail the cellular mechanisms involved in bone formation

is greatly enhanced by the use of bone cell culture. In culture conditions, cell attachment, cell

secretion, and matrix formation and mineralization can be selectively inhibited or enhanced. The

cellular responses to these changing activities can then be compared to the response found in the

whole-animal studies following space flight.

The quantitation of enzyme activities, especially for the alkaline phosphatase activity, is

especially important for analysis and description of the mechanism of matrix mineralization.

This analysis will help to separate the mineralization process from the collagen secretion process

and thus improve our understanding of how space flight or non-weight bearing affects new bone

formation.

Publications

Boskey, A.L., Camacho, N.P., Mendelsohn, R., Doty, S.B., and Binderman, I. 1992."FT-IR

microscopic mappings of early mineralization in chick limb bud mesenchymal cell cultures.

Calcified Tissue International 51: 443-448.

Boskey, A.L., Pleshko, N., Doty, S.B., and Mendelsohn, R. 1992. Applications of Fourier

transform infrared (FT-IR) microscopy to the study of mineralization in bone and cartilage.

Cells and Materials 2: 209-220.

Boskey, A.L., Stiner, D., Doty, S.B., Binderman, I., and Leboy, P. 1992. Studies of

mineralization in tissue culture: Optimal conditions for cartilage calcification. Bone and Mineral

16: I 1-36.

Boskey, A.L., Ziecheck, W., Guidon, P., and Doty, S.B. 1993. Gallium nitrate inhibits alkaline

phosphate activity in a differentiating mesenchymal cell culture. Bone and Mineral 20:179-192.

Doty, S.B., Morey-Holton, E.R., Durnova, G.N., and Kaplansky, A.S. 1992. Morphological

studies of bone and tendon (Suppl.). Journal of Applied Physiology 73: 10S-13S.

Horowitz, S.M., Doty, S.B., Lane, J.M., and Burstein, A.H. 1993. Studies of the mechanism by

which mechanical failure of polymethylmethacrylate leads to bone resorption. Journal of Bone

and Joint Surgery 75A: 802-813.

Lee, J-M., Salvati, E.A., Betts, F., Doty, S.B., Dicarlo, E.F., and Bullough, P.G. 1992. Size of

metallic and polyethylene debris particles in failed cemented total hip replacements. Journal of

Bone and Joint Surgery 74B: 380-384.

Szulczewski, D.H., Meyer, U., Moiler, K., Stratmann, U., Doty, S.B., and Jones, D.B. 1993.

Characterization of bovine osteoclasts on an ionomeric cement in vitro. Cells and Materials

3: 83-92.

221
ALTERED CHONDROCYTE DIFFERENTIATION IN RESPONSE TO ALTERED

G-LEVEL

Pauline Jackie Duke

University of Texas Health Science Center

Dental Branch, Department of Orthodontics

Houston, TX 77225

Description of Research

Changes in loading, including those resulting from changes in the gravitational field, produce

corresponding changes in connective tissue (bone, cartilage, tendon). Our laboratory uses pre- and

postnatal exposures to altered gravity to determine the effects of such exposures on skeletal

development, with an emphasis on cartilage formation and endochondral ossification.

Many defects of the skeleton are due to defects in the chondrogenic process. The use of

gravitational fields to modulate cartilage development and, thus, to study its regulation are

enormously important, not just to development and human health in space, but also to the

understanding and eventual elimination of the myriad chondrodysplasias that occur on Earth.

Accomplishments

(1) Micromass cultures of embryonic mouse limb mesenchyme were flown aboard the

first International Microgravity Laboratory (IML-1). Both scanning and transmission electron

microscopy studies of flight culture show that little, if any, matrix was produced during space

flight. Moreover, until late in the mission (4--5 days), the flight cells were abnormally smooth,

having no matrix associated with the cells.

(2) Continuing histomorphometric studies of growth plate found that: (a) Histological

changes in growth plates of rats exposed to 14 days of continuous centrifugation (Cosmos 2G

experiment) were reduced in a manner similar to that seen in rats of comparable age and weight

flown aboard Spacelab 3. (b) Growth plates of older rats flown on Cosmos 2044 were found to

be less responsive to unloading by space flight or suspension than those of younger rats from

previous missions. (c) Collagen fibrils in both flight (Cosmos 2044) and control 15-week-old

rats, when viewed by electron microscopy, often appeared to be unraveling.

(3) Rats flown on Spacelab 3 and on Cosmos 2044 were found to have more metaphyseal

vessels at the level of the primary trabeculae than their controls. Rats flown on Cosmos 1887 did

not differ from controls in number of metaphyseal vessels, probably because of the long postflight

time at 1-g.

(4) At the level of the epiphyseal-metaphyseal junction, most differences in vascularization

were found to be due to the age of animals rather than to space flight. Younger rats (Spacelab 3 and

1887 basal controls) were found to have more cells in the last row of the calcification zone, an

estimate of column number, and more vessels that appeared to be actively penetrating a lacunae. In

both Spacelab 3 and Cosmos 1887, the cell number in the last row of the calcified zone was

increased, indicating a slowing of normal development. There was slight increase in the number of

vessels in the process of invasion of lacunae in all space flight rats, but this was significant only

for Cosmos 2044.

222
Significance of the Accomplishments

Finding 1: The "CELLS" experiment flown in January of 1992, was the first culture of skeletal

cells flown in space and remains the best. This experiment had the largest number of medium

changes and fixations ever carried out in space, and the hardware developed for "CELLS" is being

used to grow other cell types and even small organisms. The lack of chondrogenesis in cell

cultures is similar to that seen in studies of growth plate, and involves both direct and indirect

effects.

Finding 2: This was the first study to compare growth plates of centrifuged rats with growth plates

of flight rats and demonstrates the response of growth plate to an increase in baseline loading,

which, according to orthopedic principles, will slow growth plate activity. The similarities in

direction of changes in a microgravity environment and the hypergravity tested demonstrate the

need to look for a g-threshold that will maintain normal growth of the epiphyseal plate.

Our electron microscopy observations are of great significance because they constitute the first set

of ultrastructural observations of the aging growth plate. Also of major significance for

developmental studies is the fact that older growth plates respond less to unloading, either by

suspension or microgravity.

Finding 3: The effects of space flight on the metaphyseal vasculature had not been previously

studied. Our results demonstrate the relationship between vasculadzation at_h'abeculae number in

the metaphyseal region of the tibia.

Finding 4: This first study of neovascularization at the metaphyseal epiphyseal junction of the tibia

indicates that space flight slows normal development.

Publications

Campbell, M., Daane, E., and Duke, P.J. 1991. Ultrastructural aspects of chondrogenesis in

"CELLS" flight hardware. The Physiologist 34: 221.

Campbell, M., Daane, E., and Duke, P.J. 1991. Ultrastructural aspects of chondrogenesis in

"CELLS" flight hardware. The Physiologist 35: $39-$40.

Daane, B., Montufar-Solis, D., Arizpe, J., and Duke, J. 1992. Alteration in limb cell attachment

and membrane topology in microgravity: Direct or indirect effects? 5th International Congress on

Cell Biology, July 26-31, 1992, Madrid, Spain, p. 478.

Daane, E., Duke, P.J., and Campbell, M. 1991. Chondrogenesis of limb mesenchymal ceils

cultured on microcarrier beads. TSEM Journal 22: 52.

Duke, J. 1991. Effect of altered g on chondrocyte differentiation. 1989-90 NASA Space Biology

Accomplishments, p. 103-104.

Duke, P.J., Daane, E., Campbell, M., and Prewett, T. 1991. Preliminary studies of

chondrogenesis on microcarrier beads. ASGSB Bulletin 5: 82.

Duke, P.J., Daane, E., Montufar-Solis, D., and Arizpe, J. 1992. Chondrocyte topology and

matrix production in the "CELLS" space flight experiment. ASGSB Bulletin 6: 58.

Duke, J., Montufar-Solis, D., and Daane, E. 1992. Teratogenic effects of gravitational changes.

The World Space Congress, August 28-September 5, 1992, Washington, DC, p. 534.

223
Duke, P.J., Montufar-Solis, D., and Daane, E. 1993. Studies of chondrogenesis in rotating

systems. Journal of Cellular Biochemistry 5 I: 274-282.

Duke, P.J., Montufar-Solis, D., and Daane, E. 1993. Teratogenic effects of AG: The concept of

allometric growth. The Physiologist 36: $34-$37.

Duke, P.J., Montufar-Solis, D., and lmrek, A. 1993. Changes in extracellular matrix area and

cell size in tibial epiphyseal growth plates of SL3 rats. ASGSB Bulletin 7: 95.

Greer, B.D., Barreto, P.D., Duke, J., and Durban, E.M. 1991. Epidermal growth factor

production is a gravity sensitive process in the mouse submandibular salivary gland. ASGSB

Bulletin 5: 56.

Joubert, A., Duke, P.J., and Montufar-Solis, D. 1991. Metaphyseal vascularization of tibial

growth plates from Spacelab 3 rats. ASGSB Bulletin 5: 58.

Montufar-Solis, D. and Duke, P.J. 1991. Proteoglycans in micromass cultures of embryonic

mouse limb mesenchymal cells: Preliminary studies for the "CELLS" space flight experiment.

The Physiologist 34: 221.

Montufar-Solis, D. and Duke, P.J. 1992. Proteoglycans in micromass cultures of embryonic

mouse limb mesenchymal cells: Preliminary studies for the "CELLS" space flight experiment. The

Physiologist 35:$41-$42.

Montufar-Solis, D., Duke, P.J., and Durnova, G. 1992. Spaceflight and age affect tibial

epiphyseal growth plate histomorphometry. Journal of Applied Physiology 73 (Suppl.): 19S-25S.

Straight, C.L. and Duke, P.J. 1991. On the development of a space flight biological cell culture

system using a silicone gas permeable "bubble" chamber. ASGSB Bulletin 5: 89.

Vergara, J.0 Montufar-Solis, D., and Duke, P.J. 1992. Space flight affects metaphyseal

vascularization of rat tibias. ASGSB Bulletin 6: 50.

224
SYNAPSE DEVELOPMENT

STUDY OF THE EFFECTS

DEVELOPMENT

Raphael Gruener

University of Arizona

College of Medicine

Department of Physiology

Tucson, AZ 85724-0001

IN ALTERED GRAVITY: A MODEL FOR THE

OF SPACE MICROGRAVITY ON EMBRYONIC

Description of Research

Our continuing research program is designed to accomplish the following goals:

1. Examine specific cell processes that axe affected by perturbed gravity on Earth using the

clinostat paradigm.

2. Examine the feasibility of using primary co-cultures of embryonic nerve and muscle cells in

space flight to determine whether the reduced gravity of space affects cellular processes.

3. Develop a model nerve cell culture system suitable for flight experiments in which to verify

findings from preliminary flight data and from clinostat results.

We have previously reported (Gruener & Hoeger, 1990 and 1991) that co-cultures of nerve and

muscle cells from Xenopus embryos show specific sensitivity to altered gravity after rotation in a

slow clinostat. The alterations in cell properties included changes in cell shape, size of the nucleus,

reduced accumulation of acetylcholine receptors at the junction between the nerve and the muscle

cell, and the formation of neuritic swellings. We interpreted our results to suggest that synapse

development will be altered if it were to proceed in the microgravity of space. We hypothesized

that our results may be explained by invoking a common pathway--the cytoskeleton--as the

cellular organelle through which mechano-information is indirectly or directly transduced. To test

this possibility, we have examined cytoskeletal markers (tubulin, actin filaments, vinculin) in both

neurons and myocytes after clinorotation, and more recently after space flight. Our preliminary

results strongly suggest that consistent changes in cytoskeletal structure may correlate with the

changes we have observed both in the clinostat and after space flight. Taken together, our data

lead us to believe that specific cellular processes can be examined in space flight and that findings

from space flight experiments can be used to test the validity of the clinostat paradigm. To the

extent that the clinostat paradigm is verified, it would appear prudent, cost effective and

scientifically compelling to use this Earth-bound simulation in order to define, refine, and optimize

experiments to be carried out in space flight where access is restricted and where the cost of

carrying out experiments is considerably higher.

Accomplishments

(1) Effects of Vector-Averaged Gravity on Bead-Induced Receptor Accumulation in

Embryonic Muscle Cells. In preparation for flight experiments, where the objective is to obtain

maximum critical data from a sunplifled model system, we used polystyrene beads to replace

neurons as the trigger agent for receptor accumulation in muscle cells. Beads act like neurons in

that they induce receptor aggregation at the point of contact between the bead and the myocyte. We

have previously shown that nerve-induced receptor aggregation is defective after clinostat rotation.

Using beads served two purposes: First, it would narrow down the source of the defect,

furthermore, it would simplify the cell culture system (only one cell type would be needed) as a

model in which to test synapse development in the microgravity of space. Our results indicate that

bead-induced receptor accumulation is significantly reduced; and, therefore, we conclude that in the

altered gravity of the clinostat the myocyte processing of the signal (from either the nerve or the

225
bead)is defective. Second,our results encouraged us to use the bead-myocyte culture system in

space flight experiments (vide ircfra).

We have repeated this experiment in space flight (STS-54) and found a reduction of about 50

percent in the number of acetylcholine receptor clusters associated with bead contacting myocytes.

Further, we found that the size of the clusters from flight cells, where they did aggregate near the

beads, was significantly smaller than in the ground control cells. These data are in complete

concordance with our reported findings from clinostat simulations examining either nerveassociated clusters or bead-associated receptor clusters. These results are of significant importance

as they validate the use of the clinostat as a model for examining the effects of altered gravity on

cellular processes.

(2) Effects of Vector-Averaged Gravi_ on Single Channel Properties of the Acetylcholine

Receptor in Cultured Myocytes. As a followup on our earlier findings of reduced receptor

accumulation after clinostat rotation (vector-averaged gravity), we tested the possibility that not

only was receptor accumulation at the neuromuscular synapse defective, but that the apparent

disconnection of receptors from their anchoring cytoskeleton also produced changes in single

receptor channel behavior. To test this possibility, we examined the AChR channel properties after

clinostat rotation of myocytes with, and without, neurons. We found that there was a reduction in

the channel open time in receptors located close to the nerve. Figure 1 shows single channel events

recorded from a control (1A) and a rotated myocyte (1B). The reader should note the overall

reducti()n in open-time event duration in the rotated myocytes. A compilation of all open tirre,.s into

a frequency histogram (Figure 2) shows that open-time events from a rotated cell cluster at shorter

times than in a control cell (the time constant is shortened from 4.0 to 2.2 msec). There was,

however, no effect on channel properties in non-junctional receptors, and channel conductance was

unaffected. These data further support the idea that vector-free gravity produces specific alterations

in cellular properties which may significantly affect synapse development and efficacy in the

microgravity of space.

1331.2 ms ,_A,w_ __4_4_ i_

1433.6 ms _

1638.4 rns __ r_qd__i _d,d

1740.8 ms _

2252.8 ms _

11.674 s

11.878 s

1'1.981s

12.083 s _

12186 s

2355.2 ms_

2457.6 ms

Trace: 1024 samples (10_.40 ms) 9.0_-_----I

Plotnotes: 48 h, control,junctional 10 pA'

12.288 s _

12.390 s b __ p_l_bd_q_--

Trace: 1024 samples (10_.40 ms) 9.0_-'_

Plot notes: 48 h,llrpm, junctional 10 pA'

Figure 1 Junctional AChR single channel activity from a control

(A) and a rotated myocyte [48 hr at 1 rpm] (B)

226
F,le _2et?rOS

770 COU_tS

30 Bin*

t5 N/_k_

.1-4 O4

D_,II I.vel _

Hl_toQrlm

. co_ro unctlo..

o'o ',o% ',5S' 2olo _!o --' _oo

Timl (1 II/d_v)

File- R2625FI0

296 Counts

30 Bins

10 N/dlv

W_-269.52

71-2,19

Dwell level i

Htstogcam

4B h. | rpm _unction=]

I

/

l

0 6,0 t2.0

Figure 2.A Figure 2.B

.... i"" , ,

180

Time (I ms/o_v)

24.0

Figure 2 Open-time histograms for junctional AChRs from a

control (A) and a rotated myocyte [48 hr at 1 rpm] (B)

(3) Effects of Spaceflight on Cultured Myocytes. We had an unprecedented opportunity

to participate in the STS-52 and STS-54 Shuttle flights through the courtesies of Mr. John

Cassanto (ITA Inc.) and Dr. Marian Lewis (University of Alabama, Huntsville). Our myocyte

cultures were placed in the Material Dispersion Apparatus (MDA) hardware wells (ITA, Inc.) and

fixed onboard the Shuttle 10 days after launch. The ceils survived the flights very well and

provided a wealth of information on the behavior of these cells in the microgravity of space. This

information can now be correlated with the changes from clinostat simulations as we previously

reported. Preliminary analysis of flown cells, when compared to synchronous ground controls,

shows that flight cells had a significantly smaller surface area than any of the controls (Figures 3

and 4). In addition, flight cells also had the smallest nuclei compared with all controls. Because of

fixation, we were unable to measure cell thickness and therefore cannot estimate cell volume.

Therefore, to determine whether the change in surface and nuclear areas are actual, we calculated

the nuclear-to-cell-area ratios. This calculation shows that the ratio for any of the cells, including

the flight cells, remained constant. This implies that the changes we observed in flight cells are due

primarily to reduced cell spreading.

7000

6000

5000

6"

4000

300C

2000

1000

0

average cell area

S L T G F

Figure 3

average number of yolk platelets

(per W2 myocyte area)

015

[] S-Standard Control(n=24)

rr_L;L_b coo_,o,(o:81) %

_, o;o

[] T Transport Control(n:25) o_

I_ 0:Grou-dCo,_t_o,(n:109)

I F Fight ceils(rl 1!9} O- 005

o

S L T G F

Figure 4

]S-Standard Cantro(n 24:1

[]_[ lab Ccntrol(r 81)

[] t Transport ControJ(n 2S)

I_ G-Ground Cont r ol(n= lOg'l

• F-F hght ce_Is(r-tl9_

Next, we estimated the metabolic and developmental status of the myocytes based on the number

and surface area of yolk platelets in myocytes. The yolk platelets are the source of metabolic

energy of the cell. They are consumed as a function of development. Initially, the cells are packed

full with yolk platelets; their number decreases steadily with development, and normally they are

almost absent after 6-8 days in culture. Flight cells had a larger area occupied by yolk platelets.

i

30.0

227
Figure5: A typicalControl(top)andspace-flown(bottom)Xenopusmusclecell.Notethe

smaller size of the flighted ceils and the over-abundance of yolk platelets (phase bright bodies) that

indicate a significant slowing of development in space-flown cells.

This suggests that the flight cells' development was arrested at a stage equivalent to ca. 2 days.

Further confh'mation of this finding comes from the analysis of the number of yolk platelets which

we found to be significantly higher in flight cells than any of the control cells (Figures 3 and 5).

This finding is further substantiated by our finding that the absolute number of yolk platelets

calculated per square micron area of myocyte is the highest in flight cells.

Further, we measured cell shape and correlated this shape to the length and linearity of actin

f'daments, a major component of the cytoskeleton. We found that flight cells were shorter than all

control cells and the length of actin filaments was also shorter than controls. The relative linearity

indicates that control cells had a more linear array of actin filaments. Finally, flight cells were

found to have a significantly reduced number of vinculin clusters (a cytoskeletal marker associated

with the infrastructure of acetylcholine receptors) and that the average size of the clusters was

smaller than normal.

228
Theresultsof theNERMUS experimentonthe STS-52Shuttleflight hasdemonstratedthat the

MDA hardwareis most suitablefor cell-attachedcells. Cell recovery wasadequate. Cells

appearedto haveremainedviablebutshoweda numberof changesconsistentwith theeffectsof

microgravity(reportedalsofor othercell typesandfor our cellsin theclinostat). Fromthatpoint

of view,theexperimentwasasignificantsuccess.

Significance of Accomplishments

Our results to date indicate that nerve and muscle cells axe sensitive to alterations in the gravity field

whether through use of the clinostat or in actual space flight. Our prehminary results from the two

space flight experiments indicate that the clinostat is a very useful paradigm through which

experimental design can be focused and through which cellular processes likely to be affected by

altered gravity may be defined. Thus, the clinostat is a useful tool for preparing rigorous

experiments for space flight. In addition, our results show that there is considerable concordance

between results obtained in the clinostat and those from actual space flight. Specifically, the

retardation of cellular development (measured in the density of yolk platelets), and the alterations in

cytoskeletal organization (presence of vinculin clusters and linearity of actin filaments) appear to be

affected in a similar manner in the clinostat and in space. On the other hand, cell size and nuclear

size showed the opposite effects, becoming larger in the clinostat and smaller (than the appropriate

controls) in space-flown cells.

Taken together, these results provide further support for our earlier conclusion that synapse

development and the development of excitable cells (nerve and muscle) are likely to be significantly

affected by the microgravity of space. Further flight experiments, in which specific cellular

processes are rigorously examined, are therefore warranted. Our preliminary results show that

such experiments are highly feasible even under suboptimal conditions (e.g., no intervention

during flight, unexpected problems such as launch delays, and temperature variations). We hope

to have the opportunity to continue with these experiments in the near future.

Publications

Gruener, R. and Hoeger, G. 1991. Bead-Induced AChR Receptor Aggregation is Defective in

Clino-Rotated Embryonic Muscle (Abstract). ASGSB Bulletin, Vol. 5(1), p. 70.

Gruener, R. and Hoeger, G. 1991. Vector-Averaged Gravity Alters Myocyte and Neuron

Properties in Cell Culture. Aviation and Space Environmental Medicine 62:1159-1165.

Reitstetter, R. and Gruener, R. 1992. Vector-Averaged Gravity Alters Kinetics of Acetylcholine

Receptor Channels in Cultured Embryonic Muscle Cells (Abstract). ASGSB Bulletin, Vol. 6(1),

p. 60.

229
MICROGRAVITY-INDUCED EFFECTS ON PITUITARY GROWTH HORMONE

CELL FUNCTION: A MECHANISM FOR MUSCLE ATROPHY IN MANNED SPACE

FLIGHT

W. C. Hymer

Department of Molecular and Cell Biology

Penn State University

University Park, PA 16802

Description of Research

Growth Hormone (GH) secreted from the anterior pituitary gland has a number of effects on

body tissues. These include, but are not limited to, increased protein synthesis in the liver,

increases in muscle mass, increased fat metabolism, stimulation of immune tissues, and increases

in blood cells. The rationale for this research stems from a series of experiments which indicate

that the production or release of GH is inhibited in microgravity (ttg). The first indication that

GH is inhibited in space occurred in 1983 on a cell culture experiment on the STS-8 mission.

This was followed by a series of experiments utilizing whole animals flown on the U.S. Space

Shuttle and the Russian COSMOS biosatellites, all of which continued to demonstrate a

deficiency in the secretion of biologically active GH after space flight.

Since growth hormone has so many important metabolic effects on organ systems of interest to

NASA, our goals were to: (a) definitely establish whether or not a secretory defect does indeed

exist, (b) determine the mechanisms involved, and (c) attempt to determine if the effect is

reversible.

To accomplish the goal of identifying the nature of the secretory defect, a pituitary cell culture

experiment was designed that utilized 165 cell culture vials that would be incubated in a middeck

locker aboard the space Shuttle. The large number of vials would allow us to examine many

different variables at one time, perhaps allowing us to identify which of the variables were

important in the observed hormone reductions.

Accomplishments

(1) The experiment was flown on the STS-46 mission in July-August 1992. This

experiment utilized 165 culture vials inoculated with rat pituitary ceils in a middeck incubator

and the 165 corresponding ground controls. Tested were pituitary cell fractions enriched in cells

which store a highly active form of GH, fractions depleted of this cell type, cells from various

regions of the pituitary which demonstrate different activities, and several types of cell culture

media. After flight, the cell cultures were returned to the laboratory and processed. This

processing included hormone analysis on all the flight and ground control media, extended cell

cultures for some of the cells to test the time needed for the cells to recover, morphometric

analysis of recovered cells from the culture vials, and biological stimulation of GH release. This

experiment yielded over 1800 separate samples to be analyzed.

(2) A separate experiment was conducted several months later to duplicate the

temperature profile of the flight experiment as recorded by flight instruments. This experiment

again resulted in over 1800 separate samples to be analyzed.

231}
(3) Preliminary analysis of all the samples has been completed. These tests included

assay of growth hormone and prolactin (PRL) levels on over 3600 samples, cell response to

growth hormone releasing hormone (GHRH), determining the biological activity of GH and

PRL, morphometric analysis of the cells that were recovered, and the distribution of hormone

within the cells.

Significance of the Accomplishments

Finding 1: The significance of the experiment lies in the fact that this was the first cell culture

experiment flown that specifically targeted the cellular basis of hormone reduction seen in intact

animals. It may be possible to begin to identify the molecular changes which result from

exposure to microgravity. Any future design for treatment of the hormone reduction in animals

will obviously require knowing where the defect is. This may have importance in future longterm manned flights.

Finding 2: The cell culture system worked. This system allows large numbers of variables to be

tested while minimizing the space and power needed, an important consideration in space flight.

