Response of tobacco tissue cultures growing in contact with lunar fines

During the quarantine periods following each Apollo mission to the Moon, various biological systems were placed in the presence of lunar material to determine if pathogenic agents were present. Although no detrimental effects resulted, various responses by the several plant systems tested were noted. One such response was the increased pigmentation observed in the callus tissue cultures of tobacco. Further investigations revealed that these tissues grown in the presence of lunar material resulted in as much as a 35% increase in total pigments while differences in fatty acid and sterol concentrations were also noted when compared to the controls. It is believed that these changes brought about by the lunar material can be attributed to a change in the nutritional environment caused by its dissolution. Lunar Science Institute Contribution.     

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     

Lipid composition of slash pine tissue cultures grown with lunar and earth soils

Lipid analyses were conducted on slash pine tissues grown in culture in the presence of lunar (Apollo 15) and Earth soils. Significant reductions in the total lipids, fatty acids, and sterol components were found in the tissues grown in contact with each of the soils employed when compared to the control. Tissues grown with lunar soil showed the greatest reductions. These results are discussed with respect to previous ultrastructural studies on similarly treated slash pine tissues and lipid analyses on tobacco tissue cultures.     

The effect of retinoic acid on proteoglycan turnover in bovine articular cartilage cultures

This paper describes proteoglycan catabolism by adult bovine articular cartilage treated with retinoic acid as a means of stimulating the loss of this macromolecule from the extracellular matrix of cartilage. Addition of retinoic acid (10(-12)-10(-6) M) to adult bovine articular cartilage which had been labeled with [35S]sulfate for 6 h after 5 days in culture, resulted in a dose-dependent increase in the rate of loss of 35S-labeled proteoglycans from the matrix of the tissue. Concomitant with this loss was a decrease in the proteoglycan content of the tissue. Incubation of cultures treated with 1 microM retinoic acid, at 4 degrees C, or with 0.5 mM cycloheximide, resulted in a significant decrease in the rate of retinoic acid-induced loss of proteoglycans and demonstrated cellular involvement in this process. Analysis of the 35S-labeled proteoglycans remaining in the matrix showed that the percentage of radioactivity associated with the small proteoglycan species extracted from the matrix of articular cartilage explants labeled with [35S]sulfate after 5 days in culture was 15% and this increased to 22% in tissue maintained in medium alone. In tissue treated with 1 microM retinoic acid for 6 days, the percentage of radioactivity associated with the small proteoglycan was 58%. Approximately 93% of the 35S-labeled proteoglycans released into the medium of control and retinoic acid-treated cultures was recovered in high density fractions after CsCl gradient centrifugation and eluted on Sepharose CL-2B as a broad peak with a Kav of 0.30-0.37. Less than 17% of these proteoglycans was capable of aggregating with hyaluronate. These results indicate that in both control and retinoic acid-treated cultures the larger proteoglycan species is lost to the medium at a greater rate than the small proteoglycan species. The effect of retinoic acid on proteoglycan turnover was shown to be reversible. Cartilage cultures maintained with retinoic acid for 1 day then switched to medium with 20% (v/v) fetal calf serum for the remainder of the culture period exhibited decreased rates of loss of 35S-labeled proteoglycans from the matrix and increased tissue hexuronate contents to levels near those observed in tissue maintained in medium with 20% (v/v) fetal calf serum throughout. Furthermore, following switching to 20% (v/v) fetal calf serum, the relative proportions of the 35S-labeled proteoglycan species remaining in the matrix of these cultures were similar to those of control cultures.     

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.     

