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NASA NTRS2001

Anaerobic Psychrophiles from Alaska, Antarctica, and Patagonia: Implications to Possible Life on Mars and Europa

Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryo-preserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 T. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of Antarctica. The microbial extremophiles recovered from permafrost, ice, cold pools and deep sea sediments may provide information relevant to the question of how and where we should search for evidence of extant or extinct microbial life elsewhere in the Cosmos.

Hoover, Richard B.↗

NASA NTRS1996

Searching for Life: Early Earth, Mars and Beyond

We might be entering a golden age for exploring life throughout time and space. Rapid gene sequencing will better define our most distant ancestors. The earliest geologic evidence of life is now 3.8 billion years old. Organic matter and submicron-sized morphologies have been preserved in the martian crust for billions of years. Several new missions to Mars are planned, with a high priority on the search for life, past or present. The recent discovery of large extrasolar planets has heightened interest in spacecraft to detect small, earth-like planets. A recent workshop discussed strategies for life detection on such planets. There is much to anticipate in the near future.

DesMarais, David J.↗

NASA NTRS2002

Anaerobic Psychrophiles from Alaska, Antarctica, and Patagonia: Implications to Possible Life on Mars and Europa

Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryopreserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 C. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep-sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of Antarctica. The microbial extremophiles recovered from permafrost, ice, cold pools and deep-sea sediments may provide information relevant to the question of how and where we should search for evidence of extant or extinct microbial life elsewhere in the Cosmos.

Hoover, Richard B.↗

NASA NTRS2002

Cyanobacterial Community Structure In Lithifying Mats of A Yellowstone Hotspring-Implications for Precambrian Stromatolite Biocomplexity

Denaturing Gradient Gel Electrophoresis (DGGE) of partial 16S rRNA gene sequences was used to investigate the molecular biodiversity of cyanobacterial communities inhabiting various lithified morpho-structures in two hotsprings of Yellowstone National Park. These morpho-structures - flat-topped columns, columnar cones, and ridged cones - resemble ancient stromatolites, which are possibly biogenic in origin. The top, middle and bottom sections of these lithified morpho-structures, as well as surrounding non-lithified mats were analyzed to determine the vertical and spatial distribution of cyanobacterial communities. Results from DGGE indicate that the cyanobacterial community composition of lithified morpho-structures (flat-topped columns, columnar cones, and ridged cones) were largely similar in vertical distribution as well as among the morpho-structures being studied. Preliminary results indicate that the cyanobacterial communities in these lithified morpho-structures were significantly different from communities in surrounding non-lithified mats. These results provide additional support to the theory that certain Phormidium/Leptolyngbya species are involved in the morphogenesis of lithifying morpho-structures in hotsprings and may have played a role in the formation of ancient stromatolites.

Lau, Evan↗

NASA NTRS2002

Mechanisms of Gravity-Evoked Neuronal Plasticity

The grant focuses on a gene we identified called, serum and glucocorticoid- induced kinase (SGK), during a previously funded NASA project. The abundance of SGK messenger RNA (mRNA) and protein is increased in CNS tissues from animals reared in microgravity in comparison with 1G reared animals. In the funded proposal we had three aims: 1) characterize the distribution of SGK mRNA in the developing and adult rat CNS, 2) determine if expression of enzymatically active or inactive forms of SGK in cells influenced cell morphology (neurite growth), and 2) determine if SGK is a CREB kinase - that is, a protein kinase that adds phosphate groups to the transcription factor CREB. Over the past year we have made strong progress in the two most difficult parts of the project, namely specific aims 2 and 3. In specific aim #2 we planned to express a dominant negative or a constitutively active form of SGK in PC12 cells and assay the effects on neurite growth. Several methods are available for examining the effects of a transgene on PC12 neurite growth. Relevant variables include the performance of the assay +/- serum, +/- NGF, substratum for growth, timing between transfection and assay. Over the past 8 months we have customized the assay to enable us to most readily determine the effects of transgene expression on neurite growth. We have also compared the relative utility of transfecting DNA as opposed to protein itself. We are now well positioned to study the effects of SGK on neurite growth. We have also made progress in parallel studies in primary neurons. We have made constructs which will lead to transgene expression in cultures of spinal cord neurons. Co-transfection of a reporter and the SGK constructs can now be performed.

