Review of methods used in lunar organic analysis: Extraction and hydrolysis techniques

Extraction, hydrolysis and crushing procedures have proven useful in analyzing for some of the carbon-containing compounds which are present in Apollo 11 and 12 lunar samples. Three main extraction methods employed with aqueous and nonaqueous solvents were refluxing in open and closed systems, Soxhlet extraction, and sonication. With water and acids, refluxing was the method of choice. Of the various nonaqueous solvent systems used, benzene: methanol mixtures were most often selected, and sonication was favored over Soxhlet extraction. Extraction of lunar samples with water followed by acid hydrolysis of the water extract was found to be superior to direct acid hydrolysis of lunar material in the search for amino acids or their precursors. Direct acid hydrolysis of lunar materials did demonstrate however, that carbides or carbide-like compounds are present on the moon. Hydrolysis with deuterated acids and bases showed that lunar samples contain indigenous methane and ethane and confirmed the presence of carbide-like materials. Crushing experiments also showed that gaseous hydrocarbons can be released from lunar samples.     

Terrestrial contamination in Apollo lunar samples

The Apollo lunar samples were seen to offer a unique opportunity in the search for extraterrestrial organic matter without the ambiguity surrounding meteorite analysis due to their unknown contamination histories. The recognition that only a small amount of indigenous organic material was likely to be present in lunar samples combined with the extreme sensitivity of organic analysis methods made it clear that this opportunity could be realized only by carefully controlling the collection, processing, and analysis of the samples in order that they might remain free of significant levels of contamination. The contamination control procedures adopted are described and the analytical evidence obtained throughout the program on potential contamination sources is presented. The organic contaminants actually found in the lunar samples by the various investigators are summarized. It is shown that the program succeeded in providing investigators with samples containing less than 0.1 ppm total contamination.     

Biochemical genetics of bacterial sporulation

Summary The morphological and biochemical characters of twenty nine sporulation mutants were compared. Some of the predictions made on the basis of unidirectional pleïotropic interactions were confirmed, namely that the latest proteolytic enzymes, like elastase, are related to late morphological stages. From the cytological point of view, mutants blocked at various stages were described. Among the late mutants, both coatless mutants with normal but incomplete cortex and cortexless mutants with flexible spore coats were found. Particularly interesting is the class of abnormal late sporulation mutants which form normal mature heat-resistant spores at high frequencies, but, in addition, present various anomalies in the structure of the spore coats and various sporangial inclusions such as a spongy fibrous material, resembling the cortex, and either onion-like or rod-shaped inclusions, probably formed by spore coat components. The presence of these structures is related to the derepression of elastase activity and may reflect overproduction of spore components. Several mutants also contain abnormal, large, dark, membrane-bound mesosomes, either compact or loose, whose presence is related to the lack of oxidation of tetrazolium dyes. The morphological heterogeneity of mutant populations is also noted. These findings are discussed in relation to the theory of sequential gene activation.     

Effects of light dynamics on coral spawning synchrony

Synchrony of spawning in many hermatypic corals, typically a few nights after the full moon, is putatively dependent on solar and lunar light cycles in conjunction with other possible cues such as tides and temperature. We analyze here the contributions of separate components of light dynamics, because the effects of twilight and lunar skylight on coral spawning synchrony have previously been conflated and the alternative hypothesis that these components have differential contributions as proximate cues has not been tested. Moonlight-dependent changes in spectra during twilight, rates of decreasing twilight intensities, and changes in lunar photoperiod were experimentally decoupled using programmed light-emitting diodes and compared for their separate effects on spawning synchrony in Acropora humilis. Effects on synchrony under the control of synthetic lunar cues were greatest in response to changes in lunar photoperiod; changes in light intensities and spectra had lesser influence. No significant differences among treatment responses were found at the circa-diel time scale. We conclude that spawning synchrony on a particular lunar night and specific time of night is a threshold response to differential periods of darkness after twilight that is primarily influenced by lunar photoperiod and secondarily by discrete optical components of early nocturnal illumination.     

Alcian Blue staining of cartilage for electron microscopy. Application of the critical electrolyte concentation principle.