This system should be adaptable to other cell-culture-based experiments.

Finding 3: The analysis of the experiment is still underway, and the significance of the

experimental results has not yet been determined.

231
ELECTROPHORETIC SEPARATION OF CELLS AND PARTICLES FROM RAT

PITUITARY AND RAT SPLEEN

W. C. Hymer

Department of Molecular and Cell Biology

Penn State University

University Park, PA 16802

Description of Research

This experiment is designed to explore the kinetics and molecular basis of endocrine deficits

which occur in microgravity (lag). The first part is a pituitary cell culture experiment to

determine changes in pituitary cell function in lag as a function of time. The second part utilizes

the Japanese-designed Free Flow Electrophoresis Unit (FFEU) to separate pituitary cells into

functional subtypes with subsequent cultures. The third part utilizes the FFEU to separate

subcellular components obtained from pituitary cells cultured in lag.

In man, both Growth Hormone (GH) and Prolactin (PRL) are multifunctional hormones

produced by the pituitary that are essential for life. While their existence has been known for 25-

50 years, the biochemical details and functional significance of molecular events involved in

their synthesis and release from pituitary cells are still incompletely understood. Moreover, the

body targets for these two hormones are numerous, including bone, muscle, fat, blood, and the

immune system. Since GH and PRL affect a number of organs in the body that are known to

change in lag, there is a strong rationale for evaluating functional changes in these hormones.

Rats and rat pituitary cells have been flown under both lag and lag-mimicked conditions. The

results of these experiments demonstrate repeatable and statistically significant changes in the

.pituitary GH and PRL cell systems. While most of the studies have utilized whole animals flown

m space, an experiment conducted on STS-46 in July 1992 utilized cultures of rat pituitary cells.

While analysis of this experiment is still underway, key findings to date show that lag-induced

changes occur within the pituitary GH/PRL cell, and strongly support the idea that the changes

seen m pituitaries of space flown rats are explicable by changes within the pituitary cells

themselves.

The value of utilizing the FFEU for this experiment lies in the realization that, while flow

electrophoresis has been used to separate cells and organelles in the past, there is agreement that

gravity limits resolution of separation because of sedimentation during exposure to the electric

field, zone sedimentation due to density gradients (droplet effect), and convection due to heating

of the sample in the electric field. The only environment where all these instabilities can be

overcome while operating with higher sample loads is in lag. This fortuitous circumstance allows

us to utilize a research technique uniquely adapted to lag and to examine biological changes that

occur in lag.

Accomplishments

(1) We have cultured rat pituitary cells, recovered them from the culture vessel, and

electrophoretically separated them utilizing the FFEU. Cells separated in this manner

demonstrated a differential mobility, with an enrichment of GH cells in the most mobile

fractions, and allows for extended culture of the separated subtypes of cells.

232
(2) Cells culturedin the vesselshavebeensuccessfullylysed,thelysateconcentrated,

and the concentratedlysate separatedon the FFEU. This separatedlysatecan be detected

utilizing the FFEU ultraviolet absorptiondetector. The successof the separationhasbeen

documentedby assayingboththeimmunodetectableandbioassayablelevelsof GH andPRL.

Significance of the Accomplishments

Finding 1: The recovery of the pituitary cells from the culture vessels and subsequent

electrophoretic separation indicates that the experiment carries a high probability of success, and

that the anticipated enrichment of the GH cells and PRL cells will permit detailed analysis of the

functionality of the FFEU. The enriched GH and PRL cell fractions are expected to yield

valuable clues as to the cellular source of the hormone disturbances.

Finding 2: The successful separation of the lysate indicates that with this experiment we can

begin to detail the changes in the molecular structure and function that occur when the hormone

is synthesized and packaged within the cell in I_g.

233
CELLS, EMBRYOS AND DEVELOPMENT IN SPACE

A.D. Krikorian

Department of Biochemistry and Cell Biology

State University of New York

Stony Brook, NY 11794-5215

Description of Research

The problems of development constitute a major part of the objectives of modern biology. One aim

of our research effort is to develop and study systems at different levels of initial organization that

will enable the effect of microgravity to be tested on the behavior of plants, in contrast or in

comparison to their performance at 1-g and in ground controls. While the initial focus is aimed at

the broad effects of gravity that operate on systems as they grow and develop, the systems are

adaptable to a variety of more specific tests that focus on mechanisms. Any of our test systems can

be used to ascertain whether there are differences in the normal rate, frequency, and pattern of cell

division or in the fidelity of partitioning of the cell chromosomes during or after exposure to space

flight; and, if so, to move toward understanding why these differences have occurred. All this

enables us to examine what I call "genome organization and variation in plants in the space

environment."

It is clear the plant genome is in a state of flux with many changes occurring during development

and reproduction. Variation can occur in response to a range of external and internal influences

and stresses. We have encountered examples of these nuclear responses and variations in spacegrown somatic embryos, tissue-culture-derived platelets, and seedling-derived materials. The

genome changes range from chromosomal perturbations of various extreme sorts such as

breakage, translocations, and deletions to ones that are milder, but very significant, such as double

nuclei.

A fuller characterization and understanding of the causes of these genomic changes is in order.

They may occur in highly repetitive fractions of the genome and are probably limited to a specific

subset(s) of DNA sequences at particular chromosomal sites. I believe that a function of these

genomic variations is the generation of a radical, but limited, reorganization of the genome in

response to stress situations in order to increase the pool of variability from which new types of

cells can be selected or emerge. These reorganizations would be of adaptive value in microgravity.

The objective in this part of the work is to study these changes in mitotic and, where possible,

meiotic cycles in space-grown materials from many viewpoints, including specificity, fate, and,

especially, the cause or the mechanism.

Our investigations in the past period have been directed at (1) those dealing with continued analysis

and interpretation of flight data, and (2) those dealing with ground-based research. The objectives

of the ground-based research include the following:

(a) A better understanding of the induction of active growth and cell proliferation and

development in otherwise mature cells as they exist in situ and as they become established as

embryogenic cultures that are amenable for use in flight experiments; and, further, as a basis

for studying how the chromosomes behave, how the genome functions in development, and to

examine any gravitational component. The approach we use for establishing cultures has

increasingly involved working with short-term pulses of growth regulators or, in the complete

absence of exogenously added growth regulators, with increased emp.hasis on physical factors

that significantly affect the induction and progression of embryogenests.

234
(b) Themultiplicationof embryogeniccell culturesandtheproductionin cultureof cell units

andtheir contrasteddevelopmentintounorganizedmasses,ontheonehand,and,ontheother,

assomatic(non-zygotic)embryosandfrom theseintoplantlets.

(c) Thegrowth,morphogenesis,andmetabolismof intactplantletsandtissue-culture-derived

propaguleswith theirestablishedgrowingregionsof shootandroot, in responseto interacting

factorswhicharebothenvironmental(i.e.,differentregimesof photoperiodicityandchanging

temperatures)andnutritionalcues.

(d) Thedevelopmentof cytological/karyologicaltestingprotocolswhichhavea highlevel of

reliability for establishingdetailedchromosomalcharacteristicsand profiles for the plant

specieswith whichweareworking;while,atthesametime,seekingto extendtheprinciplesso

gainedto a still broaderrangeof species.

(e) Themanagementof pluripotentcell culturesystemsfrom theperspectiveof beingableto

usethemeffectivelyandwithaminimumof humaninterventionin aspaceenvironmentsetting.

(f) Continuedgatheringandanalysisof datafrom CHROMEX.

(g) Further analysisof Plant Cell Culture Research(PCR) from our experimenton the

SpacelabJ mission.

(h) Continuedwork fortheimplementationof acellcultureexperimentonIML-2

(July, 1994).

Accomplishments

(1) CHROMEX.related findings. The production and growth of roots in two in vitro

generated population categories of Haplopappus gracilis, which were cultivated for 5 days aboard a

NASA Space Shuttle (STS-29) were analyzed in depth. Selective trimming of preformed roots

was used in the experimental protocol. This selective trimming allowed production of up to 50

roots per plantlet and ensured availability of roots for study in the event of an unexpectedly

curtailed period in space. Asepsis was maintained throughout the flight experiment despite the use

of relatively complicated hardware. Roots were generated (a) laterally from pre-existing roots, the

tips of which were severed at the time of plantlet insertion into the growth substrate; (b)

adventitiously from the basal or cut ends of micropropagated shoots; and (c) de novo as a purely

space-environment-derived generation of roots, which had its origin from roots that emerged

during the period in space. Root growth occurred randomly in all directions in space. In contrast,

growth was uniformly positively gravitropic in ground controls. In the spaceflight and ground

controls, both population categories produced an equivalent amount of tissue when compared to

each other and maintained their characteristic root production patterns. Seedling-derived plantlets

produced roots which were numerous but relatively short. Capitulum-derived plantlets produced

fewer roots, but they were, on average, longer than those of the seedling-derived plantlets. Thus,

the clonal root phenotype was not changed in space, at least for the short duration of the

experiment. However, both population categories exhibited total root production values that were

67-95 percent greater than those obtained in their ground control counterparts. We have proposed

that microgravity brought about an altered moisture distribution pattern within the horticultural

foam substrate that was used as the growing environment, giving a more moist and thereby more

favorable environment for root formation. The detailed data for and significance of all this for

space flight experimentation is covered in depth in a submitted manuscript.

(2) The PCR experiment on the Spacelab J mission. Embryogenic carrot and daylily cells

were exposed to space throughout an 8-day flight. The flight was launched Saturday morning

September 12, 1992, on the Shuttle Endeavour. The middeck experiment, referred to as "Gravity

235
and OrganizedDevelopmentin AsepticallyCulturedPlantCells" (PCR,PlantCell ResearchExperimentNo. 54), waslaunchedon SpacelabJ. Theobjectiveswere(a) to evaluatewhether

spaceflight affectedthe patternanddevelopmentalprogressionof embryogenicallycompetent

daylily and carrotcells from one well-definedstageto another,and (b) to determinewhether

mitosisandchromosomebehaviorweremodifiedbythespaceenvironment.

The PCR experiment was a passive one and did not involve astronaut intervention. Cultures of

embryogenic cells distributed on a semi-solid nuwient culture medium in metal Petri dishes were

prepared at Stony Brook and transported to Kennedy Space Center. The methods employed

consisted of the following: (a) Embryogenically competent cells (from a 200--400 fraction of carrot

and daylily cell cultures) were grown in plant cell culture chambers obtained from NASDA. (b)

Two dishes (plant cell culture dishes)---one for each species--were used to grow cells on a semisolid agar support. (c) Progression to later embryogenic stages occurred in space via the pHaltering metabolic activity of the cells as they grew. The timing of the setup of the experiment thus

was very important. (d) Progression to later stages in the case of such embryogenic semi-solid

cultures did not need crew intervention. (e) Living somatic embryos were returned for continued

post-flight evaluation, development, and grow-out.

Results indicate that somatic embryogenesis occurred during the flight. Analysis of data still is in

progress. When we study the temperature prof'de that the cultures experienced during the mission,

we will be able to ascertain whether the apparent increase in growth observed was due to the

elevated temperature level of the flight environment or whether there was a real increase it_ rate of

development. Significant alterations in the karyology of somatic embryos developed in space were

found. The bulk of our analysis has thus far been carried out on the daylily, but the carrot shows

similar effects as well. The responses include a substantial number in flight samples of binucleate

cells among those that are normally uninucleate. The ground control samples are uniformly

uninucleate (see Table 1 with partial data).

Table 1. Effect of Space Flight on Cell Division in Daylily Embryos

[Over 300,000 somatic cells scored]

Phase Controls, % Flight, %

Cells in division 2.9 0.5

Interpretable metaphases 32.1 1 1.36

Double nuclei 0 8.2

Chromosome breaks 0 1.4

Until we receive radiation dosimetry data promised from the National Space Development Agency

of Japan (NASDA), it will not be possible to state categorically that radiation damage was not

responsible for any of the mitotic disturbances; but, in a personal communication from Dr. S.

Nagaoka, radiation levels were said to be in the expected range.

Since our methodology for karyotype analysis involves treatment of cells and somatic embryos

with colchicine, we have taken care to eliminate the possibility of an increased sensitivity of the

flight-exposed cells to colchicine, leading to a doubling of the nuclei. Serial sampling and

examination of flight samples after recovery, beyond what we refer to as sampling 1 (i.e., the first

sampling after recovery), indicates that the number of binucleate cells are diminished in some but

are not eliminated. We are rearing embryos into plantlets of both flight and control samples to do

followup studies. In addition to the condition of double nuclei, various aberrations in chromosome

structure have been encountered as well. These will be fully quantified in due course.

An expanded version of the experiment will be carried out on IML-2 scheduled for 1994. It is

anticipated that the data from both experiments will permit us to place the events of somatic

236
embryogenesisandcell division in spaceon the firm footing that it deserves. I believe that not

only will answers obtained from space studies with such systems be of interest to developmental

plant biologists but, also, they will have significance for those seeking to use biotechnological

procedures and manipulations in space for a variety of reasons, including a Controlled Ecological

Life Support System (CELSS). Moreover, the ability to use and manipulate plant cells and other

kinds of propagules in vitro reliably in space will be a necessary prerequisite to many projected

commercialization schemes.

(3) Characterization of embryogenic cells from the perspective of their origin, potential,

and behavior continues to be a top priority so that these systems can be used with a high degree of

reliability in a space flight context. We have devised and perfected a simple method whereby it is

possible to remove a full range of units---both embryogenic structures or cells that may not have

responded--non-invasively from an assay system. With the development of this simple but

elegant procedure, it becomes possible to remove all the test units for examination with no

perturbation whatsoever. The invasive methods used to date have been to remove embryo samples

by means of forceps and to manipulate them, either singly or in small groups, depending on how

many can be handled at a time. Cells could not be removed very easily.

With our method, after cells are sorted and the appropriate fraction is ready for distribution and

testing, embryogenic cells are controllably inoculated onto dialysis membrane by means of a

pipette. This spreading of cells on various interfaces, such as polymer membranes, films, carbon

and ion exchange papers, etc., and various combinations or sequences of these, has made it

possible to follow embryogenic events in much greater detail than has hitherto been possible. For

example, cells grown in liquid culture under controlled gas environments have been spread on

membranes in Petri dishes on various media for varying periods of time and then removed by

lifting of the whole membrane and transferring to a different or challenge environment. The test

parameters first scored have focused and will continue to focus on growth rate; level of

development; number, size, and weight of organized units (somatic embryos, pre-shoots, etc.); the

stage and morphological fidelity of their development; and the level of development of epidermis

(or protoderm).

Significance of the Accomplishments

The significance of the flight data is that a clearer understanding of the effects of space flight on

plant development, at least in the near-term, is beginning to emerge. Some of the seeming

contradictions and disparities in various publications over the years can now be explained on the

basis of use of materials that have had a specific cultural history or origin. The biological nature of

the test systems, the way in which they are grown, and their predisposition, by virtue of the flight

hardware and systems, to respond to the conditions of space flight (both direct and indirect) have a

major impact on the outcome. Other discrepancies appear to derive from different strategies that

given systems have available to adapt to perturbations from ",he flight environment. A series of

cascading responses initiated from perturbations from the outside, which translate to the internal

operations of ceils, is manifest.

In the case of the behavior of the nucleus, the story seems to be more complicated, but the

implications are farreaching. One can predict that, in the longer term, growth patterns of sensitive

systems will be impacted in a major way. The development of plant tissues requires that a strict

control of the planes of cell division be in place. The fact that what appears to be a pattern of

mitotic waves without daughter cell wall development in somatic embryos indicates that the

machinery to put down wall material in the successive pattern of division was perturbed. Yet, two

patterns of cell division occurred in the cells of somatic embryos of daylily and carrot that were

grown in space. The first is a more random, normal if you will, pattern involving uninucleate

cells; the other, a successive pattern within other cells of the same complex wherein, for one reason

or another, the wail is not laid down.

237
As for the chromosomal perturbations, one is tempted to invoke effects on the spindle but this has

not yet been directly demonstrated. The role of the cortical microtubules has attracted some

interesL and it is known that a cortical system which forms just before prophase (the pre-prophase

band) plays an important role in determining the division planes, since later the phragmoplast

(wall) grows out exactly to the site of the pre-prophase band. The microtubules cannot

"memorize" the planes, since the pre-prophase band disappears before metaphase. During space

flight, spindle disturbances lead to the range of nuclear aberrations encountered. The embryogenic

cell culture system has shown that daylily and carrot somatic embryos can serve as a model for

study of the effects of the space environment on a range of cytological or karyological events. The

aseptic experiments we have done in space in NASA's Plant Growth Unit, an apparatus designed

to grow small whole plants, are labor intensive to manage. Some environmental parameters, such

as air flow and water delivery, so far, are complicated to measure, monitor, maintain, and

document with precision. We have shown that it is more convenient and more readily controllable

to use developing systems as surrogates for whole plants in certain kinds of studies.

Publications

Krikorian, A.D. 1992. Chromosomes and plant cell division in space. Presented in Cell

Responses to Microgravity (Abstracts), Workshop at 5th International Congress on Cell Biology,

27 July 1992, Madrid, Spain, pp. 470 and 479.

Krikorian, A.D., Kann, R.P., and O'Connor, S.A. 1992. An experiment to evaluate the

progression of somatic embryogenesis in space. ASGSB Bulletin 6(1): 70.

Krikorian, A.D., O'Connor, S.A., and Kann, R.P. 1993. Mitotic disturbances in daylily

(Hemerocallis) somatic embryos after an 8-day spaceflight. ASGSB Bulletin 7(1): 38.

Krikorian, A.D. and Smith, D.L. 1992. Somatic embryogenesis in carrot (Daucus carom). In:

Plant Tissue Culture Manual: Fundamentals and Application (Lindsey, K., Ed.), Dordrecht, The

Netherlands, Kluwer Academic Publishers (PTCM-A9), pp. 1-32.

Levine, H.G. and Krikorian, A.D. 1992. Chromosomes and plant cell division in space:

Environmental conditions and experimental details. Advances in Space Research 12(1): 73-82.

Levine, H.G. and Krikorian, A.D. 1992. Shoot growth in aseptically cultivated daylily and

haplopappus plandets after a 5-day spaceflight. Physiologia Plantarum 86: 349-359.

Levine, H.G. and Krikorian, A.D. 1992. Value of linear additive models for interpreting

spaceflight results. ASGSB Bulletin 6(1): 52.

Levine, H.G., Obenland, D.M., Brown, C.S., and Krikorian, A.D. 1993. Baseline data and

design considerations in plant tissue sharing effort. ASGSB Bulletin 7(1): 38.

Markert, Clement L. and Krikorian, A.D. 1993. Life: Origin and evolution on Earth; How can

we escape? In: A Scientific Role for Space Station Freedom-How Space Platforms Can Serve

Research in Life Sciences. Presented at a Symposium at AIAA Space Programs and Technologies

Conference (AIAA Paper 1348, NASA TM 4502), Huntsville, AL, March 24, 1992, pp. 35-39.

Smith, D.L. and Krikorian, A.D. 1992. Low external pH prevents cell elongation but not

multiplication of embryogenic carrot cells. Physiologia Plantarum 84: 495-501.

238
MECHANISM OF CONTROL OF BONE GROWTH IN MICROGRAVITY

Marian L. Lewis

Department of Biological Sciences

University of Alabama

Huntsville, AL 35899

Description of Research

The growth and repair of bone is a complex and poorly understood process. Over the past three

decades, it has been noted that astronauts lose large amounts of calcium and bone when exposed to

the microgravity of space flight. The mechanism by which bone growth is regulated in not known;

however, clinical observations have demonstrated that increases of endogenous cortisol, as seen in

Cushing's syndrome, are associated with bone loss and osteoporosis. In the Skylab missions,

urinary cortisol of the nine crewmembers increased almost two-fold. These data suggest that the

glucocorticoids could also play a role in the loss of bone during space flight. The laboratory of my

collaborator, Millie Hughes-Fulford (MHF), has previously shown, using osteoblast cultures, that

glucocorticoids cause a decrease in endogenous prostaglandin E2 synthesis accompanied by a

decrease in osteoblast growth and mineralization. Using a cloned osteoblast cell line as a tool to

study the cellular changes in growth, we have tested the hypothesis that space flight plays a role in

growth regulation by testing for alterations in prostaglandin synthesis, growth, and morphology of

bone cells in space flight.

Accomplishments

My collaborator (MHF) and I have selected as the bone model, the MC3T3-E1 cell line, a cloned

osteoblast line that retains synthetic functions of normal bone tissue including production of

alkaline phosphatase, prostaglandin E2, and mineralized matrix containing collagen type 1 and

hydroxyapatite. In these studies, we have serum-deprived, cloned osteoblasts pre-flight and added

serum during space flight to test the ability of serum to stimulate osteoblast cell growth in

microgravity.

(1) We developed and perfected the technique for bone cell growth on small coverslips in

microgravity on the Consort 5 sounding rocket and Shuttle flight STS-52.

(2) We have flown the MC3T3-E-I cells on STS-56 and found that mammalian

osteoblasts have reduced glucose utilization in space. These results agree with the work completed

on Skylab with the Hayflick WI-38 cells in 1973.

(3) Using microscopic methods, we have noted reduced cell growth in microgravity

conditions.

(4) In addition, the actin cytoskeleton of the flown osteoblasts has a reduced number of

actin stress fibers, an altered actin cytosketal morphology, and a changed cell morphology

compared to ground control cells.

(5) We have also shown altered prostaglandin synthesis capacity of the flown osteoblasts

as compared to the flight profile controls.

(6) These observations suggest reduced growth and DNA synthesis in bone osteoblasts

grown in microgravity. We are currently perfecting new molecular biology methods to allow us to

quantitate specific messenger RNA content in osteoblasts grown in microgravity conditions.

239
Significance of Accomplishments

Biomedical studies of crewed space flight have consistently indicated a loss of weight bearing

skeletal bone. Various lines of evidence from humans and animals suggest that this loss is due to a

lack of bone formation in the absence of gravity. Bio-mechanical properties of both cortical and

trabecular bones show strength deficits that appear to represent failure of normal increase in growth

rather than an accelerated loss of bone. Joint U.S./U.S.S.R. biosatellite flights of small animals

have shown clear morphological evidence that bone formation is abnormal and reduced during

flight.

The cause of this loss of bone has been postulated to be related to the lack of biomechanical

stimulation during flight, changes in cell surface or cytoskeleton and/or due to increases in

systemic hormones associated with space flight.

In order to test our hypothesis that microgravity plays a role in the regulation of bone growth, we

have used an osteoblast model to test changes in cell metabolism, cell cytoskeleton, and

prostaglandin synthesis. In this study, we demonstrated decreased osteoblast growth and reduced

stress fibers of actin in microgravity. We are still in the process of measuring prostaglandin

synthesis. Our results agree with Skylab studies that demonstrated that microgravity can cause

significant decreases in WI-38 cell metabolism. Also, epi-fluorescent microscopic studies have

demonstrated an alteration of actin cytoskeleton resulting in changes in cell morphology when

osteoblasts are grown in microgravity. These data add to the growing evidence that microgravity

directly plays a pivotal role in cell growth regulation.

Publications

Lewis, M.L., Kumegawa, M., Doty, S., and Hughes-Fulford, M. 1991. Characterization of

bone cell growth and differentiation in multi-dimensional culture (Abstract 0-4). ASGSB Bulletin,

October, 1991.

240
PLANT METABOLISM AND CELL

(MICROGRAVITY) AND ON EARTH

Norman G. Lewis

Institute of Biological Chemistry

Washington State University

Pullman, WA 99164-6340

WALL FORMATION IN SPACE

Description of Research

Variations in cell wall chemistry provide vascular plants with the ability to withstand gravitational

forces, as well as providing facile mechanisms for correctional responses to various gravitational

stimuli; e.g., in reaction wood formation. A principal focus of our current research is to precisely

and systematically dissect the essentially unknown mechanism(s) of vascular plant cell wall

assembly, particularly with respect to formation of its phenolic constituents; i.e., lignins and

suberins, and how gravity impacts these processes. Formation of these phenolic polymers is of

particular interest, since it appears that elaboration of their biochemical pathways was essential for

successful land adaptation. By extrapolation, we are also greatly intrigued as to how the

microgravity environment impacts "normal" cell wall assembly mechanisms/metabolism.

r

The following section describes recent progress related to disclosing the effect of gravity on plant

growth and development. The first area describes approaches to delineate the fundamental basis of

secondary cell wall assembly mechanisms (ground-based studies). The second area examines the

effect of microgravity proper (Shuttle flight studies) on plant cell wall formation/metabolism.