Kalb, Robert↗

NASA NTRS2002

Irradiated HMEC from A-T Heterozygous Breast Tissue

Women who are heterozygous for ataxia-telangiectasia (A-T) carry a single defective ATM gene in chromosome 11 q22-23, and have been statistically determined with high significance within a defined database to be approximately 5-fold more susceptible for developing breast cancer than their noma1 counterpart. Breast cancer susceptibility of these A-T heterozygotes has been hypothesized to include consequence of response to damage caused by low levels of ionizing radiation. Prophylactic mastectomy specimens were donated by a 41 year-old obligate A-T heterozygote who was located prior to her elective surgery through an existing pedigree. Harvest of that breast tissue provided an isolate of long-term growth human mammary epithelial cells (HMEC), designated WH612/3. An isolate of presumed normal long-term growth HMEC, designated 48R, was obtained from Dr. Martha Stampfer (Lawrence Berkeley Laboratory, University of California), and the A-T heterozygous HMEC were transformed with E6 and E7 oncogenes of human papilloma virus Type-16 in the laboratory of Dr. Ray White (Hunt- Cancer Institute, University of Utah) for use in this study. The objective of this study is to study the expression of end points that may bear on cancer outcome following irradiation of HMEC. Specific end points are cell survival, cell cycle, p53 expression, and apoptosis. Survival curves, immunostaining, and flow cytometery are used to examine these end points. Radiation-induced cell killing shows less shoulder development in the survival curve for WH61U3 compared to 48R HMEC, suggesting less repair of damage in the former HMEC. Additional information is included in the original extended abstract.

Richmond, Robert↗

NASA NTRS2002

Neural Correlates of Birth: Labor Contractions Induce C-Fos Expression In Newborn Rat Brain

At birth, the newborn mammal must make rapid adaptations to the extrauterine environment to survive. We have previously shown that labor contractions augment the appearance of adaptive responses at birth, viz., postpartum breathing and the onset of suckling. Since neuronal activity has been shown to upregulate the activity of immediate early genes (IEGs) in the brain, we analyzed the neural distribution of c-Fos protein expression in newborn rats using immunohistochemistry. Previous studies have reported a burst of c-Fos mRNA expression in mouse and rat brain at birth however relationships to labor and delivery have not been examined. In the present study, we exposed near-term rat fetuses to elements of the vaginal birth process: 1) Simulated labor contractions. 2) Postpartum cooling (22 deg C). 3) Umbilical cord occlusion. and 4) Stroking to mimic postpartum licking by the dam. Cardinally delivered newborns (VG) were compared with those delivered by cesarean section following either prenatal exposure to compressions (C) [simulated labor contractions], or no compressions (NC) [no labor contractions]. Similar patterns of c-fos activation were observed throughout hypothalamic and thalamic nuclei, hippocampus and cerebral cortex in VG and C newborns that were not apparent in NC newborns. Our results indicate that labor contractions play a role in the induction of widespread neural activation in the newborn brain.

Ronca, A. E.↗

NASA NTRS2002

Non-Invasive Health Diagnostics using Eye as a 'Window to the Body'

As a 'window to the body', the eye offers the opportunity to use light in various forms to detect ocular and systemic abnormalities long before clinical symptoms appear and help develop preventative/therapeutic countermeasures early. The effects of space travel on human body are similar to those of normal aging. For example, radiation exposure in space could lead to formation of cataracts and cancer by damaging the DNA and causing gene mutation. Additionally, the zero-gravity environment causes fluid shifts in the upper extremities of the body and changes the way blood flows and organ system performs. Here on Earth, cataract, age-related macular degeneration (AMD), diabetic retinopathy (DR), and glaucoma are major eye diseases and are expected to double in next two decades. To detect, prevent, and treat untoward effects of prolonged space travel in real-time requires the development of non-invasive diagnostic technologies that are compact and powerful. We are developing fiber-optic sensors to evaluate the ocular tissues in health, aging, and disease employing the techniques of dynamic light scattering (cataract, uveitis, Alzheimer's, glaucoma, DR, radiation damage, refractive surgery outcomes), auto-fluorescence (aging, DR), laser-Doppler flowmetry (choroidal blood flow), Raman spectroscopy (AMD), polarimetry (diabetes), and retinal oximetry (occult blood loss). The non-invasive feature of these technologies integrated in a head-mounted/goggles-like device permits frequent repetition of tests, enabling evaluation of the results to therapy that may ultimately be useful in various telemedicine applications on Earth and in space.

