NASA Technical Memorandum 86654 

: NASA-TM-86654 19840022595 

1983-84 NASA Space Biology 

Accomplishments 

AUGUST 1984 

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NASA Technical Memorandum 86654 

1983-84 NASA Space Biology 

Accomplishments 

Compiled by 

Thora W. Halstead 

NASA Office of Space Science and Applications 

Washington, D.C. 

F. Ronald Dutcher and Linda G. Pleasant 

The George Washington University 

Washington, D.C. 

NI\S/\ 

National Aeronautics 

and Space Administration 

Scientific and Technical 

Information Branch 

1984 
Work performed under NASA contract NASw-3165. 
Preface 

The Space Biology Program currently includes forty-two research 

tasks. The goals, objectives, accomplishments, and future plans 

of each project are described in this publication as individual 

technical summaries. The summaries cover the period from March 

1983 through March 1984. 

The intent in compiling this publication is twofold. First, we 

wish 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 task summary. 

Specific accomplishments and conclusions drawn from the past 

year's research in each task have been underlined. 

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 Commodore Roy for 

her technical assistance in preparing this report. 

iii 

Thora W. Halstead 

June 1984 

I 

Contents 

page 

Preface iii 

Introduction - The NASA Space Biology Program 3 

Plant Projects 

Robert S. Bandurski, A. Schulze, and 9 

Y. Momonoki 

Attempts to Localize and Identify the 

Gravity-Sensing Device of Plant Seedlings 

Allan H. Brown 11 

Importance of Gravity for Plant Growth and 

Behavior 

Daniel Cosgrove 14 

Biophysical Basis of Asymmetrical Plant Growth 

Induced by Gravity 

Joe R. Cowles 16 

Aromatic Biosynthesis in Pine Tissues 

Kathryn L. Edwards 18 

The Relationship of Calcium and Auxin 

Transport in Root Gravitropism 

Michael L. Evans 21 

The Role of Acid and Calcium Gradients in 

Gravitropism 

Lewis J. Feldman 24 

Gravistimulus Production In Roots of Corn 

Arthur W. Galston 26 

Starch Depletion and Gravitropic Response 

Alice Bourke Hayes 29 

The Role of Gravity in Regulation of Leaf 

Blade Form 

Takashi Hoshizaki 31 

Effects of Gravity and Light on Growth, 

Development, and Flowering in Plants: 

II. Multiple Generations on Clinostats 

M.J. Jaffe 33 

A Study of the Mechanism of Thigmomorphogenesis in Plants, with Special 

v 
Reference to the Role of Ethylene and Its 

Significance to Research with Plants in Space 

Peter B. Kaufman 36 

Gravity Perception, Transduction, and Cellular 

Response in Gravistimulated Shoots of Cereal 

Grasses 

A.D. Krikorian 39 

Plant Cells, Embryos, and Development in Space 

A. Carl Leopold and Fred D. Sack 42 

The Amyloplast as a Gravity-Sensing Device in 

Plants 

Cary A. Mitchell 44 

Mechanical Regulation of Plant Growth and 

Development 

Barbara G. Pickard 46 

Testing a Hypothesis: How the Pea Stem Senses 

Gravity, Friction, and Flexure 

David L. Rayle 48 

The Mechanism of Plant Shoot Gravitropism 

Stanley J. Roux 51 

The Cellular Bases of Gravity- and LightInduced Gravitropism 

Frank B. Salisbury 54 

Gravitropism in Leafy Dicot Stems 

Animal Projects 

Daniel D. Bikle 

The Effect of Skeletal Unloading on Bone 

Formation 

Wolf-D. Dettbarn 

Weightlessness Simulation: Physiological 

Changes in Fast and Slow Muscle 

Stephen B. Doty 

Alterations in Connective Tissue Cells Due 

to Reduced Weight Bearing 

Jackie C. Duke 

Growth and Differentiation of Mammalian 

Embryonic Tissues Exposed to Hypergravity 

In Vivo and In Vitro 

vi 

59 

62 

64 

66 
Alfred Finck 68 

Effects of Gravito-Inertial Fields on the 

Physiology of the Orb-\'leaving Spider 

Charles A. Fuller 70 

Homeostasis in Primates in Hyperacceleration 

Fields 

William F. Ganong 73 

Neural Mechanisms by Which Gravitational 

Stimuli and Stress Affect the Secretion of 

Renin and Other Hormones 

Bernard P. Halloran 75 

Changes in Bone Structure and Metabolism 

during Simulated Weightlessness: Endocrine 

and Dietary Factors 

John M. Horowitz 77 

The Effects of Hypergravic Fields on Neural 

Processing of Sensory Thermoregulatory and 

Vestibular Systems in the Rat 

George M. Malacinski and Anton W. Neff 79 

Gravity Perturbation as a Probe for Analyzing 

Pattern Specification in Early Amphibian 

Embryogenesis 

Emily Morey-Holton 81 

Structural Development and Gravity 

X.J. Musacchia, Joseph M. Steffen 84 

and Ronald D. Fell 

Regulatory Mechanism of Skeletal Muscle Atrophy 

and Fluid and Electrolyte Shifts in the Hypokinetic/Hypodynamic and Antiorthostatic Rat 

Jiro Oyama 

Hyper-Gravitational Effects on Metabolism 

and Thermoregulation 

Nello Pace and Arthur H. Smith 

Gravity, Body Mass and Composition, and 

Metabolic Rate 

V.J. Popovic 

Effect of Decreased Gravity on Circulation 

in the Rat 

W. Eugene Roberts 

Bone Cell Kinetics of Simulated Weightlessness 

vii / 

87 

89 

93 

95 
Muriel D. Ross 98 

Hammalian Gravity Receptors: Structure and 

Metabolism 

David J. Simmons and Jean E. Russell 103 

Some Aspects of Hypogravity Simulation on Bone 

Formation and Maturation 

Douglas G. Stuart 105 

Effects of Muscle Atrophy on Motor Control 

Gordon Templeton 107 

Adaptations of Rat Skeletal Muscle to 

Suspension-Hypokinesia 

Marc E. Tischler 110 

Skeletal Muscle Metabolism in Hypokinetic Rats 

Debra J. Wolgemuth 113 

Effects of Simulated weightlessness on Meiosis, 

Fertilization, and Early Embryonic Development 

in Hice 

Special Activities 

Research Associate Program 

Meetings 

viii 

119 

121 
INTRODUCTION 

THE NASA SPACE BIOLOGY PROGRAM 

Thora W. Halstead 

Life Sciences Division 

Office of Space Science and Applications 

National Aeronautics and Space Administration 

Washington, DC 20546 

Introduction 

One of the major features of the physical environment on the 

surface of Earth is the constant presence of the force of 

gravity. Terrestrial gravity has important biological 

consequences for organisms living on Earth. The phenomenon of 

weightlessness which is encountered on spacecraft provides an 

excellent 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, thereby enhancing our capability to use 

and explore space. 

Program Scope 

Research in the Space Biology Program is divided into three broad 

areas: 

1. Gravity perception. The objectives are to identify 

gravity receptors in organisms sensitive to gravity and 

determine their structure and function, and to elucidate 

the mechanisms by which gravitational stimuli are 

perceived and transmitted to a responsive site. 

3 
2. Developmental biology. The objectives are to determine 

the effects of gravity, and especially weightlessness, 

as provided by spaceflight, on the genetic integrity, 

cellular differentiation, reproduction, development, 

growth, maturation, and senescence of living systems; 

and to examine the evolutionary importance of gravity as 

a determinant of the form and function of terrestrial 

life. 

3. Biological adaptation. This area includes the use of 

gravity's physiological effects to explore biological 

problems; and achievement of an understanding of how 

gravity affects and controls the physiology, ,morphology, 

and behavior of organisms, of how gravity and other 

environmental stimuli and stresses interact in this 

control, and of the biological mechanism by which living 

systems respond and adapt to altered gravity, 

particularly that of the space environment. 

Research Opportunities 

with the proven feasibility of the Space Shuttle, we now have a 

new 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. 

Focus of Program 

The research focus of the Space Biology program is dependent upon 

4 
several dynamic factors: the requirements of NASA, the 

characteristics of flight experiment opportunities, the 

sensitivity of specific biological systems to gravity, the 

scientific value of the research, the state of knowledge and 

technology in the specific scientific areas, the interest of 

scientists in studying the biological questions, and the 

availability of funds to support the research. 

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 fertilization and development of 

plants and animals, 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 a direct effect on the embryo itself? 

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 m~chanisms 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 do gravitational and other environmental stimuli 

interact in the control and direction of living forms? 

Can the action of gravity be replaced by different 

stimuli? 

As the new opportunity to conduct biological research in space 

grows, an increasing amount of the research supported by this 

Program will contain at least some results obtained in space. 

The future of the Program is tied to space research. 

5 

PLANT PROJECTS 

ATTEMPTS TO LOCALIZE AND IDENTIFY THE GRAVITY-SENSING DEVICE OF 

PLANT SEEDLINGS 

Robert S. Bandurski, A. Schulze, and Y. Momonoki 

Department of Botany and Plant Pathology 

Michigan State University 

East Lansing, Michigan 48824-1312 

Growth Hormone Asymmetry: 

Gravity induces an asymmetric distribution of the plant growth 

hormone, indole-3-acetic acid (IAA). The intent of this work is 

to find out where, and how, the hormone asymmetry originates. 

Such knowledge should result in the identification of the 

gravity-sensing device of the plant. 

H. Dolk showed, 50 years ago, by means of a bioassay, that 

gravity induced a hormone asymmetry. To confirm this finding, an 

isotope dilution gas chromatographic-selected ion monitoring-mass 

spectrometric method was used as a definitive assay. The results 

both confirmed and extended Dolk's experiments, that is, the 

hormone was IAA and further, the IAA was asymmetrically 

distributed in the cortical cells of the shoot. 

A further and surprising conclusion resulted from this work-that 

is, that the asymmetry must have originated in the vascular 

tissue. This is so because the preponderance of free IAA is in 

the vascular tissue and yet the same percentage asymmetry was 

obtained whether cortical tissue only or cortical plus vascular 

tissue was analyzed. 

It is tentatively concluded that one cause of the asymmetric 

hormone distribution is selective and asymmetric leakage of the 

hormone from the vascular stele. If this is correct, it is an 

exciting finding since it would indicate that the gravitational 

stimulus can make the plant vascular tissue selectively leaky in 

a manner somewhat analogous to the serotonin control of capillary 

leakage. 

Growth Hormone Catabolism: 

It is now known that IAA concentration is determined by the 

summation of the inputs, including IAA conjugate hydrolysis, 

transport, and de novo synthesis--minus the outputs--including 

IAA catabolism and conjugate formation. Mr. Reinecke originally, 

and now together with H.M. Nonhebel, has discovered the major 

catabolic pathway for IAA in corn tissue. This is the oxidation 

of IAA to oxindole-3-acetic acid. 

Elucidation of the further steps in the catabolism of the growth 

hormone IAA is planned. 

9 
PUBLICATIONS 

Bandurski, R.S. Mobilization of Seed Indole-3-acetic Acid 

Reserves during Germination. In: Mobilization of Reserves in 

Germination (ed. by C. Nozzolillo, P.J. Lea, and F.A. Loewus). 

New York: Plenum Press, p. 213-228, 1983. 

Bandurski, R.S. and Schulze, A. Gravitational Effects on Plant 

Growth Hormone Concentration. Advances in Space Research 3(9): 

229-235, 1983. 

Bandurski, R.S., Schulze, A., Dayanandan, P., and Kaufman, P.B. 

Asymmetric Distribution of Endogenous Indole-3-acetic Acid in Zea 

Mesocotyl Cortex Following Gravistimulation (Abstract). Plant 

Physiology 72(1, Suppl.): 146, 1983. 

Bandurski, R.S., Schulze, A., Dayanandan, P., and Kaufman, P.B. 

Response to Gravity by Zea mays Seedlings. 1. Time Course of the 

Response. Plant Physiology 74: 284-288, 1984. 

Chijnell, J.R. and Bandurski, R.S. Translocation of [5-3H]AA and 

[5- H]IAA-Myo-Inositol from the Endosperm to the Shoot of 

Dark-Grown Zea Mays L. Seedlings (Abstract). Plant Physiology 

72(1, Suppl.): 27, 1983. 

Momonoki, Y.S. and Bandurski, R.S. The Effect of Deseeding on 

Amide lAA in Maize Seedlings (Abstract). Plant Physiology 72(1, 

Suppl.): 115, 1983. 

Momonoki, Y.S. and Bandurski, R.S. Effect of Endosperm Removal 

on 7 Normal NaOH-Labile Indole-3-acetic Acid Conjugates in Shoots 

and Roots of Zea mays Seedlings. Plant Physiology 75: 67-69, 

1984. 

Momonoki, Y.S., Schulze, A., and Bandurski, R.S. Effect of 

Deseeding on the Indole-3-acetic Acid Content of Shoots and Roots 

of Zea mays Seedlings. Plant Physiology 72: 526-529, 1983. 

Nonhebel, H.~. and Reiner~e, D.M. Synthesis of High Specific 

Activity [5- H]- and [1- C] Oxindole-3-acetic Acid. Plant 

Physiology 72(1, Suppl.): 116, 1983. 

Pengelly, W.L. and Bandurski, R.S. Analysis of Indole-3-acetic 

Acid Metabolism in Zea mays Using Deuterium Oxide as a Tracer. 

Plant Physiology 73: 445-449, 1983. 

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. 

10 
IMPORTANCE OF GRAVITY FOR PLANT GROWTH AND BEHAVIOR 

Allan H. Brown 

Department of Biology 

University of Pennsylvania 

Philadelphia, PA 19104 

Efforts during 1983 were in support of several different 

projects: 

(I) Spacelab-l HEFLEX Experiment. The principal objectives 

of the HEFLEX Experiment were accomplished. Circumnutation was 

observed in the uG environment. Therefore, the process does not 

have a mandatory requirement for a protracted G force as had been 

predicted for one particular model of the mechanism of 

circumnutation. 

(2) Preliminary work continued on measurements of leaf 

hyponastic response to an axially directed G force. The 

methodology being developed will be used to test whether 

epinastic curvatures produced by clinostatting are attributable 

to a clinostat artifact or, as proposed, to a graded response to 

any G force imposed in line with the axis of the test plant. 

Results will be important for a possibly revised interpretation 

of data from one of the earliest plant experiments flown by NASA 

on Biosatellite II. [Research Task, EPIFOG] 

(3) Preparations continued for a series of measurements of 

the liminal angle of Capsicum leaves in hypergravity. Data in 

the literature tell us what happened at 1 G and at ~G. With the 

same apparatus it will be possible to extend the experiment to G 

levels greater than unity. (It is predicted that the system will 

be relatively insensitive to the G force only above 1 G). 

[Research Task, HYGCAP] 

(4) Experimental work was completed on a study of the 

kinetics of damping out of circumnutational oscillations in 

clinostat-simulated weightlessness. A manuscript based on this 

work is now in preparation. By applying the currently most 

popular theory of mechanism of circumnutation, it was predictable 

that damping should have occurred within one period of 

oscillation. It was found that it actually required an interval 

equivalent to about 8 periods. That result, coupled with the 

more recent finding that circumnutation persisted in Spacelab ~G, 

provided strong incentive and opportunity for a constructive 

modification of the gravitropic response-with-overshoot theory of 

the mechanism driving circumnutation. [Research Task, NULYRL] 

(5) For a second year the work of a postdoctoral research 

fellow, Dr. D.G. Meyers, was supported. His research interests 

were only indirectly related to the research program but 

11 
assistance was given with the design and construction of his 

experimental apparatus and with the development of experimental 

concepts in his research area. His studies in the laboratory 

have led to publications in the literature of experimental 

limnology and to his continuing interest in further exploitation 

of the methodology he used. 

(6) At the request of the NASA HQ Life Sciences Program 

Office, samples of sunflower shoots that had been grown in 

Space lab ~G and other samples grown at 1 G on one of the HEFLEX 

centrifuges during the SL-l Mission were made available to Dr. R. 

Bandurski, a Program Grantee. Flight-type plant modules and soil 

mixture were also made available so that fully valid 1 G ground 

control plants could be cultured in Dr. Bandurski's laboratory. 

These plants will be analyzed for indoleacetic acid. 

(7) Ground-based support experiments under development for 

the Spacelab 4 (4A?) Mission include modification of the SL-l 

HEFLEX hardware for implementation of candidate experiments, 

GTHRES and FOTRAN. Since the SL-l project was not funded at a 

level that would permit fabrication of more than one flight 

hardware unit, and since that unit was not available for other 

uses during preparation and flight of SL-l, the HEFLEX hardware 

unit could not be modified to make it suitable for accomplishing 

SL-4 objectives. Therefore, the design had to be modified and 

mostly new hardware had to be fabricated for GTHRES and FOTRAN. 

By default,·a significant part of that effort had to be 

supported by grant funds. 

(8) For those experimenters who may need to make use of the 

NASA Shuttle Middeck for accommodation of biological experiments, 

the temperature profile to which those experiments may be exposed 

becomes important for planning hardware design, experimental 

protocol, identification of needed controls, and in many 

instances for the basic decision on whether it would or would not 

be scientifically worthwhile to fly a particular experiment in 

the Shuttle Middeck. Empirical data on temperatures recorded 

simultaneously in up to four Middeck lockers have been acquired 

from NASA sources and from flight packages flown in the Middeck 

on STS-2, STS-3, and STS-9. Those data are in the process of 

being collated, and, if NASA continues to acquire such 

information on a routine basis for each Shuttle mission, it 

should become possible to develop a realistic specification for 

the limits of an average thermal profile which poikilothermic 

test subjects may experience, if they are flown in the Middeck 

without active temperature control. 

PUBLICATIONS 

Brown, A.H. Resistance of Mature Arabidopsis Plants to 

Mechanical Deformation in Relation to G-force during Development. 

12 
Physiologist 26(6, Suppl.): S149-S150, 1983. 

Brown, A.H. The Significance of Gravity on Biological Systems. 

In: Proceedings of a Workshop on Space Biology, Cologne, Germany, 

March 9-11, 1983. Paris, France: European Space Agency, p. 3-9, 

1983. (ESA SP-206) 

Brown, A.D. and Chapman, D.K. Use of Simulated Hypogravity to 

Test a Model for Circumnutation. Plant Physiology 72(1, Suppl.): 

64, 1983. 

13 
BIOPHYSICAL BASIS OF ASYMMETRICAL PLANT GROWTH INDUCED BY GRAVITY 

Daniel Cosgrove 

Department of Biology 

pennsylvania State University 

University Park, PA 16802 

This research is aimed at elucidating the mechanism by which the 

growth of plant stems is altered under the influence of gravity. 

Typically, when a plant is placed in a horizontal position, the 

growing region of the stem will reorient itself into a vertical 

position by unequal growth of the upper and lower sides of the 

organ (a response called gravitropism). This project examines 

the biophysical basis for this growth asymmetry induced by 

gravity. To change the growth rate of a plant cell, gravity must 

alter at least one of the cell properties which control water 

absorption and irreversible expansion of the cell wall. These 

properties include the cell hydraulic conductance, cell turgor 

pressure, the gradient in osmotic pressure across the cell 

plasmalemma, cell wall extensibility, and cell wall yield 

threshold. The immediate question is, which of these properties 

is altered by horizontal placement in a gravitational field. 

This project began in September 1983. During the 4 mo. in 1983 

in which this project was active, an apparatus was assembled for 

high resolution measurements of the growth and curvature along 

the axis of the stem. Such measurements are essential (l) for 

quantitating the location and magnitude of the growth response to 

gravity, and (2) for subsequent studies on the mechanism of the 

growth response. The approach taken for these measurements 

involves painting very fine black marks on the upper and lower 

sides of the stem at approximately 2-mm. intervals and 

photographing the marked stem at 15- to 3D-min. intervals. The 

photographs are projected onto a digitizing tablet which is 

interfaced with a microcomputer, and the X/Y positions of the 

marks on the upper and lower surfaces of the stem are recorded by 

the computer. Various algorithms for calculating the distance 

and average curvature between any two marks on the stem were 

evaluated. The best technique appears to be to fit the data to a 

cubic spline and to estimate distance and curvature from standard 

formulas using the coefficients of the cubic spline. Tests have 

shown this apparatus and method to have the high accuracy and 

repeatability necessary for this work. 

Initial measurements were made using pea (Pisum sativum L.) 

plants, but because of their rather slow and variable response to 

horizontal placement, more recent work has been carried out with 

cucumber (Cucumis sativus L.) plants. Two surprising findings, 

still tentative, have come from the measurements made so far. 

The initial growth response appears to be an acceleration of 

growth on the lower side of the cucumber stem. This burst in 

14 
growth is most prominent during the time from 10 to 30 min. after 

the start of horizontal placement and occurs along the entire 

length of the growing region. Little change in the upper side of 

the stem is seen at first. The growth rate on the lower side, 

however, quickly returns to the prestimulus value while at the 

same time the growth of the upper side rapidly declines. Thus, 

the curvature which develops during the first 30 min. is caused 

primarily by enhanced growth on the lower side. Subsequent 

curvature is caused by a reduced growth rate on the upper side. 

These observations could be of major importance for other studies 

which are examining the role of gradients in hormones, calcium, 

pH, and other substances in mediating the effect of gravity on 

plant growth. ·It is possible, for example, that the initial 

growth burst on the lower side might be caused by one type of 

gradient, while the subsequent growth suppression on the upper 

side might be mediated by a different factor. These findings are 

also significant since models of gravitropism need to account for 

not only the development of curvature, but also must be 

consistent with the detailed kinetics of how this curvature 

develops temporally and spatially. 

Work has also begun to examine the possibility that the altered 

growth is due to an alteration in the osmotic pressure of the 

cell contents. Cucumber stems were placed horizontal and after 

45 min. were bisected, separating upper and lower halves. Cell 

sap was expressed from these bisected halves and measured with a 

cryoscopic osmometer. No difference in the osmolality of the two 

sides was detected (mean value was 210 mOs./kg.). Measurements 

to be made at other points in the gravity response are in 

progress. 

PUBLICATIONS 

Cosgrove, D.J. Photocontrol of Extension Growth: A Biophysical 

Approach. Philosophical Transactions of the Royal Society, 

London, Series B 303: 453, 1983. 

15 
AROMATIC BIOSYNTHESIS IN PINE TISSUES 

Joe R. Cowles 

Department of Biology 

University of Houston 

Houston, TX 77004 

Higher plants synthesize more aromatic compounds than any other 

group of living organisms. While the biosynthetic pathways for 

synthesis of many of the aromatic compounds are known, the 

regulation of aromatic biosynthesis remains mostly obscure. The 

principal goal of this research is to gain an understanding of 

the regulation of aromatic biosynthesis in higher plants. 

Principal focus is on synthesis of the aromatic structural 

polymer, lignin, in young developing pine and mung bean 

seedlings. The principal area studied in 1983 concerned the 

effect of light on induction of lignification in pine seedlings. 

When germinating pine seedlings are grown in the dark they become 

more elongated and less fibrous than light-grown seedlings. If 

etiolated pine seedlings are analyzed for lignin after a 13-day 

dark treatment, they contain only about 30% of the lignin of 

light-grown seedlings. If etiolated seedlings are then placed in 

light, there is a rapid increase in lignification. It is 

concluded that light is critical for normal lignification. In 

fact, low levels of lignification in etiolated seedlings appears 

to be a survival phenomenon which enhances the plant's ability to 

search for light through increased elongation. 

The basic questions regarding the effect of light on 

lignification are: 

(1) Is the light requirement principally as a source of 

energy for lignification? 

(2) Is the light requirement principally for substrate 

synthesis and specific enzyme induction? 

(3) How does light alter aromatic metabolism to enhance 

lignification? 

(4) Does light activate specific genes required for 

lignification? 

There are data pertaining to the first three questions. It 

appears, from experiments, that light is very important as an 

energy source for lignification. When etiolated seedlings are 

placed in light, the increased rate of lignification is closely 

correlated to light intensity. This suggests that light is not 

principally a trigger but an energy source for lignification. In 

addition, experiments in which pine seedlings were exposed to up 

to 60 min. of light daily did not significantly increase their 

rate of lignification. Again, this suggests that light is 

required for biosynthetic energy rather than for enzyme 

16 
induction. Still to be determined are substrate and aromatic 

intermediate levels in response to light in an effort to 

establish how light affects substrate production and intermediate 

pools. 

One of the critical enzymes for lignin synthesis is 

phenylalanine ammonia lyase (PAL). PAL is known to be induced by 

light. Experiments on PAL activity in pine in response to light 

have shown a 3- to 4-fold increase in PAL activity in 2 days. 

Activity of peroxidase, another enzyme required for 

lignification, does not increase appreciably after exposure of 

etiolated pine seedlings to light. These results support the 

suggestion that PAL but not peroxidase is light induced. 

However, based on information discussed in the previous paragraph 

along with other experiments on PAL induction in pine, the 

increase in PAL activity may not be primarily due to light 

directly, but to other factors such as changes in available 

substrate. It appears that peroxidase activity is already 

sufficient to accommodate the increase in lignification that 

results from exposure to light. Again, there is a need to 

quantitate the substrate pool during light induction. 

The conclusions at this point are: (1) that light is critical for 

lignification by supplying metabolic energy; (2) that the 

activity of at least one enzyme, PAL, increases when etiolated 

seedlings are exposed to light; and (3) that the lignin 

polymerizing enzyme, peroxidase, is not rate limiting in young 

pine seedlings. 

PUBLICATIONS 

Cowles, J.R., ScheId, H.W., Peterson, C., and LeMay, R. Plant 

Growth and Development in Near Weightlessness (Abstract). Plant 

Physiology 72(1, Suppl.): 97, 1983. 

17 
THE RELATIONSHIP OF CALCIUM AND AUXIN TRANSPORT IN ROOT 

GRAVITROPISM 

Kathryn L. Edwards 

Biology Department 

Kenyon College 

Gambier, OH 43022 

Auxin, indole-3-acetic acid (IAA), is a prime candidate for 

regulating and modulating the differential growth response of 

primary corn roots to gravity. IAA can promote and inhibit root 

elongation rapidly within a narrow concentration range. Thus, 

growth regulation would require only small changes in auxin flux 

and cellular auxin concentration which in turn could be rendered 

in the short lag period for initiation of gravitropism. Since 

auxin is transported to/through the zone of elongation toward the 

meristem, it may serve as a direct communication link between the 

zone of elongation--site of gravitropic response--and the root 

cap (RC}--site of gravity perception. When auxin transport is 

inhibited, gravitropism is also inhibited. Napthylpthalamic acid 

(NPA) is one such inhibitor. It inhibits gravitropism only when 

applied to the apical growing and dividing region of the root. 

