NASA-TM-86244 19840005138

NASA Technical Memorandum 8624~&-----------'

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!;~.~ 10 ~J~ 1982 NASA Space Biology

Accomplishments

Thora W. Halstead and Linda G. Pleasant

DECEMBER 1983

NI\SI\

NASA Technical Memorandum 86244

1982 NASA Space Biology

Accomplishments

Thora W. Halstead

NASA Office of Space Science and Applications

Washington, D.C.

Linda G. Pleasant

The George Washington University

Washington, D.C.

NI\S/\

National Aeronautics

and Space Administration

Scientific and Technical

Information Branch

1983

Preface

The Space Biology Program currently includes forty-five research

projects. The goals, objectives, accomplishments, and future plans of

each project are described in this publication as individual technical

summaries.

The intent in compiling this pUblication is twofold. First, we would

like to provide the scientific community with an annual summary of the

accomplishments resulting from research pursued under the auspices of

NASA's Space Biology Program. Secondly, we hope to stimulate the

exchange of information and ideas among scientists working in the

Program. To facilitate this exchange process, a list of pUblications has been included with each project summary.

We would like to thank all the participants in the Space Biology

Program for their cooperative response to our requests for

information. We would also like to thank April Roy and Chris Bolcik

for their technical assistance in preparing this report.

Thora W. Halstead

May 1983

iii

Contents

page

Preface

Introduction - The NASA Space Biology Program

Animal Projects

Daniel D. Bikle

Alterations in Gut Transport of Mineral and

in Binding Proteins during Simulated Weightlessness

Wol f-D. Dettbarn

Weightlessness Simulation: Physiological Changes

in Fast and Slow Muscle

Stephen B. Doty

Morphological and Histochemical Studies of Bone

and Cartilage during Periods of Simulated

Weightlessness

iii

3

9

11

12

C.D.R. Dunn 14

Regultaion of Hematopoiesis in the Suspended Rat

as a Model for Spacefl ight

Al fred Fi nck 16

Effects of Gravito-inertia1 Fields on the Physiology of the Orb-weaving Spider

Charles A. Fuller 18

Thermoregulation in Primates in Hyperacce1eration

Fields

Bernard Halloran

Changes in Bone Structure and Metabolism during

Simulated Weightlessness: Endocrine and Dietary Factors

21

John M. Horowitz 23

Thermoregulation in Rats: Effects of Hypergravic Fields

J. Richard Keefe 25

Development and Functional Differentiation of the

Vestibular System in Normal, Rotated, and Centrifuged

Rats and Mice

R.W. Mah and J. Oyama 28

Development of Requirements for Inf1ight Control and

Research Centrifuge

v
G.M. Malacinski 30

Role of Gravity Perception in the Establishment of

the Dorsal/Ventral Axis of the Amphibian Egg

Jaime Miquel 32

Effects of Weightlessness and Hypergravity on Development and Aging of Drosophila

Emily Morey-Holton 34

Computer-assisted Video Histomorphometry of Animal

Species

Emily Morey-Holton 35

Structural Development and Gravity

X.J. Musacchia 39

Renal Function, Water and Electrolyte Balance, and

Intestinal Transport in Hypokinetic Animals

George W. Nace 41

A Ground-based Comparative Study of the Effect of

Weightlessness on the Development of Amphibian Eggs

Jiro Oyama 44

Hyper-gravitational Effects on Metabolism and

Thermoregulation

Nello Pace 47

Primate Hemodynamics and Metabolism Under Conditions

of Weightlessness

Nello Pace and Arthur H. Smith 49

Gravity, Body Mass and Composition, and Metabolic

Ra te

V.P. Popovic 51

Effect of Decreased Gravity on Circulation in the

Ra t

Eugene Roberts 53

Bone Cell Kinetics of Simulated Weightlessness

M.D. Ross 54

Mammalian Gravity Receptors: Structure and Metabolism

David J. Simmons 58

Ground-based Models of Hypokinesia

Douglas G. Stuart 60

Effects of Muscles Atrophy on Motor Control

vi
G.H. Templeton and J.L. Sutko

Influence of Suspension-hYPokinesis on Skeletal

Muscle

Marc E. Tischler

Skeletal Muscle Metabolism in Hypokinetic Rats

Debra Wolgemuth

Effects of Simulated Weightlessness on Meiosis,

Fertilization, and Early Development of Mice

Plant Projects

62

64

66

Robert S. 8andurski 71 Localiz~ and Identify Gravity Sensing Mechanisms

of Plants

A.H. Brown 74

Plant Morphogenesis and Physiological Behavior in

Relation to Gravity

Joe R. Cowles 76

Aromatic Biosynthesis and Gravity

Michael L. Evans 78

The Role of Acid and Calcium Gradients in Gravitropism

Lewis J. Feldman 81

Geotropic Stimulus Production in Roots of Corn

Arthur W. Galston 83

Mechanisms of Graviperception and Tropistic Response in Pea Seedlings

Ali·ce Bourke Hayes 86

The Role of Gravity in Regulation of Leaf Blade

Form

Takashi Hoshizaki 88

Effects of Gravity and Light on Growth, Development, and Flowering in Plants

William P. Jacobs 90

Effect of Gravity and Weightlessness on the Development of Giant Coenocyte, Caulerpa

Mordecai J. Jaffe 92

Mechanism of Thigmomorphogenesis in Plants, and Its

Significance to Research with Plants in Space

vii
Peter B. Kaufman 94

Investigation of the Negative Geotropic Response fn

Shoots of the Oat Plant (Avena sativa)

Abraham D. Krikorian 97

Cells, Embryos, and Development in Space--Morphology of Plant Cells in Space

A.C. Leopold 99

The Amyloplast as a Gravity-sensing Device in Plants

Cary A. Mitchell 102

Mechanical Regulation of Plant Growth and Development

Barbara Picard 104

How the Pea Stem Senses Gravity, Friction, and

Flexure

David L. Rayle 105

Mechanism of Gravitropic Curvature in Plant Shoots

Stanley J. Roux 107

The Cellular Bases of Gravity- and Light-Induced Geotropism

Frank B. Salisbury 110

An Examination of the Clinostat, as a Simulator of

Weightlessness Using Leafy Plants

Special Projects

X.J. Musacchia

Research in Space Biology

Meetings

viii

115

119
INTRODUCTION

THE NASA SPACE BIOLOGY PROGRAM

Thora W. Halstead

Office of Space Science and Appl ications

National Aeronautics and Space Administration

Washington, D.C. 20546

Introduction

One of the major features of the physical environment on the

surface of the Earth is the constant presence of the force of

gravity. Terrestrial gravity has important biological

consequences for the living organisms of the Earth. Among the

effects of spaceflight, the phenomenon of weightlessness,

therefore, provides the greatest biological. research opportunity

both because of its uniqueness to space and because of the

importance of gravity to life on Earth. Access to space provides

an opportunity to manipulate gravity from its norm of one down to

almost zero, effectively providing the full spectrum of

gravitational research capability for the first time. This

capability, combined with the stability and pervasiveness of

gravity on Earth, its obvious impact on biological evolution, and

its continuing effect on the morphology, physiology, and behavior

of living organisms, has led the Space Biology Program to

concentrate its efforts and resources on investigating the

biological significance of gravity.

Program Goals

The goals of the Space Biology Program are to: use the unique

characteristics of the space environment, particularly microgravity, as a tool to advance knowledge in the biological sciences; understand how gravity has shaped and affected life on

Earth; and understand how the space environment affects both

plant and animal species.

Program Scope

The research in the Space Biology Program is divided into three

broad areas:

3
1. Gravity receptor mechanisms. This inclurles the identification of the organ or site of gravity reception and

the biological systems and mechanisms that transmit the

information to responsive site.

2. The physiological effects of gravity. This includes the

use of gravity's physiological effects to explore

biological problems; an understanding of how gravity affects and controls the physiology, morphology, and

behavior of organisms; how gravity and other environmental stimuli and stresses interact in this control; and

the biological mechanism by which living systems respond

and adapt to altered gravity, particularly that of the

space environment.

3. The role of gravity in development. Specifically, the

effect of gravity on reproduction, development, maturation, and evolution.

Research Opportunities

With the proven feasibility of the Space Shuttle, we now have

capability of performing biological experiments in space. The

opportunity has arrived to use the locker space within the

Shuttle orbiter on a continuing space available basis. This will

provide a valuable augmentation to the ongoing ground-based

research program.

Spaceflight will provide the validation for many

experimental hypotheses developed in ground-based research, while

gravitational experiments on Earth will continue to hone the

questions, provide the necessary baseline data, and develop spaceflight experimental protocol.

The experimental approach of the ground-based studies in the

Space Biology Program is to manipulate gravity on Earth and

develop weightless simulation models to: (1) develop and test

gravitational hypotheses, (2) identify gravity-sensitive biological systems and interacting environmental response

mechanisms, (3) analyze biological systems and mechanisms known

to be gravity-sensitive, (4) analyze flight experiment data and

iteratively expand ground research capability, and (5) plan and

design future space experiments. In addition, research is

conducted to understand how the uncontrollable biodynamic factors

of the spacecraft will affect the results of the various flight experiments.

Weightlessness, and physiological conditions similar to those

induced by weightlessness, have traditionally been partially simulated in the ground-based laboratory by a variety of

techniques.

In animal research, the attempt is to simulate the reduction

in apparent weight and alteration in hydrostatic pressure that

4
occurs under weightless conditions. Simulation is accomplished

in the Program's research by the use of a unique animal support

system that reduces loading on the hind limbs and produces a

cephalad fluid shift. This gentle and delicate method has been

developed, as well as used, by scientists associated with the

Space Biology Program and has been shown to successfully induce

some of the physiological changes associated with weightlessness.

The c1inostat is an apparatus which, by its motions, modifies

or equalizes the directional input from gravity to an attached

organism. The rotation of experimental material on a c1inostat

is useful only for those physiological phenomena that have

relatively long exposure thresholds (or presentation times), so

that it is possible to rotate the organism slowly enough to avoid

the complications produced by centrifugal acceleration, but fast

enough so that the time spent in anyone position does not result

in an induced georesponse.

To achieve hypergravity conditions, gravity loads above one

are produced by slow centrifugation and are especially useful in

identifying gravity-sensitive biological systems and mechanisms.

Changing the directional input of gravity to an organism has also

proved to be a successful technique to study the biological

effects of gravity.

Present Focus of Program

The research focus of the Space Biology Program is dependent

upon several dynamic factors: NASA requirements, the

characteristics of flight experiment opportunities, the

sensitivity of specific biological systems to gravity, the

scientific value of the research, the state of knowledge and

technology in the specific scientific areas, the interest of

scientists in studying the biological questions, and the

availability of funds to support the research.

Within the scope of the Space Biology Program, the current

Program is focused on answering the following basic scientific

questions:

1. What are the components of the gravity-sensing mechanisms

of plants and animals? How do they perceive information?

How is the information transmitted to evoke responses?

2. Does gravity influence plant and animal fertilization and

development, and can fertilization and development

proceed normally in a near zero gravity environment? If

gravity does affect fertilization and development, what

are the sensitive physiological systems and how are they affected? If early development is affected by gravity,

is it a result of an effect on the parent or the direct

effect on the embryo itself?

5
3. What is the role of gravity in the formation of

structural elements such as lignin, cellulose, silica,

chitin, and bone calcium phosphates at the molecular

level as well as at more complex organizational levels?

4. What role does gravity play in calcium-mediated

physiological mechanisms and in calcium metabolism?

5. How does gravity as an environmental factor interact with

other environmental factors to control the physiology,

morphology, and behavior of organisms? Or, how rlo

gravitational and other environmental stimuli interact in

their control and direction of living forms? Can the

action of gravity be replaced by different stimul i?

Future Focus of Program

As longer flight missions become available, biological

questions that require longer periods of micro-G, such as

multigeneration and radiation experiments, will increase in

importance. It has been recognized for some time that ionizing radiation causes cumulative damage in all living things, but

knowledge of the biological effects of the cosmic radiati~n

encountered in space, which contains high energy, heavy particles, is poorly understood. Furthermore, the bioloqical

consequences of the interaction of this unique radiation and

gravity are less known. Since long-term space exposure is needed

to adequately investigate the effects of radiation, such research

is well suited for the space flights that a space station will

provide.

6
ANIMAL PROJECTS

7

ALTERATIONS IN GUT TRANSPORT OF MINERALS AND IN BINDING PROTEINS

DURING SIMULATED WEIGHTLESSNESS

Daniel D. Bikle

University of California, San Francisco

3rd and Parnassus

San Francisco, CA 94143

SUMMARY

The objectives of this research program are: (1) to establish an

animal model on Earth by which the effects of weightlessness on

bone mineral homeostasis can be studied (i.e., simulated

weightlessness), (2) to determine whether simulated

weightlessness alters intestinal calcium transport, (3) to

determine whether simulated weightlessness has a generalized or

selective effect on bone mineral content, and (4) to determine

whether changes in dietary calcium or hormone levels can modulate

the effects of simulated weightlessness on bone mineral

homeostasis.

The original rat support model (employing a back harness) has

been modified by changing the point oS support to the base of the

tail. The rats are supported at a 40 angle such that their

forepaws continue to bear weight but their hind paws do not.

Such rats appear to be relatively unstressed. They gain weight

continuously and at the same rate as pair-fed controls. Support times of 15 days are readily achieved. Besides providing

healthy, happy, unstressed rats, this support system provides an

important internal control for our studies--the hindlimbs are

unweighted, the forelimbs are not. Thus comparison of the

effects on calcium content in the loaded humerus (forelimb bone)

and the unloaded tibia (hindlimb bone) will help determine

whether this model of weightlessness produces generalized or

local effects on bone.

Measurement of intestinal calcium transport by several different

techniques revealed no alterations using this model of simulated

weightlessness. When dietary calcium was altered to provide a

range of very low calcium to very high calcium content, the

absorption of calcium changed appropriately and similarly in both

the supported and pair-fed control rats; rats fed a low calcium

diet had a greater ability to absorb calcium than rats fed the

high calcium diet regardless of whether they were supported.

When the calcium content of a variety of bones from the supported

rats was compared with the calcium content in the same bones in

pair-fed controls, several points became clear: (1) Supported

rats had a significantly lower calcium content only in the

unweighted bones such as the tibia and lumbar vertebra compared

with pair-fed controls. The normally weighted bones such as the

9
humerus and mandible did not show differences between supported and control rats. (2) The differences in bone calcium content

between supported and control rats was progressive with time of

support; no significant differences were seen after 5 days, whereas the differences were greatest at 15 days (the longest time evaluated). (3) Despite the progressive disparity in bone

calcium content between the tibia and lumbar vertebra of

supported rats compared with control rats, calcium uptake by such

bones showed a biphasic pattern; e.g., initially (2 to 5 days),

and by 15 days, calcium uptake had rebounded to higher than

control values. These data suggest an initial inhibition of bone

formation followed by an increase in bone formation possibly

secondary to increased bone resorption when the rats were

initially supported.

The effects of dietary calcium on bone mineral content was

studied in supported and pair-fed control rats. The following observations were made: (1) The calcium content of bone was

directly proportional to the calcium content of the diet in both

supported and pair-fed controls. Rats fed the very low calcium

diet had very fragile bones regardless of whether they were or

were not supported. (2) The differences between the unweighted

bones of the supported and pair-fed control rats appeared to

decrease as the dietary calcium content was raised. Thus, it is

conceivable from these' data that the bone losses occurring during

spaceflight can be reduced or eliminated by increasing dietary

calcium intake.

PUBLICATIONS

Bikle, D.O., Globus, R.K., and Morey, E.R. Calcium Transport from the Intestine and into Bone in a Rat Model Simulating Weightlessness. Physiologist 25(6, Suppl.}: S143-S144, 1982.

Bikle, D.O. and Herman, R.H. Calcium Potentiates the Cyclic Nucleotide and Phosphaturic Response to Parathyroid Hormone

Infusion. Journal of Clinical Endocrinology and Metabolism 56:

11-17, 1983.

10
WEIGHTLESSNESS SIMULATION: PHYSIOLOGICAL CHANGES IN FAST AND SLOW

MUSCLE

Wolf-D. Dettbarn

School of Medicine

Vanderbilt University

Nashville, TN 37232

SU MMARY

This is a new project. In recent years there has been an

increased interest in the adaptation of muscle to disuse, in

particular to changes that occur with weightlessness during spaceflights. This laboratory will examine the effect of

simulated weightlessness on the biochemical characteristics of

fast and slow muscle alterations in the rat. In the model to be

used, the rat will be supported so that the hindlimbs are

unweighted, the head will be tilted downward, and all body weight will be borne by the fore~aws which will also be used for

locomotion.

During hypokinesia, biochemical parameters will be investigated in slow muscle, such as the soleus, and in fast muscle, such as

the extensor digitorum longus. Of particular interest is the

effect of disuse on the control of acetylcholinesterase (AChE)

and its molecular forms. Other proteins to be investigated are

those involved in neuromuscular transmission such as

cholineacetyltransferase (CAT) and the acetylcholine receptor.

In addition, the effects of weightlessness on axoplasmic

transport of AChE and CAT and on the rate of denervation- and

reinnervation-induced changes will be studied. The period of

disuse will vary between 1 to 5 weeks followed by a another 1 to

5 week period to study the readaptation of the rat to normal

conditions.

11
MORPHOLOGICAL AND HISTOCHEMICAL STUDIES OF BONE AND CARTILAGE

DURING PERIODS OF SIMULATED WEIGHTLESSNESS

Stephen B. Doty

Departments of Anatomy and Orthopedic Surgery

Col umbia University

New York, NY 10032

SUMMARY

The primary objective of this study is to determine how

weightless conditions influence the metabolism of skeletal

tissues. When disuse of the skeleton occurs, such as during prolonged bed rest, or during low gravity or weightless periods,

the skeleton undergoes a loss of integrity and mechanical

strength. Development of animal models to mimic these conditions

has been difficult, but the model provided by Emily Morey-Holton

shows that rats exposed to 2 to 3 weeks of non-weight-bearing in

a head-down position undergo bone reduction in a manner similar

to animals subjected to spaceflight conditions. This lab has

applied a combination of techniques using electron and light microscopy, histochemistry, and quantitative enzymology to

determine how the skeletal tissues respond to this model system.

Alkaline phosphatase activity has been classically associated

with the rate of new bone formation in all skeletal tissues.

When this lab quantitated this enzyme activity from femurs of

non-weight-bearing animals, there was a slight but statistically significant reduction in this activity in metaphyseal bone.

However, histochemical methods for alkaline phosphatase show that

this enzyme is found: (1) within the bone-forming osteoblast

population, (2) within the calcifying cartilage of the femur, (3)

along the fibrous components of the periosteum, and (4) within

the cytoplasm of the polymorphonuclear leucocytes. Are all of

these sites responding to the experimental treatment? By

combining microscopy and histochemistry this lab is accumulating

evidence that the osteoblasts (on bone surfaces) and fibroblasts

(along the periosteum) are the major sites affected by the

non-weight-bearing condition.

Electron microscopy of the fibrous sheath surrounding bone (i .e.,

the periosteum) has resulted in unexpected findings. First,

special "gap" or communicating junctions between the cells of

this fibrous tissue are reduced in numbers and size as a result

of the reduced weight-bearing. This communicating system of

junctions also occurs in bone and may function as a "nervous

system" between connective tissue cells which directs the

synchronous activity of these cells. Secondly, the periosteal fibroblasts appear to be resorbing the collagen fibers of the

periosteum, indicating that the mechanical stress on the

periosteum has been affected by the non-weight-bearing. This

suggests that variation in mechanical stress probably affects all

components of the musculoskeletal system.

12
PUBLICATION

Doty, S.B. and Morey-Holton, E.R. Changes in Osteoblastic

Activity Due to Simulated Weightless Conditions. Physiologist 25(6, Supp1.): S141-S142, 1982.

13
REGULATION OF HEMATOPOIESIS IN THE RAT WEIGHTLESSNESS SIMULATION

MODEL

C.D.R. Dunn

Division of Experimental Biology Baylor College of Medicine

Houston, TX 77030

SUMMARY

A decrease in the number of circulating red blood cells appears

to be consistent sequelae of spaceflight. Likewise, changes in

the number and function of white blood cells have been frequently reported. The cause(s) of these alterations is unknown and the

biological significance is uncertain. The objective of the

studies supported by this grant is to investigate the regulation of blood cell production in rats exposed to antiorthostatic

hypokinesia. Data from these investigations will be added to the

other information derived from antiorthostatic hypokinesic rats

that suggests this system is a good model for the bone, muscle,

and cardiovascular effects of spaceflight.

The approach to these investigations can be divided into two

distinct sections. The first phase of the study is concerned

with data collection to ascertain if the model simulates the

hematopoietic effects of spaceflight. The second phase will be

directed at mechanistic considerations.

The major accomplishment of the 1982 research has been to

validate antiorthostatic hypokinesia in the rat as a surrogate for the zero gravity of space. Thus, from the hematological viewpoint the investigations have shown:

1. A biphasic change in body weight. A rapid loss occurs

during the first 24 hours of support followed by a more

protracted phase of either zero growth or continued loss of body weight.

2. Reduced food and water consumption.

3. Transient hemoconcentration, i.e., reduction in the

plasma component of the blood.

4. Reduced production of red blood cells.

5. Decrease in average size of the red blood cells.

6. Reduced blood volume, a large part of which occurs in the

first 24 hours of support.

7. Reduced number of circulating red blood cells, i.e.,

"anemia."

14
8. A postsupport increase in the number of circulating

white blood cells.

All of the above changes are directly comparable with available

data from humans and rats subjected to spaceflight. In addition,

we have also shown functional changes in the hemoglobin in the

red blood cells of supported rats. Such changes have been

predicted to occur during spaceflight but have not yet been

documented. Further, the lab's data are consistent and do not

seem to be markedly dependent on the mechanism of support or on

the site from which the blood samples are obtained.

It is concluded from these investigations that rats exposed to

anti orthostatic hypokinesia show a variety of hematological effects directly comparable with those that occur in humans and

rats exposed to spaceflight. Thus, antiorthostatic hypokinesia

appears to be validated as a useful paradigm for zero gravity in

which to investigate the hematological effects of spaceflight.

PUBLICATION

Dunn, C.D.R., Johnson, P.C., and Leach, C.S. Fluid Shifts and

Erythropoi esi s: Rel evance to the "Anemi a II of Space Fl ight.

Physiologist 25(6, Suppl.): S79-S80, 1982.