Ansari, Rafat R.↗

NASA NTRS2002

The Hematopoietic Stem Cell Therapy for Exploration of Space

Astronauts experience severe/invasive disorders caused by space environments. These include hematological/cardiac abnormalities, bone and muscle losses, immunodeficiency, neurological disorders and cancer. While the cause of these symptoms are not yet fully delineated, one possible explanation could be the inhibition of hematopoietic stem cell (HSC) growth and hematopoiesis in space. HSCs differentiate into all types of blood cells, and growing evidence indicates that the HSCs also have the ability to transdifferentiate to various tissues, including muscle, skin, liver, neuronal cells and possibly bone. Therefore, a hypothesis was advanced in this laboratory that the hematopoietic stem cell-based therapy, herein called the hematopoietic stem cell therapy (HSCT), could mitigate some of the disorders described above. Due to the magnitude of this project our laboratory has subdivided it into 3 sections: a) HSCT for space anemia; b) HSCT for muscle and bone losses; and c) HSCT for immunodeficiency. Toward developing the HSCT protocol for space anemia, the HSC transplantation procedure was established using a mouse model of beta thalassemia. In addition, the NASA Rotating Wall Vessel (RWV) culture system was used to grow HSCs in space condition. To investigate the HSCT for muscle loss and bone loss, donor HSCs were genetically marked either by transfecting the beta-galactosidase-containing plasmid, pCMV.SPORT-beta-gal or by preparing from b-galactosidase transgenic mice. The transdifferentiation of HSCs to muscle is traced by the reporter gene expression in the hindlimb suspended mice with some positive outcome, as studied by the X-gal staining procedure. The possible structural contribution of HSCs against muscle loss is being investigated histochemically.

Roach, Allana Nicole↗

NASA NTRS2003

Two Strategies for Microbial Production of an Industrial Enzyme-Alpha-Amylase

Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments including hot springs, soda lakes and arctic water. This ability of survival at extreme conditions has rendered extremophiles to be of interest in astrobiology, evolutionary biology as well as in industrial applications. Of particular interest to the biotechnology industry are the biological catalysts of the extremophiles, the extremozymes, whose unique stabilities at extreme conditions make them potential sources of novel enzymes in industrial applications. There are two major approaches to microbial enzyme production. This entails enzyme isolation directly from the natural host or creating a recombinant expression system whereby the targeted enzyme can be overexpressed in a mesophilic host. We are employing both methods in the effort to produce alpha-amylases from a hyperthermophilic archaeon (Thermococcus) isolated from a hydrothermal vent in the Atlantic Ocean, as well as from alkaliphilic bacteria (Bacillus) isolated from a soda lake in Tanzania. Alpha-amylases catalyze the hydrolysis of internal alpha-1,4-glycosidic linkages in starch to produce smaller sugars. Thermostable alpha-amylases are used in the liquefaction of starch for production of fructose and glucose syrups, whereas alpha-amylases stable at high pH have potential as detergent additives. The alpha-amylase encoding gene from Thermococcus was PCR amplified using carefully designed primers and analyzed using bioinformatics tools such as BLAST and Multiple Sequence Alignment for cloning and expression in E.coli. Four strains of Bacillus were grown in alkaline starch-enriched medium of which the culture supernatant was used as enzyme source. Amylolytic activity was detected using the starch-iodine method.