Accomplishments and Significance

(1) Ground-Based Studies: Our most recent research endeavors have addressed

development of a model system to investigate early stages of lignin/secondary cell wall formation,

the delineation of the sequential steps involved in lignin assembly, and regulation of the pathway

itself. The following has been discovered: (a) Using cell cultures of Pinus taeda (Eberhardt, et al.,

Journal of Biological Chemistry, 1993), it has been possible to attain a cell line capable of

undergoing a developmental-like transition from an unlignified primary wall to a lignified

secondary wall (~S1 deposition). Judicious carbon-13 labeling established that the lignin so

formed was a relatively high-fidelity copy of a softwood gymnosperm lignin. We have also

discovered that lignin synthesis in these cell cultures can be totally inhibited using H202

scavengers; e.g., KI (Nese, et al., Phytochemistry, 1994). In such instances, only the

monolignols, p-coumaryl, and coniferyl alcohols were formed, but without lignin synthesis itself

occurring. During lignification/secondary wall synthesis, it was also observed that p-coumaryl and

coniferyl alcohols had different metabolic fates prior to polymerization (Bernards, et al.,

Phytochemistry, 1994). Thus, coniferyl alcohol underwent facile dimer formation prior to lignin

synthesis proper;, whereas, p-coumaryl alcohol did not react until polymerization was initiated. (b)

Using Forsythia species, we have shown that three types of phenol-coupling enzymes are present.

The most remarkable is a hitherto uncharacterized stereoselective oxidase which catalyzes the

coupling of two E-coniferyl alcohols to give (+)-pinoresinol (Par_, et al., Tetrahedron Letters,

1994); this is being purified to apparent homogeneity. The other coupling enzymes are

O2-requiring lactase(s) and H202-dependent peroxidase(s), respectively, both of which afford

racemic products.

Significance: It has long been known that lignins vary with cell type and species and that

gravitational corrections result in altered lignin compositions in the tissues so affected; e.g.. a

higher p-coumaryl alcohol content. Our recent findings now provide a means to systematically

define how these compositional changes are regulated; i.e., what factors differentially control

241
individual monomericsynthesisand how precisemechanismsof assemblyare controlled in

differentcell types.A detailedunderstandingof suchprocessesis essentialif weareto definehow

lignin synthesisis inducedduringboth"normal"and"gravitationalcorrectional"developmental

processes.

(2) Space Shuttle Studies: Previous difficulties in growing plants in space have been

partially overcome by development of a nutrient agar pack (Heyenga, et al., 1994) suitable for the

uninterrupted growth/development of plants in space (Figure 1). This obviated the need for

sporadic replenishment of water/nutrients, the fluid parameters of which have been extremely

difficult to control in microgravity.

1. Polypropylene envelope

2. Gas diffusion membrane

3. Seed support matrix

4. Support column

5. Air phase

6. Germination medium

7. Basic nutrient medium

8. Enriched medium

Figure 1. WSU "Nutrient Pack."

Using this approach, it was found that T. aestivum wheat seedlings in the nutrient pack grew very

well in space with little differences observed in shoot and root tissue growth/fresh weight over the

10-day flight when compared to the corresponding ground controls. Moreover, electron

microscopic examination of cross-sections of serial sections of wheat shoots and roots revealed

essentially no differences. That is, the organelles in the cytosol were very similar, and in the root

cell walls there were no measurable changes in the cellulose microfibril orientation/cell wall

thickness as revealed by freeze-fracture/transmission electron microscopic (TEM)/scanning electron

microscopic (SEM) analyses (Figures 2 and 3). This latter observation is particularly important,

since it strongly implies that either the process guiding microfibril orientation is fully sensitive to

even a microgravity stimulus, or it occurs independent of the g-force experienced.

242
Panel A: l-g control Panel B: Microgravity

Figure 2. Freeze-fractured and deep-etched image of the outer surfaces of the parenchyma cell wall

of wheat primary root after methylamine treatment.

1-g control

N

Microgravity

, : _. _..._'_,-

Figure 3. Inner surfaces of parenchyma cell wall (see Figure 2 for other details). Arrows show

orientation of cellulose microfibrils.

Significance: This is the f'trst data set, of which we are aware, where excellent growth, relative

to the 1-g controls, was attained in space over a relatively long duration. Importantly, cell wall

assembly was not significantly affected; these results are in direct contradiction to previous studies

in which plants, it can now be hypothesized, suffered from various stresses in microgravity.

Consequendy, these results underscore the growing need for development of reliable plant growth

systems for the microgravity environment. Note also that the greater significance of these findings

must await more detailed chemical and biochemical analyses.

243
Publications

Chu, A., Dinkova, A., Davin, L.B., Bedgar, D., and Lewis, N.G. 1993. Stereospecificity of

benzylic aryl ether reductions in lignan formation: Reduction of (+)-pinoresinol and

(+)-lariciresinol. Journal of Biological Chemistry 268: 27026-27033.

Davin, L.B., Umezawa, T., and Lewis, N.G. 1991. Enantioselective separations in

phytochemistry, modem phytochemical methods. In: Recent Advances in Phytochemistry

(Stafford, H.A. and Fischer, K., Eds.). New York: Plenum Press, 25:75-112.

Eberhardt, T.L., Bemards, M., Davin, L.B., Yamamoto, E., Wooten, J.B., and Lewis, N.G.

1993. Lignification in cell suspension cultures of Pinus taeda during early stages of cell wall

thickening. Journal of Biological Chemistry 268:21088-21096.

Lewis, N.G. 1993. Plant phenolics. In: Antioxidants in Higher Plants (Hess, J.L. and Alscher,

R., Eds.). CRC Press, pp. 135-169.

Lewis, N.G. and Davin, L.B. 1992. Phenylpropanoid metabolism: Biosynthesis of

monolignols, lignans, lignins and suberins. In: Recent Advances in Phytochemistry (Stafford,

H.A., Ed.). New York: Plenum Press, 26: 325-375.

Lewis, N.G. and Davin, L.B. 1992. Stereoselectivity in polyphenol biosynthesis. In: Plant

Polyphenols: Synthesis, Properties and Significance (Hemingway, R.W. and Laks, P.E., Eds.).

New York: Plenum Press, pp. 73-95.

Lewis, N.G. and Ryan, C.A. 1992. Microgravity in plant biological systems: Realizing the

potential of microgravity research in molecular biology. A/AA 92:1347.

Umezawa, T., Davin, L.B., and Lewis, N.G. 1991. Formation of lignans,

(-)-secoisolariciresinol and (-)-rnatairesinol with Forsythia intermedia cell-free extracts. Journal of

Biological Chemistry 266: 10210-10217.

244
DEVELOPMENTAL AND PHYSIOLOGICAL PROCESSES

SEED PRODUCTION IN MICROGRAVITY

Mary E. Musgrave

Department of Plant Pathology and Crop

Louisiana State University

Louisiana Agricultural Experiment Station

Baton Rouge, LA 70803

Physiology

Shirley C. Tucker

Department of Botany

Louisiana State University

Baton Rouge, LA 70803

INFLUENCING

Description of Research

To date, the growth of plants in space has been poor. A particularly sensitive time in the life cycle

of a plant growing in microgravity seems to be the transition from the vegetative to the reproductive

phase. Most plants grown full term in space failed to produce any seed at all; and, in a Soviet

experiment in which seeds were produced, the seed quality was poor. Dosimetry readings taken in

flight have failed to explain this ubiquitous stertl|ty in terms of a radiation load; thus, some

developmental failure seems to be triggered by the microgravity environment itself.

Reproductive events in angiosperms have a number of stages that could potentially be influenced

directly by gravity. Pollen production, formation of the embryo sac, pollination, and fertilization

are all complex developmental events. No studies exist tracking the success of any of these

processes in microgravity.

Aside from the possibility of direct effects of microgravity on seed set events, secondary effects are

possible through changes in the space environment which may be brought about in microgravity.

For example, poor root zone aeration caused by the lack of directed water movement in

microgravity might restrict root respiration to such an extent that metabolites and hormones

necessary for successful development during the reproductive cycle would not be available. As a

second example, restricted gas exchange in the aerial portion of space flight plants might lead to a

buildup of volatile products which could have a negative influence on reproduction. Simultaneous

monitoring of developmental and physiological events during the reproductive cycle of plants in

space is necessary to determine whether such a relationship might exist.

In this project, microgravity effects on developmental and physiological processes affecting seed

set events are being probed with flight experiments. Arabidopsis thaliana (L.) Heynh., a

cruciferous plant used in molecular biology experiments, has been selected for the flight

experiments because of its short life cycle, minimal light and space requirements, and history of

utilization in flight experiments by the Soviets. A full life cycle is not possible on a 6-9 day Shuttle

flight, so our initial research focuses on early events in seed production. Plants for the flight

experiments are loaded into the Plant Growth Unit (I_U) flight hardware at preflowering stage.

During exposure to microgravity, pollen and embryo sac development, fertilization, and early

embryogenesis occur. The success of these processes in microgravity is determined by

comparison of the flight plants with ground control plants using ultrastructural and physiological

analyses.

245
Accomplishments

Work has been closely geared to the successful completion of the first flight experiment, SEEDEX

(Chromex.03), which flew on STS-54 in January of 1993. Preliminary observations suggest that

flowers were aborting at an early stage of development. Anomalous root tip morphology was also

observed.

(1) At Kennedy Space Center, we successfully completed two Science Verification Tests

(SVTs) of the procedures to be used in the flight experiments. Material from these studies has

served as a preliminary ground control for our flight material.

(2) In January of 1993, we successfully performed the flight experiment, SEEDEX

(Chromex.03), on STS-54. Plants flowered during the 6-day flight, and reproductive material was

fixed immediately upon return to the ground. This material is now being sectioned and analyzed in

our laboratory.

(3) We have adapted our procedures to accommodate a longer duration experiment

(9 days) on STS-51, which will be part of Chromex.04. Procedures were also developed which

allow for delay and in-flight activation of Arabidopsis for a long-duration seed-to-seed experiment

planned for Mir.

(4) Ground-based studies have investigated embryo sac and pollen developnaent in

Arabidopsis thaliana and the role of oxygen in pollen development.

Significance of the Accomplishments

In the material collected from the first flight experiment, flowering was apparently aborted at an

early stage. Through comparison with ground control material, it should be possible to ascertain

which stage(s) of early reproductive development are sensitive to rnicrogravity.

The agar medium which is used in our growth protocol provided intact root systems for postflight

observation and analysis. This will be useful for both morphological and physiological studies.

By developing a growing pr_edure that permits the utilization of preflowering plants in the PGU,

we have extended the flexibility of this flight hardware to use in all stages of the plant life cycle. It

should now be possible to address microgravity effects on all stages of plant growth and

development if the subject plant used is small and possesses a rapid enough life cycle.

Publications

Cummins, D.B. and Musgrave, M.E. 1992. Delay and in-flight activation of germination in

Arabidopsis thaliana during seed-to-seed experiments on Mix (Abstract). ASGSB Bulletin

6(1): 70.

Daugherty, C.J.

rapa differing

University.

1992. Selection and characterization of populations of rapid-cycling Brassica

in their response to rootzone hypoxia. Ph.D. dissertation, Louisiana State

Daugherty, C.J. and Musgrave, M.E. 1991. Evaluation of rapid-cycling Brassica rapa

populations exposed to hypoxic conditions (Abstract). ASGSB Bulletin 5(1): 77.

Murgia, M., Tucker, S.C., and Musgrave, M.E. 1991. Ultrastructure of the embryo sac of

Arabidopsis thaliana (Abstract). ASGSB Bulletin 5(1): 78.

246
Murgia,M., Tucker,S.C.,andMusgrave,M.E. 1992.Viability ofArabidopsisthaliana pollen in

low oxygen (Abstract). ASGSB Bulletin 6(1): 52.

Musgrave, M.E. 1991. Phenolic foam support for growing Arabidopsis to flowering stage in the

Plant Growth Unit (Abstract). ASGSB Bulletin 5(1): 75.

Musgrave, M.E. 1992. Alternative respiration and plant responses to cytokinins. In: Physiology

and Biochemistry of Cytokinins in Plants (Kaminek, M., Mok, D.W.S., and Zazimalova, E.,

Eds.). The Hague, The Netherlands: SPB Academic Publishing b.v. pp. 295-299.

Musgrave, M.E., Hopkins, A.G., Jr., and Daugherty, C.J. 1991. Oxygen insensitivity of

photosynthesis by waterlogged Apios americana. Environmental and Experimental Botany 31 ( 1):

117-124.

Schneider, R.W. and Musgrave, M.E. 1992. The Soil Atmosphere. In: Methods for Research

on Soilborne Phytopathogenic Fungi (Singleton, L.L., Mihail, J.D., and Rush, C.M., Eds.). St.

Paul, MN: APS Press, pp. 215-224.

247
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., remodeling) which appears to be due to a decrease in the functions of the osteoblast. In the

present studies, the clonal osteoblastic cell line, UMR 106-01, has been used to investigate the

regulation of collagenase and tissue inhibitors of metalloproteases (TIMPs). This project has

shed some light on the comprehensive role of the osteoblast in the remodeling process and, in so

doing, provided some insight into how the process might be disrupted under conditions of

microgravity.

Cloning of Rat TIMP.2. We, together with Dr. John Jeffrey, purified the rat collagenase

inhibitory activities secreted by the UMR 106-01 cells and showed them to be two TIMP

molecules of 20 and 30 kDa. Amino-acid sequence of the inhibitor molecules from medium

conditioned by the rat UMR cells showed that one of these is clearly homologous to TIMP-1 in

other species. The second, and predominant, inhibitor molecule produced by UMR 106-01 cells

has been identified as rat TIMP-2, by virtue of its identity to human TIMP-2 in the amino

terminal region. We screened a rat genomic library with several different degenerate

oligonucleotides and obtained a positive clone. However, in addition, we obtained a human

TIMP-2 cDNA, which successfully hybridized to RNA from UMR cells. This indicated that

there was enough sequence homology between the two sequences to use it as a probe to clone rat

TIMP-2. We used this to screen our UMR cDNA library and obtained numerous positive clones.

These were then sequenced, and it was confirmed that we have obtained a rat TIMP-2 clone

which has 98 percent amino acid sequence identity with human TIMP-2.

Regulation of Collagenase Gene. We previously demonstrated that stimulation of collagenase

by parathyroid hormone (PTH) involves a substantial increase in collagenase mRNA via

transcription. We have also concluded that this effect occurs primarily through the cAMPdependent pathway in UMR cells and requires the expression of other genes. Our next goals are

to identify the regulatory regions of the rat collagenase gene. To do this, we have obtained a

genomic clone containing 6.5 kbp of sequence 5' of the transcriptional start site. This region has

been subcloned upstream of the reporter gene, and chloramphenicol acetyl transferase (CAT),

and processive deletions have been made to determine the minimum amount of sequence

necessary for PTH induction. The largest of these constructs has been transfected stably into

UMR cells and will be characterized. These cells could be used in flight experiments to assess

changes in the transcriptional rate of the collagenase gene under microgravity conditions.

To assess the signal transduction pathway involved in PTH regulation of collagenase gene

expression, we investigated changes in immediate early gene expression after PTH treatment.

We demonstrated that c-fos and c-jun mRNA levels are increased 20-fold and 2- to 5-fold 30 min

after addition of PTH. These effects precede the changes in transcription of collagenase, 2- to

5-fold suggesting that these transcription factors may be the proteins which induce transcription

of the collagenase gene.

248
Bone Cell Research (BCR) Experiment on Spacelab.Japanese (SL.J) Mission. We were

recruited to Spacelab-J in November 1990 and asked to test whether our rat osteoblastic cells,

UMR 106-01, would adhere and proliferate normally in the NASDA cell culture hardware for the

Spacelab-J mission. After many preliminary growth experiments, we found that, with some

manipulations, cells are viable, and will grow and generally appear normal throughout the period

which was planned for space flight using this hardware.

Many other preflight experiments were also conducted in a very short time. For instance, to

alleviate the problem of lack of CO2 in the Spacelab incubator, we tested, and found suitable, a

CO2-independent medium from Gibco. The cells were also tested to determine whether they

would resist the effects of vibration due to liftoff and showed no problems. We also

demonstrated that the hormone we were to use, PTH-related peptide, is quite stable to freezing,

thawing, and incubation at 4 ° C for an extended period of time. We also examined the stability

of collagenase in medium at various temperatures and resolved that the enzyme remains

unchanged at 15 ° C for 4 days, while at 25 ° C, the enzyme is stable for 24 and 48 h, but is

degraded by incubation at this temperature for 72 h. These criteria established our limitations for

storage of our collected media samples on the Shuttle. Many Enzyme Linked Immunosorbent

Assays (ELISA) were conducted to test each of these parameters. In May 1992, Cheryl Johanns,

my technician, was able to participate in a science simulation at Kennedy Space Center (KSC)

which involved handover and scrub turnaround procedures. It was only at this time that we

learned the protocol for cleaning and seeding the chambers from the Japanese investigators.

Before-this, we had only been provided with very limited written notes on the use of the

chambers, which had led to us developing procedures that may have not been optimal.

At about the same time, the flight chambers were sent to us to bio-condition and were used in a

full simulation in the lab. We also sent chambers with growing cells for a crew simulation in

which media exchange and photography were done. In the lab we conducted numerous

photography sessions to determine the methods that should be followed for good

photomicroscopy of the cells.

Launch was set for September 11, 1992. The launch was only a day late (with plenty of notice),

and most of the flight went smoothly. The Shuttle landed at KSC, which meant we received our

samples within 3 hours of landing and they were in very good condition. The limitations on the

amount of flight-certified hardware kept us from conducting the best ground controls. We

conducted further ground control experiments in the same flight chambers used on Spacelab-J,

since the ones performed during Spacelab-J were not in optimal hardware. One of the astronauts

misinterpreted the instructions on the first medium exchange, which led to many mitotic cells

being sloughed off the chamber surface. This resulted in far less cells being present than we

anticipated. This has made it difficult to measure collagenase produced in the medium, since the

cell-to-medium ratio is so low. We have found methods of concentrating the media so that we

can still measure the enzyme by our ELISA. We will also measure cellular proteins such as

alkaline phosphatase in the cellular lysates.

The photos taken by the astronauts are extremely good and we think there are slight differences

in the morphology of the flight cells compared to our ground controls (Figure 1).

249
a) Ground control cells.

b) s.

Figure 1. UMR cells cultured in NASDA chambers.

Overall, we would have to conclude that this hardware has severe limitations. The large size

limits the number of chambers which can be tested, the syringes are not ideal for media

exchanges, and the surface must be treated for use by various cells. Our general experience with

flight experiments is that more time is needed between experiment acceptance and flight so that

best use of the hardware can be determined sooner, along with complete training of the

astronauts.

Accomplishments

(1)

(2)

constructs.

(3)

constructs.

Cloned rat TIMP-2.

Used our genomic clones for rat collagenase to make collagenase promoter/CAT

Isolated UMR ceils stably transfected with one of our collagenase promoter/CAT

25{_
(4) Showedthat changesin immediateearly geneexpression,c-fos and c-jun correlate

with changes in coUagenase.

(5) Conducted the flight experiment, Bone Cell Research, on Spacelab and showed a

change in morphology of osteoblastic cells during flight.

Significance of the Accomplishments

Finding 1: Shows that rat TIMP-2 has close similarity to those identified in other species. The

clone will enable us to examine regulation of this gene in osteoblastic cells.

Finding 2: These constructs will allow us to determine the cis and trans elements regulating the

collagenase gene.

Finding 3: These stably transfected cells will be characterized and could be used in future flight

experiments.

Finding 4: Supports the notion that Fos is the mediator of PTH action causing the induction of

collagenase. This will lead us to determine how nuclear proteins activated by protein kinase A

(PKA) may regulate expression of Fos.

Finding 5: Suggests that microgravity causes slight retraction of osteoblastic cells from the

substratum.

Publications

Burke, J.S., Cook, T.F., and Partridge, N.C. 1991. Human osteosarcoma cell line production of

metalloproteinases and an inhibitor (Abstract 483). Journal of Bone and Mineral Research

6(Suppl.): 1.

Clohisy, J.C., Connolly, T.J., Quinn, C.O., and Partridge, N.C. 1992. Prostanoid-induced

expression of the matrix metaUoproteinase gene in rat osteoblastic cells (Abstract 109). ASGSB

Bulletin 6:1.

Clohisy, J.C., Connolly, T.J., Quinn, C.O., and Partridge, N.C. 1992. Prostanoid-induced

expression of the matrix metaUoproteinase- 1 gene in rat osteoblastic cells (Abstract 32). Journal

of Bone and Mineral Research 7(Suppl.): 1.

Clohisy, J.C., Scott, D.K., Brakenhoff, K.D., Partridge, N.C., and Quinn, C.O. 1991.

Parathyroid hormone stimulation of c-los and c-jun mRNA in osteoblastic cells (Abstract 0-1 ).

ASGSB Bulletin 5:1.

Clohisy, J.C., Scott, D.K., Brakenhoff, K.D., Partridge, N.C., and Quinn, C.O. 1991.

Parathyroid hormone stimulation of c-los and c-jun mRNA in osteoblastic cells: An immediate

early response (Abstract 206). Journal of Bone and Mineral Research 6(Suppl.): 1.

Clohisy, J.C., Scott, D.K., Brakenhoff, K.D., Quinn, C.O., and Partridge, N.C. 1992.

Parathyroid hormone induces c-fos and c-jun mRNA in rat osteoblastic cells. Molecular

Endocrinology 6:1834-1842.

Connolly, T.J., Clohisy, J.C., Partridge, N.C., and Quinn, C.O. 1992. Retinoic acid stimulates

matrix metalloproteinase-1 mRNA in rat osteoblastic cells (Abstract 120). Journal of Bone and

Mineral Research 7(Suppl.): 1.

251
Omura,T.H., Noguchi,A., Quinn, C.O., Jeffrey, J.J., and Partridge, N.C. 1992. Receptormediated binding of collagenase to osteoblasts. In: Calcium Regulating Hormones and Bone

Metabolism (Cohn, D.V., Gennari, C., and Tashjian Jr., A.H., Eds.). pp. 356-360.

Partridge, N.C., Brakenhoff, K.D., Kahn, A.J., Clohisy, J.C., and Scott, D.K. 1991. Parathyroid

hormone decreases procollagen mRNA abundance by decreasing its stability (Abstract 810).

Journal of Bone and Mineral Research 6(Suppl.): 1.

Partridge, N.C., Scott, D.K., Clohisy, J.C., Brakenhoff, K.D., and Quinn, C.O. 1991. Parathyroid

hormone induces transcription of collagenase in rat osteoblastic ceils by a mechanism utilizing

cAMP and requiring protein synthesis (Abstract 0-2). ASGSB Bulletin 5:1.

Puccinelli, J.M., Omura, T.H., Strege, D.W., Jeffrey, J.J., and Partridge, N.C. 1991. A serum

factor promotes collagenase synthesis by an osteoblastic cell line. Journal of Cellular

Physiology 147: 505-513.

Roswit, W.T., McCourt, D.W., Partridge, N.C., and Jeffrey, J.J. 1992. Purification and

sequence analysis of two rat tissue inhibitors of metalloproteinases. Archives of Biochemistry

and Biophysics 292: 402-410.

Scott, D.K., Brakenhoff, K.D., Clohisy, J.C., Quinn, C.O., and Partridge, N.C. 1992.

Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a

mechanism utilizing cAMP and requiring protein synthesis. Molecular Endocrinology 6:2153-

2159.

Scott, D.K., Brakenhoff, K.D., Clohisy, J.C., Weaver, W.R., Kahn, A.J., and Partridge, N.C.

1992. Regulation of an H-ras-related transcript by parathyroid hormone in rat osteosarcoma

cells. Molecular Endocrinology 6: 1425-1432.

Shalhoub, V., Conlon, D., Tassinari, M., Quinn, C., Partridge, N., Stein, G.S., and Lian, J.B.

1992. Glucocorticoids promote development of the osteoblast phenotype by selectively

modulating expression of cell growth and differentiation associated genes. Journal of Cellular

Physiology 50: 425-440.

252
CELL KINETIC AND HISTOMORPHOMETRIC ANALYSIS OF

MICROGRAVITATIONAL OSTEOPENIA

Eugene Roberts and Lawrence P. Garetto

Bone Research Laboratory

Indiana University School of Dentistry

W. Michigan Street

Indianapolis, IN 46202

Description of Research

Past studies of cell differentiation supporting bone formation have shown that osteoblast (bone

forming cell) histogenesis is inhibited by decreased skeletal loading and microgravity. To date,

analysis of rat maxillary molar periodontal ligament (PDL) from multiple flight experiments

(Cosmos I 129, Spacelab-3, Cosmos 1187 and Cosmos 2044) has suggested that the recovery of

osteogenic potential following space flight is of high priority. The continued goals and objectives

for research in this area are to (l) further define the cellular regulation of osteoblast production, (2)

determine how this process is suppressed in microgravity, (3) continue to assess the recovery of

osteoblast histogenesis following return to l-g, (4) begin to assess preosteoblast differentiation in

vitro using a precisely defined mechanical loading paradigm, and (5) initiate in vitro molecular

biological studies of mRNA expression for steroid hormone receptors in cultured osteogenic cells.

Using kinetically and/or morphometrically distinguishable cell compartments, we have described

the following osteoblast histogenesis sequence: (1) self-perpetuating, less differentiated precursor

cells (A type); (2) committed osteoprogenitor cells (A' type); (3) nonosteogenic cells (B type); and

(4) preosteoblasts (C and D type). The osteoblast (Ob) histogenesis sequence is A/E A'2E C/E

D/E Ob (Figure 1). The increasing nuclear volume (A'2E C) is believed to be a morphological

manifestation of change in genomic expression (differentiation) indicative of maturation of

osteoblast cells. The nuclear volume assay appears applicable to all skeletal sites tested; i.e.,

periodontal ligament (PDL), tibial metaphysis, mandibular condyle, and mandibular periosteum.

