The evolution of lipids.

Living organisms on the Earth which are divided into three major domains--Archaea, Bacteria, and Eucarya, probably came from a common ancestral cell. Because there are many thermophilic microorganisms near the root of the universal phylogenetic tree, the common ancestral cell should be considered to be a thermophilic microorganism. The existence of a cell is necessary for the living organisms; the cell membrane is the essential structural component of a cell, so its amphiphilic property is vital for the molecule of lipids for cell membranes. Tetraether type glycerophospholipids with C40 isoprenoid chains are major membrane lipids widely distributed in archaeal cells. Cyclization number of C40 isoprenoid chains in thermophilic archaea influences the fluidity of lipids whereas the number of carbons and degree of unsaturation in fatty acids do so in bacteria and eucarya. In addition to the cyclization of the tetraether lipids, covalent bonding of two C40 isoprenoid chains was found in hyperthermophiles. These characteristic structures of the lipids seem to contribute to their fundamental physiological roles in hyperthermophiles. Stereochemical differences between G-1-P archaeal lipids and G-3-P bacterial and eucaryal lipids might have occurred by the function of some proteins long after the first cell was developed by the reactions of small organic molecules. We propose that the structure of lipids of the common ancestral cell may have been similar to those of hyperthermophilic archaea.     

The biological component of the life support system for a Martian expedition.

Ground-based experiments at RF SSC-IBMP RAS (State Science Center of Russian Federation--Institute of Biomedical Problems of Russian Academia of Science) were aimed at overall studies of a human-unicellular algae-mineralization LSS (life support system) model. The system was 15 m3 in volume. It contained 45 L of algal suspension with a dry substance density of 10-12 g per liter; water volume, including the algal suspension, was 59 L. More sophisticated model systems with partial substitution of unicellular algae with higher plates (crop area of 15 m2) were tested in three experiments from 1.5 to 2 months in duration. The experiments demonstrated that LSS employing the unicellular algae play not only a macrofunction (regeneration of atmosphere and water) but also carry some other functions (purification of atmosphere, formation of microbial cenosis etc.) providing an adequate human environment. It is also important that functional reliability of the algal regenerative subsystem is secured by a huge number of cells able, in the event of death of a part of population, to recover in the shortest possible time the size of population and, hence, functionality of the LSS autotrophic component. For a long period of time a Martian crew will be detached from Earth's biosphere and for this reason LSS of their vehicle must be highly reliable, robust and redundant. One of the approaches to LSS redundancy is installation of two systems with different but equally efficient regeneration technologies, i.e. physical-chemical and biological. At best, these two systems should operate in parallel sharing the function of regeneration of the human environment. In case of failure or a sharp deterioration in performance of one system the other will, by way of redundancy, increase its throughput to make up for the loss. This LSS design will enable simultaneous handling of a number of critical problems including adequate satisfaction of human environmental needs.     

Molecular aspects of adaptation to extreme cold environments

Here are reviewed and summarized the strategies adopted by living organisms to survive low temperatures, from a molecular and membrane point of view. The presentation is aimed at a wide variety of readers.

Two prime examples of connections between biological cold adaptation and the molecular level are (1) antifreeze proteins in fish from cold sea water, (the DNA sequence of the protein gene is now known) (2) the fluidity characteristics of cell membranes in a wide variety of organisms. In model membranes of phospholipids, stabler “s-phases” have recently been found to form at low temperatures. Antarctic endolithic organisms, living just under the surface of rocks, are exposed to long periods of low temperatures, and may develop such phases in their membranes. In the saturated phosphatidyl cholines, only lipids with a restricted range of acyl chain lengths show simultaneously s-phases and a main transition : This restricted range is about the restricted range found in natural membranes. The s-phases also form in the presence of natural cryoprotectants, and may be connected with botanical vernalization.     

CNS development under altered gravity: cerebellar glial and neuronal protein expression in rat neonates exposed to hypergravity.

The future of space exploration depends on a solid understanding of the developmental process under microgravity, specifically in relation to the central nervous system (CNS). We have previously employed a hypergravity paradigm to assess the impact of altered gravity on the developing rat cerebellum. The present study addresses the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of selected glial and neuronal cerebellar proteins in rat neonates exposed to hypergravity (1.5 G) from embryonic day (E)11 to postnatal day (P)6 or P9 (the time of maximal cerebellar changes) comparing them against their expression in rat neonates developing under normal gravity. Proteins were analyzed by quantitative Western blots of cerebellar homogenates; RNA analysis was performed in the same samples using quantitative PCR. Densitometric analysis of Western blots suggested a reduction in glial (glial acidic protein, GFAP) and neuronal (neuronal cell adhesion molecule, NCAM-L1, synaptophysin) proteins, but the changes in individual cerebellar proteins in hypergravity-exposed neonates appeared both age- and gender-specific. RNA analysis suggested a reduction in GFAP and synaptophysin mRNAs on P6. These data suggest that exposure to hypergravity may interfere with the expression of selected cerebellar proteins. These changes in protein expression may be involved in mediating the effect of hypergravity on the developing rat cerebellum.     