"5
EFFECTS OF GRAVITO-INERTIAL FIELDS ON THE PHYSIOLOGY OF THE

ORB-WEAVING SPIDER

A1 fred Fi nck

Department of Psychology

Temple University

Philadelphia, PA 19122

'SUMMARY

Mechanical receptors for the detection of gravity stimuli have

evolved to respond to linear and/or angular acceleration.

Perhaps the most complex system of gravity (G) receptors is seen

among the vertebrates in which the utricular and semicircular

canal systems have become exquisitely dedicated to gravito- inertial forces. Among the invertebrates, a progenitor of the

linear acceleration detector can be seen in the statocyst.

Statocysts (e.g., in the crayfish) employ a mass within an

enclosed cavity such that changes in linear acceleration or

position stimulate different hair cells. Spiders do not present

such organs. A different receptor mechanism evolved to inform

the animal about its orientation in the gravito-inertia1 field.

A primary objective of this research project is to expose the

possible mechanism for gravity detection in the spider and to

describe its operating characteristics. During the 1982 research

year, it was demonstrated that the G function is logarithmic having an estimated absolute threshold of perhaps 1.0008 Gz • This surprisingly small increment to normal gravity is

tentatively explained by the exquisite sensitivity of the

1yriform organ on the spider leg. Other research has shown that

the 1yriform can respond to mechanical displacement on the order

of millimicrons. In this work, the gravity sensitivity of the

spider is determined by examining the change in its heart rate

following centrifugation.

During these first experiments it had often been observed that

the vigor of the spider heart pulse changed with changes in G and

with changes in tilt (i.e., a change in the direction of G).

During the latter part of 1982 a tilt-system was designed and

computer programs were written to analyze the amplitude density

of the spider pulse as a function of tilt. At the time of this

writing there is initial evidence to suggest a functional

relationship between tilt and amplitude density of the pulse. It

can be demonstrated that the spider's absolute sensitivity to

tilt may be in the region of 2 degrees or less.

This growing knowledge about the spider gravity response system has suggested a model in which spiders detect the local G vector

according to strains in their exoskeletons. Reflex mechanisms of

the heart respond to this sensory input to produce a blood

16
pressure change in the legs. In this fashion the spider may be

able to maintain postural homeostasis when either the magnitude

or the direction of the G field changes.

PUBLICATION

Finck, A. Gravito-inertial Sensitivity of the Spider; Araneus

sericatus. Physiologist 25(6, Suppl.): S121-S122, 1982.

17
THERMOREGULATION IN PRIMATES IN HYPERACCELERATION FIELDS

Charles A. Fuller

Division of Biomedical Sciences

University of California

Riverside, CA 92521

SUMMARY

Three experimental studies have been performed since this program

started slightly less than 12 months ago. These studies are

examining the responses of a small diurnal primate, the squirrel monkey, to exposures to hypergravic fields. The first two of

these studies involved examinations of acute exposures to 2 and

3G

z

fields. The third study examined responses to chronic

exposure to 1.5 and 2G z .

In the first study, an examination of the thermoregulatory

responses of the squirrel monkeys to 70-minute 2G z exposures, both during the day and night, was made. The respgnse of the

animal during the day was demonstrated to be a 1.5 C fall in

colonic temperature, mediated at least in part by an increase in

vasomotor heat loss. This response was highly reproducible and

seen in all animals. In contrast, at night the temperature

response was completely eliminated. Individual animals showed

small deviations in temperature either in an upward or downward

direction but on an average there was no change in colonic

temperature. Similarly, the skin temperatures did not show any

major deviations in vasomotor heat loss. These results

demonstrate that although the response in body temperature is

very prominent and reproducible during the day, physiologically these animals have the ability to regulate their body

temperatures independent of this gravitational force during the

night.

This day-night difference in a series of rats was also

investigated. Historically, all rats exposed to hyper- gravitational fields have been examined during the day,

which is the rest phase of these nocturnal animals. The

investigators wanted to determine whether the light vs. the dark

had a significant influence on the response or whether the

magnitude of the response was the function of the activity pbase

vs. the rest phase of the animal. The previously reported 2 C

response was observed when centrifuging the animals during the

day. However, upon exposure to 2G at night, which is the

animal's active phase, the body te~perature fall was even larger. This indicates that although the monkeys were able to conserve

body temperature more readily than the rats, both species showed

an increased ability to maintain body temperature during their

rest phase when their circadian body temperatures were at a

minimum.

18
The second study examined the primate sleep responses to 70-

minute exposures to hypergravic fields. The investigators had

previously noted that centrifuged animals, when observed

visually, demonstrated a sleeping behavior periodically when in a

hypergravic field. This study further examined this observation

with e1ectrophysio10gica1 recording of the EEG of a group of

primates. It was noted that during a Rrecentrifugation phase, the animals showed various amounts of 'napping" behaviors in

which slow-wave sleep was noted to occur on a periodic basis.

(Rapid eye movement sleep was not observed during this daytime study.) Upon centrifugation, however, the amount of sleep was

inhibited markedly for several minutes after which the slow-wave

sleep pattern began to recur. The amount of sleep never reached

the baseline level in the precentrifugation phase. Postcentrifugation, the amount of sleep increased significantly

and was noted to be maintained for the entire 70-minute

postcentrifugation phase. This response was relatively

proportional in the animals studied at both 2 and 3G. At 3G z • the animals showed an increased response such that t~ere was a

greater reduction in slow-wave sleep during the centrifugation phase, and an increased amount of slow-wave sleep during the

postcentrifugation phase. It is interesting to note that the

amounts of slow-wave sleep have always been inversely correlated

with the level of body temperature. That is, lower body

temperatures are usually correlated with increased sleep. In

·this experiment, this correlation did not hold since during centrifugation the amount of sleep observed during the fall in

body temperature was less than the precontrolled phase where body

temperature was higher.

The third study was an examination of the circadian response of

these primates to chronic exposure of 1.5 and 2G z ' In this

experiment, the animals were maintained on the centrifuge for 90

days at different G levels. On the centrifuge with the animals

was a small microcomputer which monitored the animals' feeding

and drinking responses in half-hour intervals throughout this

period of time. The animals were initially exposed to lG for 3

weeks followed by l.SG for approximately 2 weeks. This was

followed by 2G for S weeks and then the animals were returned to

lG for the remainder of the study. In each phase, the animals

were exposed to a normal 24-hour light-dark cycle. Additionally, during the precentrifugation and postcentrifugation lG phases and

during the 2G phase, the animals were exposed to constant light where the circadian system was allowed to free-run. In all

instances, the animals maintained the normal 24-hour circadian

rhythm when exposed to the light-dark cycle. There is some

suggestion, however, that the synchronization was not as precise in the hypergravic fields. Further, these preliminary da,ta

suggest that there may have been a period change in the

free-running rhythm at 2G. Further work needs to be performed to

determine the exact responses.

19
PUBLICATIONS

Full.er, C.A. and Williams, B.A. Primate Thermal Sensitivity to

Short Hyperacceleration Profiles (Abstract). Physiologist 25(4): 231, 1982.

Fuller, C.A. and Williams, B.A. Short Hyperdynamic' Profiles

Influence Primate Temperature Regulation. Physiologist 25(6, Suppl.): S91-S92, 1982.

20
CHANGES IN BONE STRUCTURE AND METABOLISM DURING SIMULATED

WEIGHTLESSNESS: ENDOCRINE AND DIETARY FACTORS

Be rnard Ha 11 oran

Medical Center

University of California

San Francisco, CA 94143

SU MMARY

Bone mineral is lost from both the rat and human skeletons under

conditions of weightlessness that occur during spaceflight. This

loss in turn weakens the skeleton, increasing the likelihood of

bone fractures. In addition, the disruption of normal mineral

metabolism may adversely affect renal function.

The long-term objective of this project is to determine the cause

of this mineral loss and at the same time investigate the

dynamics of bone metabolism. The present goal is to examine the

roles of vitamin D, parathyroid hormone (PTH), stress

(corticosterone), and dietary calcium and phosphorus in the

skeletal alterations induced by weightlessness. In this study the ground-based rat model developed at NASA Ames Research Center

is used to simulate the weightlessness of spaceflight.

This program was initiated in August 1982. At present this lab

is in the process of completing the first series of experiments

designed to examine the relationships between dietary calcium and

phosphorus, the bone changes induced by simulated weightlessness

and the serum concentrations of calcium, inorganic phosphate, PTH, and the vitamin D metabolites. Four separate experiments have been completed utilizing different dietary levels of calcium

and phosphorus. Measurement of the serum concentrations of

calcium and inorganic phosphate and total bone ash of the tibia

and humerus have been completed. Moreover, approximately 50~ of

the bone histomorphometric measurements designed to identify the

specific changes in bone structure and metabolism associated with

simulated weightlessness have been completed. Measurement of the

serum concentrations of PTH and the vitamin D metabolites is

underway.

The results of these initial experiments indicate that the bone

mineral loss that occurs during simulated weightlessness on a

diet with normal levels of calcium and phosphorus can be reduced

by increasing the dietary intake of calcium and phosphorus. On a

normal diet, tibia from animals undergoing simulated

weightlessness showed a highly significant (p < .005) 13~

decrease in total bone weight when compared with tibia from

control animals. On a high calcium/phosphorus diet no

significant differences in bone weight were observed. When the

measurements of PTH and the vitamin 0 metabolites are completed,

21
it should be possible to relate the apparent protective effect of

increased dietary calcium and phosphorus to the primary hormones

regulating mineral metabolism. Once this has been accomplished, the next plan is to determine whether or not PTH, either directly

or indirectly, is mediating the bone mineral loss that occurs

under normal dietary conditions.

22
THERMOREGULATION IN RATS: EFFECTS OF HYPERGRAVIC FIELDS

John M. Horowitz

Department of Animal Physiology

University of California

Davis, CA 95616

SUMMARY

Experiments were completed to further characterize neural

processing of sensory information under conditions of altered

gravity. One set of experiments provided data on the

thermoregulatory system of rats exposed to hypergravic fields. A

second set of experiments involved measuring auditory brainstem

responses in rats exposed to hypergravic fields. These

experiments provide complementary information on how altered

gravitational fields affect information processing involving the

brainstem and hypothalamic areas of the central nervous system.

In addition, the results of the experiments serve as the basis

for future studies in hypogravic fields, particularly experiments

on a space station.

A major objective during 1982 was to further elucidate the

mechanisms underlying the impairment of temperature regulation in

mammals exposed to hypergravic fields. Experiments involved

measurement of the rate of oxygen consumption and core and tail

temperatures in cOld-exposed rats from 1 to 6G. To briefly summarize the results of the studies, the data were consistent

with the proposal that the thermoregulatory system is composed of

a parallel arrangement of neural controllers that can be

reversibly uncoupled by placing the animal in a hypergravic field. In hypergravic fields, the parallel controllers for both

modes of heat production (shivering and nonshivering

thermogenesis) fail to adequately activate thermogenic effectors,

even though these parallel controllers for thermogenesis are

responsive to lowered ambient temperature. Moreover, acclimation

to cold prior to acute cold exposure does not prevent the

decrease in core temperature seen in animals acclimated to an

ambient temperature of 23 C. Similar experiments in a

hypogravic environment would serve to characterize a mammalian

neural regulatory system over a range of 0 to 1G.

A secondary objective was to determine the effects of

hypergravity on auditory brainstem responses (ABRs). It was

found that hypergravic fields of 6G, but not 3G, altered the

ABRs. lhe ABRs have a series of negative peaks (iN, 2N, •••• ) and

positive peaks (lP, 2P, ..•• ) and at 6G there were significant

increases in central conduction time for peaks 3N, 4P, 4N, and

5P. The delay of peaks later than 3P suggests that structures

above the midpontine level were impaired in 6G fields. Secondary

changes reflect lowered brain temperature rather than a direct

effect of the gravitational field. These data indicate that the

auditory system is less sensitive to hypergravic fields than is

23
the thermoregulatory system. Several aspects of these studies on

ABRs will serve as useful background studies for experiments on

vestibular far-field potentials. The studies have also pointed

out the temperature sensitivity of ABRs. Finally, this lab

"developed techniques to measure ABRs, and to mask ABRs, which

will prove useful in control experiments in vestibular studies.

PUBLICATIONS

Horowitz, J.M., Monson, C.B., and Horwitz, B.A. Effects of

Restraint on Two Modes of Heat Production of Cold-exposed Rats in

Hypergravic Fields (Abstract). Physiologist 25(4): 212, 1982.

Jones, T.A., Hoffman, L., and Horowitz, J.M. Altered Auditory

Function in Rats Exposed to Hypergravic Fields. Physiologist

25(6, Suppl.): S93-S94, 1982.

Jones, T.A. and Horowitz, J.M. Bone-conducted Auditory

Stimulation in Unrestrained, Unanesthetized Animals. Journal of

Neuroscience Methods 7: 261-267, 1983.

Kott, K.S. and Horwitz, B.A. Photoperiod and Pinealectomy Do Not

Affect Cold-Induced Deposition of Brown Adipose Tissue in the

Lon g- Evan s Rat. Cry 0 bi 01 0 gy 20: 100 -1 05, 198 3.

Monson, C.B., Horowitz, J.M., and Horwitz, B.A.

Hyrothermia in Cold-exposed Rats at 3G and 1G.

25 6, Suppl.): S89-S90, 1982.

24

Restraint

Physiologist
DEVELOPMENT AND FUNCTIONAL DIFFERENTIATION OF THE VESTIBULAR

SYSTEM IN NORMAL, ROTATED, AND CENTRIFUGED RATS AND MICE

J. Richard Keefe

Department of Anatomy University of Louisville

Louisville, KY 77058

SUMMARY

The timing of the processes of cellular mitoses and

differentiation in the developing mammalian central nervous

system (CNS) is a highly regulated process controlled in four

dimensions. Each adult neuron is the direct result of a series

of mitotic divisions by a particular cell-line in a precisely

controlled positional and temporal sequence. It appears that

each eNS develops in a particularly descriptive sequence

reflecting its primitive embryonic derivation as well as its

phylogenetic relationship. Thus, the so-called primitive neural

systems appear to develop the earliest in embryonic age.

Each neural system consists of cell bodies, located in nuclei,

precisely interconnected with either other nuclei or with

peripheral receptors/effectors by the establishment of precisely

regulated levels and patterns of synaptic connections. The

patterns of synaptic contact are regulated by the singular timing

sequence present during early development in which sensory,

interpretive, and effector neurons must be generated within a

close time frame for their mutual interaction to occur.

Geometrical relationships must also be facilitated during early

system generation to increase the probability of the

establishment of the proper set of synaptic interconnections

among the plethora of newly developing neurons. Finally, to

ensure the probability of establishing a set of highly integrated and precisely patterned synaptic contacts, an excess of neurons

is mitotically generated in the germinal region providing each

eNS nucleus. Many of these are lost during the process of

postmitotic migration from their birthplace to their final neural

location. Those neurons that arrive within the proper time frame

at the proper location attempt to establish a proper set of

synaptic contacts and, if successful, continue their

differentiation.

Stockard (1921) developed the critical period concept which in

its modern expression holds that each neuron is specifically programmed to establish synaptic connections with another

specifically programmed neuron and to do so only during a brief

period of its early life. The failure to establish such profer synaptic contact during this critical period leads to the ce 1

death of that neuron.

25
This laboratory is attempting to determine the timing patterns of

cellular birth, migration, and differentiation in two

phylogenetically primitive systems--proprioceptive and

vestibular--in Wistar rats and two strains of mice. Included as

an integral part of this study is the determination of the timing

patterns and levels of cell death occurring in each element of

each system during prenatal and early postnatal development.

During 1982 this laboratory has continued timed breeding in each

of these species to achieve a number of staged specimens under

normal (nonstimulated) conditions. The sectioning, autoradiographic processing, and serial microscopic analysis of

these specimens is continuing. The accomplishments to be

described are part of a continuing study and, as such, must be

considered preliminary in nature. However, the data presently available do allow the following observations:

1. The data clearly indicate a limited time frame for the

generation of elements of the peripheral and central integrative

elements of the vestibular system.

2. This critical period of primary neuronal generation occurs at

the same time that the developing embryo is undergoing

implantation and maternal placentation is established. This

event appears to be most susceptible to alterations in maternal

cardiovascular function, a pathology to be expected under

conditions of null-gravity adaptation.

3. The only nuclei of the lower brainstem (involved in

proprioception) that we have sufficient data on are the

second-order sensory relay nuclei gracilis, cuneatus, and the

ext. cuneate and these do not appear to show a timing gradient that would reflect the normal adult topographic patterns although there does appear to be a size gradient with the larger cells

(believed involved in muscle proprioception) being generated earlier than the smaller cells.

4. Cell death in the sensory regions of the vestibular

membranous labyrinth is almost nonexistent (probably diffuse and

missed).

5. Centrally, the onset of significant cell death in the

vestibular system occurs in the superior and inferior vestibular

nuclei on Embryonic Day 14, reaching a peak by Day E16 and

persisting at low levels into the postnatal period. The lateral

vestibular nucleus, receiving predominantly utricular input, shows a marked level (15 to 20t) of cell death on days El6 and

Ell, roughly 3 days after the peak generation times observed for

the Maculae of the utricles, while the medial vestibular nucleus,

receiving inputs from a variety of peripheral receptors, shows

the lowest level of cell death through the fetal and early postnatal periods.

26
PUBLICATION

Keefe, J.R. Timing of Neuron Development in the Rodent

Vestibular System. Physiologist 25(6, Suppl.): Sl15-S116,

1982.

27
DEVELOPMENT OF REQUIREMENTS FOR INFLIGHT CONTROL AND RESEARCH

CENTRIFUGE

R.W. Mah and J. Oyama Ames Research Center

Moffett Field, CA 94035

SUMMARY

The initial objective of the animal research portion of this

study was to determine whether the thermoregulatory responses

effected by hypergravitational exposures in rats are due to the

gravitational intensity (G) alone or whether they are also

affected by the rotational rates and radii of the centrifuge

used. Can a smaller-sized centrifuge operating at a higher speed be used instead of a larger-sized centrifuge operating at a

slower speed to produce the same kind of biological effect? What

minimum radius and maximum rotational speed can be used in a

spaceflight centrifuge which can provide satisfactory inflight 1G

control data to compare with the weightlessness-exposed group of

animals? Are there differences in the biological responses in

animals exposed to the same G-intensity using different

rotational rates and radii over longer exposure periods even

though shorter exposures may produce no differences? These are

among the important questions for which answers are being sought.

A series of experiments was run in which different groups of rats

were exposed to 1.41G using various combinations of rotational

rates (RPM) and radii over a 30-minute period. A comparison of

both the rectal temperature response (decrease) and tail

temperature response (increase) between the various groups showed

that essentially the same degree of change in temperature was

effected in all of the groups. The RPM ranged from 22.6 to 44.0

while the radius ranged from 173 cm to 46 cm. We conclude from

this study that the changes in body temperature in hyper-Gexposed rats are due solely to the G-intensity and are

independent of the radius and RPM. It remains to be determined

whether this will hold up for even shorter radii (less than 46

cm) and even higher RPM rates. Hence it appears that with

respect to thermoregulatory responses identical responses are

obtained using as small a centrifuge as 46 cm (1.5 ft.) or a much

larger centrifuge (173 cm = 5.7 ft).

These temperature studies preempted the use of the centrifuge for

long exposure duration studies; these studies will be conducted

in the near future. These longer duration studies will be

concerned with growth rate comparisons of rats exposed to the

same G-intensity but employing different radii and RPM rates as

was done in the short exposure temperature studies. In addition

to thermoregulatory function studies, a limited number of

experiments have been completed in which simultaneous

28
measurements of blood constituents (glucose and corticosterone) have been performed in an attempt to see whether changes in body

temperature could be associated with changes in these two

constituents. Both glucose and corticosterone elevations are

associated with the generalized stress response. Although preliminary results indicate an apparent correlation of the

temperature response with changes in blood and corticosterone

levels, the studies clearly show that the temperature response is

far more sensitive and reliable as a stress index than the other

two parameters~

29
ROLE OF GRAVITY PERCEPTION IN THE ESTABLISHMENT OF THE

DORSAL/VENTRAL AXIS OF THE AMPHIBIAN EGG

G.M. Malacinski

Department of Biology Indiana University Bloomington, IN 47401

SU MMARY

The main goal of this research program is to understand the role

gravity plays in establishing the pattern of early amphibian

embryogenesis. The amphibian egg has been chosen as a model

system. It offers several distinct experimental advantages: (1)

it is easily obtained in large numbers, (2) it displays a

distinct and reproducible dramatic response to gravity when

fertilized (it "rotates" so its darkly pigmented hemisphere

opposes gravity), and (3) it can be manipulated in the laboratory in several straightforward ways (e.g., it can be held in abnormal

gravity orientations).

During 1982 a number of observations have been made on pattern form at ion in" i nve r ted II egg s, i. e ., egg s t hat we r e he1d ups ide

down from the period before fertilization until the neurulation

stages (by which time pattern formation is essentially completed). Remarkably, several aspects of early pattern formation (e.g., cleavage pattern, involution, etc.) were normal.

That is, the external features of morphogenesis proceeded in a

normal fashion. When examined by conventional histological approaches, however, it was observed that although some major cytoplasmic components shifted almost completely to accommodate

gravity, not all components did. Yet the pattern of

morphogenesis was surprisingly normal.

For example, large yolk platelets displayed a substantial shift,

while the germ plasm (a collection of granules localized in the

egg's original vegetal-facing gravity-hemisphere) does not shift

at all. Those observations are being employed to develop an

appreciation for the extent to which the amphibian egg cytoplasm is rigidly organized for early embryonic pattern specifications. At the present time it appears that the amphibian egg is less

rigidly organized than believed by earlier embryologists.--rKe

egg is capable of tolerating a substantial amount of gravity perturbation. It is still not possible, however, to predict

whether frog eggs will be able to develop normally in space (see

below).

Careful examination of the cytoplasmic rearrangements that occur

in normal orientation eggs and inverted eggs suggests that under

normal fertilization conditions, cytoplasmic components may, in

fact, rearrange according to their intrinsic buoyant densities.

30
It is not yet known whether inverted eggs compensate for gravity effects, since they appear to develop normally; but under natural

(ground-based) conditions it appears that gravity-driven

cytoplasmic rearrangements may occur. We have formulated those

observations into a "density compartment" model. This model

proposes that upon activation (fertilization), internal egg

components stratify according to their densities.

Further experiments are being designed to test a set of

predictions that this model offers. The outcome of these

experiments should generate a sound basis for predicting whether

amphibian eggs will be able to polarize (following fertilization)

and develop normally in space.

