Thymine photoproduct formation and inactivation of intact spores of Bacillus subtilis irradiated with short wavelength UV (200-300nm) at atmospheric pressure and in vacuo.

Vacuum exposure renders the survival of spores of Bacillus subtilis approximately five times more sensitive to ultraviolet light irradiation than exposure under atmospheric conditions. The photoproduct formation in spores irradiated under ultrahigh vacuum (UHV) conditions is compared to the photoproduct formation in spores irradiated at atmospheric pressure. Compared to irradiation at atmospheric pressure, where only the "spore photoproduct" 5-thyminyl-5,6-dihydrothymine (TDHT) can be detected, two additional photoproducts, known as the c,s and t,s isomers of thymine dimer (T<>T) are produced in vacuo. The spectral efficiencies for photoproduct formation in spores under atmospheric and vacuum conditions are compared. Since there is no increased formation of TDHT after irradiation in vacuum, TDHT cannot be made responsible for the observed vacuum effect. "Vacuum specific" photoproducts may cause a synergistic response of spores to the simultaneous action of ultraviolet light (UV) and UHV. Three different mechanisms are discussed for the enhanced sensitivity of B. subtilis spores to UV radiation in vacuum. The experiments described contribute valuable research information on the chance for survival of microorganisms in outer space.     

Physical determinants of radiation sensitivity in bacterial spores.

Several factors modifying radiation sensitivity in dry bacterial spores are described and discussed. Vacuum inducing the loss of critical structural water, very low dose rates of radiation from which the cell may recover, radiations of high linear energy transfer, and the action of temperature over long periods of time on previously irradiated cells are recognized from extensive laboratory work as important in determining survival of spores exposed to low radiation doses at low temperatures for long periods of time. Some extensions of laboratory work are proposed.     

Early development of fern gametophytes in microgravity.

Dormant spores of the fern Ceratopteris richardii were flown on Shuttle mission STS-93 to evaluate the effects of micro-g on their development and on their pattern of gene expression. Prior to flight the spores were sterilized and sown into one of two environments: (1) Microscope slides in a video-microscopy module; and (2) Petri dishes. All spores were then stored in darkness until use. Spore germination was initiated on orbit after exposure to light. For the spores on microscope slides, cell level changes were recorded through the clear spore coat of the spores by video microscopy. After their exposure to light, spores in petri dishes were frozen in orbit at four different time points during which on earth gravity fixes the polarity of their development. Spores were then stored frozen in Biological Research in Canister units until recovery on earth. The RNAs from these cells and from 1-g control cells were extracted and analyzed on earth after flight to assay changes in gene expression. Video microscopy results revealed that the germinated spores developed normally in microgravity, although the polarity of their development, which is guided by gravity on earth, was random in space. Differential Display-PCR analyses of RNA extracted from space-flown cells showed that there was about a 5% change in the pattern of gene expression between cells developing in micro-g compared to those developing on earth.     

Survival of microorganisms in space: A review

Spores of $\text{Bacillus subtilis}$ were exposed to selected factors of space (vacuum, solar UV radiation, heavy ions of cosmic radiation), and their response was studied after recovery. These investigations were supplemented by ground-based studies under simulated space conditions. The vacuum of space did not inactivate the spores. However, vacuum-induced structural changes in the DNA, and probably in the proteins, caused a supersensitivity to solar UV radiation. This phenomenon is caused by the production of specific photoproducts in DNA and protein, which cannot be removed by normal cellular repair processes. In vegetative bacterial cells, exposed to vacuum, cell dehydration led to damage of the cell membrane, which could be partly repaired during subsequent incubation. The high local effectiveness of the cosmic heavy ions further decreases the chance that spores can survive for any length of time in space. Nonetheless, a spore travelling through space and protected from ultraviolet radiation could possibly survive an interplanetary journey. Such a situation favors panspermia as a possible explanation for the origin of life.     

Biological space experiments for the simulation of Martian conditions: UV radiation and Martian soil analogues.