The critical electrolyte concentration principle was applied to the Alcian Blue staining of rat epiphyseal cartilage proteoglycans for electron microscopy. The distribution and structure of material in glutaraldehyde-fixed cartilage stained at pH 5.8 without MgCl2 and in the presence of 0.05, 0.4, 0.5, 0.9 and 1.0 M MgCl2 was compared with that produced by simultaneous staining and fixation at neutral pH. Both methods resulted in staining of intracellular material within vacuoles as well as staining of non-collagenous matrix material. The structure and distribution of Alcian Blue-positive matrix material consisted of rounded or polygonal granules which accumulated around cells in the proliferative and hypertrophied zones. A similar pattern of distribution was observed in samples stained in the presence of 0.4 or 0.5 M MgCl2. In these cases, however, the stained material exhibited a ribbon-like configuration and granules were few in number. Increasing the MgCl2 concentration to 1.0 M resulted in a marked reduction of Alcian Blue stained material. No ribbon-like structures were observed, and matrix granules were reduced in both number and size. The decreased staining associated with increased electrolyte concentration lends support to the concept that epiphyseal cartilage matrix granules are composed primarily of chondroitin sulphate, and suggest that this same material is present in vacuoles associated with the Golgi apparatus in chondrocytes of the proliferative and hypertrophying zones.     

Effect of bile acids on formation of the mutagen, quercetin, from two flavonol glycoside precursors by human gut bacterial preparations

Human fecal cultures, induced with either of the flavonols, quercitrin or rutin, were grown in the presence of various concentrations of chenodeoxycholic acid, deoxycholic acid or cholic acid. Cell-free preparations (fecal preparations) from these cultures were then incubated with rutin or quercitrin. The formation of the aglycone, quercetin, was monitored by the Ames assay using tester strain TA98. The presence of chenodeoxycholic or deoxycholic acids in the quercitrin-induced culture resulted in a fecal preparation which enhanced the mutagenesis of quercitrin approximately two-fold at optimal concentrations of 0.6 mM and 0.8 mM respectively. Higher concentrations of these bile acids decreased the activity of the fecal preparations. Cholic acid gave similar results except a much higher concentration (3.0 mM) was required to achieve this effect. Analogous results with rutin-induced cultures were less clear cut: considerable variation in bile acid effect was noted among volunteers. The authors propose that bile acid in the medium may enhance the ability of rutin- and quercitrin-glycosidase elaborating organisms to successfully compete with other microbial populations. Additionally, the greater variation in results using rutin as inducer may reflect more heterogeneous populations of organisms active against this substrate. The possible role of bile acids and flavonols in bowel cancer is discussed.     

The influence of bacteria of the Burkholderia cepacia and Pseudomonas aeruginosa complex on cell-mediated immune reactions in experimental animals

The evidence has been obtained that various species, as well as individual strains having pathogenicity factors, produced different effect on the functional activity of immunocompetent B and T lymphocytes of mice infected intraperitoneally. The injection of live P. aerruginosa PA 103 and B. cepacia 8240 cells resulted in imunosuppression of antibody-forming cells, synthesizing antibodies to heterologous antigens. On the contrary, in the animals infected with B. cepacia 8236 the functional activity of B lymphocytes increased. An increase in the proliferative activity of spleen cells was noted in the presence of T and B mitogens after the infection of mice with P. aeruginosa PA 103 in comparison with B. cepacia 8236 and B. cepacia 8240 which produced a faintly pronounced modulating effect. The pathogenesis mechanisms of infections induced by these microorganisms as well as the development of chronic, persisting forms of the infectious process are discussed.     

Influence of sperm and phytoplankton on spawning in the echinoid Lytechinus variegatus

The cues triggering large-scale broadcast-spawning events in marine invertebrates are not fully understood. Using the sea urchin Lytechinus variegatus, we tested the effectiveness of a variety of potential spawning cues in eliciting a spawning response. In the laboratory, during two consecutive spawning seasons, about 400 isolated sea urchins were exposed to phytoplankton, sperm, or eggs, singly or in combination. The likelihood of spawning, time to spawning, and spawning behavior were recorded for both sexes. Sperm was most successful at inducing spawning. No response to eggs was noted. Phytoplankton alone did not trigger spawning, but when a phytoplankton cue was followed by the addition of sperm, spawning behavior was induced, the time between addition of sperm and spawning was reduced, and the variance among individuals in the time of spawning initiation was reduced. Males spawned sooner in response to cues than females and rarely spawned spontaneously in phytoplankton or control treatments. A semilunar pattern in the sensitivity to spawning cues was noted. During time periods when sea urchins were less ripe, the ratio of spawning males to spawning females increased. Our results indicate that seasonal and lunar cycles, together with the presence of phytoplankton, increase the sensitivity of these sea urchins to spawning cues and the precision of their responses to conspecific sperm.     