Investigation of fungal biomolecules after Low Earth Orbit exposure: a testbed for the next Moon missions

The Moon is characterized by extremely harsh conditions due to ultraviolet irradiation, wide temperature extremes, vacuum resulting from the absence of an atmosphere and high ionizing radiation. Therefore, its surface may provide a unique platform to investigate the effects of such conditions. For lunar exploration with the Lunar Gateway platform, exposure experiments in Low Earth Orbit are useful testbeds to prepare for lunar space experiments and to understand how and if potential biomarkers are influenced by extra-terrestrial conditions. During the BIOMEX (BIOlogy and Mars EXperiment) project, dried colonies of the fungus Cryomyces antarcticus grown on Lunar Regolith Analogue (LRA) were exposed to space conditions for 16 months aboard the EXPOSE-R2 payload outside the International Space Station. In this study, we investigated the stability/degradation of fungal biomarkers in LRA after exposure to (i) simulated space and (ii) real space conditions, using Raman spectroscopy, gas chromatography–mass spectrometry and DNA amplification. The results demonstrated that fungal biomarkers were detectable after 16 months of real space exposure. This work will contribute to the interpretation of data from future biological experiments in the Cislunar orbit with the Lunar Gateway platform and/or on the lunar surface, in preparation for the next step of human exploration.     

Microbial contamination associated with the Apollo 6 spacecraft during final assembly and testing

The National Aeronautics and Space Administration (NASA) requires that microorganisms which could contaminate the surface of the moon as the result of lunar missions be enumerated and identified so that life forms in lunar materials returned to earth may be more easily recognized as being of native or terrestrial origin.Assessment of microbial contamination in the intramural environments used for the assembly and test of the manned lunar spacecraft (Apollo) was made using fallout strips and air samplers. Microbial contamination on the surfaces of Apollo Command and Lunar Modules was determined by use of the swab-rinse method.Preliminary results indicate that the levels of microbial contamination which accumulated on exposed stainless steel surfaces, as well as airborne microbial contamination in the high bay assembly areas, were similar to those encountered in the unmanned spacecraft assembly areas. However, higher levels of microbial contamination were detected on the Apollo spacecraft than on the unmanned lunar spacecraft.     

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.     

Lunar Philia in a Nocturnal Primate

The influence of moonlight on behavior has been well documented for many nocturnal mammals, including rodents, lagomorphs, badgers and bats. These studies have consistently shown that nocturnal mammals respond to bright moonlight by reducing their foraging activity, restricting their movement, and reducing their vocalizations. Lunar phobia among nocturnal mammals is generally believed to be a form of predator avoidance: numerous studies indicate that predation increases during moonlit nights. A study I conducted at Tangkoko Nature Reserve in Sulawesi, Indonesia, demonstrates that spectral tarsiers, (Tarsius spectrum), are not lunar phobic, but are lunar philic; they become more active during full moons. During full moons, spectral tarsiers increased foraging, decreased resting, increased travel (distance traveled per unit time, nightly path length, and home range size), increased the frequency of group travel and decreased the frequency of olfactory communication. I explore several potential hypotheses to account for the lack of lunar phobia and potential increased risk of predation resulting from this unusual behavior. Two hypotheses that may account for the behavior are that: 1) foraging efficiency increases during full moons and outweighs the increased risk of predation, and 2) predation risk is not greater during full moons. Instead, predation risk increases during new moons.     

Lunar synchronization of spawning in cichlid fishes of the tribe Lamprologini in Lake Tanganyika

Lunar synchronization of spawning was investigated for eight substrate brooding cichlid fishes belonging to the tribe Lamprologini. Their spawning activities all peaked during the second quarter of the lunar cycle. Comparison between their breeding styles and the degrees of spawning synchronization suggested that the nocturnal guarding-efficiency of eggs, especially exposed ones, is improved by the maximal amount of moonlight during full moon and, in species whose young leave the breeding sites about 2 weeks after spawning, the survival of dispersing young is enhanced by the darkness of moonless nights.     

Effect of lunar periodicity on the locomotor activity of silver-stage Japanese eel,Anguilla japonica

Lunar periodicity has been thought to influence the onset of the spawning migration of anguillid eels. In this study, we measured daily locomotor activity of 8 silver-phase Japanese eels Anguilla japonica in outdoor tanks to examine the effect of lunar periodicity on their activity and the following seaward migration. The activity of silver eels was highest around the new moon during the early part of the experiment, which is the ordinary season of seaward migration in Japan. The observed patterns of activity may reflect the importance of the lunar cycle for the onset of the spawning migration in anguillid eels.     