PUBLICATIONS

Malacinski, G.M. Reversal of Early Pattern Formation in Inverted

Amphibian Eggs (Abstract). Physiologist 25(4): 258, 1982.

Neff, A.W. and Malacinski, G.M. Reversal of Early Pattern

Formation in Inverted Amphibian Eggs. Physiologist 25(6,

Suppl.): Sl19-S120, 1982.

31
EFFECTS OF WEIGHTLESSNESS AND HYPERGRAVITY ON DEVELOPMENT AND

AGING OF DROSOPHILA

Jaime Miquel

Ames Research Center

Moffett Field, CA 94035

SUMMARY

The fundamental mechanisms of development and aging in Drosophila melanogaster (fruit fly) were examined under normal laboratory

conditions in comparison with fruit flies housed in clinostats

and centrifuges during various periods of their life cycles or

exposed to weightlessness onboard Cosmos biosatellites.

Investigators completed the study of the relation between the

rate of oxygen consumption and lifespan in three wild strains

(OR-R, 0-32, Sw-C) and two mutants (w/w and w/mei-41-D5) of

Drosophila. (The w/mei-41-D5 is a mutant whose embryonic tissue

showed deficiency during post-replication repair.) There was an

inverse correlation between median lifespan and oxygen

consumption for flies having normal DNA repair. In contrast the

w/mei-41-D5 showed an abnormally short lifespan, significant deficiency in mating performance, and a depressed metabolic rate,

even during the first days of adult life. In contrast to a

widely held opinion, these results suggested that flies, who are

deficient in DNA repair, are not suitable for gerontological

studies because their short lifespan does not result from

accelerated aging, but from specific genetic diseases.

Morphology of worker bees exposed to weightlessness during the

Space Shuttle Columbia's third flight was compared to Drosophila. This analysis was conducted in collaboration with the Electron

Microscopy Laboratory at Ames and NASAls Shuttle Student

Involvement Program. This preliminary investigation on the

effects of near-zero gravity on the biology of social insects of

the order Hymenoptera showed that the Shuttle flight did not

result in morphological injury to the bees, in contrast to the

wing injuries seen previously in Drosophila flown on Cosmos. The

difference between the responses of flies and bees to

weightlessness resulted from the bees' inability to fly in

weightlessness, as shown by movies taken onboard the Shuttle.

Therefore, bees were not likely to injure their wings in

weightlessness. Unlike bees, the highly sophisticated flying mechanisms of Drosophila incorporate gyroscope-like halteres, and

may have allowed fruit flies a certain amount of poorly controlled flight in weightlessness, resulting in injury due to

impact against the walls of the housing unit.

32
PUBLICATIONS

Fleming, J.E., Miquel, J., Cottrell, S.F., Yengoyan, L.S., and

Economos, A.C. Is Cell Aging Caused by Respiration-dependent

Injury to the Mitochondrial Genome? Gerontology 28: 44-53, 1982.

Miquel, J. and Economos, A.C. Space Gerontology, Proceedings of

a Workshop, NASA Ames Research Center, Moffett Field, California, January 30-31, 1978. washlngton, D.C.: NASA, 1982.

Miquel, J., Fleming, J.E., and Economos, A.C. Antioxidants,

Metabolic Rate and Aging in Drosophila. Archives of Gerontology and Geriatrics 1: 159-165, 1982.

33
COMPUTER-ASSISTED VIDEO HISTOMORPHOMETRY OF ANIMAL SPECIES

Emily Morey-Holton Ames Research Center

Moffett Field, CA 94035

SUMMARY

Investigators successfully completed a method of computer analysis to measure the cross-sectional growth in the cortical

bone of rats. To relieve the time-consuming and tedious

point-counting of microscopic specimens, computer software was

developed to digitize a bone image into a frame buffer display on

a TV screen to trace tetracycline labels. The operator corrects

the trace if it goes astray. Perimeter and area measurements,

generated from the traced labels, are used to calculate bone

formation and apposition rate. Tests with multiple bone

sections, analyzed by point-counting and with a digitizing table,

were within 5% of computer measurements. In addition, a

statistics package calculated the mean, standard deviation, and

t-statistic for each experimental parameter. This technique of

image processing and analysis compared favorably with other

techniques, and demonstrated very low variability of data due to

the observer.

PUBLICATION

Globus, A. and Morey-Holton, E. Computer-assisted Image Analysis of Cross-sectional Bone Growth. Calcified Tissue International

34: S7, 1982.

34
STRUCTURAL DEVELOPMENT AND GRAVITY

Emily Morey-Holton

Ames Research Center

Moffett Field, CA 94035

SUMMARY

The major thrust of this research is to investigate bone changes

in a rat model which simulates the lack of weightbearing (only in

the rear limbs) and the shift of body fluids into the chest and

head which occur during spaceflight. Animals of various ages and

strains will be used in this research; thus, the first series of

studies will establish normal bone parameters in two different

strains of rats throughout their lifespan. Bone formation

decreases with age but this chan~e varies from strain to strain

and from bone to bone and even dlfferent sites within the same

bone; thus, the first objective of this research is to define

changes in bone formation rate and bone structure with age in

normal rats so that realistic research goals can be established

for rats of various ages and strains. A second objective of this

research program deals with defining the type and extent of

changes in bone structure and mineral metabolism that might occur

in young rats during a routine shuttle mission (2-10 days) by using the rat model. A third objective of the program is to

determine whether interpretation of data from the model is

compromised by excessive amounts of physiological stress. This

objective is addressed by comparing date from animals exposed to

a known chronic physiological stress (4 C) with animals on the

model.

The accomplishments in the first objective include processing

bones from rats at 1 month, 2 months, 4 months, or 8 months after

birth. The rats remaining will be 16 months of age in August. Two different strains of rats are being used; one strain attains

a mature mass of about 800 grams while the other group only

weighs about 400 grams at maturity.

In support of the first objective, bones have been processed for

histomorphometric analysis from 1-, 2-, 4- and 8-month-old rats

and will be processed from 16-month-old rats in August 1983.

X-rays have been taken of the 16-month group at 1, 2, 4 and 8

months of age to determine approximate growth rates of entire

long bones, vertebra, etc., and to determine time that growth in

length stops. Rats are weighed three times each week to provide

a growth curve of each strain of rat. These analyses allow

definition of the processes occurring and the rates of bone

formation at specific sites within each bone without perturbing the animals experimentally.

35
For the second objective, various radioisotopes that monitor cell

activity and cell synthesis of certain or~anic substances are

being given to determine the most approprlate time to label and

the maximum number of isotopes that can be given to get meaningful information from a single animal. Urine and fecal

samples were collected and analysis of calcium, phosphorus, and

other minerals in these samples are in progress. These metabolic

studies will determine whether mineral excretion is altered in

the rat model with time. The studies lasted 3 weeks with

collections being taken every day the first week and three times

a week thereafter.

Rats cold exposed for 8 and 24 hours/day (2 rats per plastic cage

with sawdust bedding) did not show a stress response. Thus, a

final experiment was conducted in which the rats were

individually housed in the cold in metal cages. A basal group

was used at the beginning of the experiment to determine muscle

mass and dynamic bone parameters. Animals (1) fed at will and

(2) fed the average amount of food consumed by the rats on the

model were the experiment controls. Preliminary data suggest the

following:

1) Model animals ate about 17 grams of food/day the first week, about 20 grams per day during the second week, and about 22 grams

per day during the third week of the experiment. Cold-stressed

rats consumed about 42 grams per day during the third week while

the ad libitum rats (those allowed to feed at will) ate about 26

gramS-per day during the third week. Interestingly, despite the

differences in food intake, the cold-stressed animals gained weight at about the same rate as the group-mean-fed controls and

supported animals with all groups having a mean weight of about

233 grams during the third week of the experiment. The ad lib

rats weighed an average of 275 grams at the same time perTo~

The cold-exposed rats may have been shredding, rather than

eating, some of their food.

2) Thymus weight and adrenal mass, indicators of stress, changed

only in cOld-exposed animals.

3) The mass of the extensor digitorum longus (EDL) and

gastrocnemius (G) muscles in the le~ were less in the

experimental than in the correspondlng control groups, but

greater than the basal controls. In contrast, although the

cold-stressed animals also demonstrated about a 20% decrement in

soleus (5) muscle mass at all time periods as compared to

corresponding controls and a gain over the basal level, the

"model" rats showed at least a 50% decrement in the 5 with the

mass always being less than the basal group.

4) Bone parameters from this experiment are presently being processed.

36
Although not included in the actual objectives of this research,

three other programs have been supported and data obtained. In a

study to support the Cosmos monkey spaceflight, our results from

the proximal femurs showed that bone formation was reduced in

casted monkeys. Osteons, the basic units in the bone that

consist of a central canal and a concentric radiation of bone

cells, slowed their rate of depositing bone mineral. The

percentage of osteons in the bone did not change significantly, however. The supporting trabecular surfaces in the humerus and

femur were more severely retarded than the osteon, or Haversian,

system which is found in bone shaft. The second study was in

support of the Student Shuttle Involvement Project (SSIP).

Surprisingly, model rats did not develop the systemic arthritic

inflammation to the same extent as control rats. Although the

mechanism for this difference is unknown, the data suggest that

fluid shifts and/or unloading the sensitive paws in some way

interferes with the disease process and, presumably, the immune

system. The answers to such alterations in the immune system may

be found in the third study. A dramatic decrease (80%) in

production of alpha and beta interferon was observed in those

animals exposed to simulated weightlessness as compared to

control rats. These data suggest that weightlessness simulation

may alter certain immunological functions in the rat.

PUBLICATIONS

Holton, E.M. Effects of Weightlessness on Bone and Muscle of

Rats. In: Space Gerontology (ed. by J. Miquel and A.C.

Economos). washlngton, D.C.: NASA, p. 59-66, 1982.

Jee, W.S.S., Wronski, T.J., Morey, E.R., and Kimmel, D.B.

Effects of Spaceflight on Trabecular Bone in Rats. American

Journal of Physiology 244: R310-R314, 1983.

Morey-Holton, E.R., Bomalaski, M.D., Enayati-Gordon, E.,

Gonsalves, M.R., and Wronski, T.J. Is Suppression of Bone

Formation During Simulated Weightlessness Related to

Glucocorticoid Levels? Physiologist 25(6, Suppl.): S145-S146,

1982.

Pace, N., Morey-Holton, E., Rahlmann, D.F., and Smith, A.H. Body Composition of 80-day-old and 270-day-old Rats After 14 Days or--

Hindquarter Suspension. u. California, Berkeley, Environmental

PhYS10logy Laboratory, 79 p., December 1982. (EPL-82-2)

Wronski, T.J. and Morey-Holton, E.R. Alterations in Calcium

Metabolism and Bone Structure During Space Flight in Man and Rats

(Abstract). In: Abstracts, 24th COSPAR Meeting, Ottawa, Canada,

May 16-June 2, 1982. Paris: CasPAR, p. 539, 1982.

37
Wronski, T.J. and Morey, E.R. Effect of Spaceflight on

Periosteal Bone Formation in Rats. American Journal of

Physiology 244: R305-R309, 1983.

Wronski, T.J. and Morey, E.R. Skeletal Abnormalities in Rats

Induced by Simulated Weightlessness. Metabolic Bone Diseases and

Related Research 4: 69-75, 1982.

38
~ENAL FUNCTION, WATER AND ELECTROLYTE BALANCE, AND INTESTINAL

TRANSPORT IN HYPOKINETIC ANIMALS

X.J. Musacchia

Department of Physiology and Biophysics

University of Louisville

Louisville, KY 40208

SUMMARY

Research in this laboratory has focused on three areas during 1982: (1) muscle composition and function during hypokinesia and

hypodynamia (H/H), (2) fluid and electrolyte shifts as they impinge on cardiovascular changes, and (3) modification of the

animal support system to utilize large adult rats and the

laboratory mouse.

Research is concluding in the area of muscle physiology which

involves the protein, DNA and RNA relationships in four hind-limb

muscles: soleus, gastrocnemius, plantaris, and extensor

digitorum longus (EDL). They represent the range of muscle

function, from antigravity (load-bearing) to non-load bearing. The soleus responded with the greatest degree of atrophy and the

most pronounced protein and RNA decrease during a 1- and 2-week

period of H/H. At the same time, the EDL, a non-load bearing muscle, exhibited little or no change in these biochemical

parameters. The gastrocnemius and plantaris show intermediary

changes, indicative of their mixed functional roles in terms of

antigravity functions. The DNA contents in all four muscles

showed relatively little or no change during a 2-week support

period. It was concluded that there was a decrease in muscle

protein synthesis as a reflection of the protein/RNA

relationship, principally in the soleus.

Studies are currently in progress to determine the mechanisms

which underlie the protein/RNA changes. A Space Biology Research

Associate working in this laboratory has a program of research

dealing with the involvement of glucocorticoid receptor levels in

muscles undergoing H/H atrophy due to unloading. Evidence of a

potential role for glucocorticoids in disuse atrophy is found in

reports that with H/H there is adrenal hypertrophy and increased

circulating corticosterone; also, rats exposed to weightlessness showed adrenal hypertrophy. The current experiments are aimed at

identifying changing levels of tissue sensitivity to

glucocorticoids by assessing receptor concentrations in the four

muscles identified above. Initial results showed a 400% increase

in receptor levels in soleus, about 50% in the gastrocnemius and

plantaris, and no change in EDL.

Having identified that diuresis and natriuresis occur only in

head-down-tilted suspended antiorthostatic (AOs) subjects, but

39
not in orthostatic (Os) rats, this laboratory utilized the

Gauer/Henry concept that AOs animals are responding to an

expanded thoracic blood volume and that cardiopulmonary stretch

receptors have responded to those changes in central blood

volume. It was reasoned that blood pressures could reflect AOs

and 0 positioning. Investigations we~e aimed at assessing cardiovascular resBonses of AOs and Os rats, and to a rapid head-up tilt of 70. Direct blood pressures, i.e., mean arterial

pressure (MAP), systolic, diastolic, and pulse pressures and

heart rates were taken from aortic cannulations. There was a

significant increase in blood pressure during Day 1 in both Os

and AOs rats. In the Os rats, the MAP returned to control levels

on Day 3; in contrast, the MAP remained elevated in AOs rats.

This difference was chiefly the result of diastolic pressure

changes. It was reasoned that the elevated blood pressures on

Day 3 are a reflection of the AOs positioning and relatable to

fluid and electrolyte changes which are evidenced after the 2nd

day of support in AOs subjects. As anticipated, during Day 1, a

rapid head-up tilt showed increases in MAP in both Os and AOs

rats and also increased heart rates. However, the response was

less pronounced in the AOs subjects, and on Day 3 there was a

lack of MAP response. This suggests an adaptive response, probably increased vasomotor tone, as a result of AOs

positioning, and the elevated fluid and electrolyte losses. Upon

return to a preti1t position, blood pressures returned to the

preti1t levels. These initial experiments support the previous hypothesis of significant differences between the

antiorthostatic-positioned and orthostatic-positioned supported

rats.

Two additional series of experiments were conducted. The support

apparatus was adapted for mature adult rats weighing 450 to 500

gm. They responded in much the same manner as 180 to 200 gm animals used in previous experiments, i.e., an initial body weight loss, a loss of muscle mass (soleus> gastrocnemious =

plantaris> EDL). A highly modified support system was adapted

to the laboratory mouse. This is a collaborative experimental series in which the role of support H/H on interferon production is being ascertained.

PUBLICATIONS

Musacchia, X.J. and Steffen, J.M. Short Term (1 and 3 Day)

Cardiovascular Adjustments to Support Antiorthostasis in Rats.

Physiologist 25(6, Suppl.): S163-S164, 1982.

Steffen, J.M. and Musacchia, X.J. Effect of Support Hypokinesia/Hypodynamia on Glucocorticoid Receptor Levels in Rat

Hindlimb Muscles. Physiologist 25(6, Suppl.): S151-S152, 1982.

40
A GROUND-BASED COMPARATIVE STUDY OF THE EFFECT OF WEIGHTLESSNESS

ON THE DEVELOPMENT OF AMPHIBIAN EGGS

George W. Nace

Division of Biological Sciences and

Center for Human Growth and Development

University of Michigan

Ann Arbor, MI 48109

SUMMARY

Bilaterality is a basic theme of animal organization and seems to

have arisen from animals of radial symmetry. This phylogeny is

reiterated as the first step in the development of every bilateral animal, including man, that starts life as a radially

symmetrical egg. Dating from the 19th century, hypotheses

relating this change in symmetry to the action of sperm or of

gravity have been debated without resolution. With the advent of

space biology the means to resolve this question have become

available, and, in preparation for in-space studies, a rich body

of new evidence confirms that both sperm entry and gravity are

involved, but "how" remains uncertain.

This laboratory has examined the dynamics of the initial rotation

of the frog egg, and by the use of clinostat techniques has more

nearly pinpointed the short period relative to this rotation when

the action of gravity is critical. Now detailed study at the

physical/chemical level is needed to understand the mechanisms by which gravity influences bilateralization.

Basic to the examination of this question is the recognition that

all of evolution has been in the presence of gravity--a fact that

has received little attention from biologists who have generally

taken gravity as a constant and thus consider it irrelevant to

questions concerning biological variation. However, gravity acts

on every mass, and every organism has had to adapt to its

presence, even at the initial stages of development. Moreover,

its local action is not mediated through a receptor-transducer

system, the typical mechanism by which organisms deal with

environmental variables.

With this recognition, an expression was derived to describe the

torque produced on eggs by the action of gravity on the cytoplasm which is heterogeneously distributed within the egg. It was

immediately seen that all cells and defined structures in the 13

body are subiect to torque, and that this varies from 2.5 x 10-

to 8.3 x 10- dyne-em for cells ranging from 6.4 ~m to 31 ~m in

diameter. Of major significance was the discovery that this

torque is proportional to the fourth power of the diameter of the

body.

41
One finds that (1) gravity imparts torque to cells; (2) since

this torque is proportional to gravity, it is reduced to zero as

gravitational acceleration is reduced to zero; and (3) the torque is highly sensitive to the size of the cell (structure) and the

distribution of its subcellular particulates (components).

Considering that the orientation of every cell (structure) relative to its neighborhood is critical to its normal function

and that gravity tends to distort this orientation, one is led to

the previously unlabeled concept of positional homeostasis and to

two major hypotheses:

Hypothesis 1: Energy, to the extent needed to counteract the

action of gravitational force, must be expended by the cell

(structure) to maintain its position dynamically or to create and

maintain the structures involved in holding it in position.

Hypothesis 2: Evolution in a lG enviroment has been accompanied

by the elaboration of machinery for the maintenance of positional

homeostasis and, energetically, this is a costly investment.

These concepts and hypotheses find experimental support in the

apparent decrease in glucose utilization (58% less) by tissue

culture in spaceflight (Montgomery, et al., 1977). They are not

contradicted by evidence that Brownian movement dominates over

the action of gravity on bacterial cells, diameter <5 ~m

(Pollard, 1977).

Various secondary hypotheses derive from the above. Interesting

among these is that Brownian movement and gravity define the

lower and upper limits of cell size for cells subject to the

principle of positional homeostasis. One of the recen~tasKs on

thlS proJect has been to examine cell size in this context. It

has been found that few metazoan cells are less than 10 ~m or

greater than 40 ~m in diameter and that the functions attributed

to them can be related to their location in this size range.

Other secondary hypotheses concern the mechanisms which protect cells from displacement by the action of gravity. Examination of

these hypotheses has led to interesting new interpretations of

cell and tissue structural organization.

Examining these hypotheses for physiological significance leads

to the prediction of marked metabolic changes associated with

exposure to either hypo- or hypergravity, and the reexamination

of the sparse relevant literature reveals that indeed there is

strong evidence of metabolic changes that can be attributed to

the influence of gravity on the positional homeostasis of cells

and organs or their parts. The next task is to conduct

experiments to confirm this relationship.

42
This laboratory is also examining the influence of gravity on

cells resulting from its generation of other quantities: tension,

compression, shear, strain, and simple positional displacement. The calculations and evidence all point in a direction indicating that gravity may be one of the most significant environmental

variables influencing the structure and function of organisms and

that exposure to changes in the value of this variable will lead

to fundamental structural and physiological changes, many of

which may be expressed on a time line which has only now begun to

be accessible to manipulation.

PUBLICATONS

Nace, G.W. Exploration of Possible Gravitational Influences on

Fertilization and Initiation of Development (Abstract). In:

Abstracts, 24th CaSPAR Meeting, Ottawa, Canada, May 16-June 2,

1982. Paris: CaSPAR, p. 536, 1982.

Nace, G.W. Gravity and Positional Homeostasis of the Cell

(Abstract). Physiologist 25(4): 258, 1982.

Nace, G.W. and Manders, E.K. Marking Individual Amphibians.

J 0 urn a1 0 f Her peto logy 16: 3a9- 311, 1982.

43
HYPER-GRAVITATIONAL EFFECTS ON METABOLISM AND THERMOREGULATION

Jiro Oyama Ames Research Center

Moffett Field, CA 94035

SUMMARY

The main objectives of this research are to determine how changes in gravitational fields affect the ability of mammalian animals

to maintain their body temperature and blood sugar level. Both

show rapid and substantial departures from the levels observed in

normal-gravity-maintained animals when subjected to hypergravity conditions. The deviations observed have been closely related to

the gravitational intensity (G) which is imposed on the animal

and the duration of exposure.

The blood sugar deviations have been investigated by studying

r

rocesses that control the rate at which sugar leaves the blood

utilization rates) and the rate at which it enters the blood

(formation rates). Both neural "(sympathetic nervous system) and

hormonal (insulin, glucagon, catecholamines, corticosterone)

systems are being studied in rats under hypergravitational

conditions.

In a similar manner, body temperature regulation is being studied

by determining the rate at which animals lose heat as well as

generate it under hypergravity. The balance between the two

rates determines the body temperature. Both neural and hormonal

mechanisms which regulate heat loss and heat production are under

investigation. Contraction and dilation of blood vessels which

help regulate the rate of heat loss are being monitored through

the use of surface temperature probes while heat production is

being determined through measurements of oxygen consumption

rates. The basic studies are directed toward developing a better

understanding of how gravity and gravitational changes affect

systems that help maintain the internal environment of the animal

at a balanced state in spite of marked changes in the external

environment.

