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NASA NTRS1999

The Role of Calcium in the Response of Osteoblasts to Mechanical Stimulation

A major biomedical concern in the exploration and development of space is the rapid loss of bone associated with extended periods of spaceflight. Mineral content, bone formation, matrix protein production and total body calcium are all reduced during long-term periods of weightlessness. These effects of weightlessness appears to be due to decreases in the anabolic function of osteoblasts and osteocytes rather than changes in the resorptive activity of osteoclasts. Conversely, subjecting the skeleton to exogenous mechanical loading increases matrix protein synthesis and bone formation rate, a process which also appears mediated through osteogenic cells. Osteoblasts have been shown to respond to a number of types of mechanical stimulation. However recently we have demonstrated that osteoblasts respond to fluid shear, but not physiologic levels of mechanical strain, with increases in expression of the matrix protein, osteopontin. We have also shown similar responses in other markers for the anabolic response in bone. The expression of the early response gene, c-fos, and the inducible-isoform of the prostaglandin synthetic enzyme, cyclooygenase-2 (COX-2), both increase rapidly in response to fluid shear, but not strain. How osteoblasts and osteocytes perceive mechanical stimuli and convert this stimulus into a biochemical event within the cell is still unknown. However, examination of the cellular events following mechanical stimulation indicate that two of the earliest responses are a rapid increase in intracellular calcium ([Ca(2+)](sub i)) and a reorganization of the actin cytoskeleton. The increase in [Ca(2+)](sub i) is dependent on the presence of extracellular Ca(2+), suggesting the activation of membrane Ca(2+) channel. We have previously characterized a mechanosensitive, cation-selective channel (MSCC) in osteoblast-like clonal cells, which we postulate is important in this early response to mechanical loading. Using an antisense oligodeoxynucleotide strategy, we have tentatively identified this channel as an isoform of the alc subunit of the dihydropyridine-sensitive, voltage sensitive Ca(2+) channel (VSCC). However, a major component in this mechanically induced rise in [Ca(2+)](sub i) is the release of Ca(2+) from intracellular stores. The actin cytoskeleton also rapidly responds to fluid shear with an increase in stress fiber formation and a realignment of the cell parallel to the direction of flow. To ascertain whether these two observations are related and how they effect shear-induced gene expression, we examined the role of Ca(2+) channels and intracellular Ca(2+) release on cytoskeletal reorganization and the resultant increases in the expression and production of c-fos and COX-2 in response to fluid shear.

Duncan, R. L.↗

NASA NTRS1999

GH/IGF-I Transgene Expression on Muscle Homeostasis

We propose to test the hypothesis that the growth hormone/ insulin like growth factor-I axis through autocrine/paracrine mechanisms may provide long term muscle homeostasis under conditions of prolonged weightlessness. As a key alternative to hormone replacement therapy, ectopic production of hGH, growth hormone releasing hormone (GHRH), and IGF-I will be studied for its potential on muscle mass impact in transgenic mice under simulated microgravity. Expression of either hGH or IGF-I would provide a chronic source of a growth-promoting protein whose biosynthesis or secretion is shut down in space. Muscle expression of the IGF-I transgene has demonstrated about a 20% increase in hind limb muscle mass over control nontransgenic litter mates. These recent experiments, also establish the utility of hind-limb suspension in mice as a workable model to study atrophy in weight bearing muscles. Thus, transgenic mice will be used in hind-limb suspension models to determine the role of GH/IGF-I on maintenance of muscle mass and whether concentric exercises might act in synergy with hormone treatment. As a means to engineer and ensure long-term protein production that would be workable in humans, gene therapy technology will be used by to monitor muscle mass preservation during hind-limb suspension, after direct intramuscular injection of a genetically engineered muscle-specific vector expressing GHRH. Effects of this gene-based therapy will be assessed in both fast twitch (medial gastrocnemius) and slow twitch muscle (soleus). End-points include muscle size, ultrastructure, fiber type, and contractile function, in normal animals, hind limb suspension, and reambutation.