Application at the basal end of the root does not influence 

gravitropic curvature. NPA causes upward curvature when applied 

to the upper surface of horizontal, 2-day-old, intact corn roots. 

This effect is countered by application of IAA to the opposite 

side. NPA affects auxin accumulation in I-mm. slices of apical 

root tissue but does not affect abscissic acid (ABA, another 

possible mediating hormone) net uptake. 

Such evidence strongly implicates IAA and its transport within 

the apical region in the molecular control of root gravitropism. 

Calcium applied asymmetrically to the root tip will elicit 

curvature towards the calcium side. In addition, calcium is 

laterally and polarly translocated downwards at the RC of 

horizontal roots (Lee, Mulkey, and Evans 1983 Science 220: 

1375-1376; Lee, Mulkey, and Evans 1983 Plant Physiology 73: 

874-876). In light of this accumulating evidence implicating the 

importance of calcium in the RC in determining the direction of 

gravitropic curvature in roots, the relationships between IAA 

transport and apical calcium have been investigated. In order to 

establish a mechanism for communication/regulation between the 

elongation zone and the RC of graviresponding roots, the effects 

of calcium on lateral transport, acropetal/basipetal transport, 

and asymmetric distribution of IAA are being researched. 

Corn seedlings were germinated vertically at 30 0 C for 2 days in 

light. Seedlings with straight roots 1-3 cm. long were selected. 

Since both removal of the RC and EDTA (a calcium chelator) 

applied to the RC can prevent gravitropic curvature, it has been 

18 
suggested that the source of calcium that elicits an effect on 

gravitropism is in the RC (Lee et al.) To test the effect of 

calcium and EDTA on acropetal lAA transport, the RC was gently 

teased off the meristem and replaced with an agar receiver block 

containing calcium, EDTA, or buffer alone. An apical 6-mm 

segment was excised from the decapped root and held vertically in 

a Plexiglas holderS the apical end on the receiver block. Donor 

blocks containing H-IAA were applied at the base. After a 

I.S-hr. transport period the receivers were collected. Calcium 

(1 mM) in the receiver did not affect efflux of label into the 

receiver. However, EDTA significantly reduced the label in the 

receivers whereas ABA enhanced the label content. EDTA only 

inhibited label effluxing from the meristem. Acropetal transport 

investigated in 6-mm. segments taken 3 mm. behind the RC was not 

effected by EDTA. The effect of EDTA at the meristem could be 

reversed by replacing EDTA with a calcium block. Calcium in the 

RC may regulate lAA translocation from the meristem to the RC. 

Does asymmetrical calcium applied at the tip influence asymmetric 

distribution of acropetally or basipetally transported IAA? To 

test the former, apical 6-mm. segments were excised from decapped 

roots and oriented horizontally in Plexiglas holders. 

Radiolabeled donor blocks were applied at the basal ends. One 

calcium and one buffer agar block were placed in opposition on 

the upper and lower surfaces of the root tip. After I.S hr. 

there was no significant difference in the radioactivity of these 

upper and lower receiver blocks at the tip. Asymmetrical calcium 

at the tip did not affect acropetal transport and asymmetrical 

distribution of label at the tip. 

Calcium did strongly affect asymmetric distribution of 

basipetally translocated IAA. In this case donor blocks were 

applied at the meristematic apex, a calcium block was placed on 

the upper or lower surfaces of the tip, and receivers were 

positioned on the upper/lower surface of the basal end of the 

segment. Asymmetric calcium at the tip resulted in greater 

accumulation of label in the basal receiver block corresponding 

to the calcium. Furthermore, these data reveal that calcium can 

be preferentially translocated across the meristem. Lee et ale 

found that radiolabeled calcium was only translocated polarly 

across the RCi when the RC was removed so was the polarity of 

lateral translocation. Thus, since no preferential movement of 

calcium exists in the meristem, then one would expect that the 

ratios of radiolabel found in the basal receivers (receiver on 

the side of calcium/receiver opposite calcium) with calcium on 

the upper side and with calcium on the lower side would be equal. 

However, these ratios are significantly different (p=O.0066), 

indicating that calcium is translocated laterally downwards 

across the meristem. The identity of the radiolabel collected in 

the receivers has been identified as IAA using high performance 

liquid chromatography (confirmation by mass spectroscopy is 

intended). 

19 
In summary, calcium has been found to affect auxin acropetal 

efflux from the meristem and to elicit asymmetric transport of 

auxin traveling basipetally from the meristem. Calcium research 

has suggested that graviperception results in asymmetric calcium 

accumulation on the lower side of the root cap and, it is 

proposed, may impose calcium asymmetry in the meristem. It is 

suggested that this calcium redistribution and asymmetry effects 

a reciprocal asymmetric distribution of auxin in the elongation 

zone by affecting basipetal transport of auxin toward the site of 

gravitropic response. This is the first evidence suggesting a 

mechanism for communication between the site of gravity 

perception and the site of gravity response in roots. 

PUBLICATIONS 

Edwards, K.L. Implications for IAA in Gravitropism Control of 

Corn Roots (Abstract). Plant Physiology 72(1, Suppl.): 145, 

1983. 

20 
THE ROLE OF ACID AND CALCIUM GRADIENTS IN GRAVITROPISM 

Michael L. Evans 

Department of Botany 

Ohio State University 

Columbus, OH 43210 

Research is directed toward understanding the influence of 

gravity (or lack of gravity) on plant growth--in particular the 

mechanism by which roots become oriented in a gravitational field 

and grow perpendicular to the direction of gravity. This 

phenomenon is called gravitropism. 

Work during 1982 implicated three major factors in the control of 

root gravitropism: the growth hormone, auxin; hydrogen ions 

secreted from the roots; and calcium ions present in the root 

cap, the region where gravity detection occurs. The evidence 

implicating calcium was: (1) calcium-immobilizing agents applied 

to the cap caused roots to lose gravisensitivity, and (2) 

establishment of a calcium gradient across the root cap caused 

gravitropiclike curvature toward the calcium. 

During 1983 calcium involvement in root gravitropism was 

investigated. The following questions were addressed: (1) Does 

gravistimulation cause redistribution of calcium in the root? If 

so, does this occur in the gravity-detecting portion of the root 

(the cap), in the gravity-responding portion of the root (the 

growth zone), or both? (2) Is calcium movement linked to auxin 

movement? (3) Can calcium gradients at the root tip influence 

auxin gradients in the growth zone? (4) Is calcium involved in 

the gravity-detection mechanism or in the growth response 

mechanism? (5) Is there a correlation between the effect of 

environmental parameters on gravitropism and on calcium 

physiology? These questions were studied by using radioactive 

calcium and auxin to measure the influence of gravity on calcium 

movement, the influence of the hormone on calcium movement, and . 

the influence of calcium on the movement of the hormone. 

The major findings from these studies are: (1) Gravistimulation 

induces polar transport of calcium toward the bottom of the root 

cap of gravisensitive roots. This is the most important finding 

from the research. It is especially significant because it 

relates a natural consequence of gravistimulation (development of 

a transorgan calcium gradient) to the observation that applied 

calcium gradients induce gravitropiclike curvature without 

gravistimulation. This suggests that a gravity-induced calcium 

gradient across the cap links gravity detection in the cap to the 

asymmetric growth that causes curvature. This link has eluded 

plant physiologists for decades. (2) The polar transport of 

calcium in caps of gravistimulated roots is linked to movement of 

auxin. Inhibitors of auxin transport prevent gravity-induced 

21 
polar movement of calcium. This relates this study to the work 

of others who have shown that polar transport of auxin in shoots 

depends upon calcium. The results raise the possibility of 

coupling calcium asymmetry to the asymmetry of auxin, the hormone 

most likely to regulate the gravitropic growth response. (3) The 

calcium antagonist, flunarizine, and the calmodulin inhibitor,---

chlorpromazine, interfere with the uptake of calcium by root cell 

protoplasts, and they prevent gravitropism. This strengthens the 

hypothesis that calcium movement is critical to gravitropism and 

raises the possibility that calmodulin is involved in the action 

of calcium in gravitropism. (4) Application of calcium to one 

side of the cap of an intact vertical root enhances the movement 

toward the tip of label from radioactive auxin applied to the 

elongation zone. This is significant because it indicates that 

calcium gradients in the cap may lead to auxin gradients in the 

elongation zone, a factor long thought to control gravitropic 

curvature. (5) When roots made nongraviresponsive by treatment 

with EDTA are gravistimulated, they show gravitropic curvature 

when returned to the vertical and supplied with calcium in the 

absence of EDTA. This shows that the role of calcium is not in 

the detection of gravity but in the response of the roots to a 

perceived gravity signal. 

Toward the end of 1983, work began on the investigation of two 

other aspects of calcium involvement in gravitropism: (I) 

Examination of the changes .in gravistimulated calcium movement 

which accompany the induction by light of gravitropic sensitivity 

in dark-grown roots. The results indicate that graviinduced 

calcium movement across root tips is light-dependent. This is 

further evidence that calcium movement is a primary factor in 

root gravitropism. (2) The redistribution of radioactive calcium 

within tissues of the elongation zone of gravistimulated roots 

was studied. Although no polar movement of radioactive calcium 

was seen across the elongation zone (in contrast with the root 

cap), there was, however, a strong asymmetry of labeled calcium 

within the elongation zone, with calcium accumulating along the 

lower side. These recent findings open up new ways of 

investigating the role of calcium in root gravitropism. 

Experiments on photomodification of graviinduced calcium movement 

and on calcium redistribution within tissues of the elongation 

zone will constitute a major part of the research effort in the 

coming year. 

PUBLICATIONS 

Evans, M., Lee, J., and Mulkey, T. Gravity-Induced Polar 

Transport of Calcium Across Root Tips of Maize. Plant Physiology 

72(1, Suppl.): 63, 1983. 

Evans, M.L. The Mechanism of Action of Auxin in the Promotion of 

Cell Elongation. In: Aspects of Physiology and Bio-chemistry of 

22 
plant Hormones (ed. by S.S. Purohit). New Delhi: Kalyani 

Publishers, p. 69-92, 1983. 

Evans, M.L. and Mulkey, T.J. A Reevaluation of the Role of 

Abscisic Acid in Root Gravitropism. Physiologist 25(6, Suppl.): 

S109-Sl10, 1983. 

Lee, J.S., Mulkey, T.J., and Evans, M.L. Gravity-induced Polar 

Transport of Calcium Across Root Tips of Maize. Plant Physiology 

73: 874-876, 1983. 

Lee, J.S., Mulkey, T.J., and Evans, M.L. Reversible Loss of 

Gravitropic Sensitivity in Maize Roots after Tip Application of 

Calcium Chelators. Science 220: 1375-1376, 1983. 

Mulkey, T., Evans, M., Lee, J., and Kuzmanoff, K. Effects of 

Calcium Entry Blockers on Growth and Gravitropism of Maize Roots 

(Abstract). Plant Physiology 72(1, Suppl.): 63, 1983. 

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. 

23 
GRAVISTIMULUS PRODUCTION IN ROOTS OF CORN 

Lewis J. Feldman 

Department of Botany 

University of California 

Berkeley, CA 94720 

The perception of gravity by roots occurs in a specialized region 

of the root known as the root cap. To understand the biochemical 

steps involved in translating the gravity stimulus into a growth 

response, the occurrence in the root cap of two specific 

processes has been examined: (1) protein synthesis and turnover 

and (2) carotenoid synthesis and turnover. Evidence has been 

obtained supporting the notion that both of these groups of 

compounds are involved in the gravity translation process. 

For this research, a variety of corn has been used which, if 

germinated and maintained in darkness, fails to exhibit any 

response by the primary root to gravity. Illumination of the 

root, specifically illumination of the root cap, with white 

light causes the root to respond to gravity and to bend downward. 

It had been shown that inhibiting protein synthesis within the 

root cap prevents the root from responding to gravity when 

illuminated. During 1983 the involvement of protein synthesis 

within the cap in the processing of the gravity stimulus was 

examined in more detail in order to determine whether the gravity 

translation mechanism involves: 

(1) de novo synthesis of one or more unique proteins, 

(2) a-general enhancement in the levels of many proteins, 

or 

(3) an enhancement in the levels of selected proteins. 

This work has shown that light markedly stimulates the levels of 

several groups of proteins which appear to be important in 

processing the gravity stimulus. However, since no de novo 

synthesis was shown, it is concluded that light, in part, affects 

gravitropic root bending by enhancing the levels of selected 

preexisting proteins in the root cap. Furthermore, it has been 

shown that in the dark these light-enhanced proteins turn over 

relatively rapidly, usually within a half an hour after returning 

the roots to the dark. 

A second and now major effort has focused on carotenoid synthesis 

and turnover in the root cap. It has been shown that light 

causes a 50-60% increase in carotenoid levels in caps, compared 

with levels in caps maintained in continuous darkness. Not all 

carotenoids follow this pattern, however. Within 5 min. of 

illumination, a specific carotenoid, violaxanthin, decreases to a 

level 50% of that observed in caps maintained in continuous 

darkness. In caps illuminated for periods in excess of 5 min., a 

24 
60-70% reduction occurs in the original level of violaxanthin. 

If roots are illuminated and returned to the dark, violaxanthin 

levels increase to that observed in dark control root caps. This 

light-stimulated turnover of violaxanthin is particularly 

intriguing because: 

(1) violaxanthin has been reported in other systems to be 

converted to compounds with plant growth inhibitor 

properties, and 

(2) current hypotheses suggest that roots bend in response 

to gravity because of the production and asymmetric 

redistribution of a growth inhibitor. 

The analysis of violaxanthin synthesis and turnover is 

continuing. In addition, examination of whether inhibitory 

growth substances increase in the cap concomitant with the 

turnover of violaxanthin has begun. 

A third aspect has focused on establishing whether an association 

exists between carotenoids and amy1oplasts, the organelles 

traditionally associated with gravity perception in roots. For 

this work, amyloplasts have been successfully isolated from root 

caps. To date the yields have been lower than hoped 

(approximately 30% intact). During 1984, improving the isolation 

procedure for amylop1asts will continue in order to increase the 

percent intact amyloplasts. 

PUBLICATIONS 

Feldman, L.J. Light-enhanced Protein Synthesis in 

Gravitropically Stimulated Root Caps of Corn. Plant Physiology 

72: 833-836, 1983. 

Feldman, L.J. and Gildow, V. Effects of Light on Protein 

Patterns in Gravitropically Stimulated Root Caps of Corn. Plant 

Physiology 74: 208-212, 1984. 

25 
STARCH DEPLETION AND GRAVITROPIC RESPONSE 

Arthur W. Galston 

Department of Biology 

Yale University 

New Haven, CT 06511 

Considerable correlational evidence supports the contention that 

especially dense starch-containing amyloplasts function as 

statoliths for gravity perception in higher plants. The data 

come from three types of experiments: (1) When 

amyloplast-containing root caps are surgically excised, 

gravitropic sensitivity disappears, but sensitivity reappears 

when the starch-containing caps are regenerated. (2) Genetic 

mutants with smaller amyloplasts show decreased gravisensitivity. 

(3) Depletion of amyloplast starch by hormonal treatments leads 

to decreased gravitropic sensitivity. While this paints a 

generally convincing picture, correlations alone do not give 

proof, and more evidence is required. 

It has been reported previously that gravisensitive stems of 

etiolated peas contain two types of amyloplasts: small 

unigranular amyloplasts (UGA), found extensively in cortex and 

pith, which do not visibly displace in a 1 x G field; and larger, 

multigranular amyloplasts (MGA), limited to bundle sheath 

parenchyma, cells that displace readily within 2-3 min. in a 

1 x G field. Methods have been developed for isolating each of 

these in bulk, and some biochemical data have been provided for 

the MGA, the putative gravireceptors in this plant (Gaynor and 

Galston, 1983). While preparing for further molecular 

investigations on these MGAs another technique has been used to 

get more physiological evidence for the essential role of the MGA 

in graviperception. 

It has been known for more than a century that plants exposed to 

near-freezing temperatures massively degrade starch to sugar. 

This effect has been used by several investigators to induce 

decreased gravitropic response and then to study recovery of full 

response. This technique has been used in conjunction with 

starch analyses to get more precise quantitative correlations 

between starch content and response to gravity. Individual pea 

plants in vials were exposed to 4 0 C for various periods, given a 

40-min. horizontal induction in the cold, then returned to the 

vertical position and allowed to curve for 1 hr. at 27 0 C. 

Figure 1 shows that the gravitropic response decreases with 

increasing duration of cold storage, with 12 hr. of cold showing 

near maximal effect. Cold storage caused alpha-amylase activity 

to increase and amyloplast starch content to decrease, although 

some MGA were still visible after 6 1/2 days at 4 0 C. 

Cotylectomy coupled with cold treatment induced both a more rapid 

loss of gravitropic sensitivity and a virtually complete absence 

26 
of MGA. When cotylectomized, cold-treated seedlings were 

returned to 27 0 C before gravitropic induction, their response to 

gravity was partially restored within 2 hr. if the initial cold 

period had not lasted too long (Figure 2). This early recovery 

of gravisensitivity precedes detectable starch increase, but 

later recovery is correlated with more starch. 

The most striking finding is that the presentation time (minimal 

induction time necessary to elicit a statistically significant 

curvature) rises as starch content declines during cold storage 

(Table 1). This establishes a precise quantitative correlation 

between these two parameters, and shows that only a -20% 

reduction in amyloplastic starch is sufficient to trigger a 

4-fold loss in ~ravitropic sensitivity. This experiment is to be 

repeated in an even more definitive way, that is, by analyzing 

MGA and UGA separately. 

PUBLICATIONS 

Britz, S.J. and Galston, A.W. Physiology of Movements in the 

Stems of Seedling Pisum sativum L. cv. Alaska. III. Phototropism 

in Relation to Gravitropism, Nutation, and Growth. Plant 

Physiology 71: 313-318, 1983. 

Dumortier, F.M., Flores, H.E., Shekhawat, N.S., and Galston, A.W. 

Gradients of Po1yamines and Their Biosynthetic Enzymes in 

Coleoptiles and Roots of Corn. Plant Physiology 72: 915-918, 

1983. 

Dumortier, F.M., Flores, H.E., Shekhawat, N.S., and Galston, A.W. 

Reverse Polyamine Gradients in Etiolated Pea and Corn Seedlings 

(Abstract). Plant Physiology 72(1, Suppl.): 110, 1983. 

Flores, H.E. and Galston, A.W. Osmotic Stress-induced Polyamine 

Accumulation (Abstract). Plant Physiology 72(1, Suppl.): 106, 

1983. 

Galston, A.W. Leaflet Movements in Samanea. In: The Biology of 

Photoreception (ed. by D.J. Cosens and D. Vince-Price). New 

York: Cambridge University Press, p. 541-559, 1983. (Society for 

Experimental Biology Symposium 36). 

Galston, A.W. Polyamines as Modulators of Plant Development. 

BioScience 33: 382-388, 1983. 

Galston, A.W., Flores, H.E., and Young, N.D. Putrescine 

Formation by Arginine Decarboxylase: A Stress Response in Cereal 

Leaves (Abstract). Plant Physiology 72(1, Suppl.): 105, 1983. 

Gaynor, J.J. and Galston, A.W. Purification and Characterization 

of Amyloplasts from Etiolated Epicotyls of Pisum sativum. Plant 

and Cell Physiology 24(3): 411-421, 1983. 

27 
Table 1. Effect of Incubation at 4°C on the Presentation Time and 

the Starch Content of Coty1ectomized Pea Seedlings 

Treatment Presentation time 

Control 27°C 3.4 + 

- 1.1 

1.0 day 4°C 12.3 + at 4.9 + 3.5 days at 4°C 22.8 + 9.6 

6.0 days at 4°C 60.4 - 11. 2 

CUR\.lATURE 36. 

r (DEGREES) 

AFTER 1 HR 30. 

~~ 

20. ~ 1 1 1 

10. l'l-t-t- -1_ -I 1 1 

0. 

0. 1. 2. 3. 4. 5. 6. 

Figure 1. 

Ct.lRUAruRE 

(DEGREES) 

AFTER 1 HR 

Figure 2. 

TIME (CAYS) 

-0- 27 C CONTROL -~- COLO TREATED (4 C) 

20. 

10. 

~--1 1 

0. 1. 2. 3. 4. 5. 

HOURS OFRECOVERV AT 27 C 

-0- 2 HR COLD TREATED 

-0- 3 DAYS COLD TREATED -~- G DAYS COLO TREATED 

28 

Starch content 

(ll g starch/ mg pro te in) 

118 + 6 

94 + 10 + 72 

+ 

4 

56 - 9 

7. 

6. 
THE ROLE OF GRAVITY IN REGULATION OF LEAF BLADE FORM 

Alice Bourke Hayes 

Department of Natural Science 

Loyola University of Chicago 

Chicago, IL 60611 

The objective of this research is to understand how gravity 

influences the mechanisms that maintain the flat form and 

horizontal position of the leaf. The system studied is the 

nastic curvature of the primary leaves of the pinto bean 

(Phaseolus vulgaris L. var pinto) in response to auxin treatment, 

clinostat rotation, and other chemicai and environmental factors. 

The basic question being addressed is: What causes the unequal 

growth that produces curvature? The related questions are: What 

cells are involved? Why do some cells respond and not others? 

How do they respond--elongation or inhibition? What are the 

roles of auxin and ethylene in the cellular response? How do 

cellular responses relate to the responses of the whole leaf in 

determining blade form and position? 

The following methods have been used: 

(1) Whole leaf responses to various treatments have been 

quantitated by bioassay techniques developed by this laboratory 

to monitor curvatures of blade, petiole, and pUlvinus; (2) 

standard gas chromatography has been used to determine the time 

course, location, and stimuli for ethylene production by the 

leaf; (3) cytological studies, using scanning electron 

microscopy, epidermal films, and histological sections, have 

helped identify and localize cellular responses; and (4) 

transport of labeled indole-3-acetic acid (IAA) through leaves 

has been studied by standard donor-receiver techniques and 

quantitated with a liquid scintillation counter. 

Comparison of hyponastic with epinastic leaves has revealed that 

both the upper and lower epidermal cells can respond to auxin 

treatment by differential elongation, producing curvature. 

However, the upper epidermis does not respond to auxin treatment 

unless an inhibitory agent, such as an auxin transport inhibitor 

(TIBA, NPA), ethylene, or clinostat rotation, affects the 

distribution of auxin in the leaf. Auxin distribution and the 

differential sensitivity of upper and lower epidermal cells 

determine whether the response will be epinastic or hyponastic. 

Studies during 1983 have also shown that it is the interaction of 

blade, pulvinus, and petiole, rather than the response of anyone 

part of the leaf, that constitutes the significant response to 

orientation to gravity. It has been demonstrated that leaf blade 

response is auxin regulated, leaf angle response is ethylene 

requiring, and that the two responses are linked by the 

29 
relationship between auxin and ethylene synthesis in the blade. 

The important observation that dorsally applied 14C-IAA 

accumulates in ventral receivers when a blade tissue section is 

in a normal orientation to gravity, but that it does not so 

accumulate when the blade is inverted, is the first demonstration 

of dorsiventral transport through the blade. Earlier studies 

with auxin transport inhibitors, inversion and clinostat 

treatments, and other incubation conditions had anticipated this 

observation. 

Other factors that affect nastic curvature were also 

investigated. It was found that low pH buffer did not promote 

hyponasty in statistically significant amounts, nor did sodium 

vanadate, an inhibitor of ATP-driven proton secretion, produce 

significant epinasty. However, when applied to the lower surface 

of the leaf opposite the IAA upper, it did inhibit hyponasty. 

Also, it had been noted earlier that dark treatment promotes 

hyponasty and bright light inhibits it. Responses to red, far 

red, blue, green, and white light are now being examined. 

Preliminary studies show blue light inhibits hyponasty and far 

red promotes it. 

PUBLICATIONS 

Hayes, A.B. Auxin Transport Inhibitors and Ethylene in Pinto 

Bean Leaf Epinasty (Abstract). Plant Physiology 72(1, Suppl.): 

106, 1983. 

30 
EFFECTS OF GRAVITY AND LIGHT ON GROWTH, DEVELOPMENT, AND 

FLOWERING IN PLANTS: II. MULTIPLE GENERATIONS ON CLINOSTATS 

Takashi Hoshizaki 

Jet Propulsion Laboratory 

California Institute of Technology 

Pasadena, CA 91109 

The effect of a long-term microgravity environment on the life 

cycle of plants is poorly understood. Whether higher plants have 

evolved to a stage where removal or reduction of gravity is 

detrimental to the plant life cycle and thus fatal to the plant 

species is an unanswered question. In space, plants have been 

successfully grown through the various stages of their life 

cycle. Attempts to grow plants in space as a continuous integral 

process from seed to seed through one generation have not been 

successful until recently. 

Culture of plants through multiple generations has not been 

accomplished in space or in ground-based simulated weightlessness 

studies. The effect of long-term simulated weightlessness is 

being investigated by growing three consecutive generations of 

plants continuously on clinostats using the cruciferous plant 

Arabidopsis thaliana (L.) Heynh. 

Three generations of Arabidopsis have been grown on clinostats. 

Two generations (Fl and F2 ) were successfully grown from seed-toseed on clinostats. The third (F 3 ) generation is now being 

tested. In analyzing leaf, stem, and seed pod development of the 

Fl and F2 generations, a significant delay in the formation and 

development of seed pods was found in both generations of 

clinostatted plants, compared with vertically rotated and 

stationary upright controls. Attention will be focused on the 

delay response in the Fl and F generations to see if the delay 

response appears again in the ~3 generation. Soviet workers have 

recently reported a similar delay in Arabidopsis grown in space 

through one generation. This delay found in plants grown both in 

space and on clinostats may indicate that gravity is a 

requirement during the reproductive process of Arabidopsis. 

Seeds representing the FI generation were tested for viability. 

The crop was divided into those seeds from dry mature pods and 

those from pods still green at harvest. The germination rate was 

significantly higher for seeds from dry pods (95.9 ~ 0.9%) than 

those from green pods (83.6 + 2.5%). Abnormal seedlings were 

significantly lower in the dry pod population (0.37 + 0.26%) than 

in the green pod population (1.69 ~ 0.21%). These results will 

be compared later with seeds from the F3 generation grown on 

clinostats. 

The effect of closed cultures on the growth and development of 

31 
Arabidopsis was studied. The closed-culture environment 

represents typical cultural conditions found in plant experiments 

conducted in space. Closed cultures sealed with vinylidene 

polymer film had little or no gas exchange with the ambient 

atmosphere. Cultures plugged with polyurethane foam and having 

ample gas exchange were used as controls. Closed-culture plants, 

compared with controls, produced a larger number of leaves, 

stems, and flowers but did not form seed pods. It was not known 

whether low CO2 concentration or accumulation of metabolic 

products such as ethylene or excess moisture were singly or in 

combination responsible for the developmental and morphological 

differences. Thus, a CO 2 analysis of the in vitro head gas of 

closed Arabidopsis cultures was conducted. Instead of the 

expected concentrations of less than 300 ppm of CO , 

concentrations of around 146,000 ppm and a concomitant decrease 

of 02 concentrations were found in closed 8-wk.-old cultures. 