Protein crystal growth aboard the U.S. space shuttle flights STS-31 and STS-32.

The first microgravity protein crystal growth experiments were performed on Spacelab I by Littke and John. These experiments indicated that the space grown crystals, which were obtained using a liquid-liquid diffusion system, were larger than crystals obtained by the same experimental system on earth. Subsequent experiments were performed by other investigators on a series of space shuttle missions from 1985 through 1990. The results from two of these shuttle flights (STS-26 and STS-29) have been described previously. The results from these missions indicated that the microgravity grown crystals for a number of different proteins were larger, displayed more uniform morphologies, and yielded diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth. This paper presents the results obtained from shuttle flight STS-32 (flown in January, 1990) and preliminary results from the most recent shuttle flight, STS-31 (flown in April, 1990).     

Controlled environments alter nutrient content of soybeans.

Information about compositional changes in plants grown in controlled environments is essential for developing a safe, nutritious diet for a Controlled Ecomological Life-Support System (CELSS). Information now is available for some CELSS candidate crops, but detailed information has been lacking for soybeans. To determine the effect of environment on macronutrient and mineral composition of soybeans, plants were grown both in the field and in a controlled environment where the hydroponic nutrient solution, photosynthetic flux (PPF), and CO2 level were manipulated to achieve rapid growth rates. Plants were harvested at seed maturity, separated into discrete parts, and oven dried prior to chemical analysis. Plant material was analyzed for proximate composition (moisture, protein, lipid, ash, and carbohydrate), total nitrogen (N), nonprotein N (NPN), nitrate, minerals, amino acid composition, and total dietary fiber. The effect of environment on composition varied by cultivar and plant part. Chamber-grown plants generally exhibited the following characteristics compared with field-grown plants: 1) increased total N and protein N for all plant parts, 2) increased nitrate in leaves and stems but not in seeds, 3) increased lipids in seeds, and 4) decreased Ca:P ratio for stems, pods, and leaves. These trends are consistent with data for other CELSS crops. Total N, protein N, and amino acid contents for 350 ppm CO2 and 1000 ppm CO2 were similar for seeds, but protein N and amino acid contents for leaves were higher at 350 ppm CO2 than at 1000 ppm CO2. Total dietary fiber content of soybean leaves was higher with 350 ppm CO2 than with 1000 ppm CO2. Such data will help in selecting of crop species, cultivars, and growing conditions to ensure safe, nutritious diets for CELSS.     

Extreme dryness and DNA-protein cross-links.

Exposure of fungal conidia (Aspergillus ochraceus) or spores of Bacillus subtilis to extreme dryness or vacuum induces DNA lesions, including strand breaks and the formation of DNA-protein cross-links. In wet cells only a small amount of protein is bound to DNA, but exposure to conditions of lowered water activity results in an increasing number of cross-links between DNA and proteins. In fungal conidia these cross-links are detected after selective iodination (125 J) of the DNA-bound proteins followed by gel electrophoresis and subsequent autoradiography. Another approach is the labelling of DNA with 32P by means of nick translation and the detection of differences in the electrophoretic mobility of DNA before and after digestion with proteinase K of proteins bound to DNA.     

Growth of wheat under one tenth of the atmospheric pressure.

Wheat plants were grown in twin closed growth chambers under normal and reduced atmospheric pressures. For the first 22 days from sowing, the reduced pressure was maintained at 200 hPa, and at 100 hPa for the remaining 27 days until harvest. These pressures were obtained by evacuation of the chamber and adding oxygen (170 and 79 hPa respectively) and carbon dioxide (0.65 and 1.0 hPa respectively; about 2 and 3 times above the control). Eighty-seven per cent of the final dry mass was produce under 100 hPa treatment. Growth and development of wheat are not negatively affected by low pressure treatment. Compared to the control, final dry mass increased by 76%, leaf number by 133%, and ear number by 35%, probably due to elevation of CO2. Shortening of shoot parts and increases in chlorophyll and proteins content are not in accordance with a predicted CO2 effect and could be attributed to the N2 removal and the subsequent alteration in gas diffusion rate.     