Schwartz, Robert J.↗

NASA NTRS1999

Radiation-Induced Cytogenetic Damage as a Predictor of Cancer Risk for Protons and Fe Ions

We have successfully completed the series of experiments planned for year 1 and the first part of year 2 measuring the induction of chromosome aberrations induced in multiple cell types by three model space radiations: Fe-ions, protons and photons. Most of these data have now been compiled and a significant part subjected to detailed data analyses, although continuing data analysis is an important part of our current and future efforts. These analyses are directed toward defining the patterns of chromosomal damage induction by the three radiations and the extent to which such patterns are dependent on the type of cell irradiated. Our studies show significant differences, both quantitatively and qualitatively, between response of different cell types to these radiations however there is an overall pattern that characterizes each type of radiation in most cell lines. Thus our data identifies general dose-response patterns for each radiation for induction of multiple types of chromosomal aberrations but also identifies significant differences in response between some cell types. Specifically, we observe significant resistance for induction of aberrations in rat mammary epithelial cells when they are irradiated in vivo and assayed in vitro. Further, we have observed some remarkable differences in susceptibility to certain radiation-induced aberrations in cells whose genome has been modulated for two cancer- relevant genes, TP53 and CDKNIA. This data, if confirmed, may represent the first evidence of gene-specific differences in cellular metabolism of damage induced by densely-ionizing radiation that confers substantial sensitivity to protons compared to photons.

Williams, Jerry R.↗

NASA NTRS1998

Simulated Microgravity Induced Cytoskeletal Rearrangements are Modulated by Protooncogenes

Microgravity is the environment living systems encounter during space flight and gravitational unloading is the effect of this environment on living systems. The cell, being a multiphasic chemical system, is a useful starting point to study the potential impact of gravity unloading on physiological function. In the absence of gravity, sedimentation of organelles including chromosomes, mitochondria, nuclei, the Golgi apparatus, vacuoles, and the endoplasmic reticulum may be affected. Most of these organelles, however, are somewhat held in place by cytoskeleton. Hansen and Igber suggest that intermediate filaments act to stabilize the nuleus against rotational movement, and integrate cell and nuclear structure. The tensegrity theory supports the idea that mechanical or physical forces alters the cytoskeletal structures of a cell resulting in the changes in cell: matrix interactions and receptor-signaling coupling. This type of stress to the cytoskeleton may be largely responsible regulating cell shape, growth, movement and metabolism. Mouse MC3T3 El cells under microgravity exhibited significant cytoskeletal changes and alterations in cell growth. The alterations in cytoskeleton architecture may be due to changes in the expression of actin related proteins or integrins. Philopott and coworkers reported on changes in the distribution of microtubule and cytoskeleton elements in the cells of heart tissue from space flight rats and those centrifuged at 1.7g. Other researchers have showed that microgravity reduced EGF-induced c-fos and c-jun expression compared to 1 g controls. Since c-fos and c-jun are known regulators of cell growth, it is likely that altered signal transduction involving protooncogenes may play a crucial role in the reduced growth and alterations in cytoskeletal arrangements found during space flight. It is clear that a microgravity environment induces a number of changes in cell shape, cell surface molecules, gene expression, and cytoskeletal reorganization. However the underlying mechanism for these cellular changes have not been clearly defined. We examined alterations in endothelial migration, and cytoskeleton architecture (microfilamentous f-actin and vimentin-rich- intermediate filaments) following wounding under simulated microgravity. We also examined the possibility that altered signal transduction pathways, involving protooncogenes, may play a crucial role in microgravity-induced retardation of cell migration and alterations in cytoskeletal organization. We hypothesize that, based on the tensegrity theory, cytoskeletal organization respond to gravitational unloading and through this response, cell behavior, function and gene expression are modified.