These results are compatible with those from a I-wk. STS-3 flight 

where unexpectedly high levels of CO2 were detected (50,000 ppm). 

In the experiments reported here, high CO2 levels appeared to 

coincide with bolting and flowering. A daily rhythm in C02 

concentration was also found. Preliminary tests for bacterial 

contamination have proven negative. Further studies are 

necessary to identify the source of the CO2 in closed cultures. 

Growth and morphological data have been obtained for Arabidopsis 

plants grown for two generations on clinostats. A significant 

delay in seed pod formation was found in plants subjected to 

simulated microgravity. Since no information is available on the 

effect of continuous true weightlessness on the growth, 

flowering, and seed production in sequential generations of 

plants, plans are being made to carry out studies in space using 

Arabidopsis thaliana (L.) Heynh. 

PUBLICATIONS 

Hoshizaki, T. 

Arabidopsis. 

Clinostat Effects on Shoot and Root of 

Physiologist 26(6, Suppl.): S15l-Sl52, 1983. 

Hoshizaki, T. Seed to Seed Growth of Arabidopsis on a Clinostat 

(Abstract). Plant Physiology 72(1, Suppl.): 62, 1983. 

32 
A STUDY OF THE MECHANISM OF THIGMOMORPHOGENESIS IN PLANTS, WITH 

SPECIAL REFERENCE TO THE ROLE OF ETHYLENE AND ITS SIGNIFICANCE TO 

RESEARCH WITH PLANTS IN SPACE 

M.J. Jaffe 

Biology Department 

Wake Forest University 

Winston-Salem, NC 27109 

The objective of this research project is to gain a thorough 

understanding of the response of plants to physical force. 

Emphasis is placed on those forces th~t result in mechanical 

stress and strain on the plant tissues. The most common forms of 

such forces are wind blowing through plants, the resistance of 

soil particles to emerging seedlings, animals and other plants 

rubbing against the plants, and the force of gravity, omnipresent 

on the surface of the Earth, pulling on plant tissues. The 

adaptive response by plants to such mechanical perturbations is 

known as thigmomorphogenesis. Gravitropism, the directional 

response of plants to changes in gravitational orientation, has 

many characteristics in common with thigmomorphogenesis and may 

be a result, in part, of a common mechanism. 

During 1983, research has primarily focused on the role of 

ethylene in thigmomorphogenesis and its relationship with the 

rapid deposition of the beta 1-3, glucan, callose, and the 

production of elicitors. Also, since any handling constitutes a 

mechanical perturbation of the plants, development of 

nonintrusive methods of examination using computer-assisted video 

image analysis have continued. 

Evidence now quite clearly indicates a role for ethylene in the 

response of plants to mechanical force. As a result of exposure 

to mechanical perturbations, ethylene is produced from increased 

synthesis of l-aminocyclopropane-I-carboxylic acid (ACC). ACC 

production increases within 20 min. of mechanical perturbation 

and is converted to ethylene which becomes detectable about 45 

min. after perturbation and peaks at 4-5 hr. The ethylene 

produced triggers a wide variety of changes in growth and 

development of the plant, generally resulting in a plant of 

shorter final height and increased girth. 

Analysis of these changes in growth pattern has been facilitated 

by a computer-assisted image analyzer which can quantify callose 

deposition and measure changes in cell size and shape, as well as 

whole plant dimensions. Examination of pine seedlings has 

indicated an increase in bark and resin production, following 

mechanical perturbation, as well as decreased shoot and needle 

growth. In bean and tomato plants, mechanical perturbation not 

only reduces elongation but inhibits the formation of a hollow 

33 
stem (pithiness development). This appears to reduce the effects 

of drought stress. 

To better understand these types of developments, an examination 

of subcellular structure is continuing. Of particular interest 

has been the observation of numerous changes in the membrane 

lipid and protein complement of the cells. Mechanical 

perturbation results in reduction of the unsaturated to saturated 

ratio of the fatty acids. Phosphat idyl choline levels decrease 

and phosphat idyl ethanolamine levels increase. Membraneassociated protein levels increase as does the activity of 

Golgi-associated IDPase, but cytochrome C reductase levels 

decline. 

Little is yet known about the events leading to the increased 

production of ethylene in mechanically stressed plants. However, 

at least two early observable events, preceding or concurrent 

with the production of ethylene, appear closely tied to ethylene 

production. The deposition of callose in the cell walls occurs 

almost immediately following any stress. This deposition peaks 

5-6 hr. later and then callose levels in the tissue slowly 

decline over the next 12-24 hr. An inhibitor of protein 

glycosylation (DDG) has been shown to block callose deposition as 

well as ethylene synthesis and thigmomorphogenesis. This 

compound has also been shown to block gravitropism in etiolated 

corn and pea shoots. Furthermore, examination of the activity of 

beta 1-3 glucanase, the callose degrading enzyme, indicates a 

rise in this enzyme's activity following mechanical perturbation, 

which may account for the decline in callose levels commencing 

about 6 hr. after mechanical perturbation. 

Elicitors are relatively small molecules of varied structure 

that are released by plants in response to some biotic or abiotic 

stress. They are called elicitors because they induce the 

production of "stress metabolites." One of these metabolites is 

ethylene. Elicitors have been shown to be produced by bean 

plants in response to mechanical perturbation. Furthermore, an 

elicitor extracted from bean plant cell wall material has been 

further shown to induce ethylene production in nonperturbed 

plants and result in an increased thickening of the stems. Known 

elicitors, from other systems, have also been shown to promote 

ethylene production in beans and increased radial growth. 

In experiments begun at the end of 1983, attempts were made to 

treat plants with beta 1-3 glucans to see if they will induce 

ethylene production. So far, indications are that beta 1-3 

glucans, when applied exogenously, do induce ethylene production. 

PUBLICATIONS 

Erner, Y. and Jaffe, M.J. Thigmomorphogenesis: Membrane Lipid 

and Protein Changes in Bean Plants as Affected by Mechanical 

34 
Perturbation and Ethrel. Physiologia Plantarum 58: 197-203, 

1983. 

Pressman, E., Huberman, M., Aloni, B., and Jaffe, M.J. 

Thigmomorphogenesis: The Effect of Mechanic~l Perturbation and 

Ethrel on Stem Pithiness in Tomato [Lycopersicon esculentum 

(Mill.) Plants]. Annals of Botany 52: 93-100, 1983. 

Takahashi, H. and Jaffe, M.J. Thigmomorphogenesis: Elicitor 

Activity in Response to Mechanical Perturbation and its Role in 

the Accumulation of Phytoalexin-like Stress Metabolite Via 

Ethylene Production (Abstract). Plant Physiology 72(1, Suppl.): 

64, 1983. 

Telewski, F. and Jaffe, M.J. On the Mechanism of 

Thigmomorphogenesis in Conifers (Abstract). Plant Physiology 

72(1, Suppl.): 64, 1983. 

Telewski, F.W., Wakefield, A.H., and Jaffe, M.J. Computerassisted Image Analysis of Tissues of Ethrel-treated Pinus taeda 

seedlings. Plant Physiology 72: 177-181, 1983. 

35 
GRAVITY PERCEPTION, TRANSDUCTION, AND CELLULAR RESPONSE IN 

GRAVISTIMULATED SHOOTS OF CEREAL GRASSES 

Peter B. Kaufman 

Department of Cellular and Molecular Biology 

Division of Biological Sciences 

University of Michigan 

Ann Arbor, MI 48109 

The specific objectives in this project are to shed light on how 

gravity is perceived in cereal grass shoot pulvini, learn how and 

when hormone asymmetry is established during transduction in 

gravistimulated pulvini, and what the physiological and 

biochemical bases are for the asymmetric growth response that 

occurs during upward bending of gravistimulated cereal grass 

pulvini. 

In an effort to learn how gravity is perceived in grass shoot 

leaf-sheath pulvini, the kinetics for fall of the presumed 

graviperceptive organelles, the starch statoliths, were 

determined. In oats and barley, they start to descend in 

gravistimulated shoots within 15 sec., and essentially all (ca. 

50 per cell) have reached the bottoms of the statenchyma cells 

(located just inside the ring of vascular bundles) within 2 min. 

This is well before the pulvini start to bend upwards, namely, 

15-20 min. after first being gravistimulated. 

In order to find out whether starch statoliths in the pulvini 

are, in fact, the graviperceptive organelles, pulvini were 

treated with alpha amylase (from hog pancreas, which hydrolyzes 

only alpha l,4-linked glucan = starch). After a 24-hr. treatment 

on a slow shaker in the dark, the amyloplasts were observed, 

microscopically, to be devoid of all starch (based on I2KI 

staining). Furthermore, such pulvini did not show upward bending 

when placed horizontally. Such alpha amylase-treated pulvini 

were then placed in 0.1 M sucrose for a second 24-hr. period, in 

an upright position. They were found not only to have 

resynthesized starch in the amyloplasts, but also to have 

regained the ability to bend upwards when gravistimulated. It is 

thus seen that starch statoliths in the leaf-sheath pulvini are 

indeed the graviperceptive organelles, as they are in root caps. 

Further progress was made in learning which hormones are 

asymmetrically distributed in gravistimulated pulvini of both 

oats and maize. They include three types of hormones: 

gibberellins (GA3 , GA4 , and GA7 in oats: GAl' GA4 , GAS' and GA20 

in maize): auxin (indole-3-acetic acid, or AA): and ethylene. 

The ethylene asymmetry in oat leaf-sheath pulvini is only first 

seen 6 hr. after shoots are gravistimulated, almost 5.5 hr. after 

shoots first start to bend upwards. So the production of 

ethylene is the result of gravistimulation, probably resulting 

36 
from either the stress imposed by gravitropic bending, or more 

likely, the asymmetry in IAA which occurs in gravistimulated 

pulvini. The latter is believed to be true because in 

gravistimulated maize seedlings, auxin asymmetry (top vs. bottom 

half, with significantly more in the bottom half), as expressed 

by amounts of free IAA present, is seen as early as 15 min. after 

seedlings are gravistimulated. Tissue fractions of pulvini of 

oats were prepared to determine the precise time course for 

establishment of asymmetry of both native GAs and free IAA and 

their respective conjugates from the time that bending first 

begins until it ceases. It is already known that the asymmetry 

in free GAs and free IAA is well established after 24 hr. of 

bending, when the shoots have bent upwards to about 30 0 (and that 

GA conjugates show the reverse ratio as the free GAs--more on the 

tops for the conjugates and more on the bottoms for the free 

GAs). It is now essential to determine when the asymmetry is 

first established for both hormones in relation to the time 

course of upward bending--a major project started in 1983 and due 

to be completed in the summer of 1984. 

In connection with elucidating the biochemical and physiological 

bases for the asymmetric growth response in gravistimulated grass 

shoot pulvini, extensive polyacrylamide gel electrophoresis and 

isoelectric focusing were done to analyze protein changes, 

especially since it is already known that protein synthesis is 

essential for the bending response to occur. As early as 2 hr. 

after shoots are first gravistimulated, changes in protein occur 

in tops vs. bottoms of horizontally placed pulvini, as compared 

with "left" and "right" halves of upright pulvini. A significant 

increase is seen in amounts of five specific acidic proteins in 

the bottom halves, as compared with the top halves. They are 

currently being identified. One may be a cellulase; another may 

be invertase. Wall-loosening enzymes are likely candidates, as 

are enzymes involved in cell wall synthesis (the latter process, 

like protein synthesis, is essential for gravitropic bending to 

occur). Finally, methods have been devised for analyzing the 

activities of key wall-loosening enzymes during the entire time 

course of upwards bending in gravistimulated oat shoot pulvini. 

PUBLICATIONS 

Bandurski, R.S., Schulze, A., Dayanandan, P., and Kaufman, P.B. 

Asymmetric Distribution of Endogenous Indole-3-acetic Acid in Zea 

Mesocotyl Cortex Following Gravistimulation (Abstract). Plant---

Physiology 72(1, Suppl.): 146, 1983. 

Dayanandan, P. and Kaufman, P.B. Analysis and Significance of 

Gravity-induced Asymmetric Growth in the Grass Leaf-sheath 

Pulvinus. Annals of Botany 53: 29-44, 1984. 

Dayanandan l P. and Kaufman, P.B. Evidence for a Radial, 

Centrifugal Transport of Growth-promotors in the Leaf-sheath 

37 
Pulvini of Grasses (Abstract). Plant Physiology 72(1, Suppl.): 

146, 1983. 

Harrison, M.A. and Kaufman, P.B. Estimates of Free and Bound 

Indole-3-acetic Acid and Zeatin Levels in Relation to Regulation 

of Apical Dominance and Tiller Release in Oat Shoots. Journal of 

Plant Growth Regulation 2: 215-223, 1983. 

Kaufman, P.B. Physiology and Biochemistry of Gibberellins in 

Developing Shoots of Grasses. In: Aspects of Physiology and 

Bio-Chemistry of Plant Hormones (ed. by S.S. Purohit). Ludhiana, 

India: Kalyani Publishers, p. 125-137, 1983. 

Song, I. and Kaufman, P.B. Changes in Protein Patterns during 

the Cource of GA3-promoted Growth in Oat Stem Segments 

(Abstract). Plant Physiology 72(1, Suppl.): 83, 1983. 

Thomas, R.J., Harrison, M.A., Taylor, J., and Kaufman, P.B. 

Endogenous Auxin and Ethylene in Pellia (Bryophyta). Plant 

Physiology 73: 395-397, 1983. 

38 
PLANT CELLS, EMBRYOS, AND DEVELOPMENT IN SPACE 

A.D. Krikorian 

Department of Biochemistry, Division of Biological 

Sciences 

State University of New York at Stony Brook 

Stony Brook, NY 11794 

The ultimate objective of the overall research plan has been to 

ascertain whether flowering plants can carry out their full 

growth, development, and reproduction in a near-zero or 

hypogravity environment. Highly responsive experimental systems 

are being developed at different developmental or organizational 

levels--that is; (I) free protoplasts from higher plant cells 

which can deposit new cellulose walls, divide, multiply, embark 

upon organized development, and ultimately give rise to organized 

plantlets; (2) free somatic cells which by division under aseptic 

and heterotrophic conditions may express morphogenetic competence 

and form somatic embryos which can, in turn, develop into 

plantlets; and (3) at the level of plants as they develop from 

seeds or predetermined growing points. 

The daylily (Hemerocallis) monocotyledonous system which has been 

developed provides an excellent model for studying development in 

space because plant lets can now be generated equally efficiently 

from aseptically cultured cells, protoplasts, and tissues. The 

chromosomes are relatively large and few in the diploid state 

(n=ll); these have been studied in detail each step along the way 

to full development. Efforts have continuously been made to 

improve precision in these procedures. But it has not been 

possible to carry out chromosome analysis on materials that have 

not been treated with cytostatic agents such as colchicine, 

vinblastine, or l-bromonaphthalene. These cytostatic agents can 

easily be used in an on-Earth setting but it is impossible to 

envision their use in space without a sophisticated piece of 

hardware being developed. This hardware would have to expose 

materials (root tips, for example) to a predetermined level of 

the cytostatic agent for a specific period of time at a 

relatively low temperature (say 100 C). This procedure would be 

followed by a thorough rinsing and then another exposure to a 

fixative (generally comprised of ethanol and hydrochloric acid) 

at a still different temperature and environment. All this, in 

turn, requires still other procedures. So while it may sound 

easy to say materials can or will be fixed in space, the 

connotation of "fixation" varies with the task to be achieved. 

Straightforward fixation, as performed for light or electron 

microscopy, may be difficult enough in itself to achieve in a 

space environment, but for karyological or chromosomal 

investigations, inflight fixation presents problems at a still 

greater level of difficulty. 

39 
The utilization of cold treatment as a cytostatic agent has been 

investigated. This would permit vastly greater degrees of 

flexibility for studying cytological events in the space 

environment. Materials could be chilled prior to flight to 

suppress or reduce their cell division activity. Once in orbit, 

ambient or physiologically permissive temperatures could be set 

in motion, and, once again, at critical stages in development, 

plant materials could be chilled or exposed to cold to arrest the 

processes. The material once chilled to a sufficient level could 

be maintained quiescent until recovery when the so-called 

"fixation" stage could be carried out. Numerous tests carried 

out during 1983 demonstrate that this is a viable approach, and 

efforts have been made to define precisely the level and length 

of cold exposure required to arrest oats, daylily, and sunflower 

in metaphase, and to evaluate the quality of the preparations in 

terms of acceptability for karyotype preparation and measurement. 

Some 72 hr. of cold (loC) is needed to fully bring about 

metaphase arrest in oats. In terms of potential Shuttle 

investigations, this means that an experiment could, for all 

practical purposes, be terminated in space well before recovery; 

similarly, just before deorbit, materials could be exposed to 

cold. The potential means whereby the effects of reentry, etc., 

vs. actual microgravity can be distinguished seem feasible. The 

daylily and sunflower similarly appear amenable to cold as well. 

It is anticipated that the successful use of cold can be extended 

equally well to cells and protoplasts. Preliminary experiments 

indicate that this will be possible. 

Another feature of the work during 1983 has been the rigorous 

establishment of an explantation procedure for daylily from 

meristems of karyotyped plants which permits exact chronological 

scheduling of fresh suspension cultures for study without the 

intervention of a phase of growth on semisolid media. Explants 

are directly placed in liquid. The precision with which the 

final desired product can now be achieved is thus shortened; this 

eliminates any undesired changes in the cell types. 

It is hoped, further, that knowledge of the use of cold will 

permit clarification of the kind of instrument that might best be 

designed for cytological studies. 

PUBLICATIONS 

Cronauer, S.S. and Krikorian, A.D. Multiplication of Musa from 

Excised Stem Tips. Annals of Botany 53: 321-328, 1984-.---

Fitter, M.S. and Kirkorian, A.D. Generation of Daylily Plant lets 

from Cells Grown in Suspension (Abstract). In: Genetic 

Engineering: Applications to Agriculture (ed. by L.D. Owens). 

Totowa, N.J.: Rownan: Allanheld, p. 5, 1983. 

40 
Keefe, J.R. and Krikorian, A.D. Gravitational Biology on the 

Space Platform. Paper presented at the 13th Intersociety 

Conference on Environmental Systems, July 11-13, 1983, San 

Francisco, CA, 23 p., 1983. (SAE Technical Paper Series 831133) 

Krikorian, A.D. Aseptic Culture Systems for Experimentation in 

Space (Abstract). Plant Physiology 72(1, Suppl.): 64, 1983. 

Krikorian, A.D. Developing High Plant Systems in Space. In: The 

Regulatory Functions of Calcium and the Potential Role of Calcium 

in Mediating Gravitational Responses in Cells and Tissues (ed. by 

S.J. Roux). Washington, D.C.: NASA, p. 83-96, 1983. (NASA 

Conference Publication 2286) 

Nwankwo, B.A. and Krikorian, A.D. Aseptic Storage of Elaeis 

guineensis form pisifera seeds. Principes 27: 34-37, 1983. 

Nwankwo, B.A. and Krikorian, A.D. Morphogenetic Potential of 

Embryo- and Seedling-derived Callus of Elaeis guineensis Jacq. 

var. pisifera Becc. Annals of Botany 51: 65-76, 1983. 

41 
THE AMYLOPLAST AS A GRAVITY-SENSING DEVICE IN PLANTS 

A. Carl Leopold and Fred D. Sack 

Boyce Thompson Institute 

Ithaca, NY 14853 

During 1983, the primary focus has been determining the dynamics 

of the gravity-induced reorientation of amyloplasts in the 

statocytes of corn seedlings. While there is a large body of 

evidence that the movement of amyloplasts plays a central role in 

gravity sensing, the transduction mechanism for converting 

amyloplast movement into a physiological gradient has not been 

identified. Knowledge of the parameters of the dynamics of 

amyloplast movement, especially during the presentation time, is 

essential in order to develop relevant hypotheses about the 

nature of the transduction mechanisms. 

In corn coleoptiles, the presentation time (the minimum time that 

a seedling needs to be horizontal to induce subsequent 

gravitropic curvature) has been determined using a log plot of 

the gravistimulation period against curvature (measured 90 min. 

after the start of gravistimulation). Because the amount of 

curvature increases roughly logarithmically with increased 

gravistimulation, this type of plot approximates a straight line. 

The intercept with the abscissa, which will be the minimal time 

for a gravitropic response, yields a value of 40 sec. Thus, the 

dynamics of amyloplast movement within this 40-sec. period will 

be of particular interest. 

Utilizing coleoptiles that were fixed after various periods of 

gravistimulation and then sectioned, a similar plot for 

amyloplast redistribution in the cell has been made. The 

intercept of the log of gravistimulation time against the number 

of amyloplasts close to the new lower wall gain yields a straight 

line, and the intercept of this line with the abscissa yields a 

value of 11 sec. for the cells on the upper side of the 

coleoptile and 15 sec. for those on the lower side. Thus, the 

commencement of amyloplast arrival to the new lower wall during 

gravistimulation occurs in less than half of the presentation 

time. Furthermore, up to 21% of the total sedimentation occurred 

within the first 30 sec., and this amount of sedimentation was 

statistically significant when compared with zero time (vertical) 

control plants. 

Observation of amyloplast movement was also made in living 

statocytes using a horizontal microscope equipped with Nomarski 

optics and a video camera and recorder. These observations 

confirmed that rapid sedimentation occurs in living cells as 

well. 

Collectively, these measurements of sedimentation dynamics are 

42 
consistent with the classical statolith hypothesis that the 

transduction of amyloplast movement into a physiological gradient 

may occur near the plasmalemma of the new lower wall. 

Living cells were also examined by video microscopy to observe 

the process as well as the kinetics of amyloplast sedimentation. 

Sedimentation is decidely not a simple case of inert spheres 

falling in a Newtonian fashion in response to gravity. 

Amyloplasts move in a heterogeneous fashion and cytoplasmic 

streaming is clearly involved in producing this variability. 

Amyloplasts move fastest when falling through cytoplasmic streams 

flowing in a downward direction. Conversely, sedimentation may 

be deferred for a substantial period of time if the amyloplast 

faces an upward s~ream. The range of the rates of amyloplast 

movement (0-210 gm./min.) was found to be much larger than had 

been calculated by previous workers from fixed material (0.3-1.2 

~./min.). This high range in rates would not be detected in 

fixed tissue where average rates are determined for whole 

popUlations of amyloplasts rather than by direct calculation of 

rates for individual amyloplasts in living cells. 

Streaming affects not only the rates of sedimentation, but also 

the pathway that individual amyloplasts follow during the gravity 

response. It is common for amyloplasts to be swept along by the 

streaming cytoplasm into a lateral or even an upward direction. 

These perambulations of amyloplasts bring them into contact with 

numerous other organelles, especially the vacuole. Interaction 

of moving amyloplasts with other cytological structures such as 

endoplasmic reticulum or possibly microtubules or microfilaments 

has not yet been observed in living cells. 

Cytochalasin B, which halts cytoplasmic streaming, has been 

employed to explore the relationship between cytoplasmic 

streaming and amyloplast movement. Methods have been developed 

for introducing this poison into living sections while they are 

under observation with the video microscope. When the poison 

suspends streaming, the rate of amyloplast sedimentation is 

somewhat reduced. These results suggest that cytoplasmic 

streaming may play a role as an amplifier of the amyloplast 

component of gravity sensing in the corn coleoptile. 

PUBLICATIONS 

Sack, F.D. and Leopold, A.C. Interaction of Amyloplast 

Sedimentation and Streaming in Coleoptile Cells of Corn 

(Abstract). Plant Physiology 72(1, Suppl.): 89, 1983. 

Sack, F.D., Priestley, D.A. and Leopold, A.C. Surface Charge on 

Isolated Maize Coleoptile Amyloplasts. Planta 157: 511-517, 

1983. 

43 
MECHANICAL REGULATION OF PLANT GROWTH AND DEVELOPMENT 

Cary A. Mitchell 

Department of Horticulture 

Purdue University 

West Lafayette, IN 47907 

On Earth, gravity is the predominant physical force governing 

plant orientation, and contributes to the pattern and amount of 

mechanical tissue deposition within the plant as well. Seismic 

(shaking) and vibric (vibrational) forces resulting from wind 

action alter patterns of mechanical tissue formation, and 

certainly limit plant growth in the outdoor environment. 

Furthermore, traumatic (wounding), haptic (contact), and thigmic 

(rubbing) stimuli caused by certain types of precipitation, 

animal movements, etc., certainly contribute to the overall 

effects of mechanical stress on plant growth and development. In 

space, the static force of gravity is lacking, but dynamic 

physical forces such as shaking and vibration may assume an even 

more important role in altering or directing the growth of plants 

within an orbiting spacecraft, compared with on the ground. On 

the one hand, there is concern that spacecraft vibration, 

particularly if vectorial, may mimic the force of gravity and 

confound attempts to determine effects of true weightlessness on 

plant development; on the other hand, a little physical 

stimulationlTIay benefit plants by stimulating the development of 

mechanical support tissue that might be needed for normal 

development. The net effects of mechanical stresses on plants 

growing under otherwise microgravity conditions awaits the 

results of experiments to be performed under real spaceflight 

conditions. 

Soybean (Glycine max (L.) Merr. cv. Wells II) plants grown and 

shaken in a g~eenhouse or growth chamber on the ground develop 

shorter and thinner internodes, smaller leaves, and roots and 

shoots with less fresh and dry weight than do undisturbed control 

plants. 

Seismic stress is applied by shaking soybean plants at 220-240 

rpm on a gyratory platform shaker. Five minutes of gyratory 

shaking applied three times daily retarded dry weight gain of 

vegetative soybean plants by 25% and leaf area growth by 27% over 

a l2-day treatment period in a growth chamber. Growth dynamics 

analysis revealed that a concomitant decrease in relative growth 

rate (RGR) was due entirely to a reduction in net assimilation 

rate (NAR) in the growth chamber. An inhibition of NAR implies a 

decline in photosynthetic efficiency caused by seismic stress. 

I Five minutes of gyratory shaking caused a temporary decrease in 

whole plant transpiration rate of 15-17% that lasted for at least 

90 min., as well as a 39% increase in leaf water potential 30 

44 
min. after treatment. Both effects are consistent with a 

transitory, stress-induced reduction in stomatal aperture. 