Bernhardsdotter, Eva C. M. J.↗

NASA NTRS2003

Growth and Metabolism of the Green Alga, Chlorella Pyrenoidosa, in Simulated Microgravity

The effect of microgravity on living organisms during space flight has been a topic of interest for some time, and a substantial body of knowledge on the subject has accumulated. Despite this, comparatively little information is available regarding the influence of microgravity on algae, even though it has been suggested for long duration flight or occupancy in space that plant growth systems, including both higher plants and algae, are likely to be necessary for bioregenerative life support systems. High-Aspect-Ratio Rotating-Wall Vessel or HARV bioreactors developed at Johnson Space Center provide a laboratory-based approach to investigating the effects of microgravity on cellular reactions. In this study, the HARV bioreactor was used to examine the influence of simulated microgravity on the growth and metabolism of the green alga, Chlorella pyrenoidosa. After the first 2 days of culture, cell numbers increased more slowly in simulated microgravity than in the HARV gravity control; after 7 days, growth in simulated microgravity was just over half (58%) that of the gravity control and at 14 days it was less than half (42%). Chlorophyll and protein were also followed as indices of cell competence and function; as with growth, after 2-3 days, protein and chlorophyll levels were reduced in modeled microgravity compared to gravity controls. Photosynthesis is a sensitive biochemical index of the fitness of photosynthetic organisms; thus, CO2-dependent O2 evolution was tested as a measure of photosynthetic capacity of cells grown in simulated microgravity. When data were expressed with respect to cell number, modeled microgravity appeared to have little effect on CO2 fixation. Thus, even though the overall growth rate was lower for cells cultured in microgravity, the photosynthetic capacity of the cells appears to be unaffected. Cells grown in simulated microgravity formed loose clumps or aggregates within about 2 days of culture, with aggregation increasing over time. Presently, the basis for, or significance of, the cell aggregation is unknown. The results from this study suggest that cell growth and morphological characteristics of green algae may be altered by culture in simulated microgravity. The data obtained to date should provide a solid basis for additional experimentation regarding the influence of modeled microgravity on cell morphology, physiological activity, protein production and possibly gene expression in algal and plant cell systems. The final aim of the study is to provide useful information to elucidate the underlying mechanism for the biological effects of microgravity on cells.

Mills, W. Ronald↗

NASA NTRS2003

Thermococcus Thioreducens sp. nov., A Novel Hyperthermophilic, Obligately Sulfur-Reducing Archaeon from a Deep-Sea Hydrothermal Vent

A novel hyperthermophilic organo-heterotrophic archaeon, strain OGL-20P(sup T), was isolated from 'black smoker' chimney material from the Rainbow hydrothermal vent site on the Mid-Atlantic Ridge (36.2 N; 33.9 W). The cells of strain OGL-20P(sup T) have an irregular coccoid shape and are motile with a single flagellum. Growth was observed to occur within the pH range 5.0-8.5 (optimum pH 7.0), NaCl concentration range 1-5 % (w/v) (optimum 3 %), and temperature range 55-94 C (optimum 83-85 C). Novel isolate is strictly anaerobic and obligately dependent from elemental sulfur as electron acceptor, but it cannot reduce sulfate, sulfite, thiosulfate, iron (III) or nitrate. Proteolysis products that can be utilized as substrates during sulfur-reduction are: peptone, bactotryptone, casamino-acids, and yeast extract. Strain OGL-20P(sup T) is resistant to ampicillin, chloramphenicol, kanamycin, and gentamycin, but sensitive to tetracycline and rifampicin. The G+C content of DNA is 57.1 mol% . Comparative 16S rRNA gene sequence analysis revealed that strain OGL-20P(sup T) is most closely related to Thermococcus celer and 'T. barossii', but no significant homology by DNA-DNA hybridization was observed between those species and the new isolate. On the basis of physiological and molecular properties of the new isolate, the name Thermococcus thioreducens sp. nov., is proposed. The type strain is OGL-20P(sup T) (= ATCC BAA-394(sup T) = DSM 1498(sup T)).