Environmental impacts on the developing CNS: CD15, NCAM-L1, and GFAP expression in rat neonates exposed to hypergravity.

We have previously reported that the developing rat cerebellum is affected by hypergravity exposure. The effect is observed during a period of both granule and glial cell proliferation and neuronal migration in the cerebellum and coincides with changes in thyroid hormone levels. The present study begins to address the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of cerebellar proteins that are known to be directly involved in cell-cell interactions [protein expressing 3-fucosyl-N-acetyl-lactosamine antigen (CD15), neuronal cell adhesion molecule (NCAM-L1)] and those that affect cell-cell interactions indirectly [glial fibrillary acidic protein (GFAP)] in rat neonates exposed to centrifuge-produced hypergravity. Cerebellar mass and protein expression in rat neonates exposed to hypergravity (1.5 G) from gestational day (G) 11 to postnatal day (P) 30 were compared at one of six time points between P6 and P30 against rat neonates developing under normal gravity. Proteins were analyzed by quantitative western blots of cerebellar homogenates prepared from male or female neonates. Cerebellar size was most clearly reduced in male neonates on P6 and in female neonates on P9, with a significant gender difference; differences in cerebellar mass remained significant even when change in total body mass was factored in. Densitometric analysis of western blots revealed both quantitative and temporal changes in the expression of selected cerebellar proteins that coincided with changes in cerebellar mass and were gender-specific. In fact, our data indicated certain significant differences even between male and female control animals. A maximal decrease in expression of CD15 was observed in HG females on P9, coinciding with maximal change in their cerebellar mass. A shift in the time-course of NCAM-L1 expression resulted in a significant increase in NCAM-L1 in HG males on P18, an isolated time at which cerebellar mass does not significantly differ between HG and SC neonates. A maximal decrease in expression of GFAP was observed in HG males on P6, coinciding with maximal change in their cerebellar mass. Altered expression of cerebellar proteins is likely to affect a number of developmental processes and contribute to the structural and functional alterations seen in the CNS developing under altered gravity. Our data suggest that both cerebellar development and its response to gravitational manipulations differ in males and females.     

Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level.

The changes of [Ca2+]i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station "Salyut 6". These results: 1) indicate that observed Ca(2+)-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca2+ influx through membranes. In model presented, I propose that Ca(2+)-activated channels in plasma membrane in response to microgravity allow the movement of Ca2+ into the root cells, causing a rise in cytoplasmic free Ca2+ levels. The latter, in its turn, may induce the inhibition of a Ca2+ efflux by Ca(2+)-activated ATPases and through a Ca2+/H+ antiport. It is possible that increased cytosolic levels of Ca2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca2+]i. Plant cell can response to such a Ca2+ rise by an enhancement of membranous Ca(2+)-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca(2+)-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca2+ to plant cell.     

Subcritical and supercritical water oxidation of CELSS model wastes.

Controlled-Ecological-Life-Support-System (CELSS) model wastes were wet-oxidized at temperatures from 250 to 500 degrees C, i.e., below and above the critical point of water (374 degrees C and 218 kg/cm2 or 21.4 MPa). A solution of ammonium hydroxide and acetic acid and a slurry of human urine, feces, and wipes were used as model wastes. Almost all of the organic matter in the model wastes was oxidized in the temperature range from 400 to 500 degrees C, i.e., above the critical conditions for water. In contrast, only a small portion of the organic matter was oxidized at subcritical conditions. Although the extent of nitrogen oxidation to nitrous oxide (N2O) and/or nitrogen gas (N2) increased with reaction temperature, most of the nitrogen was retained in solution as ammonia near 400 degrees C. This important finding suggests that most of the nitrogen in the waste feed can be retained in solution as ammonia during oxidation at low supercritical temperatures and be subsequently used as a nitrogen source for plants in a CELSS while at the same time organic matter is almost completely oxidized to carbon dioxide and water. It was also found in this study the Hastelloy C-276 alloy reactor corroded during waste oxidation. The rate of corrosion was lower above than below the critical temperature for water.     

Studies on precellular evolution: the encapsulation of polyribonucleotides by liposomes.