Photosynthetic rate (Pn) fell 10% during the 5-min. shake period, 

and continued to decline to 16% below controls 15 min. after 

treatment ended. Pn of shaken plants still averaged 12% less 

than that of controls 50 min. after treatment. Seismic stress 

also caused an immediate leaf droop of 310 from the equilibrium 

angle at the secondary pulvini immediately after shaking, a 

response from which the plants did not fully recover for as much 

as 2 hr. Leaflets of soybean plants never having been shaken 

previously recovered to their equilibrium position more slowly 

than did those of previously shaken plants. The extent to which 

the seismic stress-induced decline in RGR, NAR, and Pn are due to 

leaf reorientation vs. leaf resistance 'is under investigation. 

PUBLICATIONS 

Heuchert, J.C., Marks, J.S., and Mitchell, C.A. Strengthening of 

Tomato Shoots by Gyratory Shaking. Journal of the American 

Society for Horticultural Science 108: 801-805, 1983. 

Heuchert, J.C. and Mitchell, C.A. Inhibition of Shoot Growth in 

Greenhouse-grown Tomato by Periodic Gyratory Shaking. Journal of 

the American Society for Horticultural Science 108: 795-800, 

1983. 

Pappas, T. and Mitchell, C.A. Mechanical Stress Regulation of 

Growth, Transpiration, and Photosynthetic Productivity of Glycine 

max Merr. C.V. Wells II (Abstract). Plant Physiology 72{1, 

Suppl.): 62, 1983 

45 
TESTING A HYPOTHESIS: HOW THE PEA STEM SENSES GRAVITY, FRICTION, 

AND FLEXURE 

Barbara G. Pickard 

Biology Department 

Washington University 

Saint Louis, MO 63130 

A new model for reception by plant stems of gravity and of the 

related stimuli of friction and flexure is being tested. The 

mode12~pecifies (1) that all these stimuli increase the leakage 

of Ca across the plasmalemma and that in consequence certain 

proteins are phosphorylated; (2) that gravitropic phosphorylation 

activates a protein which transports auxin out of the lower sides 

of the cells, leading to a lateral migration of auxin across the 

stem and t~ differential stem elongation; and (3) that reception 

of friction and flexure involves phosphorylation of alpha- and 

beta-tubulins, which polymerize to form a "DeLorenzo polymer," 

ultimately causing synthesis of ethylene and consequent 

thickening and strengthening of the stem. 

The first phase of work, now completed, was testing the 

electrophysiological predictions of the model. A postulated 

consequence of the phosphorylation of alpha- and beta-tubulin is 

exocytosis of vesicles containing material which, upon release, 

depolarizes cell membranes. Transients, ranging in size up to 

600 ~V. and possessing risetimes (10-90%) of approximately 200 

msec. are now demonstrated t21be elicited by friction and by 

AG2-187, an iono hore for Ca ,as weI as b indoleacetic acid. 

Accor lng to the mode , gravltropic Ca leakage occurs through 

more specialized channels and the ion binds immediately to the 

proteins effecting phosphorylation; nevertheless a little leakage 

to the cytosol is expected. A 1.6-fold increase in voltage 

transients is now documented. This result, which is 

statistically reliable, is considered to be a striking success 

for the model. 

Current experimentation addresses phosphorylation occurring 

during gravitropism and f~2xural stimulation. After pretreating 

etiolated pea stems with Pi, stimulating, freezing in liquid 

nitrogen, extracting with appropriate buffers and inhibitors of 

proteolysis and dephosphorylation, running SDS polyacrylamide 

gels, and exposing the gels to X-ray film, there have been strong 

hints that phosphorylation of certain proteins increases. 

However, by letting the extracts sit for a few minutes before 

preparing samples for the gels, it could be demonstrated that 

phosphatase activity was not adequately inhibited. Thus, it is 

not known whether the increase in radioactivity that has been 

seen in certain protein bands is a product of preferential 

phosphorylation or preferential dephosphorylation. Consultation 

with others working on phosphorylation in plants has revealed 

46 
that problems with phosphatases are quite general. Efforts are 

being extended to find suitable phosphatase inhibitors, but the 

experiments with other plants are also being repeated in the hope 

of finding material with less vigorous phosphatase activity. It 

is felt that these efforts are valuable, for it seems evident 

that phosphorylation is an important regulatory mechanism in 

plants, and the technical difficulties must be overcome. 

PUBLICATIONS 

Pickard, B.G. Voltage Transients Elicited by Sudden Step-up of 

Auxin. Plant, Cell and Environment 7: 171-178, 1984. 

47 
THE MECHANISM OF PLANT SHOOT GRAVITROPISM 

David L. Rayle 

Department of Botany 

San Diego State University 

San Diego, CA 92182 

Research is directed toward understanding how gravity influences 

plant growth. More specifically, interest is focused on how 

plant shoot systems detect a gravitational field and transduce 

this information into an oriented pattern of growth. During 

1983, research was focused in two areas: (1) What is the role of 

the plant growth hormone auxin in shoot gravitropism? and (2) 

What role, if any, does calcium play in the ability of plants to 

detect and respond to a gravitation stimulus? 

In order to assess whether auxin plays a key role in shoot 

gravitropism, an attempt was made to kinetically dissect three 

seemingly interwoven phenomena: asymmetric growth, auxin 

redistribution, and asymmetric proton efflux. Using light-grown 

sunflower seedlings, it was established that gravitropic 

curvature is initiated in 25-30 min. This curvature response 

results from enhanced growth on the lower surface and reduced 

growth on the upper surface, relative to vertical controls. In 

gravitropicallY3stimulated hypocotyls, auxin redistribution 

(followed with H-IAA) is detectable in 20-25 min. This 

redistribution occurs simultaneously and uniformly along the 

growing axis. 

The above data suggest but do not prove that auxin is the 

gravitropism-mediating hormone. That is, one might argue that 

asymmetric growth causes auxin redistribution rather than vice 

versa, since the kinetics of both phenomena are quite similar. 

In an attempt to sort out cause from effect, auxin redistribution 

was followed in hypocotyls submerged in neutral buffers of high 

and low molarity. (High molarity buffers prevent asymmetric acid 

efflux and gravicurvature, while shoots in low molarity buffers 

respond normally.) It was found that auxin redistribution was 

kinetically identical in both cases. These results, therefore, 

demonstrate that auxin redistribution can occur in the absence of 

asymmetric growth and asymmetric acid efflux. It appears, then, 

that hormone redistribution is directly initiated by gravity 

perception mechanisms and not by a secondary event. Using a 

variety of other treatments (e.g., osmotic shock, inhibitors of 

auxin transport, etc.), it was confirmed that auxin 

redistribution could occur under conditions that prevented 

curvature. Further, no conditions under which the reverse 

situation occurred--that is, gravicurvature without concurrent 

auxin redistribution--were observed. Collectively, these results 

indicate the following seguence of key events in shoot 

48 
gravitropism: gravity perception ) auxin redistribution) 

asymmetric acid efflux ) asymmetric growth. 

During 1983, there has been increasing interest in the 

possibility that calcium may playa role in the gravitropic 

response of plant organs. Three kin~~ of observations tend to 

support this view: (1) asymmetric Ca movement has been detected 

in gravistimulated roots and shoots, (2) application of a Ca++ 

gradient can initiate c¥~vatures even in the absence of gravity 

stimulation, and (3) Ca chelators such as EGTA can stimulate 

straight growth and, when applied asymmetrically, initiate 

gravitropismlike curvatures. In the latter part of 1983, studies 

focusing on the mechanism by which EGTA stimulates shoot growth 

were begun. As this chelator is being ~sed extensively in 

tropism-related studies, it was considered important to know if 

indeed its gr~~th-promoting activity derived from removing 

wall-bound Ca or by some unrelated phenomenon. 

Using Avena coleoptile sections, it was confirmed that EGTA does 

indeed stimulate growth provided the sections are abraided to 

remove the cuticle. However, during the course of these studies 

it was also observed that at neutra*+pHs EGTA liberates protons 

(from the nitrogens) as it binds Ca • As protons are well known 

to stimulate growth, it seemed possible previous workers were 

simply observing acid-promoted growth in response to EGTA 

application-+$ phenomenon which would have no direct implications 

regarding Ca and the physical properties of plant cell walls. 

This possibility was tested using a model system employing 

frozen-thawed coleoptile sections and an exte~~al force to 

simulate turgor. Data indicate removal of Ca from cell walls 

by EGTA does not in itself stimulate cell extension. Rather, it 

can be shown that EGTA-induced growth is indeed simply due to 

wall acidification and subsequent acid growth. In light of this 

finding, extreme caution is needed in evaluating the effect of 

EGTA on root and shoot gravitropism as the observed responses may 

be indirect and thus misleading. 

PUBLICATIONS 

Hasenstein, K.-H. and Rayle, D. Cell Wall pH and Auxin Transport 

Velocity (Abstract). Plant Physiology 72(1, Suppl.): 145, 1983. 

Migliaccio, F. and Rayle, D. Relationship Between H+ Efflux, 

Auxin Redistribution and Shoot Gravitropism (Abstract). Plant 

Physiology 72(1, Suppl.): 90, 1983. 

Migliaccio, F. and Rayle, D.L. Sequence of Key Events in Shoot 

Gravitropism. Plant Physiology 75: 78-81, 1984. 

Taiz, L., Rayle, D.L., and Eisinger, W. Ethylene-Induced Lateral 

Expansion in Etiolated Pea Stems. plant Physiology 73: 413-417, 

1983. 

49 
wright, L.Z. and Rayle, D.L. Evidence for a Relationship Between 

H+ Excretion and Auxin in Shoot Gravitropism. Plant Physiology 

72: 99-104, 1983. 

50 
THE CELLULAR BASES OF GRAVITY- AND LIGHT-INDUCED GRAVITROPISM 

Stanley J. 

Department 

University 

Austin, TX 

Roux 

of Botany 

of Texas at Austin 

78712 

Light greatly accelerates the positive gravitropic response of 

certain roots and alters the gravitropic sensitivity of 

coleoptiles and stems. Thus, it is generally believed that some 

cellular response initiated by light is the same as, or affects, 

one of the gravity-induced cellular responses necessary for 

gravitropism. The objective of this research is to elucidate the 

specific fundamental processes that are altered by gravity and 

light in the induction of gravitropic growth in plants. 

Previous results have suggested that at least one of these 

fundamental processes is the movement of calcium ions in the 

stimulated cells and organs. Calcium ions have repeatedly been 

shown to play an important role in the regulation of plant growth 

and development, and both gravity and light induce calcium fluxes 

in responding tissues. During 1983, research was focused on 

several major questions pertinent to the proposed role of calcium 

in gravity- and light-induced gravitropism: (I) What is the 

subcellular distribution of the calcium-activated regulatory 

protein calmodulin in plant cells? {2} Can calcium chelators 

alter the gravitropic responsiveness of plants in a specific 

fashion? {3} Does one observe a rapid redistribution of calcium 

in roots, as is found in shoots, early during the induction of 

gravitropic growth? 

At least some calcium-mediated responses in plants are controlled 

by the calcium-binding regulatory protein, calmodulin. Earlier 

published reports of ours had indicated a possible role for 

calmodulin in mediating light and gravitropic responses in oat 

coleoptiles. To help further evaluate how calmodulin could 

affect plant growth, calmodulin was isolated and characterized 

from oats and its content was estimated, both in intact tissue 

and in isolated subcellular fractions, by radioimmunoassay. The 

results indicated that calmodulin is present in large quantities 

in plant cells and that it is specifically associated with 

mitochondria, etioplasts, and nuclei. In an analysis of a 

specially prepared extract of soluble wall proteins, calmodulin 

was found to be the major component of these proteins, too. If 

further research conclusively demonstrates that calmodulin is 

indeed a wall protein, this would provide a plausible regulator 

target for the calcium which moves asymmetrically into walls 

early during gravitropism. 

The calcium chelator EGTA may be used as a probe to test whether 

calcium movements are important for gravitropism, since it could 

51 
both chelate wall calcium and possibly inhibit the activation of 

calcium-dependent plasma membrane pumps. The research group of 

M. Evans, funded by NASA, found that application of EGTA to the 

tips of corn roots caused a loss of gravitropic sensitivity, 

which could be restored by replacing the chelator with calcium 

chloride. The effect of EGTA on the gravitropism of etiolated 

oat coleoptiles was investigated. It was found that a treatment 

period as brief as 2 hr. in 1 roM EGTA completely blocked 

gravitropism in 50-60% of the treated coleoptiles without 

inhibiting growth. Only about 10% of the plants perfused in 

water failed to exhibit gravitropism. Subsequent perfusion of 

EGTA-treated plants with calcium completely restored 

gravitropism; postperfusion with water did not. Controls showed 

that the effects of EGTA could not be attributed to its weak 

capacity to buffer the pH of the treatment solution. The fact 

that calcium reversed the inhibitory effects of EGTA on 

gravitropism indicated that the inhibition was probably due to a 

reduction in the availability of free calcium required for one or 

more of the transduction steps of gravitropism. 

As of 1983, it is not known whether a gravitropic stimulus 

induces a redistribution of calcium in roots as it apparently 

does in coleoptiles and shoots. The work of Evans on the 

inhibition of root gravitropism by calcium chelators, mentioned 

above, and his finding that calcium transport through root tips 

is altered early during root gravitropism suggested that calcium 

movements might be important for root gravitropism. This led to 

an investigation of whether a gravitropic stimulus of corn roots 

would result in a redistribution of calcium in this organ and, if 

so, how soon and in what cells would this redistribution occur. 

Antimonate staining procedures were used to determine the 

patterns of localization of calcium in nonstimulated and 

gravistimulated corn roots. It was found that in the region of 

the developing bend there was a change in the staining pattern 

from that principally localized within cells of the stele to 

asymmetric staining within the vacuoles in the cortical cells 

along the upper surface of the root. There was very little 

staining in the walls. This staining pattern is quite different 

from that seen in gravistimulated coleoptiles, but it does 

represent an asymmetric redistribution of calcium induced by a 

gravitropic stimulus, and it does occur rapidly enough (within 10 

min.) to playa potential role in the regulation of root 

gravitropism which becomes visible only after about 20 min. This 

question needs to be investigated further. 

PUBLICATIONS 

Biro, R.L. and Roux, S.J. Quantification of Calmodulin in 

Isolated Organelles from Oat by Radioimmunoassay and Analysis of 

Chlorpromazine Binding to Calmodulin in vivo (Abstract). Plant 

Physiology 72(1, Suppl.}: 66, 1983. 

52 
Roux, S.J. (ed.). Regulatory Functions of Calcium and the 

Potential Role of Calcium in Mediating Gravitational Responses in 

Cells and Tissues, Proceedings of a Workshop, FASEB, Bethesda, 

MD, Sept. 16-18, 1982. Washington, D.C.: NASA, 228 p., 1983. 

(NASA Conference Publication 2286) 

Roux, S.J. A Possible Role for ca2+ in Mediating Phytochrome 

Responses. In: The Biology of Photoreception (ed. by D.J. Cosens 

and D. Vince-Price). New York: Cambridge University Press, p. 

561-580, 1983. (Society for Experimental Biology Symposium 36) 

Roux, S.J., Biro, R.L., and Hale, C.C., II. Calcium Movements 

and the Cellular Basis of Gravitropism. Advances in Space 

Research 3(9): 221-227, 1983. 

Serlin, B.S •• Sopory, S.K., and Rou 2f S.J. Modulation of Oat 

Mitochondrial ATPase Activity by Ca and Phytochrome. Plant 

Physiology 74: 827-833, 1984. 

Slocum, R.D. and Roux, S.J. Cellular and Subcellular 

Localization of Calcium in Gravistimulated Oat Coleoptiles and 

Its Possible Significance in the Establishment of Tropic 

Curvature. Planta 157: 481~492, 1983. 

Sun, D. and Roux, S.J. Inhibition 

Coleoptiles by a Calcium Chelator, 

Inhibition by Calcium (Abstract). 

Suppl.): 66, 1983. 

53 

of Gravitropism in Oat 

and the Reversal of this 

Plant Physiology 72(1, 
GRAVITROPISM IN LEAFY DICOT STEMS 

Frank B. Salisbury 

Utah State University 

Logan, UT 84322 

Objectives 

(I) To understand mechanisms of gravity perception. 

(2) To consider the possible role of plant growth substances in 

transduction of the perceived stimulus to actual bending. 

(3) To study the mechanics of stem bending: kinetics of the 

process, the forces involved, the mechanical properties of 

cell walls, etc. 

Salisbury spent most of 1983 on sabbatical in Austria and Israel. 

This report encompasses his research plus that of two students in 

Utah. 

The Mechanics of Gravitropic Stem Bending. Cocklebur (Xanthium 

strumarium) stems were subjected to four treatments: (I) 

horizontal restrained stems (stem placed between two stiff wires 

and wrapped with thread), (2) horizontal free bending, (3) 

vertical controls, and (4) stems restrained for 48 hr. or more 

and then released by cutting the threads. Stems bent 

immediately, typically to about 1350 • Both tops and bottoms of 

horizontal stems were photographed with two cameras (stereo 

photogrammetry). Length measurements were taken between dots of 

india ink on stems by projecting the negatives onto a digitizer 

interfaced with a microcomputer. 

Growth nearly stops on the top of horizontal free-bending stems 

and is accelerated on the bottom compared with controls. In the 

bending regions on the top, there is often a significant 

shrinkage. Upon release of a horizontal restrained stem, the top 

shrinks considerably, and the bottom elongates, emphasizing the 

cessation of growth in upper tissues when stems are turned to the 

horizontal. (Wesley Mueller, completed Ph.D.) 

When a vertical stem is split from the tip toward the base, the 

two halves bend away from each other, indicating that internal 

stem tissues are under pressure compared with surface tissues. 

This was studied in castor bean (Ricinus communis) by making 

horizontal cuts at 2.5-mm. intervals halfway around one side of 

the stem. Depth of the cuts was controlled by the extent to 

which a razor blade protruded from between two pieces of a holder 

and was measured on free-hand sections after the degree of 

bending (always away from the side of the cuts) had been 

measured. Plotting stem bending as a function of depth of the 

cuts showed that maximum bending was achieved when the cuts were 

about 0.6 mm. deep, extending through epidermis, collenchyma, 

cortex, and at least into the phloem of the vascular bundles. 

54 
Thus, pressure seems to be localized in the pith (even in these 

hollow stems), and tensions apparently exist in all the remaining 

tissues. 

A device was built to cut longitudinal sections of measured 

thickness from the center of plant stems, giving increased 

bending of the two halves (usually equal for a vertical stem). 

When horizontal stems are bending upward, the upper half of the 

cut central section bends about 20 0 more than the lower half, 

emphasizing the importance of the gradient in tissue 

tension/pressure in the upper half of free-bending stems. Split 

central sections were placed in sorbitol solutions of varying 

concentrations. Sections placed in solutions hypotonic to the 

cell sap continued to bend as pith cell~ took up water, while 

hypertonic solutions caused an inward bending (a straightening) 

of the two stem halves. Response in solutions close to isotonic 

suggest that water potential in the bottom pith is less negative 

than in the top. (Salisbury) 

Effects of unilateral Applications of Ethephon. Solutions of 

ethephon were painted on one side of stems and hypocotyls of 

young tomato (Lycopersicon esculentum), cocklebur, castor bean, 

and sunflower (Helianthus annuus) plants. Vertical stems and 

hypocotyls (especially tomato) bent away from the side of 

application within 2-3 hr. When ethephon was neutralized to 

pH 6, vertical stems did not bend away from the side of 

application, but leaves developed epinasty, a typical ethylene 

response. When buffered acid or even unbuffered HCl was applied 

to one side of vertical stems, bending occurred away from the 

side of application, epinasty did not develop, but stems were 

damaged in a manner similar to that produced by unbuffered 

ethephon. 

When ethephon (pH 6) was applied to the bottom of stems just 

turned to the horizontal, gravitropic bending was delayed. Acid 

solutions applied to the bottom of horizontal stems greatly 

delayed bending--an unexpected result in light of the experiments 

just described. Ethephon (pH 6) applied to the upper surface of 

horizontal stems caused a small delay in gravitropic bending, but 

acid solutions applied to the top of horizontal stems caused 

downward bending. (Rosemary White) 

PUBLICATIONS 

Harris, C.S. and Salisbury, F.B. Plant Responses to Clinostating 

and Mechanical Stress: Growth and Flowering Responses (Abstract). 

Plant Physiology 72(1, Suppl.): 63, 1983. 

Mueller, W.J. and Salisbury, F.B. Dimensional Changes of 

Xanthium strumarium L. (Cocklebur) Stems during Gravitropic 

Bending (Abstract). Plant Physiology 72(1, Suppl.): 62, 1983. 

55 
White, R.G. and Salisbury, F.B. Effects of a Unilateral 

Application of Ethephon on Gravitropic Bending in Dicot Stems 

(Abstract). Plant Physiology 72(1, Suppl.): 63, 1983. 

56 
ANIMAL PROJECTS 

THE EFFECT OF SKELETAL UNLOADING ON BONE FORMATION 

Daniel D. Bikle 

Department of Medicine 

Veterans Administration Medical Center 

and University of California 

San Francisco, CA 94121 

Available data suggest that bone mass is a result of the balance 

of bone formation and bone resorption. Skeletal unloading as 

occurs in spaceflight, bedrest, or immobilization affects this 

balance and leads to osteoporosis, negative calcium balance and 

osteopenia. A role for both hormones and local factors in 

maintaining this balance seems likely, but it is not clear to 

what extent skeletal unloading alters the balance through effects 

on hormones vs. local factors. A model in which growing rats are 

suspended so that their hind limbs are unweighted but their 

forelimbs are normally weighted is being used to test the role of 

hormones in mediating the local disturbances in bone formation 

caused by selective skeletal unloading. 

Growing rats (160-200 g) have been suspended by their tails for 

at least 4 wk. without altering their growth patterns. No 

difference in weight gain between such animals and their pair-fed 

controls was observed. The suspended rats in comparison to the 

pair-fed controls showed a decrease in fat free weight, ash 

weight, and calcium content of the unloaded bones (tibia, lumbar 

vertebra) but not of the normally loaded bones (humerus and 

mandible). Most of the difference in bone mass between suspended 

and pair-fed controls occurred progressively between days 2-10, 

after which no further difference between suspended and pair-fed 

controls was observed. The effect of su~gension on two 

biochemical measures of bone3 formation-- calcium uptake 

presumably into mineral and H-proline uptake presumably into 

matrix--has been examined. The proximal portion of the long 

bones, which contains a higher percentage of endosteal bone than 

the shaft (which is primarily cortical bone and has a higher 

percentage4 gf calci~m/mg fat free weight) had a 3-fold greater 

amount of Ca and H proline incorporation. with suspension, 

both portions of the tibia showed a decrement in calcium content, 

calcium uptake, and proline incorporation by 5-7 days when 

compared with the tibia from pair-fed controls. Afte45 10 days, 

no f~rther decrement in calcium content was seen and calcium 

and H-proline uptake rebounded to control or above control 

levels. The bone formation rate was determined at the 

tibia-fibula junction (cortical bone) in suspended rats and pairfed controls using three tetracycline labels administered on days 

3, 9, and 14 to mark two periods of bone formation: period 1 = 

days 3-9, period 2 = days 9-14. The results indicate that 

suspension unloading leads to a 50% reduction in bone formation 

59 
in the tibia during period 1, but with continued suspension these 

measurements of growth return to normal. 

The initial approach to determining whether the calciotropic 

hormones were involved in signaling the unloaded bone to stop 

growing was to measure calcium, phosphorus, PTH, and 1,25(OH)2D 

in the serum of suspended and pair-fed control rats. The results 

indicate no differences in the serum levels of any of these ions 

or hormones at 15 days. However, small increases in serum 

calcium were observed at days 5-7 of suspension which returned to 

pair-fed control values by day 15. The rise in serum calcium was 

associated with a fall in serum 1,25(OH)2D values. Furthermore, 

a small reduction in intestinal calcium transport (a process 

regulated by 1,25(OH)2D) occurred between 5-10 days of suspension 

which returned to control levels by day 15. These observations 

suggest that cyclic changes in the serum levels of the 

calciotropic hormones during skeletal unloading may provide at 

least part of the mechanism by which bone formation is inhibited 

by day 5 and is restored to normal by day 10. 

These data are also consistent with the possibility that skeletal 

unloading alters the response of bone cells to calciotropic 

hormones. Such an alteration in bone cell response would explain 

why bone cell formation ceases in the tibia but not in the 

humerus of the suspended rat. An attempt is currently being made 

to determine whether the receptors for 1,25(OH)2D and PTH in bone 

are altered by skeletal unloading. An assay to measure 1,252D 

receptors in the cytosol extract of pulverized bone from which 

marrow has been carefully removed has been developed. The 

1,25(OH)2D receptor from bone is similar to the 1,25(OH)2D 

receptor from other tissues in that it is 3.7S in size and 

specific for 1,25(OH)2D. 

Finally, isolation of bone cells from the long bones of the 

growing rat has ~ust begun. Initial experiments indicate that 

approximately 10 cells can be isolated per bone (tibia, femur) 

which in culture develop into typical appearing bone cells. In 

future experiments, such preparations will be tested for their 

biologic function and response to PTH and 1,25(OH)2D in an effort 

to evaluate whether cells obtained from a bone in which formation 

has been inhibited by tail suspension differ from cells obtained 

from a bone in which bone formation is unimpaired. 

PUBLICATIONS 

Bikle, D.D. 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. 

Bikle, D.D. and Munson, S.J. Calcium Transport by Duodenal Brush 

Border Membranes as a Function of Position on the Villus: 

60 
Differential Regulation by 1,25(OH)2D (Abstract). Clinical 

Research 31: 92A, 1983. 

Simmons, D.J., Grazman, B., Russell, J.E., Walker W.V., Bikle, 

D.O., and Morey, E.R. Simulating Certain Aspects of Hypogravity: 

Effects on Bone Maturation in the Non-weight Bearing Skeleton. 

Aviation, Space, and Environmental Medicine 54: 1080-1084, 1983. 

61 
WEIGHTLESSNESS SIMULATION: PHYSIOLOGICAL CHANGES IN FAST AND SLOW 

MUSCLE 

Wolf-D. Dettbarn 

Department of Pharmacology 

Vanderbilt University 

Nashville, TN 37212 

Weightlessness during spaceflight produces numerous physiological 

changes in virtually all organ systems. Particularly affected is 

the musculo-skeletal system. Changes in bone turnover and muscle 

atrophy have been well described. So far, however, there have 

been no systematic reports available based on biochemical, 

physiological, and pharmacological approaches to studying the 

cholinergic system of nerve and muscle during hypokinetic 

conditions. The key enzymes, choline acetyltransferase (ChAT) 

and acetylcholinesterase (AChE), involved in the synthesis and 

hydrolysis of neurotransmitter, acetylcholine (ACh), are reported 

to be synthesized in the cell bodies of cholinergic motor neurons 

which innervate skeletal muscles. Following synthesis of both 

these enzymes, they are transported by axoplasmic flow to nerve 

terminals. ChAT is thought to remain highly localized in nerve 

terminals with low presence in the muscles proper, whereas, AChE 

is present in the axon in equally high or higher concentration 

than in the muscle. Its presence in muscle depends to a large 

extent on innervation. 