The survivability of resistant terrestrial microbes, bacterial spores of Bacillus subtilis, was investigated in the BIOPAN facility of the European Space Agency onboard of Russian Earth-orbiting FOTON satellites (BIOPAN I -III missions). The spores were exposed to different subsets of the extreme environmental parameters in space (vacuum, extraterrestrial solar UV, shielding by protecting materials like artificial meteorites). The results of the three space experiments confirmed the deleterious effects of extraterrestrial solar UV radiation which, in contrast to the UV radiation reaching the surface of the Earth, also contains the very energy-rich, short wavelength UVB and UVC radiation. Thin layers of clay, rock or meteorite material were shown to be only successful in UV-shielding, if they are in direct contact with the spores. On Mars the UV radiation climate is similar to that of the early Earth before the development of a protective ozone layer in the atmosphere by the appearance of the first aerobic photosynthetic bacteria. The interference of Martian soil components and the intense and nearly unfiltered Martian solar UV radiation with spores of B. subtilis will be tested with a new BIOPAN experiment, MARSTOX. Different types of Mars soil analogues will be used to determine on one hand their potential toxicity alone or in combination with solar UV (phototoxicity) and on the other hand their UV protection capability. Two sets of samples will be placed under different cut-off filters used to simulate the UV radiation climate of Mars and Earth. After exposure in space the survival of and mutation induction in the spores will be analyzed at the DLR, together with parallel samples from the corresponding ground control experiment performed in the laboratory. This experiment will provide new insights into the principal limits of life and its adaptation to environmental extremes on Earth or other planets which and will also have implications for the potential for the evolution and distribution of life.     

Differentiation of Dictyostelium discoideum vegetative cells into spores during Earth orbit in space.

We reported previously that emerged amoebae of Dictyostelium (D.) discoideum grew, aggregated and differentiated to fruiting bodies with normal morphology in space. Here, we investigated the effects of space radiation and/or microgravity on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space in a radiation-sensitive strain, gamma s13, and the parental strain, NC4. In gamma s13, there were hardly spores in the fruiting bodies formed in space. In NC4, we found a decrease in the number of spores, a delay in germination of the spores and delayed start of cell growth of the spores formed in space when compared to the ground control. However, the mutation frequency of the NC4 spores formed in space was similar to that of the ground control. We conclude that the depression of spore formation might be induced by microgravity and/or space radiation through the depression of some stage(s) of DNA repair during cell differentiation in the slime mold.     

Early and late mammalian responses to heavy charged particles.

This overview summarizes murine results on acute lethality responses, inactivation of marrow CFU-S and intestinal microcolonies, testes weight loss, life span shortening, and posterior lens opacification in mice irradiated with heavy charged particles. RBE-LET relationships for these mammalian responses are compared with results from in vitro studies. The trend is that the maximum RBE for in vivo responses tends to be lower and occurs at a lower LET than for inactivation of V79 and T-1 cells in culture. Based on inactivation cross sections, the response of CFU-S in vivo conforms to expectations from earlier studies with prokaryotic systems and mammalian cells in culture. Effects of heavy ions are compared with fission spectrum neutrons, and the results are consistent with the interpretation that RBEs are lower than for fission neutrons at about the same LET, probably due to differences in track structure. Issues discussed focus on challenges associated with assessments of early and late effects of charged particles based on dose, RBE and LET, and with the concordance or discordance of results obtained with in vivo and in vitro model systems. Models for radiation damage/repair and misrepair should consider effects observed with in vivo as well as in vitro model systems.     

Inactivation, mutation induction and repair in Bacillus subtilis spores irradiated with heavy ions.

Studies on the response of bacterial spores to accelerated heavy ions (HZE particles) help in understanding problems of space radiobiology and exobiology. Layers of spores of Bacillus subtilis strains, differing in repair capabilities, were irradiated with accelerated boron, carbon and neon ions of linear energy transfer (LET) values up to 14000 MeV cm2/g. Inactivation as measured by loss of colony forming ability and induction of mutations as measured by reversion to histidine prototrophy and resistance to 150 micrograms/ml sodium azide were tested, as well as the influence of repair processes on these effects. For inactivation, the cross-sectional values sigma plotted as a function of LET follow a saturation curve. The plateau, which is reached around a LET of 2000 MeV cm2/g, occurs at 2.5 x 10(-9) cm2, a value in good agreement with the dimensions of the spore protoplast. Lethal damage produced at LET values < 2000 MeV cm2/g is reparable. Recombination repair is more effective than excision repair. At higher LET values, lethal damage could not be reconstituted by the repair mechanisms studied. In addition, at these high LET values, the frequency of induced mutations was drastically decreased. The data support the assumption of at least two qualitatively different types of lesion, depending on the LET of the affecting heavy ion.     

Inactivation probability of heavy ion-irradiated Bacillus subtilis spores as a function of the radial distance to the particle's [correction of paricle's] trajectory.

