Embryonic development of the freshwater snail Biomphalaria glabrata under microgravity conditions (STS-89 mission)

The embryonic development of the fresh-water snail Biomphalaria glabrata was examined under microgravity-conditions and compared with the ground control and standard embryos, putting special emphasis on the shell formation. The process of shell formation may be particularly sensitive to the change of gravitational forces. The project aimed at determining whether the processes of mineralization during the formation of the exoskeleton in the growing snail embryo take place normally under microgravity conditions. Twenty-four adult individuals of the tropical freshwater snail B. glabrata were maintained 9 days in the Closed Equilibrated Biological Aquatic System (CEBAS Minimodule) on Space Shuttle flight STS-89. The animals produced spawning packs throughout the duration of the mission so that embryos of all developmental stages were achieved. The embryos developed slightly slower in the CEBAS than under standard conditions, and in older embryos a decreased mineralization of the shell was detected. These phenomena, however, were observed in the flight module as well as in the ground control specimens and was not an effect caused by the microgravity conditions. Embryos of B. glabrata showed a correct morphogenesis under microgravity, no teratological effects were noticed, and the shell formation proceeded normally.     

Gravitational Symmetry Breaking Leads to a Polar Liquid Crystal Phase of Microtubules In Vitro

Recent space-flight experiments performed by Tabony's team provided further evidence that a microgravity environment strongly affects the spatio-temporal organization of microtubule assemblies. Characteristic time and length scales were found that govern the organization of oriented bundles under Earth's gravitational field (GF). No such organization has been observed in a microgravity environment. This paper discusses physical mechanisms resulting in pattern formation under gravity and its disappearance in microgravity. The subtle interplay between chemical kinetics, diffusion, gravitational drift, thermal fluctuations, electrostatic interactions and liquid crystalline characteristics provides a plausible scenario.     

Collagenase digestion of bone fragments in microgravity

The effect of a quiescent microgravity fluid environment on the activity of collagenase directed at demineralized bone fragments was investigated over a period of 10 days. Enzyme treatment resulted in greater mass loss in microgravity, with nearly three times the loss of mass during Space Shuttle mission STS-62 compared to the stationary ground control. Clinorotation enhanced the loss of mass relative to a stationary control, but this increase was still significantly less than the increase with exposure to microgravity. This suggests the detrimental influence of turbulence on the enzyme function and the benefit of using microgravity to provide both low turbulence and uniformity of unequally dense materials within the reaction chamber. The results are considered for their general applicability to a variety of bioprocessing applications that may be enhanced in microgravity. (c) 1996 John Wiley & Sons, Inc.     

Cell-wall architecture and lignin composition of wheat developed in a microgravity environment.

The microgravity environment encountered during space-flight has long been considered to affect plant growth and developmental processes, including cell wall biopolymer composition and content. As a prelude to studying how microgravity is perceived - and acted upon - by plants, it was first instructive to investigate what gross effects on plant growth and development occurred in microgravity. Thus, wheat seedlings were exposed to microgravity on board the space shuttle Discovery (STS-51) for a 10 day duration, and these specimens were compared with their counterparts grown on Earth under the same conditions (e.g. controls). First, the primary roots of the wheat that developed under both microgravity and 1 g on Earth were examined to assess the role of gravity on cellulose microfibril (CMF) organization and secondary wall thickening patterns. Using a quick freeze/deep etch technique, this revealed that the cell wall CMFs of the space-grown wheat maintained the same organization as their 1 g-grown counterparts. That is, in all instances, CMFs were randomly interwoven with each other in the outermost layers (farthest removed from the plasma membrane), and parallel to each other within the individual strata immediately adjacent to the plasma membranes. The CMF angle in the innermost stratum relative to the immediately adjacent stratum was ca 80 degrees in both the space and Earth-grown plants. Second, all plants grown in microgravity had roots that grew downwards into the agar; they did not display "wandering" and upward growth as previously reported by others. Third, the space-grown wheat also developed normal protoxylem and metaxylem vessel elements with secondary thickening patterns ranging from spiral to regular pit to reticulate thickenings. Fourthly, both the space- and Earth-grown plants were essentially of the same size and height, and their lignin analyses revealed no substantial differences in their amounts and composition regardless of the gravitational field experienced, i.e. for the purposes of this study, all plants were essentially identical. These results suggest that the microgravity environment itself at best only slightly affected either cell wall biopolymer synthesis or the deposition of CMFs, in contrast to previous assertions.     