The transmission of impulses from nerve to muscle is caused by 

the release of ACh from nerve terminals which interacts with the 

nicotinic ACh receptor in the muscle fiber. Only the endplate 

membrane directly beneath the nerve terminal is highly sensitive 

to ACh and also has the highest density of ACh receptors. 

Denervation causes the entire muscle fiber to become sensitive, 

while reinnervation restricts this ACh sensitivity once again to 

the endplate membrane. 

The overall aims of this proposal are directed to study alterations in muscle biochemical, morphological, and physiological 

characteristics as they are produced by simulated weightlessness 

and the time course of recovery after release from weightlessness. 

During 1983, the research has focused on three major questions. 

(1) What is the effect of disuse on AChE activity in muscle and 

peripheral nerve and how does it affect the regulation of the 

molecular forms, 4S, IDS, 12S, and l6S, in slow and fast muscle. 

(2) How is the enzyme that regulates acetylcholine (ACh) 

synthesis, i.e., ChAT, affected in nerve and muscle. (3) What are 

the effects of prolonged disuse on the ACh receptor in slow and 

fast muscle. (4) How does prolonged disuse affect morphological 

as well as biochemical characteristics in slow and fast muscle? 

62 
To produce experimental weightlessness, rats were suspended by 

their tails in such a way that their hind legs were completely 

unloaded, while the animals were able to move freely on their 

forelegs throughout the cage. Animals were suspended from 1 to 3 

wk. Activities of ChAT and AchE were measured with highly 

sensitive radiochemical assays, and the molecular forms were 

separated by velocity sedimentation and quantitated with the 

radiometri~ assay. To study the quantitative changes in ACh 

receptors H-ACh was used as the natural ligand for nicotinic 

receptor sites. 

With disuse induced by hind limb suspension, the predominantly 

slow soleus (SOL) lost 60% of wet weight while the fast extensor 

digitorum longus (EDL) lost 29% compared with their respective 

control muscles. The activity of ChAT was markedly elevated in 

sciatic nerve (38%), soleus, and EDL (79% and 78%, respectively) 

compared with controls. The increase in ChAT activity was timedependent in both muscles as the maximal change was noticed at 

the end of 3 wk. In soleus, AChE activity increased by 250% 

involving all molecular forms such as 4S, lOS, 12S, and 16S 

forms, while there was only a minimal change in the EDL. Total 

protein concentrations in nerve and muscle were not significantly 

2ifferent in control and hind limb suspended animals. By using 

H-ACh as the ligand for the nicotinic receptor, there was 

evidence of increased nicotinic AChR binding in both SOL and EDL 

muscles. Fiber typing by actomyosin ATPase reaction showed a 

shift in fiber type profile in the soleus towards a larger 

proportion of fast (Type II) fibers. 

It is evident from the present findings that some properties of 

skeletal muscles are strongly dependent on pattern and level of 

load-bearing and motor unit activity. The antigravity muscle 

such as the SOL appears to be more affected by hind limb 

suspension than the fast EDL. Whether all the observed changes 

are due to fiber type changes, as observed in SOL, remains to be 

seen. 

Future studies will investigate the effects of weightlessness on 

axoplasmic transport, the electrophysiology of neuromuscular 

transmission, and the reversibility of the observed changes 

during recovery from hind limb suspension. 

PUBLICATIONS 

Gupta, R.C., Misulis, K.E., and Dettbarn, W.-D. Status of 

Enzymes Catalyzing Synthesis and Hydrolysis of Acetylcholine in 

Hypokinetic Rat (Abstract). Federation Proceedings 43(3): 548, 

1984. 

63 
ALTERATIONS IN CONNECTIVE TISSUE CELLS DUE TO REDUCED WEIGHT 

BEARING 

Stephen B. Doty 

Departments of Orthopedic Research 

and Anatomy/Cell Biology 

Columbia University 

New York, NY 10032 

The research objectives are: (1) to determine and describe 

effects that occur to connective tissue cells as a result of 

reduced gravitational or mechanical forces, and (2) to increase 

the understanding of normal cell function by perturbing normal 

gravitational or mechanical forces and searching for mechanisms 

that will describe the cellular behavior. 

The cells being studied are derived from skeletal, cartilage, and 

dense connective tissues. Tissues have been collected from rats 

subjected to spaceflight conditions (e.g., Cosmos 1129) and from 

animals placed under non-weight-bearing conditions in a l-G 

environment (the Morey-Holton suspended animal model). The 

methods of cell study include light, fluorescence, and electron 

microscopy, autoradiography, histochemistry, and some biochemical 

measurements. 

Results that have been obtained relating to Objective 1 indicate: 

(1) An extracellular cell membrane-associated alkaline 

phosphatase enzyme has shown reduced activity as a result of 

reduced mechanical stress. (2) Golgi-associated enzymes have 

reduced activity in those cells actively synthesizing-collagen. 

(3) Lysosomal enzyme activity is increased in cOllagensynthesizing cells, evidently in response to less need for matrix 

collagen. (4) Cell-cell communication maintained through special 

junctional complexes between cells appear to be reduced in size 

and number as a result of reduced weight bearing or hypogravity 

conditions. 

These studies are continuing in order to determine whether these 

results are possibly due to some systemic factor (e.g., a change 

in circulating hormones, altered blood flow, production of 

inhibitory substances, or other unknown blood-borne factors), or 

whether the cellular changes are a direct response to altered 

mechanical forces. 

During 1983 work began on Objective 2. It has been found that a 

calcium-stimulated ATPase can be localized in bone cells, which 

appears by light microscopy to be within the cell cytoplasm and 

may be located at the site of junctional complexes. This would 

be exactly opposite to the localization of membrane-associated 

alkaline phosphatase. In fact, electron microscopy shows that 

alkaline phosphatase is on the extracellular surface of the cell 

64 
membrane and does not exist at the site of junctional complexes. 

It was also noted that calcium ion at 2-5 mM produces the 

greatest ATPase activity whereas IO-mM calcium inhibits this 

enzyme. The interrelationship between ATPase and alkaline 

phosphatase is not yet known. However, there is now the exciting 

possibility that an ATPase is regulated by calcium ion and. a 

phosphatase is controlled by phosphate ions. Thus, mechanisms 

exist for controlling cellular activity through changes in 

environmental ion products. 

In addition to stimulation of ATPase, calcium ion also influences 

the cytoskeleton of connective tissue cells. This cytoskeleton 

functions importantly not only in mechanisms of cellular 

secretion but also in the regulation of cell motility. 

Therefore, by fluorescence localization of fibrous actin, it has 

been determined that actin content of bone cells has been reduced 

as a result of skeletal non-weight-bearing. The next step is 

determining whether the actin content and cytoskeletal or 

microfilament contractility is controlled by calcium ion levels 

or by direct transduction of external mechanical force across 

cell membranes into the cytoskeletal network. These studies will 

provide important insight into how environmental factors may 

exert control over normal cell functions. 

65 
GROWTH AND DIFFERENTIATION OF MAMMALIAN EMBRYONIC TISSUES EXPOSED 

TO HYPERGRAVITY IN VIVO AND IN VITRO 

Jackie C. Duke 

Dental Science Institute 

University of Texas Dental Branch 

P.O. Box 20068 

Houston, TX 77225 

The specific aims of this research are (1) to determine the 

effects of centrifugation on the cytology and biochemistry of 

differentiating systems; (2) to ascertain whether a quantitative 

relationship exists between gravitational force and development; 

(3) to identify gestational ages and basic cellular processes 

susceptible to gravitational forces; (4) to determine the 

duration of exposure needed to effect a change; and, (5) to 

investigate the effectiveness of the clinostat as a zero G 

simulator in a mammalian system. 

The in vitro studies have shown that excess gravity (2.6 G) is 

able to suppress morphogenesis in embryonic mouse limbs 

developing in vitro by promoting early differentiation. These 

earlier studies were carried out with BGJ b medium containing 25% 

fetal calf serum and have been repeated wIth serum-free medium 

(BGJ b plus 25% salt solution) to insure that the changes seen in 

centrifuged limbs are not due to sedimentation of necessary serum 

components. Morphogenetic scoring of the various elements in 

fixed whole mounts of control limbs showed that the serum-free 

medium supported limb development just as well as did medium with 

serum. Also, centrifuged limbs attained scores similar to those 

of centrifuged limbs in the previous study, showing that the 

possible unavailability of serum components involved in growth 

regulation was not a factor in the lower morphogenetic scores 

seen in centrifuged limbs. Other observations seen in the 

previous study were repeated: (1) there is a proximo-distal 

gradient of sensitivity to gravity's teratological effects with 

distal elements (i.e., less differentiated ones) being more 

susceptible and proximal ones less so; (2) only certain stages in 

limb development exhibit geosensitivity and these stages are the 

same as for other (i.e., chemical) teratogens; (3) centrifugation 

may cause limb elements present at explantation to resorb; and 

(4) finally, the lack of morphogenesis seen in centrifuged limbs 

is due to early differentiation of cells. 

In addition to the cytologic and morphometric studies, 

histochemical analyses of excess gravity on chondrogenesis have 

been carried out on micromass cultures of embryonic mouse limb 

cells using type-specific anticollagen antibodies and various 

immunological techniques (HRP, fluorescence). 

The major biochemical transitions that occur during cartilage 

66 
differentiation are the initiation of synthesis and disposition 

of macromolecules of the cartilage-specific extracellular matrix. 

Therefore, the studies also propose to characterize transitions 

in phenotypic expression of differentiating limb buds in 

chondrogenic stages using biochemical markers such as chondroitin 

sulfate and collagen type. 

For biochemical studies, the limb buds are rinsed, blotted, 

lyophilized, and then weighed to obtain dry weights. Lyopholized 

samples are dissolved in O.lN NaOH prior to determination of 

collagen, glycosaminoglycan, and noncollagen protein 

accumulation. The initial results of 12-day limb bud cultures 

indicate that no significant difference in content of collagen is 

exhibited by exposed vs. nonexposed limb buds, but a 23% decrease 

in glycosaminoglycan concentration is seen in limbs developing 

under excess gravity. 

Recently, the effects of exposure to excess gravity on fusion of 

the embryonic mouse secondary palate have been studied. During 

fusion, the palatal shelves first adhere by means of 

glycoproteins appearing along the medial epithelial edge. The 

contacting epithelia then reorganize and undergo programmed cell 

death, allowing the underlying mesenchymes to corne in contact. 

The process of cell death occurs in vitro at about the same time 

that it occurs in vivo. When embryonic palates were exposed to 

excess G in vitro for 24 hr., and then examined by light and 

electron microscopy, palatal cell death and fusion were 

accelerated under 2.6 G. 

With the recent completion of a small animal centrifuge 

examination of the effect of hypergravity on in utero mammalian 

development is now possible. The centrifuge is able to house up 

to 80 mice, 20 at each of four different gravities. The animals 

are provided with chow ad libitum and water is provided through a 

nuzzle valve system. A~present, 60 young mice are undergoing 

adaptation prior to mating, having been placed on the centrifuge 

at 4 wk. of age. Control animals are housed in the centrifuge 

room. 

Besides the in vivo and in vitro centrifuges for excess gravity 

studies, a clinostat is being developed for zero G studies. 

PUBLICATIONS 

Duke, J.C. Suppression of Morphogenesis in Embryonic Mouse Limbs 

Exposed In Vitro to Excess Gravity. Teratology 27: 427-436, 

1983. 

67 
EFFECTS OF GRAVITO-INERTIAL FIELDS ON THE PHYSIOLOGY OF THE 

ORB-WEAVING SPIDER 

Alfred Finck 

Department of Psychology 

Temple University 

Philadelphia, PA 19122 

This project is directed toward identifying the gravity receptor 

of the spider and evaluating its role in the maintenance of 

equilibrium. Prior to this NASA-supported work, gravity and the 

spider had not been studied. This research also evaluates the 

operating characteristics of the orb-weaving spider: the absolute 

threshold to hyper-Gz and the dynamic range of increments to 

gravity. Improvements in experimental methodology and data 

analysis during 1983 have permitted the investigation of issues. 

in gravitational physiology of the spider heretofore not 

possible. 

The initial work on hypergravity produced by centrifugation 

indicated a system having a dynamic range of sensitivity of at 

least four orders of magnitude in which the absolute threshold to 

hyper-Gz was less than 1.001. In the first experiments it was 

necessary to measure Gz effects postrotation. During 1983 an 

"on-board" system, employing a laser diode, permitted the cardiac 

reflex to be taken during centrifugation. Replication of the 

postrotation study confirms the logarithmic relationship between 

the response of the spider heart and Gz intensity, as well as the 

low absolute threshold to increases in gravity. 

During the year the centrifuge drive system was modified so that 

it is now under computer control. Thus, small or large 

decelerations or accelerations, with exposure time held constant 

or systematically varied, can be programmed. An issue that can 

now be attacked directly concerns the differential sensitivity of 

the spider to changes in Gz (delta Gz). The procedure can be 

viewed as producing a Corioluslike stimulus, with the animal 

remaining motionless but the gravito-inertial field changing in 

magnitude over highly controlled time periods. Its good 

sensitivity to constant radial acceleration suggests that the 

spider response to delta Gz might also be a small value. Most 

probably the Weber Ratio (delta Gz/Gz) is a relatively constant 

value for low intensity gravito-inertial fields. This aspect of 

the research is just beginning. 

The lyriform organ on the spider legs has been identified as a 

source of vibratory and proprioceptive information. It 

apparently has a dual role in sensory transduction, providing the 

animal with relevant cues as to the magnitude and direction of 

the Gz stimulus. Removal of the patellar lyriform organs results 

in a decrease of sensitivity to Gz and a statistically 

68 
significant increase in the rate of the cardiac response. Small 

changes in Gz and/or in tilt are also accompanied by a reduction 

in the rate and/or amplitude of the beat over a period of time. 

The recovery time from this bradycardia is not yet known. Thus 

the lyriforms are involved with Gz detection and there is an 

indication that they may provide an inhibitory input to the 

neural cardiac pacemaker. During 1983, a brief communication 

from another laboratory reported that the spider heart was 

innervated by neurons traversing the abdominal nerve possibly 

arriving from the suboesophageal ganglion (SOG). Within the SOG 

are the centers that integrate the sensory input from the 

lyriform organs. These may be the morphological bases for 

observations that sensory stimuli can produce a bradycardia of 

the spider heart. This is important because of postulations that 

the neurogenic heart is an active element in the spider 

equilibrium system. That is, blood serum pumped into the 

cephalothorax can produce leg extension and the spider is 

supported under changed gravity conditions. 

A test of the hypothesis that the lyriform organs exert 

inhibitory control on the neurogenic heart can be made by 

substituting a vibratory stimulus for the Gz stimulus. If, as is 

supposed, the lyriform encodes both vibration and gravity, then a 

small displacement of the legs should inhibit the cardiac 

response. In the latter part of 1983 the spider heartbeat was 

recorded by laser-plethysmograph with animals placed on a 

vibration table. A variety of stimulus frequencies (from DC to 

about 150 Hz) delivered at suprathreshold amplitudes (circa less 

than 1 micrometer) produced bradycardias. This is evidence that, 

in the spider, gravity and vibration have a similar physiological 

effect. It also implies that the spider contains a frequency 

analysis mechanism for discriminating the stimulus input. 

The relationship between gravity/vibratory stimuli and the beat 

of the neurogenic heart of the spider seems clear. If the 

mechanical input serves to inhibit cardiac activity then in the 

microgravity of orbital flight the heart should beat at an 

inordinately high rate, but sensory control should be reinstated 

by vibration of the legs. An application to test this hypothesis 

during a "shuttle flight" has been submitted to the NASA Space 

Biology Program. 

PUBLICATIONS 

Finck, A. The Gravity Sense of Araneus sericatus (Abstract). 

Paper Presented at the 1983 American Arachnological Society 

Eastern Division Meeting, Ohio University, Athens, June 24-27, 

1983. 

69 
HOMEOSTASIS IN PRIMATES IN HYPERACCELERATION FIELDS 

Charles A. Fuller 

Division of Biomedical Sciences 

University of California at Riverside 

Riverside, CA 92521 

The homeostatic capabilities of animals are sensitive to changes 

in the ambient acceleration environment. Such changes in 

centrifuged rats and dogs include depressed body temperatures, 

alterations in circadian timekeeping, and changes in body 

composition. To date, however, little work has been done 

examining these changes in man or any other primate. During 

1983, various homeostatic responses of a nonhuman primate, the 

squirrel monkey (Saimiri sciureus), to acute changes in the 

acceleration environment were examined. When these animals were 

exposed to a hyperdynamic field, the body temperature was 

consistently depressed and the animals showed behavioral 

indications of increased drowsiness. Further, time of day played 

a significant role in influencing these responses. 

Initially, loosely restrained squirrel monkeys were exposed to 

hyperdynamic fields. The centrifugation consisted of a 60-min. 

step change in the acceleration environment from 1 G to 2 G in 

the z axis (head-to-toe). All animals demonstrated significant 

depressions of body temperature while in the hyperdynamic field. 

After the centrifuge was started, there was generally a small 

increase in body temperature. Within 5-10 min., however, a 

continuous decline in temperature became apparent. The average 

decrease in body temperature was 1.4 0 C and the body temperature 

depression was maintained for the duration of the step change. 

Approximately 5 min. after the animal was returned to 1 G the 

body temperature began to rise. 

Next, the primate sleep responses to 70-min. exposures to the 2 

Gz hyperdynamic fields were examined. Initial observations on 

centrifuged animals, when observed visually, suggested a sleeping 

behavior when in the hyperdynamic field. This phenomenon was 

examined further with electrophysiological recording of the EEG 

in a group of chronically implanted primates. During the 

precentrifugation phase, the animals showed various amounts of 

"napping" behavior in which slow wave sleep occurred on a 

periodic basis. (Rapid eye movement sleep was never observed 

during this daytime study.) Upon centrifugation, however, sleep 

was inhibited for several minutes after which the slow wave sleep 

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

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

maintained above baseline levels for the entire 70-min. 

postcentrifugation phase. This response was proportional to 

field intensity in the animals studied at both 2 and 3 G

z

• At 3 

70 
Gz, the animals showed an increased response in that there was a 

greater rebound in slow wave sleep during the postcentrifugation 

phase. It is interesting to note that slow wave sleep has always 

been inversely correlated with body temperature. That is, lower 

body temperatures are usually associated with increased sleep. 

In this experiment, the correlation did not hold. During 

centrifugation, the amount of sleep observed during the fall in 

body temperature was less than that seen in the control phase 

where body temperature was higher. 

Finally, the thermoregulatory responses of squirrel monkeys to 

70-min. 2-Gz exposures during the day and night were compared. 

Again6 the response of the animal during the day was, on average, 

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 the average there was no 

change in colonic tempera~ure. Similarly, the skin temperature 

did not show any major deviations in vasomotor heat loss. These 

results demonstrate that although the centrifuge-induced changes 

in body temperature are very prominent and reproducible during 

the day, these animals can regulate their body temperature 

independently of the gravitational field during the night. 

This day-night difference in body temperature response was also 

investigated in a series of rats. Historically, all rats exposed 

to hypergravitaitonal fields have been examined during the day, 

which is the rest phase of these nocturnal animals. An attempt 

was made to determine whether the light vs. the dark had a 

significant influence on the response or whether the magnitude of 

the response was a function of the activity level of the animal. 

The previously reported 20 C response was observed when 

centrifuging the animals during the day. However, upon exposure 

to 2 Gz at night, which is the animal's active phase, the body 

temperature fall was even larger. This indicates that although 

the monkeys were able to regulate body temperature more precisely 

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. 

Current efforts are proceeding to examine the influence of 

chronic acceleration on these and other homeostatic systems, with 

special interest in examining the new steady-state levels of 

these homeostatic variables and their 24-hr. rhythmicity. 

Finally, protocols are being developed to further describe the 

mechanisms by which these responses are produced. 

PUBLICATIONS 

Fuller, C.A. Ac~te Physiological Responses of Squirrel Monkeys 

71 
Exposed to Hyperdynamic Environments. Aviation, Space, and 

Environmental Medicine 55(3}: 226-230, 1984. 

Fuller; C.A. Sleep-wake Responses of Squirrel Monkeys Exposed to 

Hyperdynamic Environments. Physiologist 26(6, Suppl.}: S90-S91, 

1983. 

Fuller, C.A., Griffin, D.W., and Horowitz, J.M. Circadian 

Responses of Mammals to the Hyperdynamic Environment. 

Physiologist 25: A32, 1983. 

72 
NEURAL MECHANISMS BY WHICH GRAVITATIONAL STIMULI AND STRESS 

AFFECT THE SECRETION OF RENIN AND OTHER HORMONES 

William F. Ganong 

Department of Physiology 

University of California 

San Francisco, CA 94143 

This project was activated August 1, 1983. The goal of the 

research is delineation of the neural pathways and transmitters 

that mediate the changes in the secretion of renin and the 

antidiuretic hormone produced by gravitational and other stimuli. 

Evidence indicates that stimulation of serotonergic neurons in 

the dorsal raphe nucleus of the midbrain increases renin 

secretion, and that these neurons project to the mediobasal 

hypothalamus (Karteszi, et al., Neuroendocrinology 34: 323-326, 

1982). The initial goal of the present project has been to 

determine how the message gets from the hypothalamus to the 

renin-secreting cells in the kidneys. 

A pharmacological study was undertaken to determine if the 

pathway from the hypothalamus to the kidney was sympathetic. 

Catecholamines secreted from the renal nerves and the adrenal 

medulla are known to mediate sympathetic stimulation of renin 

secretion via beta-adrenergic receptors located on the reninsecreting cells. The serotonin-releasing drug 

p-chloroamphetamine (PCA) was used to initiate serotonergic 

discharge in rats and the sympathetic effector pathway was 

blocked by administration of the beta-adrenergic blocking drug 

L-propranolol. The D isomer of propranolol, which has all the 

properties of the L-propranolol except that it has only weak 

beta-adrenergic blocking activity, was used as a control. 

L-propranolol but not D-propranolol prevented the increase in 

plasma renin activity produced by PCA. Because propranolol 

penetrates the brain, and it could be argued that it was exerting 

its effects on the brain rather than the kidney, the experiment 

was repeated with sotalol, a beta-adrenergic blocking drug which 

does not cross the blood-brain barrier. Sotalol also prevented 

the renin response to PCA. 

In other experiments, the effects of PCA were tested after 

transmission in sympathetic ganglia was blocked by administration 

of the ganglionic-blocking drug, chlorisondamine. 

Chlorisondamine increases plasma renin activity by itself because 

it interrupts the tonic sympathetic discharge to the blood 

vessels that maintain blood pressure. The resultant decrease in 

blood pressure acts directly on the kidneys to increase renin 

secretion. However, PCA plus chlorisondamine produced no greater 

increase in plasma renin activity than chlorisondamine alone. 

Failure of plasma renin activity to increase was not due to renin 

secretion being maximal after chlorisondamine because a further 

73 
increase in renin secretion could be produced by injecting 

isoproterenol, a drug that bypasses the block to act directly on 

the beta-adrenergic receptors on the renin-secreting cells in the 

kidneys. Thus, both the beta-adrenergic blocking experiment and 

the ganglionic-blocking experiment indicate that the 

renin-stimulating effect of serotonergic discharge-rs mediated 

via the sympathetic nervous system. A paper reporting these 

results is in press. 

Detailed investigation of the parts of the hypothalamus and 

brainstem involved in regulating the renin response to PCA and 

other stimuli have now begun. Discrete lesions are being 

produced in various parts of the hypothalamus and the brainstem 

and the response to PCA is being tested. These experiments will 

subsequently be expanded to determine the effects of these 

lesions on the renin response to tilting and psychological 

stimuli. 

Some preliminary experiments, have also been conducted to see 

under what conditions head-up tilting, one of the gravitational 

stimuli to be used in experiments, increases plasma renin 

activity. Preliminary experiments aimed at discovering a 

reliable and reproducible psychological stress that increases 

renin secretion in rats are also underway. 

74 
CHANGES IN BONE STRUCTURE AND METABOLISM DURING SIMULATED 

WEIGHTLESSNESS: ENDOCRINE AND DIETARY FACTORS 

Bernard P. Halloran 

Department of Medicine 

University of California 

San Francisco, CA 94143 

The objective of this research program is to determine the role 

of specific endocrine factors in the bone changes associated with 

simulated weightlessness and at the same time investigate the 

dynamics of bone metabolism. A rat model, in which the animal is 

suspended by its tail, is used to simulate weightlessness. 

Specific objectives were to: (1) determine the relationships 

between the circulating concentrations of calcium (Ca), inorganic 

phosphorus (p.), parathyroid hormone (PTH), 1,25-dihydroxyvitamin 

D (1,25 (OH)20), and the bone changes induced by simulated 

weightlessness; and, (2)determine the temporal changes in the 

serum concentration of 1,25(OH)2D following suspension. 

The first objective was accomplished by manipulation of dietary 

Ca and P. Four diets were used ranging from low Ca (0.1%), 

normal P (0.3%) to high Ca (2.4%), high P (1.2%). The animals 

were studied after 2 wk. of suspension (simulated 

weightlessness). The results of these experiments indicate that 

the bone mineral loss which occurs during simulated 

weightlessness on a diet with normal levels of Ca and P can be 

reduced by increasing the dietary intake of these elements. On a 

normal diet, tibia from animals undergoing simulated 

weightlessness showed a highly significant (P(.005) 13% decrease 

in total weight when compared with tibia from control animals. 

On a high Ca/P diet no significant differences in bone weight 

were observed. 

The serum concentrations of Ca, P., PTH, and 1,25 (OH)2D were 

virtually identical in suspended ~nd pair-fed control rats on all 

diets except the high Ca/P diet. In this case, serum Pi was 

elevated in suspended rats (6.0 + 1.5 mgldl vs. control of 4.8 + 

0.6 mg/dl) and serum 1,25 (OH)2D-was depressed (66 + 18 pglml vs. 

control of 112 + 19 pg/ml). Tfie similarities (except in the case 

of the high Ca/P diet) in the mineral and hormonal composition of 

serum from suspended and control animals after 2 wk. of 

suspension seem to suggest that changes in the circulating 

concentrations of PTH, and 1,25 (OH}2D are not responsible for 

the differences in bone mineral content observed at this time. 