The understanding of the radiobiological action of heavy ions requires the knowledge of the dependence of the inactivation probability on the distance between the particle's trajectory and the biological test organism (the impact parameter). Spores of Bacillus subtilis with a cytoplasmic core of about 0.22 micrometer cross section are suitable test objects for the study of this radial inactivation probability in its microscopic details. The spores are irradiated at low fluences of some 10(6) ions/cm2 with very heavy ions at different specific energies up to 10 MeV per atomic mass unit u while in fixed contact with visual nuclear track detectors. The methods are described by which the biological response of individual cells can be evaluated and the impact parameter be determined with an accuracy typically better than 0.2 micrometer. The results demonstrate that the common characteristics of inactivation, e.g., an effective range of inactivation extending to at least 3 micrometers, a nonmonotonic dependence of the inactivation probabilities on the radial distance, and the fact that the inactivation probability even for direct central hits on the cytoplasmic core is substantially below one, are nearly independent of the particle energy and type. The results are incompatible with the assumption that the radiobiological effectiveness can be attributed to the dose of secondary electrons as currently understood. They also demonstrate that the widely held notion of an "overkill" at low impact parameters does not apply for the spores even with the most densely ionizing ions.     

Quantification of biologically effective environmental UV irradiance.

To determine the impact of environmental UV radiation on human health and ecosystems demands monitoring systems that weight the spectral irradiance according to the biological responses under consideration. In general, there are three different approaches to quantify a biologically effective solar irradiance. (i) weighted spectroradiometry where the biologically weighted radiometric quantities are derived from spectral data by multiplication with an action spectrum of a relevant photobiological reaction, e.g. erythema, DNA damage, skin cancer, reduced productivity of terrestrial plants and aquatic foodweb, (ii) wavelength integrating chemical-based or physical dosimetric systems with spectral sensitivities similar to a biological response curve, and (iii) biological dosimeters that directly weight the incident UV components of sunlight in relation to the effectiveness of the different wavelengths and to interactions between them. Most biological dosimeters, such as bacteria, bacteriophages, or biomolecules, are based on the UV sensitivity of DNA. If precisely characterized, biological dosimeters are applicable as field and personal dosimeters.     

Cellular responses to gravity: extracellular, intracellular and in-between.

Our understanding of gravitational effects (inertial effects in the vicinity of 1 x g) on cells has matured to a stage at which it is possible to define, on the basis of experimental evidence, extracellular effects on small cells and intracellular effects on eukaryotic gravisensing cells. Yet undetermined is the nature of response, if any, of those classes of cells that are not governed solely by extracellular physical events (as are prokaryotes) and are devoid of obvious mechanical devices for sensing inertial forces (such as those possessed by certain plant cells and sensory cells of animals). This "in-between" class of cells needs to be understood on the basis of the combination of intracellular and extracellular gravity-dependent processes that govern experimentally-measurable variables that are relevant to the cell's responses to modified inertial forces. The forces that certain cell types generate or respond to are therefore compared to those imposed by approximately 1 x g in the context of cytoskeletal action and symmetry-breaking pathways.     

Quantitative interpretation of heavy ion effects: comparison of different systems and endpoints.

For a quantitative interpretation of biological heavy ion action the following parameters have to be taken into account: variations of energy depositions in microscopical sites, the dependence of primary lesion formation on local energy density and changes in reparability. They can be studied in objects of different size and with different sensitivities. Results on survival and mutation induction in yeast and in mammalian cells will be compared with theoretical predictions. It is shown that shouldered survival curves of diploid yeast can be adequately described if the final slope is adjusted according to the varying production of primary lesions. This is not the case for mammalian cells where the experiments show a rapid loss of the shoulder with LET, contrary to theoretical expectations. This behaviour is interpreted to mean that the reparability of heavy ion lesions is different in the two systems. Mutation induction is theoretically expected to decrease with higher LET. This is found in yeast but not in mammalian cells where it actually increases. These results suggest a higher rate of misrepair in mammalian cells.     

Biological responses to space: results of the experiment "Exobiological Unit" of ERA on EURECA I.