Effect of lunar materials on plant tissue culture

Lunar material collected during the Apollo 11, 12, 14, and 15 missions has been used to treat 12 species of higher plant tissue cultures. Biochemical and morphological studies have been conducted on several of these species. Tobacco tissue cultures treated with 0.22 g of lunar material exhibited increased greening more complex chloroplasts, less cytoplasmic vacuolation and greater vesiculation. Pine tissue cultures reacted to treatment by an increased deposition of tannin-like materials. The percentage of dry weight and soluble protein was increased in cultures treated with either lunar or terrestrial rock materials. Lunar Science Institute Contribution.     

Relative Contributions of Bacteria, Protozoa, and Fungi to In Vitro Degradation of Orchard Grass Cell Walls and Their Interactions

To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.     

Moonlighting? - Consequences of lunar cues on anuran reproductive activity

While the influence of environmental variables, particularly temperature and rainfall, on the breeding behavior of amphibians is widely recognized, relatively few studies have addressed how the moon affects amphibian behavior. Yet, the lunar cycle provides several rhythmic temporal cues that animals could use to time important group events such as spawning, and the substantial changes in light levels associated with the different moon phases may also affect the behavior of nocturnal frogs. Using seven years of field observation data, we tested for lunar effects on the reproductive activity of male and female Eastern Gray Treefrogs (Hyla versicolor). We found that chorusing and breeding activity was statistically more likely to occur around the first quarter of the moon and during intermediately bright nights, but that reproductive activity also occurred during various other times during the lunar cycle. We discuss these findings in relation to the two main hypotheses of lunar effects on animals: predator avoidance and temporal synchronization of breeding.     

Variation in lunar neutron dose estimates

The radiation environment on the Moon includes albedo neutrons produced by primary particles interacting with the lunar surface. In this work, HZETRN2010 is used to calculate the albedo neutron contribution to effective dose as a function of shielding thickness for four different space radiation environments and to determine to what extent various factors affect such estimates. First, albedo neutron spectra computed with HZETRN2010 are compared to Monte Carlo results in various radiation environments. Next, the impact of lunar regolith composition on the albedo neutron spectrum is examined, and the variation on effective dose caused by neutron fluence-to-effective dose conversion coefficients is studied. A methodology for computing effective dose in detailed human phantoms using HZETRN2010 is also discussed and compared. Finally, the combined variation caused by environmental models, shielding materials, shielding thickness, regolith composition and conversion coefficients on the albedo neutron contribution to effective dose is determined. It is shown that a single percentage number for characterizing the albedo neutron contribution to effective dose can be misleading. In general, the albedo neutron contribution to effective dose is found to vary between 1-32%, with the environmental model, shielding material and shielding thickness being the driving factors that determine the exact contribution. It is also shown that polyethylene or other hydrogen-rich materials may be used to mitigate the albedo neutron exposure.     

Lunar synchronization of testicular development and steroidogenesis in rabbitfish

Lunar synchronization of testicular development in the golden rabbitfish, Siganus guttatus, was assessed by measuring changes in sperm motility and conditions in the seminal plasma, and by in vitro production of steroid hormones in testicular fragments and sperm preparations. The duration and percentage of sperm motility was low 1 week before spawning (the new moon), but increased significantly on the day of spawning (the first lunar quarter). During the first lunar quarter, the osmolality decreased, but Ca(2+) concentration increased in the seminal plasma. These results suggest that spermiation occurs rapidly towards the specific lunar phase. Testicular fragments and sperm preparations were incubated with human chorionic gonadotropin (hCG) and two precursor steroid hormones, 17alpha-hydroxyprogesterone (17alpha-OHP) and testosterone (T), during the two lunar phases. The production of 11-ketotestosterone (11-KT) increased significantly when the testicular fragments were incubated with hCG at the first lunar quarter, while incubation of sperm preparations with 17alpha-OHP during the same moon phase resulted in a significant increase in 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) production in the medium. These results suggest that 11-KT is produced in the somatic cells of the testis under the influence of gonadotropin, and that sperm can convert 17alpha-OHP to DHP. Additionally, steroidogenic activity was considered to increase toward the specific lunar phase. The synchronous increase in testicular activity supports the hypothesis that lunar periodicity is a major factor for the testicular development of S. guttatus.     