However, la4Sr studies, which examined bone formation rates 

(uptake of Ca into the bone) as a function of time after 

suspension, indicated that bone formation falls dramatically 

during the first week of suspension and then returns to 

approximately normal during the second week. At 14 days of 

suspension, even though there is less total bone mass, formation 

75 
is nearly normal. This is consistent with the fact that serum 

concentrations of Ca, Pi' PTH, and 1,25(OH)2D (with the exception 

of the high Ca/P diet) are also normal. 

To examine the relationship between bone formation and the serum 

concentrations of Ca, P., PTH, and 1,25 (OH)2D (the second 

objective) during the e~rly phases of suspension, animals were 

sacrificed at 2, 5, 7, 10, 12, and 15 days after being suspended. 

Although not all data are available (PTH has not yet been 

measured), the results indicate that suspension causes a 

transient hypercalcemia and a transient fall in the circulating 

concentration of 1,25 (OH)2D from 130 + 13 pglml to 50 ± 12 

pg/ml. This occurs between days 2-7 of suspension and is 

followed by normalization of both variables. At the same time 

that animals are hypercalcemic and have low serum concentrations 

of 1,25 (OH)2D, bone formation is inhibited. 

These findings can be interpreted in at least two ways. On the 

one hand, suspension may cause a transient inhibition of bone 

formation (primary defect). The reduction in Ca demand by the 

bone may in turn cause a transient hypercalcemia which could lead 

to a transient reduction in circulating 1,25 (OH) D 

concentration. On the other hand, the primary defect may be a 

reduction in renal 1 alpha-hydroxylase activity, the enzyme 

primarily responsible for maintaining the serum concentration of 

1,25 (OH)2D. A reduction in serum 1,25 (OH)2D may reduce bone 

formation which in turn could cause a transient hypercalcemia. 

Further experiments are presently underway to examine both of 

these hypotheses. 

76 
THE EFFECTS OF HYPERGRAVIC FIELDS ON NEURAL PROCESSING OF SENSORY 

THERMOREGULATORY AND VESTIBULAR SYSTEMS IN THE RAT 

John M. Horowitz 

Department of Animal Physiology 

University of California 

Davis, CA 95616 

A major objective during 1983 was the continuation of studies on 

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 

in hypergravic fields from 1.5 to 6 G. One series of studies was 

completed, and additional experimental studies are partially 

completed. Data obtained during 1983 from the ongoing studies 

include the following: 

First, heat conservation and production were studied in rats at 3 

G and at 1 G. A closed-circuit system that includes a Krogh-type 

spirometer for the continuous measurement of oxygen consumption 

was used to determine heat production. At the same time, core 

temperature (Tc) and tail temperature (T t ) were also measured. 

During the first 20 min. at 3 G, oxygen consumption increased by 

at most 18% in some of the rats and fell as much as 15% in the 

remaining rats. However, in all rats at 3 G, there were falls in 

Tt and Tc during this time. Thus, the initial fall in Tc at 3 G 

was independent of concurrent changes in the rate of oxygen 

consumption. Furthermore, the impaired mechanism responsible for 

the rapid fall in T during the initial exposure to hypergravity 

was not reduced heat production (since oxygen consumption was 

variable and in some rats increased) but an increase in heat 

loss. However, even after 3 hr. at 3 G, when heat conservation 

mechanisms had recovered, the rate of oxygen consumption in rats 

was not significantly increased relative to the 1 G rate; hence 

thermogenesis was not activated to rewarm the animal. 

Second, data were also obtained on rats acclimated to a 2.1 G 

field and then cold exposed while in a hypergravic field of the 

same magnitude (2.1 G). Data obtained at this stage clearly 

indicate that acclimation to hypergravic fields enhances the 

ability of a rat to cope with cold exposure during centrifugation. In this experiment, 9th generation rats born and 

reared at 2.1 G were cold exposed at 2.1 G, and their rates of 

oxygen consumption were compared with those of rats born and 

reared at 1 G. This study is being performed in collaboration 

with Dr. Jiro Oyama at Ames Research Center. 

Third, experiments on the resetting of the set-point for 

shivering and nonshivering thermogenesis are also in progress. 

The experiments are designed to test the proposal that in 

hypergravic fields, rats regulate their core temperature, but at 

77 
an abnormally low level. Preliminary data indicate that while 

core temperature is regulated, this regulation is complex in 

depending not only on the hypergravic field but also on the 

ambient temperature. 

All three of the above studies center on the mechanisms 

underlying impaired temperature regulation in mammals exposed to 

hypergravic fields. This information is crucial to an assessment 

of this mammalian neural control system and provides background 

data for future studies in a hypogravic environment. 

In addition to the neural processing of thermal signals, studies 

have been initiated on processing brainstem vestibular signals in 

the rat. Electrical circuits and associated mechanical elements 

to provide a precise vestibular stimulus to rats have been } 

constructed and tested. In addition, the software for averaging 

individual vestibular responses has been developed. In 

preliminary experiments, vestibular evoked responses were 

recorded from rats using the vestibular stimulator and a 

laboratory computer. 

PUBLICATIONS 

Giacchino, J.L., Schertel, E.R., Horowitz, J.M., and Horwitz, 

B.A. Effect of E-chlorophenylalanine on Thermoregulation in 

Unrestrained Rats. American Journal of Physiology 244: 

R299-R302, 1983. 

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

in Cold- and Noncold-acclimated Rats Cold Exposed in Hypergravic 

Fields. Physiologist 26(6, Suppl.): S169-S172, 1983. 

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

Parallel Thermocontrollers in Rats Exposed to Hypergravic Fields 

(Abstract). Federation Proceedings 42: 1124, 1983. 

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

Fields and Parallel Controllers for Thermoregulation. Journal of 

Applied Physiology: Respiratory, Environmental and Exercise 

Physiology 55: 990-995, 1983. 

Monson, C.B., Horowitz, J.M., and Oyama, J. Thermoregulation 

during Cold Exposure at 2.1 G in Rats Adapted to Hypergravic 

Fields (Abstract). Federation Proceedings 43(4): 907, 1984. 

78 
GRAVITY PERTURBATION AS A PROBE FOR ANALYZING PATTERN 

SPECIFICATION IN EARLY AMPHIBIAN EMBRYOGENESIS 

George M. Malacinski 

Department of Biology 

Indiana University 

Bloomington, IN 47405 

Anton W. Neff 

Medical Sciences Program 

Indiana University 

Bloomington, IN 47405 

Gravity perturbation is being employed as a probe to study the 

manner in which early pattern (e.g., animal/vegetal polarity; 

dorsal/ventral polarity; primary axis morphogenesis) is 

specified. The eggs of most common laboratory amphibia (e.g., 

Xenopus, Rana, Ambystoma) display a prompt and immediate response 

to gravity, following activation (fertilization). Within minutes 

after sperm penetration, the typical amphibian egg rotates from 

its random orientation vis-a-vis gravity so that the darkly 

pigment animal hemisphere opposes gravity. Under natural 

conditions, amphibian eggs develop through the early pattern 

specification stages in that same orientation (animal hemisphere 

"up"). 

In order to employ gravity perturbation as a probe to understand 

the significance to pattern specification of egg rotation, a 

series of detailed analyses on morphogenesis in inverted Xenopus 

eggs, was carried out. Specifically, the cytoplasmic 

rearrangements which follow fertilization in both naturally 

oriented and inverted eggs were monitored. A set of yolk 

compartments was resolved by cytological analyses. Those 

compartments were characterized by their yolk platelet 

compositions and the manner in which they moved during egg 

inversion. During egg inversion several (but not all) of the 

yolk compartments shift. Those observations were coupled with 

additional observations on the pattern of morphogenesis in 

inverted eggs: T~ relocation of the major yolk compartments, but 

not some of the minor ones, is a prerequisite for normal pattern 

specification. Those findings were interpreted in terms of a 

novel "density compartment model" as a coherent way to view the 

organization of the egg cytoplasm and the specification of early 

pattern. 

As an additional subcellular marker, the germ plasm (cytoplasmic 

localization involved in primordial germ cell (PGC) specification) was monitored in inverted eggs. Inverted eggs typically 

contain reduced numbers of PGCs. Although most of the major 

cytoplasmic compartments shift in inverted eggs, the germ plasm 

does not. Reduced PGC numbers might be due to the inability of 

the germ plasm compartment to adjust (by movement) to the novel 

gravity orientation of egg inversion. 

As a further attempt to employ gravity as a probe for analyzing 

early pattern specification, Xenopus eggs were clinostatted. The 

79 
constant rotation of the clinostat serves to "cancel" the effects 

of natural gravity orientation. Several variations of previous 

clinostat protocols were employed, including fertilizing eggs 

directly on the clinostat (while rotating), and beginning the 

rotation after eggs were oriented in regard to the sperm entrance 

site. 

Pattern specifications were monitored up to the tadpole stage. 

Briefly, although the normal orientation of the dorsal lip 1800 

opposite the sperm entrance site was not observed, morphogenesis 

was normal. Hence, it is predicted that the major pattern 

specification events of early morphogenesis will be normal in the 

microgravity of outer space. 

PUBLICATIONS 

Neff, A.W., Wakahara, M., Jurand, A., and Malacinski, G.M. 

Response of Amphibian Egg Cytoplasm to Novel Gravity Orientation 

and Centrifugation. Physiologist 26{6, Suppl.): S143-Sl44, 1983. 

80 
STRUCTURAL DEVELOPMENT AND GRAVITY 

Emily Morey-Holton 

Biomedical Research Division 

NASA-Ames Research Center 

Moffett Field, CA 94035 

During 1983, research concentrated on two major objectives. In 

addition to research goals, the laboratory was involved with the 

Weber Student Shuttle Involvement Project (SSIP) which was flown 

aboard STS-41B in February 1984. 

The first major research objective was to define changes in bone 

formation rates and bone structure with age in the rat. 

Characterization of bone structure with age is essential since 

each bone appears to have a rate of formation and elongation and 

shape appropriate for its particular function and age. Two 

strains of rats were studied: (1) Fischer 344 and (2) 

Sprague-Dawley derived; these strains should obtain significantly 

different adult body size with the Fischer strain being smaller 

than the Sprague-Dawley. Total adult bone mass should be 

directly related to the size of the animal. This study was 

initiated in 1982 and was completed in August 1983. Rats were 

studied at 6, 10, 18, 36, and 68 wk. of age. Bone formation in 

the distal shaft of the leg bone (tibia-fibula junction) was most 

rapid at 6 wk. of age and precipitously decreased until between 

18-36 wk. of age when bone formation appeared to plateau. The 

bone formation rate curve for both strains was very similar 

although the initial rate of bone formation was higher in the 

Sprague-Dawley animals and, hence, this strain had slightly 

larger bones. This curve was also essentially superimposeable on 

the curve reported by Pace and co-workers for basal metabolic rat 

vs. age suggesting that skeletal maturation and metabolic 

maturation occur simultaneously. Data from bone elongation and 

other bone sites are in progress. 

The second research objective was to validate and refine the rat 

model used to simulate some of the physiological effects of 

spaceflight. Animals on the model for 1, 2, or 3 wk. were 

compared with animals exposed to 4 0 C for the same period of time 

to determine whether endocrine stress (elevated glucocorticoid 

levels) might be contributing to the changes in muscle mass or 

bone mass produced by unloading the hindquarters of the rats on 

the model. Only animals exposed to the cold responded with an 

increase in adrenal weight and a decrease in thymus weight, data 

indicative of increased glucocorticoid activity. Only animals on 

the model showed true atrophy (actual loss of muscle mass) of the 

soleus muscle (an antigravity leg muscle) although the 

cold-stressed rats showed decreased muscle growth. Bone 

formation rate was decreased similarly during the first week in 

both groups, but only the cold-stressed animals tended to return 

81 
to normal rates during the subsequent times. These data 

demonstrate that animals on the model for 1-3 wk. are minimally 

stressed as they do not show alterations in glucocorticoid 

sensitive tissues as do cold-stressed rats. Thus, elevated 

glucocorticoids play only a minor, if any, role in the 

physiological responses of unweighting rat hindquarters; the 

major factor influencing such changes is unloading the 

hindquarters and/or fluid shifts induced by the head-down 

position of animals on the model. 

The laboratory assisted with the Weber SSIP which was to 

determine whether the pathogenesis of arthritis involves a 

gravity component. The hardware test of the animal enclosure 

module (AEM) for housing the rats in the middeck of the shuttle 

was conducted aboard STS-8 which flew August 30-September 4, 

1983. Six, healthy, male, Lewis gnotobiotic rats were included 

in this test which was managed by NASA-Johnson Space Center with. 

the assistance of NASA-Ames Research Center. Following the 

successful hardware test, the experiment was scheduled for 

STS-41B. Dr. Holton was appointed NASA scientist/project manager 

for the Weber SSIP and established a biweekly newsletter for the 

project beginning in October 1983. The outcome of this project 

will be detailed in next year's progress report. 

PUBLICATIONS 

Holton, E. Bone and Calcium Alterations during Spaceflight. In: 

The Regulatory Functions of Calcium and the Potential Role of 

Calcium in Mediating Gravitational Responses in Cells and Tissues 

(~d. by S.J. Roux). Washington, D.C.: NASA, p. 111-126, 1983. 

(NASA Conference Publication 2286) 

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. 

Patterson-Allen, P.E., Shakes, D.C., Cann, C.E., Grindeland, 

R.E., and Morey-Holton, E.R. Effects of Stress and 

Immobilization on Serum Osteocalcin in Rats (Abstract). 

Calcified Tissue International 35: 647, 1983. 

Simmons, D.J., Grazman, B., Russell, J.E., Walker, W.V., Bikle, 

D.O., and Morey, E.R. Simulating Certain Aspects of Hypogravity: 

Effects on Bone Maturation in the Non-weight Bearing Skeleton. 

Aviation, Space, and Environmental Medicine 54: 1080-1084, 1983. 

Spector, M., Turner, R.T., Morey-Holon, E., Baylink, D.J., and 

Bell. N.H. Arrested Bone Formation during Spaceflight Results in 

a Hypomineralized Skeletal Defect. Physiologist 26(6, Suppl.): 

SlID-SIll, 1983. 

82 
Spengler, D.M., Morey, E.R., Carter, D.R., Turner, R.T., and 

Baylink, D.J. Effects of Spaceflight on Structural and Material 

Strength of Growing Bone. Proceedings of the Society for 

Experimental Biology and Medicine 174: 224-228, 1983. 

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

and Bone during Actual and Simulated Spaceflight. Medicine and 

Science in Sports and Exercise 15: 410-414, 1983. 

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. Inhibition of Cortical and 

Trabecular Bone Formation in the Long Bones of Immobilized 

Monkeys. Clinical Orthopaedics and Related Research 181: 

269-276, 1983. 

Wronski, T.J. and E.R. Morey. Recovery of the Rat Skeleton from 

the Adverse Effects of Simulated Weightlessness. Metabolic Bone 

Disease and Related Research 4: 347-352, 1983. 

83 
REGULATORY MECHANISM OF SKELETAL MUSCLE ATROPHY AND FLUID AND 

ELECTROLYTE SHIFTS IN THE HYPOKINETIC/HYPODYNAMIC AND 

ANTIORTHOSTATIC RAT 

X.J. Musacchia and Joseph M. Steffen 

Department of Physiology & Biophysics 

Ronald D. Fell 

Exercise Physiology Laboratory 

School of Medicine 

University of Louisville 

Louisville, KY 40292 

The focus of this research is the development and utilization of 

a dual purpose animal model to effectively plan experiments for 

weightlessness in orbital flight. Two areas are under 

investigation: (1) differential muscle atrophy resulting from 

nonloading suspension and the processes of recovery, using 

hind limb muscles (soleus, gastrocnemius, plantaris, and extensor 

digitorum longus (EDL» in hypokinetic/hypodynamic (H/H) 

suspended rats; and (2) relationships between fluid and 

electrolyte imbalance and cardiovascular response in head-down 

suspended rats. 

These studies extend from previous results of differential muscle 

atrophy (soleus> gastrocnemius = plantaris> EDL) from this lab 

which served to illustrate that the H/H rat model mimics and 

compares favorably with the results of rats exposed to 

weightlessness in the COSMOS series of experiments. The 

objectives of the muscle projects are currently focused on 

examination of contractile function and the endurance (i.e., the 

rate and extent of muscle fatigue) of hind limb muscles from H/H 

rats at various work intensities. In addition, several indices 

of metabolic activity before and after contractile activity are 

also measured (namely, glycogen content, lactate production, and 

oxidative capacity). 

Examination of the endurance capacity of muscles from one-wk. 

suspended H/H rats showed that the atrophied gastrocnemius 

fatigues significantly faster than the soleus. The loss of 

endurance capacity in the gastrocnemius, but not the soleus, of 

H/H rats suggests a differential involvement of fiber types. 

Evolution of the limb musculature in relation to functional 

adaptation is readily illustrated in the soleus, which is 

predominantly a slow twitch, highly oxidative muscle, in 

comparison with the gastrocnemius which is a mixture of 

fast-twitch oxidative glycolytic and fast twitch glycolytic 

fibers. In addition, this raises a most interesting question, 

namely, despite the greater loss of soleus muscle mass the 

remaining nonatrophied soleus fibers are highly' functional. In 

contrast, atrophy in the gastrocnemius, a mixed fiber muscle, 

84 
responds with a loss of some endurance capacity. Another series 

of experiments showed that cytochrome c concentrations (index of 

oxidative capacity) as well as activity of citrate synthase were 

decreased in gastrocnemius during one-wk. H/H suspension. Thus, 

the increased fatigability of the gastrocnemius may be due to a 

selective loss of the sarcoplasmic protein fraction. These and 

other speculative aspects will continue to receive attention in 

the 1984-1985 phase of this program. 

The general conclusion to be drawn from these investigations is 

that despite muscle atrophy with short periods (lor 2 wk.) of 

nonloading H/H, the muscle remains functional, provided that the 

work intensity is kept low. Future experiments will test this 

hypothesis by doubling the work intensity. In addition, recovery 

tests will be aimed at discovering the time it takes for full 

recovery of endurance capabilities. Since muscle fiber type has 

been found to be related to the fatigue reactions, an analysis of 

specific fiber type alterations in response to atrophy will be 

done using image analysis, histochemistry, and electron 

microscopy techniques. 

The second area of research, involving previously demonstrated 

disturbances in fluid and electrolyte excretion will be examined 

in terms of correlated cardiovascular responses. The basic 

hypothesis has been that animals in the model, that is, head 

down, whole body suspended rats, respond with a cephalad fluid 

shift. This fluid shift, in response to an altered gravitational 

orientation, is one of the chief characteristics of orbital 

flight. Since no direct experiments have been performed with 

experimental animals under conditions of weightlessness, the only 

relatable studies are those with the human subject. In the 

astronaut exposed to weightlessness, for varying periods, there 

is a recognized excess output of urine and electrolytes. The 

same conditions were obtained in water-immersed animals and 

humans, and in prolonged bed-rested humans. Since the animal 

model, the bed-rested human and the weightless human can be said 

to experience unusual gravitational vector orientation, there is 

a working model for future understanding of the disruption of the 

normal gravitational vector in the mammal. 

Two areas of investigation are underway. In one series of 

experiments, vascular pressure measurements showed increased 

blood pressures (mean, systolic, and diastolic) in head-down 

tilted rats. This could, in general terms, be characterized as a 

hypertensive response. such a response is also characteristic of 

an increased atrial blood volume and suggests ·a response to 

baroreceptor reflexes. Some preliminary studies have been 

published. 

A second series of studies involves the role of hormone 

regulators, namely, aldosterone (regulating sodium retention) and 

antidiuretic hormone (regulating water retention). It is 

85 
proposed that in the antiorthostatic rat, that is, the head-down 

tilted subject, these hormones may play an important role in the 

genesis of the fluid and electrolyte disturbances (e.g., 

increased excretion of urine and electrolytes). Similar 

conditions have been demonstrated in human studies during 

spaceflight and now can be simulated with the animal model. It 

has been hypothesized that the level of both hormones should be 

depressed to account for the diuretic and natriuretic effects. 

Preliminary results, however, have demonstrated an early (after 

1-3 days of suspension) increase in plasma aldosterone 

concentration, suggesting that the fluid and electrolyte 

disturbance may result from a complex series of hormonal events. 

Detailed studies for 7- and l4-day subjects are underway and will 

continue in the 1984-1985 phases of this program. 

In summary, the animal model used in this laboratory has proven 

valuable for investigating disturbances in basic muscle 

physiology and in fluid and electrolyte regulation. Future 

studies are intended to further elucidate specific mechanisms in 

order to plan crucial flight experiments and to determine the 

mechanisms involved in the course of recovery phenomena. 

PUBLICATIONS 

Musacchia, X.J. Introduction--Gravitational Physiology. 

American Journal of Physiology 244: R303, 1983. 

Musacchia, X.J. and Steffen, J.M. The Validity of an Animal 

Model for Experiments Related to Weightlessness. Physiologist 

26(6, Suppl.): S37-S40, 1983. 

Musacchia, X.J., Steffen, J.M., and Deavers, D.R. Rat Hindlimb 

Muscle Responses to Suspension Hypokinesia/Hypodynamic. 

Aviation, Space, and Environmental Medicine 54(11): 1015-1020, 

1983. 

Robb, R., Steffen, J.M., Sonnenfeld, G., Musacchia, X.J., 

Dombrowski, M.J. Mouse Model for Studies of Hypokinesia and 

Antiorthostasis. Federation Proceedings 42: 1123, 1983. 

Steffen, J.M. and Musacchia, X.J. Altered Numbers of 

Glucocorticoid Receptors in Cardiac Muscle from Suspended 

Hypokinetic/Hypodynamic Rats. Federation Proceedings 42: 1123, 

1983. 

86 
HYPER-GRAVITATIONAL EFFECTS ON METABOLISM AND THERMOREGULATION 

Jiro Oyama 

NASA-Ames Research Center 

Moffett Field, CA 94035 

The objective of the research program is to develop a more 

comprehensive and basic understanding of the effects and 

mechanisms of action of acute and long duration hypergravity 

exposures on mammalian organisms. Investigations are focused on 

systems regulating blood glucose and body temperature of the rat. 

Studies during 1983 have been aimed at deterimining: (I) the 

effects of acute hyper-G stress on gluconeogenesis and glucose 

homeostasis, (2) basal metabolic rates of hyper-G-adapted rats, 

and (3) the relationship between G intensity and the hypothermic 

response induced by hyper-G stress exposures. 

Results from these studies have further delineated the important 

role of the sympathetic-adrenal system on the initial rapid rise 

in blood glucose during hyper-G stress exposures. Results from 

gluconeogenic rate measurements using labeled lactate, alanine, 

and glycerol in hyper-G-stressed rats have shown that the initial 

surge in blood glucose is due mainly to an increase in 

gluconeogenesis from lactate (from catecholamine-induced muscle 

glycogenolysis). Since the initial increase in plasma 

catecholamine levels is only transient returning to control 

levels after 1 hr. of hyper-G exposure, the sustained increase in 

gluconeogenesis and in blood glucose levels with longer exposure 

durations appears to be mediated by glucagon action. The 

increased plasma glucagon levels found correlate well with the 

increased gluconeogenic rates and the increase in blood glucose 

levels found in hyper-G-stressed rats. 

A considerable amount of effort was expended during 1983 to gain 

experience with and to improve upon the previously developed 

procedures to isolate hepatocytes from livers of hyper-G-stressed 

rats for in vitro gluconeogenic rates measurements. Problems 

affecting~he viability of the hepatocytes as well as yields were 

finally resolved, and gluconeogenic rate measurements using 

labeled lactate and alanine were initiated on livers from 

hyper-G-stressed (0.5 hr.; 3.1 G) and control rats. The method 

used differs from all other published methods in that cannulation 

and perfusion are performed immediately after decapitating the 

rat thereby obviating any possible effects of anesthetic drug 

actions on the rate measurements. 

Data obtained over a number of years on oxygen consumption rates 

of different groups of hyper-G-adapted and normal gravity control 

rats have been analyzed along with data on body composition. 

Results of the analysis, which takes into account differences in 

87 
body mass, body composition, and age, provides additional 

evidence that the resting metabolic rates of hyper-G-adapted rats 

are significantly higher (as much as 30%) than comparable rats 

reared under normal gravity. A paper on this study will be 

submitted for publication shortly. 

An extensive series of acute hyper-G exposure studies were 

completed in which the effects of centrifuge radius, rotational 

rate, and G intensity on rectal and tail temperature responses of 

rats were determined. The important finding was that the changes 

observed in rectal temperature (decrease) and tail (increase) are 

directly and solely related to the G intensity imposed on the 

animal and are independent of the radius and rotational rates 

used. Identical temperature responses were obtained for the same 

G intensity when the radius ranged between 0.46-1.73 m and the 

rotational rate ranged between 22-44 RPM. 

PUBLICATIONS 

Scibetta, S.M., Caren, L.D., and Oyama, J. The Effects of 

Hypergravity on the Rate of Antibody Formation in the Rat. 

Physiologist 26(6, Suppl.): S135-S136, 1983. 

88 
GRAVITY, BODY MASS AND COMPOSITION, AND METABOLIC RATE 

Nello Pace and Arthur H. Smith 

Environmental Physiology Laboratory 

University of California 

Berkeley, CA 94720 

The purpose of this project is to examine energy metabolism and 

body composition characteristics of mammals as a function of body 

size in order to assess the evolutionary influence of 

gravitational loading on present-day scaling relationships. Five 

species of common laboratory mammals--the mouse, hamster, rat, 

guinea pig, and rabbit--have been selected for examination. This 

series provides a 100-fold range of mature body mass from 0.05 to 

5 kg. 

Six male and six female animals of each of eight age cohorts from 

I mo. to 24 mo. for each of the five species form the basic 

animal matrix of the study. The oxygen consumption rate of each 

animal is measured under standard conditions to provide metabolic 

rate, and the animal is then killed for organ mass measurements 

and biochemical analysis for body contents of water, fat, 

potassium, sodium, magnesium, calcium, phosphorus, nitrogen, and 

creatine. From these parameters it is also possible to derive 

fat-free body mass, body cell mass, intracellular and 

extracellular water masses, bone mineral mass, and skeletal 

muscle mass. Quantitative scale relationships of the parameters 

to body mass are determined by allometric analysis. Comparison 

of the data among the various age groups also permits separation 

of growth effects from loading effects. 

By the end of 1983 the data for the animal matrix was about 90% 

completed. All the metabolic rate, body water content and body 

fat content measurements were in hand, and the biochemical 

analyses of the dried, fat-free, whole body powders for all but 

two age cohorts were done. Although definitive treatment of the 

data awaits completion of all the analyses, several preliminary 

findings have emerged. For example, the three major body 

components--skin, viscera, and skinned, eviscerated carcass--have 

been considered separately. The skinned, eviscerated carcass 

comprises all of the body skeleton and practically all of the 

skeletal muscle, and may be regarded as the weight-bearing organ 

of the body. It has been shown that for the series, mouse to 

rabbit, the allometric relationship between carcass mass and 

total body mass (TBM) is: 

Carcass: kg = 0.503(TBM, kg)I.055 j r = 0.999. 