Spores of different strains of Bacillus subtilis and the Escherichia coli plasmid pUC19 were exposed to selected conditions of space (space vacuum and/or defined wavebands and intensities of solar ultraviolet radiation) in the experiment ER 161 "Exobiological Unit" of the Exobiology Radiation Assembly (ERA) on board of the European Retrievable Carrier (EURECA). After the approximately 11 months lasting mission, their responses were studied in terms of survival, mutagenesis in the his (B. subtilis) or lac locus (pUC19), induction of DNA strand breaks, efficiency of DNA repair systems, and the role of external protective agents. The data were compared with those of a simultaneously running ground control experiment. The survival of spores treated with the vacuum of space, however shielded against solar radiation, is substantially increased, if they are exposed in multilayers and/or in the presence of glucose as protective, whereas all spores in "artificial meteorites", i.e. embedded in clays or simulated Martian soil, are killed. Vacuum treatment leads to an increase of mutation frequency in spores, but not in plasmid DNA. Extraterrestrial solar ultraviolet radiation is mutagenic, induces strand breaks in the DNA and reduces survival substantially; however, even at the highest fluences, i.e. 3 x 10(8) J m-2, a small but significant fraction of spores survives the insolation. Action spectroscopy confirms results of previous space experiments of a synergistic action of space vacuum and solar UV radiation with DNA being the critical target.     

High temperature effect on microflora of radish root-inhabited zone and nutrient solutions for radish growth.

The effect of high temperatures (35 and 45 degrees C) on microflora of the root zone of radish plants grown in phytotron was evaluated by the response of microorganisms from 9 indicator groups. Phytotron air temperature elevated to 35 degrees C for 20 hours caused no significant changes in qualitative and quantitative composition of the root microflora in experimental plants. By the end of the experiment, the species diversity of microflora had changed. The amount of phytopathogenic microorganisms decreased which can be interpreted as more stable co-existence of microflora with plants. The numbers of microbes from other indicator groups was in dynamic equilibrium. The plants' condition did not deteriorate either. Exposure to the temperature of 45 degrees C for 7 hours have been found to change the numbers and species diversity in the radish root zone microflora. The microorganisms were observed to increase their total numbers at the expense of certain indicator groups. Bacteria increased spore forms at the stage of spores. Colon bacillus bacteria of increased their numbers by the end of experiment by an order. By the end of experiment the roots of experiment plants had microscopic fungi from Mucor, Aspergillus, Trichoderma, Cladosporium genera. The observed changes in the microbial complex seem to be associated with the changes of root emissions and general deterioration of the plants' condition. It is suggested that the response of the microorganisms can be indicative of the condition of plants under investigation.     

Stratospheric ozone loss, ulraviolet effects and action spectroscopy

The major effect of stratospheric ozone loss will be an increase in the amount of ultraviolet radiation reaching the ground. This increase will be entirely contained within the UV-B (290–320nm). How this will impact life on Earth will be determined by the UV-B photobiology of exposed organisms, including humans. One of the analytical methods useful in estimating these effects is Action Spectroscopy (biological effect as a function of wavelength). Carefully constructed action spectra will allow us to partially predict the increase in bio-effect due to additional UV exposure. What effect this has on the organism and the system in which the organism resides is of paramount importance. Suitable action spectra already exist for human skin cancer, human cell mutation and killing, and for one immune response. Comprehensive and widely applicable action spectra for terrestrial and aquatic plant responses are being generated but are not yet suitable for extensive analysis. There is little data available for animals, other than those experiments completed in the laboratory as model systems for human studies. Some polychromatic action spectra have proven useful in determining the possible impact of ozone loss on biological systems. The pitfalls and limits of this approach will be addressed.     

Spore dosimetry of solar UV radiation: applications to monitoring of daily irradiance and personal exposure.

Environmental UV radiation can be quantified using spore dosimetry, which measures the inactivation of repair-deficient Bacillus subtilis spores dried on a membrane filter. The system exhibits highly selective sensitivity to UV radiation, not being affected by various environmental adversities, such as high and low temperature and humidity. Biologically-effective dose rate and cumulative dose of ambient radiation are measurable under various conditions at various places on the earth, including tropical, temperate, and polar sites. Applications to monitor the exposure at the surface of organisms including humans and plants have also been advanced.     

Mammalian cell cultivation in space.