Melhado, C. D.↗

NASA NTRS2000

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

Hong, Y. S.↗

NASA NTRS2001

Distribution and Spectroscopy of Green Fluorescent Protein and Acyl-CoA: Cholesterol Acytransferase in Sf21 Insect Cells

Acyl-CoA: cholesterol acyltransferase (ACAT) is thought to significantly participate in the pathway of cholesterol esterification that underlies the pathology of artherosclerosis. This enzyme is a membrane protein known to be preferentially bound within the endoplasmic reticulum of mammalian cells, from which location it esterifies cholesterol derived from low density lipoprotein. Cultures of insect cells were separately infected with baculovirus containing the gene for green fluroescent protein (GFP) and with baculovirus containing tandem genes for GFP and ACAT. These infected cultures expressed GFP and the fusion protein GCAT, respectively, with maximum expression occurring on the fourth day after infection. Extraction of GFP- and of GCAT-expressing cells with urea and detergent resulted in recovery of fluorescent protein in aqueous solution. Fluorescence spectra at neutral pH were identical for both GFP and GCAT extracts in aqueous solution, indicating unperturbed tertiary structure for the GFP moiety within GCAT. In a cholesterol esterification assay, GCAT demonstrated ACAT activity, but with less efficiency compared to native ACAT. It was hypothesized that the membrane protein ACAT would lead to differences in localization of GCAT compared to GFP within the respective expressing insect cells. The GFP marker directly and also within the fusion protein GCAT was accordingly used as the intracellular probe that was fluorescently analyzed by the new biophotonics technique of hyperspectral imaging. In that technique, fluorescence imaging was obtained from two dimensional arrays of cells, and regions of interest from within those images were then retrospectively analyzed for the emission spectra that comprises the image. Results of hyperspectral imaging of insect cells on day 4 postinfection showed that GCAT was preferentially localized to the cytoplasm of these cells compared to GFP. Furthermore, the emission spectra obtained for the localized GCAT displayed a peak blue shift from 518nm obtained in neutral aqueous solution to 505nm obtained in localized regions within the cells. This blue shift indicates change in the fluorescence coupling of the GFP moiety of GCAT. It is hypothesized that change in tertiary environment of GCAT, coincident with intracellular deposition of GCAT, follows from intracellular trafficking of GCAT leading to membrane interactions with the ACAT moiety, and/or self-assembly of GCAT, that alters the chromophore environment of the GFP moiety of GCAT. These findings introduce a new technique of biophotonic imaging to studies of intracellular protein trafficking and interactions. This technique of hyperspectral imaging could contribute to advancing the emergent field of proteomics. Because of the noninvasive nature of this technique, kinetic processes associated with intracellular protein trafficking, and interactions of proteins within cellular domains, can be considered for investigation within a single cell as well as a cell population.

Richmond, R. C.↗

NASA NTRS2001

Expression of the Acyl-Coenzyme A: Cholesterol Acyltransferase GFP Fusion Protein in Sf21 Insect Cells

The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth. Protein crystals grown in microgravity are often larger and have fewer defects than those grown on earth. The analysis of higher quality space-grown crystals will assist in structure-based drug design. We have successfully grown GCAT-infected Sf21 cells in both adhesion and suspension cultures. Expression levels of GCAT in cell lines such as Sf9 and High Five appear to be reduced. We intend to replicate GCAT expression in all three cell lines using the NASA rotating wall bioreactor which effectively duplicates a microgravity environment. The bioreactor itself could be launched to study the expression of the GFP and GCAT proteins in the actual microgravity environment achieved in orbit.

Mahtani, H. K.↗

NASA NTRS2002

Choosing Between Yeast and Bacterial Expression Systems: Yield Dependent

Green fluorescent protein (GFP) is a naturally occurring fluorescent protein isolated from the jellyfish Aequorea victoria. The intrinsic fluorescence of the protein is due to a chromophore located in the center of the molecule. Its usefulness has been established as a marker for gene expression and localization of gene products. GFP has recently been utilized as a model protein for crystallization studies at NASA/MSFC, both in earth-based and in microgravity experiments. Because large quantities of purified protein were needed, the cDNA of GFP was cloned into the Pichia pastoris pPICZ(alpha) C strain, with very little protein secreted into the media. Microscopic analysis prior to harvest showed gigantic green fluorescent yeast, but upon harvesting most protein was degraded. Trial fermentations of GFP cloned into pPICZ A for intracellular expression provided unsatisfactory yield. GFP cloned into E, coli was overexpressed at greater than 150 mg/liter, with purification yields at greater than 100mg/liter.