The mammalian response to lunar particulates

The response of germfree mice to subcutaneous (SC) and intraperitoneal (IP) injection of aqueous suspensions of lunar fine material (LFM) was evaluated. Both uninjected mice and mice injected with dry heat sterilized LFM were included as controls. After injection, the majority of mice were subjected to serial sacrifice to assess the time course of the tissue response. A smaller group of animals were held for lifespan studies. Following IP injection lunar material was rapidly dispersed throughout the peritoneum. Aggregates of LFM could be identified adhering to the serosa of the viscera, omentum and peritoneum. The initial inflammatory response to IP injected material was mixed with both neutrophil and mononuclear leukocytes participating (Figure 4). Five to seven days after injection, the IP lesions consisted of almost pure populations of histiocytes and scattered lymphocytes IFigure 5). Lunar particulates injected IP were disseminated widely via lymphatics and intracellular macrophage transport. Evidence of this transport was provided by the appearance of LFM in the mediastinal lymph nodes of mice within 4 hr of IP injection (Figure 6). A classic acute inflammatory response to SC injected material was observed. Initially, the response was characterized by edema, vascular congestion, and neutrophil leukocyte infiltration (Figure 7). The edema quickly resolved and by Day 5 post injection, the response was characterized by an almost pure population of mononuclear cells. Rare foreign body giant cells were observed as a component of the SC response after Day 9 (Figure 8). LFM was observed to persist for the life of the animal (over 20 months). A low grade inflammatory reaction and the absence of significant fibroplasia characterized the end stage lesion. These observations suggest that LFM is relatively insoluble in tissue and that, while acting as a low grade irritant, it has little tendency to evoke reactive fibrosis. The significance of such a chronic low level stimulus and the various factors governing the retention, elimination, and turnover of LFM in mammalian tissue are not clear at this time. Further definitive study will be necessary to determine whether or not the unique chemical and physical properties of LFM will affect such life processes as senescence, disease resistance, or tumorigenesis. Lunar Science Institute Contribution     

Craniofacial dysmorphogenesis in transgenic mice.

While using transgenic mice to study the regulation of alpha-fetoprotein (AFP) it was noted that two different alpha-fetoprotein-chloramphenicol acetyl transferase (CAT) transgenes resulted in the appearance of craniofacial anomalies in 11% of the offspring derived from crosses between transgenic mice and nontransgenic mates. A total of 13 fetuses exhibited abnormalities; two are described in detail. Ninety-two percent of the affected fetuses had some form of mandibular abnormality while zygomatic and ossicular defects appeared in more than 40% of the specimens. Aglossia and aberrant musculature were also present in the most severely involved specimen. Eight of the affected fetuses were screened for the presence of the AFP-CAT plasmid and all were found to be heterozygous for the transgene. Since the probability that all 8 of the abnormal fetuses known to carry the CAT gene would have done so by chance was only 1 in 256, it may be assumed that these anomalies did not appear spontaneously, but were somehow created by the transgenic procedure. It is not known how the transgenic material led to the observed dysmorphogenetic pattern, but theoretically introduction of the AFP-CAT plasmid could have disrupted morphogenesis through the presence of the "foreign" CAT protein or a decrease in the availability of AFP. Since AFP levels were found to be normal in both the liver and the yolk sac of transgenic fetuses, it appears that the presence of CAT was responsible for the craniofacial anomalies described here.     