Equipment used in space for the cultivation of mammalian cells does not meet the usual standard of earth bound bioreactors. Thus, the development of a space worthy bioreactor is mandatory for two reasons: First, to investigate the effect on single cells of the space environment in general and microgravity conditions in particular, and second, to provide researchers on long term missions and the Space Station with cell material. However, expertise for this venture is not at hand. A small and simple device for animal cell culture experiments aboard Spacelab (Dynamic Cell Culture System; DCCS) was developed. It provides 2 cell culture chambers, one is operated as a batch system, the other one as a perfusion system. The cell chambers have a volume of 200 microliters. Medium exchange is achieved with an automatic osmotic pump. The system is neither mechanically stirred nor equipped with sensors. Oxygen for cell growth is provided by a gas chamber that is adjacent to the cell chambers. The oxygen gradient produced by the growing cells serves to maintain the oxygen influx by diffusion. Hamster kidney cells growing on microcarriers were used to test the biological performance of the DCCS. On ground tests suggest that this system is feasible.     

Solar wind-driven electron radiation belt response functions at 100-min time scales

We present a simple yet numerically robust technique, using autoregressive linear filters, to remove unwanted “colored noise” from solar wind and radiation belt electron data at sub-daily resolution. The remaining signal is then studied using finite impulse response linear prediction filters to represent the driven portion of the linear dynamics that describe the coupling between solar wind speed and electron flux. Sub-daily resolution response profiles covering magnetic L-shells between 1.1 and 8.0 R_E are presented which are consistent with daily resolution response functions. Namely, while there is strong global coherence governing electron flux dynamics, there are at least two distinct responses. The first response is an immediate dropout of electrons between L = 4 and L = 7 that is at least a partly adiabatic effect associated with enhancements in the ring current. This is followed by a 1–2 day delayed enhancement across the same L-shells that is likely a result of increased radial diffusion. The second response is an immediate enhancement seen between L = 3 and L = 4 with a typical duration of less than one day. Plausible explanations for this second response are briefly discussed, but neither empirical nor theoretical evidence can establish conclusively a definite physical cause. Finally, the response profiles show significant solar cycle and seasonal dependencies, indicating that better model output might be achieved with: (1) additional simultaneous solar wind inputs; (2) more sophisticated dynamical model structures capable of incorporating non-linear feedback; and/or (3) time-adaptive linear filters that can track non-stationary dynamics in time.     

Responses of ionospheric foF2 to geomagnetic activities in Hainan

Responses of low-latitude ionospheric critical frequency of F2 layer to geomagnetic activities in different seasons and under different levels of solar activity are investigated by analyzing the ionospheric foF2 data from DPS-4 Digisonde in Hainan Observatory during 2002–2005. The results are as follows: (1) the response of foF2 to geomagnetic activity in Hainan shows obvious diurnal variation except for the summer in low solar activity period. Generally, geomagnetic activity will cause foF2 to increase at daytime and decrease at nighttime. The intensity of response of foF2 is stronger at nighttime than that at daytime; (2) seasonal dependence of the response of foF2 to geomagnetic activity is very obvious. The negative ionospheric storm effect is the strongest in summer and the positive ionospheric storm effect is the strongest in winter; (3) the solar cycle has important effect on the response of foF2 to geomagnetic activity in Hainan. In high solar activity period, the diurnal variation of the response of foF2 is very pronounced in each season, and the strong ionospheric response can last several days. In low solar activity period, ionospheric response has very pronounced diurnal variation in winter only; (4) the local time of geomagnetic activities occurring also has important effect on the responses of foF2 in Hainan. Generally, geomagnetic activities occurred at nighttime can cause stronger and longer responses of foF2 in Hainan.     

UV photobiochemistry under space conditions

The response of spores of Bacillus subtilis, cells of Deinococcus radiodurans and conidia of Aspergillus ochraceus to actual and simulated space conditions (UV in combination with long-term exposure to extremely dry conditions, including vacuum) has been studied: The following effects have been analyzed: decrease of viability, occurrence of DNA double strand breaks, formation of DNA-protein cross-links and DNA-DNA cross-links. All organisms show an increased sensitivity to UV light in extreme dryness (dry argon or vacuum) compared to an irradiation in aqueous suspension. The UV irradiation leads in all cases to a variety of DNA lesions. Very conspicuous is the occurrence of double strand breaks. Most of these double strand breaks are produced by incomplete repair of other lesions, especially base damages. The increase in DNA lesions can be correlated to the loss in viability. The specific response of the chromosomal DNA to UV irradiation in extreme dryness, however, varies from species to species and depends on the state of dehydration. The formation of DNA double strand breaks and DNA-protein cross-links prevails in the case of B. subtilis spores. In cells of Deinococcus radiodurans DNA-DNA cross-links often predominate, in conidia of Aspergillus ochraceus double strand breaks. The results obtained by direct exposure to space conditions (EURECA mission and D2 mission) largely agree with the laboratory data.