Liposomes are 5 to 50 micron vesicles with an internal aqueous environment, whose amphiphilic lipidic components self-assemble into systems with at least one double-layered membrane. Liposomes have been suggested as possible models of precellular systems formed in the early Archean Earth from lipids of non-enzymatic origin. Since it is generally accepted that RNA molecules preceded double-stranded DNA molecules as genetic material, we have studied the encapsulation of polyribonucleotides within liposomes made from dipalmitoyl phosphatidylcholine, and from egg yolk phosphatidylcholine to which cholesterol was added in some cases. The liposomes were prepared under anoxic conditions following the reverse phase evaporation method described by Szoka and Papahadjopoulos. Quantitative determinations show that approximately 50% of the available lipids form liposomes, and that up to 5% of the polyribonucleotides can be entrapped by them. We have also studied the encapsulation of polyribonucleotides in the presence of 1) urea and cyanamide, two non-electrolytes that have been used as prebiotic condensing agents, and 2) of Zn++ and Pb++, two cations employed in the non-enzymatic template-directed synthesis of polyribonucleotides from activated nucleotides.     

Protein expression profile changes in human fibroblasts induced by low dose energetic protons

Extrapolation of known radiation risks to the risks from low dose and low dose-rate exposures of human population, especially prolonged exposures of astronauts in the space radiation environment, relies in part on the mechanistic understanding of radiation induced biological consequences at the molecular level. While some genomic data at the mRNA level are available for cells or animals exposed to radiation, the data at the protein level are still lacking. Here, we studied protein expression profile changes using Panorama antibody microarray chips that contain antibodies to 224 proteins (or their phosphorylated forms) involved in cell signaling that included mostly apoptosis, cytoskeleton, cell cycle and signal transduction. Normal human fibroblasts were cultured until fully confluent and then exposed to 2 cGy of 150 MeV protons at high-dose rate. The proteins were isolated at 2 or 6 h after exposure and labeled with Cy3 for the irradiated cells and with Cy5 for the control samples before loading onto the protein microarray chips. The intensities of the protein spots were analyzed using ScanAlyze software and normalized by the summed fluorescence intensities and the housekeeping proteins. The results showed that low dose protons altered the expression of more than 10% of the proteins listed in the microarray analysis in various protein functional groups. Cell cycle (24%) related proteins were induced by protons and most of them were regulators of G1/S-transition phase. Comparison of the overall protein expression profiles, cell cycle related proteins, cytoskeleton and signal transduction protein groups showed significantly more changes induced by protons compared with other protein functional groups.     

An extraterrestrial habitat on Earth: the algal mat of Don Juan [correction of Jaun] Pond.

On the edge of Don Juan Pond in the Wright Valley of Antarctica lies a mat of mineral and detritus cemented by organic matter. In spite of a CaCl2 concentration of about 33% (w/v), the mat contains Oscillatoria and other cyanobacteria, unicellular forms, colonial forms rich in carotenoids, and diatoms. Bacteria are rare; fungal filaments are not. Oscillatoria showed motility but only at temperatures <10 degrees C. Acetone extracts of the mat and nearby muds yielded visible spectra similar to those of laboratory grown O. sancta, with 50- to 70-fold molar ratio of chlorophyll a to b. Although rare, tardigrades were also found. The algal mat had enzymatic activities characteristic of peroxidase, catalase, dehydrogenase, and amylase. Cellulose, chitin, protein, lipid and ATP were present. Previously, algae in the Wright Valley have been described in melt water, not in the brine itself. Wright Valley has been used as a near sterile Martian model. It obviously contains an array of hardy terrestrial organisms.     

Electrophoretic separation of kidney and pituitary cells on STS-8.

A Continuous Flow Electrophoresis System (CFES) was used on Space Shuttle flight STS-8 to separate specific secretory cells from suspensions of cultured primary human embryonic kidney cells and rat pituitary cells. The objectives were to isolate the subfractions of kidney cells that produce the largest amounts of urokinase (plasminogen activator), and to isolate the subfractions of rat pituitary cells that secrete growth hormone, prolactin, and other hormones. Kidney cells were separated into more than 32 fractions in each of two electrophoretic runs. Electrophoretic mobility distributions in flight experiments were spread more than the ground controls. Multiple assay methods confirmed that all cultured kidney cell fractions produced some urokinase, and five to six fractions produced significantly more urokinase than the other fractions. Several fractions also produced tissue plasminogen activator. The pituitary cells were separated into 48 fractions in each of the two electrophoretic runs, and the amounts of growth hormone (GH) and prolactin (PRL) released into the medium for each cell fraction were determined. Cell fractions were grouped into eight mobility classes and immunocytochemically assayed for the presence of GH, PRL, ACTH, LH, TSH, and FSH. The patterns of hormone distribution indicate that the specialized cells producing GH and PRL are isolatable due to the differences in electrophoretic mobilities.     