G1 Presosteoblast

Stress

and/or

Strain

G2 Presosteoblast

' I

Committed Precursor

Osteopregenitor Cell

Figure 1. Schematic depiction of osteoblast histogenesis model.

253
The rate-limiting osteogenic induction step (A',,E C) is stimulated by mechanical force and appears

to be inhibited following exposure to microgravity. To date, the kinetic distinction of these cell

populations has required the use of 3H-thymidine, a radioactive tracer for DNA synthesis. During

a previous accomplishment period, the ability to utilize 5-bromo-2'-deoxyuridine (BrdU; a

nonradioactive marker for DNA synthesis) was initially demonstrated.

Research conducted in the past year has included both flight projects and ground-based preflight

experimental development. This work has specifically focused on (1) examination of rat maxillary

PDL and mandibular condyle specimens flown aboard the Space Life Sciences-1 mission (SLS-1);

(2) verification of 5-bromo-2'-deoxyuridine as a nonradioactive, immunohistochemical labeling

technique for cellular DNA synthesis; and (3) assessment of mechanical induction of preosteoblast

differentiation, in vitro, using a precisely defined mechanical loading paradigm.

Accomplishments

(1) SLS-1 was a 9-day space flight with a 2- to 7-hour recovery period at l-g.

Immediately postflight (R+O), no differences were seen in PDL osteoblast precursor cells (Figure

2) from flight (F) and control (C) rats housed in both the Animal Enclosure Modules (AEM, group

housed) and Research Animal Holding Facility (RAHF and Viv-S, 1 individually housed).

0

A+A' B C D C÷D

Flight AEM _ Control AEM [_ Flight RAHF _ VIV'S I

Figure 2. Distribution of PDL osteoblast precursor cells from ground-based controls and SLS- 1 flight animals.

(2) PDL osteoblast precursor cell kinetic compartments data from SLS-1 control animal

groups do not match previous control results (abnormally depressed A+A' and elevated C+D

cells).

(3) Comparison of SLS-1 histomorphometric data (volume percent) from mandibular

condyle bone and cartilage likewise revealed no significant differences between flight and groundbased control groups (Figure 3).

1Viv-S is the acronym for the vivarium animals that were used as synchronous controls for the F-RAHF.

254
6O

40

r, 30

2o

CorUcal Trabecular Marrow

Bone Bone Space

ll..-, [] R*OCAEM II]._o E]R+OVIV'S FAEM[]"÷O 17IR.MLC-AEM F-RAHF 5]"."L I_R+MLVIV-S F-AEM i_"÷M" F-_HF

Figure 3. Comparison of SLS-1 mandibular condyle histomorphometric volume percent data for

comcal bone, trabecular bone, and marrow space from individually and group-housed flight and

control groups. No significant differences were observed.

Significance of the Accomplishments

Findings 1 and 2: The lack of a significant flight effect is disturbing. Both the control data and the

apparent lack of a significant flight effect are inconsistent with previous results. The data suggest a

block in proliferation of both preosteoblasts and less-differentiated precursor cells in flight and

control animals. The reason for this block is not clear. Further data will be gathered from the

PARE.03B experiment flown in April 1993.

Finding 3: Previous data describing the effect of microgravity on rat mandibular condyle

(Spacelab-3) indicated a diminished magnitude of condylar growth and differences in the

distribution of cortical and trabecular bone. The results of the present experiment do not confirm

the previous results. Further data will be gathered from the PARE.03B experiment flown m April,

1993.

References

1Viv-S is the acronym for the vivarium animals that were used as synchronous controls for the

F-RAHF.

Publications

Chen, J., Lu, X., Paydar, N., Akay, H., and Roberts, W.E. 1991. An investigation of

mechanical environment within the human mandible with and without endosseous implant. ASME

BED 22: 301-304.

Chen, J., Paydar, N., Akay, H.U., and Roberts, W.E. 1992. Mechanical simulation of the

human mandible with an endosseous implant. Journal of Dental Research 71: 528.

Garetto, L.P., Jennermann, C., and Roberts, W.E. 1991. 5-Bromo-2'-Deoxyuridine as a

nonradioactive immunohistochemical label for DNA synthesis in osteoblast precursor cells.

ASGSB Bulletin 5: 34.

255
Garetto, L. P., Morey, E. R., Durnova, G. N., and Roberts, W.E. 1992. Preosteoblast

production in Cosmos 2044 rats: Short term recovery of osteogenic potential. Journal of Applied

Physiology 73(Suppl): 14S- 18S.

Garetto, L.P., Morey-Holton, E.M., and Roberts, W.E. 1992. Preliminary analysis of rat

periodontal ligament osteoblast histogenesis from SLS- 1. ASGSB Bulletin 6: 58.

Paydar, N., Akay, H.U., Katona, T.R., and Roberts, W.E. 1991. Finite element analysis of the

orthodontic response in rat molar periodontium. ASME BED 20: 159-162.

Paydar, N., Akay, H.U., Poyraz, C.L., and Roberts, W.E. 1991. Finite element model of a

human mandible for investigating joint reactions and bone stresses during mastication. ASME

BED 20: 163-166.

Paydar, N., Roberts, W.E., Akay, H.U., Katona, T.R., and Guthrie, B. 1991. Finite element

model of osteoblast differentiation in periodontal ligament. Journal of Dental Research 70: 552.

Roberts, W.E., Garetto, L.P., Arbuckle, G.R., Simmons, K.E., and DeCastro, R. 1991. Bone

physiology: Evaluation of bone metabolism. Journal of the American Dental Association 122: 59-

61.

Roberts, W.E., Simmons, K.E., Garetto, L.P., and DeCastro, R.A. 1992. Bone physiology and

metabolism in dental implantology: Risk factors for osteoporosis and other metabolic bone

diseases. Implant Dentistry 1:11-21.

256
MICROGRAVITY EFFECTS DURING FERTILIZATION, CELL

DEVELOPMENT AND CALCIUM METABOLISM IN SEA URCHINS

Heide Schatten

Department of Zoology

University of Wisconsin-Madison

1117 West Johnson Street

Madison, WI 53717

DIVISION,

Description of Research

The long-term goal of this research is to explore the role of microgravity during fertilization, cell

division, and early development on cytoskeletal organization and skeletal calcium deposition in a

model developmental system: the sea urchin. Gravity has already been shown to affect bone

calcium, and it has been reported that it alters fertilization and embryogenesis in several

developmental systems. The egg at fertilization performs virtually all the events of any higher cell,

many of which involve spatially and temporally separated sequestration of calcium. The metabolic

activation of the unfertilized egg is triggered by an intracellular release of calcium which also

initiates the secretion of cortical granules leading to a modification of the cell surface. Sperm

incorporation is mediated by an eruption of egg microvilli formed by the assembly of

microfilaments. Once the sperm has entered the egg cytoplasm, the dynamic assembly of

microtubules on the base of the sperm head is responsible for uniting the genomes of sperm and

egg. Cell division, involving the chromosomal movements during cytokinesis, also requires the

sequential activity of microtubules and microfilaments. Though the precise mechanisms are still

unclear, intraceUular membranes, competent to sequester and release intracellular calcium, are the

primary regulatory systems of the mitotic events. Most striking is the deposit of calcium in the

spicules of sea urchin embryos that occurs as a result of the deposition of calcium carbonate on an

organic matrix. The utility and speed of this example of the formation of extracellular skeletal array

will serve as a significant model in which to evaluate the effects of gravity on calcium deposition,

particularly with quantitive analytical scanning transmission electron microscopy. Notwithstanding

the global importance of gravity on development, it has only recently been possible to begin to

design experiments which will directly investigate the specific effects of this vector. The objectives

for these studies are met by developing specific molecular probes, to identify specific structures

and analyze their formations under terrestrial and microgravity conditions. Monoclonal antibodies

were raised to characterize cytoskeletal components such as microtubules, microfilaments, and

centrosomes. The use of these molecular probes, together with valuable collaborations with the

NASA space program to design the most suitable conditions for experiments on the Space Shuttle,

have enabled us to gain insights into normal developments. By extrapolating our present

techniques to the conditions on the Shuttle Orbiter, we will further the understanding of basic

events during fertilization, cell division, and development, as well as answer critical questions

regarding the space biology of cytoskeletal and skeletal organizations.

Accomplishments

(1) We have used a variety of monoclonal antibodies for the characterization of

microtubules (designated E7) and centrosomes (designated Ah-6 and 4D2), as well as polyclonal

antibodies raised in rabbits, to characterize microtubules and centrosomes, and human antibodies to

characterize centrosomes (designated 5051 and SPJ). DNA could be detected with the

commercially available fluorescent dye DAPI. These probes allowed us to document the dynamic

microtubule reorganizations necessary for the union of sperm and egg nuclei, and for the formation

of the mitotic apparatus, the machinery for cell division. It also allowed us to gain insights in the

highly dynamic interactions between centrosomes and microtubules. Centrosomes are the major

257
organellesin thecell for microtubuleorganizations.By usinginhibitorsof microtubuleformation,

suchascolcemid or griseofulvin, we could demonstratethat microtubules,are important for

structuralchangesof centrosomes.In theabsenceof microtubules,centrosomeswereunableto

unfold. Additionally, it wasdemonstratedthatchromosomesdid notundergodecondensationas

requiredduringcell division. Anotherinhibitor of metabolicprocesseswasusedto interferewith

calcium metabolism. Membrane fusions require proper calcium concentrations. When

dithiothreitolwasused(whichis suspectedto interferewith metabolicprocessesrequiringcalcium)

membranefusionsasnecessaryfor pronuclearfusionandcell divisiondid notoccur.

(2) Major efforts during the past2 yearswere devotedto identify the most suitable

biologically compatiblematerialandhardwareto allow for optimalconditionsfor theconductof

experimentson the SpaceShuttle. Successfulcollaborationswith MPB Technologies,the

CanadianSpaceAgency, and the Kennedy SpaceCenter have led to testsdetermining the

compatibility of constructedmaterialswith seaurchin gametes,ability to maintainlife support

parameters,propergameteratiosand volumesto attainfertilization, properfixation ratiosand

volumesto preservespecimensfor post-flight analysis,and reliability of dilution and mixing

hardwarecomponents. Severalprototypesof hardwarewere tested. The materialstestedfor

biocompatibility wereSilastic,Eccosil 5877andTeflon. Teflon was chosento give the most

reliableresults,andwasthereforeselectedfor experimentationon a0-g KC-135testflight. The

resultsof the testflight concludedthat spermandeggsdo not mix readily undermicrogravity

conditions and devicesshould be designedwhich would allow for enforcedmixing of both

gametes.

Significance of the Accomplishments

The work with the antibodies directed towards microtubules and centrosomes and dyes to detect

DNA employing fluorescence microscopy has led to major insights into the complicated and

intrinsic interactions of cytoskeletal organizations and reorganization during fertilization, cell

division, and further development. Proper calcium concentrations are required for the formation of

the cytoskeleton and membrane fusion and for calcium deposition in the spicules during later

development. Investigations of these fundamental biological calcium sequestration processes wiU

lead towards understanding the remarkable loss of skeletal calcium by astronauts and cosmonauts,

which can amount to up to 460 mg/day. The experiments using sea urchins conducted under

microgravity conditions will help us understand problems affecting human health, including

osteoporosis. The significance of the research is rather farreaching and may have important

implications in understanding the skeletal problems associated with space travel, as well as aging

on Earth. New information on fertilization and cell division may well provide useful approaches to

solving problems of infertility, contraception, and abnormal cell proliferation during cancer. The

development of hardware during the conduct of these experiments will provide improved living

conditions for developmental systems. The hardware designs invented in collaboration with MPB

Technologies, the Canadian Space Agency, and the Kennedy Space Center, with the goal to culture

live cells in space, are reusable and will contribute to further technical developments.

Publications

Schatten, G., Steffen, S.L., and Simerly, C. 1992. Fertilization during an aircraft parabolic

flight. In: The World Space Congress, Washington, DC, USA: p. 531.

Steffen, S., Fiser, R., Simerly, C., Schatten, H., and Schatten, G. 1992. Microgravity effects

on sea urchin fertilization and development. Vol. 12, Advances in Space Research No. 1: p.

(1)167-(1)173.

258
STL.01 AND STL.02 EXPERIMENTS

IMMUNOLOGY

Gerald Sonnenfeld and Edwin S. Miller

Department of Microbiology & Immunology

School of Medicine

University of Louisville

Louisville, KY 40292

Description of Research

The purpose of the STL.01 experiment was to test the ability of the Space Tissue Loss (STL)

tissue culture module designed by the Walter Reed Army Institute of Research to support growth

of macrophage cell lines in flight. The STL.02 experiment was a repeat of the STL.01 experiment.

Our experiments involved the study of the effects of flight on two cell lines of immunologic

importance. We wished to determine if space flight using the STL module would result in

alterations in immune function of P388D 1 ceils (a murine macrophage line) and would alter the

ability of HL-60 cells (a human stem cell line) to differentiate into macrophage-like cells.

Accomplishments

On the STL.01 experiment, unforeseen technical difficulties were encountered. The temperature

on the rail containing only our cells was not regulated properly and remained at 39°C throughout

the flight. In addition, medium supplements to stimulate the ceils were not released during the

flight. As a result of these difficulties, we lost all of our P388D1 cells. Nevertheless, we were

able to recover HL-60 cells and tried to stimulate them to differentiate into macrophage-like cells

(Table 1). Differentiation was not observed (Table 2), and the nutritional content of the medium

was analyzed by Dr. David Epstein of Life Sciences Technologies to ensure there was cell growth

during flight (Table 3).

TABLE 1

CELL YIELDS AFTER RECOVERY

(STL.01 FLIGHT)

CELL LINE INOCULUM YIELD

P388D1

HL-60

50,000 cells/cartridge

100,000 cells/cartridge

No viable cells

5,000 cells

259
TABLE 2

DIFFERENTIATIONOF HL-60CELLSFOLLOWINGSTIMULATION

WITH VITAMIN D3AFTERRECOVERY

(STL.01FLIGHT)

SAMPLE

GROUNDCONTROLS

FLIGHT SAMPLES

NUMBEROFDIFFERENTIATEDCELLS

-lz.L_YIr2xtdI_

0/600 (0%)

O/600 (0%)

vI._.E/ZAMI_ +

382/600 (64%)

0/600 (0%)

TABLE 3

UTILIZATION OF SELECTED MEDIUM COMPONENTS BY HL-60 CELLS

(STL.01 FLIGHT)

Medium Component Unused DMEM Ground Control Flight Sample

GLUCOSE (g/l)

GLUTAMINE (mg/l)

ARGININE (mg/1)

HISTIDINE (mg/ml)

METHIONINE (mg/ml)

TRYPTOPHAN (mg/ml)

LYSINE (m_/ml)

4.50

584.00

69.60

31.20

30.00

16.00

117.10

3.55

433.70

73.50

29.76

23.80

12.40

97.80

4.08

115.10

47.20

23.60

24.30

11.70

98.80

For the STL.02 experiment, the flight module functioned without major problems; however, an

apparent cennal source of fungal contamination in the media flow path of the STL apparatus caused

contamination of both our P388D1 and HL-60 cells, as well as other cell lines that were flown by

other investigators. It was apparent that the P388D1 cells and the HL-60 cells had grown and

survived in the STL module during flight, but further analysis of the data was impossible because

of the fungal contamination. The conclusion from this experiment is that the STL module can

support the growth of our cells in flight but still had teething difficulties.

26(I
Significance of the Accomplishments

The results of the current study suggest that space flight can inhibit the differentiation of precursor

cells to mature macrophages. This result is in accordance with previous human and animal studies

which suggest that the macrophage is a key cell in space-flight-induced immune dysfunction. The

current study suggests a possible mechanism for the effects seen in vivo. It must be noted that this

study has been performed successfully only once on STL in an experiment fraught with technical

problems with the STL module. Repetition and confirmation are required.

Publications

Sonnenfeld, G., Miller, E.S., Klein, J., McLeish, K., Epstein, D., Kubiak, J., and Wiesmann,

W. 1993. Effect of space flight on immune function - Macrophage studies (Abstract). Fifth

International Congress of Cell Biology Abstracts, p. 466.

261
SKELETAL MUSCLE METABOLISM IN HYPOKINETIC RATS

Marc E. Tischler

Department of Biochemistry

University of Arizona

Tucson, AZ 85724

Description of Research

This work has concerned the mechanisms of atrophy and metabolic alterations associated with the

lack of load-bearing (unweighting), which may occur with prolonged bedrest or weightlessness.

For these studies, young rats were used to assess the potential role of gravity in growing muscles.

Our general approach has been to utilize both unweighting and denervation (interrupted nerve

supply), to better understand atrophy in general. We have found important differences between

unweighted and denervated muscles, and such comparisons have proved to be very fruitful. Other

more specific comparisons have led to successful results during this time period. Most important

was the opportunity to compare muscle atrophy and glucose transport data from flight and

ground-based, hindlimb-suspended animals. Secondly, we have made further use of our ability to

compare in vitro and in situ (intramuscular injections) approaches to evaluate the physiological

significance of our findings. Finally comparisons of the myofibriUar (structural) and sarcoplasmic

(non-structural) protein responses to unweighting have helped to further complete the picture.

Accomplishments

(1) Flight experiment. (a) Soleus (back of leg) of flight animals atrophied to a similar

extent as that of hindlimb-suspended animals. (b) Plantaris and gastrocnemius (back of legs) of

flight animals showed slower growth, just as these muscles did in hindlimb-suspended animals.

(c) Extensor digitorum longus and tibialis muscles (front of the leg) showed no response from

flight, just as on the ground. (d) The soleus, but not the extensor digitorum longus muscle,

showed increased glucose transport in the presence of insulin after flight, just as after hindlimb

suspension on the ground. (e) Space flight increased the interstitial fluid volume of the soleus

muscle, as occurred in the soleus of hindlimb-suspended animals.

(2) Myofibrillar vs. sarcoplasmic proteins. (a) Loss of protein during unweighting

atrophy of the soleus is due to a diminished pool of the structural (myofibrillar) proteins. (b)

Disappearance of myofibrillar proteins is due to slower formation and accelerated destruction. (c)

The pool of sarcoplasmic proteins remains constant during unweighting atrophy. (d) Sarcoplasmic

proteins are spared owing to a slowing of their rate of destruction.

(3) Physiological significance studies. (a) Following intramuscular injection with

insulin, unweighted muscles take in more glucose than do normal soleus muscles. (b) Insulin-like

growth factor-1 has similar effects as insulin on glucose uptake by the unweighted soleus

following in vitro incubation intramuscular injection. (c) Catecholamines show enhanced effects

on the unweighted soleus in vivo just as in vitro.

Significance of the Accomplishments

Flight Experiment. The most critical significance of the flight experiment was our

demonstration the hindlimb unweighting model used in our and numerous other laboratories can

reproduce the changes in muscle size and biochemistry which occur in space. The ground-based

studies support the notion that the major site of protein destruction with unweighting atrophy

occurs in the cytoplasm. The parallel results from spaceflight suggest that a similar mechanism is

262
true, at leastfor atrophyof the soleusof young animals. In space,ason Earth, unweighting

producesselectiveeffectson musclesize,dependingon theparticularphysiologicalfunctionof

eachmuscle.

Myofibrillar vs. Sarcoplasmic Proteins, Unlike denervated muscle, which loses all

proteins at a constant rate, the unweighted soleus loses only the myofibrillar (structural) proteins.

Thus, a significant amount of protein (sarcoplasmic pool) in the unweighted muscle is spared from

such loss. These sarcoplasmic proteins include those on the membrane which are responsible for

attracting hormones to the cell surface (receptors) or which are involved in transport processes

(e.g., for getting glucose into the cell). As the slowing of myofibrillar formation precedes the

destruction of these proteins, the two processes are clearly controlled independently by

unweighting. It is of further interest that one pool of proteins undergoes accelerated destruction,

while the sarcoplasmic proteins show slower destruction. This supports the idea of separate

compartments in the cell for protein destruction, which is under independent control.

Physiological Significance Studies, The development of the ability to study biochemical

responses by intramuscular injection afforded the opportunity to test the physiological significance

of our in vitro findings. The discovery of the greater effect of insulin and catecholamines on

metabolic processes in the unweighted muscle led to the conclusion that there is a sparing of the

receptors on the cell surface, in accord with the sparing of sarcoplasmic proteins. Our ability to

demonstrate this phenomenon following intramuscular injection and, therefore, under

physiological conditions shows the in vivo significance of these results.

Publications

Henriksen, E.J., Ritter, L.S., Munoz, K.A., and Tischler, M.E. 1992. Enhanced effect of

insulin-like factors on glucose transport activity in the unweighted rat soleus muscle. ASGSB

Bulletin 6(1): 93.

Henriksen, E.J., Tischler, M.E., Woodman, C.R., Munoz, K.A., Stump, C.S., and Kirby, C.R.

1993. Elevated interstitial fluid volume in soleus muscles unweighted by spaceflight or

suspension. Journal of Applied Physiology 75(4): 1650-1653.

Isaias, T., Truesdell, M., Rodriguez, S., and Tischler, M.E. 1992. Effects of modified

gravitational orientation on adult development of Manduca sexta. ASGSB Bulletin 6(I): 46.

Kirby, C.R., Woodman, C.R., Woolridge, D., and Tischler, M.E. 1991. Unweighting but not

denervation increases muscle hormone sensitivity in vitro and in vivo. ASGSB Bulletin 5( 1): 36.

Kirby, C.R., Woodman, C.R., Woolridge, D., and Tischler, M.E. 1992. Cyclic AMP

accumulation and B-adrenergic binding in unweighted and denervated rat soleus muscle.

Metabolism 41: 793-799.

Munoz, K.A., Satarug, S., and Tischler, M.E. 1992. Time course of the response of myofibrillar

and sarcoplasmic protein metabolism to unweighting of the soleus. ASGSB Bulletin 6( 1): 34.

Munoz, K.A., Satarug, S., and Tischler, M.E. 1993. Time course of the response of myofibrillar

and sarcoplasmic protein metabolism to unweighting of the soleus muscle. Metabolism 42(8):

1006-1012.

Munoz, K.A. and Tischler, M.E. 1991. A novel approach to measuring in vivo protein synthesis.

ASGSB Bulletin 5(1): 55.

263
Munoz,K.A. andTischler, M.E. 1991. The effect of a space food bar diet on body and muscle

mass in normal and hind limb suspended rats. Aviation and Space Environmental Medicine 62:

875-878.

Tischler, M.E. 1992. Estimation of protein synthesis and proteolysis in vitro. In: Modern

Methods in Protein Nutrition and Metabolism (Nissen, S., Ed.). San Diego, CA: Academic Press,

pp. 225-248.

Tischler, M.E., Henriksen, E.J., Munoz, K.A., Stump, C.S., Woodman, C.R., and Kirby, C.R.

1992. Spaceflight and earth-based unweighting produce similar effects on muscle of young rats.

ASGSB Bulletin 6(1): 57.

Tischler0 M.E., Henriksen, E.J., Munoz, K.A., Stump, C.S., Woodman, C.R., and Kirby, C.R.

1993. Spaceflight on STS-48 and Earth-based unweighting produce similar effects on skeletal

muscle of young rats. Journal of Applied Physiology 74(5): 2161-2165.

Tischler, M.E. and Kirby, C.R. 1991. Space Travel (Biochemistry and Physiology). In:

Volume 7: Encyclopedia of Human Biology (Dulbecco, R., Ed.). New York: Academic Press,

pp. 143-152.

264
SOFT DENSE FIBROUS CONNECTIVE

UNLOADING

Arthur C. Vailas

Department of Kinesiology

School of Education

University of Wisconsin

Madison, WI 53706-1189

TISSUE RESPONSE TO HINDLIMB

Accomplishments

The connective tissue research program has devoted a considerable amount of time in

demonstrating that collagen plays a major role in the plasticity of muscle. In particular, the

maturation rate and compartmentalization of collagen are affected by load and the rate of muscle

growth. Collaboration with the Mayo Clinic (see publications by Gosselin, et al.) clearly

demonstrates that load during growth and development plays an important role in affecting the

maturation rate of muscle type I collagen, thus effecting passive tensile properties of muscle and

supporting the hypothesis that unloading has the potential of reducing passive muscle stiffness,

which makes the muscle more likely to tear or damage during eccentric loading.

A second major accomplishment is that the flight studies on COSMOS 2044 report that animal age

and endocrine status alter the responsiveness of bone to microgravity. In particular, the skeletally

mature rat is less responsive to microgravity as compared to the rapidly growing rat. In addition,

there were significant changes in bone microstructure and geometry of growing animals exposed to

2-g. Therefore, animal endocrine status (age) modifies significantly the adaptive potential of bone

to various stresses.