Lunar phase-dependent expression of cryptochrome and a photoperiodic mechanism for lunar phase-recognition in a reef fish, goldlined spinefoot

Lunar cycle-associated physiology has been found in a wide variety of organisms. Recent study has revealed that mRNA levels of Cryptochrome (Cry), one of the circadian clock genes, were significantly higher on a full moon night than on a new moon night in coral, implying the involvement of a photoreception system in the lunar-synchronized spawning. To better establish the generalities surrounding such a mechanism and explore the underlying molecular mechanism, we focused on the relationship between lunar phase, Cry gene expression, and the spawning behavior in a lunar-synchronized spawner, the goldlined spinefoot (Siganus guttatus), and we identified two kinds of Cry genes in this animal. Their mRNA levels showed lunar cycle-dependent expression in the medial part of the brain (mesencephalon and diencephalon) peaking at the first quarter moon. Since this lunar phase coincided with the reproductive phase of the goldlined spinefoot, Cry gene expression was considered a state variable in the lunar phase recognition system. Based on the expression profiles of SgCrys together with the moonlight's pattern of timing and duration during its nightly lunar cycle, we have further speculated on a model of lunar phase recognition for reproductive control in the goldlined spinefoot, which integrates both moonlight and circadian signals in a manner similar to photoperiodic response.     

Epifaunal colonization of the Loch Linnhe artificial reef: influence of substratum on epifaunal assemblage structure

A one-year study was carried out off the west coast of Scotland to compare the epifaunal colonization of concrete material used in the construction of the Loch Linnhe artificial reef with that on four other types of artificial substrata (preservative treated wood, rubber, steel and PVC). Settlement panels made from each of the materials were submerged in a vertical orientation during four seasonal exposure periods. There were clear seasonal trends across the four exposure periods with higher epifaunal biodiversity on all types of panel in the spring and summer exposure periods. Epifaunal assemblage structure was significantly different between the five types of material after each three-month exposure period. Concrete, preservative treated wood and PVC tended to have the highest species diversities. A successional study was also carried out. Over a 12-month exposure period epifaunal biodiversity increased on all five materials. After 12 months of exposure, the epifaunal assemblage structure was still significantly different between materials but had become more similar indicating a successional change towards a stable assemblage on all panels. The results indicate that material type and season have a significant effect on epifaunal assemblage structure after short (three-month) periods of submersion but that these effects are reduced with increasing length of exposure. The study concludes that the choice of construction material for an artificial reef will have little effect on the long-term epifaunal community structure, as long as the material is physically stable, non-toxic and offers a high degree of habitat complexity.     

Epifaunal colonization of the Loch Linnhe artificial reef: Influence of substratum on epifaunal assemblage structure

A one-year study was carried out off the west coast of Scotland to compare the epifaunal colonization of concrete material used in the construction of the Loch Linnhe artificial reef with that on four other types of artificial substrata (preservative treated wood, rubber, steel and PVC). Settlement panels made from each of the materials were submerged in a vertical orientation during four seasonal exposure periods. There were clear seasonal trends across the four exposure periods with higher epifaunal biodiversity on all types of panel in the spring and summer exposure periods. Epifaunal assemblage structure was significantly different between the five types of material after each three-month exposure period. Concrete, preservative treated wood and PVC tended to have the highest species diversities. A successional study was also carried out. Over a 12-month exposure period epifaunal biodiversity increased on all five materials. After 12 months of exposure, the epifaunal assemblage structure was still significantly different between materials but had become more similar indicating a successional change towards a stable assemblage on all panels. The results indicate that material type and season have a significant effect on epifaunal assemblage structure after short (three-month) periods of submersion but that these effects are reduced with increasing length of exposure. The study concludes that the choice of construction material for an artificial reef will have little effect on the long-term epifaunal community structure, as long as the material is physically stable, non-toxic and offers a high degree of habitat complexity.     