Miller, Rebecca S.↗

NASA NTRS2002

Calcium/Calmodulin-Mediated Gravitropic Response in Plants

The goal of this project was to gain a fundamental understanding of how calcium/calmodulin-mediated signaling is involved in gravity signal transduction in plants. During the period of support, significant progress was made in elucidating the role of calmodulin and its target proteins in gravitropism. This laboratory has made breakthroughs by cloning and characterizing genes that are involved in calcium/calmodulin-mediated signaling. Some of these genes show altered expression under hypergravity and simulated microgravity conditions. A major advance was made in our attempts to understand gravity signal transduction by cloning and characterizing a catalase which requires calcium/calmodulin for its activation. Our results suggest that calcium/calmodulin have dual roles in regulating the level of hydrogen peroxide (H202), a signal molecule that plays a major role in gravitropism. It is well established that auxin plays a major role in gravitropism. Our results indicate that there is a 'cross-talk' between calcium/calmodulin-mediated signaling and auxin-mediated signal transduction. Auxin-regulated SAUR proteins that are involved in gravitropism bind to calmodulin in a calcium-dependent manner. A novel chimeric calcium/calmodulin-dependent protein kinase was cloned and characterized and its role in gravity signal transduction was investigated. These studies have provided some answers to the fundamental questions about how signal molecules such as calcium, H202, and hormones such as auxin bring about the ultimate gravitropic response and the integral role of calmodulin in gravity signal transduction. This NASA-funded study has led to some spinoffs that have applications in solving agricultural problems. The Washington State University Research Foundation has obtained several patents related to this work.

Poovaiah, B. W.↗

NASA NTRS2002

Hemichordates and the Origin of Chordates

At the start of the period of the NASA grant three years ago, we had no information on the organization and development of the body axis of the hemichordate, Saccoglossus kowalevskii. Now we have substantial findings about the anteroposterior axis and dorsoventral axis, and based on this information, we have new insights about the origin of chordates from ancestral deuterostomes. We found ways to obtain and preserve large numbers of embryos and hatched juveniles. We can now collect about 40,000 embryos in the month of September, the time of S. kowalevskii spawning at Woods Hole. Excellent cDNA libraries were prepared from three developmental stages. From these libraries, we directly isolated about 30 gene ortholog sequences by screening and pcr techniques, all of these sequences of interest in the inquiry about the animal's organization and development. We also performed a mid-sized EST project (60,000 randomly picked clones, many of these arrayed). About half of these have been analyzed so far by blastx and are suitable for direct use of clones. We have obtained about 50 interesting sequences from this set. The rest still await analysis. Thus, at this time we have isolated orthologs of 80 genes that are known to be expressed in chordates in conserved domains and known to have interesting roles in chordate organization and development. The orthology of the S. kowalevskii sequences has been verified by neighbor joining and parsimony methods, with bootstrap estimates of validity. The S. kowalevskii sequences cluster with other deuterostome sequences, namely, other hemichordates, echinoderms, ascidians, amphioxus, or vertebrates, depending on what sequences are available in the database for comparison. We have used these sequences to do high quality in situ hybridization on S. kowalevskii embryos, and the results can be divided into three sections-those concerning the anteroposterior axis of S. kowalevskii in comparison to the same axis of chordates, those concerning the dorsoventral axis of S. kowalevskii in comparison to the same axis of chordates, and those concerning the signals and transcription factors found in the endoderm, of S. kowalevskii compared to the signals and transcription factors in the endo-mesodermal cells of Spemann's organizer of chordates.