Future Goals

The program will continue to study bone plasticity and address adaptive potential to stress by using

the dwarf rat model and growth hormone supplementation as a countermeasure. Also, muscle

studies are being proposed to actually test the passive material properties at a time where there are

significant collagen changes. In addition, we have adapted some microtechniques to adequately

determine the resorption rate of type I collagen by measuring the pyridinoline levels in various

body fluids of the rat. This marker work has been applied to major clinical studies of primates and

humans.

Publications

Gosselin, L., Betlach, M., Vallas, A., Greaser, M., and Thomas, P. 1992. Myosin heavy chain

isoforms of young and senescent rat diaphragm: effect of exercise training. Journal of Applied

Physiology 73:1282-1286.

Gosselin, L.E., Betlach, M.B., Vailas, A.C., and Thomas, D.P. 1992. Training-induced

alterations in young and senescent rat diaphragm muscle. Journal of Applied Physiology 72: 1506-

1511.

Gosselin, L.E., Martinez, D.A., Vailas, A.C., and Sieck, G.C. 1992. Collagen maturation in

developing rat diaphragm. Journal of Applied Physiology 72:1506-1511.

Gosselin, L.E., Martinez, D.A., Vailas, A.C., and Sieck, G.C. 1993. Interstitial space and

collagen alterations of the developing rat diaphragm. Journal of Applied Physiology 74: 2450-

2455.

265
Gosselin, L.E., Martinez, D.A., Vailas, A.C., and Sieck, G.C. 1993. Passive length-force

properties of the senescent diaphragm: relationship to collagen characteristics. Journal of Applied

Physiology 74: 2450-2455.

Graf, B.K., Fujisaki, K., Vanderby, R., and Vailas, A.C. 1992. Effect of in situ freezing on

rabbit patellar tendon: A histological, biochemical and biomechanical analysis. American Journal of

Sports Medicine 20: 401-405.

Ortho M.W., Martinez, D.A., Cook, M.E., and Vailas, A.C. 1993. The presence of

lysylpyridinoline in the hypertrophic cartilage of newly hatched chicks. Biochemistry Biophysics

Acta 1157: 229-232.

Salem, G.J., Zernicke, R.F., Martinez, D.A., and Vailas, A.C. 1993. Adaptations of immature

trabecular bone to moderate exercise: geometrical, biochemical and biomechanical correlates. Bone

14: 647-654.

Vailas, A.C., Vanderby, R., Martinez, D.A., Ashman, R.B., Ulm, M. J., Grindeland, R.E.,

Durnova, G.N., and Kaplansky, A. 1992. Humerus bone adaptation to space flight in the adult

rat. Journal of Applied Physiology 73: 4-9.

266
EARLY DEVELOPMENT OF A GRAVITY-RECEPTOR ORGAN IN MICROGRAVITY

Michael L. Wiederhold

Department of Otolaryngology--Head and Neck Surgery

The University of Texas Health Science Center

San Antonio, TX 78284-7777

Description of Research

Adult and embryonic Japanese red-bellied newts, Cynops pyrrhogaster, will be flown on the

International Microgravity Laboratory-2 Shuttle mission. Our project on this flight concerns the

effects of rearing in microgravity (lag) on the development of both the gravity-sensing and

angular-acceleration sensing portions of the inner ear. Adult female newts, which were

inseminated in the wild the previous fall, will be injected with hormone to induce fertilization

and egg laying. Thus, we will be able to observe development of embryos fertilized in lag.

Later-stage fertilized eggs will be reared in lag, starting either before the inner ear begins to

develop or before the start of production of the stones (otoconia) in the gravity-sensing organs.

Our work in the past year has documented the normal developmental sequence and schedule of

the inner ear, which had not previously been performed systematically in any amphibian. We

have also studied the form and crystal pattern of the otoconia and performed behavioral studies

of the reactions of larvae at several developmental stages to transient lag during parabolic'tlight.

Accomplishments

Published descriptions of the embryonic development of Cynops, as well as many other

amphibian species, fail to mention the otolith, the collection of stones upon which gravity and

linear acceleration act in the inner ear. After experimenting with several techniques, we are now

convinced that this is due to the nature of the chemical fixatives traditionally used, which can

easily dissolve the otoconia. We have developed rapid fixation and dehydration techniques

which do not dissolve the stones but adequately preserve the neural tissue. Using these

techniques, we find that the inner ear is first seen at 5 days after the eggs are laid, and the

otoconia are first seen at 8 days. The otolith continues to grow throughout development. A

relatively mature arrangement of sensory receptor cells (hair cells) and nerve fibers is seen in the

two gravity-sensing organs (the utricle and saccule) and the lateral semicircular canal (which

detects angular acceleration in the horizontal plane) at 13 to 14 days. The posterior semicircular

canal is innervated at about 19 days and the anterior canal another week later. Thus, the basic

schedule of development of each component of newt's vestibular system has been established in

ground-based control animals.

In several amphibian species it has been reported that the otoconia in the utricle axe composed of

calcium carbonate in the calcite crystal form, whereas those in the saccule are made of the same

mineral but in the aragonite form. We have found this to be true in the adult newt also.

However, when the otolith organs first develop, the saccule and utricle cannot be distinguished

from one another. Thus, it was difficult to understand how the two crystal forms could be

produced in the same organ. Recently, we have shown that in the early larval stages (before 19

days) both the utricle and saccule have otoconia which appear to be of the calcite form. It is only

later that the aragonite-appearing otoconia appear. This suggests that the aragonite otoconia

might be produced outside of the saccule itself, perhaps in the endolymphatic sac, and

transported into the saccule. In adult animals, the entire endolymphatic system, which in

amphibians extends to the surface of the spinal cord, is filled with aragonite otoconia. These

267
havebeensuggestedto serveasa sourceof metaboliccalciumfortheanimal. Thus,it will be of

interest to see if these otoconia are depleted in adult animals which have been in space for 14

days on IML-2.

In conjunction with colleagues in Japan, we have been developin.g a non-invasive X-ray

microfocus tube system, which currently allows an effective beam s_ze of 10 _tm. With this

system, we have been able to visualize the otoliths in early embryos, as young as 8 days after egg

laying. A striking finding with this system is that the otolith appears to be the first system

calcified in the embryo. The system may also allow us to quantitate the amount of calcium in the

endolymphatic system. It will allow us to follow the growth of the otoliths after space flight

without having to sacrifice the animals.

In Japan, we had the opportunity to observe newt larvae of several developmental stages in

parabolic flight, to study their reaction to lag. Larvae at stages 42 to 44, in the first three days

after hatching, were found to react very little to lag, although they did swim significantly more in

the light than in the dark. Larvae at stages 54 to 58, approximately 10 to 14 days after hatching,

swam quite vigorously throughout their container, particularly at the onset of lag, in either light or

dark. These responses correlate with the development of the newt's vestibular system: at stages

42-44, the otolith organs and lateral semicircular canal are present with well-developed sensory

innervation. Both the utricle and saccule are in a horizontal position. Thus, all three organs are

suited to sensing movements in the horizontal plane but not upward swimming. In the dark these

larvae would have no information about where they had gone if they did swim upward. We have

interpreted the results to mean that the animals refrain from making movements which cannot be

accurately monitored. At stages 54-58, all three semicircular canals are well innervated and the

saccule rises to assume a vertical position. Thus, the saccule, as well as the anterior and posterior

semicircular canals, could sense upward and downward movements. With a relatively complete

vestibular system, these animals can measure and keep track of any movements and thus are

equipped to monitor their exploratory movements in the novel environment of _.g. At the early

stages (42-44) the limited spatial information provided by the developing visual system allows

the larvae to make limited movements at the onset of lag.

Significance of the Accomplishments

Despite several qualitative studies of the vestibular apparatus in animals reared in space, there

are conflicting conclusions in the literature. As we contemplate deep-space probes, perhaps

lasting many years, we must think about having animals, including humans, conceived and

reared in lag. Although there are obvious concerns about how the musculoskeletal system will

develop without the constant influence of gravity present on Earth, similar concerns are relevant

to the vestibular system, and in particular the gravity-sensing organs. At one level, we know

practically nothing about what regulates the production of otoconia to form the otolith. In

general, small animals have small otoliths and large animals have large ones. In those species

which grow throughout their lives, the otolith continues to grow as long as the animal lives.

Thus, there appears to be an ideal mass of otolith for a given size (and probably locomotory

style) of animal. If the regulation is based on the weight of the otolith, we would expect larger

otoliths to be produced in space. Indeed, this has been found in one of the more quantitative

Russian studies. In order to study regulation of otolith production, the studies described here will

form the basis of ground-based control studies for comparison with development occurring, in

space. Other studies we are initiating now will address the mechanisms of otoconial producuon

and assembly.

On another level, even if the otolith were to develop completely normally in lag, the reflex

connections in the brain, by which the otolith acts to keep the eyes in a proper orientation during

head flit, will probably develop differently in lag. During development on Earth, the otolith

organs always experience the steady pull of gravity, in addition to forces of linear acceleration,

268
on the massof the otolith. In space,thesteadyinput of gravity is missing,sothe reflexeswill

developwith only linear-accelerationforcespresent. We arecurrently developingtestsof the

otolith-ocularreflex in newtlarvae. With this, we will beableto comparethe developmentof

otolith functionin space-rearedandgroundcontrolanimals.

Publications

Wiederhold, M.L., Yamashita, M., and Asashima, M. 1992. Development of the gravity-sensing

organs in the Japanese red-bellied newt, Cynops pyrrhogaster. In: Proceedings of the Eighteenth

International Symposium on Space Technology and Science, Kagoshima, Japan, 18:2103-2108.

Wiederhold, M.L., Yamashita, M., Izumi-Kurotani, A., Larsen, K., Koike, H., Nagaoka, S., and

Asashima, M. 1992. Developmental changes in the vestibular system of Imori. In: Proceedings

of the Ninth Space Utilization Symposium, 9: 127-131.

Yamashita, M., Wiederhold, M.L., Izumi-Kurotani, A., Koike, H., Asashima, M., Mogami, Y.,

Okuno, M., Kawasaki, Y., and Komada, S. 1992. Preparatory work for astronewt experiment.

In: Proceedings of the Ninth Space Utilization Symposium, Tokyo, 9: 132-134.

269

SPECIAL ACTIVITY

SPACE BIOLOGY RESEARCH ASSOCIATES PROGRAM

Gerald Sonnenfeld

Department of Microbiology &

School of Medicine

University of Louisville

Louisville, KY 40292

Immunology

Description of Program

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 young biological scientists by offering

them Research Associate Awards. These awards offer opportunities to work on projects directly

related to Space Biology in laboratories that provide the necessary facilities and a relevant research

environment. It is anticipated that Research Associates will develop 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.

Accomplishments

The program began on June 1, 1980, with funding to support several Research Associates each

year. As of May 1, 1993, awards have been granted to 59 individuals, of whom 33 have received a

second year of funding. Table I shows the variety of projects submitted and institutions to which

the 59 Research Associates have been assigned. These scientists represent different disciplines

including: animal physiology, botany, cellular and molecular biology, developmental biology,

immunology, plant physiology, and zoology.

Significance of Accomplishments

Many of the Research Associates have been asked to participate in NASA panels, national

workshops, and national meetings. Each year, in the fall, the Research Associates attend the

annual meeting of the American Society for Gravitational and Space Biology (ASGSB), where

they are active participants, presenting papers and posters along with their senior colleagues. The

Research Associates are also 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 private industry, and with NASA. Table II shows the location of the

Research Associates today.

A complete list is provided of the 59 Research Associates, the title of their research projects, the

host laboratory and director, and their current location.

273
Table I. SPACE BIOLOGY RESEARCH ASSOCIATES

ANIMAL PROJECTS

NAME INSTITUTION

G. ADAMS

S. BAIN

W. BERRY

M. BINDER

S. BLACK

H. BLAIR

J. BUCKEY

G. BURROWS

D. CLOHISY

J. CLOHISY

M. COOPER

D. DICKMAN

S.GLOTZB ACH

E. GOOLISH

C. GOULD

M. GRAY

E.GREENFIELD

T. JONES

T. KERR

C. KIRBY

D. KLIGMAN

A. LYSAKOWSKI

K. MCLEOD

D. MEYERS

E. MILLER

L. MINOR

D. MURAKAMI

S. PERKINS

K. POTE

G. RADICE

R. REITSTETI'ER

F. ROBINSON

J. STEFFEN

J. SZILAGYI

D. THOMASON

J. THOMPSON

L. THOMPSON

Y. TORIGOE

U. Cal., Irvine

SUNY, Stony Brook

U. Louisville

Dartmouth Col.

U. Cal., Berkeley

Washington U.

U. Texas, Dallas

National Institutes of Health

Washington U.

St. Louis U.

U. Cal., Berkeley

U. Texas, Galveston

Stanford U.

U. Michigan

U. of Louisville

Tufts U.

Washington U.

U. Cal., Davis

Wayne State U.

U. Texas, Houston

National Institute of

Mental Health

U. Chicago

SONY. Stony Brook

U. Pennsylvania

U. Louisville

U. Chicago

U. Cal.,Davis

Washington U.

U. Virginia

Indiana U.

U. Arizona

U. Pittsburgh

U. Louisville

Cleveland Clinic

U. Texas, Houston

Med. Col., WI.

Marquette U.

U. Cal., Irvine

RESEARCH AREA

Muscle plasticity/Signal pathways

Skeletal bone remodeling

Role of vitamin D/Immune function

Cardiacpathology

Amphibian developmentalorientation

Cellular/Boneatrophy

CzdiovascularResponses

Symptogenesis/Neuropathoiogy

Osteoclastogeuesis/B one atrophy

Bone/space flight and gene expression

Osteopomsis/Bone atrophy

Semicircular muscle canals

Neurophy siolog y/Circadian rhythms

Weightlessuess/Swim bladder function

Immunology/Interferon functions

Mechanical enviroranent in bone smJcmre

Osteoblast role/Osteoclast activity

Neurophysiology/Brainstempotentials

Mammalian vestibular system

Weightlessness/Swim bladder function

Neurite extension factor responses

Vestibular hair ceUs/Synaptic relations

Electrical fields in bone remodeling

Gravityperception - Microcntstacean (FW)

Immunological memory/Infection

Responses of secondary vestibular neurons

HyperdynamiaNisual systems

Vitamin D/Osteoclast differentiation

Otoconia Ca binding Wotein

Gravity-sensors/Amphibian embryology

Microgravity/Cell interaction

Sensory motor properties in uvula

Gl_coid receptms/Muscle responses

Hypodynamic response_Animal model

Decreased actin synthesis/Muscle atrophy

Muscle fibers following atrophy

Muscle fatigability/cross-bridge

Neurophysioiogy of gut

274
TABLE I. SPACE BIOLOGY RESEARCH ASSOCIATES (CONTINUED)

PLANT PROJECTS

NAME INSTITUTION RESEARCH AREA

S. BARSEL

T. BJORK/_AN

T. BROCK

M. DESROSIERS

J. GARAVELLI

J. GAYNOR

M. HARRISON

G. JAHNS

J. KISS

W. KROEN

K.KUZMANOFF

V.LING

M. MATILSKY

M. MUSGRAVE

D.REINECKE

M. RICE

B. SERLIN

R. SLOCUM

J. SLONE

L. TALBO'Iff

L. WIMMERS

Michigan St. U.

U. Washington

U. Michigan

Michigan St. U.

Texas A&M U.

Yale U.

Washington U.

U. Houston

U. Colorado

N.C. State U.

Stanford U.

Harvard U.

Princeton U.

Duke U.

Michigan St. U.

Oregon St. U.

U. Texas, Austin

Yale U.

Washington U.

Washington U.

U. CA., Davis

Plant cell physiology

Electrical responses/Gravity sensing

Auxin & protein synthesis in gravitropism

Electrical potential in hormone transport

Plant/Algaecllchemistry

Amyloplast/Gravitational sensitivity

Environmentalethylene/Gravitropism

Ligninbiosynthesis inplantdevelopment

Chara rhizoids gravitropic response

CO 2 enrichment and root/shoot ratios

Enzyme regulators in plant cell wall

Gravity signal transduction/Ca-calmodulin

Gravity perception/Coenocyte

Plant respiratory metabolism/Space flight

IAA distribution/Plant geosensing

Gravicurvatme/Auxins

Cell wall growth/Corn roots

Role of calcium/Gravistimulation

Auxin transpon/Gravitropism

Stem gravicurvature/Specific polymers

Gravitropism/Ca- ATPase

275
TABLE II.

LOCATION OF RESEARCH ASSOCIATES TODAY

59

6

33

1

3

11

4

1

1

= Total Number

= Current Recipients of this Award

= Faculty Members in Universities

= Located in NASA Facilities

= Other Government & Private Research

Foundation Facilities

- Other Research Capacities

- Industry

-- Deceased

= One of the Faculty Members was the

Alternate Payload Specialist

Candidate for the SLS-2 Flight

276
RESEARCH ASSOCIATE AWARDEES

The 59 awardees are listed alphabetically, including their award terms (in parenthesis after their

name), host laboratory, and current location. Current professional appointments are indicated in

parenthesis as: Current Research Associate (C); Faculty (F); NASA facility (N); Other Government

& Private Research Foundation facilities (G); Other Research Capacities (R); Industry (I); and

Deceased (D).

GREGORY ADAMS, Ph.D. (11/1/92 - 10/30/93) is working on "Metabolic Modulation of

Skeletal Muscle Phenotype During Unweighting Induced Atrophy and Compensatory

Hypertrophy" in Dr. Kenneth Baldwin's laboratory at the University of California, Irvine, CA. (C)

STEVEN BAIN, Ph.D. (6/1/88 - 5/30/90) worked on "The Interaction of Skeletal Remodeling

with Systemic Disorders: An Obstacle to Extended Space Flight?" in Dr. Clinton Rubin's

laboratory at SUN'Y, Stony Brook, NY. He is now Senior Scientist of Biomedical Research at

ZymoGenetics, Seattle, WA. (I)

SARA-ELLEN BARSEL, Ph,D. (6/1/87 - 5/30/88) worked on "Molecular and Genetic and

Phototropism in Arabidopsis thaliana" in Dr. Kenneth Poff's laboratory at Michigan State

University, East Lansing, MI. She is working for Chemical Abstracts in Columbus, OH. (I)

WALLACE BERRY, Ph.D. (7/1/88 - 6/30/90) worked on "Lymphokine Producing Capacity

of Antiorthostatically Suspended Rats: Relationship to 1,25-dihydroxy-vitamin D3" in Dr. Gerald

Sonnenfeld's laboratory at the University of Louisville, Louisville, KY. He is now an Assistant

Professor in the Boyd Research Center, University of Georgia, Athens, GA. (F)

MICHAEL BINDER, M.D. (1/1/83 - 12/30/83) worked on "Congenital Heart Malformations

and Situs Inversus" in Dr. W. M. Layton's laboratory at Dartmouth Medical School. He is now

an Assistant Professor in the Department of Anatomy, Dartmouth College, Hanover, NH. (F)

THOMAS BJORKMAN, Ph.D. (10/1/86 - 9/30/88) worked on "The Mechanism of Gravity

Sensing in Plants" in Dr. Robert Cleland's laboratory at the University of Washington, Seattle,

WA. He is now an Assistant Professor in the Department of Horticultural Sciences at Cornell

University, Geneva, NY. (F)

STEVEN BLACK, Ph.D. (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 now an Associate Professor in the Department of

Biology at Reed College, Portland, OR. (F)

HARRY BLAIR, M.D. (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, MO. He is now an Assistant Professor in the Department of Pathology

at the University of Alabama, Birmingham, AL. (F)

THOMAS BROCK, Ph.D. (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 b4vena sativa

L.)" in Dr. Peter Kaufman's laboratory at the University of Michigan. He is now a Research

Associate in the Department of Pediatrics at the University of Michigan Medical School, Ann

Arbor, MI. (R)

277
JAY BUCKEY, JR., M.D. (7/1/82 - 6/30/84) worked on "Three-Dimensional

Echocardiography For Measuring Ventricular Volume" in Dr. C. Gunnar Blomqvist's laboratory at

the University of Texas Health Sciences Center, Dallas. He an Assistant Professor of Medicine at

the University of Texas Health Sciences Center, Dallas, TX. He was also the Alternate Payload

Specialist for the SLS-2 flight. (F,N)

GEORGE H. BURROWS, Ph.D. (7/1/81 - 6/30/83) worked on "Studies of Synaptogenesis"

in Dr. Marshall Nirenberg's laboratory at National Institute Health, Bethesda, MD. His last

known position was as a staff member of the National Heart, Lung, and Blood Institute, Bethesda,

MD. (G)

DENIS CLOHISY, M.D. (7/1/86 - 6/30/87) worked on "Mechanisms of Osteoclast Precursor

Differentiation" in Dr. Steven Teitelbaum's laboratory at Washington University Medical Center,

St. Louis, MO. He is now an Assistant Professor in the Department of Orthopaedic Surgery,

University of Minnesota, St. Paul, MN. (F)

JOHN CLOHISY (7/1/91 - 6/30/92) worked on "Regulation of Gene Expression in Bone

Remodeling" in Dr. Nicola Partridge's laboratory at St. Louis University Hospitals, St. Louis,

MO. He is completing his clinical residency at SL Louis University Hospital, St. Louis, MO. (G)

MARK COOPER, Ph.D. (1/1/85 - 12/30/86) worked on "Osteoporosis of Weighdessness and

the Electrophysiology of Bone" in Dr. John Miller's laboratory at The University of Calif, omia at

Berkeley, CA. He is now an Assistant Professor in the Department of Zoology, University of

Washington, Seattle, WA. (F)

MARK DESROSIERS, Ph.D. (7/1/86 - 6/30/88) worked on "A Search for Voltage-gating of

Plant Hormone Transport Channels" in Dr. Robert Bandurski's laboratory at Michigan State

University. He is continuing to work with Dr. Bandurski at Michigan State University, East

Lansing, MI. (R)

J. DAVID DICKMAN, Ph.D. (6/1/87 - 5/30/89) worked on "High Frequency Response

Properties of Semicircular Canal Fibers" in Dr. Manning Correia's laboratory at the University of

Texas, Galveston, TX. He is now an Assistant Professor in the Department of Surgery, Division

of Otolaryngology at the University of Mississippi, Jackson, MS. (F)

JOHN S. GARAVELLI, Ph.D. (111182 - 12/30/82) 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 Data Base Coordinator for Protein Information Resource at the National

Biomedical Research Foundation, Washington, DC. (G)

JOHN GAYNOR, Ph.D. (1/1/81 - 12/30/82) worked on "Purification and Characterization of

Amyloplasts from Pisura sativum" in Dr. Arthur Galston's laboratory at Yale University. He is

now an Associate Professor in the Department of Biology at Montclair State College, Upper

Montclalr, NJ. (F)

STEVEN GLOTZBACH, Ph.D. (1/1/84 - 12/30/84) worked on "Neurophysiological Studies

of Circadian Rhythm Control Mechanisms" with Dr. H. Craig Heller at Stanford University. He is

a Senior Research Scientist in the Department of Pediatrics; Co-Director, Pediatric SIDS Research

Program and Program Coordinator of the Perinatal Emphasis Research Center at Stanford

University, Stanford, CA. (R)

278
EDWARD GOOLISH, Ph.D. (8/1/90 - 7/30/91) worked on "The Effects of Simulated

Weightlessness on Swimbladder Function and Buoyancy Regulation in Fish" in Dr. Paul

Webber's laboratory at the University of Michigan, Ann Arbor, MI. He is a Research Associate at

Scripps/NOAA in LMolla, CA. (R)

CHERYL GOULD, Ph.D. (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, KY. She is now an Assistant

Professor in Medical Technology at the University of Dayton, Dayton, OH. (F)

MARTHA GRAY, Ph.D. (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 invivo Bone Preparation" in Dr. Clinton Rubin's laboratory at Tufts University School of Veterinary

Medicine, North Grafton, MA. She is an Associate Professor in the Department of Electrical

Engineering and Computer Science at M.I.T. and in the Harvard-M.I.T. Division of Health

Sciences and Technology in Cambridge, MA. (F)

EDWARD GREENFIELD, Ph.D. (7/1/88 - 6/30/90) worked on "Regulations of Osteoclastic

Bone Resorption by Osteoblasts" in Dr. Steven Teitelbaum's laboratory at Washington University,

St. Louis, MO. He is now an Assistant Professor in the Department of Orthopaedics, Case

Western Reserve University, Cleveland, OH. (F)

MARCIA HARRISON, Ph.D. (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. She is now an Assistant Professor in the Biology Department at Marshall University in

Huntington, WV. (F)

GARY JAHNS, Ph.D. (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 working at NASA-Ames, Moffett Field, CA. (N)

TIMOTHY JONES, Ph.D. (1/1/81 - 12/30/82) worked on "The Effects of Hypergravic Fields

on Brainstem Auditory-evoked Potentials" in Dr. John Horowitz's laboratory at the University of

California, Davis. He is now an Assistant Professor at the University of Nebraska, Lincoln, NE.