Among the significant accomplishments during 1982 have been the

completion of studies to quantify the deep body (rectal)

temperature decrease and surface (tail) temperature increase

responses in hypergravity-exposed rats. Incremental changes of

both have been found between 1.0G and 1.5 to 1.8G. Higher G

intensities produced no further decrease in rectal temperature or

increase in tail temperature. The tail temperature response

which actually decrelsed with higher G loads indicates that

mechanisms to constrict the blood vessels of the tail

(sympathetic nervous system) and decrease the rate of heat loss

become activated at the higher G intensities but not at the lower

44
intensities. There appears to be a threshold for activating the

sympathetic system. Investigations are currently underway to

study this interesting response further.

The results obtained lend further support to the view that the

rectal and tail temperature response system is one of the most

reliable and practical means of monitoring and quantifying the

stress of hypergravity exposures in rats. It is a sensitive

system with as little 0.1G difference effecting a different

rectal and tail temperature change. Another interesting finding is that exposing rats to hypergravity in either a head-downward

or head-upward position (tilt angle = 29 degrees) had no

significant effect on the rectal or tail temperature response. These results indicate that thermoregulation under hypergravity conditions is not affected by changes in body position but is

sensitive to the field intensity.

Accomplishments dealing with the metabolic aspects (glucose

balance) in hypergravity-exposed rats include the completion of

blood lactate and glycerol response patterns for periods ranging

up to 24 hours of exposure. Hormonal patterns have also been

established for insulin, glucagon, and catecholamines. These

hormones are major determinants in regulating the utilization of

blood sugar as well as its production by the liver. The latter

process appears to be the primary regulator of blood sugar elevation during the initial stages of hypergravitational

exposures. What role this process has in sustaining the

evaluation of blood sugar over more extended exposures remains to

be elucidated. During 1982, techniques have been developed to

prepare viable liver cells from hypergravity-exposed rats for

testing the state of the liver tissue to form new sugar from

noncarbohydrate sources, i.e., amino acids, and from the

breakdown of fat (glycerol) and muscle glycogen (lactate). By

using this isolated cell system preparation, many types of

studies which cannot be conducted in the intact, living animal

can be performed under well-defined conditions. In developing

the procedure, an elaborate system for perfusing the liver tissue

under carefully controlled conditions was assembled and

successfully placed into operation. The new procedure will

permit study of the effects of individual hormones such as

glucagon on sugar formation capacity by the liver cells of

hyper-G stressed rats separated from the effects of other

hormones which are normally present in the intact animal.

PUBLICATIONS

Daligcon, B.C. and Oyama, J. Increased Gluconeogenesis in

Hyper-G Stressed Rats. Physiologist 25(6, Suppl.): S87-S88, 1982.

45
Oyama, J. Metabolic Effects of Hypergravity on Experimental

Animals. In: Space Gerontology (ed. by J. Miquel and A.C.

Economos). Washlngton, D.C.: NASA, p. 37-51, 1982.

(NASA-CP-2248)

46
PRIMATE HEMODYNAMICS AND METABOLISM UNDER CONDITIONS OF

WEIGHTLESSNESS

Nello Pace

Environmental

University of

Berkeley, CA

SUMMARY

Physiology Laboratory California

94720

A major accomplishment during 1982 was the preparation of a

formal Flight Verification Analysis of a Primate Metabolic System in collaboration with the McDonnell Douglas Astronautics Company, Huntington Beach, California. The report defines an engineering configuration of a system capable of maintaining a 10 to 14 kg

adult macaque in comfortable seated restraint in the mid-deck of

the Shuttle during flights of up to 7 days' duration, or longer. The design is based on several years of development and testing

of a monkey restraint habitat in this laboratory and in NASA

flight payload simulations.

The system provides appropriate ventilation, food and water, and

excreta coll~ction, as well as continuous physiological

monitoring of the animal during the period of the flight. The

design emphasizes the use of components already developed, and in

some instances already flown, by NASA in other flight programs in

order to minimize costs.

A plan is proposed to construct an Engineering Development Unit

to test the system concept with a live animal in a NASA Mid-Deck

simulator for several days before a decision is made to proceed with fabrication of a Flight Unit. It is further proposed that

the Fl ight Unit be tested in a short duration, 2- to 3-day

Shuttle flight, followed by a longer, 5- to 7-day Shuttle flight using monkeys with no surgical preparation. This plan provides

conservative examination of the potential for use of large

primates in space life sciences research in a cost-effective

manner.

During 1982 substantial progress was also made in developing a

battery of radioimmunoassay procedures for assessment of the

status of thyroid function in macaques. It has been known for

many years that the metabolic rate of the body is regulated by thyroid hormones circulating in the blood. More recently, it has

become evident that there is a complex feedback relationship between metabolic rate, the blood thyroid hormone levels, and the

activities of the pituitary and thyroid glands. Thus, in

evaluating changes in metabolic rate it is necessary to define

the correlative changes in thyroid system activity.

The seven principal blood parameters evaluated were: thyroid

47
stimulating hormone (TSH) produced by the pituitary gland, thyroxine (T4) produced by the thyroid gland, triiodothyronine (T3) produced in part by the thyroid gland and in part from T4 in

the body tissues, and a T3 isomer (rT3) produced in the body

tissues. TSH stimulates the thyroid gland to produce T4 and T3.

In turn, both T4 and T3 stimulate oxygen consumption by body

cells, T3 being six times as potent as T4, while rT3 has no

effect on oxygen consumption. T4 and T3 are largely bound to

serum proteins and, because only the free form of these hormones

is physiologically active, it is also necessary to measure the

serum concentration of thyroxine-binding globulin (TBG), of free

T4 (FT4), and of free T3 (FT3).

The close phylogenetic relationship of macaques to man permits the use of radioimmunoassay procedures developed for human blood,

and commercially available kits are available for all the thyroid

parameters noted above with one important exception--FT3. Accordingly, in 1982 the project was successful in developing a

radioimmunoassay procedure for FT3 in small volumes of macaque

serum. A protocol was evolved for measuring the seven principal thyroid hormone parameters in 1.5 ml of macaque serum, so that

sequential evaluation of thyroid function status in a particular

animal is practically possible.

In one 1982 study, measurements were made of five of the seven

parameters (all but TBG and FT3) in blood samples collected 3

weeks apart from 17 of our colony (adult male, Macaca

nemestrina). Good test-retest reliability was demonstrated. In

another study, it was demonstrated in three monkeys that TSH, T4,

T3, and FT4 serum levels were unaffected by continuous seated

restraint for 8 days in our Environmental Physiology Laboratory

(EPL) monkey pod.

PUBLICATION

Second, T. Primate Metabolic System (PMS) Flight Verification

Analysis: Final Report. Huntington Seach, California, McDonnell

Douglas Astronautlcs Co., 1982. (Report MDC-H00056)

48
GRAVITY, BODY MASS AND COMPOSITION, AND METABOLIC RATE

Nello Pace and Arthur H. Smith

Environmental Physiology Laboratory

University of California

Berkeley, CA 94720

SUMMARY

It is common knowledge that the colder the surroundings in which

an animal finds itself, the greater is the metabolic energy

expenditure rate the animal requires to maintain body

temperature. Less appreciated is the fact that as the

surrounding temperatures approach and exceed body temperature, a

greater metabolic energy expenditure by the animal is likewise

required to maintain body temperature. Thus, there exists a

region of surrounding temperatures in which the metabolic rate of

an animal is at a minimum. This temperature range is known as

the zone of thermoneutrality. It also follows that if

comparative studies of metabolic rate among animals are made,

care must be taken to standardize the ambient temperature conditions.

Under this grant, a four-species model--hamster, rat, guinea pig, and rabbit--has been developed to study the scaling of metabolic

rate on body size among mammals. Although previous investigators have examined the thermoneutrality zone of three of

these species, the work has largely been done on young, immature

animals which generally have a higher metabolic rate than mature

animals. During 1982, a series of measurements of metabolic ra~e

by dire8t calorimetry at different ambient temperatures from 20

C to 35 C were carried out in six mature animals of each of the

four species in order to define the thermoneutrality zone.

Results show that the metabolic rate remained w&thin 10% of the

minimum ~alue in the temperatube range 27 to 31 C for hamsters6 24 to 30 C for rats, 26 to 3a C for guinea pigs, and 25 to 35

C for rabbits. Hence, 28 + 1 C represents an optimal

temperature for all four species to pursue comparative metabolic

rate studies, and probably represents the most comfortable

ambient temperature for these animals.

Measurements have also been made of metabolic rate in the

four-species model during chronic centrifugation for 6 weeks at

2.0G. Although the experiment is not yet completed, preliminary

results indicate that the metabolic rate of hamsters with a mean

body mass of 0.11 kg was increased by 6% after 2 weeks at 2.0G,

that of O.SO-kg rats by 16%, that of O.70-kg guinea pigs by 11%,

and that of 3.3-kg rabbits by 36%. Because the metabolic rate of

the larger animals was increased proportionally more than that of

the smaller animals, it may be concluded that gravitational

loading definitely influences the scaling of metabolic rate on

body mass such that at 2.0G the scaling is probably close to the

0.80 power rather than to the classic 0.75 power seen under

49
normal gravity conditions. The implication is strong that the

scaling under conditions of weightlessness will be significantly less than the 0.75 power.

The development of a method for estimating skeletal muscle mass

of mature animals of our four-species model from the body

creatine content was completed. The appropriate conversion

factors were derived experimentally by analysis of creatine

content of fat~free skeletal muscle samples. For hamsters, body

skeletal muscle mass in grams is given by multiplying body creatine content in grams by 291, for rats multiplying by 227,

for guinea pigs by 237, and for rabbits by 217. It was also

shown that the four species display consistently different

degrees of muscularity, the skeletal muscle content of the

fat-free body mass ranging from 43% in hamsters to 53% in

rabbits.

A collaborative study with Emily Morey-Holton of the NASA Ames

Research Center on the effects of 14 days of hindquarter unloading on body composition of 80-day-old and 270-day-old rats

was completed in 1982. The results were compared with similar

data obtained earlier on rats from Cosmos 1129 after 18.5 days of

weightlessness. It was concluded that hindquarter unloading mimics the effects of weightlessness in some respects, such as

the skeletal changes in young, growing animals, but that

important differences also exist. Therefore, results from

suspended animal experiments should be interpreted with caution.

PUBLICATIONS

Pace, N., Morey-Holton, E., Rahlmann, D.F., and Smith, A.H. Body

Composition of 80 day-old and 270-day-old Rats After 14 Days or--

Hindquarter Suspension. U. California, Berkeley, Environmental

Physiology Lab., 79 p., December 1,1982. (EPL-82-2)

Pace, N. and Rahlmann, D.F.

from Body Creatine Content.

1982.

Estimation of Skeletal Muscle Mass

Physiologist 25(6, Suppl.): S83-S84,

Pace, N., Rahlmann, D.F., Smith, A.H., Pitts, G.C., Ushakov,

A.S., and Smirnova, T.A. Body Composition Data from the Rat

Subjects of Cosmos 1129 Experiment K-316. U. California,

Berkeley, Environmental Physiology Lab., 63 p., January 15,1982.

(EPL-82-1; NASA-CR-168678)

50
EFFECT OF DECREASED GRAVITY ON CIRCULATION IN THE RAT

V.P. Popovic Medical School

Emory University Atlanta, GA 30322

SUMMARY

Gravity has a profound effect on some processes in mammals. The

effect of gravity on cardiovascular systems is especially great,

(e.g., blood pressure in the small blood vessels (arteries or

veins) in the legs of a standing man is increased to 120 to 220

mm Hg instead of the much smaller values observed when man is in

a horizontal position, as in bed). Thanks to many cardiovascular

(mostly reflex) mechanisms and many adaptations developed during millions of years of biological history, blood does not pool in

the legs of a standing man but returns toward the heart. In a

weightless state the blood returns easily toward the heart

because the high hydrostatic pressure in the lower extremities

disappears and the mentioned circulatory mechanisms are not

needed or used. Circulatory (orthostatic) intolerance and a

decreased work capacity are observed after return to Earth, and

these changes (shifts in blood distribution, overloading of the

atria of the heart because of the movement of blood toward the

chest, neurohumoral stimulation leading to excessive water loss,

and a profound blood volume loss concomitant with other

circulatory changes) persist postflight and require readaptation

of the circulatory system to lG.

In order to better understand circulatory changes induced by weightlessness and to identify physiological mechanisms

responsible for these changes, an animal model was developed that

mimics circulatory effects of weightlessness. The cardiovascular

measurements were performed in control experiments on Earth in

unrestrained, unanesthetized rats; and in the same animals in

head-down hypokinetic conditions; and during readaptation of the

same animals to free activity. The aortas and right atria of the

animals were permanently cannulated 15 days before experiments

and an electromagnetic flowmeter was implanted around each

animal's aorta. Arterial and central venous blood pressures,

heart rate, cardiac output, and other cardiovascular parameters

were measured after 15 days (full recovery of the animals). The

animals were undisturbed and seemed to be unaware that any work

was being done on them. Seven-day experiments (corresponding to

7-day Space Shuttle/Space Lab experiments) have shown that the

hypokinetic rat initially had an increased right heart atrial

pressure (congestion of blood in the chest), a decreased arterial

pressure, an increased stroke volume, and an increased cardiac

output. After 2 days these parameters returned to normal. After

release from the harness the rats showed a decreased ability to

51
"work" (exercise on a treadmill), and their heart rate was very much elevated while their cardiac output was significantly decreased. In order to study the effect of stress (induced by harness and head-down position) on cardiac output increase,

levels of "stress hormones" were measured in the blood of the

antiorthostatic rats.

On the basis of the results it was concluded that stress induced

during early exposure to antiorthostatic hypokinesia is one of

the reasons for the reported increased cardiac output. However,

the animals adapted to the new situation quickly. After a few

days of exposure the increased plasma hormonal levels returned to

control values.

PUBLICATIONS

Popovic, V., Popovic, P., and Honeycutt, C. Hormonal Changes in

Antiorthostatic Rats. Physiologist 25(6, Suppl.): S77-S78, 1982.

Popovic, V., Popovic, P., and Honeycutt, C. Pretreatment with

Levodopa and Aspirin Beneficial in Experimental Decompression Sickness. In: Preprints of 1982 Annual Scientific Meeting, Aerospace Medical Association, Sal Harbour, FL, Ma 10-13, 1982.

as lng on, erospace Me lca Assoclatlon, p. 2 1-2

1982.

52
BONE CELL KINETICS OF SIMULATED WEIGHTLESSNESS

Eugene Roberts

School of Dentistry

University of the Pacific

San Francisco, CA 94115

SUMMARY

This is a new project. Studies of rat weight-bearing bones

indicate that the osteopenia of weightlessness is due to

inhibited bone formation with no appreciable effect on

resorption. Cosmos 1129 results suggest suppression of bone

formation may be more generalized because preosteoblast

differentiation is depressed in the periodontal ligament (PDL) of

rat molars in nonweightlessness maxilla. The hypothesis to be

tested is that weightlessness is associated with a generalized

(systemic) suppression of osteoblast precursor cell proliferation and differentiation, resulting in marked inhibition or arrest of

bone formation. The number of preosteoblasts will be determined,

utilizing the nuclear morphometric assay, in the diaphysis and

metaphysis of ulna bones from Cosmos 1129 rats (18.5 days of

weightlessness). Results will be compared with similar data

previously reported for maxillary molar POL. Bone precursor cell

proliferation osteoblast activity and 03teoclast activity will be

assessed autoradiographically by using H-thymidine, -proline,

and -leucine in the maxilla and long bones of rats subjected to

jim¥!ated weightlessness (head-down support). A double labeling H/ C method will be developed for simultaneously assessing bone

cell activities with two labeled compounds. A suitable double

marker technique for labeling animals immediately after return

from space would be advantageous in future missions for studying

reinitiation of normal bone modeling/remodeling.

PUBLICATION

Roberts, W.E., Smith, R.K., and Cohen, J.A. Change in Electrical

Potential Within Periodontal Ligament of a Tooth Subjected to

Osteogenic Loading. In: Factors and Mechanisms Influencing Bone

Growth (ed. by A.D. Dixon and B.G. Sarnat). New York: Alan R.

[iss p. 527-534, 1982.

53
MAMMALIAN GRAVITY RECEPTORS: STRUCTURE AND METABOLISM

M.D. Ross

Department of Anatomy

University of Michigan School of Medicine

Ann Arbor, MI 48109

SUMMARY

The long-term goal of this research is to shed light on

mechanisms underlying calcium metabolism by gravity receptors.

This will in turn lead to a better understanding of the otoconial

complexes they contain.

Otoconial complexes consist of tiny, microscopic-size crystals of

a polymorph of calcium carbonate (calcite in mammals) together

with a small complement of organic material. Organic material

also binds the crystals together and to the underlying receptor

area. The crystals are called "otoconia" (literally, "ear dust")

and the organic substance connecting them to the receptor sites

in the saccule and the utricle of the inner ear is the otoconial

membrane.

Organic material is continuous from the otolithic membrane to the

inside of the crystals, but it is unclear whether the two

materials, inside and outside the otoconia, are identical in

chemical composition. Embryologically, the organic material is

incorporated into the crystals before a well-defined otoconial

membrane is evident.

It has been accepted for many years that the otoconia, by virtue

of the mineral they contain, add mass to the otoconial membrane,

making the sensory cells of the receptors more sensitive to

linear acceleration. It is thought that an inertial force acting

upon the more dense crystals changes their position relative to

the sensory cells as acceleration is applied. Research on this

project, however, has begun to cast doubt on this simplistic explanation of otoconial function, as the following points should

help illustrate:

1. Otoconial complexes take up and release calcium ions within

relatively short periods of time. Uptake is nearly complete within 1 hour, which compares favorably with uptake in bone

mineral; while release is nearly complete within 4 days, which is

much faster than in bone. Thus, otoconial complexes are dynamic,

not static, structures. The speed of uptake and release is too

fast to be accounted for on the basis of new calcite formation by

ordi nary mechani sms of crystal growth, impl i cati ng the organi c

material of the complexes in short-term ion uptake and release.

2. Fetal studies in the rat have shown that the organic and

54
mineral phases are closely aligned during otoconial crystal

growth and that multiple seeding sites are present. Coalescence

of the crystallites and organic material into adult cryst~l form

takes place later. These findings suggest that the organlc material serves as seeding material for the crystallites and

guides their further growth in a relatively strict pattern; it

may also be responsible for limiting their growth.

3. Micro-disc gel electrophoresis studies of the proteins of the

complexes revealed that a protein with the molecular weight of

16,000 to 17,000 daltons is present and is extractable with EDTA.

Calmodulin, a calcium-regulating protein present in both plant

and animal cells, has a molecular weight in this same range (15,000 to 19,000 daltons, depending upon experimental conditions) and is similarly EDTA extractable. It is possible that calmodulin is a component of otoconial complexes even though

the latter are extracellular constituents of gravity receptors. The surrounding fluid in the compartment in which the otoconia

function is high in potassium ions and low in sodium ions,

mimicking intracellular ionic conditions.

4. Ultrahigh resolution studies of the otoconia demonstrated

that the otoconia are not single crystal in nature, as was

previously thought. Instead, they are highly ordered composites

of organic and inorganic materials, with the inorganic material

consisting of crystallites that are not comparable in appearance

to those of pure calcite. This again suggests that the mineral

is seeded and ordered by the organic phase. Moreover, the

morphology of the crystallites is not symmetrical. This lack of

symmetry as well as the ordering of the crystallites in layers strongly suggests that the otoconia are piezoelectric. Thus, the

crystals may function to alter the electric field around them

during linear acceleration and, if so, need not move at allover

the underlying receptor area to stimulate the sensory cells. It

is also possible that the organic phase is a piezoelectric, or

semiconducting material, contributing to the piezoelectric effect

or being solely responsible for it. It is strongly suspected, for example, that it is the organic component in bone mineral

that is responsible for the piezoelectric effect in that

biomineralized material.

The short-term goal of this study is to carry out amino acid

analysis of the organic phase of the otoconial complexes. This

project was chosen as the first goal, because a body of

literature already exists concerning the amino acid composition of other related substances, namely, calcite-containing shells

and otoliths of fish. Fish otoliths consist largely of a

different polymorph of calcium carbonate, aragonite, but are

homologous with the otoconia of other vertebrate gravity

receptors. Additionally, we are dealing with a very small mass.

A pair pf saccular otoconial complexes weighs about 9.5

micrograms while a pair of utricular complexes weighs about 14.5

55
mi crograms. Thi s i ncl udes the mi neral! It seemed more rewardi ng

to attempt amino acid analysis first and to accomplish protein separation and amino acid analysis of each protein later. This

is because analysis of each protein represents a step into a more

difficult dimension quantitatively. Moreover, methods for amino

acid analysis would have been perfected and the size of the

sample necessary for such analysis would have been determined

prior to this step. Sugar analysis will also be required, because some sugars are important in calcium binding in other

systems. Again, the amounts of material required for sugar

analysis can be estimated based upon the amino acid analyses.

Because of the level of sensitivity required, high pressure liquid chromatography (HPLC) was selected for this phase of work,

and postreaction with a chemical (ortho-phthalaldehyde, OPAl that

makes the amino acids fluoresce has been utilized. A

fluorescence detector then determines the amount of fluorescence

given off by each amino acid component. The method is sensitive

in the low picomolar range (10 or fewer picomoles for each amino

ac i d) •

The project l s HPLC equipment was put in place fall 1982.

Achievements since that time are as follows:

1. Organic material of otoconial complexes can be analyzed by

HPLC in spite of the small mass, using a relatively small sample size. This is, perhaps, the most significant achievement.

2. This research has shown that quantitative and qualitative amino acid analysis is possible using a sample pooled from eight animals. Moreover, this sample apparently could be halved.

3. Amino acid profiles of th~ organic phase of the otoconia have

been produced and procedures are under refinement.

4. Subsequent studies of the organic material utilizing alkaline

hydrolysis for detection of gamma-carboxyglutamate, an amino acid

found in other calcium-sequestering systems such as bone and

thought to be important in inhibition of mineral deposition, showed the amino acid to be present in otoconial complexes.

(Gamma-carboxyglutamate does not survive acid hydrolysis.) This

work also demonstrated that hydroxyproline was present, making the otoconial complexes more comparable to otoliths than to

shells in composition.

PUBLICATIONS

Kerr, T.P., Ross, M.D., and Ernst, S.A. Cellular Localization of

Na+, K+-ATPase in the Mammalian Cochlear Duct: Significance for

Cochlear Fl uid Balance. American ,Journal of Otolaryngology 3:

332-338, 1982.