Gerhart, John↗

NASA NTRS2002

ASTROCULTURE(tm) Commercial Plant Growth Unit and Glove Box Insert

Two commercial plant investigations will be conducted during the STS-107 mission: living flower essential oil production and gene transfer. The research will be done using the ASTROCULTURE (trademark) hardware, which builds on similar experiments flown in the past on the space shuttle. This research will investigate how microgravity might affect the formation of the volatile chemical compounds - the essential oils - produced by two different types of living flowers. The flowers will be cultured in the ASTROCULTURE (trademark) plant chamber, which provides an enclosed and controlled environment. As the flowers bloom in space, they will produce essential oils, and these volatile compounds will be collected using International Flavors and Fragrance's proprietary Solid Phase Micro Extraction (SPME) technology. The gene transfer experiment examines a newly developed transformation system to see if it operates efficiently in the microgravity environment. This research is important for the development of genetically engineered crops, also known as transgenic crops.

Wei-Jia Zhou↗

NASA NTRS2003

Biotechnology System Facility: Risk Mitigation on Mir

NASA is working with its international partners to develop space vehicles and facilities that will give researchers the opportunity to conduct scientific investigations in space. As part of this activity, NASA's Biotechnology Cell Science Program (BCSP) at the Johnson Space Center (JSC) is developing a world-class biotechnology laboratory facility for the International Space Station (ISS). This report describes the BCSP, including the role of the BTS. We identify the purpose and objectives of the BTS and a detailed description of BTS facility design and operational concept, BTS facility and experiment-specific hardware, and scientific investigations conducted in the facility. We identify the objectives, methods, and results of risk mitigation investigations of the effects of microgravity and cosmic radiation on the BTS data acquisition and control system. These results may apply to many other space experiments that use commercial, terrestrial-based data acquisition technology. Another focal point is a description of the end-to-end process of integrating and operating biotechnology experiments on a variety of space vehicles. The identification of lessons learned that can be applied to future biotechnology experiments is an overall theme of the report. We include a brief summary of the science results, but this is not the focus of the report. The report provides some discussion on the successful 130-day tissue engineering experiment performed in BTS on Mir and describes a seminal gene array investigation that identified a set of unique genes that are activated in space.

Gonda, Steve R., III↗

NASA NTRS2003

Evaluation of Genetic Algorithm Concepts using Model Problems: Single-Objective Optimization - Part 1

A genetic-algorithm-based optimization approach is described and evaluated using a simple hill-climbing model problem. The model problem utilized herein allows for the broad specification of a large number of search spaces including spaces with an arbitrary number of genes or decision variables and an arbitrary number hills or modes. In the present study, only single objective problems are considered. Results indicate that the genetic algorithm optimization approach is flexible in application and extremely reliable, providing optimal results for all problems attempted. The most difficult problems - those with large hyper-volumes and multi-mode search spaces containing a large number of genes - require a large number of function evaluations for GA convergence, but they always converge.

Holst, Terry L.↗

NASA NTRS2003

Physiological and Molecular Genetic Effects of Time-Varying Electromagnetic Fields on Human Neuronal Cells

The present investigation details the development of model systems for growing two- and three-dimensional human neural progenitor cells within a culture medium facilitated by a time-varying electromagnetic field (TVEMF). The cells and culture medium are contained within a two- or three-dimensional culture vessel, and the electromagnetic field is emitted from an electrode or coil. These studies further provide methods to promote neural tissue regeneration by means of culturing the neural cells in either configuration. Grown in two dimensions, neuronal cells extended longitudinally, forming tissue strands extending axially along and within electrodes comprising electrically conductive channels or guides through which a time-varying electrical current was conducted. In the three-dimensional aspect, exposure to TVEMF resulted in the development of three-dimensional aggregates, which emulated organized neural tissues. In both experimental configurations, the proliferation rate of the TVEMF cells was 2.5 to 4.0 times the rate of the non-waveform cells. Each of the experimental embodiments resulted in similar molecular genetic changes regarding the growth potential of the tissues as measured by gene chip analyses, which measured more than 10,000 human genes simultaneously.