Pikuta, Elena V.↗

NASA NTRS2003

Gelidivirgula Patagoniensis Gen. Nov., Sp. Nov., A Novel Psychrotolerant, Sporeforming Anaerobe Isolated from Magellanic Penguin Guano in Patagonia, Chile

A novel obligately anaerobic, psychrotrophic bacterium, strain PPP2(sup T), was isolated from guano of the Magellanic penguin (Spheniscus magellanicus) in Patagonia, Chile. The Gram-positive, sporeforming, straight rods with sizes 0.6-0.9 x 3.0-5.0 microns, are motile by peritrichous flagella. Growth was observed to occur within the pH range 6.0-9.5 (optimum pH x), and temperature range 2-28 C (optimum 20 C). The novel isolate does not require NaCl for growth, but is halotolerant and growth was observed between 0 and 7 % NaCl (w/v) with optimum at 0.5 % (w/v). The new isolate is a catalase negative chemoorganohetherotroph with fermentative metabolism and uses as substrates: peptone, Bacto-tryptone, Casamino acids, and yeast extract. The major metabolic products are: acetate, butyrate, ethanol, and hydrogen is a minor gas product.. Strain PPP2 was sensitive to ampicillin, tetracycline, chloramphenicol, rifampin, kanamycin, and gentamycin. The G+C content of the DNA is 43.6 mol%. On the basis of 16S rDNA gene sequences and phenotypic characteristics, it is proposed that the strain PPP2(sup T) (= ATCC BAA-755(sup T) = JSM ...(sup T)) is assigned to the new genus Gelidivirgula gen. nov., as a representative of the new species, Gelidivirgula patagonensis sp. nov.

Pikuta, Elena V.↗

NASA NTRS2003

Cellular Bases of Light-regulated Gravity Responses

This report summarizes the most significant research accomplished in our NAG2-1347 project on the cellular bases of light-regulated gravity responses, It elaborates mainly on our discovery of the role of calcium currents in gravity-directed polar development in single germinating spore cells of the fern Ceratopteris, our development of RNA silencing as a viable method of suppressing the expression of specific genes in Ceratopteris, and on the structure, expression and distribution of members of the annexin family in flowering plants, especially Arabidopsis.

Roux, Stanley J.↗

NASA NTRS2003

The Application of Advanced Cultivation Techniques in the Long Term Maintenance of Space Flight Plant Biological Systems

The development of the International Space Station (ISS) presents extensive opportunities for the implementation of long duration space life sciences studies. Continued attention has been placed in the development of plant growth chamber facilities capable of supporting the cultivation of plants in space flight microgravity conditions. The success of these facilities is largely dependent on their capacity to support the various growth requirements of test plant species. The cultivation requirements for higher plant species are generally complex, requiring specific levels of illumination, temperature, humidity, water, nutrients, and gas composition in order to achieve normal physiological growth and development. The supply of water, nutrients, and oxygen to the plant root system is a factor, which has proven to be particularly challenging in a microgravity space flight environment. The resolution of this issue is particularly important for the more intensive crop cultivation of plants envisaged in Nasa's advanced life support initiative. BioServe Space Technologies is a NASA, Research Partnership Center (RPC) at the University of Colorado, Boulder. BioServe has designed and operated various space flight plant habitat systems, and placed specific emphasis on the development and enhanced performance of subsystem components such as water and nutrient delivery, illumination, gas exchange and atmosphere control, temperature and humidity control. The further development and application of these subsystems to next generation habitats is of significant benefit and contribution towards the development of both the Space Plant biology and the Advanced Life Support Programs. The cooperative agreement between NASA Ames Research center and BioServe was established to support the further implementation of advanced cultivation techniques and protocols to plant habitat systems being coordinated at NASA Ames Research Center. Emphasis was placed on the implementation of passive-based water and nutrient support systems and techniques, which can be used to minimize demands on power, mass, and operational complexity in space flight studies. This effort has direct application to the development of next-generation space flight plant chambers such as the Plant Research Unit (PRU). Work was also directed at the development of in-flight plant preservation techniques and protocols consistent with the interest in applying recent developments in gene chip micro array technologies. Cultivation technologies and protocols were evaluated in a 55 day space flight plant growth study, conducted on the ISS, mission 9A (10/7/02 - 12/7/02).