56
Parker, S.B., Mann, S., Ross, M.D., Skarnulis, A.J., and

Williams, R.J.P. A High Resolution Electron Microscopic Study of

the Balance Organs of the Inner Ear. In: Biomineralization and

Biological Metal Accumulation (ed. by P. Westbroek and E.W.

DeJong). Dordrecht, Holland: D. Reidel, 1982.

Rarey, K.E. and Ross, M.D. A Survey of the Effects of Loop Diuretics on the Zonulae Occludentes of the Perilymph-Endolymph Barrier by Freeze Fracture. Acta Otolaryngologica 94: 307-316,

1982.

Ross, M.D. Gravity and the Cells of Gravity Receptors

(Abstract). In: Abstracts, 24th COSPAR Meeting, Ottawa, Canada,

May 16-June 2, 1982. Paris: CaSPAR, p. 536, 1982.

Ross, M.D. Striated Organelles in Hair Cells of Rat Inner Ear

Maculas: Description and Implication for Transduction.

Physiologist 25(6, Suppl.): S113-S114, 1982.

Ross, M.D., Ernst, S.A., and Kerr, T.P. Possible Functional

Roles of Na+, K+-ATPase in the Inner Ear and Their Relevance to

Men i ere ISOi sea s e . Ame ric a n J 0 urn a1 0 f 0 to 1a ry ngo logy 3:

353-360, 1982.

57
GROUND-BASED MODELS FOR HYPOKINESIA

David J. Simmons

Department of Surgery Division Orthopedic Surgery Washington University School of Medicine

St. Louis, MO 63110

SUMMARY

In order to understand the effects of spaceflight on the

weight-bearing and non-weight-bearing skeletons, an animal model

system that at Earth's gravity produces changes in bone structure

similar to those recorded after exposure to O-G conditions was

employed.

The effect of simulated spaceflight on the growth and maturation

of the non-weight-bearing skeletons was compared in young (41-day) and old (i-year) rats. The animals were supported in a

head-down tilt mode for 15 days, after which the lower jaws wer~

saved and prepared for analyses of the distributions of calcium,

phosphorus, and hydroxyproline (= bone .collagen matrix). The

jaws were subsequently ground into 40 ~m particles and these were

separated into four fractions according to their specific gravity. The fractions represent a maturation gradient for bone

mineral and matrix, such that the bone packets with specific gravity of 1.3 to 1.7 are very immature, and those with a

specific gravity of 2.2 to 2.8 are the most mature. The animals

also had received injections of tetracycline to mark the growing

surfaces of their lower incisors and supporting jaw bone.

At either age, the support system failed to alter total jaw calcium, phosphorus, and hydroxyproline concentrations, or their

profiles within the density gradient system in both the younger and older rats. The rates of bone growth and growth of dentin

(16 to 17 ~m/day) in the incisors remained at control levels.

The results of this study did not mirror the effect of actual

spaceflight on the maturation of mineral and matrix moieties in

the rat jaws. For the supported rat model to produce changes which faithfully mimic all the effects of spaceflight in rats,

the skeletal response should include evidence of a delay in the

maturation of bone mineral and matrix moieties. This would be

seen as a shift in the concentrations of calcium, phosphorus, and

hydroxyproline toward the lower specific gravity fractions.

How should one try to account for the inability of this

land-based rat model to produce spaceflight-specific effects on

mandibular bone maturation? The most appropriate answer to this

question would seem to be that the head-down tilt model does not

interfere with the continued normal functioning of the jaw·s

58
antigravity muscles. Unlike the situation during spaceflight, the land-based experiments do not seem to moderate these normal

biomechanical-structural interrelationships which are so

essential to the normal pace of bone growth and remodeling.

PUBLICATIONS

Simmons, D.J., Grazman, B., Chang, S.-L., Russell, J.E., Smith,

K.C., Nussbaum, N., and Oloff, C.M. Some Skeletal Effects of

Land-based Models for Hypogravity in the Rhesus Monkey Mandible:

Effects of Chronic Tetracycline Treatment vs Postcranial

Immobilization (Abstract). Physiologist 25T4): 302,1982.

Simmons, D.J., Russell, J.E., Winter, F., Tran Van, P., Vignery, A., Baron, R., Ros,enberg, G.D., ,and Walker, W.V. Effect of

Spaceflight on the Non-weight-bearing Bones of Rat Skeleton.

Ame ric an J 0 urn a1 0 f Physi 0 logy 244: R319-R326, 1983.

59
EFFECTS OF MUSCLE ATROPHY ON MOTOR CONTROL

Douglas G. Stuart

Arizona Health Sciences Center

University of Arizona

Tucson, AZ 85724

SUMMARY

As a consequence of U.S. and Soviet space program experiences, it

is apparent that weightlessness reduces both the cardiovascular

and muscular capabilities of astronauts and cosmonauts. Of these

post-flight-observed weightlessness-induced responses, this

project has focused on (1) the effects on muscle, and (2) the

implications of these changes for the control of movement. While

numerous investigators have noted the loss in whole-muscle weight and functional capability that accompanies weightlessness, little

attention has been directed toward the effects upon the

constituent units of muscle. Since muscle does not comprise homogeneous units, it is unlikely that whole-muscle changes

represent uniform effects experienced by all the units. To

extrapolate these effects from one muscle to another and to

elucidate the mechanisms involved in atrophy, it is necessary to

characterize the differential effects at the unit level.

Muscle represents but one component of the system responsible for

movement production, the others being a neural element to command

the movement and a sensory element(s} to provide information to

the nervous system on the execution of the movement.

Accumulating evidence suggests that should the integrity of one

component be altered the ot~ers will be similarly influenced.

Thus, the second objective of this project addresses the effect

of muscle atrophy on these neural and sensory elements.

The experimental paradigm involves a suspension model which has

been demonstrated to induce changes that parallel the responses of space-flight-subjected animals. In this protocol, the

hindlimbs of rats are supported above the ground by a harness for

up to 16 weeks. The animals are able to move about the cage by using their forelimbs. Following the support, measurements are

made on some of the muscles and associated neural and sensory

elements which contribute to movement of the foot.

The intended strategy for the research project was to quantify

the extent of support-induced atrophy before beginning an

analysis of the differential effect of the atrophy on the muscle

units. This was to be accomplished by comparing the whole-muscle

physiologic status (e.g., speed and force of muscle contraction,

fatigability) of control to supported animals. However, after

several experiments it became apparent that the neuromuscular

connection in the experimental hindlimb muscle (medial

gastrocnemius) was less than anticipated prior to support. For

example, a standardized fatigue test produced more substantial

60
reductions in force and neural input (EMG; 79 and 66%,

respectively) than we expected based on theoretical calculations

(62 and 23%, respectively). The evidence suggested an impairment of the process that couples the muscle-directed neural input to

the force produced by the muscle.

As a result of this finding, the initial phase of the project has

been redirected to examine the fatigue profiles of muscles in

exercise-deprived (small cage-reared), exercise-exposed

(near-free environment-reared), and wild rats. The working hypothesis is that the cage-reared rat is inappropriate for the

study of the weightless state whereas a rat of the same species

reared in a near-free environment is as suitable as a wild rat.

Upon conclusion of these experiments, the investigators will

return to the initial goals of the project with the acceptable

animal model.

PUBLICATION

Stuart, D.G. and Enoka, R.M. Weightlessness Hypokinesia: Significance of Motor Unit Studies. Physiologist 25(6, Suppl.): S157-S158, 1982.

61
INFLUENCE OF SUSPENSION-HYPOKINESIS ON SKELETAL MUSCLE

G.H. Templeton and J.L. Sutko

Department of Physiology University of Texas Health Science Center

5323 Harry Hines Blvd.

Dallas, TX 75235

SUMMARY

Upon return from space, both humans and experimental animals

exhibit a number of changes, particularly in skeletal muscle,

which shows marked atrophy and altered functional and

bioenergetic characteristics. The goal of this project is to

identify the physiological, histochemical, and biochemical

parameters of skeletal muscle which are most sensitive to zero

gravity and best characterize the influence of zero gravity on

muscle.

To accomplish these goals, this investigation is currently

evaluating the Morey-Holton rat model for weightlessness and h~s

characterized rats supported for up to 4 weeks and rats that have

recovered for 1 week after support for 2 weeks.

The most significant accomplishment during 1982 has been the

demonstration that the Morey-Holton model evokes speeding of the

soleus, an antigravity muscle in the rat. Speeding refers to a

decline in times required for the muscle to contract (contraction

time) and relax (one-half relaxation time). The histochemical

and biochemical investigations of the soleus have also provided a

possible explanation for this soleus speeding.

The soleus is normally composed of from 70 to 90% Type I

(slow-twitch) muscle fibers; the remaining fibers are Type II

(fast-twitch). Measuring the relative amounts of Type I and II

myosin in the soleus, rat suspension was found to cause a

significant decline in Type I myosin without changing the Type II

component. In agreement, the histochemical investigation, which

used ATP-ase staining to identify Type I and II fibers in the

soleus, found a change in the normal distribution of fiber types during rat suspension from the normal Type I predominance toward

a greater perc~ntage of Type II fibers.

The other histochemical data indicate that rat suspension elicits

changes in the soleus muscle that are characteristic of

denervation. These changes include esterase positive fibers,

which are indicative of increased enzymatic activity, and pale

centers or moth-eaten fibers on nicotinamide-adenine-dinucleotide

phosphate staining, which indicate irregular mitochondrial

distribution.

Recovery from rat suspension has been studied by supporting rats

for 2 weeks and allowing recovery for 1 week. The soleus muscles

62
from these rats displayed a return toward control values in their

contractile parameters and their Type I myosin concentration. In

contrast, their histopathic changes characteristic of denervation

persisted after the 1 week recovery period.

Comparing this project's data from suspended rats with that of

space-traveled rats indicates that the Morey-Holton support model

is worthy of consideration as a weightlessness model. Although

only limited data from space-traveled rats is available for

direct comparison with these results, similarities between the

data do exist. These similarities include decreased muscle

function and speeding of the soleus.

PUBLICATIONS

Templeton, G.H., Manton, J., Silver, P., Glasburg, M., and Sutko,

J. Influence of Suspension-hypokinesia on Rat Skeletal Muscle

(Abstract). Physiologist 25(4): 303,1982.

Templeton, G.H., Padalino, M., Glasberg, M., Manton, J., Silver,

P., and Sutko, J. Evaluation of the Response of Rat Skeletal

Muscle to a Model of Weightlessness. Physiologist 25(6, Suppl~):

S153-S154, 1982.

63
SKELETAL MUSCLE METABOLISM IN HYPOKINETIC RATS

Marc E. Tischler

Department of Biochemistry Arizona Health Sciences Center

University of Arizona

Tucson, AZ 85724

SUMMARY

Weightlessness abolishes weight-bearing requirements for skeletal

muscles in the legs of animals. In both astronauts and rats,

this condition leads to loss of muscle mass; i.e., atrophy of the

muscle ensues. Although it is known that this loss of muscle can

be attributed to the disappearance of protein, the principal

component of muscle, little is known about the causes for this

loss and whether there are attendant biochemical alterations as

well. Therefore, this project is using a suspension hypokinesia model in rats, which induces leg muscle atrophy, to understand

better the biochemical adaptations of muscle to non-weightbearing conditions. Futhermore, using this model, this lab hopes

to develop a means for preventing the marked atrophy of certain

leg muscles while maintaining normal muscle function.

A suspension-hypokinesia model has been used for studying the

effects of reduced weight-bearing on the metabolism of skeletal

muscle in the rat hindlimb. The rats are supported from the base

of their tails to make the hindlimbs non-weight-bearing. Two

different types of control animals are used for comparison with

the experimental hypokinetic (supported non-weight-bearing)

animals. One group is kept normally with one animal per cage (nonsupported). The other control group has their tails attached

to the support apparatus but are permitted to walk on their

hindlimbs so they are weight-bearing.

Research during 1982 has focused on two main questions .. The

first considered what other alterations in the biochemistry of

the muscle might accompany the loss of muscle protein. This lab

showed previously that the formation of protein decreases and the

destruction of protein increases in certain leg muscles of the

hypokinetic rats. This response leads to a greater availability

in the muscle of amino acids, which are the basic units of

protein. Therefore, the study of the fates of these excess amino

acids in muscle was an important consideration. Two of these

amino acids, alanine and glutamine, are of particUlar import because they provide a vehicle for removing from the muscle

nitrogenous waste which is generated by the destruction of other

amino acids.

Since soleus muscle undergoing protein loss in hypokinesia is

likely to have excess nitrogen waste, a marked increase in the

output of alanine and glutamine was expected. Instead, alanine

output was unchanged and the output of glutamine was diminished

64
markedly. Normally the output of glutamine in muscle is

controlled by glucocorticoid hormones produced in the adrenal

glands. However, even after removal of these glands from control

and hypokinetic animals a large difference was still observed.

Hence, in muscle of the hypokinetic animals there is an impaired ability to manufacture glutamine. This observation is important

because it suggests that such muscles will have difficulty removing nitrogen waste materials, which can in turn lead to

impairment of certain aspects of muscle function.

The second question addressed was whether there is a way to

prevent the marked atrophy of the soleus muscle and its attendant

lost capacity for producing glutamine. It is well-known that

constant passive stretch of a muscle can increase muscle mass;

i.e., cause hypertrophy. Therefore, this lab tested whether

passive stretch of one soleus muscle in the hypokinetic rat would

show a diminution of the atrophy response. The results were most

encouraging! The soleus muscle in the leg stretched passively

during 6 days of hypokinesia showed not only no loss of mass but

amazingly increased in size to a greater extent than did this

muscle in normal rats. Furthermore, the passively stretched

muscle seemed to show a more normal capacity to produce glutamine. Hence in hypokinesia, simple passive stretching seems

to maintain muscle size and perhaps even normal muscle function.

Such results suggest that passive stretch may be a useful

approach for preventing muscle atrophy and loss of muscle

function in astronauts.

PUBLICATIONS

Jaspers, S. and Tischler, M.E. Correlation of Quantity and the

Metabolism of Protein in Hindlimb Muscles of Hypokinetic Rats

(Abstract). Federation Proceedings 41: 867, 1982.

Tischler, M.E. and Jaspers, S. Synthesis of Amino Acids in

Weight Bearing and Non-weight Bearing Leg Muscles of Suspended Rats. Physiologist 25(6, Suppl.): S155-S156, 1982.

65
EFFECTS OF SIMULATED WEIGHTLESSNESS ON MEIOSIS, FERTILIZATION,

AND EARLY DEVELOPMENT OF MICE

Debra Wolgemuth College of Physicians and Surgeons

Col umbi a Un i ve r si ty

New York, NY 10032

SUMMARY

This is a new project. The long-range goal of this research

effort is to examine the effects of one aspect of spaceflight, specifically, an altered gravitational field, on critical stages

of reproduction in animals. The aspects which will be examined

over the next 3 years include (1) the reduction divisions

involving the female germ cells' genetic information that occur

during the process of meiosis; (2) the fertilization process

wherein the sperm enters the egg and sets the stage for embryonic development; and (3) early embryonic development up to the

eight-cell stage.

The laboratory mouse is used as a model system since the cellular

biology and genetics of the mouse are the best understood of

possible animal models. Altered gravitational conditions are

achieved using a clinostat, an instrument that simulates a zero

gravitational state for organisms, or as in the present case, for

cells or groups of cells in an in vitro culture system. All

experiments utilize in vitro cuTture conditions in order to

separate direct effects on the cells being studied from indirect

effects that altered gravitational states could have via maternal

factors. It is anticipated that these studies will provide important new information on the effects of altered gravitational

fields on basic biological processes such as cell division,

proper segregation of the chromosomes that carry the genetic information, and recognition of one cell by another during

fertilization and early embryonic development. Such information

will be extremely useful in predicting the effects of both 10ng- and short-term spaceflight on animal reproduction.

In the few months since this project was initiated, the following have been accomplished:

1. Adaption of Standard Tissue Culture Conditions for Use in the

C1inostat: In this lab's standard laboratory conditions, oocytes

are recovered under sterile conditions from the ovaries of mature

mice and placed in a petri dish with the appropriate medium to

support the metabolic functioning of the cells during the

reduction (meiotic) division. Tbe cultures are loosely covered

and placed in an incubator at 37 C and 5~ CO in air for 16

hours. It was necessary to alter this proced~re so that the

cultures might be attached to the c1inostat and rotated while

still permitting gas exchange and equilibration with the culture.

This required assessing several different types of culture

66
dishes, various volumes of media, and various overlaying substances to prevent leakage. The investigators'believe that

they have found an appropriate culture system using Falcon No.

3072 microtitre plates, 0.4 ml of McCoy's Sa culture medium, and

a 20-lambda overlay of gas-equilibrated paraffin oil. Under

these conditions, the oocytes can progress to the second meiotic

metaphase, which is the endpoint routinely achieved under our

standard conditions.

2. Construction of a Clinostat for Use in Culturing Mammalian

Oocytes: The clinostat constructed is modeled after that

developed by J. Tremor and K. Souza for use in their studies on

amphibian eggs and development (Space Life Sciences 3: 179-191,

1972.) The single greatest difference is that, as mentioned

above, this lab's studies require an enviranment of 5% CO 2 in air

and a humidified ambient temperature of 37 C. This has Deen

achieved by constructing a clinostat that will fit into a Forma

Model No. 3325 incubator in which temperature, gaseous

environment, and humidity can be regulated. The initial

construction uses a motor that produces rotation of 1 revolution

per minute of the culture dish containing the eggs. Additional

motors, with a range of rotational speeds, will now be examined.

The clinostat is designed to permit easy exchange of motors, to

minimize vibration of the rotating shaft, and to permit rotation

of the cultures in horizontal and vertical planes.

These studies are currently focusing on determining the effects

of various rotation conditions on the meiotic process and will

include observations on cellular morphology.

67

PLANT PROJECTS

69

LOCALIZE AND IDENTIFY GRAVITY SENSING MECHANISMS OF PLANTS

Robert S. Bandurski

Department of Botany and Plant Pathology

Michigan State University East Lansing, MI 48824

SUMMARY

Plant stems grow up and plant roots grow down. If the plant is

displaced from its vertical orientation to the earth1s surface,

it responds by growing back into a vertical position. The plant accomplishes this response to gravity by altering the relative

amounts of growth hormone, indole-3-acetic acid (IAA), on the

upper and lower si·des of the stem. How the plant alters IAA

concentration in the tissue and how quickly this asymmetry is

accomplished is unknown.

This laboratory, in cooperation with that of Peter Kaufman,

studied the length of time required for the plant to begin its

growth response to gravity and the length of time required for a

change in the relative amounts of IAA in the upper and lower

sides of the stem. The results of this study are shown in the

figure below:

x-x-----x

50*/

40

.... 30

I

.c

(I)

...

j

.....

co 20 >...

j

u

0

10

o 0.25 o.s 1.0 1.5

time (h)

71
As can be seen, from the solid line, the plant begins curving back into a vertical position in about 5 to 10 minutes after

being placed in a horizontal position and reaches its maximum

rate of straightening in something more than 1 hgur. In 1.5

hours it is straightening itself at a rate of 50 per hour--

almost 10 per minute. The change in distribution of the hormone,

shown by the line with Xs, is far more rapid in that in 0.25

hours, 55% of the IAA is on the lower, more rapidly growing, side

of the stem. Clearly, the maximum change in hormone

concentration precedes the attainment of the maximum rate of

straightening. Thus, the change in hormone concentration could

be the cause for the change in growth rate. However, other

factors must also be involved since the growth response and the

change in hormone concentration do not coincide exactly in time.

The above-described studies are continuing and are being extended

to measure other forms of the growth hormone--IAA forms in which

the IAA is conjugated to a sugar. From these studies it is

expected to learn how a plant can alter its growth rate in

response to gravity and al so how, in general, pl ants can control

and alter their growth rate.

PUBLICATIONS

Bandurski, R.S. Auxin Biosynthesis and Metabolism. In: Plant

Growth Substances 1982. New York: Academic Press, p. 3-11, 1982.

Bandurski, R.S. Gravitational Effects on Pl ant Growth Hormone

Concentration (Abstract). In: Abstracts, 24th COSPAR Meeting,

Ottawa, Canada, May 16-June 2, 1982. Paris: COSPAR, p. 533,

1982.

Ban durski, R. S. Met abo1ism 0 f My 0 - i nos ito1 Este r s 0 f

Indole-3-acetic Acid (Abstract)-.--Federation Proceedings 41: 865,

1982.

Bandurski, R.S. and Schulze, A. Gravitationally Induced

Assmmetry in the Distribution of Indole-3-acetic Acid (Abstract).

Physiologist 25(4): 233, 1982.

Chisnell, J.R. and Bandurski, R.S. Isolation and Characterization of Indol-3-yl-acetyl-myo-inositol from Vegetative Tissue

of Zea mays (Abstract). Plantl'"hysiology 69(4 Suppl.): 55,1982. --- --- Cohen, J.D. Identification and Quantitative Analysis of

Indole-3-acetyl-L-aspartate from Seeds of Glycine max L. Plant

Phy s i 01 ogy 70: 749 -7 53, 1982. -

Cohen, J.D. and Bandurski, R.S. Chemistry and Physiology of the

Bound Auxins. Annual Review of Plant Physiology 33: 403-430, 1982.

72
Corcuera, L.J. and Bandurski, R.S. Biosynthesis of Indol-3-yl- acetyl-myo-inositol Arabinoside in Kennels of Zea mays L. Plant

Physiology 70: 1664-1666, 1982.

Corcuera, L.J., Michalczuk, L., and Bandurski, R.S. Enzymic

Synthesis of Indol-3-ylacetyl-myo-inositol Galactoside.

Biochemical Journal 207: 283-290, 1982.

Michalczuk, L. and Bandurski, R.S. Enzymic Synthesis of

1-0-indol-3-ylacetyl-beta-D-glucose and Indol-3-ylacetyl- myo-inositol. Biochemical Journal 207: 273-281, 1982.

Mishalczuk, L. and Chisnell, J.R3 Enzymatic Synthesis of

5- H-in~ole-3-acetic acid and 5- H-indole-3-acetyl-myo-inositol from 5- H-L-tryptophan. Journal of Labelled Compouncrs and

Radiopharmaceuticals 19: 121-12B, 19B2.

Pengelly, W.L., Hall, P.J., Schulze, A., and Bandurski, R.S.

Distribution of Free and Ester Indole-3-acetic Acid in the Cortex

and Stele of the Zea mays Mesocotyl. Plant Physiology 69:

1304-1307, 1982. -------

Reinecke, D.M. and Bandurski, R.S. Oxindole-3-acetic Acid, an

Indole-3-acetic Acid Catabolite in Zea mays. Plant Physiology

71: 211-213, 1983.