The Biomineralization Proteome: Protein Complexity for a Complex Bioceramic Assembly Process

There are over 62 different biominerals on Earth and a diverse array of organisms that generate these biominerals for survival. This review will introduce the process of biomineralization and the current understanding of the molecular mechanisms of mineral formation, and then comparatively explore the representative secretomes of two well‐documented skeletal systems: vertebrate bone (calcium phosphate) and invertebrate mollusk shell (calcium carbonate). It is found that both skeletal secretomes have gross similarities and possess proteins that fall into four functional categories: matrix formers, nucleation assisters, communicators, and remodelers. In many cases the mineral‐associated matrix former and nucleation assister sequences in both skeletal systems are unique and possess interactive conserved globular domains, intrinsic disorder, post‐translational modifications, sequence redundancy, and amyloid‐like aggregation‐prone sequences. Together, these molecular features create a protein‐based environment that facilitates mineral formation and organization and argue in favor of conserved features that evolve from the mollusk shell to bone. Interestingly, the mollusk shell secretome appears to be more complex compared to that of bone tissue, in that there are numerous protein subcategories that are required for the nucleation and organization of inner (nacre) and outer (prismatic) calcium carbonate regions of the shell. This may reflect the organizational and material requirements of an exoskeletal protective system.     

Cell structure and mobilization of lipids and proteins from cotyledon under microgravity influence

The structural-functional organization of cotyledon parenchyma cells of 6-day soybean (Glycine max L.) seedlings that were grown on board the space Shuttle Columbia (STS-87) have been studied. The purafil (KMnO4) was used in the experiment for the removing of some part of ethylene that secretes out from seedlings. There were four variants of the experiment: ground control (+purafil), ground control (-purafil), microgravity (+purafil) and microgravity (-purafil). The electron microscopy, srereological, and pyroantimonate cytochemical methods have been used. It is established the some indices of changes of storage substances in cotyledon parenchyma cells under influence of microgravity. It is displayed in the change of cell ultrastructure, the decrease of relative volume of storage cytoplasmic lipid bodies, a disappearance of storage protein body into vacuole and the redistribution of ionized calcium in cell. It was supposed that microgravity is influenced on the acceleration of storage substances catabolism.     

Spicule matrix protein LSM34 is essential for biomineralization of the sea urchin spicule.

Biomineralized skeletal structures are composite materials containing mineral and matrix protein(s). The cell biological mechanisms that underlie the formation, secretion, and organization of the biomineralized materials are not well understood. Although the matrix proteins influence physical properties of the structures, little is known of the role of these matrix proteins in the actual formation of the biomineralized structure. We present here results using an antisense oligonucleotide directed against a spicule matrix protein, LSM34, present in spicules of embryos of Lytechinus pictus. After injection of anti-LSM34 into the blastocoel of a sea urchin embryo, LSM34 protein in the primary mesenchyme cells decreases and biomineralization ceases, demonstrating that LSM34 function is essential for the formation of the calcareous endoskeletal spicule of the embryo. Since LSM34 is found primarily in a specialized extracellular matrix surrounding the spicule, it is probable that this matrix is important for the biomineralization process.     

Early root cap development and graviresponse in white clover (Trifolium repens) grown in space and on a two-axis clinostat.

White clover (Trifolium repens) was germinated and grown in microgravity aboard the Space Shuttle (STS-60, 1994; STS-63, 1995), on Earth in stationary racks and in a slow-rotating two-axis clinostat. The objective of this study was to determine if normal root cap development and early plant gravity responses were dependent on gravitational cues. Seedlings were germinated in space and chemically fixed in orbit after 21, 40, and 72 h. Seedlings 96 h old were returned viable to earth. Germination and total seedling length were not dependent on gravity treatment. In space-flown seedlings, the number of cell stories in the root cap and the geometry of central columella cells did not differ from those of the Earth-grown seedlings. The root cap structure of clinorotated plants appeared similar to that of seedlings from microgravity, with the exception of three-day rotated plants, which displayed significant cellular damage in the columella region. Nuclear polarity did not depend on gravity; however, the positions of amyloplasts in the central columella cells were dependent on both the gravity treatment and the age of the seedlings. Seedlings from space, returned viable to earth, responded to horizontal stimulation as did 1 g controls, but seedlings rotated on the clinostat for the same duration had a reduced curvature response. This study demonstrates that initial root cap development is insensitive to either chronic clinorotation or microgravity. Soon after differentiation, however, clinorotation leads to loss of normal root cap structure and plant graviresponse while microgravity does not.     