Virus protein assembly in microgravity.

The coat of polyomavirus is composed of three proteins that can self-assemble to form an icosahedral capsid. VP1 represents 75% of the virus capsid protein and the VP1 capsomere subunits are capable of self assembly to form a capsid-like structure. Ground-based and orbiter studies were conducted with VP1 protein cloned in an expression vector and purified to provide ample quantities for capsomere-capsid assembly. Flight studies were conducted on STS-37 on April 5-9, 1991. Assembly initiated when a VP1 protein solution was interfaced with a Ca+2 buffer solution (pH 5.0). After four days a second alignment terminated the assembly process and allowed for glutaraldehyde fixation. Flight and ground-based samples were analyzed by electron microscopy. Ground-based experiments revealed the assembly of VP1 into capsid-like structures and a heterogenous size array of capsomere subunits. Samples reacted in microgravity, however, showed capsomeres of a homogenous size, but lack of capsid-like assembly.     

Radiation effects in nematodes: results from IML-1 experiments.

The nematode Caenorhabditis elegans was exposed to natural space radiation using the ESA Biorack facility aboard Spacelab on International Microgravity Laboratory 1, STS-42. For the major experimental objective dormant animals were suspended in buffer or on agar or immobilized next to CR-39 plastic nuclear track detectors to correlate fluence of HZE particles with genetic events. This configuration was used to isolate mutations in a set of 350 essential genes as well as in the unc-22 structural gene. From flight samples 13 mutants in the unc-22 gene were isolated along with 53 lethal mutations from autosomal regions balanced by a translocation eT1(III;V). Preliminary analysis suggests that mutants from worms correlated with specific cosmic ray tracks may have a higher proportion of rearrangements than those isolated from tube cultures on a randomly sampled basis. Right sample mutation rate was approximately 8-fold higher than ground controls which exhibited laboratory spontaneous frequencies.     

Cellular responses by exposure to heavy-ions.

To better understand cellular responses in human lymphoblastoid cell TK6 after exposure to C-ion (22 keV/micrometer) and Fe-ion (1000 keV/micrometer), both protein induction and cell-cycle progression have been extensively analyzed by the recently developed techniques. While proceeding this line of analyses, we realized the importance of studying low-dose effect, in relation to the genetic alterations. Adaptive response by 5~20 cGy of such C- or Fe-ion irradiation to both lethal and mutagenic effects of the challenging X-ray exposure (1~3 Gy) was difficult to be seen in this TK6 cells, but surprisingly, a relatively high level of p53 and its related proteins induction was observed after low-dose irradiations of heavy-ions. Here, we focus to introduce the above results of genetic and biochemical studies to elucidate the adaptive response.     

Behavioral toxicity of selected radioprotectors.

Effective radioprotection with minimal behavioral disruption is essential for the selection of protective agents to be used in manned spaceflight. This overview summarizes the studies on the behavioral toxicity of selected radioprotectors classified as phosphorothioates (WR-2721, WR-3689), bioactive lipids (16, 16 dimethylprostaglandin E2(DiPGE2), platelet activating factor (PAF), leukotriene C4), and immunomodulators (glucan, synthetic trehalose dicorynomycolate, and interleukin-1). Behavioral toxicity was examined in laboratory mice using a locomotor activity test. For all compounds tested, there was a dose-dependent decrease in locomotor behavior that paralleled the dose-dependent increase in radioprotection. While combinations of radioprotective compounds (DiPGE2 plus WR-2721) increased radioprotection, they also decreased locomotor activity. The central nervous system stimulant, caffeine, was able to mitigate the locomotor decrement produced by WR-3689 or PAF.     

Toluene removal from air by Dieffenbachia in a closed environment.

Higher plants are likely to play a major role in bioregeneration systems for food, air and water supplies. Plants may also contribute by the removal of toxic organic substances from the air of a closed environment. Dieffenbachia amoena plants were exposed to 0 to 1.2 x 10(6) micrograms toluene m-3 at light intensities of 35 and 90 micromoles m-2 s-1 in sealed chambers. Toluene removal, photosynthesis and respiration were measured. An increased light intensity increased the rate of toluene removal five-fold over the rate at the lower intensity; the kinetics suggest active regulation by the plant. The removal rate saturated at 2700 micrograms toluene h-1 at the lower intensity and failed to saturate at the higher intensity. Toluene exposure inhibited photosynthesis and respiration only transiently and without correlation to toluene concentration. These plants can act as efficient scavengers of toluene in a contaminated environment.