Reinecke, D.M. and Bandurski, R.S.

acetic Acid in Zea mays (Abstract). Suppl.): 55, 19~----

73

Quantitation of Oxindol-3-ylPlant Physiology 69(4,
PLANT MORPHOGENESIS AND PHYSIOLOGICAL BEHAVIOR IN RELATION TO

GRAVITY

A.H. Brown

Department of Biology University of Pennsylvania Philadelphia, PA 19104

SUMMARY

Gravity is one of the major environmental factors that influence

plant growth. Even so, the basic mechanisms that enable plants

to detect, measure quantitatively, and respond to perceived

gr avita t ion ali nform ation are not yet we 11 un de r stoo d . The

objectives of our studies are to learn how earth ls gravity and

other g forces affect plant growth processes, as well as to

better understand how plants grow.

A Helianthus Flight Experiment (HEFLEX), which is designed to

e1ucldate the contribution of gravity to the phenomenon of

circumnutation, is planned for inclusion on Spacelab-l. An

empirical verification test, the HEFLEX Bioengineering Test

(HBT), was carried out in preparation for this experiment. The

effect of possible alterations in moisture content conditions in

microgravity is of particular concern, since shoot height--which

shows a correlation with the amplitude and frequency of

nutational osci11ations--is markedly dependent on soil moisture

content. HBT was also a verification test of the hardware and

processes planned for HEFLEX.

HBT was the first test of plant growth on the Shuttle. An HBT

experiment that was carried out on STS-2 was repeated on STS-3

because the shortened 56-hour duration of STS-2 did not permit sufficient growth to occur. A 1 g control test, with a duration

and temperature profile simulating that of the STS-3 experiment,

was sUbsequently carried out.

The objectives of HBT were achieved. There was no significant

difference between the trend of the data of shoot height versus

soil moisture content, as measured on ST~-3 and on the ground.

Based on the results, the hardware and the 70% moisture content

chosen for HEFLEX are judged to be satisfactory. In 45% of the

flight seedlings, one or more roots were exposed above the soil

line. (The normal incidence of such exposed roots at 1 g is no

more than 1%). There were also a few instances of elevated

shoots in which the shoot was "driven" out of the soil as the

plant axis elongated. We have recently completed an analysis of

this elevated shoot anomaly.

Experiments to determine how quickly the imposition of simulated

weightlessness causes damping of circumnutation to minimal

amplitude and period have been completed. Another series of

74
tests, consisting of comparisons of different clinostat

configurations simulating specific g-force conditions, are

continuing. The goal is to learn whether the biological results

of clinostat simulations of particular g conditions are the same;

circumnutation is the test parameter.

Our plans include completing the above work. In addition,

measurements of the hyponastic response of horizontally clinostatted plants to a range of centrifuge-induced axial g

forces will be made. This should help determine the extent to

which flexing or flopping of stems contributes to

hyponasty/epinasty, since the clinostatted plants should

experience much the same flexing irrespective of the presence or

absence of the axial force. We also plan to measure the angular displacement of the shoot axis under a range of transverse g

loads. This is ~elated to the previous task; the application of

transverse g forces to a vertically oriented plant should shed

light on the question of sag of the plant axis that occurs during

horizontal clinostat rotation. We then plan to determine the

magnitude of plant "sag" to be expected at 1 g transverse

load--and to translate that information into the expected change

of leaf angle (and change in other parameters as well), based on

the results of the two experiments just described. Finally,

indirect support of the planned HEFLEX (SL-1), GTHRES (SL-4), and

FOTRAN (SL-4) flight experiments will continue. The plan of

GTHRES is to establish a gravitropic threshold through the

delivery of g-pulses, by means of centrifugation, during the

flight. FOTRAN will investigate the nature of phototropism in

microgravity.

PUBLICATION

Brown, A.H. and Chapman, O.K. The First Plants to Fly on

Shuttle. Physiologist 25(6): S5-S8, 1982.

75
AROMATIC BIOSYNTHESIS AND GRAVITY

Joe R. Cowles

Department of Biology

University of Houston

Houston, TX 77004

SUMMARY

Gravity is believed to be a primary controlling stimulus for

lignification. The approach that has been followed in our

investigations of the gravity-lignification relationship has been

to elucidate the regulation of aromatic biosynthesis related to

lignification in higher plants, to achieve an understanding of

the role of lignin and protein synthesis in plant orientation,

and to study the effect of near-weightlessness on lignification

in higher plants.

Lignin synthesis in young «10-day-old) pine seedlings responding

to geostimulation is not altered in the bend region. Synthesis is increased in the region of the stem below the bend zone,

however (this region supports most of the seedling mass, even in

a horizontal position). In our experiments, no lignin synthesis

gradient across this region has been detected. We have

demonstrated a difference in protein content between the upper

and lower side of the bend region as a result of geostimulation. No quantitative difference in one-dimensional electrophoresis has

been found. Another parameter that distinguishes geostimulation from photostimulation has been successfully separated; bending due to geostimulation is blocked by cyclohexamine whereas

photostimulation is not.

The principal accomplishment was the successful flight of a

carry-on plant experiment. This was achieved on STS-3 in March,

1982. The specific objectives of this experiment were to test

the functioning and effectiveness of a "mini" plant growth unit

(PGU), in terms of its ability to support plant growth in space;

to observe overall growth and development of young seedlings exposed to near-weightlessness; and to utilize the PGU to

investigate the effect of near-weightlessness on the synthesis of

lignin.

Thirty-two 4-day-old pine seedlings and 32 oat and 32 mung bean

seeds were the starting materials for the experiment. The seeds

and seedlings were planted in small chambers which were

subsequently inserted into the PGU. The PGU was loaded onto the

Shuttle 10 hours prior to launch; a second PGU was prepared as a

1 g control. After the 194-hour flight, the seedlings were

observed, measured, photographed, and analyzed.

76
The experiment demonstrated that the PGUs can be used to support

plant growth in space, and that plants can germinate and grow in

space (at least during the initial stages). The seedlings appeared healthy but the roots and stems of flight seedlings were

shorter than those of the ground controls. Some of the

difference, however, was likely due to differences between the

two PGUs themselves. Some disorientation was encountered: a

substantial number of oat and mung bean roots grew upwards and

several mung bean stems grew horizontally; these phenomena were

not observed in the ground controls. Neither light nor the water

source was adequate to orient all roots down or stems up.

The results of the lignin and other chemical analyses were

complicated by the seedling height differences. Expressed on a

per cm basis, lignin content of flight seedlings was some 4-8%

greater than for ground control seedlings. In pine and mung

bean, the differences in lignin content decreased in the upper

parts of the stems. There may have been a problem with data

expression, however. Baseline work has shown that shorter stems

generally contain more lignin on a per cm stem length basis, and,

as was pointed out, differences in the height of stems of flight

and control seedlings were found. It may be more accurate,

therefore, to express the data on a per stem basis. On a per

stem basis, total lignin content of flight stems of pine and mung

bean was about 4% and 25% less, respectively, than for controls.

The activities of two enzymes in the lignin biosynthetic pathway,

phenylalanine ammonia lyase (PAL) and peroxidase, were reduced in

space-flown pine seedlings, compared with the controls. PAL

activity in flight seedlings was reduced by 15% (per cm basis)

and 31% (per stem); peroxidase activity in flight seedlings was

1-2% (per cm) and 22% (per stem) less.

Our continuing studies in this area will focus on searching for

specific proteins that are associated with geostimulated bending in pine seedlings and on examining the selective inhibition of

cyclohexamide on different bending responses. The search for

proteins associated with bending will utlilize two-dimensional

electrophoresis. The cyclohexamide studies will involve

incorporation of radioactive precursors into end products.

Attempts will also be made to develop additional analytic tools

for the quantitation of early lignification in plant cell walls.

Among the analytical methods under consideration is a possible

monoclonal antibody-type probe.

PUBLICATIONS

Cowles, J.R., Scheld, H.W., Peterson, C., and LeMay, R. Response

of Young Plant Seedlings to Space Flight on STS-3. Physiologist 25(4): 284, 1982.

COWles, J.R., Scheld, H.W., Peterson, C., and LeMay, R.

Lignification in Young Plants Exposed to the Near-zero Gravity of

Space Flight. Physiologist 25(6, Suppl.): S129-S130, 1982.

77
THE ROLE OF ACID AND CALCIUM GRADIENTS IN GRAVITROPISM

Michael L. Evans

Department of Botany

Ohio State University

Columbus, OH 43210

SUMMARY

Research on this project is directed toward the understanding of

the influence of gravity (or lack of gravity) on plant growth--

in particular, the mechanism by which plant roots orient . themselves in a gravitational field and grow perpendicular to the

direction of gravity, a phenomenon known as gravitropism. During

1982 research was focused on several major questions: (1) What is

the role of the two plant growth hormones, auxin and abscisic

acid, in the response of roots to graVity? (2) What is the time

course of plant responses to gravity and what does this tell us

about the response mechanisms? and (3) What roles do secondary growth factors such as hydrogen ions and calcium play in the

response of roots to graVity?

In order to facilitate the study of these problems, three methods

of recording gravity effects on plant growth with high precision

were devised: (1) a highly sensitive "root auxanometer" which

allows a computer to record the straight growth of roots

continuously with resolution sufficient to detect growth over

a period of a few seconds, (2) a special plant growth chamber in

which color time-lapse movies of plant responses to gravity can

be made for frame-by-frame analysis, and (3) a computer-operated video camera which allows computer analysis of surface extension

in plant roots responding to gravity.

Using this specially developed equipment, substantial progress

was made in studying the three major questions posed above

(hormone involvement, growth kinetics, and roles of protons and

calcium). In a detailed study of the comparative effects of

auxin and abscisic acid on root growth, it was learned that the

effects of auxin are closely parallel to effects to be expected of a gravitropism-mediating hormone. The effects of abscisic

acid, by contrast, are not. This is a major findirtg since it

greatly reduces the number of viable models of gravitropism and

focuses research along more promising lines.

Using time-lapse cinematography and computer analysis of root

surface extension, it was learned that gravitropic curvature is

initiated within 15 minutes by enhanced growth on the top and

reduced growth on the bottom of the roots. This information is

crucial to an assessment of physiological changes in top and

bottom cells which might lead to coordination of root responses

to gravity.

78
Among the most striking results obtained in 1982 are ~he findings

that asymmetric gradients of protons and calcium ions parallel precisely the asymmetric growth patterns responsible for root

gravitropism. This research has shown a close correlation

between proton efflux and growth rates in growing root cells, and

has shown that this proton efflux is under the control of the

plant growth hormone, auxin. In gravity-stimulated roots there

is enhanced proton efflux on the rapidly growing upper cells,

indicating a close link between auxin redistribution, asymmetric

proton efflux, and gravitropic curvature. This is an especially significant finding, since it ties the action of the plant

hormone auxin to the apparent immediate cause of asymmetric

growth during gravitropism, i.e., asymmetric efflux of protons

from the roots.

In studies begun toward the end of 1982, it was learned that

gravitropismlike curvatures can be induced in roots without

gravity stimulation by simply applying a calcium gradlent at the

tip of the root. Additionally, it was found that calcium binding

agents applied to the root tip cause the root to lose its

sensitivity to gravity. This strongly suggests that internal

calcium gradients playa key role in either the detection of

gravity or the early response to gravity. This notion is also

supported by other recent studies of calcium and gravitropism.

This is perhaps the project's most important finding during the

past year. It provides, for the first time, a possible

physiological tip asymmetry which may mediate the action of

gravity on root growth. This lab can induce gravitropismlike

curvature without gravity stimulation, and this ability should be

invaluable in testing the involvement of the other physiological

parameters under study (especially auxin redistribution and

proton effl ux asymmetry) in the normal control of growth by gravity.

PUBLICATIONS

Evans, M.L. and Mulkey, T.J. Asymmetric Efflux of Protons as a

Mediator of Gravitropism in Roots (Abstract). Physiologist 25

(4): 234, 1982.

Evans, M.L. and Mulkey, T.J. Comparative Effects of Auxin and

Abscisic Acid on Growth, Hydrogen Ion Efflux and Gravitropism in

Primary Roots of Maize. In: Plant Growth Substances 1982. New

York: Springer-Verlag, p. 33-42, 1982.

Evans, M.L. and Mulkey, T.J. The Effect of ABA on Growth in Corn

Roots (Abstract). Plant Physiology 69(Suppl. 4): 76, 1982.

Evans, M.L. and Mulkey, T.J. A Reevaluation of the Role of

Abscisic Acid in Root Gravitropism. Physiologist 25(6, Suppl.): S109-S110, 1982.

79
Kuzmanoff, K.M. and Evans, M.L. A Calmodulin-like Protein in

Corn Roots: Evidence for Its Existence and Potential Involvement

in Root Growth (Abstract). Plant Physiology 69(Suppl. 4): 56,

1982.

Mulkey, T.J. and Evans, M.L. Suppression of Differential Acid

Efflux and Geotropism in Corn Roots Treated with Auxin Transport Inhibitors (Abstract). Plant Physiology 69(Suppl. 4): 55, 1982.

Mulkey, T.J., Evans, M.L., and Kuzmanoff, K.M. The Kinetics of

Abscisic Acid Action on Root Growth and Gravitropism. Planta

157: 150-157, 1983.

80
GEOTROPIC STIMULUS PRODUCTION IN ROOTS OF CORN

Lewis J. Feldman

Department of Botany University of California

Berkeley, CA 94720

SWMMARY

Plants orient their root and shoot axes with respect to the

gravitational field.

In roots the gravity stimulus is perceived in a specialized

tissue of the root called the root cap. It is hypothesized that

once gravity is p~rceived by the cells of the root cap, a series

of biochemical processes occur in the cap and ultimately lead to

downward growth in roots. This project is interested in studying

the biochemical processes that occur in the cap in the interval

of time between the perception of gravity and the downward growth of the root.

For this investigation seedling roots of corn were used. Such

roots are particularly suited for this work because if grown in

total darkness, roots of corn do not respond to gr~vity and fail

to reorient their axes with respect to the gravitational field.

A brief flash of light, however, initiates the bending response.

Using light as a trigger for the bending process, this lab has

begun to characterize those biochemical processes occurring in

the cap that are believed to be involved with translating the

perception of gravity into downward growth.

Protein synthesis is necessary for this translation process. If

protein synthesis is prevented in the caps, roots fail to bend

downward when illuminated. Thus far, work indicates that no new

proteins are formed as a result of the light treatment. Rather,

the results suggest that several preexisting proteins selectively increase in amount as a result of the light treatment. This

would imply that the effect of the light on proteins is

quantitative and not qualitative. So if proteins are involved in

the translation process we would at present conclude that the

gravity translation processes operate in part by selectively

enhancing the levels of specific proteins (enzymes?) without

affecting the types of proteins present. Currently this lab is

using a more sophisticated technique to confirm these preliminary

observations, and in particular, to examine the notion that small

amounts of new proteins may be produced as a result of the light and/or gravity stimulus.

PUBLICATIONS

Feldman, L.J. Formation and Partial Characterization of Growth

Inhibitors from Cultured and Intact Root Caps. Annals of Botany 50: 747-756, 1982.

81
Feldman, L.J. Protein Synthesis in Geostimulated Root Caps. Physiologist 25(6, Suppl.): S105-S106, 1982.

82
MECHANISMS OF GRAVIPERCEPTION AND TROPISTIC RESPONSE IN PEA

SEEDLINGS

Arthur W. Galston

Department of Biology Yale University

New Haven, CT 06511

SUMMARY

In order for a plant cell to sense gravity, it must have some

physical mechanism that can convert the energy of a displaced

organelle into some signal that will alter the direction of

growth. There is considerable evidence that especially dense

starch grains, contained in amyloplasts, serve this function in

higher plants. Such bodies had never before been isolated intact

and clearly identified by chemical markers. This project succeeded in this task in 1982.

Falling amyloplasts exist only in special gravisensitive cells,

called statocytes. Using cell-wall digesting enzymes, this lab

has succeeded in isolating such cells in a viable condition.

When such cells are mounted on a vertical microscope slide, their

amyloplasts fall repeatedly to the bottom of the cell as it is

rotated. Electrical and chemical studies are planned on these

isolated cells to determine the sequence of events following amyloplast displacement.

Samples of oat and mung bean seedlings flown on the third flight of the space shuttle in 1982 in the experiment of J. Cowles were

prepared and examined by electron microscopy, making it the first

detailed fine structure analysis of space-grown plant material.

No anatomical differences were found.

Plants in space are plants under stress. It is thus of interest

that the investigators have found a marked rise in the level of

putrescine, a common polyamine, shortly after application of

various stresses including dehydration, osmotic shock, low pH, high salt, and no potassium. In all cases, the extra putrescine

comes from extra, newly synthesized, arginine decarboxylase activity. Investigation of the relevance of this system to

gravitational and mechanical displacement is planned.

PUBLICATIONS

Britz, S.J. and Galston, A.W. Light-enhanced Perception of

Gravivy in Stems of Intact Pea Seedlings. Planta 154: 189-192,

1982.

Britz, S.J. and Galston, A.W. Physiology of Movements in Stems

of Seedling Pisum sativum L. cv. Alaska. I. Experimental

Separation of Nutation from Gravitropism. Plant Physiology 70:

264-271, 1982.

83
Britz. S.J. and Galston. A.W. Physiology of Movements in Stems

of Seedling Pisum sativum L. cv. Alaska. II. The Role of the

Apical Hook and of Auxin in Nutation. Plant Physiology 70:

1401-1404. 1982.

Britz. S.J. and Galston. A.W. Physiology of Movement in the

Stems of Seedling Pisum sativum L. cv. Alaska. III. Phototropism in Relation to Gravltropism. Nutation. and Growth. Plant

Phy s i 01 0 gy 71: 313 - 318. 1983.

Dai. Y.-R. and Galston. A.W. Kinetics of Arginine Decarboxylase Induction Following Gibberellin Treatment (Abstract). Plant

Phys i 01 0 gy . 69 (4. Sup pl. ): 83. 1982.

Flores. H.E. and Galston. A.W. Analysis of Polyamines in Higher

Plants by High Performance Liquid Chromatography. Plant

Phy s i 01 0 gy 69: 701 - 706. 1982.

Flores, H.E. and Galston. A.W. Effect of Osmotic Stress and

Gravistimulation on Polyamine Titer and Distribution (Abstract). Plant Physiology 69(4. Suppll: 57. 1982.

Flores, H.E. and Galston, A.W. Polyamines and Plant Stress:

Activation of Putrescine Biosynthesis by Osmotic Shock. Science

217: 1259-1261, 1982.

Fuhrer. J .• Kaur-Sawhney. R.• Shih. L.-M., and Galston. A.W.

Inhibition of Ethylene Biosynthesis and Senescence in Excised Oat

Leaves by 1.3-Diaminopropane and Spermidine (Abstract). Plant

Physiology 69(4. Suppl.): 137,1982.

Galston. A.W., Flores. H.E. and Kaur-Sawhney. R. Polyamine Formation by Arginine Decarboxylase as a Transducer of Hormonal

Environmental and Stress Stimuli in Higher Plants. Physiologist 25(6. Suppl.): S137-S138. 1982.

Galston. A.W. and Kaur-Sawhney. R. Polyamines: Are They A New

Class of Plant Growth Regulators? In: Plant Growth Substances

1982. New York: Academic Press. p. 451-461. 1982.

Gaynor. J.J. and Galston. A.W. Isolated Statocytes from

Etiolated Pea Epicotyls: A Model System for the Study of

Graviperception at a Cellular Level (Abstract). Physiologist 24

(4): 232. 1982.

Gaynor. J.J. and Galston. A.W. Purification and Characterization

of Amyloplasts from Etiolated Epicotyls of Pisum sativum

(Abstract). Plant Physiology 69(4. Suppl.): 99. 1982.

Shekhawat. N.S •• Stove. B.B. and Galston. A.W. Lipid Release

during Oat Leaf Protoplast Isolation (Abstract). Plant

Phy s i 01 0 gy 69 (4. Sup P1 ): 147. 1982.

84
Slocum, R.D. and Galston, A.W. A Comparative Study of Monocot

and Dicot Root Development in Normal (Earth) and Hypogravity (Space) Environments. Physiologist 25(6, Suppl): S131-S132,

1982.

Young, N.D. and Galston, A.W. Acid Stress Induces Accumulation

of Putrescine in Excised Oat Leaf Segments (Abstract). Plant

Physiology 69(4, Suppl.): 58, 1982.

85
THE ROLE OF GRAVITY IN REGULATION OF LEAF BLADE FORM

Alice Bourke Hayes Department of Natural Science

Loyola University of Chicago Chicago, IL 60611

SUMMARY

Major accomplishments in 1982 have been: (1) the identification

of gravity and auxin-sensitive cells in the leaf blade; (2)

observations on the role of ethylene, abscisic acid, and other

growth factors in regulation of leaf blade form; and (3)

characterization of the separable roles of the petiole, pulvinus,

and blade in leaf response to gravity.

This lab completed gross anatomical and histological studies and

identified specific cell layers in the blade (lower epidermis and

vascular bundle sheaths) whose expansion controls the form of the

leaf. Radioautographic studies have shown that labeled material

applied as the plant hormone indole-acetic acid, or auxin,

becomes distributed across the entire blade, consistent with this

lab's observation of response in the lower epidermis. Studies of

transport of labeled auxin have shown that there is a pronounced dorsi ventral gravipositive movement of labeled material but that

movement in the opposite direction is minimal.

Ethylene production by leaves given a variety of chemical,

physical, and environmental stimuli has been studied.

Quantitative analysis of ethylene production throughout the time

course of blade response to auxin shows that ethylene production follows blade hyponasty and precedes petiole and pulvinus

epinasty. Rotation on a clinostat promotes ethylene production and blade, petiole, and pulvinus epinasty. Treatments that

inhibit ethylene production (silver nitrate, aminoethoxyvinyl glycine) inhibit petiole-pulvinus epinasty but not blade

curvature. Treatments that do not promote ethylene production (2,3,5-triiodobenzoic acid, Na-N-1-naphthylphthalamic acid,

abscisic acid, gibberellic acid) do not promote leaf angle change. Ethylene appears to be required for leaf angle change (petiole-pulvinus epinasty), and auxin appears to be required for

blade curvature.

This project is now studying the hypothesis that the blade is the

part of the leaf that senses a change in gravitational orientation, and that the system regulating this is auxin

transport. The pulvinus is the part of the leaf that responds to

a change in gravitational orientation, and the system regulating this is ethylene synthesis. Auxin-induced ethylene synthesis is

the link between blade perception and leaf angle response to

gravity.