Heyenga, A. G.↗

NASA NTRS2003

A Biodosimeter for Multiparametric Determination of Radiation Dose, Radiation Quality, and Radiation Risk

Predicting risk of human cancer following exposure of an individual or a population to ionizing radiation is challenging. To an approximation, this is because uncertainties of uniform absorption of dose and the uniform processing of dose-related damage at the cellular level within a complex set of biological variables degrade the confidence of predicting the delayed expression of cancer as a relatively rare event. Cellular biodosimeters that simultaneously report: 1) the quantity of absorbed dose after exposure to ionizing radiation, 2) the quality of radiation delivering that dose, and 3) the risk of developing cancer by the cells absorbing that dose would therefore be useful. An approach to such a multiparametric biodosimeter will be reported. This is the demonstration of a dose responsive field effect of enhanced expression of keratin 18 (K18) in cultures of human mammary epithelial cells irradiated with cesium-1 37 gamma-rays. Dose response of enhanced K18 expression was experimentally extended over a range of 30 to 90 cGy for cells evaluated at mid-log phase. K18 has been reported to be a marker for tumor staging and for apoptosis, and thereby serves as an example of a potential marker for cancer risk, where the reality of such predictive value would require additional experimental development. Since observed radiogenic increase in expression of K18 is a field effect, ie., chronically present in all cells of the irradiated population, it may be hypothesized that K18 expression in specific cells absorbing particulate irradiation, such as the high-LET-producing atomic nuclei of space radiation, will report on both the single-cell distributions of those particles amongst cells within the exposed population, and that the relatively high dose per cell delivered by densely ionizing tracks of those intersecting particles will lead to cell-specific high-expression levels of K18, thereby providing analytical end points that may be used to resolve both the quantity and the quality of the radiation dose absorbed by individual cells. The principal value of this reported potential multiparametric cellular biodosimeter is suggested to be that it justifies a search for similar but more robust radiogenic assays. That is, K18 is only one radiation dose-sensitive expressed protein, whereas analytical techniques of genomics and proteomics can be used to simultaneously analyze multiple gene and protein expressions resulting from radiation-dose absorption. The potential usefulness of multiparametric cellular biodosimeters will be best realized from quantitatively profiling these multiple markers using these modern techniques.

Richmond, Robert↗

NASA NTRS2003

Identification and Characterization of Extremophile Microorganisms with Significance to Astrobiology

It is now well recognized that microorganisms thrive in extreme ecological conditions such as geothermal vents, polar region, acid and alkaline lakes, and the cold pressurized depth of the ocean floor of this planet. Morphological, physiological, biochemical and genetic adaptations to extreme environments by these extremophile microorganisms have generated immense interest amongst astrobiologists who increasingly believe in the existence of extraterrestrial life. The evidence collected by NASA's space probe Galileo suggested the presence of liquid water and volcanic activity on Mars and Jupiter's satellite Europa. Volcanic activity provides some of the heat necessary to keep the water on Europa from freezing that could provide important dissolved chemicals needed by living organisms. The possibility of the existence of hypersaline alkaline lakes and evaporites confined within closed volcanic basins and impact craters on Mars, and a layer of liquid water under the ice on Europa provide sufficient 'raison d'etre' to study microorganisms in similar extreme environments on Earth, which could provide us with a model that would help establish the existence of extraterrestrial life on other planetary bodies. The objectives of the summer research project were as follows: (1) application of molecular approaches to help establish new species of extremophile microorganisms isolated from a hypersaline alkaline lake; and (2) identification of a major cold-shock gene (cspA) homolog from a psychrotolerant microorganism, PmagG1.

Bej, Asim K.↗

NASA NTRS2003

Evolutionary Origin of Body Axis Segmentation in Annelids and Arthropods

During the period of this report, we have made a number of important discoveries. To date this work has led to 4 peer-reviewed publications in primary research journals plus 1 minireview and 1 chapter in the proceedings of a meeting. Publications resulting from this grant support are enumerated at the end of the report. Two additional, on-going studies also described. 1. Using laser cell ablation, we have obtained evidence that an annelid - the leech Helobdella robusta - patterns the anteroposterior (AP) polarity of its nascent segment primordia independent of cell interactions oriented along the AP axis. 2. We cloned a Helobdella homologue (hro-hh) of the Drosophila segment polarity gene hedgehog, and used in situ hybridization and northern blots to characterize its expression in the embryo. 3. We have used laser cell ablations to examine the possible role of cell interactions during the developmental patterning of the 4 rostralmost "head" segments of the leech Helobdella robusta.

Shankland, S. Martin↗