Whereas, the relationship for viscera mass to TBM is 

Viscera: kg = 0.158(TBM, kg)0.871; r = 0.995, 

89 
and for skin it is 

Skin: kg = 0.139(TBM, kg)0.942; r = 0.993. 

Thus, Galileo's classic theory that the scale of the weightbearing components of the body must increase proportionately to 

body mass as animals become larger, and by implication the 

visceral components must diminish proportionately, has been 

confirmed. Allometric analysis of the relationships of the body 

elements to body mass will permit further definition of the 

influence of gravity on the evolution of body characteristics. 

Another preliminary finding has been that when metabolic rate is 

expressed as metabolic intensity, !pat is, .in the.mass-specific 

terms of kcal· hr. ·kg body mass ,and 1S exam1ned as a 

function of age in this series the relationship is hyperbolic in 

form. Metabolic intensity is high in young animals, and falls 

asymptotically to a relatively constant value by age 5-8 mo. in 

these species. Thus, it is possible to define metabolic maturity 

quantitatively, and to demonstrate that the interspecific 3/4 

power allometric scaling of metabolic rate on TBM holds only for 

metabolically mature mammals. 

During 1983, study of the intracellular concentration of creatine 

in skeletal muscle tissue from the series of animal species has 

been extended. The development of a method for estimating 

skeletal muscle mass of mature animals 8 mo. or more in age from 

their body creatine content has been reported previously. 

Skeletal muscle tissue from 2-, 3-, and 5-mo.-old animals is now 

being examined with the objective of expanding the method to 

include these younger animals. It is anticipated that the 

results will make possible interpretation of the body creatine 

data from the basic animal matrix in terms of skeletal muscle 

mass as a function of species, sex, and age. 

In other experiments, the effects of ambient temperature change 

and of increased gravitational loading by chronic centrifugation 

on the allometric relationship between metabolic rate and TBM in 

met~bolically mature animals of the series have been examined. 

It is well known that the metabolic rate of mammals is at a 

minimum in a relatively narrow ambient temperature range known as 

the thermoneutral zone. When ambient temperature falls below, or 

rises above, the thermoneutral zone, the metabolic rate 

increases. The precise range of the thermoneutral zone is a 

function of species, age, and sex differences, and has been 

established only for a relatively few cases. Hence it was 

necessary for the thermoneutral zone range to be determined for 

the animals of this series. It has been found that 28 + 10 C 

represents the ambient temperature range within which the 

metabolic rate remains within + 10% of the minimum value for 

metabolically mature animals of the species used. 

90 
During 1983 it was shown that in this ambient temperature range 

the interspecific allometric relationship between metabolic rate 

and TBM for the series is given by 

Metabolic Rate, kcal·hr.-l = 2.91(TBM, kg)O.766; 

r = 0.979, 

in good agreement with the classic Kleiber 3/4 power relationship 

for mammals generally. 

However, when the same animals were measured in an ambient 

temperature environment of 24 ~ 10 C, the allometric relationship 

became 

Metabolic Rate, kcal' hr.-l = 3.51 (TBM, kg)O.676; 

r = 0.987. 

The positioning constant of the equation is elevated because of 

the higher metabolic rate at the lower temperature, and the 

exponent is lessened because the metabolic rate of the smaller 

animals was increased proportionately more at the lower 

temperature than was that of the larger animals. Hence, it may 

be concluded that the condition of thermoneutrality is important 

for experimental studies of the effect of altered gravitational 

loading on metabolic scale effects. 

Such a study was completed during 1983 of the effect of 6 wk. of 

chronic centrifugation at 2.0 G on the allometric scale 

relationship between metabolic rate and TBM in metabolically 

mature animals of the species. The relationship was found to be 

Metabolic Rate 2. 0G , kcal·hr.-l = 3.30(TBM, k9)O.813; 

r = 0.993. 

This finding has led to the conclusion that augmented 

gravitational loading shifts the relationship between metabolic 

rate and TBM by an increase in both allometric parameters, as was 

predicted previously, and that therefore gravitational loading is 

an important contributor to mammalian metabolic energy 

reguirements. A continuing prediction is that abatement of 

gravitational loading in spaceflight will result in a lowering of 

both allometric parameters in the classic 1.0 G 3/4 power 

relationship between metabolic rate and TBM. 

PUBLICATIONS 

Pace, N. and Rahlmann, D.F. Thermoneutral Zone and Scaling of 

Metabolic Rate on Body Mass in Small Mammals. Physiologist 26(6, 

Suppl.): S51-S52, 1983. 

Pace, N. and Smith, A.H. Scaling of Metabolic Rate on Body Mass 

in Small Mammals at 2.0 G. Physiologist 26(6, Suppl.): 

91 
S125-S126, 1983. 

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

D.F., arid Smirnova, T.A. Effects of weightlessness on Body 

Composition in the Rat. American Journal of Physiology 244: 

R332-R337, 1983. 

92 
EFFECT OF DECREASED GRAVITY ON CIRCULATION IN THE RAT 

V.J. Popovic 

Department of Physiology 

School of Medicine 

Emory University 

Atlanta, GA 30322 

Limiting movement, or hypokinesia, in the antiorthostatic 

(head-down) position mimics the circulatory effects of 

weightlessness. Circulatory processes in the rats were studied 

during their exposure to head-down (-20 0 ) hypokinesia, and during 

readaptation of the rats to control conditions. The 

cardiovascular measurements were done in control experiments on 

unrestrained, unanesthetized rats, and in the same animals in 

hypokinetic conditions, and during readaptation to free activity. 

Because surgery and anesthesia drastically decrease cardiac 

output and other circulatory parameters in rats, measurements 

were taken from unanesthetized rats. For sampling blood during 

the study, the aortas and right ventricles of the animals were 

permanently cannulated 10-15 days before the experiment. 

Investigators measured the hormones ACTH, corticosterone, and 

prolactin by radioimmunoassay to monitor the stress imposed on 

the rats by placement of the suspension harness and by the 

head-down position. These stress hormones rose in the early 

exposure to antiorthostatic hypokinesia, but after 6-7 days of 

exposure the animals adapted to the unusual position, as judged 

on the return of these three hormones to their normal levels. 

Plasma catecholamines were also elevated on days 1 and 3 of the 

antiorthostatic exposure. The plasma growth hormone level was 

decreased on day 1. On day 7 of the exposure, the plasma levels 

of both hormones were only slightly above the control levels. 

After release from the harness and after return to their own 

cages, the hormone levels were again elevated, although not as 

much as during the antiorthostatic exposure. The elevation 

lasted 2-3 days. In harnessed animals that were orthostatic (not 

positioned head-down), the increase in the plasma level of stress 

hormones was very small and lasted only a few hours on the first 

day. The level of hormones was at or near control values during 

the next 6 days. Harness removal did not induce any increase of 

studied hormones. 

Investigators studied hormonal stress levels during 30-min. 

immobilization in rats, repeated 17 times during a 17-day period. 

Adaptation processes were completely absent during repeated 

immobilization, while during exposure to antiorthostatic 

hypokinesia, the animals adapted after 3-4 days, and in the case 

of orthostatic hypokinesia this process was only a few hours 

long. 

93 
In other studies the ADH levels decreased the first day of 

exposure to antiorthostatic hypokinesia, but angiotension II 

levels were only slightly elevated. Also, cardiac output in 

antiorthostatic rats followed circadian patterns 4-7 days after 

initiation of hypokinesia. 

94 
BONE CELL KINETICS OF SIMULATED WEIGHTLESSNESS 

W. Eugene Roberts 

Department of Orthodontics 

University of the Pacific 

School of Dentistry 

San Francisco, CA 94115 

In general, this project addresses the influence of gravity on 

mammalian cell differentiation (specialization). Specifically, 

the focus is on the mechanism by which production of osteoblasts 

(bone-forming cells) is inhibited in weightlessness. Osteoblast 

differentiation is studied in weight-bearing long bones (tibia 

and ulna) as well as in maxillary periodontal ligament, adjacent 

to the roots of teeth in the non-weight-bearing upper jaw. The 

cytological marker for distinguishing preosteoblasts (the 

immediate predecessors of osteoblasts) from less differentiated 

and nonosteogenic cells is nuclear size. Differentiation of a 

committed osteoprogenitor cell (A') to preosteoblasts (C/D cells) 

involves a 200-300% increase in nuclear volume. This increase in 

nuclear size is a morphological manifestation of change in 

differentiation. 

During 1983, focus was on the following objectives: (I) define 

the proliferation and differentiation sequence for osteoblast 

production (histogenesis); (2) determine the effect of simulated 

weightlessness, via head-down suspension of a rat by its tail, on 

osteoblast histogenesis in the periodontal ligament (PDL); (3) 

develop an autoradiographic technique (histologiI~l dete3tion of 

radioactive tracers) for distinguishing between C and H labels 

in the same specimen; (4) utilize the nuclear morphometric assay 

for osteoblast precursors to assess the effects of 18.5 days of 

weightlessness on the ulna of Cosmos 1129 rats; and (5) in a 

related project with pilot funds from another source, develop a 

method of using rigid, endosseous implants to test osseous 

adaptation to a defined interval load, in bone of a true 

remodeling species (rabbits). 

Progress 

1. Osteoblast Histogenesis Sequence 

Extensive analysis of previously published and unpublished cell 

kinetic data has resulted in identification of a five-compartment 

model for osteoblast histogenesis {proliferation and 

differentiation seguence for forming a specialized cell}: (I) 

self-perpetuating, relatively undifferentiated precursor cells 

(A); (2) committed osteoprogenitor cells (A'); (3) GI stage 

(pre-DNA synthesis) preosteoblasts (C cells); (4) G2 stage 

(post-DNA synthesis) preosteoblasts (D ceIJs); (5) mature 

osteoblasts. Nuclear volumes are 40-80 ~m , for A and A' cells, 

95 
120-169 ~m3 for C cells, and> 170 ~m3 for D cells. Mathematical 

simulation of osteoblast production, based on the numbers of each 

cell type present in the rat PDL, has resulted in a close 

approximation of two sets of experimental data. This suggests 

the present model of osteoblast differentiation has identified 

all major osteogenic components among fibroblastlike cells of the 

POL. As far as is known, this is the first osteoblast 

differentiation sequence which has been worked out in any boneforming tissue. 

2. weightlessness vs. Simulated Weightlessness 

Both weightlessness (COSMOS) and simulated weightlessness (30 0 

head-down suspension by the tail) results in significant 

decreases in the numbers of preosteoblasts (C/D cells). This 

suggests an inhibition of the key rate limiting step in 

osteoblast histogenesis, which is the increase in nuclear size 

(A' + C) to forma preosteoblast. Since both weightlessness and 

simulated weightlessness have a similar net effect, of decreasing 

preosteoblast production, the block in the A' +C shift is not a 

direct effect of weightlessness. It may be related to a 

generalized disturbance in cell physiology, associated with the 

well-documented fluid shift and loss of extracellular water. 

3. 3H/14C Tracer Studies 

A low dose (0.1 ~Ci/gm) of Ijc-thymidine (DNA precursor) and a 

higher dose (3.0 ~Ci/gm) of H-Iysine, successfully distinguished 

between labeled nuclei (DNA synthesis) and labeled cytoplasm 

(protein synthesis). Routine autoradiography with 7-10 days of 

exposure produces a good assessment of protein synthe!~s, which 

is not significantly contaminated by the low dose of C which is 

restricted lar~~ly to the nucleus. A special technique for 

detecting the C labeled nuclei was developed. Histological 

sections receive a 3-5 ~m coat of gelatin prior t03 dipping in 

nuclear tract (autoradiographic) emulsion. Since H has a 

disintegration range of only about 1.0 ~m it is not registered in 

t~e emulsion. Exposure of 3 mo. is necessary to record 

C-Iabeled nuclei because of the low injected dose. 

The only major problem is that 14C-Iabeled nuclei cannot be 

distinguished from unlabeled nuclei in al~ense population of 

cells, like bone marrow. The energy of C is sufficiently high 

that a halo of disintegrations is registered for 4-5 ~m around 

each labeled nucleus. This problem would significantly interfere 

with analysis of some populations of bone-lining cells. In 

conclusion, it is best to use separate animals to assess DNA and 

protein synthesis, but it is possible to simultaneously assess 

both in the same animal using the present double label technique, 

at least in areas of relatively low cell density. This method 

presents an option for maximizing the scientific yield from a 

small number of animals. 

96 
4. Osteoblast Differentiation of Long Bones 

Nuclear size analysis of fibroblastlike cells in the primary 

spongiosa (just beneath the growth cartilage) of rat ulna 

specimens from Cosmos 1129 (18.5 days of spaceflight) show a 

similar net depression of the number of C/D cells as PDL. These 

results indicate: (1) the osteoblast differentiation sequence 

developed in PDL is applicable to other skeletal sites, and 2) 

osteoblast histogenesis is inhibited in the growing long bones 

in weightlessness. 

5. Osseous Adaptation in Rabbits 

A titanium endosseous implant has been devised which develops a 

rigid, osseous interface with cortical bone. A spring delivering 

100 gm of compression is applied 6 wk. after implants are placed. 

Periosteal areas exposed to concave fle~ure display osseous 

hypertrophy to compensate for the load. This is a well-defined 

means of assessing adaptation to a known load. The response can 

be assessed with radiographs and multiple colors of bone-staining 

dyes which mark areas of bone formation at the time of injection. 

This method could be utilized to test skeletal adaptation to 

applied loads (form/function interaction) under conditions of 

weightlessness and simulated weightlessness. It is readily 

adaptable to quantitative procedures such as histomorphometry 

(measurement of bone turnover using vital labels) and nuclear 

morphometric assessment of osteoblast histogenesis. 

PUBLICATIONS 

Roberts, W.E. Endosseous Implants as Anchorage (Abstract). 

American Journal Orthodontics 83(2): 163, 1983. 

Roberts, W.E., Mozsary, P.G., and Smith, R.K. Bone Modeling and 

Remodeling Response to Loads Applied via Rigid Endosseous 

Implants (Abstract). Journal of Dental Research 62: 686, 1983. 

97 
MAMMALIAN GRAVITY RECEPTORS: STRUCTURE AND METABOLISM 

Huriel D. Ross 

Department of Anatomy and Cell Biology 

University of Michigan 

Ann Arbor, HI 48109 

The long-term goal of this research is to elucidate calcium 

metabolism in mammalian gravity receptors. To accomplish this 

objective it is necessary to study both the mineral repositories 

of the receptors, the otoconia, and the sensory areas themselves, 

the saccular and utricular maculas. The main focus of this 

research project has been on the otoconia. 

Mammalian otoconia (literally "ear dust") are minute particles of 

calcite that mimic the form and symmetry of naturally occurring 

single crystals. Otoconia contain organic material in addition 

to calcite, and organic material also binds the particles to one 

another and to the underlying macula. The organic material below 

the otoconia has, in particular, been referred to as the 

"otoconial (or otolithic) membrane." It is commonly accepted 

that otoconia simply increase the mass of the organic membrane to 

magnify the inertial force acting upon the receptors (hair cells) 

under conditions of linear acceleration (or deceleration). Early 

results of this study demonstrated, however, that otoconia are 

dynamic mineral deposits and suggested that they function in 

calcium regulation in the system. The rate of uptake and release 

of radioactive calcium in vivo and in vitro strongly indicated 

that organically directed mechanismS; rather than processes 

involved in inorganic crystal growth, were responsible. 

These early findings led to the investigation of the role of 

organic material in facilitating the seeding and growth of th~ 

mineral deposits and in controlling their form and mass. Fetal 

studies in the rat indicated that deposition of organic and 

inorganic otoconial phases was nearly simultaneous 

developmentally. The two phases appeared identical in 

organization within the crystals. These results prompted more 

recent explorations of: (1) the structural organization of 

otoconia and related otoliths, (2) the evolution of the mineral 

deposits in vertebrates, and (3) the precise chemical composition 

of the organic otoconial phase in various vertebrate species. 

This research is being carried out through the use of ultrahigh 

resolution transmission electron microscopy to determine the 

lattice structure of the mineral phase; scanning electron 

microscopy of otoconia of species representative of the various 

vetebrate orders; and high performance liquid chromatography of 

the amino acid and carbohydrate composition of the organic 

material of inner ear mineral deposits. 

A comparative, ultrahigh resolution transmission electron 

98 
microscopic study of fish otolith, frog otoconia, and rat 

otoconia was reported during 1983. This study showed that none 

of the otoconia investigated were single crystals nor were the 

otoliths polycrystalline, as had been suggested by previous 

crystallographic research. All were composites of highly ordered 

crystallites. In the case of rat otoconia, the crystallites were 

typically _80 nm in broadest diameter and had some curved and 

some sharp edges. In contrast, naturally occurring calcite 

(Iceland spar) broke into fragments of nonuniform sizes having 

sharp edges. Electron beam diffraction patterns obtained from 

all the inner ear mineral deposits showed numerous imperfections 

in comparison to their inorganic counterparts. The results taken 

together were interpreted to indicate that organic material was 

important in the seeding and growth of the unusual crystallites 

included in an otoconium or otolith, and for ordering them into 

the final form. An important, coincidental conclusion based upon 

crystallite asymmetry was that otoconia might function 

piezoelectrically, that is, by altering the electric field around 

the hair cells rather than by mechanical bending of the hair 

bundles of the receptor cells. 

Another observation was that the diffraction patterns sometimes 

obtained from fragmented frog otoconia, which typically contain 

aragonite rather than calcite, matched the hexagonal patterns 

yielded by some rat otoconial fragments. The spacings in 

hexagonal aragonite and calcite diffraction patterns (001 faces) 

are nearly identical and make it impossible to distinguish 

between the two polymorphs of calcium carbonate on these grounds 

alone. Nevertheless, the observation raised the questions 

whether calcitic otoconia were present in the frog, or whether 

the calcitic configuration evolved from a prior, aragonitic form 

which it mimicked. 

The questions raised led to more recent, scanning electron 

microscopy of otoconia from a number of species representative of 

the various vertebrate classes. In June 1983 it was reported 

that only calcitic-type otoconia were present in turtle utricles. 

In the American alligator, calcitic-type otoconia occurred in all 

its gravity receptors. These reptiles were studied because they 

descended from the same vertebrates that served as ancestors for 

mammals and for birds. Occasional calcitic-type otoconia in 

amphibian gravity receptors, have also been observed but the 

study is incomplete. 

The scanning electron microscope results clearly showed that the 

calcitic configuration preceded the evolution of birds and ---

mammals, but it has not yet been demonstrated whether the mineral 

in such otoconia is calcite or aragonite. The findings led to 

deeper questions: Precisely what determimes whether aragonite or 

calcite is to be deposited in a particular gravity receptor? What 

difference, if any, does this make to the functioning system? 

The chemical composition of the organic material and the number 

99 
of proteins involved needs to be known to learn the answers to 

these questions. Although this line of research began several 

years ago, using microdisc gel electrophoresis methods, high 

performance liquid chromatography (HPLC) and a sensitive 

fluorescence detector have been used more recently to analyze 

otoconial masses for their amino acid and carbohydrate 

constituents. 

The first analysis of the amino acid composition of otoconia was 

presented during the summer of 1983 at an international meeting 

on biomineralization. The findings were that otoconial complexes 

(otoconia plus otoconial membrane) were high in acidic and low in 

basic amino acids. The original method employed for amino acid 

analysis involved postcolumn derivitization with a substance 

called O-phthaldialdehyde (OPA). This method did not permit 

detection of proline and hydroxyproline, which are significant 

components in collagen. (Collagen is important in 

biomineralization of bone.) In late 1983, therefore, analyses 

using hypochlorite (clorox bleach) in addition to OPA were 

carried out to determine whether proline and hydroxyproline were 

important constituents of otoconial organic material. The 

results, reported in February 1984, were that small amounts of 

these two amino acids occur in otoconial matrix, but they are 

relatively insignificant. 

In collaborating with a colleague in the Department of 

Pharmacology, Dr. Fulvio Perini, otoconial organic material was 

analyzed for the presence of amino and neutral sugars. It was 

demonstrated that otoconial organic material contains relatively 

large amounts of glucosamine and galactosamine as well as 

galactose, and smaller quantities of mannose. The findings could 

indicate that presence of glycoproteins containing N-linked 

oligosaccharides, possibly with a repeating galactose-Nacetylglucosamine sequence, as well as Q-linked oligosaccharides 

of the type sialic acid-galactose-N-acetylglucosamine. 

The results of the HPLC analyses are exciting. They show that 

otoconial complexes, like other biomineralized materials (such as 

shells and otoliths) that contain a polymorph of calcium 

carbonate, have organic material that is high in acidic and low 

in basic amino acids. Moreover, the carbohydrate findings 

indicate that the organic matrix is, or contains, 

glycoprotein(s). In many other biomineralizing systems, it is a 

soluble, highly sulfated acidic glycoprotein that is considered 

to be important in the seeding and growth of crystallites, and in 

inhibiting crystallite growth beyond a certain size. That is, 

the acidic, aspartic acid residues of the glycoprotein attract 

calcium, then carbonate ions to seed a crystallite by ionotropy. 

The crystallite grows to a certain size whereupon acidic 

glycoprotein in solution in the surrounding medium is adsorbed, 

stopping further growth of that small crystal. Free acidic, 

aspartic acid residues of the adsorbed glycoprotein are, however, 

100 
able to attract calcium ions to repeat the process, enabling the 

biomineralized material to grow in total size. While not all 

aspects of otoconial growth and inhibition are thus explained, 

the hypothesis would fit the nearly simultaneous deposition of 

organic and inorganic materials observed in developing otoconia. 

Muriel Ross gave a paper on her new findings concerning the 

complexity of innervative patterns in rat gravity receptors at 

the American Medical Association Meeting in San Diego in May 

1984. Her ultrastructural results indicate that the two kinds of 

receptor cells in the maculas are integrated functionally through 

shared afferents and that there exists a system of efferent type 

nerve fibers and terminals of intramacular (mostly calyces) 

origin. Additionally, there is a plexus-like arrangement of 

afferent and efferent-type terminals at many sites in both 

maculas. On the basis of her finding, Dr. Ross suggested that 

sensory processing of information concerning linear acceleration 

begins peripherally. The finding of an intramacularly 

originating system of efferents may provide anatomical evidence 

for lateral inhibition in the macula, to improve signal to noise, 

and for peripheral adaptation to constant acceleration. Because 

of the complexity of the neural patterns both on the afferent and 

the efferent neural side, Dr. Ross hypothesized that naturally 

occurring asymmetry in macular neural organization may be common, 

and that variability in degree of asymmetry may provide a 

peripheral contribution to the expression of space adaptation 

syndrome in the novel environment of microgravity. The results 

provide an anatomical basis for complex sensory processing in the 

maculas, and suggest that macular asymmetry may be common. 

PUBLICATIONS 

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

Williams, R.J.P. The Ultrastructure of the Calcium Carbonate 

Balance Organs of the Inner Ear: An Ultra-high Resolution 

Electron Microscopy Study. Proceedings of the Royal Society of 

London, Series B: Biological Science 218: 415-424, 1983. 

Ross, M. Calcium Ions, Stores, and Modulators: What is the 

Gravity Receptor Connection? In: The Regulatory Functions of 

Calcium and the Potential Role of Calcium in Mediating 

Gravitational Responses in Cells and Tissues (ed. by S.J. Roux). 

Washington, D.C.: NASA, p. 147-163, 1983. (NASA Conference 

Publication 2286) 

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

Mammals. Advances in Space Research 3(9): 179-190, 1983. 

Ross, M.D. and Boune, C. Interrelated striated Elements in 

Vestibular Hair Cells of the Rat. Science 220: 622-624, 1983. 

Ross, M.D. and Pote, K.G. Some Properties of Otoconia. 

101 
Philosophical Transactions of the Royal society of London, Series 

B: Biological Sciences 304: 445-452, 1984. 

102 
SOME ASPECTS OF HYPOGRAVITY SIMULATION ON BONE FORMATION AND 

MATURATION 

David J. Simmons and Jean E. Russell 

Department of Surgery 

Division of Orthopedic Surgery 

Washington University school of Medicine 

st. Louis, MO 63110 

Research is directed toward an understanding of the use of 

land-based hypokinetic systems (LBHS) to model the effects of 

hypogravity on the skeleton. The particular focus has been to 

determine the applicability of LBHS on the non-weight-bearing 

skeletal parts which do not show the severe osteopenic 

consequences of spaceflight and unloading observed in the 

weight-bearing bones. However, the non-weight-bearing bones are 

not unaffected by spaceflight. The jaws and teeth of rats flown 

in the Cosmos-1129 Biosatellite showed a defect in the rate of 

maturation of the mineral and matrix moieties, indicating that 

the changes in these bones might discriminate between specific 

effects of unloading and specific effects of gravity. 

This report deals with three experimental land-based hypokinetic 

systems--a rat suspension model and two primate restraint 

systems. In the suspension model, the limb bones ar5 pastially 

unloaded by raising the hindquarters of rats at a 30 -40 angle 

(NASA-Ames Research Center). The primate restraint systems 

involve postcranial whole body plaster casts, or chair-restraint 

on a roto-positioner (Wright Patterson AFB). All systems, then, 

mimic the effects of skeletal unloading on the weight-bearing 

bones, but none of them should have any special influence on 

skeletal parts such as the jaws which are already "unloaded" save 

for the stresses placed on the bones by antigravity muscles. 

Thus, where the jaws are concerned, these systems then test more 

specifically for an effect 6f gravity. 

Several techniques were applied to this work. First, 

tetracycline labeling was used so that the rates of appositional 

bone growth could be followed accurately during the periods of 

immobilization and postimmobilization recovery. Second, a 

bromoform-toluene density gradient fractionation method was used 

to follow the rate of maturation of newly forming mineral 

(calcium and phosphorus) and matrix (hydroxyproline) moieties. 

It was the latter method which defined the failure of maturation 

in the jaws of rats actually flown in space. 

Rat Suspension Model: The most striking result from this series 

of studies was the complete absence of abnormal patterns of 

appositional bone growth (i.e., mineralization rates) and tissue 

maturation in the jaws of rats entered into the suspension model. 

Because this model did not mimic the effect of spaceflight, there 

103 
is reason to believe that at least some of the processes involved 

in tissue maturation have an important gravity component. 

Primate Immobilization Model: During postcranial immobilization 

(plaster casting), the freely moving jaws of immature 4-6 kg 

Rhesus monkeys exhibited normal rates of lamellar (periosteal and 

endosteal) bone formation. However, a population of osteons 

showed a failure of normal appositional growth (tetracycline 

labeling), and the more trabecular regions of the jaw exhibited 

abnormal patterns (sp.gr. distribution) of mineral and matrix 

maturation. The effect on osteon growth and trabecular bone 

maturation was not observed in older 6-10 kg monkeys. Therefore, 

this model of hypokinesia is most sensible to the youthful, 

rapidly growing skeleton. 