(F)

THOMAS KERR, Ph.D. (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' laboratory at the

University of Michigan and the second year in Dr. Dennis Drescher's laboratory at Wayne State

University. He was an Assistant Professor at Wayne State University, Detroit, MI. (D)

CHRISTOPHER KIRBY, Ph.D. (11/1/90 - 10/30/92) is working on "Eccentric Exercise as a

Countermeasure to Unweighting Atrophy" in Dr. Frank Booth's laboratory at The University of

Texas Medical School, Houston, TX. He is continuing to work with Dr. Booth at The University

of Texas Medical School, Houston, TX. (R)

JOHN KISS, Ph.D. (9/1/90 - 8/30/91) worked on "Gravitropism and Golgi apparatus function

in Chara rhizoids" in Dr. L. Andrew Straelin's laboratory at the University of Colorado, Boulder,

CO. He is now an Assistant Professor in the Department of Biology at Hofstra University,

Hempstead, IVY. (F)

279
DOUGLAS KLIGMAN, Ph.D. (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's

laboratory at the National Institute of Mental Health (NIMH), Bethesda, MD. The last known

position was as a staff member at NIMH, Bethesda, MD. (G)

WILLIAM KROEN, Ph.D. (1011/90 - 8/30/91) worked on "Balancing Reproductive and Root

Demands for Carbohydrates and Nitrogen in Atmospheres with High CO 2 Concentrations" in Dr.

Mary Peet's laboratory at North Carolina State University, Raleigh, NC. He is now an Assistant

Professor in the Department of Natural Sciences at Wesley College, Dover, DE. (F)

KONRAD KUZMANOFF, Ph.D. (7/1/83 - 7/30/85) worked on "Isolation and Identification

of B-glucan Synthetase: A Potential Biochemical Regulator of Gravistimulated Differential Cell

Wall Loosening" in Dr. Peter Ray's laboratory at Stanford University. He is now a Consultant

with Zoeticon, Berwyn, IL. (I)

VINCENT LING, Ph.D. (11/01/92 - 10/30/93) is working on "Calcium and Calmodulin

Mediation of the Gravitropic Signal" in Dr. Sarah Assmann's laboratory at Harvard University,

Cambridge, MA. (C)

ANNA LYSAKOWSKI, Ph.D. (7/1/89 - 6/30/91) worked on "Synaptic Relations of Type I

and Type II Vestibular Hair Cells" in Dr. Jay Goldberg's laboratory at the University of Chicago,

Chicago, IL. She is a Research Assistant Professor in the Department of Neurobiolog)_ at the

University of Chicago, Chicago, IL. (F)

MICHAEL MATILSKY, Ph.D. (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 the Director of the Laboratory and Sperm Bank Fertility Unit in the Department of Obstetrics and

Gynecology at Central Emek Hospital, Afula, Israel. (I)

KENNETH MCLEOD, Ph.D. (11/1/87 - 10/30/89) worked on "Regulation of Bone

Remodeling Activity through the Control of Stress Generated Electric Fields" in Dr. Clinton

Rubin's laboratory, SUNY, Stony Brook, NY. He now has a joint appointment as an Associate

Professor in the Department of Orthopaedics and in the Department of Biophysics and Physiology

at SUNY, Stony Brook, N'Y. (F)

DEWEY MEYERS, Ph.D. (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 is now a

medical student in the Medical School, Marshall University, Huntington, WV. (R)

EDWIN MILLER, Ph.D. (7/1/92-6/30/93) is working on "Influence of Antiorthostatic

Suspension on the Generation and Expression of Immunological Memory" in Dr. Gerald

Sonnenfeld's laboratory at the University of Louisville, Louisville, KY. (C)

LLOYD MINOR, Ph.D. (7/1/87 - 6/30188) 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. He is now a Clinical Instructor in the Department of Otolaryngology at

Vanderbilt University in Nashville, TN. (F)

DEAN MURAKAMI, Ph.D. (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 now an Assistant Researcher in the

Department of Animal Physiology at the University of California, Davis, CA. (R)

280
MARY MUSGRAVE, Ph.D. (6/1/86 - 5/30/88) worked on "Studies of Respiratory

Metabolism" in Dr. Boyd Strain's laboratory at Duke University, Durham, NC. She is now an

Associate Professor at Louisiana State University, Baton Rouge, LA. (F)

SHERRIE LYNN PERKINS, M.D. (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, MO. She is now an Assistant Professor in the Department of Pathology,

University of Utah School of Medicine, Salt Lake City, UT. (F)

KENNETH POTE, Ph.D. (6/1/88 - 5/30/90) worked on "An Otoconial Calcium Binding

Protein; Its Temporal Expression and Tissue Distribution" in Dr. Robert Kretsinger's laboratory at

the University of Virginia, Charlottesville, VA. He is now an Assistant Professor in the

Department of Otolaryngology, Harvard Medical School, Boston, MA. (F)

GARY RADICE, Ph.D. (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. He

is now an Assistant Professor in the Department of Biology, University of Richmond, Richmond,

VA. (F)

DENNIS REINECKE, Ph.D. (10/1/88 - 9/30/89) worked on "Does Indole-3-Acetic Acid

Turnover Correlate with Topically-induced Asymmetric Growth?" in Dr. Robert Bandurski's

laboratory at Michigan State University, East Lansing, MI. He is now working as a Research

Associate in the Department of Plant Biology at the University of Minnesota, St. Paul, MN. (R)

RAVEN REITSTETTER, Ph.D. (10/1/91 - 9/30/92) is working on "Effects of Altered

Gravity on Single Channel Acetylcholine Receptor Kinetics" in Dr. Raphael Gruener's laboratory

at the University of Arizona, Tucson, AZ. He is continuing to work with Dr. Gruener at the

University of Arizona, Tucson, AZ. (R)

MARGARET S. RICE, Ph.D. (1/1/92 - 12/30/92) is working on "Immunolocalization and

Molecular Characterization of an IAA Binding Protein" in Dr. Terri Lomax's laboratory at Oregon

State University, Corvallis, OR. (C)

FARREL R. ROBINSON, JR., Ph.D. (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, PA. He is now working as an Assistant Professor in

the Department of Physiology & Biophysics at the University of Washington School of Medicine,

Seattle, WA. (F)

BRUCE SERLIN, Ph.D. (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 in the Department of Biological

Sciences at DePauw University, Greencastle, IN. (F)

ROBERT SLOCUM, Ph.D. (1/1/81 - 12/30/82) 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 in the Department of Biological

Sciences at Goucher College, Baltimore, MD. (F)

281
J. HENRY SLONE, Ph.D. (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 an Assistant Professor in the

Department of Biology at Francis Marion University, Florence, SC. (F)

JOSEPH STEFFEN, Ph.D. (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 Associate Professor in the Department of Biology at the University of Louisville,

Louisville, KY. (F)

JULIANNA SZILAGYI, Ph.D. (7/I/81 - 12/30/81) worked on Progressive Hemodynamic

Changes in Simulated Weightlessness" in Dr. C. Ferrario's laboratory at the Cleveland Clinic. She

is now an Assistant Professor in the Department of Pharmacology at the University of Houston,

Houston, TX. (F)

LAWRENCE TALBOTT, Ph.D. (7/1/89 - 12/30/89) worked on "Actuation of gravicurvature

in pea stems by alterations of specific wall polymers" in Dr. Barbara Pickard's laboratory at

Washington University, St. Louis, MO. He is now a Research Associate at the University of

California, Los Angeles. (R)

DONALD THOMASON, Ph.D. (11/1/89 - 8130190) worked on "Mechanisms of decreased

actin synthesis during rodent hindlimb unweighting" in Dr. Frank Booth's laboratory at the

University of Texas Medical School, Department of Physiology and Cell Biology, Houston, TX.

He is now an Assistant Professor in The Department of Physiology & Biophysics at the University

of Tennessee, Memphis, TN. (F)

JOYCE THOMPSON, Ph.D. (7/1/90 - 6/30/92) worked on "Targetoid and Type IIC Fibers in

Hypodynamic and Recovering Rat Muscle - Histochemistry, Ultrastructure and Myosin Isozyme

Composition" in Dr. Danny Riley's laboratory at the Medical College of Wisconsin, Milwaukee,

WI. She is a Research Assistant Professor in the Department of Cellular Biology and Anatomy in

the Medical College of Wisconsin, Milwaukee, WI. (F)

LADORA THOMPSON, Ph.D. (7/1/92 - 6/30/93) is working on "The Role of H + and

Inorganic Phosphate in the Fatigability of the Soleus Following Hindlimb Suspension" in Dr.

Robert Fitts' laboratory at Marquette University, Milwaukee, WI. (C)

YASUHIRO TORIGOE, Ph.D. (1/1/84 - 12130/85) worked on "Anatomical correlated

underlying vestibulo-autonomic outflow to the gut" with Dr. Robert H.I. Blanks at the University

of California, lrvine. He is now an Assistant Adjunct Professor in the Department of Anatomy and

Neurobiology at the University of California, Irvine, CA. (F)

LARRY WIMMERS, Ph.D. (11/1/92 - 10130/93) is working on "Role of the E.R.-Localized

Ca2+-ATPase in Higher Plant Gravity Perception" in Dr. Alan Bennett's laboratory at the

University of California, Davis, CA. (C)

282
ECCENTRIC EXERCISE TRAINING AS A COUNTERMEASURE TO NON-WEIGHT

BEARING MUSCLE ATROPHY

Christopher R. Kirby

Department of Physiology and Cell Biology

University of Texas Medical School

Houston, TX 77225

Description of Research

The goal of this research is to examine the control of muscle gene expression during states of

altered contractile activity. The specific aims of this research are: 1) to determine if eccentric

resistance (lengthening contractions) exercise training can maintain normal protein content in

non-weight bearing soleus, and 2) to examine whether the duration of an acute bout of aerobic

exercise is associated with elevated skeletal muscle cAMP content post-exercise.

Muscle atrophy resulting from non-weight bearing is one of the principle alterations associated

with space flight conditioning. Normal locomotion, posture, and activity incorporate a

significant degree of eccentric contraction, which is absent during non-weight bearing. This

provides a convincing rationale for testing eccentric resistance exercise as a countermeasure to

non-weight bearing (e.g., space flight) muscle atrophy.

Increased aerobic work capacity in skeletal muscle undergoing chronic endurance training

results, in part, from increases of mitochondrial density. Although, the mechanochemical link(s)

between chronic endurance exercise and the increase in mitochondrial density is unknown,

cAMP is one possible signal under consideration, cAMP concentration in skeletal muscle was

doubled in the first couple of minutes following an acute bout of running at all exercise durations

ranging from 5 to 30 minutes. Since 5 to 10 minute bouts of aerobic exercise, repeated daily, are

insufficient to stimulate increased mitochondrial density, a dilemma exists: "If cAMP is a

mechano-chemical link between endurance exercise and increased mitochondrial density, why is

cAMP elevated after 5 or 10 minute runs, which when performed daily do not increase skeletal

muscle mitochondrial density?"

Accomplishments

(1) Eccentric exercise training during non-weight bearin.g attenuated, but did not prevent,

the loss of soleus muscle wet weight and noncollagenous protein by 77 percent and 44 percent,

respectively.

(2) No increases in skeletal muscle cAMP were noted for the first 4 hours after a single

10 or 60 minute bout of running by untrained rats.

Significance of the Accomplishments

Because the soleus muscle exhibits the greatest degree of atrophy during non-weight bearing, it

has been the focus of most exercise countermeasure studies. Virtually all exercise modalities

attenuate soleus muscle atrophy. However, compared with other forms of exercise, resistance

training is at least equal in its effectiveness and superior in its efficiency. This is supported by

the finding that eccentric exercise during only 0.035 percent of the total non-weight bearing

period attenuated 44 percent of soleus muscle noncollagenous protein loss.

283
Theabsence of change in skeletal muscle cAMP in the 4 hour time period after running for either

10 or 60 minutes in the present study could suggest the following: 1) cAMP values by

themselves, axe not a function of run duration, 2) elevated muscle cAMP levels could occur at

higher exercise intensities, 3) increases in cAMP may require multiple bouts of exercise, or 4)

increases in muscle cAMP may be transient, initiating more prolonged increases in factors distal

to cAMP in the adenylate cyclase cascade. Future studies should investigate the correlative

relations between run duration and protein kinase A, or some protein phosphorylated by protein

kinase A, rather than cAMP.

284
COMPETITION BETWEEN FRUIT DEVELOPMENT AND ROOT GROWTH IN

TOMATOES GROWN AT HIGH CO2

William K. Kroen

Department of Horticultural Science

North Carolina State University

Raleigh, NC 27695

Description of Research

Long-term growth of plants at elevated CO2 (approximately 1000 I.tl 1-1) generally leads to

increased growth despite only transient increases in photosynthetic carbon uptake. We have

previously shown increased growth of leaves, stems, and roots during the first 2 weeks of

enrichment in two varieties of tomato; this growth was accompanied by slight increases in carbon

exchange rate. Leaves and stems remained larger in plants grown at high CO2, though growth

rate of plants in the two CO2 treatments was similar after the first 2 weeks. Root growth slowed

or stopped at the time of fruit set in plants growing at elevated CO2, resulting in similar or

reduced total root biomass relative to ambient-grown plants. This decreased rooting ability was

hypothesized to account for nutrient deficiency-like symptoms exhibited by plants grown at high

CO2. Tomato fruit weight, but not necessarily number, significantly increased at increased CO2.

Decreased root biomass and increased fruit weights would be beneficial in a CELSS system,

since less non-edible material would be produced in systems anticipated to operate at high CO2

concentrations.

A second set of experiments, conducted during the summer of 1991, examined plant biomass,

fruit production, photosynthetic carbon uptake, and nutrient and carbohydrate partitioning during

growth of the small cherry tomato variety "Florida Petite" at normal (380 i.tl 1-1) and elevated

(1000 lal 1"1 ) CO2 concentrations when the plants were subjected to root restriction or fruit

removal. We were interested in testing the hypothesis that there was internal competition for

carbon and nutrients between fruit and root systems.

Accomplishments

(I) Plants grown at high CO2 had significantly higher biomass than those grown at

ambient levels of CO2. When growth was reduced by tight spacing and root restriction, biomass

of all plant parts was significantly reduced at both CO2 levels. However, leaves and stems of

root-restricted plants growing at high CO2 were larger than those at ambient CO2, though root

biomass remained the same. (See accompanying figure of plant biomass after 6 weeks of

growth.) There was no significant reduction in growth of unripe fruit due to root restriction at

either CO2 level. Space and funding constraints precluded running the experiment until fruit had

ripened.

(2) Removal of fruit significantly increased the remaining vegetative biomass of leaves

and roots in plants grown at both ambient and elevated CO2, though the plants exhibited severe

leaf deformations. Photosynthetic carbon uptake declined in defruited plants under both CO2

regimes.

285
L

o

n

leO

n

O

rJ_

e

L

e.s

25

20

15

10

5

0

8

8

4

2

0

8

4

360 ,_L L-_ L-1 1000 ,uL

2

0

Z n •

E E

(3) Plants grown at high CO2 had significantly higher levels of leaf starch and soluble

sugars than did leaves of plants grown at ambient levels of CO2. There were no changes in

carbohydrate concentrations of stems and roots. We have previously described both of these

results. No significant changes in nutrient concentrations were found in any part of plants

growing at high CO2 at the time of fruit ripening.

(4) Fruit removal at each CO2 level resulted in significant reductions in Leaf starch and

hexose concentrations (relative to plants bearing fruit) particularly at high CO2, whereas there

were significant accumulations of hexose and sucrose in roots. Fruit removal caused an increase

in phosphorus concentration and a decrease in calcium concentration in leaves. Stems

accumulated significant amounts of nitrogen, phosphorus, and magnesium, while roots also

accumulated magnesium.

286
Significance of Accomplishments

(1) Root restriction and tight spacing at elevated CO2 may not severely reduce fruit

production while bringing about less vegetative growth in bushy tomatoes. This line of research

needs to be explored further in reference to potential nutrient delivery systems and their effect on

root growth and viability, particularly since there is evidence in the literature suggesting that

root-restricted plants show symptoms of water deficiency.

(2) Fruits are such potent sinks for the products of photosynthesis that root growth

appears to be limited by internal competition with fruit. Removal of this carbon sink allows for

increased root growth, even when photosynthetic carbon uptake decreases to compensate for the

reduced demand for carbon.

(3) The reduced root-to-shoot ratio during fruit development in these plants grown at

elevated CO2 does not appear to result in deficiencies for carbohydrates or nutrients within

leaves. Leaves of high CO2 -grown plants still accumulate massive amounts of carbohydrates.

Whether growth of plants at elevated CO2 has any effect on the nutritional value of tomato fruit

is still unknown, though we found no difference in carbohydrate or nutrient concentrations of

ripening fruit due to elevated CO2.

(4) Fruit removal, despite the apparent sink-limited reduction in carbon uptake, allows

for increased levels of carbohydrates to be moved to the roots under either CO2 regime.

Nutrients also accumulate in the stem when not required by ripening fruit. All three results bear

out the hypothesis that root growth in tomatoes is restricted by fruit load.

Publications

Alp, W.J. 1991. Effects of source-sink relations on photosynthetic acclimation to elevated CO2.

Plant, Cell and Environment 14: 869-875.

Kroen, W.K. and Peet, M.M. 1991. High CO2 concentration alters carbohydrate partitioning in

favor of shoots in tomato (Abstract). Plant Physiology 96(Suppl): 636

Peterson, T.A., Reinsel, M.D., and Krizek, D.T. 1991. Tomato (Lycopersicon_esculentum Mill.,

cv. 'Better Bush') plant response to root restriction. Journal of Experimental Botany 42:1233-

1240.

Starck, Z. and Witek-Czuprynska, B. 1991. Fruit growth as factor controlling photosynthesis

and carbon partitioning in tomato plants. Acta Physiologiae Plantarum 13:105-113.

Tripp, K.E., Peet, M.M., Pharr, D.M., Willits, D.H., and Nelson, P.V. 1991. CO2 -enhanced

yield and foliar deformation among tomato genotypes in elevated CO2 environments. Plant

Physiology 96:713-719.

287
CHARACTERIZATION OF A CALMODULIN-BINDING PROTEIN FROM PLANT

ROOTS

Vincent Ling

Department of Organismic and Evolutionary Biology

The Biological Laboratories

Harvard University

Cambridge MA 02138

Description of Research

The long-term objective of this research is to dissect the biochemical components comprising the

calcium-mediated signal transduction pathways within plant cells. The current focus is on the

characterization and purification of a calcium-dependent calmodulin-binding protein

preferentially localized in plant roots.

Although the process of development in plants is greatly affected by a variety of environmental

conditions, relatively little is known about the mechanisms which link perception of external

signals to alterations in growth. Stimuli such as light and gravity, when applied to plants, result

in trar_.sient and localized increase of subcellular calcium ion concentration prior to growth

response. This system appears to be analogous to the transient calcium fluxes that occur during

the stimulation of mammalian cells, especially those of neural origin. In addition, pivotal

proteins involved in neural signal transduction pathways have also been found in plant ceils,

most notably calmodulin.

The calmodulins are a small family of highly conserved calcium-binding proteins that function as

activators of disparate regulatory pathways. Calmodulin has no intrinsic enzymatic property.

Through calmodulin, transient calcium increases result in the temporal and indirect activation of

many calcium-calmodulin dependent enzymes (including protein kinases and

phosphatases)[Figure 1]. In this nascent field of plant signal transduction, only 4 calmodulindependent enzymes have been described, as opposed to the dozens documented in animal

systems. Due to this paucity of information, a detailed model of the calcium signaling network

in plants has yet to emerge. Clearly, further elucidation of calmodulin-dependent enzymes in

plant systems is necessary.

Stimuli (gravity, light, etc)

Altered Growth

Ca2+-CaM (_-6) ? \ ?\ X \ ?

Ca _CaM Enzyme1

Ca _CaM Enzyme2

Ca =-*CAM Enzyme3

Ca _aM Enzyme4

CaM-dependent enzymes ...... ?????

Figure 1.

288
Protein extractsof various plant cell protoplasts,tissues,and organswere analyzedfor the

presence of these target calmodulin-binding proteins by electrophoretic separation,

immobilizationontoPVDF membranes,andexposureto radiolabeledcalmodulin. In this way,

proteinsboundto the taggedcalmodulin were detected. With this assay,thesecalmodulinbinding proteins could then be subjected to biochemical purification and subsequent

identification. Thecharacterizationof theseproteinswouldthusleadto abetterunderstandingof

the biochemicalsignaling networks that link sensoryperceptionand physiological response

within plants.

Accomplishments

(1) Calmodulin levels and calmodulin-binding proteins are not distributed uniformly

between protoplast types, tissues, or organs in Viciafaba (fava bean).

(2) Two categories of calmodulin-binding proteins are present in plants. One set is

ubiquitously distributed throughout the plant, and the others appear to be unique and found in

root, stem, or guard cell protoplasts.

(3) One prominent calmodulin-binding protein with a molecular weight of

approximately 62 kilodalton was detected in both the cortex and stele of Vicia roots. This

protein was detected along the axis of the root but not at the root tip.

(4) Other major calmodulin-binding proteins were detected in stems and nodules.

These proteins were similar in mass but not identical to the root protein.

(5) Development of a purification protocol for the root 62 kilodalton calmodulinbinding protein resulted in a method that offered an overall 200-fold purification of this protein

from crude extracts. This protein was characterized as a calmodulin-stimulated enzyme,

glutamate decarboxylase.

Significance of Accomplishments

Finding 1: Differential distribution of calmodulin and calmodulin-binding proteins suggests that

calcium signaling pathways are not identical in all parts of the plant. This finding is consistent

with the evidence that stimuli-induced calcium increases lead to disparate physiological

responses in plants, depending on tissue type.

Finding 2: The presence of two categories of calmodulin-binding proteins suggests two classes

of calmodulin-regulated activity: The first class being proteins involved with "housekeeping"

functions and the second class involved with responses unique to that tissue.

Finding 3: The preferential accumulation of the 62 kilodalton calmodulin-binding protein in the

mature regions of the root suggest a developmental regulation of this protein.

Finding 4: Discovery of other similar, but not identical, calmodulin-binding proteins in stems

and nodules suggests post-translational modification(s) of proteins or presence of different

protein isoforms may be involved in specialized signaling activity in specific tissues.

Finding 5: Identification of this calmodulin-stimulated protein as glutamate decarboxylase within

the framework of calcium signal transduction offers insight into the mechanism(s) by which the

root responds to gravity and other signals via the production of gamma-arninobutyrate (GABA).

289
Publication

Ling, V. and Assmann, S.M. 1992. Cellular distribution of calmodulin and calmodulinbinding proteins in Viciafaba L. Plant Physiology 100: 970-978.

290
SYNAPTIC RELATIONS OF TYPE I AND TYPE

MAMMALIAN CRISTA AMPULLARIS

Anna Lysakowski

Department of Pharmacological and Physiological Sciences

The University of Chicago

Chicago, IL 60637

II HAIR CELLS IN THE

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 studies to date have concentrated primarily on the crista ampullaris, but preliminary data

from the utriculus indicate similar regional synaptic organization.

Previous work at the light microscopic level has characterized three types of afferent nerve fibers

innervating two types of receptor cells (type I and type II hair cells). The three types of nerve

fibers are: (1) calyx (or chalice) fibers, which germinate 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. In contrast, the three types of nerve fibers show an unequal

distribution. Calyx fibers (10 percent of the total) are found primarily in the central zone.

Bouton fibers (20 percent of the total) are found primarily in the peripheral zone. Dimorphic

fibers (70 percent of the total) are found throughout the epithelium. These three types of fiber

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. Dimorphic fibers 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 earlier studies, it

was concluded that the physiology of an afferent fiber 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.

Building upon these studies, we have been making progress on three projects: (1) ultrastructural

analysis of physiologically characterized afferent fibers; (2) further analysis of the regional

variations in synaptic organization; and (3) comparative studies on a primate model, the squirrel

monkey.

In the first project, we have observed some morphological features that may serve to distinguish

hair cells contained in the two different types of afferents in unlabeled material. Previously, it

has not been possible to distinguish type I hair cells belonging to pure calyx units from those

belonging to dimorphic units without some form of dye labeling of the afferent fiber. Based

upon serial section analysis of l0 samples from cristae in four animals, there appear to be at least

two classes of type I hair cells. We have confirmed this difference in several horseradish

peroxidase (HRP)-labeiled calyx and dimorphic units.

291
Figtn'¢ 1. An HRP-injected physiologically characterized calyx

unit. This unit had a cv of 0.63 and a low gain to head rotation

at 2 Hz. Note the large subcuticular mitochondria and thick

stereocilia on the type l hair cell enclosed with the unlabeled

calyx.

292
In our previous studies of the chinchilla cristae, we found it convenient to divide the

neuroepithelium into central, intermediate, and peripheral zones of equal areas. The synaptic

ultrastructure of these three zones differed in several ways. We have now found that the central

zone can, itself, be divided longitudinally into two subzones. Conclusions are based on a serial

ultrastructural examination of 10 samples, each 2 t.tm thick. Four of the samples were cut in the

longitudinal plane of the cristae and the others were sectioned transversely. Stereological

methods were used to estimate the number of synaptic features per hair cell.