Effects of human carbonic anhydrase III (CA III) on synovial and muscle fibroblast glycosaminoglycan metabolism

We investigated the ability of CA III, isolated from adult human skeletal muscle, to regulate cell growth and glycosaminoglycan (GAG) formation in connective tissue cells derived from various human tissues. Unlike muscle, dermal, and cartilage fibroblasts, synovial connective tissue cells were substantially activated by CA III and showed enhanced hyaluronic acid (HA) synthesis. Cell culture experiments showed that CA III induced a 2- to 11-fold increase in [14C]HA synthesis by human synovial fibroblasts (SF) in a dose-dependent manner (P less than 0.001); erythrocyte CA I and CA II were inactive. Exposure of SF and muscle fibroblasts to CA III also resulted in a 20-45% and 16-70% increased 35S incorporation into proteoglycans, respectively. When adult human skin and cartilage fibroblasts were studied in the presence of CA III, no differences in the level of DNA and GAG formation were noted. These latter cell types were clearly activated by a platelet (CTAP-III) growth factor. The potential physiological implications of these observations are discussed.     

Hypobaric biology: Arabidopsis gene expression at low atmospheric pressure

As a step in developing an understanding of plant adaptation to low atmospheric pressures, we have identified genes central to the initial response of Arabidopsis to hypobaria. Exposure of plants to an atmosphere of 10 kPa compared with the sea-level pressure of 101 kPa resulted in the significant differential expression of more than 200 genes between the two treatments. Less than one-half of the genes induced by hypobaria are similarly affected by hypoxia, suggesting that response to hypobaria is unique and is more complex than an adaptation to the reduced partial pressure of oxygen inherent to hypobaric environments. In addition, the suites of genes induced by hypobaria confirm that water movement is a paramount issue at low atmospheric pressures, because many of gene products intersect abscisic acid-related, drought-induced pathways. A motivational constituent of these experiments is the need to address the National Aeronautics and Space Administration's plans to include plants as integral components of advanced life support systems. The design of bioregenerative life support systems seeks to maximize productivity within structures engineered to minimize mass and resource consumption. Currently, there are severe limitations to producing Earth-orbital, lunar, or Martian plant growth facilities that contain Earth-normal atmospheric pressures within light, transparent structures. However, some engineering limitations can be offset by growing plants in reduced atmospheric pressures. Characterization of the hypobaric response can therefore provide data to guide systems engineering development for bioregenerative life support, as well as lead to fundamental insights into aspects of desiccation metabolism and the means by which plants monitor water relations.     

Aqueous two-phase extraction of plant enzymes from sources containing large amounts of tannins and anionic mucilages.

The isolation of plant enzymes is frequently hampered by the presence of phenolic compounds, pigments and mucilages. Recently, extraction procedures based on aqueous two-phase systems were used to overcome these problems. Two-phase systems have a great advantage in respect to yield, product purity and processing time. Two-phase systems may open many new avenues in research as well as for application of enzymes from plant material, especially making available enzymes of sources avoided till now, due to the difficulties to work with.     

Partial purification of trimethylamine-N-oxide (TMAO) demethylase from crude fish muscle microsomes by detergents

Detergent treatments were examined for their efficacy in purifying trimethylamine-N-oxide (TMAO) demethylase activity from fish muscle microsomes. Tritons X-100 and X-45, deoxycholate, Brijs, Tweens 20, 65, and 80, and SDS were generally ineffective in solubilizing demethylase activity from this membrane fraction, at concentrations up to 10 mg detergent per mg protein. In all of these cases, specific activity became enriched in the particulate fraction obtained post-treatment. Highest fold-purification was achieved by using 10 mg SDS per mg protein in 5 mM histidine, pH 7.0 at 10-14 degrees C. Activity was relatively stable to the presence of SDS at this level, and with this treatment, TMAO demethylase activity became purified in the resultant particulate fraction 28- and 58-fold for activity stimulated by ascorbate-iron-cysteine and FMN-NADH, respectively. The presence of urea or 2-mercaptoethanol, or sonication of the SDS-microsome suspension during purification resulted in significant losses of recovered activity. This partially purified fraction represented about 1% of the original microsomal protein and SDS-PAGE revealed the presence of several protein components. The partially purified demethylase could utilize the same two cofactor systems as the native microsomes. It displayed a curvilinear dependence on iron for activity and a sigmoidal response for cysteine. Utilization of NADH, FMN, and ascorbate differed for the purified fraction as compared to the microsomes. Substrate inhibition by TMAO was observed for the partially purified preparation, whereas saturation kinetics were previously noted for microsomal activity.(ABSTRACT TRUNCATED AT 250 WORDS)