The benthos of a natural West African lake, with emphasis on the diel migrations and lunar and seasonal periodicities of the Chaoborus populations (Diptera, Chaoboridae)

SUMMARY. 1. Three Chaoborus species dominated in abundance and biomass the mid-lake benthic fauna of a small, natural Nigerian lake. Each population showed distinct seasonal varialitions. C. anomalus being most abundant during the early rainy season, while ( ceratopogones and C edults were most abundant during the dry season. A fourth species. C. fuscinervis (not reported previously from West Africa), was collected only in the adult stage. Also abundant at mid-lake were the Chironomidae. Chironomus seydeli, Prodadius brevipetiolatus and Tanypus brevipalpis. all of which showed a seasonal pattern most resembling that of C. anomalus .
2. Second. third and fourth instar larvae and pupae of the Clutoborus species underwent a diel vertical migration between the lake sediments (diurnal) and the water column (nocturnal) which was strongly related to prevailing light conditions.
3. Lunar periodicities of Chaoborus emergence determined from emergence trap collections are described for the first time. Corresponding lunar periodicities of larval abundance were observed in two of the species. The statistical techniques employed in describing the lunar periodicities permit the estimahon of generation times.
4. C. anomalus is believed to be parthenogenetie in the study lake (no males collected). while the other Chaoborus species are bisexual (1:1 sex ratios). The biology of this species is discussed in relation to the parthenogenetie condition.     

SHORT-TERM REGULATION OF CATECHOLAMINE BIOSYNTHESIS IN A NERVE GROWTH FACTOR RESPONSIVE CLONAL LINE OF RAT PHEOCHROMOCYTOMA CELLS

Abstract— A clonal cell line (designated PC12) has been previously established from a transplantable rat adrenal medullary pheochromocytoma. Tissue cultures of PC12 cells synthesize, store, release and take up catecholamines. PC12 cells also respond to nerve growth factor (NGF) protein by cessation of mitosis and extension of neurites. The present studies concern the comparison of several aspects of catecholamine metabolism in PC12 cultures with that in normal noradrenergic tissues. One question was why the ratio of dopamine to norepinephrine in PC12 cultures (in contrast to that in normal noradrenergic tissue) is considerably more than one. The presence of exogenous reduced ascorbate (a cofactor for dopamine-β-monooxygenase) enhanced by 5–10-fold the rate at which PC12 cultures converted [³H]tyrosine to [³H]norepinephrine. Under such conditions, the rate of synthesis of [³H]do-pamine was unchanged. It was also found that the ratio of norepinephrine to dopamine increased by 10-fold when the cells were grown in vivo as tumors. Since tissue culture medium is essentially free of reduced ascorbate, it is likely that the absence of this cofactor is responsible for the low norepinephrine to dopamine ratio in PC12 cultures. Experiments were also carried out on short-term regulation of catecholamine synthesis in PC12 cultures. These studies revealed the following: (1) The rate of conversion of [³H]tyrosine to [³H]catechols was increased 2–3-fold (as compared with controls) in the presence of depolarizing levels of K⁺ (51.5 mM), and by 2-fold in the presence of 0.5–2 mM-dibutyryl cyclic adenosine 3′, 5’monophosphoric acid (db-cAMP). (2) Similar increases occurred in cultures which had been treated with (and had responded to) nerve growth factor. (3) The stimulatory effects of 51.5 mM-K⁺ rapidly returned toward control levels when the cultures were returned to control medium and (4) required the presence of Ca²⁺ in the extracellular medium. (5) Stimulation of catechol synthesis by 51.5 mM-K⁺ and db-cAMP also occurred in the presence of an inhibitor of DOPA decar-boxylase. Thus, the ultimate effects of these agents were probably at the level of conversion of tyrosine to dopa by tyrosine 3-monooxygenase. (6) Simultaneous exposure of cultures to 51.5 mM-K⁺ and mM-db-cAMP gave additive levels of stimulation. Such findings demonstrate that catecholamine synthesis in cultures of PC12 cells undergoes short-term regulation which is similar to that previously demonstrated in normal monoaminergic tissues. As a homogeneous tissue culture line, the PC12 bears certain advantages for studying the primary mechanisms of such effects.     