Comparison of the three-dimensional structures of a human Bence-Jones dimer crystallized on Earth and aboard US Space Shuttle Mission STS-95

Crystals of a human (Sea) Bence-Jones dimer were produced in a capillary by vapor diffusion under microgravity conditions in the 9 day US Space Shuttle Mission STS-95. In comparison to ground-based experiments, nucleation was facile and spontaneous in space. Appearance of a very large (8 x 1.6 x 1.0 mm) crystal in a short time period is a strong endorsement for the use of microgravity to produce crystals sufficiently large for neutron diffraction studies. The Sea dimer crystallized in the orthorhombic space group P2(1)2(1)2(1), with a = 48.9 A, b = 85.2 A, and c = 114.0 A. The crystals grown in microgravity exhibited significantly lower mosaicities than those of ground-based crystals and the X-ray diffraction data had a lower overall B factor. Three-dimensional structures determined by X-ray analysis at two temperatures (100 and 293 K) were indistinguishable from those obtained from ground-based crystals. However, both the crystallographic R factor and the free R factor were slightly lower in the models derived from crystals produced in microgravity. The major difference between the two crystal growth systems is a lack of convection and sedimentation in a microgravity environment. This environment resulted in the growth of much larger, higher-quality crystals of the Sea Bence-Jones protein. Structurally, heretofore unrecognized grooves on the external surfaces of the Sea and other immunoglobulin-derived fragments are regular features and may offer supplementary binding regions for super antigens and other elongated ligands in the bloodstream and perivascular tissues.     

Microgravity and clinorotation cause redistribution of free calcium in sweet clover columella cells

In higher plants, calcium redistribution is believed to be crucial for the root to respond to a change in the direction of the gravity vector. To test the effects of clinorotation and microgravity on calcium localization in higher plant roots, sweet clover (Melilotus alba L.) seedlings were germinated and grown for two days on a slow rotating clinostat or in microgravity on the US Space Shuttle flight STS-60. Subsequently, the tissue was treated with a fixative containing antimonate (a calcium precipitating agent) during clinorotation or in microgravity and processed for electron microscopy. In root columella cells of clinorotated plants, antimonate precipitates were localized adjacent to the cell wall in a unilateral manner. Columella cells exposed to microgravity were characterized by precipitates mostly located adjacent to the proximal and lateral cell wall. In all treatments some punctate precipitates were associated with vacuoles, amyloplasts, mitochondria, and euchromatin of the nucleus. A quantitative study revealed a decreased number of precipitates associated with the nucleus and the amyloplasts in columella cells exposed to microgravity as compared to ground controls. These data suggest that roots perceive a change in the gravitational field, as produced by clinorotation or space flights, and respond respectively differently by a redistribution of free calcium.     

Suppression of Hydroxycinnamate Network Formation in Cell Walls of Rice Shoots Grown under Microgravity Conditions in Space

Network structures created by hydroxycinnamate cross-links within the cell wall architecture of gramineous plants make the cell wall resistant to the gravitational force of the earth. In this study, the effects of microgravity on the formation of cell wall-bound hydroxycinnamates were examined using etiolated rice shoots simultaneously grown under artificial 1 g and microgravity conditions in the Cell Biology Experiment Facility on the International Space Station. Measurement of the mechanical properties of cell walls showed that shoot cell walls became stiff during the growth period and that microgravity suppressed this stiffening. Amounts of cell wall polysaccharides, cell wall-bound phenolic acids, and lignin in rice shoots increased as the shoot grew. Microgravity did not influence changes in the amounts of cell wall polysaccharides or phenolic acid monomers such as ferulic acid (FA) and p-coumaric acid, but it suppressed increases in diferulic acid (DFA) isomers and lignin. Activities of the enzymes phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) in shoots also increased as the shoot grew. PAL activity in microgravity-grown shoots was almost comparable to that in artificial 1 g-grown shoots, while CW-PRX activity increased less in microgravity-grown shoots than in artificial 1 g-grown shoots. Furthermore, the increases in expression levels of some class III peroxidase genes were reduced under microgravity conditions. These results suggest that a microgravity environment modifies the expression levels of certain class III peroxidase genes in rice shoots, that the resultant reduction of CW-PRX activity may be involved in suppressing DFA formation and lignin polymerization, and that this suppression may cause a decrease in cross-linkages within the cell wall architecture. The reduction in intra-network structures may contribute to keeping the cell wall loose under microgravity conditions.     