Primate Immobilization (Roto-positioning) Model: In this system, 

9 juvenile 4 kg monkeys were restrained in a supine position and 

rotated (using the Primate Rotational System, Patent No. 4120266) 

90 0 every 30 min. through a full 360 0 arc for 14 days (RP/14). 

These animals maintained normal growth. Six were allowed to 

recover unrestrained in metabolism cages and were killed after 28 

days (RP/14+28) and 56 days (RP/14+56). Three monkeys were 

maintained as controls (C) in metabolism cages. Osteon growth 

was normal in all groups, and roto-positioning did not effect 

changes in the porosity on the inferior mandible or change the 

normal frequency of tetracycline-labeled osteons with different 

microradiographic mineral densities. The patterns of mineral and 

matrix maturation have not yet been investigated. 

The data to date do not suggest that it is feasible to mimic the 

effects of spaceflight on bone maturation in the jaw by imposing 

hind limb unloading hypokinesia at I-G conditions. The data 

support the concept that bone growth and maturation have a 

gravity component. 

PUBLICATIONS 

Simmons, D.J., Grazman, B., Russell, J.E., Walker, W.V., Bikle, 

D.D.; and Morey, E.R. Simulating Certain Aspects of Hypogravity: 

Effects on Bone Maturation in the Non-weight Bearing Skeleton. 

Aviation, Space and Environmental Medicine 54: 1080-1084, 1983. 

Simmons, D.J., Russell, J.E., Walker, W.V., Grazman, B., Oloff, 

C., and Kazarian, L. Growth and Maturation of Mandibular Bone in 

Otherwise Totally Immobilized Rhesus Monkeys. Clinical 

orthopaedics and Related Research 182: 220-230, 1984. 

Simmons, D.J. Russell, J.E., Winter, F., Tran Van, P., Vignery, 

A., Baron, R., Rosenberg, G.D., and Walker, W.V. Effect of 

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

American Journal of Physiology 244: R3l9-R326, 1983. 

104 
EFFECTS OF MUSCLE ATROPHY ON MOTOR CONTROL 

Douglas G. 

Department 

University 

Tucson, AZ 

Stuart 

of Physiology 

of Arizona 

85724 

One of the goals of the Space Biology Program is to assess the 

physiological effects of gravity. Much of the animal data 

addressing this concern have been obtained from rats that were 

raised in small laboratory cages. The current objective of the 

research program is to determine the appropriateness of this 

small-cage rat as an animal model for the study of physiological 

adaptations to reduced-use conditions. Since the physiological 

status of muscle is traditionally quantitated by its speed of 

contraction, the maximum force it can exert, and its 

fatigability, these criteria were used to address two issues: (1) 

Given that the muscles of small-cage-reared rats are more 

fatigable than theoretically expected does transmission failure 

at the neuromuscular junction contribute to this impairment? (2) 

Does cage size during rearing affect the physiological status of 

selected rat muscles? 

With regard to the first issue, the maximum force elicited by 

stimulation of the nerve was compared with that produced by 

stimulation of the muscle directly. Any difference between the 

two forces is typically interpreted as an impairment of 

transmission of the electrical signal across the neuromuscular 

junction. Although use of this paradigm has been reported in the 

literature for at least 35 yr., the inadequacy of this 

interpretation has not been well documented. In particular, when 

the muscle is stimulated directly it appears that the primary or 

initial site of initiation of the muscle action potential is the 

neuromuscular junction via the intramuscular branches of the 

nerve and not the sarcolemma as is generally supposed. In 

confirmation of this, experiments revealed no difference in the 

maximum force produced by stimulation of the nerve relative to 

the muscle. This does not, unfortunately, resolve the issue of 

neuromuscular junction-transmission failure. 

In subsequent preliminary experiments, an attempt was made to 

compare the force exerted in response to nerve stimulation with 

that produced by stimulation of the muscle once it had been 

curarized~ since curare blocks neuromuscular junction 

transmission, initiation of the action potential in response to 

direct-muscle stimulation in a curarized muscle cannot occur at 

the neuromuscular junction. Thus, any difference in force in 

these two conditions would reflect a neuromuscular-junction 

effect. Preliminary results indicate that direct-muscle 

stimulation occurs through the intramuscular branches of the axon 

and not through the sarcolemma. However, completion of these 

105 
experiments awaits the acquisition of some necessary equipment. 

To address the cage-size issue, 30 Sprague-Dawley weanling male 

rats were randomly divided into two groups and raised for 98-lSS 

days in either of two sized cages. One group of ;S was raised in 

the conventional small cages while the other group was reared in 

a cage that was 476X larger. For comparison with previous data 

in the literature, the physiological tests were performed on the 

soleus and extensor digitorum longus muscles. As an antigravity 

muscle, soleus is representative of muscles which serve both 

postural and movement functions and hence is thought to be more 

susceptible to the demands of usage. In contrast, extensor 

digitorum longus is less active and perhaps less adaptable. 

Although the analyses are still in progress, preliminary results 

tend to support this generalization. Cage size did not affect 

either the weight of extensor digitorum longus or the maximum 

force it could exert. The soleus of large-cage animals, however, 

exhibited a doubling of the cross-sectional area of some of its 

muscle fibers and hence was heavier and stronger than the soleus 

of the small-cage rats. In addition, cage size had a substantial 

effect on the fatigability of both muscles; the muscles of the 

small-cage rats were about twice as fatigable. Of the three 

physiological criteria, therefore, preliminary data indicate that 

cage size influences the maximum force and the fatigability of 

muscle. 

Given that these observations will be further substantiated as 

experiments continue, the next determination must be whether the 

adaptations of large-cage rats to reduced-use conditions (e.g., 

suspension hypokinesia) are comparable to those of small-cage 

animals. The outcome of this comparison will determine the 

appropriate animal model that will be used in subsequent 

experiments which explore the mechanisms associated with these 

adaptations. 

PUBLICATIONS 

Joyner, M.J., Enoka, R.M., Rankin, L.L., Volz, K.A., and Stuart, 

D.G. Neural Factors in Whole-muscle Fatigue of Rat Soleus and 

Medial Gastrocnemius Muscles (Abstract). Clinical Research 32: 

S6A, 1984. 

Stuart, D.G. and Enoka, R.M. Motoneurons, Motor Units and the 

Size Principle. In: The Clinical Neurosciences Section S: 

Neurobiology (ed. by R.N. Rosenburg and W.D. Willis). New York: 

Churchill Livingstone, p. 471-S17, 1983. 

106 
ADAPTATIONS OF RAT SKELETAL MUSCLE TO SUSPENSION-HYPOKINESIA 

Gordon Templeton 

University of Texas Health Science 

at Dallas 

5323 Harry Hines Blvd. 

Dallas, TX 75235 

A well-documented consequence of spaceflight is skeletal muscle 

atrophy. Research goals are to investigate the physiological 

changes associated with the influence of zero gravity on muscle 

function by first establishing a model for simulating zero 

gravity. Subsequent goals are (1) to investigate the underlying 

mechanisms responsible for the muscular changes caused by the 

model and by spaceflight and (2) to develop suitable conditioning 

regimens both to possibly preclude changes in muscular function 

for the purpose of facilitating the performance of man in space 

and to enhance recovery to normal muscle function upon returning 

from space. 

Muscle atrophy has been investigated extensively using techniques 

that limit function to isometric contractions by limb 

immobilization and consequently, prevent passive limb movement, 

which is a characteristic of zero gravity. Fortunately, an 

alternative method for evaluating reduced muscular activity, 

was developed by Morey-Holton and permits unrestricted limb 

movement while reducing the loading influence of gravity on hind 

limb muscles. The Morey-Holton method suspends a rat so that its 

hind limbs are non-weight bearing. 

The initial goal of the research was to determine whether the 

Morey-Holton technique simulates the influence of zero gravity on 

skeletal muscle. Although this validation procedure continues to 

be a part of current research, the data acquired indicate the 

Morey-Holton method is a valid model for weightlessness: (1) 

Atrophy of the soleus muscle and lack of atrophy of the 

gastrocnemius muscle were common consequences both of Cosmos 605, 

690, and 782, and of rat suspension by the Morey-Holton method. 

(2) Both zero gravity and the Morey-Holton method evoke greater 

atrophy of slow-twitch fibers as compared with fast-twitch fibers 

in both the soleus and gastrocnemius muscles. This finding 

supports the accepted contention that slow-twitch fibers function 

to resist gravity or maintain posture while fast-twitch fibers 

are responsible for locomotion requiring greater force and 

velocity. (3) Soleus contractile function is reduced in both 

space-flown and Morey-Holton-suspended rats. (4) Although data 

are not conclusive, speeding of the soleus contraction suggested 

by Cosmos 605 and 609 is a significant result of rat suspension 

by the Morey-Holton technique. 

In view of the resulting cited evidence of previous investi107 
gations that the Morey-Holton technique provides a valid model 

for simulating zero gravity, the 1983 research became more 

involved with investigating the underlying mechanisms responsible 

for the muscular changes evoked by both spaceflight and the 

Morey-Holton method. 

One such goal is to investigate the mechanisms involved in the 

greater atrophy of the slow-twitch fibers as compared with 

fast-twitch fibers. From cross sections of soleus muscles 

stained for myosin ATPase, the greater area occupied by all the 

slow-twitch fibers as compared with that of the fast-twitch 

fibers (slow-twitch fiber predominance) in control muscles was 

reversed by the suspension to a greater area occupied by 

fast-twitch fibers (fast-twitch fiber predominance). 

Additionally, measurement of individual fiber areas showed that 

slow-twitch fibers had a greater area in control soleus muscles, 

but that after 2 wk. of rat suspension, the fiber areas of the 

fast-twitch and slow-twitch fibers were equal. These data raise 

the possibilities that the change to fast-twitch fiber 

predominance with rat suspension could result either from greater 

atrophy of slow-twitch fibers and from conversion of slow-twitch 

to fast-twitch fibers. Research in 1984 is attempting to discern 

between these two possibilities by counting the number of fibers 

in the soleus muscle in control and suspended rats. 

Although the speeding of the soleus contraction with rat 

suspension and spaceflight can partially be explained by the 

observed change in the proportion of fast-twitch and slow-twitch 

fibers, a change in the rate of calcium uptake by the 

sarcoplasmic reticulum (SR) could also contribute to th~5change. 

Accordingly, fragmented SR vesicles were prepared, but Ca++ 

uptake was found to be unaffected by rat suspension. 

Consequently, it was conclude~+that soleus speeding did not 

result from a change in SR Ca uptake. 

Part of the decline in muscle weight in spaceflight and the 

Morey-Holton method for rat suspension is caused by a decrease in 

muscle protein. This decline in protein could result from either 

decreased protein synthesis or increased protein degradation. 

Since degradation may involve lysosomal (acid) proteases, the 

activities of these proteases were measured and found to be 

elevated in suspended rats. 

PUBLICATIONS 

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

C.J., Silver, P., DeMartino, G., LeConey, T., Klug, G., Hagler, 

H., and Sutko, J.L. Influence of Suspension Hypokinesia on Rat 

Soleus Muscle. Journal of Applied Physiology: Respiratory, 

Environmental and Exercise Physiology 56: 278-286, 1984. 

108 
Templeton, G.H., Padalino, M., Manton, J., LeConey, T., Hagler, 

H., and Glasberg, M. The Influence of Rat Suspension-hypokinesia 

on the Gatrocnemius Muscle. Aviation, Space, and Environmental 

Medicine 55: 381-386, 1984. 

109 
SKELETAL MUSCLE METABOLISM IN HYPOKINETIC RATS 

Marc E. Tischler 

Department of Biochemistry 

Arizona Health Sciences Center 

Tucson, AZ 85724 

Research focuses on the metabolic response of skeletal muscle to 

disuse. A broad range of metabolic pathways are being analyzed 

to determine which ones may be adversely affected with decreased 

muscular activity. The severity of these changes will be 

considered by evaluating the rapidity with which they return to 

normal when muscle use is restored. 

In muscle, glutamine, together with alanine, serves as an 

important vehicle for transporting nitrogenous waste out of the 

tissue. Since muscle of growing animals in disuse shows growth 

failure and in some instances atrophy, excess nitrogen is 

available in these muscles. Hence, the role of glutamine takes 

on extra import under such conditions. In light of these ideas, 

it was rather surprising to find that the production of glutamine 

was slower in the disused soleus muscle. This slower production 

was characterized by a decreased ratio of tissue glutamine to 

glutamate due to a fall in tissue glutamine. 

In muscle, a major source of nitrogen for the synthesis of 

alanine and glutamine are the branched chain amino acids, which 

transaminate with ketoglutarate to produce glutamate. One 

possible reason for the decreased synthesis of glutamine and 

glutamate might be decreased metabolism of the branched amino 

acids, which were present in the medium. In the soleus muscle, 

non-weight-bearing for 6 days increased the rate of flux through 

leucine aminotransferase. This effect could be accounted for by 

increased flux through ketoisocaproate dehydrogenase, the rate 

limiting step in the leucine degradative pathway. No differences 

were seen for the extensor digitorum longus muscles over the same 

period. This result is contrary to the data for decreased 

synthesis of glutamine. In fact, the data suggest that the 

soleus muscle could have a severe problem in removing nitrogen 

waste derived from the increased degradation of the 

branched-chain amino acids and protein. 

Also considered, as part of the study of leucine metabolism, was 

the extent to which leucine was oxidf~ed completel¥4 This can be 

donr4 by comparing the production of CO from [1- C] and 

[U- C] leucine. If the ratio of produc€ion is only 0.17 then 

none of the leucine molecule is completely oxidized. Since both 

soleus and extensor digitorum longus muscles showed ratios 

significantly above this value leucine catabolism in soleus 

muscles of hypokinetic animals would increase the supply of 

acetyl CoA oxidized in the citric acid cycle. 

110 
The slower production of glutamine could have been due to several 

factors: (1) decreased concentrations of glutamate or ammonia, 

which are substrates for the glutamine synthetase reaction; (2) 

less ATP as an energy source for the reaction; or (3) a lower 

activity of glutamine synthetase. As noted above, tissue 

glutamate levels are not lowered so that this is not a limiting 

factor. Furthermore, the tissue concentration of ATP is elevated 

and not reduced in the soleus muscle in disuse. 

In light of these findings it seemed likely that the activity of 

glutamine synthetase might be reduced. However, assays of this 

activity showed a 3-fold higher activity in the 

hypokinetic/hypodynamic soleus muscles. Despite a greater amount 

of enzyme, these muscles produced less glutamine. The remaining 

possibility as a defect in the ammonia availability. 

Determination of the dose-response curve for extracellular 

ammonium chloride during production of glutamine showed that 

indeed this must be true. At a high concentration of ammonium 

chloride (lmM) the hypokinetic soleus muscle produced 

considerably more glutamine. In fact the ratios of these 

apparent Vmax values were similar to the ratio of enzyme 

activities. Calculation of the Km values for extracellular 

ammonia showed a much higher value for the disused muscle. This 

result suggests that either ammonia transport is altered in 

hypokinesia or basal tissue ammonia concentration is lower, thus 

requiring more external ammonia to achieve the intracellular Km. 

In support of the latter idea, total adenine nucleotides are 

higher in hypokinesia, which is indicative of decreased ammonia 

production via the purine nucleotide cycle. 

The effects of passive stretch in reducing the metabolic 

responses to disuse have also been studied by casting one limb of 

the hypokinetic rats in a dorsal flexed position to stretch the 

soleus muscle. Passive stretch prevented atrophy and growth 

failure of the muscle and actually caused a significant 

hypertrophy. This enlargement of the soleus muscle was borne out 

by more rapid rates of protein synthesis and slower rates of 

protein degradation in the stretched compared with the 

contralateral leg muscle. Passive stretch also prevented growth 

failure of the plantaris and gastrocnemius muscles. Preliminary 

results suggest that passive stretch may also reduce other 

metabolic responses such as the changes in the metabolism of 

ammonia and glutamine. 

PUBLICATIONS 

Jaspers, S.R., Fagan, J.M., and Tischler, M.E. Effect of Limb 

Immobilization on Protein Turnover and Redox State in Muscles of 

Hypokinetic Rats (Abstract). Federation Proceedings 42: 1815, 

1983. 

Jaspers, S.R. and Tischler, M.E. Regulation of Glutamine 

III 
Production in Soleus Muscles from Hypokinetic/Hypodynamic Rats 

(Abstract). Federation Proceedings 43(6): 1549, 1984. 

Tischler, M.E., Jaspers, S.R., and Fagan, J.M. Prevention of 

Metabolic Alterations Caused by Suspension Hypokinesia in Leg 

Muscles of Rats. Physiologist 26{6, Suppl.): S98-S99, 1983. 

112 
EFFECTS OF SIMULATED WEIGHTLESSNESS ON MEIOSIS, FERTILIZATION, 

AND EARLY EMBRYONIC DEVELOPMENT IN MICE 

Debra J. Wolgemuth 

Department of Human Genetics and Development 

Columbia University College of Physicians and Surgeons 

New York, NY 10032 

Research is directed toward elucidating the extent to which 

reproduction in mammals will (or will not) be affected during 

spaceflight conditions. The specific aspect being focused on is 

microgravity; the reproductive stages under study include 

meiosis, fertilization, and early embryonic cell division. These 

studies in turn will contribute to understanding of the 

environmental factors important for proper cell functioning 

during development and differentiation. 

In order to experimentally simulate a microgravity environment, 

the initial objective was to develop a clinostat suitable for 

mammalian tissue culture; in particular, for culture of mouse 

oocytes, ova, and embryos. In 1983, this approach was first used 

to ask what effect rotating mouse oocytes in the clinostat would 

have on: (1) germinal vesicle breakdown, (2) progression to 

Metaphase I and Metaphase II of meiotic prophase, and (3) 

extrusion of the first polar body. Second, the original 

clinostat was modified to include a vertically rotating culture 

dish as a control for vibration, etc., in the experimental 

condition (horizontal rotation). Third, these studies are being 

extended to examine the effects of this altered gravity 

environment on in vitro fertilization in the mouse. 

For the studies on the effects of clinostat rotation on meiosis, 

oocytes were recovered from ovarian follicles from CD-l mice and 

placed immediately «10 min.) into the tissue culture system. 

Incubation was conducted under clinostat rotation or control 

static conditions for 16 hr. The oocytes were then removed, 

examined under a dissecting microscope for polar body formation 

and gross morphological appearance, and then processed by 

cytogenetic techniques which permit a precise assessment of 

meiotic stage and chromosomal integrity. Previous experiments 

had shown that mouse oocytes cultured in the conditions adapted 

for use on a clinostat, but without rotation, yielded meiotic 

maturation indistinguishable from oocytes cultured under routine 

laboratory conditions. 

A total of 59 experiments have been analyzed, representing 

rotations at a (control static), 1/4, 1, 10, 30, and 100 rpm. 

Qualitative evaluation of the condition of the cells immediately 

after culture on the clinostat revealed that there were no 

obviously damaged or fragmented cells, even at a rotation of 100 

113 
rpm. Cellular features such as the integrity of the zona 

pellucida, granularity of the cytoplasm, and clumping or necrosis 

of the granulosa cells were similar in the rotated vs. control 

oocytes. To date, there are no statistically significant 

differences in the percentage of germinal vesicle breakdown 

between static oocytes and oocytes cultured on the clinostat at 

any of the rotation speeds. This observation is consistent with 

what is known about meiotic maturation in other systems and has 

added to the understanding of which stages of meiotic maturation 

might be sensitive to changes in the environment (here, an 

altered gravitational field). That is, germinal vesicle 

breakdown is believed to be initiated within 30 min. of release 

from the ovarian follicle. Therefore, an effect would have to 

act very quickly to alter these events. In contrast, the period 

of chromosome alignment and movement is much more extended 

(_14-16 hrs.). These events are also highly dependent on 

cytoskeletal structure and relative orientation; thus they might 

be more susceptible to environmental changes. 

In contrast, there is an indication of a slight difference in 

rate of progression through to Metaphase II among the rotated 

oocytes, particularly those rotated at 1 rpm. To determine if 

this trend is statistically significant, additional experiments 

are being completed. However, it was considered important to 

rule out the possibility that such differences could result from 

a feature of the clinostat culture system other than rotating the 

cells relative to their orientation in a gravitational field. 

Vibration could be such a feature. Therefore, a modification of 

the clinostat was developed this year. It consists of the 

addition of two bevel gears set at a perpendicular angle to each 

other, such that simultan"eous horizontal and vertical rotations 

are achieved. This modified clinostat is now being used in all 

experiments. 

Finally, experiments examining the effect of rotating oocytes on 

in vitro fertilization have just begun. As before, the first 

step has been to determine if the standard culture conditions can 

be m9dified for use on the clinostat. Fortunately, it was found 

that both the atmospheric conditions and the culture vessel used 

in the meiotic maturation experiments can be used in 

fertilization experiments. The major change lay in the selection 

of a different culture medium, a modified Whittens medium adapted 

from Hoppe and Pitts. For these experiments, oocytes are 

recovered from the ampulae of B6D2Fl mice (Fl of C57Bl/6J x 

DBA/2J) which had been superovulated. The oocytes, surrounded by 

their cumulus mass, ar~ placed with capacitated sperm at a 

concentration of -4xlO sperm/ml and rotated for 4 hr. The 

cultures are then removed from the clinostat, transferrred to 

fresh culture dishes, and rotated for an additional 4-8 hr. The 

embryos are fixed with glutaraldehyde and then formalin, stained 

with toluidine blue, and examined for the following endpoints: 

(1) extrusion of the second polar body; (2) formation of both a 

114 
male and female pronucleus; and (3) percentage of artificially 

activated eggs. Based on observations to date on meiotic 

maturation, it might be predicted that later rather than earlier 

events in fertilization would be more likely affected, since they 

would be exposed to longer rotation times. 

PUBLICATIONS 

Wolgemuth, D.L. Synthetic Activities of the Mammalian Early 

Embryo: Molecular and Genetic Alterations Following 

Fertilization. In: Mechanism and Control of Animal 

Fertilization. New York: Academic Press, p. 415-452, 1983. 

115 
SPECIAL ACTIVITIES 
RESEARCH ASSOCIATE PROGRAM IN SPACE BIOLOGY 

The NASA Shuttle Program is currently providing a unique 

opportunity to conduct biological research in outer space and to 

continue relevant ground-based Space Biology research. To 

maximize the potential for Space Biology as an emerging 

discipline, there is a need to develop a cadre of scientists who 

are interested in working in Space Biology. The program utilizes 

a competitive grant process and awards are made to the more 

promising applicants. It is anticipated that these scientists 

will develop their research careers in the newly evolving 

discipline of Space Biology. 

The grant is designed to make awards on January 1 and July 1 of 

each year, following semiannual announcements. An Award 

Committee reviews the proposals and recommends appointments. The 

awards are for a I-yr. period with the possibility of renewal for 

a second year. The program began on June 1, 1980, and since then 

32 awards have been made. The recipients have been 20 

scientists, including zoologists, developmental biologists, 

botanists, and physiologists (animal and plant). Twelve of these 

individuals have received a second year of funding. These 

scientists work in NASA-funded laboratories or laboratories that 

can provide the necessary facilities and environment for 

specialized Space Biology projects. Originally (June 1980) there 

were 19 laboratories participating. Presently (April 1984), 

there are 46 laboratories participating. 

In addition to the salary stipend, the awardees are encouraged to 

attend and present papers at two national meetings: 1) the annual 

AlBS/NASA meeting, and 2) a national society meeting of their 

choice. 

The success of the program is readily measured in terms of the 

quality and quantity of publications as well as the job 

opportunities and placements of the Research Associates. 

Individuals have obtained positions in college or university 

settings and in research laboratories. A large number of 

publications have resulted from this program. 

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. 

119 
Heetings 

Organized or participation by the Space Biology Program: 

o "Space Biology Workshop," DFVLR-Institute of Aerospace 

Medicine, Cologne, Germany, March 9-11, 1983 

o "SL-2 Plant Experiment Meeting," AIBS, Rosslyn, Virginia, USA, 

March 21, 1983 

o "Primate Workshop,", FASEB, Bethesda, Maryland, USA, April 

17-19, 1983 

o "Spaceflight Centrifuge Workshop," FASEB, Bethesda, Maryland, 

USA, April 26, 1983 

o "Thirteenth Intersociety Conference on Environmental Systems," 

San Francisco, California, USA, July 11-13, 1983 

o "Fifth Annual Meeting of the IUPS Commission on Gravitational 

Physiology," Moscow, USSR, July 26-29, 1983 

o "Fifty-ninth Annual Meeting of the American Society for Plant 

Physiologists," Colorado State University, Fort Collins, USA, 

August 7-11, 1983 

o "Symposium on Gravitational Physiology," Syndey, Australia, 

August 28-September 3, 1983 

o "Annual Symposium of the NASA Space Biology Program," Rosslyn, 

Virginia, USA, October 12-14, 1983 

o "Space Plant Growth unit Workshop," Rosslyn, Virginia, USA, 

February 8, 1984. 

121 
1. Report No. I 2. Government Accession No. 3. Recipient's Catalog No. 

NASA TM-86654 

4. Title and Subtitle 5. Report Date 

August 1984 

1983-84 NASA SPACE BIOLOGY ACCOMPLISHMENTS 6. Performing Organization Code 

7. Author(sl 8. Performing Organization Report No. 

" Thora W. Halstead, F. Ronald Dutcher, 

and Linda G. Pleasant, Compilers 10. Work Unit No. 

9. Performing Organization Name and Address 

NASA Office of Space Science and Applications 

Washington, Contract or Grant No. 20546 11. DC 

and NASW-3165 

The George Washington University 

Washington, DC 20036 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 EBR 

15. Supplementary Notes 

Thora ·W. Halstead: NASA Office of Space Science and Applications, Washington, D.C. 

F. Ronald Dutcher and Linda G. Pleasant: The George Washington University, 

Washington, D.C. 

For previous edition, see NASA TM-86244. 

16. Abstract 

Summaries 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 summaries. Also includes bibliographies for 

each project. 

17. Key Words (Suggested by Author(sll 18. Distribution Statement 

Gravity Animal Unclassified - Unlimited 

Space Plant 

Weightlessness Hypogravity 

Simulated Hypergravity 

weightlessness Life sciences Subject Category 51 

19. Security Classif. (of this report I 20. Security Classif. (of this pagel 21. No. of Pages 22. Price . Unclassified Unclassified 130 A07 

• For sale by the National Technical Information Service, Springfield, Virginia 22161 

NASA-Langley, 198 
End of Document