Having completed our studies of the ultrastructural synaptic organization of the chinchilla crista,

we initiated a parallel study in the monkey. We had previously shown that the ratio of type I to

type I1 hair cells was higher in the monkey (3:1), than in the chinchilla (1:1). We were interested

in how the difference in hair-cell ratios would be reflected in the synaptic organization of the

crista in the two species. Our results are based on five samples from two cristae in two squirrel

monkeys.

Accomplishments

(1) There appear to be two classes of type I hair ceils in mammalian cristae. These two

classes are distinguished by certain ultrastructural features. The first class has large

mitochondria subjacent to the cuticular plate and thicker stereocilia. These mitochondria were

twice as large as those found elsewhere in the same cell. Cells containing these larger

mitochondria were found mainly in the central zone at the apex of the crista and occasionally in

the intermediate zone. So far they have not been observed in the peripheral zone.

The second class of type hair cells has smaller subcuticular mitochondria and thinner stereocilia.

This second class was found in units identified as dimorphic units by the presence of calyceal

collaterals. We have confirmed this difference in several units unlabeled by extracellular

injections of HRP and in a small number of HRP-unlabeled and physiologically characterized

calyx and dimorphic units. Type 1I hair cells had subcuticular mitochondria similar in size to the

second class, but their stereocilia were thinner than those of type I hair cells in any region of the

crista. Preliminary studies of the utricular macula indicate that the same two classes of type I

hair cells are also present in the striolar region.

(2) In the chinchilla, the central zone can be divided longitudinally into two subzones:

one (CA) occupying the apex of the crista, and the other (CS) located along its slopes. The CA

region differs from the CS region in having (1) larger, more widely space hair cells; (2) thicker

calyx endings; (3) most of the complex calyx endings present in the neuroepithelium; (4) larger

numbers of calyceal invaginations per type I hair cell; (5) a higher concentration of ribbon

synapses between type II hair cells and calyx outer faces; and (6) fewer afferent boutons per type

II hair cell. Within the CA zone, type I hair ceils are innervated by complex, as compared to

simple, calyx endings that have more calyceal invaginations and fewer outer-face ribbon

synapses.

(3) From our ultrastructural studies in the monkey we found that there are 6-10 type

ribbons per type I hair cell and some indication that central type I hair cells have more ribbons

that peripheral type I hair cells. In addition, each central type I hair cell had _=20 calyceal

invaginations, whereas each peripheral type I hair cell had -2 invaginations. Although

qualitatively similar gradients were observed in the chinchilla, the numbers of ribbon synapses

and calyceal invaginations were smaller in the monkey.

Type II hair cells in the monkey had similar numbers of ribbons and afferent boutons; there were

fewer boutons and ribbons centrally, than peripherally (_--13 vs. ---40 per type II hair cell). Very

few ribbons from type 1I hair cells onto the outer surface of calyces were seen in the monkey as

compared to the chinchilla. As in the chinchilla, the majority of these were found in the central

293
zone. Suchribbonsconstitutedonly 1percent(12/952)of thetotalnumberof synapticribbonsin

the monkey;whereasin the chinchilla, they constituted11 percent(456/4007)of the central

outer-face ribbons. In the chinchilla, outer-facesynapsesare so common that most calyx

afferentscouldbefunctionallydimorphic;this isnotthecasein themonkey.

In the monkey,the numberof efferent boutonsper hair cell differs betweenthe central and

peripheral zones with = 2 efferent boutons per type I hair cell in the central zone, and _=0.5 per

hair cell in the periphery. Type II hair cells had = 4 in the central zone and _=_10 in the periphery.

In contrast, the efferent innervation of type I and type II hair cells was more uniform in the

chinchilla.

Significance of the Accomplishments

Finding 1: The use of purely morphological features to distinguish subclasses of type I hair

cells belonging to different afferent classes would provide a significant contribution to studies of

the vestibular periphery. Such criteria would obviate the necessity of physiological

characterization, such that space flight or human material could be analyzed. The functional

significance of the features used to determine the two different classes of type I hair cells is yet to

be determined; but, e.g., it is conceivable that the thicker stereocilia found on type I hair cells in

calyx afferents are responsible for the lower gain of these calyx afferents due to their greater

viscous drag and mechanical loading on the hair cell.

Finding 2: It may be of functional importance that 75--80 percent of the calyx fibers found in

the central zone in the chinchilla are located in CA, the subdivision along the apex of the crista.

We have yet to determine whether the synaptic differences are a reflection of the specific

innervation of calyx fibers. In addition, we are currently attempting to determine whether this

subdivision of the central zone also holds for the squirrel monkey.

Finding 3: The general trends in regional synaptic innervation that we observed in the crista of

the chinchilla, a rodent model, seem to be valid, as well, in the monkey, a primate model. This

has implications for studies of human synaptic innervation of the vestibular periphery.

However, two features were different in the monkey: (1) The numbers of outer-face synapses.

In the chinchilla, outer-face synapses are so common that most calyx afferents could be

functionally dimorphic; this is not the case in the monkey. This means that most likely the only

innervation that a calyx unit in the monkey receives is from the hair cells enclosed within it. (2)

Regional variations in efferent innervation. The most significant data that we have on efferent

effects on afferent discharge rates are from the squirrel monkey. These data show that efferents

exert a greater effect upon irregular afferents. Subsequent studies have shown that irregular

afferents are found predominantly in the central zone. The present findings suggest that the

greater numbers of efferent boutons per central type I hair cells may be responsible for the

greater effects of efferent innervation on irregular afferents.

An understanding of the synaptic relations of hair cells with their afferent and efferent endings is

fundamental to understanding alterations during space flight, as well as during development of

the vertebrate vestibular apparatus in extended space flights. These changes will probably be

subtle, at best; and, so, quantitative baseline data from normal adult tissue, including non-human

primates, such as that shown in the present study, are needed for comparison to space flight

experiments.

294
Publications

Fernandez, C. A., Lysakowski, A., and Goldberg, J. M. 1991. Comparison of hair-ceU

populations and afferent innervation patterns in the cristae of the squirrel monkey and the

chinchilla. Society of Neuroscience Abstracts 17:312.

Goldberg, J. M., Lysakowski, A., and Fernandez, C. 1992. Structure and function of vestibular

nerve fibers in the chinchilla and squirrel monkey. Annuals of the New York Academy of

Sciences 656: 70-80.

Lysakowski, A. 1992. Possible morphological features to distinguish calyx vs. dimorphic type I

vestibular hair ceils in the chinchilla cristae. Society of Neuroscience Abstracts 18: 1400.

Lysakowski, A. and Goldberg, J. M. 1993. Cellular and synaptic architecture of the central zone

of the chinchilla cristae (Abstracts). ARO 16th Midwinter Meeting, p. 116.

Lysakowski, A., McCrea, R. A., and Tomlinson, R. D. 1992. Anatomy of the Vestibular

Endorgans and Neural Pathways. In: Otolaryngology: Vol. 4, Ear and Cranial Base (Cummings,

C.W., Ed.). St. Louis: Mosby-Year Book Inc., p. 2525-2547.

295
INFLUENCE OF ANTIORTHOSTATIC SUSPENSION

INFECTION

Ed Miller

Department of Microbiology and Immunology

School of Medicine

University of Louisville

Louisville, KY 40292

ON RESISTANCE TO

Description of Research

This research project was designed to evaluate how antiorthostatic suspension, a ground-based

modeling system that simulates certain aspects of weightlessness that occur during space flight,

influences the capacity of mice to resist microbial infection.

When humans are exposed to pathogenic microorganisms, either through childhood vaccination

programs or through natural contact with environmental microorganisms, the immune system

generates protective immunity to that pathogen. Sometimes this immunity lasts for the life span of

the host (e.g., polio vaccine), and in other instances the immunity lasts only for several years

(e.g., tetanus vaccine). Long-term immunological memory plays an important role in the health of

adult humans.

Since space flight has been shown to alter many of the effector mechanisms of the adult immune

system, some of which include those responsible for mediating resistance to infection and

immunological memory, this research project has addressed the question of whether space flight,

which is a stressful event, could alter the ability of the immune system to resist microbial infection

and generate and express immunological memory. If space flight inhibits the capacity of the

immune system to resist infection and generate and express immunological memory, this could

predispose subjects exposed to space flight to an increased risk of microbial infections.

The antiorthostatic suspension model has become the most widely accepted ground-based

modeling system employed to simulate certain changes in immune cell effector function that occur

as a result of space flight, and has been utilized in this project. This model entails suspending

rodents with a head-down tilt of -20", such that their hind limbs no longer bear weight. This

induces a forward shift in body fluids similar to that which occurs as a result of weightlessness

during space flight, and also induces some changes in immunological function that have been

reported during and following space flight.

Mice were suspended in the antiorthostatic suspension system or in the control orthostatic position,

in which there is no head-down tilt, to distinguish the immunological effects induced by the stress

of the head-down tilt from the effects induced by the stress of attaching the suspension apparatus,

and were infected with pathogenic microorganisms to study their ability to resist infection and

generate and express protective immunological memory.

Accomplishments

It is well documented that stressful events can depress certain facets of immunological function.

However, the relevance of these alterations in the context of resistance to infection have yet to be

studied in any detail. The predominant number of reports in the literature contend that since stress

can depress some functional capacities of the immune system, a correlated decrease in resistance to

infection should accompany it.

296
The datageneratedin this project have,in fact, shown the exactopposite. Mice have been

suspendedin the antiorthostaticposition and infected at various timepoints throughoutthe

suspension.UsingthepathogenicbacteriumListeria monocytogenes, a microorganism commonly

used to evaluate cell-mediated resistance to infection, we found that resistance to the challenge

infection was not altered if mice were infected concurrently with the onset of the suspension.

However, if the infection was started on day 2 or 4 of the suspension, which represents the most

stressful period of the suspension experiments, mice were completely resistant to the challenge

infection. In fact, mice infected on day 2 or 4 of the suspension were completely resistant to a

lethal dose of L. monocytogenes. Control mice infected with L. monocytogenes typically require 7

to 10 days to clear the challenge infection. Mice infected on day 2 or 4 of the suspension were able

to clear the infection within 1 day, which represents a remarkable enhancement in resistance to

infection, even though the subjects were being subjected to the stressful event of antiorthostatic

suspension. As the mice adapted to being in the antiorthostatic position, an attenuation of the

enhancing effect was observed. Mice infected on day 7 of the suspension no longer displayed the

same magnitude of enhanced resistance to L. monocytogenes, and the immune system required 7

days to clear the infection.

Unfortunately, the enhancement of resistance was not always viewed as advantageous, in that mice

infected on day 2 or 4 of the infection failed to generate a long-lived state of protective

immunological memory to the listeria infection. This has been attributed to the host being able to

clear the challenge infection so rapidly that stimulation of effector T-lymphocytes, which mediate

memory immunity to L. monocytogenes, did not occur. Mice that were infected concurrently with

the onset of infection, or after adapting to the stress of antiorthostatic suspension, generated normal

levels of protective immunological memory.

.V,

t_

,..1

o

Start Primacy Infection

Concurrently with Suspension

8

7

6

5

4

3

2

1

8

7

6

5

Start Primary Infection

on Day 2 or 4 of Suspension

8-

7-

6-

5-

4.-

3-

2-

1 I i I i I

0 1 2 3 4 5

Day of Infection

Control l

O' Orthostatic

Antiorthostatic

Start Primary Infection

on Day 7 of Suspension

0 1 2 3 4 5 1 2 3 4 5

Day of Infection Day of Infection

FigureI. Growth ofa primaryL.monocytogenesinfectioninmice suspendedintheorthostatic

orantiorthestatic positions.Suspendingmicefor2-4 dayspriortotheonsetofinfectionenhanced

thecapacityoftheimmune systemtoresistinfection.

Since the macrophage isthe celllineageresponsiblefor the killingof L. monocytogenes, we

studiedmarcrophage functionalactivities.We were able tocorrelatetheenhancement of resistance

during suspensionwith increasedlevelsof rnarcrophageactivation.

297
Significance of the Accomplishments

The novel findings of this project are not consistent with the predominant reports in the literature,

which suggest that suspension, like many other forms of stress, depress immune cell function and

therefore are expected to decrease host resistance to disease. The mechanisms underlying the

capacity of antiorthostatic suspension to enhance resistance to L. monocytogenes were not

apparent, but may have involved direct macrophage activation, macrophage activation via other

cells of the immune system (such as natural kiUer INK] cells), and by other tissues and organs,

most notably the neurological and endocrine systems, which are affected by stress and can exert

immunomodulatory capacities during stressful events. While it is possible that certain forms of

stress may depress some facets of the immune system and in fact render the host more susceptible

to disease, some forms of stress may actually enhance certain immune cell effector mechanisms,

which may endow the host with increased resistance to infection by particular classes of pathogenic

microorganisms during stressful periods.

The number of animal models available for evaluating how stressful events influence

immunological function is small. Of these models, only a small percentage is utilized for studies of

how stress can influence resistance to infection. These results are among the first which

conclusively demonstrate that a physiological stressor can, in fact, enhance resistance to infection.

Identifying the mechanisms by which the enhancement to infection was mediated can contribute to

our knowledge of how other physiological systems interact with the immune system. The immune

system is not an isolated entity that works in an independent fashion; but instead, bidirectionally

communicates with other tissues and organs by mechanisms which are largely unknown. Studies

such as these can help to elucidate how the immune system participates in mediating systemic

physiological homeostasis, and how stressful events, be they on Earth or as a result of manned

space missions, can affect these normal modes of communication. By understanding what the

normal modes of communication are and how they are altered as a result of stress, it may be

possible to design and test therapeutic regimens that help the host better adapt to certain stressful

events.

Stressful

Event

Agent?

L Infection and Cancer

Figure 2. Possible mechanisms by which antionhostatic suspension, and other potential

physiological slxessors, may activate macrophages to enhance resistance to infection. If the

mechanisms by which enhanced m_rophage activation occurs can be elucidated, it may be possible

to develop chemotherapeutic agents that stimulate these mechanisms, which could be of benefit to

NASA and to basic biomedical sciences.

29_
Moreover, identifying how the enhanced resistance to infection was mediated may lead to advances

in preventing and treating microbial infections. By developing chemotherapeutic agents that can

stimulate the underlying mechanisms which enhance resistance to infection, it may be possible to

activate the immune system in persons at increased risk of infection, including astronauts, trauma

patients, and burn victims. In addition, it may be possible to stimulate macrophage activation in

patients with dysfunctional immune systems, such as transplant recipients that are receiving

tmmunosuppressive therapy, or AIDS patients, who have macrophages but lack the T-cells

necessary for stimulating macrophage activation.

Such therapies may not be restricted to effectiveness against microbial infection, but also may

include resistance to neoplastic diseases, since the macrophage can exert potent effects against

transformed cells. By altering normal events that are occurring within the host, it may be possible

to alleviate many of the toxicities associated with administering exogenous materials, such as high

levels of cytokines, that are encountered with many of the therapies used today. Indeed, there is

enthusiasm and potential for this NASA project in contributing not only to the manned space

program but also to basic biomedical sciences.

Publications

Miller, E.S. and Sonnenfeld, G. 1992. Influence of antiorthostatic suspension on cytokine

production and resistance to murine Listeria monocytogene$ infection. Tucson, AZ, 8th Annual

Meeting ASGSB .

Sonnenfeld, G. and Miller, E. 1993. Influence of antiorthostatic suspension on resistance to

bacterial infection. Annual Meeting of American Association of Immunologists, Denver, CO,

Journaloflmmunology 150: 68A.

299
DEVELOPMENTAL STUDY OF RESPONSES

DIAGEOTROPICA TOMATO MUTANT

Margaret S. Rice

Department of Botany and Plant Pathology

Oregon State University

Corvallis, OR 97331-2902

TO AUXIN IN THE

Description of Research

My research is aimed at understanding the cellular events in plants that take place after the

perception of a gravity stimulus. This signal transduction chain may involve calcium ions, growth

inhibitors, and other factors. However, it is generally behoved that during the response to gravity

in plant shoots, indole-3-acetic acid (IAA) plays an tmportant role. According to the prevailing

Cholodny-Went hypothesis, IAA is transported across the stem in the direction of the gravity

vector, which causes asymmetric growth (elongation of cells on the lower side), leading to upward

curvature of the plant.

The diageotropica (d&t) mutant of tomato offers insight into the role of IAA in gravicurvature. The

dgt is insensitive to exogenous IAA in elongation assays, and has an impaired perception of gravity

(the mature plant has a horizontal growth habit, for which it was named). Recently we have found

that dgt seedlings can respond to gravity. Within the framework of the Cholodny-Went hypothesis,

it is puzzling how dgt can respond to gravity if it is indeed insensitive to IAA. Therefore, we have

undertaken a careful developmental study to dem whether IAA indeed plays a role in the dgt

gravitropic response.

Accomplishments

(1) The gravitropic response of d&t hag been measured over a developmental time course,

and compared to that of the isogenic wild type tomato, VFN8. As shown in Figure 1, 10-day-old

d&t seedlings respond more slowly than VFNS, but after 22 hours dgt achieves essentially the

same upward curvature as wild type plants. This pattern continues for approximately l0 more

days, with dgt always slower at early time poiats (e.g., 6 hours), but then eventually achieves the

same degree of curvature as VFN8 (see Figure 2). However, 20 days after germination dgt can no

longer curve upward as well as VFNS. This impaired graviresponse corresponds to development

of the first internode of the tomato seedlings. Some developmental event apparently occurs during

formation of the intemode which makes dgt less responsive to gravity.

100-

_'e 8O

60

40-

o

-2G

0

VFN8

dgt

lb '1'5 2'0 2'5 3'o

Hours offer re-orientation

Figure 1.

3OO
PANEL A 6 Hours

110

m 90

70

50

L

30- -4--

o

? io-

-I

VFN8

dg!

-10 i , l = I

o 5 lb 15 2b 2_ 30 35 40 4S

110-

%-,

¢ 90-

G,I

L

ET;

m 70-

"o

50-

L

30-

,.e,-.

o

>

L 10-

-10

PANEL B 22 Hou_

VFN8 dg!

t I | I ] l I I I

0 5 10 15 20 25 30 35 40 45

Doys after germination

Figure 2.

20-.

r- 18-

:,_16'

¢_14-

oC12 -

"0 8-

6- -,I,--

o

E 2-

,u

"_ 0-

I -2-

¢-

"_ -4-

-I

<1:-6-

_-8

0

dgl

VFN8

I l I I I _ I I 10 15 20 25 30 5 40 45

Days offer germination

Figure 3.

301
(2) Duringour study,wetesteddgt and VFN8 for stimulation of stem elongation by IAA

solutions. Figure 3 shows that dgt never significantly responds to IAA, while VFN8 exhibits

IAA-stimulated elongation of 8 to 15 percent as compared to stem segments in buffer only.

Therefore, at no point in development do the dgt seedlings in our study elongate in response to

exogenously applied IAA.

(3) The above findings led us to investigate further whether IAA is indeed involved in the

response of young tomato seedlings to gravity. We immersed 7-day-old intact seedlings in the

buffer with various IAA transport inhibitors and, 2 hours later, reoriented the seedlings

horizontally and measured the upward curvature. Figure 4 shows that 10 and 100 _tmol 2,3,5-triiodobenzoic acid (TIBA, which specifically blocks polar IAA transport at the cellular effiux career)

greatly reduces the gravitropic response of both dgt and VFN8. N-l-naphthylphthalamic acid

(another polar transport inhibitor) and IAA produce very similar responses. The effect of IAA

transport inhibitors and high levels of IAA itself on the gravitropic response in dgt and the wild

type tomato indicates that IAA is involved in the gravitropic response of both normal tomatoes and

the diageotropica mutant.

EFFECT OF TIBA

90.0

70.0

50.0

a)

_- 30.0

"6

_ lo.o

21

90.0

70.0

._ 50.O

L_ 30.0

0

t_ 10.0

dgt L _r _ Butler

o

6 _ 1'o _'5 2'0 2'5 3b 3_

Hours oiler grovislimulafion

" I

Hours offer grovistimulolion

Figure 4.

302
Significance of the Accomplishments

We have shown that dgt does not elongate in response to exogenous IAA but does respond to

gravity in an IAA-dependent manner. Taken together, these results suggest either that IAA

perception or response is different or shifted between the elongation and curvature assays, or that

dgt may respond to gravity by inhibition of growth of the upper side, rather than elongation of the

lower side, of the hypocotyl. We are testing these hypotheses further, and confirmation of either

hypothesis would lead to a better understanding of the mechanism of the gravitational response in

plants.

Publications

Hicks, G.R., Rice, M.S., and Lomax, T.L. 1993. Characterization of auxin-binding proteins

from the zucchini plasma membrane. Planta 189: 83-90.

Lomax, T.L., Coenen, C., Gaiser, J.C., Hopkins, R., Rayle, D.L., and Rice, M.S. 1993.

Auxin perception and the regulation of tomato growth and development. In: Molecular Biology of

Tomato (ed. by J. Yoder). Lancaster, PA, Technomic Publishing Co., Inc., pp. 129-138.

Lomax, T.L., Rice, M.S., Hassell, L.A., Roelfsema, M.R.G., and Coenen. C. 1992.

Developmental analysis of growth responses in the diageotropica mutant of tomato. Plant

Physiology 99: 60.

Rice, M.S., Hassell, L.A., Ozmen, N., and Lomax, T.L. 1992. Developmentally regulated

gravitropism in an auxin sensitive tomato mutant. ASGSB Bulletin 6: 97.

Rice, M.S. and Lomax, T.L. 1993. IAA-mediated elongation and gravicurvature in the

diageotropica tomato mutant. ASGSB Bulletin 7: 33.
SARCOMERE LESIONS IN ATROPHIED RELOADED ADDUCTOR LONGUS

Joyce L. Thompson

Department of Cellular Biology and Anatomy

Medical College of Wisconsin

Milwaukee, Wisconsin 53226

Description of Research

The overall goal of the current research is to investigate alterations in muscle structure and

function during and following exposure to microgravity (space) or simulated microgravity

(hindlimb suspension unloading, HSU) which predisposes such muscle to injury on reloading.

The higher tensions produced in eccentric work and the association of eccentric work with

greater damage in normal muscle have led workers to postulate that the absence of the

high-tension eccentric component of muscle contraction during unloading or microgravity

renders such muscle more susceptible to the high-tension of eccentric contractions on reloading.

Specifically, the primary goals of the present line of investigations are to test the hypotheses that

HSU adductor longus (AL) is more susceptible to lengthening contraction damage than the

normal AL, and that elevations in tension (P) and dP/dt during the eccentric protocol correlate

with morphological damage. A secondary goal is to investigate the mechanism of any elevated

sensitivity.

Accomplishments

(1) Using the 5-minute AL eccentric contraction model (Thompson, 1993) at a Fast (F),

Medium (M), or Slow (S) lengthening rate, it has been determined that:

A. The muscle damage produced, as assessed by occurrence of myofiber

phagocytic invasion and degree of sarcorneric disruption, increases with faster

lengthening rates (rate=mm/ms) and higher rates of rise in tension

(dP/dt=g/ms) over the first minute (Table 1).

Table 1

Muscle Damage Produced as

Lengthening

Rate

2/200 0.5-0.8

' 2/500 0.4--0.5

2/800 0.3-0.4 Sarcomeres indistinguishable

from control.

F

M

s

Result of Faster Len_thenin_ Rates and l-Ii_her Rates of Rise in Tension

Damage Tetanus Tetanus

Duration Frequency

Multiple fiber phagocytic 300(ms) 30x/m

invasion.

Focal sarcomeric disruptions, 600(ms) 30x/m

no invasions.

900(ms) 15x/m

B. The distal tendon of the AL could be surgically severed, attached to the

transducer for physiological data acquisition, then reattached to its origin in

the surviving animal without causing the extensive damage to the AL that is

seen following the Medium and Fast stretch rates.

C. Application of a standardized bout of eccentric contractions (Table 2)

produced at least five distinct lesion types.

304
Table2

Eccentric Contractions

Lengthening Duration Repetitions Bout Duration Stimulation

Rate: Parameters

2 mm/400 ms The last 400 ms of a 20x/minute 5 minutes 3 v, 100 Hz

500 ms tetanus.

All five lesion types (Figure 1) were observed in AL muscles reloaded for 12-48

hours following a 14-day HSU treatment. The most prominent lesion seen in pilot

studies that eccentrically stretched normal muscle was the hypercontraction type

(lesion illustrated in Figure 1C; lesion frequency in parenthesis, Figure 2).

Figure 1. Photomicrographs of the five lesion types seen foUowing the eccentric contraction protocol: (A)

Wide-A: focal widening of A-band; (B) Opaq.: uniform opaque regions covering 3< sarcomeres; ({2) H.C.:

hypercontracted focal regions of 3< sarcomeres; (D) Miss-A: missing A-band focal regions of 3<

sarcomeres; (E) H.S.: hyperstretched focal regions of 3< sarcomeres. The abrupt nature of these disruptions

(between arrowheads), appositioning the seemingly normal sarcomeric pattern, strongly suggests

mechanical failure of some element of the muscle fiber. Bar equals 10 microns.