Features of development of fetal bone organ culture in space flight

Fetal mouse metatarsals cultured for 4 days onboard international space laboratory IML-1 (STS-42) were investigated using light microscopy and electron microscopy combined with X-ray microanalysis. Bones cultured in microgravity were equal in length to both ground and inflight (1 g) controls. Three zones: epiphyseal, proliferative, and hypertrophic chondrocytes were distinguished and measured in metatarsals isolated from 16-day-old fetuses. In bone cultures exposed to microgravity, hypertrophic zone tended to decrease and epiphyseal area was increased compared to controls. Proliferative zone has equal length both in bones cultured under microgravity and in controls. The same tendency was observed in bone cultures from 17-day-old fetuses. Metatarsals cultured in microgravity have less spreading calcification zone of diaphysis in comparison with both controls. The results suggest that maturation of chondrocytes and calcification of cartilage, but not cell proliferation, are microgravity sensitive processes in developing bones isolated from the organism.     

Structural and functional organization of mitochondria in soybean root statocytes under microgravitation

The effects of microgravity and ethylene on morphology and ultrastructural organization of mitochondria in root statocytes of soybean seedlings grown for 6 days on the board of the space shuttle Columbia during the STS-87 mission were investigated. The spaceflight seedlings and the ground-grown control seedlings were grown in BRIG (Biological Research in Canister) in the presence of KMnO4 to remove ethylene. It was revealed that irrespectively of KMnO4 treatment the mitochondria in the spaceflight seedlings were characterized by round or oviform and by low electron density of organelle matrix, whereas the organelles in the ground controls were polymorphic in shape and had higher electron density of matrix. The possible mechanisms of morphological and ultrastructural rearrangements of mitochondria that may be involved in adaptation processes of soybean seedlings to microgravity conditions are discussed.     

Biomineralization: Some complex crystallite-oriented skeletal structures

The present review focuses on some specific aspects of biomineralization with regard to the evolution of the first focused visioning systems in trilobites, the formation of molluscan shell architecture, dental enamel and its biomechanical properties and the structure of the calcified amniote egg, both fossil and recent. As an interdisciplinary field, biomineralization deals with the formation, structure and mechanical strength of mineralized skeletonized tissue secreted by organisms. Mineral matter formed in this way occurs in all three domains of life and consists of several mineral varieties, of which carbonates, phosphates and opaline silica are the most common. Animals and plants need mechanical support to counteract gravitational forces on land and hydrostatic pressure in the deep ocean, which is provided by a skeletonized framework. Skeleton architecture mainly consists of basic elements represented by small usually micrometer- to nanometer-sized crystallites of calcite and aragonite for carbonate systems and apatite crystallites for phosphatic ones, and then these building blocks develop into structured more complex frameworks. As selective pressures work towards optimizing stress and response, the orientation, morphology and structural arrangement of the crystallites indicates the distribution of the stress field of the biomineralized tissue. Large animals such as the dinosaurs have to deal with large gravitational forces, but in much smaller skeletonized organism such as the coccoliths, a few micrometer in diameter made up of even smaller individual crystallites, van der Waals forces play an increasingly important role and are at present poorly understood. Skeleton formation is dependent upon many factors including ambient water chemistry, temperature and environment. Ocean chemistry has played a vital role in the origins of skeletonization, 500 to 600 million years (ma) ago with the dominance of calcium carbonate as the principal skeleton-forming tissue and with phosphates and silica as important but secondary materials. The preservation of calcareous skeletons in deep time has resulted in providing interesting information: for example, the number of days in the Devonian year has been established on the basis of well-preserved lunar (annual) cycles, and isotope chemistry has led to an elaborate protocol for using O¹⁸/O¹⁶ stable isotopes for palaeotemperature measurements in the geological past. Stable isotopes of dental apatite have helped to establish ecological shifts (terrestrial to wholly marine) during the evolution of the Cetacea. Biomineralization as a field of specialization is still searching for its own independent identity, but gradually, its importance is being realized as a model for engineering applications especially at the nanometer scale.     

Multitalentierte Biominerale

Biominerals and their applications in medicine and technology Biomineral forming systems are widespread in nature, but have gained more attention only in recent years with regard to the development of new materials and their application in medicine and technology. A major factor were, in particular, the development of the modern nanotechnology and the discovery that the main classes of biominerals, silica, calcium carbonate and calcium phosphate/hydroxyapatite, can be formed by an enzymatic mechanism. This allows the biocatalytic production both of organic‐inorganic hybrid materials with new property combinations and of defined structures from these biominerals. These applications range from optics to surface coatings, cell encapsulation, core shell materials and implants.     