Ecology of mosquitoes (Diptera: Culicidae) in areas of Serra do Mar State Park, State of São Paulo, Brazil. III. Daily biting rhythms and lunar cycle influence

The ecology of mosquito species (Diptera: Culicidae) was studied in areas of the Serra do Mar State Park, State of S?o Paulo, Brazil. The influence of the lunar cycle and the daily biting rhythms of mosquito populations were analyzed. Systematized biweekly human bait collections were made in a silvatic environment for 24 consecutive months (January 1991 to December 1992). A total of 20,591 specimens of adult mosquitoes belonging to 55 species were collected from 545 catches. Sabethini species were captured exclusively during daylight periods, with the exception of Trichoprosopon digitatum, while members of Anophelinae predominated during nocturnal hours. Members of the subfamily Culicinae that were collected primary during nocturnal periods included Culex nigripalpus, Coquillettidia chrysonotum and Cq. venezuelensis while daytime catches included Psorophora ferox and Ps. albipes. Others members of culicines mosquitoes that were collected during both day and night included: Aedes serratus, Ae. scapularis and Ae. fulvus. Lunar cycles did not appear to influence the daily biting rhythms of most mosquito species in the area, but larger numbers of mosquitoes were collected during the new moon. Ae. scapularis were captured mainly during the full moon.     

A Neoplastic Connective Tissue Mast Cell Capable of Continuous Growth In Tissue Culture

A neoplastic connective tissue mast cell from a dog mast cell sarcoma has been grown in tissue culture for 50 passages over a period of 2 years. The cells were grown as monolayer cultures in glass bottles, using Eagle's basal medium fortified with calf serum. The cultures were contaminated with an Alkaligenes sp. for 10 months but finally were sterilized bacteriologically by treatment with specific antiserum combined with antibiotics. The cells grow in a fibroblastic pattern, and contain mitochondria, mast cell granules, and lipid granules or droplets. The mast cell granules stain basophilic with Giemsa's stain and metachromatically with azure A or toluidine blue. They also stain with Sudan black B and with periodic acid-Schiff stain. The interphase nuclei are vesicular, contain from 1 to 20 nucleoli, and frequently show bizarre outlines. Multinucleate cells are often seen, as are mitotic figures. Extracellular fibrous material occurs in all cultures and apparently originates from the cell surface. This material does not have the structure of connective tissue fibers and has not been identified. The cells develop an increased number of metachromatic granules when grown in medium containing heparin and an increased number of sudanophilic granules when grown in medium containing stearic acid. Only small amounts of histamine were present in the tumor from which this cell line was derived and in the cells grown in tissue culture.     

Enhancement of flavour biosynthesis from strawberry (Fragaria x ananassa) callus cultures by Methylobacterium species

Two important character-impact compounds of strawberry flavour, the furanones 2,5-dimethyl-4-hydroxy-2H-furan-3-one (DMHF) and 2,5-dimethyl-4-methoxy-2H-furan-3-one (mesifuran) were synthesized by strawberry tissue cultures derived from a cultivated species (Fragaria × ananassa, cv. Elsanta) after these were treated with Methylobacterium extorquens. These flavour compounds were analysed by HPLC-UV and their levels were compared in the treated and control tissues. In Methylobacterium extorquens treated callus cultures DMHF and mesifuran levels were 5.9 and 11.4 μg/g of fresh weight of callus respectively, compared to zero in the untreated ones. When Methylobacterium extorquens was fed with 1,2-propanediol, 2-hydroxy-propanal (lactaldehyde) was formed. This bacterial oxidation of 1,2-propanediol to lactaldehyde linked with the presence of 1,2-propanediol in strawberry suggests that the increased levels of the two furanones in the treated strawberry cultures is the result of Methylobacterium extorquens oxidative activity on 1,2-propanediol and the bioconversion by the plant cells of this oxidation product, lactaldehyde to DMHF and mesifuran. This revised version was published online in June 2006 with corrections to the Cover Date.