305
H.S.

0

(4.37)

(4.81)

(-)

(-)

(83.53)

(lO.68)

(-)

• (-)

(-)

(4.:37)

5 10 15 20 25

Lesion Frequency (_mm squared)

n HSU Ecc 4<6R

I1 HSU No Ecc 4<6R

Figure 2. Bar graph depicting the lesion frequency for the five types of lesions observed

following the AL eccentric contraction protocol. Wide-A, Opaq., H.C.. Miss-A, H.S. are

abbreviated as in Figure 1. HSU Ecc 4<6R: 14-day hindlimb suspension unloaded animals

exposed to eccentric protocol and reloaded for 4-6 hours. HSU No Ecc 4<6R: 14-day

hindlimb suspension unloaded animals exposed to no eccentric protocol and reloaded for 4-6

hours. Asterisk (*) indicates more elevated in HSU-Ecc-4<tR group over the HSU No Ecc

4<6R group (Wilcoxon paired sample test, 1_0.05; n=6/group, unless dotted "," indicating

n=5). Lesion frequency for a normal rat AL, exposed to the eccentric conlzaction protocol, is

parenthetically indica=ed (in progress group, n=l); (-)_<0,5/mm squared.

D. The rank in total lesion frequency was very highly correlated with the rank of

pre-lengthening contractile tension (Spearman's Rank Correlation Coefficient,

0.950) and directly and highly correlated to the rank of %-decrease in optimal

tension, Po, 15 minutes following the eccentric protocol (Spearman's Rank

Correlation Coefficient 0.829).

(2) Methacrylate embedded muscle tissue remains immunoactive to goat anti-rabbit

(GAR) or goat anti-mouse (GAM) antibodies (anti-sarcomeric myosin, GAR

lgG; anti-titin 9D 10, GAM IgM), if mildly fixed prior to embedment (Fixation

protocol: 1. Immersion fixed in 1-4 percent paraformaldehyde and 0.1-0.5

percent glutaraldehyde, or 2. Freeze substituted in 0.5 percent uranyl acetate in

methanol or acetone at -80°C).

Significance of Accomplishments

Finding 1A: Since tension has been shown to increase in contracting muscle as the speed of

eccentric lengthening increases, this implicates elevated tension in eccentric damage production.

By controlling lengthening rate, while monitoring P and dP/dt, correlation of the effect of

changes in P and dP/dt with the specific sarcomeric sites of damage may be executed.

Finding 1B: Demonstration of the ability to sever and reattach the distal tendon of the AL

without producing artifactual damage to the AL allows the collection of physiological data (Po,

dP/dt, Lo) on this muscle under controlled conditions and the ability to follow the progress over

time of subsequent lesion development and repair in normal and atrophic antigravity muscles.

Such muscles, predominately composed of slow-twitch oxidative fibers, are deep and adjacent to

bone. To investigate the eccentric component on reloading of atrophic antigravity muscles, a

surgical model is required in order to free the distal tendon and monitor physiological parameters

306
under controlled eccentric conditions for later correlation to actual damage. It has also been

observed by the sponsoring laboratory (Dr. Danny A. Riley, personal communication) that

reloading the soleus muscle in rats is a less reliable model because the conscious ambulatory rat

often behaviorally unloads the soleus by walking on its heels to avoid using the soleus when

reloaded. This is not the case with the AL; this muscle has been documented to consistently

undergo atrophic modification on unloading and to consistently demonstrate early eccentric-like

lesions that are not present in the simultaneously harvested soleus (at 6-12 hours reloading).

Finding IC: The appearance of the different lesion types may represent differing stages in a

sequel of damage (leading to segmental necrosis?), or may represent independent lesion types

arising from differing and unrelated "weak points." The sudden appearance of these sarcomeric

disruptions adjacent to seemingly normal sarcomeres, particularly the wide A-band, strongly

suggest an initial mechanical failure of some components (myofibrillar, cytoskeletal, and/or

membrane components: sarcolemma, t-tubule, or sarcoplasmic reticulum), which may later be

acted upon by other factors; i.e., enzymatic digestion (calcium activated neutral protease) and

compromised vascular supply (anoxia). Similar lesion morphology, previously seen in reloaded

14-day HSU muscle, indicates operation of a common mechanism in both eccentric HSU and

reloaded HSU muscle; therefore, this eccentric model is suitable to study eccentrically induced

lesion progression and repair in atrophic versus normal muscle. The pilot work on normal

eccentrically stretched muscle suggests that the sarcolemma is more sensitive (most prominent

lesion is hypercontracture, indicative of calcium leakage due to membrane damage), whereas, in

the atrophic muscle the sarcomeric elements are more susceptible (most prominent lesion is the

wide A-band). Atrophic muscle possesses more redundant sarcolemma than does normal

muscle, thus, atrophic muscle may be under less sarcolemmal stress and more contractile element

stress for a given level of tension than is normal muscle.

Finding 1D: The correlation between damage frequency and pre-lengthening, tension level

supports the premise that tension is a factor in lesion production. More damage Is produced in

muscle exposed to high initial tension and faster stretch rates than if the muscle is exposed to the

same faster stretch rate under lower initial tension. Such high-tension, high-stretch rate

conditions are produced in the "braking actions" of the antigravity muscles. And, finally,

correlating the damage morphology with decreased post-lengthening Po confirms the use of

15-minute post-treatment Po decrease as an index of sarcomeric damage, as opposed to fatigue.

Finding 2: At the high-magnification light microscopy (LM) level and low-magnification

electron microscopy (EM) level, the vulnerable sites of high-tension damage may be identified

using both conventional LM and EM visualization techniques and the more recently developed

immunohistochemical and immunoelectron microscopic techniques. This will assist in

visualizing the immunoantigenicity and continuity of the normally invisible sarcolemmal and

myofibrillar cytoskeletal elements (titin, desmin, dystrophin) within normal and lesioned muscle,

and will assist in the detection of isozyme shifts in contractile and regulatory proteins (myosin

and protein C isoforms) in lesioned and non-lesioned muscle.

Publications

Riley, D.A., Ellis, S., Slocum, G.R.,

Macias, M.Y., and Thompson, J.L. 1992.

aboard SLS-1. ASGSB Bulletin 6: 99.

Sedlak, F.R., Bain, J.L.W., Krippendorf, B.B.,

Postflight changes in muscle of rats flown 9 days

Thompson, J.L. 1993. Sarcomere lesions in atrophied reloaded adductor longus. NASA

Technical Memorandum 4501: 250-253.

Thompson, J.L., Balog, E.M., Fitts, R.H., and Riley, D.A. 1993. Adductor longus eccentric

contraction model. Medicine and Science in Sports and Exercise 25: $33.

307
Thompson, J.L., Fitts, R.H., McDonald, K.S., and Riley, D.A. 1992. Fast myosin

immunostaining and maximal contraction velocity in single muscle fibers of the hypokinetic rat.

ASGSB Bulletin 6: 94.

Thompson, J.L., Sabina, R.L., Ogasawara, N., and Riley, D.A. 1992. Immunofluorescence

localization of adenosine monophosphate deaminase isozymes in rat soleus and plantaris muscle.

Journal of Histochemistry & Cytochemistry 40:931-946.

Thompson, J.L., Sabina, R.L., and Riley, D.A. 1991. AMP deaminase (AMPda) isozymes in rat

muscle. Medicine and Science in Sports and Exercise 23: $57.

Thompson, J.L., Slocum, G.R., and Riley, D.A. 1991. Spatial analysis of muscle lesions in

space-flown rats. ASGSB Bulletin 5: E-8.

Thompson, J.L., Slocum, G.R., and Riley, D.A. 1992. Reloaded COSMOS-flown rats exhibit

grouped eccentric contraction-like lesions within the adductor longus. Medicine and Science in

Sports and Exercise 24 (5 Suppl.): $54.

3_)8
THE ROLE OF H + IN THE FATIGABILITY OF

HINDLIMB SUSPENSION

LaDora V. Thompson

Department of Physical Medicine and Rehabilitation

Medical School

University of Minnesota

Minneapolis, MN 55455

THE SOLEUS FOLLOWING

Description of Research

The long-term goal of our space biology research program is to understand the effects of space

travel (zero-g) on limb skeletal muscle function with particular emphasis on the cellular alterations

in single slow- and fast-twitch muscle fibers. A second goal is to develop countermeasures to

prevent the deleterious effects of zero-g travel on limb skeletal muscle. Weightlessness has been

shown to cause considerable muscle atrophy in slow-twitch antigravity muscles such as the soleus

(SOL) and to lead to an increased fatigability. The hindlimb suspension (HS) rat model has been

extensively employed by us and others to investigate the functional changes (cellular and

molecular) associated with zero-g. The model has been shown to induce muscle fiber atrophy,

reduce peak force and power, increase maximal shortening speed (Vmax), and increase fatigability

of the SOL muscle. Additionally, HS has been shown to increase the percentage of fast .'ype IIa

fibers in the SOL. Since H+ ion inhibits force to a greater extent in fast compared to slow-twitch

fibers, I hypothesized that the increased fatigability of the SOL following HS could be in part

attributed to an enhanced susceptibility of the actomyosin cross-bridge to the force inhibiting

effects of H+.

In order to test this hypothesis, the single-skinned fiber preparation was used and the H÷

concentration of the solution surrounding the contractile proteins varied such that the independent

effect of H+ on contractile function was determined. The effects of variations in pH of 7.0 and 6.2

on contractile properties were examined; these pH conditions were selected, as they mimic those

observed before and following intense contractile activity in limb skeletal muscle.

Accomplishments

HS induced significant atrophy of the SOL muscle, and the SOL muscle to body weight ratio

declined 35 percent. The change in SOL fiber diameter, peak force and tension (P0) is presented in

Table 1. Fiber diameter and peak force and tension decreased following HS. Peak tension

(kN/m2) was depressed by 15 percent at pH 6.2 for both groups.

Table 1. Diameter, Peak Force and Tension in Single Soleus Fibers.

Values are means + SE. Number of observations is in parentheses.

Diam_t_:r (ttrn) Force (x 10-4 N) _2)

Control (43) 70+2 4.7+_0.1 4.0_+0.1"* 129+6 109+5"*

2 wk HS (43) 49+2* 2.0+._0.1 * 1.7+-0.1 110+._6" 93+5**

**Significantly different from pH 7.0, !><0.05. *Significantly different from control, I><0.05.

309
The maximumshorteningvelocity of singleSOL fibers is summarizedin Table 2. FiberV0

(maximumshorteningvelocitydeterminedby theslacktesttechnique)wassignificantlyelevatedby

64percentfollowing HS. TheVmax(maximumshorteningvelocity calculatedusingthestraight

lineform of theHill equation)wasnotdifferentfrom controlsfollowing HS. After 2 weeks,HS

fiber stiffness(E0= elasticmodulus)wasdepressedby 31percent(Table3). SinceP0indicates

the forceperCSA, andE0representsthenumberof attachedcross-bridgesper CSA, theP0/E0

ratiowasmarkedlyincreasedfollowing 2 weekHS(62percent),suggestinganincreasedforceper

cross-bridge(Table3).

Table 2. Maximum Shortening Velocity.

Values are means + SE. Number of observations is in parentheses.

E0 fl.sec-la

H KZg_.

Control (43) 1.55_+0.07 1.58+0.05 (39) 0.91_-+-0.05 1.01_+0.05

2 wk HS (43) 2.54+__0.19" 2.15+_0.14"* (43) 0.96_+0.08 1.01+_0.06

**Significantly different from pH 7.0, p<0.05. *Significantly different from control, i)<0.05.

Table 3. Elastic Modulus and Tension/Elastic Modulus Ratio.

Values are means + SE. Number of observations is in parentheses.

_7 N.m-2)

atLg2,

Control (19) 1.71_-+-0.15 1.76+_.0.14 6.9+_0.4 5.7+_0.2**

2 wk HS (19) 1.19+-0.21" 1.30+_0.21 11.1+1.1" 8.1+_0.8"*

**Significantly different from pH 7.0, p<0.05. *Significantly different from control, p<0.05.

In control single SOL fibers, as pH was lowered, V0, Vmax and EO were unaltered, whereas the

P0/F4) ratio was significantly depressed by 18 percent (Tables 2 and 3). In contrast, altered pH had

a pronounced effect upon V0 following HS, with V0 decreasing 16 percent (Tables 2 and 3).

Additionally, P0/E0 ratio was reduced (Table 3). This finding suggests that the fibers from HS

animals are less resistant to H+.

As pH was lowered, the force-pCa relationship shifted to the right (i.e., to higher Ca2+

concentrations) and was depressed in amplitude, indicating a reduction in the Ca2+ sensitivity of

force development. The Ca2+ necessary to achieve half-maximal tension in fibers from both

groups increased when pH was lowered from 7.0 to 6.2 (pCa50 5.8 to 5.1). Furthermore, the

Ca2÷ threshold for force development was greater at pH 6.2 than at pH 7.0 in both groups by 1.0

pH unit. As pH was lowered, the steepness of the force-pCa curve increased in SOL control

fibers, with pCas0 values of 5.79 at pH 7.0 and 5.06 at pH 6.2. Similarly, pCa50 values of 5.87

at pH 7.0 and 5.14 were observed in SOL fibers following HS. This suggests that the apparent

co-operativity of force development had increased in both groups. Presently, the fibers are being

typed on SDS gels; therefore, these preliminary results have not been subgrouped into fast- and

slow-twitch fibers. It is anticipated that following fiber typing, HS and pH effects will be more

apparent.

310
Significance of the Accomplishments

The effects of HS on muscle function (fiber atrophy, decreased peak tension, and increased V0 of

the slow type I fibers of the SOL) are conftrmed in this study. The results of this study suggest

that an increased H+ ion concentration contributes to depressed contractile function, since both

force and velocity decreased significantly as pH changed from 7.0 to 6.2. The relationship

between V0 and peak force ratio (pH 6.2/7.0) is presented in Figure 1. HS induces a shift from

type I fibers with slower velocities to fibers with faster velocities. Importantly, the fibers

exhibiting the fastest velocities showed the greatest reduction in force. This result supports the

hypothesis that the increased fatigability following HS (and presumably zero-g travel) is in part

caused by an increased susceptibility of the contractile apparatus to H+ ions. Future efforts should

be directed at the development of exercise countermeasures that prevent the myosin isozyme

change (slow to fast) responsible for the increased sensitivity to H+.

1.O

O

r_ 0.9

¢q

"r

0.

•,--, 0.8

O

°_

t_

0.7

o

la.

0 Control

• 2 wk HS

0

eOt_g -•

t...

ooo o • oo

_0 0 O• t .

0.6 t i t , J

0 1 2 3 4 $

Vo (fl/see)

Figure 1. Relationship between V0 and peak force ratio.

Publications

Thompson, L.V. and Fitts, R.H. 1993. Effects on tension and stiffness due to reduced pH in

single soleus fibers following hindlimb suspension. ASGSB Bulletin 7(1): 99

311
MOLECULAR CHARACTERIZATION OF A PLANT Ca 2+ ATPase

Larry E. Wimmers

Mann Laboratory

University of California

Davis, CA 95616

Description of Research

The goal of our research is to elucidate the role of Ca 2+ as a second messenger in gravity

perception. We are approaching this goal using molecular, genetic, and biochemical techniques to

investigate the role of higher plant Ca 2+ translocating ATPases (Ca2+-ATPases) which are critical

to the regulation of cytosolic calcium levels.

Gravitropism, the ability of plants to grow directionally relative to the gravitational field is

universal among higher plants. Despite years of research, the mechanisms by which plants detect

and respond to gravity have not been exhaustively explored. One commonly cited model proposes

that release of endoplasmic reticulum-localized Ca 2+ to the cytoplasm acts as a second messenger

in gravity perception. According to this model, a mass moving within the gravitational field causes

Ca 2+ .channels to open, resulting in an increase in cytosolic Ca 2+, which might activate (directly,

via calmodulin, or calmodulin sensitive protein kinases) Ca2+-ATPases, H+-ATPases, anti auxin

carriers.

The participation of Ca 2+ in plant gravity perception is supported by three lines of recent evidence.

First, oat coleoptiles exposed to chlorpromazine, a calmodulin inhibitor, showed reduced

gravitropism. Second, in maize seedling roots, there is a strong correlation between the induction

of graviresponsiveness by light, and the level of calmodulin in the apical millimeter where

graviperception occurs. Third, using the fluorescent Ca 2+ dye fluo-3 and scanning laser confocal

microscopy, researchers were able to measure rapid increases in the level of cytosolic Ca 2+ in the

cells of maize coleoptile tips when horizontally oriented. Taken together, these results support a

role for changes in the cytosolic Ca 2+ concentration in graviperception.

Implicit in this model is the maintenance of a very low resting level of cytosolic Ca 2+, the

maintenance of Ca 2+ stores in the endoplasmic reticulum, and the ability to reestablish this

condition after gravity induced Ca 2+ fluxes. The regulation of cytosolic Ca 2+ levels is widely

recognized as a central element of regulatory processes in plants and is essential to the proposed

role for Ca 2+ as a second messenger in gravitropism. Active Ca 2+ transport is responsible for

maintaining low levels of cytosolic Ca 2+ and in this way interacts with other mechanisms of Ca 2+

release (channels) to effect regulated modulation of cytosolic Ca 2+ that is important in several

signal transduction pathways, including graviperception. Although active Ca 2+ transport is

catalyzed by both Ca2+-ATPases and Ca2+/H + antiports in plant cells, the high affinity of Ca 2+-

ATPase-driven transport is proposed to play the most significant role in modulating cytosolic Ca 2+

levels in the physiological range. The goal of our research is to use molecular probes to test the

hypothesis that the E.R.-localized Ca2+-ATPase is involved in the signal transduction pathway

leading to gravitropic growth of higher plants.

Ca2+-ATPases have been difficult to study biochemically in plants because of the high levels of

H+-ATPase associated with membrane preparations that also possess Ca2+-ATPase activity. To

overcome this biochemical limitation and achieve the proposed research goal, we have identified

cDNA and genomic clones encoding a Ca2+-ATPase (identified as LCA) from tomato, which may

be localized in the endoplasmic reticulum. The Ca2+-ATPase cDNA can be distinguished from

H+-ATPase cDNA clones and has provided the basis for developing both Ca2+-ATPase-specific

nucleic acid and antibody probes. The specific objectives of our research have been to:

312
Objective1:

Objective2:

Objective3:

Objective 4:

Identify Ca2+-ATPase(s) localized in the endoplasmic reticulum (ER).

Determine the tissue localization of ER Ca2+-ATPase in relation to the

graviperceptive regions of the organs.

Determine whether the expression of the gene encoding ER Ca2+-ATPase is

regulated in a tissue or cell specific manner.

Critically assess the role of the ER-localized Ca2+-ATPase in gravity perception by

the expression of an antisense Ca2+-ATPase gene in transgenic plants and

determine whether reduced Ca2+-ATPase levels interfere with gravitropic growth.

Accomplishments

(1) Genomic Sequencing: In order to finalize analysis of the primary sequence of the tomato

ER-localized Ca2+-ATPase and to identify its regulatory regions that may control tissue-specific

expression, it was necessary to complete sequencing its genomic clone. We have completed

nucleotide sequencing of the entire 10.3 kb LCA1 genomic clone (32, GenBank-accession number

M96324) which includes 3 kb upstream of the coding region.

(2) Reporter Gene Fusion: We originally proposed to analyze LCA1 gene expression at the

level of mRNA abundance in the gravity sensing region of the root tip by in situ RNA blot

hybridizations. We have discovered that the very low abundance of LCA1 transcript makes this

approach impractical. As an alternative, we have constructed a fusion gene comprising 3.0 kb of

the sequences upstream of the LCA 1 coding region linked to the B-glucuronidase (GUS) coding

sequence as a reporter gene. This construct has been transformed into tomato, and plants

expressing GUS are now being analyzed.

(3) Identification of a Second Ca2+-ATPase: We have recently identified and cloned a

second gene encoding a putative Ca2+-ATPase. The deduced sequence of this protein is

dramatically different from LCA but shares very high amino acid homology with a yeast Ca 2+-

ATPase which is localized in the endomembrane system. The subcellular localization and

functional analysis of this protein will now be carried out in tandem with LCA.

(4) Analysis of Transgenic LCA 1 Antisense Plants: In order to directly test the role of the

ER Ca2+-ATPase in gravity perception, we proposed analyzing the gravitropic response of roots in

transgenic tomato and tobacco plants expressing an antisense LCAI construct. We have

successfully regenerated tobacco plants expressing an antisense gene. The T1 plants have reached

maturity and have produced T2 seed. However, no reduction in LCA 1 sense message levels was

detected by RNA blot hybridization analysis. It is possible that the tomato and tobacco ERlocalized Ca2+-ATPase gene sequences are dissimilar enough to make antisense inhibition

ineffective.

Regeneration of transgenic antisense tomato plants has been particularly difficult. Most antisense

constructs were apparently lethal. No antisense plants could be regenerated while numerous plants

were regenerated from vector only transformations. Five antisense plants have been regenerated,

but their growth has been very slow, requiring nearly I year to reach maturity. We are now

collecting T2 seed, and phenotypic analysis will be carried out in the T2 plants.

The difficulty in regenerating transgenic antisense tomato plants could be due to a direct effect of

the antisense gene. In order to overcome this potential difficulty, we are now employing an

inducible expression system based upon the tetracycline binding protein. In this system, exposure

of plants to low levels of tetracycline activates transcription of the antisense gene.

313
Significance of the Accomplishments

Each of our accomplishments has contributed to our understanding of the role of Ca 2+ in plant cell

signal transduction, or set the stage for immediate progress in that area. Specifically, the LCA

reporter gene fusions will tell us if the expression of a gene involved in Ca 2+ homeostasis is

correlated to sensory perception. Similarly, the antisense transgenic plants will help elucidate the

importance of cytosolic Ca 2+ in gravity perception. Lasdy, the identification of a second Ca 2+-

ATPase may reform our models of plant cell Ca 2+ control and, thus, the role of Ca 2+ in signal

transduction.

Publications

Wimmers, L.E., Ewing, N.N. and Bennett, A.B. 1992. Higher plant Ca2+-ATPases: primary

structure and regulation of mRNA abundance by salt. Proceedings of the National Academy of

Sciences of the United States of America 89: 9205-9209.

314

REPORT DOCUMENTATION PAGE

I. AGENCY USE ONLY (LMve blaak)

4. TITLE AND SUBTITLE

1992-1993

I Form AR_.d OM_ No. O704-011MI 3. REI_RT TYPE AND DATES COVERED

Technical Memorandum L_ 7 [I

2. REPORT DATE

September 1994

NASA Space Biology Accomplishments

_ FUNDING NUMBERS

6. AUTHOR(S)

Thora W. Halstead, Editor

7. PERFORMING ORGANIZA_ONNAME(S)ANDADDRESS(T_)

Lockheed Engineering and Sciences Company

Washington, DC 20024 and NASA Office of

Life and Microgravity Sciences and Applications

Washington, DC 20546

9. SPONSORING/MONFFOR]NG AGENCY NAME(S) AND ADDRESS(F.S)

NATIONAL AERONAUTICS AND SPACE ADMINISTRATION

WASHINGTON, DC 20546

8.PERFORMING ORGANIZATION

REPORT ,NUMBER

I0. SPONSORINCdMONITORING AGENCY

REPORT NUMBER

11. SUPPLEMENTARY NOTES

,,. =S'nU._O,,AV_a._ _Am_r

UNCLASSIFIED- UNLIMITED

SUBJECTCATEGORY _" [

12b. DISTIIUBUTION CODE

13. ABSTL_CT (ma=m,,m2OOwordl)

This report consists of individual technical summaries of research projects of NASA's Space

Biology Program, for research conducted during tim calends" years of 1992 and 1993. This

program includes both plant and animal research, and is dedicated to understanding the role of

gravity and the effects of microgravi'ty on biological processes; determining the effects of the

interaction of gravity and other envu'onmcntal factors on biological systems; and using the

microgravity of the space environment as a tool to advance fundamental scientific knowledge in

the biological sciences to improve the quality of life on Earth and contribute to NASA's goal of

manned exploration of space. The summaries for each project include a descripdon of the

research, a list of the accomplishments, an explanadon of the significance of the

accomplishments, and a list of publications.

t4. SUBECTTERMS life sciences, microgravity, cells, plants,

animals, simulated weightlessness, biological effects,

space flight

17. SECURITY CL_IT_CATION

OF REPORT

UNCLASS

18.SECURITY CLASSIFICATION

OF THIS PAGE

UNCLASS

19.SE_CLASSlFICA_ON

O_BffI3tACT

UNCLASS

15. NUMBER OF PAGES

__3_

l& PRICE CODE

20. LIMfrATION OF ABSTRACT

UNLIMITED

National Aeronautics and

Space Administration

Code J'l-r

Washington, D.C.

20546-0001

Official Business

Penalty for Privalo Use, $300

SPECIAL FOURTH-CLASS RATE I

POSTAGE & FEES PAID J

NASA I

PERMIT No. G27 I

I

POSTMASTER: If Undeliverable (Section 158

Postal Manual) Do Not Return