Genetic variation in eggshell crystal size and orientation is large and these traits are correlated with shell thickness and are associated with eggshell matrix protein markers

The size and orientation of calcium carbonate crystals influence the structure and strength of the eggshells of chickens. In this study, estimates of heritability were found to be high (0.6) for crystal size and moderate (0.3) for crystal orientation. There was a strong positive correlation (0.65) for crystal size and orientation with the thickness of the shell and, in particular, with the thickness of the mammillary layer. Correlations with shell breaking strength were positive but with a high standard error. This was contrary to expectations, as in man-made materials smaller crystals would be stronger. We believe the results of this study support the hypothesis that the structural organization of shell, and in particular the mammillary layer, is influenced by crystal size and orientation, especially during the initial phase of calcification. Genetic associations for crystal measurements were observed between haplotype blocks or individual markers for a number of eggshell matrix proteins. Ovalbumin and ovotransferrin (LTF) markers for example were associated with crystal size, while ovocleidin-116 and ovocalyxin-32 (RARRES1) markers were associated with crystal orientation. The location of these proteins in the eggshell is consistent with different phases of the shell-formation process. In conclusion, the variability of crystal size, and to a lesser extent orientation, appears to have a large genetic component, and the formation of calcite crystals are intimately related to the ultrastructure of the eggshell. Moreover, this study also provides evidence that proteins in the shell influence the variability of crystal traits and, in turn, the shell's thickness profile. The crystal measurements and/or the associated genetic markers may therefore prove to be useful in selection programs to improve eggshell quality.     

Hypergravity stimulates collagen synthesis in human osteoblast-like cells: evidence for the involvement of p44/42 MAP-kinases (ERK 1/2).

The formation and organization of skeletal tissue is strongly influenced by mechanical stimulation. There is increasing evidence that gravitational stress has an impact on the expression of early response genes in mammalian cells and may play a role in the formation of extracellular matrix. In particular, osteoblasts may be unique in their response to gravitational stimuli since in these cells microgravity has been reported to reduce collagen synthesis, while in fibroblasts the opposite effect was observed. Here, we have investigated the influence of hypergravity induced by centrifugation on the collagen synthesis of human osteoblast-like cells (hOB) and studied the possible involvement of the mitogen-activated protein (MAP) kinase signaling cascade. Collagen synthesis was significantly increased by 42+/-16% under hypergravity at 13 x g, an effect paralleled by the enhanced expression of the collagen I alpha 2 (COL1A2) mRNA. No difference was seen in the proportion of collagen types I, III, and V synthesized by hOB. Hypergravity induced a markedly elevated phosphorylation of the p44/42 MAP kinases (ERK 1/2). The inhibition of this pathway suppressed the hypergravity-induced stimulation of both collagen synthesis as well as COL1A2 mRNA expression by about 50%. Our results show that the collagen synthesis of non-transformed hOB is stimulated under hypergravitational conditions. This response appears to be partially mediated by the MAP kinase pathway.     

Gravitropic moss cells default to spiral growth on the clinostat and in microgravity during spaceflight

In addition to shoots and roots, the gravity (g)-vector orients the growth of specialized cells such as the apical cell of dark-grown moss protonemata. Each apical cell of the moss Ceratodon purpureus senses the g-vector and adjusts polar growth accordingly producing entire cultures of upright protonemata (negative gravitropism). The effect of withdrawing a constant gravity stimulus on moss growth was studied on two NASA Space Shuttle (STS) missions as well as during clinostat rotation on earth. Cultures grown in microgravity (spaceflight) on the STS-87 mission exhibited two successive phases of non-random growth and patterning, a radial outgrowth followed by the formation of net clockwise spiral growth. Also, cultures pre-aligned by unilateral light developed clockwise hooks during the subsequent dark period. The second spaceflight experiment flew on STS-107 which disintegrated during its descent on 1 February 2003. However, most of the moss experimental hardware was recovered on the ground, and most cultures, which had been chemically fixed during spaceflight, were retrieved. Almost all intact STS-107 cultures displayed strong spiral growth. Non-random culture growth including clockwise spiral growth was also observed after clinostat rotation. Together these data demonstrate the existence of default non-random growth patterns that develop at a population level in microgravity, a response that must normally be overridden and masked by a constant g-vector on earth.     

Bacterial biofilm formation under microgravity conditions

Although biofilm formation is widely documented on Earth, it has not been demonstrated in the absence of gravity. To explore this possibility, Pseudomonas aeruginosa, suspended in sterile buffer, was flown in a commercial payload on space shuttle flight STS-95. During earth orbit, biofilm formation was induced by exposing the bacteria to sterile media through a 0.2-microm (pore size) polycarbonate membrane. Examination of these membranes by confocal microscopy revealed biofilms to be present and that these biofilms could persist in spite of vigorous agitation. These results represent the first report of biofilm formation under microgravity conditions.