86
PUBLICATIONS

Hayes, A.B. Auxin Transport Inhibitors and Ethylene in Pinto

Bean Leef Epinasty (Abstract). Plant Physiology 72(1, Suppl.}:

106, 1983.

Hayes, A.B. Gravitropic Basis of Leaf Blade Nastic' Curvatures.

Physiologist 25(6, Suppl.): S133-S134, 1982.

Hayes, A.B. Nastic Curvature of Bean Leaf Blades and Petioles

(Abstract). Plant Physiology 69(4, Suppl.): 56, 1982.

87
EFFECTS OF GRAVITY AND LIGHT ON GROWTH, DEVELOPMENT, AND

FLOWERING IN PLANTS:. I. EFFECT OF THE CLINOSTAT ENVIRONMENT

Takashi Hoshizaki

Jet Propulsion Laboratory California Institute of Technology Pasadena, CA 91109

SUMMARY

A goal of this project is to grow plants in space weightlessness

for several successive generations. In doing so, we may better

understand how the growth, shape, size, and reproduction in

plants are influenced by the force of gravity and whether gravity

is essential for the survival of plants.

The most significant finding during 1982 on this project was that

mouse-earred cress plants, Arabidopsis tha1iana (L.) Heynh., were

successfully grown through one generation (seed to seed) on the

c1inostats. Abundant seeds were produced by plants cultured

axenically in tubes sealed by urethane foam or cotton plugs. The

viability of the seeds produced by plants growing on clinostats

is being tested. It is planned to test-grow plants on the

c1inostat for three successive generations.

In sealed cultures, mouse-earred cress failed to produce seeds in

all treatments. In horizontally clinostatted sealed cultures,

the roots grew into areas where moisture condensed on the tube

walls, and thereby hindered shoot growth.

Plants, as measured by plant height, grew faster in larger containers. These results indicate that the CO 2 supply may be a

critical factor in growth and floral development rate, as

evidenced by growth in containers having free atmospheric gas exchange. We plan to grow mouse-earred cress in sealed 5-ga110n

bottles since calculations indicate enough CO 2 is present in the

bottle to provide the carbon requirements for a mature plant with

seeds.

Three varietal strains were tested for seed-to-seed growth. All

three strains failed to set seeds in sealed culture tubes (25 x

200 mm) containing 20 ml of agar medium. Good seed set occurred

for all three strains in cultures provided ample gas exchange.

More leaves (15 to 18) and stems (3 or more) were produced by plants under sealed conditions than in open cultures (7 to 10

leaves and 1 or 2 stems). The multiple leaved and stemmed plants

failed to set seeds. The prime suspects, high moisture and low

CO 2 levels, will be investigated.

88
PUBLICATIONS

Hoshizaki, T. Effect of Clinostat and Cultural Conditions on the

Seed to Seed Growth of Arabidopsis and Cardamine: Initial Results

(Abstract). Physiologist 25(4): 284, 1982.

Hoshi zaki, T. Effect of Cul tural Condi ti ons on the Seed-to-Seed

Growth of Arabidopsis and Cardamine: A Study of Growth Rates and

Reproductive Development as Affected by Test Tube Seals.

Physiologist 25(6, Suppl.): S127-S128, 1982.

89
EFFECT OF GRAVITY AND WEIGHTLESSNESS ON THE DEVELOPMENT OF GIANT

COENOCYTE, CAULERPA

William P. Jacobs

Department of Biology Princeton University Princeton, NJ 08544

SUMMARY

The organism used--Caulerpa--is one of the largest cells known to

science. Its giant size makes it of particular interest in

following what happens inside a cell when gravity responses

occur.

Gravity effects on the regeneration of parts of Caulerpa were

studied. Segments of "blade" from two species were studied under

normal orientation to gravity and inverse orientation. In

contrast to earlier findings on the site of rhizoid

differentiation on the inverted rhizome, gravity had no effect on

the location or number of regenerated organs on these "blade"

pieces. Segments of rhizome, however, failed to exhibit a strict

polarity of regeneration with respect to gravity. Hence, gravity

acts differently or not at all on the different "organs" of this

giant cell.

Investigations as to what occurs inside the cell when there is a

gravity effect resulted in the discovery that, before gravity

causes a relocation of the site of rhizoid formation, it causes

microscopic organelles inside the cell to sediment to the lower

side of the inverted r~izome. These organelles are amyloplasts,

as revealed by histochemical staining and polarized light studies. Such sedimentation of amyloplasts occurs in just the

sites where rhizoids will later form, further confirming that

they are the cause of the relocation of rhizoid formation.

Based on the hypothesis that the amyloplasts act via hormones,

two known hormones from multicellular plants have been

investigated. Treatment of the organism with gibberellic acid

increased rhizome elongation and caused more frequent formation

of rhizoids, thus making this hormone a likely candidate for the

mediating hormone in rhizoid relocation under gravity.

The other hormone that was apt to be involved was indole-3-acetic

acid (IAA). This hormone was not believed to be present in

Caulerpa, from earlier work by others, but by applying improved technlques it was demonstrated by both bioassay and

electrochemical detection after High Power Liquid Chromatography (HPLC) that IAA was present and in sizable amounts. Its action

on Caulerpa is to stimulate "blade" growth, so it seems to act in

concert wlth gibberellic acid to coordinate the development of

the giant cell.

90
PUBLICATIONS

Jacobs, W.P. and Davis, W. Differential Effects of Gibberellic

Acid on Development of the Three Organs of the Marine, Giant

Coenocyte, Caulerpa prolifera (Abstract). Physiologist 25(4):

285, 1982.

Matilsky, M.B. Redistribution of Amyloplasts in Inverted

Caulerpa prolifera Rhizome Tips (Abstract). Physiologist 25(4):

233, 1982.

91
MECHANISM OF THIGMOMORPHOGENESIS IN PLANTS, AND ITS SIGNIFICANCE

TO RESEARCH WITH PLANTS IN SPACE

Mordecai J. Jaffe

Department of Botany Wake Forest University

Winston-Salem, NC 27109

SUMMARY

One of the most important accomplishments of 1982 was developing

the computer-assisted image processing instrumentation to the

point where it is on-line and being used in experiments. It has

taken three years to reach this point. This instrument uses a

video camera as the "eye". The image produced by the camera can

be enhanced by electronic means so that it is possible to see on

the video monitor features in a subject which cannot be otherwise

visualized. By linking the image electronically with a

microcomputer, and writing special programs for the computer, the

instrument will scan the image, generate data, and plot out

computations based on the data. For example, the camera can be

pointed at a plant stem bending upward during gravitropism, scan

the image at selected intervals, and the computer will

automatically draw graphs representing the changes in curvature

and length of the upper, lower and midline parts of the stem with

time. Using another program, it is possible to automatically

count, measure the size of and the average density of objects such as cells under a microscope, birds in a flock, or trees in a

forest. This instrument is currently being used in several

projects of interest to NASA.

This laboratory has previously shown that the plant hormone

ethylene is responsible for the thickening of plant stems that

have undergone mechanical stress or perturbation; current

evidence strongly suggests that when the plant is stressed, it

produces a compound called an "elicitor" which then induces the

production of ethylene. The elicitor has not yet been

identified, but it may be of considerable importance in plant

responses to changes in the environment.

When a plant is mechanically stressed or gravitationally

stimulated, it is found to produce large amounts of a special kind of cell wall material called callose. This callose, when

measured under the microscope with the computer-assisted video

processor, may also be involved in the stimulation of the

production of ethylene. At this stage, no one knows how this

happens, but this laboratory is planning experiments to shed

light on this important problem.

92
PUBLICATIONS

Erner, Y. and Jaffe, M.J. Thigmomorphogenesis: The Involvement

of Auxin and Abscisic Acid in Growth Retardation Due to

Me c han i cal Pert u r bation • P1ant and Cell P hy s i 0 logy 2 3 : 935 - 9 41 ,

1982.

Jaffe, M.J. The Interactions of Thigmic, Gravitic and Drought

Perturbations on Callose and Ethylene Production in Plants

(Abstract). Physiologist 25(4): 285, 1982.

J a f fe, M. J ., Hub e rman, M., Pre ssm an, E., and A10 ni, B. The Use

of Computer Image Analysis to Study the Inhibition of Pithiness

by Mechanical Perturbation or Ethrel (Abstract). Plant

P hy s i 0 logy 6 9 ( 4, Sup pl. ): 58 , 19 8 2 .

Riehl, LE. and Jaffe, M.J. Coiling of Pea Tendrils with Regard

to Light and the Deposition of Callose (Abstract). Plant

Physiology 69(4, Suppl.): 87, 1982.

Riehl, T.E. and Jaffe, M.J. Physiological Studies on Pea

Tendrils. XII. Effects of Temperature on Contact Coiling and

Uncoiling. Physiologia Plantarum 55: 187-191, 1982.

Ri e h1, T. E. and J a f fe, M. J • Phy s i 01 0 g i cal Stu die son Pea

Tendrils. XIII. Respiration is Necessary for Contact Coiling.

Physiologia Plantarum 55: 192-196, 1982.

Takihashi, H. and Jaffe, M.J. The Interaction of Callose and

Ethylene in Thigmomorphogenesis. Physiologist 25(6, Suppl.):

S139-S140, 1982.

Wakefield, A.H., Telewski, F.W., Jaffe, M.J. Darwin: A

Microcomputer-Based Digitized Video Analysis System (Abstract).

P 1ant Phy s i 01 0 gy 69 ( 4, Sup pl. ): 157, 1982.

93
INVESTIGATION OF THE NEGATIVE GEOTROPIC RESPONSE IN SHOOTS OF THE

OAT PLANT (Avena sativa)

Peter B. Kaufman

Department of Cellular and Molecular Biology Division of Biological Sciences

University of Michigan Ann Arbor, MI 48109

SUMMARY

Since cereal grasses are our most important source of food, we

need to know more about the gravity responses of these plants. This knowledge is essential for growing cereal plants in outer

space as well as in improving their yield on this planet. The

most important gravitropic organ of cereal shoots is the

pulvinus. This organ keeps the plants erect and allows the

grains to develop well above ground level. Failure of this organ

would lead to grain losses of severe consequences. Knowledge gained from this study of the gravitropic response in the grass pulvinus will have equal applicability to other plant organs

involved in gravitropism.

The overall objective of this project is to shed light on how the

gravitropic stimulus is perceived by gravisensitive leaf-sheath

pulvini of grass shoots when they are subjected to a gravitropic

stimulus--especially the nature of the starch statolith-cell

membrane interaction that we have observed in this system.

A model has been formulated which explains in one simple equation

the individual response of every cell in an organ that is

responding to gravity. Important inferences from the model

include: (1) a fundamental distinction between the gravitropic

responses of multicellular and unicellular structures, (2)

cellular asymmetry as a basis for organ asymmetry, and (3)

difficulties in accepting lateral transport of hormone as a basis

for gravitropic response in plants.

During the perception phase of the gravitropic response in the

grass leaf-sheath pulvinis, it was found that the vacuolar

membrane (the tonoplast) interacts with the organelles which

sediment in response to gravistimulation, namely, the starch

statoliths. The tonoplast surrounds each starch statolith at the

bottom of each statocyte (statocyte = a cell containing statoliths). This is the first direct proof for starch statolith

interaction with a cellular component, here, the tonoplast membrane.

It was demonstrated that in barley, the starch statoliths show

intense esterase activity when stained with fluoroscein

diacetate. Such esterase increases on the lower side of a

gravistimulated pulvinus in comparison with the upper side;

staining with naphthol As-D also confirms the increase in

esterase activity on the lower side of ~ravistimulated pulvini.

94
The cellulose synthesis inhibitor, DCBN (2,6-dichlorobenzonitrile), significantly blocks gravitropic curvature in

gravistimulated grass leaf-sheath pulvini, and at the same time,

causes large thickenings to appear in the radial walls of

pulvinus cells, first in the statenchyma, then in other cells,

and with increasing frequency as one progresses from top to

bottom of the gravistimulated pulvinus. Their distribution

pattern closely follows the equation that describes the geometry of asymmetric growth in the leaf-sheath pulvinus.

With the use of continuously recording transducers, it was shown

that corn seedling coleoptile responds to gravjstimulation within

15 minutes, and curves upward at the rate of 1 per minute until

the coleoptile is vertical at 120 minutes. In systems with

barley and oats, the pulvini start to curve upward within 20 to

30 minutes after they are gravistimulated, buS here, the rate of

curvature is much slower, on the order of 1.5 per hour for a

total period of about 48 hours.

Collections were made of tops and bottoms of gravistimulated and

lllefts" and "rights" of upright pulvini at different times during

the course of gravitropic curvature in oat and barley pulvini for

cell-wall analyses; these wall polysaccharide analyses (cellulose, pectins, hemicellulose) are currently in progress.

Also, collections of similar fr~cti02~ of corn at dif~orent times

after feeding the pulvini with H-GA (gibberellin A , labeled

with tritium) to follow the time-course changes in GA metabolites

were carried out; these analyses are currently in progress. Earlier studies of oats showed that GA conjugates were at their

highest levels in the upper halves, and the free active GAs (GA 3 , GA 4 , GA 7 ) were at their highest levels in the lower halves of gravist~mulated pulvini.

PUBLICATIONS

Dayanandan, P., Franklin, C.I., and Kaufman, P.B. Control of

Gravity-induced Cell Elongation in Leaf Sheath Pulvini of Barley and Oats (Abstract). Plant Physiology 69(4, Suppl.): 87, 1982.

Dayanandan, P., Franklin, C.l., and Kaufman, P.B. Linkage between Gravity Perception and Response in the Grass Leaf-sheath

Pulvinus. Physiologist 25(6, Suppl.): S101-S102, 1982.

Harrison, M.A. and Kaufman, P.B. Does Ethylene Playa Role in

the Release of Lateral Buds (Tillers) from Apical Dominance in

Oats? Plant Physiology 70: 811-814, 1982.

95
Kaufman, P.B., Dayanandan, P., and Meudt, W. Growth Promotion by Brassinosteroid in Sheath Pul vini of Grasses (Abstract). Plant

Phy s i 01 0 gy 69 ( 4, Sup pl. ): 1 2, 1982.

Kaufman, P.B., Dayanandan, P., Thomas, R.J., Taylor, J., and

Umberfield, R.J. Comparative Analysis of Rapid Growth Responses Using three Model Systems: Conocephalum Carpocephalum-stalk, Pell i a seta, and Avena Internode. Journal of the Hattori

Botanical Laboratory 51: 195-209, 1982.

Koning, R.E. The Roles of Auxin, Ethylene, and Acid Growth in

Filament Elongation in Gaillardia grandiflora (Asteraceae).

Am e ric an J 0 urn a1 0 f Bot any 70 : 60 2- 61 0, 198 3.

96
CELLS, EMBRYOS, AND DEVELOPMENT IN SPACE--MORPHOLOGY OF PLANT

CELLS IN SPACE

Abraham D. Krikorian

Department of Biochemistry Division of Biological Sciences

State University of New York at

Stony Brook

Stony Brook, NY 11794

SUMMARY

The ultimate overall objective of this research project is to

ascertain whether higher plants can carry out their full

development, repro~uction, and growth in near-O G despite the

fact that their evolution occurred under the asymmetric 1 G

conditions of Earth.

Experimental systems are being developed at different

developmental or organizational levels to determine what a

minimum gravitationally responsive organizational unit will be in

space. Cells which have had their cellulose walls removed

(protoplasts) and free cells which derive from nonsexual body

cells (that can nevertheless behave as if they were embryos derived from the fusion of sex cells) have been the subject of

our special attention, because these represent test systems that

can develop into entire plants even as they "bypass" the sexual

process. Simultaneously, investigations on the growing points or

zones of focused cell division and growth are made since these

represent a much higher degree of organization than that -of

isolated, free cells and protoplasts. Cell theory states that

cells are the units of life, but it has not been established

whether higher plant cells in isolation act as receptors of

gravity. Morphological comPTexity, as in a discrete growing shoot or root tip, may be required.

The hope is that by using test systems at different levels of

initial organization, but which are capable of attaining the most

advanced degrees or levels of higher plant shape and form under

closely controlled environments, research will determine whether

highly asymmetric growing regions of shoot and root are really

crucial to detectable degrees of responsiveness. Moreover, since

nutrition and metabolism of higher plants supports and are

mediated by highly asymmetric systems such as shoot and root, it

is considered critical to learn whether all these events and

subsequent physiological functions can be established and if they

can be established repetitively under hypogravity conditions and

can proceed virtually continuously in the near weightless state

to establish consecutive multiple generations. This will

disclose whether there is any remnant or a retention of a sort of

"memory" of the gravity experienced on Earth.

97
Significant progress has been made in initiating non-sex-cell

suspension cultures of a number of higher plants which have been

carefully selected because they represent different kinds of

growth habits. Each of these can go on and organize into

plantlets as they mimic the events of sexual embryo formation and

growth. Recent progress has not only been made in increasing the

speed with which the embryonic structures are induced, but in the

degree of precision with which they undergo development.

Since cloning of higher plants via cells and protoplasts presumes

genetic fidelity, we have recently developed methods that permit chromosome analysis at the cell and protoplast level with more

precision than has been possible before. This methodology will

help to establish decisively whether cells and protoplasts . exposed to various "insults" respond by visible changes in their

karyotype or chromosomal profile.

PUBLICATIONS

Krikorian, A.D. Cloning Higher Plants from Aseptically Cultured

Tissues and Cells. Biological Reviews 57: 151-218, 1982.

Krikorian, A.D. Coconut "Stones" or "Pearls": Early Descriptions

by Alzina, Kamel and Rumphius. Principes 26: 107-121, 1982.

Krikorian, A.D. Plant Cells, Embryos, Growth and Development in

Space (Abstract). Physiologist 25(40): 284,1982.

Krikorian, A.D. and O'Connor, S.A. Some Karyological Observations on Plants Grown in Space. Physiologist 25(6,

Suppl.): S125-S126, 1982.

Krikorian, A.D., Singh, M., and Quinn, C.E. Aseptic

Micropropagation of Cinchona: Prospects and Problems. In: Tissue

Culture of Economically Important Plants, Proceedings of the

International Symposium, National University of Slngapore,

Singapore, April 28-30, 1981 (ed. by A.N. Rao). Singapore: The

Committee on science and Technology in Developing Countries

(COSTED) and the Asian Network for Biological Sciences (ANBS), p.

167-174, 1982.

Nwankwo, B.A. and Krikorian, A.D. Aseptic Storage of Elaeis

guineensis Form pisifera Seeds. Principes 27(1): 34-37, 1983.

Nwankwo, B.A. and Krikorian, A.D. Water as a Storage Medium for

Elaeis guineensis (pisifera) Seeds Under Aseptic Conditions.

Annals of Botany 50: 793-798, 1982.

Singh, M. and Krikorian, A.D. Inhibition of Trypsin Activity in

vitro by Phytate. Journal of Agricultural and Food Chemistry 1U:

799-800, 1982.

98
THE AMYLOPLAST AS A GRAVITY-SENSING DEVICE IN PLANTS

A.C. Leopold

Boyce Thompson Institute

Cornell University

Ithaca, NY 14853

SUMMARY

This study concerns the early events in the plant's perception of

gravity. Gravity-sensing plant cells contain starch-filled

organelles (amyloplasts) which settle under the influence of

gravity. The movement of these organelles somehow results in the

establishment of a physiological gradient across the plant organ

which ultimately results in the curvature of the organ (e.g.,

root, stem, or coleoptile) toward or away from gravity.

It is not known how amyloplast movement causes this differential

growth. This laboratory has taken two approaches to uncover what

this connection may be. The first was to try to characterize the

surface properties of these organelles. Intact amyloplasts were

successfully isolated from gravisensitive tissues. Their

intactness was confirmed with the electron microscope.

This isolation enabled a determination of whether amyloplasts

possess a strong surface charge. Some workers have proposed that

if amyloplasts were electrically charged, their settling could

create an intracellular charge redistribution that may playa

role in gravity perception. To find out, amyloplasts were placed in a small glass cell and exposed to an electric field while

being observed under the light microscope. By measuring the rate

and direction of their movement, it was discovered that there is

a significant net negative surface charge on these organelles.

This lends some support to the charge redistribution hypothesis

although it does not directly answer the question of whether the

amyloplast charge plays a role in gravity sensing.

Using various techniques, including an ion microscope and a

fluorescent indicator (the antibiotic, chlorotetracycline), it

was also determined that amyloplasts contain a substantial amount

of calcium. Some of this calcium is bound to the charge shell

that surrounds the organelle. Since experiments from other

laboratories indicate that calcium may playa role in the plant's

response to gravity, it is possible that the settling of

amyloplasts redistributes calcium intracellularly, perhaps in a

manner useful in gravity perception.

A second approach to studying gravity perception involves the

examination of the response of living cells to gravity while they

are being observed with the light microscope. Very little is

known about what occurs in living cells when amyloplasts

99
sediment. Most of the information is derived from studies of

tissues fixed (preserved in a dead state) for light and electron

microscopy. This laboratory is studying the process of

sedimentation in living cells by cutting thin sections of plant

organs and observing the cells under a microscope that is

horizontally mounted. Rotation of the microscope stage results

in a changed position of the cells and their parts with respect

to gravity.

Many of the hypotheses purporting to explain the phenomenon of

plant gravity perception are based on studies of sedimentation

after the first few minutes. However, recently it has been

determined that it takes the plant less than a minute to perceive the new gravity stimulus. Thus, it becomes particularly important to determine the path and rate of movement of the

amyloplasts during this initial period.

Many scientists think of the structure of the cells that perceive

gravity in fairly static terms. It is not widely known that the

contents of many of these cells are under constant internal

motion. The cytoplasm in healthy graviperceptive cells undergoes

active and multidirectional streaming. Very little is known

about how this streaming itself responds to gravity or of how

streaming affects the rate and direction of amyloplast movement.

This laboratory is in the process of assembling a high resolution

system that links a video camera and recorder to a light microscope so that these questions (and others) can be answered.

PUBLICATIONS

Chabot, J.F., Chandra, A., Morrison, G.H., and Leopold, A.C.

Studies on the Distribution of Calcium in Root Cap Cells Using

Ion Microscopy (Abstract). Plant Physiology 69(Suppl. 4): 77,

1982.

Hoekstra, P.A. and Leopold, A.C. Imbibitional Chilling Injury in

Pollen: Involvement of the Respiratory Chain. Plant Physiology 69(Suppl. 4): 107, 1982.

O'Neill, S.D. and Leopold, A.C. An Assessment of Phase

Transitions in Soybean Membrane Lipids and Mixed Phospholipid Vesicles (Abstract). Plant Physiology 69(Suppl. 4): 108, 1982.

OINeill, S.D. and Leopold, A.C. An Assessment of Phase

Transitions in Soybean Membranes. Plant Physiology 70:

1405-1409, 1982.

lDO
Sack, F.D. and Leopold, A.C. C~aracteristics of Statoliths from

Rootcaps and Coleoptiles. Physiologist 25(6, Suppl.): S97-S98,

1982.

Walters, C. and Leopold, A.C. Water Viscosity and Effects of

Temperature on Seed Imbibition: A Misleading Correlation

(Abstract). Plant Physiology 69(Supp1. 4): 154, 1982.

Willing, R.P. and Leopold, A.C. Cellular Expansion at Low

Temperature as a Cause of Membrane Lesions. Plant Physiology 71:

118-121, 1983.

101
MECHANICAL REGULATION OF PLANT GROWTH AND DEVELOPMENT

Cary A. Mitchell

Department of Horticulture

Purdue University

West Lafayette, IN 47907

SU MMARY

During 1982, activities in this laboratory have emphasized

effects of periodic mechanical shaking or vibration on the

photosynthetic productivity of one plant species, soybean (Glycine max L. cv. Wells II). The research of this laboratory

as well asthat of others has shown that these same mechanical

forces have an inhibitory effect on plant growth and development

on Earth, the main natural counterpart of which is represented by wind. In space, however, mechanical vibrations are measured in

the same units as gravity; i.e., they have "g" equivalents. Thus, it is imperative that well-controlled flight experiments be

conducted to determine positive as well as negative aspects of

mechanical action on plants in an environment otherwise deprived of mechnical stimul i.

Growth analysis studies with soybean in this laboratory indicate

that overall photosynthetic productivity of soybean (measured as

whole plant dry weight) is sharply reduced if the plants are

agitated for 3- to 4-minutes two or three times daily on an

orbital shaker. This means that not only is biomass accumulation

affected by mechanical disturbance, but so also is air

revitalization (0 2 generation, CO 2 scavenging) capacity. During 1982 this laboratory has conducten a number of mechanical stress

experiments in controlled environments involving growth dynamics

analysis. This approach involves sequential harvest of treated

plants over time and the calculation of growth dynamics indices

such as relative growth rate (RGR), net assimilation rate (NAR),

and leaf area ratio (LAR). RGR is the slope of the

log-transformed cumulative growth curve (measured as whole plant

dry weight) vs. time during exponential growth, and is related to

the other two growth index parameters by RGR = NAR X LAR. NAR is

a measure of photosynthetic efficiency and LAR is the ratio of

leaf area to whole plant dry weight. One of the most significant findings during the past year was that RGR of mechanically stressed plants was less than that of undisturbed control plants. Furthermore, analysis of RGR components indicated that reduction

in NAR accounted for virtually all of the decline in RGR, whereas

LAR either was unaffected or actually increased.

Further investigations of possible causes of stress-induced

decreases in NAR by means of leaf porometry and measurements of

whole plant transpiration and leaf water potential have been

102
made. Each of these three measurements reflect plant water

status, which in turn may reflect the degree of opening or

closing of stomatal pores on the leaves. Briefly shaken plants transpired significantly less water vapor out of their foliage

for at least 90 minutes following a single 3-minute shaking

treatment. Preliminary leaf water potential measurements

indicate that 30 minutes after shaking a soybean plant, both leaf

and stem water potential are higher for shaken plants than for

undisturbed control plants. All of these measurements are

consistent with the interpretation that shaking causes a

temporary closure of the stomatal pores, thereby lowering transpirationa1 water loss and causing improvement in the water

status of stems and leaf cells.

Future efforts will emphasize measurement of leaf gas-exchange

rates as a function of mechanical stress pretreatment, which will

involve direct measurement of photosynthetic CO 2 fixation and

determination of component resistances contributing to the

suspected overall increase in leaf resistance to CO 2 fixation.

Complete characterization and analysis of gas-exchange responses

to mechanical disturbance are very important for understanding the impact of such forces on the vital life support functions

( i . e " prim ary pro duc t i vi ty ) 0 f P1ants . This appro ach eve nt ua11 y

must be extended to the weightless or hypogravity situation to

appreciate its full importance in an orbiting spacecraft.

PUBLICATIONS

Brooks, C.A. and Mitchell, C.A. Action of Metabolic Inhibitors

on Lettuce Seed Germination (Abstract). Plant Physiology 69(4, Supp1.): 154, 1982.

Pappas, T. and Mitchell, C.A. Mechanical Stress Regulation of

Growth and Photosynthetic Productivity of Glycine max (L.) Merr.

cv. Wells II under Different Environmental Regimes-.-Physiologist 25(6): S135-S136, 1982.

103
HOW THE PEA STEM SENSES GRAVITY, FRICTION, AND FLEXURE

Barbara Picard

Biology Department

Washington University St. Louis, MO 63130

SUMMARY

This is a new project. It is proposed that stimulation of cells

in the pea stem by gravitational and other acceleration, by friction, and by flexure results in delicate deformation of the

plasmalemma. Consequent deformational shifts in macromolecules

controlling the permeability of the membrane for calcium ion

result in its movement into the cytosol down an actively maintained concentration gradient. As the amount of cytosolic calcium rises, it binds to the modulator protein calmodulin,

which in turn can phospory1atively activate (1) a membrane-bound

protein which transports the hormone indoleacetic acid out of the

cell, and (2) a tubu1in-type protein which, upon pOlymerization,

brings about exocytosis of vesicles containing an agent that can

stimulate ethylene production in surrounding cells. Because

gravitational acceleration is vectorial, the consequent vectorial

activation of auxin transport enzyme ultimately leads to an

asymmetry of the growth hormone across the stem and geotropic

curvature results. Because friction and flexure do not tend to

distort membranes with net asymmetry, and because ethylene is a

rapidly diffusing molecule, these stimuli symmetrically result in

the "slower" sturdier growth of the stem which ethylene induces.

To the extent that activation of the two types of protein overlaps, the responses interfere with each other; in particular, since ethylene tends to antagonize auxin effects, this explains

the prominent counterreactive phase of geotropism.

Using 32 p, phosphorylation of proteins will be studied during and

following stimulation by gravity, friction, and flexure. Any phosphorylated protein increased by the stimulation will be

isolated so that biochemical properties may be studied.

Voltage transients that may represent vesicle release have been

described following stimulation, and will be subjected to more

rigorous study. Conditions governing their frequency will be

evaluated.

The putative agent released from the vesicles for stimulation of

ethylene production will be sought.

The release of ethylene will be monitored at a fine level of

discrimination during and following gravitational stimulation,

looking for correlation between the frequency of voltage transients, the abundance of this gaseous hormone, and the

counterreactive phase of geotropism.

104
MECHANISM OF GRAVITROPIC CURVATURE IN PLANT SHOOTS

David L. Rayle

Department of Botany

San Diego State University San Diego, CA 92182

SUMMARY

A basic but as yet unresolved problem in plant physiology is the

mechanism by which plants transduce information about the

direction of gravity into an oriented direction of growth. During the past 2 years (1981-1982), this project has focused on

this problem using the gravitropic curvature of plant shoots as a

model.

Briefly, it is known that when a shoot is oriented in a

horizontal position it begins to curve upward in a smooth arc

after about 30 minutes. Reorientation is complete within 2 to 4

hours. This curvature response ultimately derives from enhanced

growth of those cells comprising the lower half of a horizontal

shoot and a retardation of cell growth near the upper surface.

This research centers on the factor or factors which are

responsible for such asymmetric growth.

In 1981, Rayle reported that a proton gradient develops across

gravitropically stimulated shoots, and that this gradient is

critical in initiating the asymmetric growth that gives rise to

negative shoot gravitropism (i .e., upward curvature). Research

during 1982 has been directed toward sorting out whether

graviperception mechanisms give rise to the proton gradient directly or whether the proton gradient is derived indirectly,

perhaps as a result of auxin (indole-3-acetic acid; IAA)

redistribution. The latter possibility would be consistent with

much existing data, while the former possibility, if correct,

would be a radical departure from classical thinking on this

subject but consistent with the chemiosmotic theory of auxin

transport. The data described below represent our approach to

resolving these alternatives.

Using 3H- IAA , this investigation has established that auxin

redistribution occurs within 30 minutes of gravistimulation. This redistribution occurs simultaneously and uniformly along the

growing axis. These data suggest the kinetics of auxin

redistribution are rapid enough to directly initiate asymmetric

proton secretion.

High molarity neutral buffers prevent shoot gravitropism by neutralizing excreted protons and thus prevent an acid asymmetry from developing across a Jhoot. However, such buffers do not

prevent development of a H-IAA asymmetry. These data thus show

a proton gradient is not necessary for IAA redistribution.

105
Rather, they are consistent with the notion that IAA

redistribution precedes asymmetric proton secretion and may in

fact initiate the proton gradient.

The fact that the lateral auxin redistribution appeared to be

qualitatively and quantitatively normal when shoots were

submerged in neutral buffers was surprising. Purely theoretical

considerations which have been formalized as the chemiosmotic

theory of auxin transport would predict otherwise. In an attempt

to explain this paradox, the investigators have begun experimentally probing the chemiosmotic theory.

This line of experimentation involves altering the cell wall pH of shoots via infiltration with buffers and/or by treating shoots

with agents that inhibit (e.~., cycloheximide) or stimulate

(e.g., fusicoccin) in vivo H secretion. Preliminary results do

not sup port the cheiiiT0 smot i c theory • La t era 1 an d pol ar auxi n

transport are not influenced by cell-wall pH.

PUBLICATIONS

Rayle, D.L., Migliaccio, F., and Watson, E. Role of Auxin and

Protons in Plant Shoot Gravitropism. Physiologist 25(6, Suppl.):

5103-5104, 1982.

Wright, L.W. and Rayle, D.L. Inhibition of Shoot Geotropism by

Neutral Buffers. Plant Physiology 69: 278-279,1982.

106
THE CELLULAR BASES OF GRAVITY- AND LIGHT-INDUCED GEOTROPISM

Stanley J.

Department

University

Austin, TX

SUMMARY

Roux

of Botany

of Texas at Austin

78712

T~e overall objective of this research project is to arrive at a

better understanding of the specific fundamental processes that

are altered by gravity and light to produce geotropic growth in

plants.

Previous results from this and other laboratories give reason to

believe that at least one of these fundamental processes may be

the movement of calcium (Ca) ions in the stimulated cells and

organs. These results indicate that a gravity stimulus promotes the asymmetric distribution of Ca across growing stems. Light activation also promotes calcium movements in plants. Calcium is

known to reduce the plasticity of plant cell walls, i.e., reduces

their ability to be stretched in response to a force, such as

intracellular turgor pressure. After a plant has been given a

new gravity stimulus by turning it to a horizontal position,

higher concentrations of Ca accumulate in the walls of cells on

the upper side of its stem. Cells on this side will grow more

slowly than those on the lower side as the stem curves upward in

response to the gravity stimulus. It has been proposed that Ca,

through its effects on wall plasticity, could playa role in

mediating this differential growth. This project is focused on

examining whether Ca does play an important role in mediating geotropism and, if so, how it does so.

Experiments during 1982 further examined the Ca hypothesis by testing inhibitors which would be expected to interfere with Ca

accumulation in walls and by testing whether Ca redistribution

could also be observed in roots as it had been observed

previously in shoots. The major findings are as follows:

1. Correlation between the amount of 14C-chlorpromazine (CPZ)

bound to calmodulin (CaM) in oat coleoptiles and how effectively

CPZ blocks geotropism in coleoptiles.

The regulatory protein CaM, controls the activity of a

plasma-membrane-localized ATPase in plants which serves to pump calcium out of cells. This pump probably plays an important role

in the asymmetric movement of Ca which has been postulated to be

one of the early transduction steps necessary for geotropism.

CPZ, a CaM antagonist, r~versibly inhibits the calcium pump. It

also inhibits geotropism in oat coleoptiles at cp~centrations

that permit normal growth rates. This lab used C-labeled CPZ

107
to photoaffinity label CaM in vivo to determine if the drug is

actually binding to some portion of endogenous CaM when it

inhibits geotropism. It was found that CPZ does bind to cellular

CaM in co1eopti1es when it blocks their geotropism and that those

co1eopti1es that showed the least geotropism after CPZ treatment

had the greatest amount of their CaM bound to CPZ. This

correlation is consistent with the hypothesis that CPZ blocks

geotropism at least in part through its blockage of the CaM

function.

2. The calcium chelator, EGTA, blocks geotropism in oat

co1eoptiles.

The chemical EGTA strongly binds, or che1ates, Ca and it is very effective in removing Ca from walls of plant cells. It was found

that perfusion of oat co1eopti1es in 1 mM EGTA for 6 hours or

more blocks geotropism in 60S of the co1eopti1es (compared with

blockage of only lOS of co1eopti1es perfused in water), but does

not inhibit growth of these co1eopti1es. These results support

the hypothesis that the accumulation of Ca in walls of cells in

geotropically stimulated coleoptiles helps to mediate their

tropic growth.

3. Rapid structural changes occur in the walls of epidermal root

cells after geostimulation.

In preliminary ultrastructural studies, we did not observe an

asymmetric buildup of Ca in the walls of geostimulated root

cells, but we did detect a rapid «30 minute) change in the a~pearance of the outer walls of epidermal cells on the upper (faster growing) sides of the roots. These walls appear less

dense and to be minus a thin outer layer present on the outer

walls of epidermal cells on the lower sides of the roots. These

results indicate that rapid chemical changes are occurring in the

upper epidermal walls, and tests are underway to determine what

they are and how they relate to geotropism.

PUBLICATIONS

Biro, R.L., Hale, C.C., II, Wiegand, O.F., and Roux, S.J.

Effects of Chlorpromazine on Gravitropism in Avena Coleopti1es. Annals of Botany 50: 737-745, 1982.

Biro, R.L., Hale, C.C., II, Wiegand, O.F., and Roux, S.J.

Effects of Chlorpromazine on Gravitropism in Avena Co1eoptiles ( Ab st r act). P1ant Phy s i 01 0 gy 69 (Sup PL 4): 92, 1982.

Roux, S.J. Calcium Movements and the Cellular Basis of

Gravitropism (Abstract). In: Abstracts, 24th COSPAR Meeting, Ottawa, Canada, May 16-June 2, 1982. Par;s: CaSpAR, p. 533,

1982.

108
Roux, S.J. and Biro, R.L. Quantitation of Chlorpromazine-bound

Calmodulin during Chlorpromazine Inhibition of Gravitropism (Abstract). Physiologist (6, Suppl.): S107-S108, 1982.

Roux, S.J. and Slocum, R.D. Role of Calcium in Mediating Cellular Functions Important for Growth and Development in Higher Plants In: Calcium and Cell Function, Vol. III (ed. by W.-Y ..

Cheung). New York: Academic Press, p. 409-453, 1982.

Slocum, R.D. and Roux, S.J. An Improved Method for the

Subcellular Localization of Calcium Using a Modification of the

Antimonate Precipitation Technique. Journal of Histochemistry and Cytochemistry 30(7): 617-629, 1982.

109
AN EXAMINATION OF THE CLINOSTAT, AS A SIMULATOR OF WEIGHTLESSNESS

USING LEAFY PLANTS

Frank B. Salisbury

Department of Plant Science UMC 48

Utah State University

Logan, UT 84322

SUMMARY

A weightless condition may be simulated in the lab by using clinostats to rotate plants about a horizontal axis, or by periodic inversion. During the past few years this laboratory has developed several treatments to separate plant responses caused by mechanical stresses, during clinostatting, from those

effects caused by gravity compensation. Several parameters were

measured, including (1) height to a point marked near the apex of

the stern, (2) height to a point about two internodes below the

above point, (3) stern diameters at the two marked points, and (~)

leaf epinasty.

Changes in stem heights and diameters showed no simple patterns

among the various treatments nor for the two species used in our

studies: cocklebur (Xanthium strumarium) and tomato

(Lycopersicon esculentum). For example, stern elongation (especlal Iy of young lnternodes) was inhibited by mechanical

stress and gravity compensation in cockleburs but promoted in

tomatoes. It may be safe to generalize that both gravity compensation and mechanical stress typically inhibit, but may sometimes produce, stern elongation. Gravity compensation may be

especially important in promoting elongation of older, more

mature sterns, and this could be related to the stimulated

elongation of grass nodes induced by clinostatting.

Studies have also been made of the mechanics of stern bending by

placing plants in a horizontal position and wrapping threads

around a wire frame so that they are unable to bend upward in

response to gravity. Changes in surface dimensions and internal

pressures of such plants were then measured before and after

release from restraint (at which time they spring to a strongly bent position), and also in vertical controls and plants laid

horizontally and allowed to bend freely. When the restrained

plants were released, stretching of bottom surfaces and shrinkage

of top surfaces was found, and pressures produced by upward bending continued to increase until the time when maximum bending

upon release had been reached. Plants that were released from

restraint, then forcibly straightened and restrained again, continued to develop bending forces.

Another experiment involved slicing cocklebur or castor bean

110
(Ricinus communis) stems. Plants were placed horizontally and

were allowed to bend freely or were restrained for 48 hours in

the dark. They were: (1) cut longitudinally, either parallel or

perpendicular to the bend; or (2) small cuts were made at right

angles to the stem along the top or the bottom of stems.

Vertical control stems were also cut in these experiments. The

results clearly showed that the surface layers of stems are under

tension compared with the inside, and also suggested that the

pressures may be located in the cortex and vascular tissues

rather than in the pith. Plants placed horizontally and allowed

to bend freely then slit through the center with a horizontal

razor blade showed strong bending in the upper half, but little

or no bending in the lower half. This again shows the tension in

the outer layers compared with pressures in the inner tissues,

and emphasizes the importance of the response in the top half of

the stem in producing normal gravitropic bending.

PUBLICATIONS

Salisbury, F.B., Mueller,W.J., Blotter, P.T., Harris, C.S.,

White, R.G., Gillespie, L.S., and Sliwinski, J.E. The Mechanics

of Gravitropic Bending in Leafy Dicot Stems. Physiologist 25(6,

Suppl.): Slll-Sl12, 1982.

Salisbury, F.B., Sliwinski, J.E., Mueller, W.J., and Harris, C.S.

How Stems Bend Up. Utah Science 43(2): 42-49, 1982.

Salisbury, F.B., ,Wheeler, R.M., Sliwinski, J.E., and Mueller,

W.J. Plants, Gravity and Mechanical Stresses. Utah Science

43(1): 14-21, 1982.

111

SPECIAL PROJECTS

113

RESEARCH IN SPACE BIOLOGY

X.J. Musacchia

Department of Physiology and Biophysics

University of Louisville

Louisville, KY 40208

SUMMARY

Within the next decade, the NASA Shuttle Program will provide a

unique opportunity to conduct biological research in outer space

and to continue relevant ground-based space biology research. To

maximize this potential, there is a need to develop a growing cadre of scientists who are knowledgeable and interested in space

biology. The purpose of this grant is to provide NASA Research

Associate Awards to young scientists who are interested in

working in space biology. These are competitive grants. It is

anticipated that these scientists will develop their research

careers in the newly evolving discipline of space biology.

The Research Associate Awards are for a I-year period of funding

with the possibility of renewal for a 2nd year of additional

funding. Since the program1s inception on June 1, 1980, 24

awards have been made. So far, 8 of the 16 scientists selected

have received a 2nd year of funding.

The award recipients work in NASA-funded laboratories or in

laboratories that can provide the necessary facilities and

environment for specialized space biology projects. In June

1980, 19 laboratories participated in this program; as of

February 1983, 42 laboratories are now participating.

The grant is administered through the University of Louisville,

Louisville, Kentucky. Dr. X.J. Musacchia, Dean of the Graduate

School, is the Project Director and Science Advisor.

In addition to the salary stipend, travel expenses are paid for

the awardees to attend and present papers at two national

meetings: (1) the annual AIBS/NASA meeting, and (2) a national

society meeting of their choice.

115

MEETINGS

117

MEETINGS

Organized or participation by the Space Biology Program

"AIBS/NASA Space Biology Panel Meeting," John F. Kennedy Space

Center, Kennedy Space Center, FL, March 1982.

"XXIV COSPAR Meeti'ng," Ottawa, Canada, May 16-June 2, 1982.

"Workshop on Plant Growth in Space," Federation of American

Societies for Experimental Biology, Bethesda, MD, May 20-21,

1982.

"Workshop on the Regulatory Functions of Calcium and the

Potential Role of Calcium in Mediating Gravitational Responses in

Cells and Tissues," Federation of American Societies for

Experimental Biology, Bethesda, MD, September 16-18, 1982.

"33rd Annual Fall Meeting of the American Physiological Society,"

San Diego, CA, October 10-15, 1982.

"Fourth Annual Meeting of the IUPS Commission on Gravitational

Physiology," San Diego, CA, October 10-15, 1982.

119
1. Report No. I 2. Government Accession No. 3. Recipient's Catalog No.

NASA TM-86244

4. Title and Subtitle 5. Report Date

1982 NASA SPACE BIOLOGY ACCOMPLISHMENTS December 1983

6. Performing Organization Code

EBT-3

7. Author(s) 8. Performing Organization Report No.

Thora W. Halstead and Linda G. Pleasant

10. Work Unit No.

9. Performing Organization Name and Address

NASA Office of Space Science and Applications 11. Contract or Grant No. Washington, D.C. 20546 and NASW-3165 George Washington U., Washington, D.C. 20037

13. Type of Report and Period Covered

12. Sponsoring Agency Name and Address Technical Memorandum

National Aeronautics and Space Administration 14. Sponsoring Agency Code

Washington, DC 20546

15. Supplementary Notes

Thora W. Halstead: NASA Office of Space Science and Applications,

Washington, D.C.

Linda G. Pleasant: The George Washington University, Washington, D.C.

1.6. Abstract

Surrmaries of NASA's Space Biology Program projects. The goals,

objectives, accomplishments, and future plans of each 'project are

described in this publication as individual technical surmnaries.

17. Key Words (Suggested by Author(s)) 18. Distribution Statement

Gravity Animal Unclassified - Unlimited

Space Plant

Weightlessness

Simulated weightlessness Subject Category 12

19. Security Oassif. (of this reportl 120. Security Classif. (of this page) 121. No. of Pages /22. Price·

Unclassified Unclassified 119 A06

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