Goodwin, Thomas J.↗

NASA NTRS2003

Physiological Maturation of Regenerating Hair Cells

The bullfrog saccule, a sensor of gravity and substrate-borne vibration, is a model system for hair cell transduction. Saccular hair cells also increase in number throughout adult life and rapidly recover after hair cell damage, making this organ an ideal system for studying hair cell development, repair, and regeneration. We have used of hair cell and supporting cell immunocytochemical markers to identify damaged hair cells and hair cell precursors in organotypic cultures of the bullfrog saccule. We then used an innovative combination of confocal, electron, and time-lapse microscopy to study the fate of damaged hair cells and the origin of new hair cells after gentamicin ototoxicity in normal and mitotically blocked saccular cultures. These studies have shown that gentamicin ototoxicity produces both lethal and sublethal hair cell damage. They have also shown that hair cell recovery in this organ takes place by both the repair of sublethally damaged hair cells and by the replacement of lost hair cells by mitotic regeneration. In parallel studies, we have used biophysical and molecular biological techniques to study the differentiation and innervation of developing, repairing, and regenerating hair cells. More specifically, we have used RT-PCR to obtain the bullfrog homologues of L-type voltage- gated calcium (L-VGCC) and large-conductance Ca(2+)-activated potassium (BK) channel genes. We have then obtained probes for these genes and, using in situ hybridization, begun to examine their expression in the bullfrog saccule and amphibian papilla. We have also used fluorescent-labeled channel toxins and channel toxin derivatives to determine the time of appearance of L-type voltage-gated calcium (L-VGCC) and Ca(2+)-activated potassium (BK) channels and to study dynamic changes in the number, distribution, and co-localization of these proteins in developing, repairing, and regenerating hair cells. Using time-lapse microscopy, we are also studying the dynamic relationship between ion channel clustering and synaptic formation in hair cells and afferent neurons. In future studies, we will determine when hair cell precursors acquire electrical tuning, and, using whole-cell patch-clamp techniques, identify and characterize their L-VGCC and BK currents. We will also use biophysical techniques to determine the number of L-VGCC and BK channels and the size and gating kinetics of their underlying L-VGCC and BK conductances, correlating these variables with the amplitude and frequency of membrane oscillations produced by intracellular current steps. We expect these studies to determine how hair cells regulate ion channel expression to achieve specific physiological responses.

Baird, Richard A.↗

NASA NTRS2004

Characterization of Two Microbial Isolates from Andean Lakes in Bolivia

We are currently investigating the biological population present in the highest and least explored perennial lakes on earth in the Bolivian and Chilean Andes, including several volcanic crater lakes of more than 6000 m elevation, in combination of microbiological and molecular biological methods. Our samples were collected in saline lakes of the Laguna Blanca Laguna Verde area in the Bolivian Altiplano and in the Licancabur volcano crater (27 deg. 47 min S/67 deg. 47 min. W) in the ongoing project studying high altitude lakes. The main goal of the project is to look for analogies with Martian paleolakes. These Bolivian lakes can be described as Andean lakes following the classification of Chong. We have attempted to isolate pure cultures and phylogenetically characterize prokaryotes that grew under laboratory conditions. Sediment samples taken from the Licancabur crater lake (LC), Laguna Verde (LV), and Laguna Blanca (LB) were analyzed and cultured using enriched liquid media under both aerobic and anaerobic conditions. All cultures were incubated at room temperature (15 to 20 C) and under light exposure. For the reported isolates, 36 hours incubation were necessary for reaching optimal optical densities to consider them viable cultures. Ten serial dilutions starting from 1% inoculum were required to obtain a suitable enriched cell culture to transfer into solid media. Cultures on solid medium were necessary to verify the formation of colonies in order to isolate pure cultures. Different solid media were prepared using several combinations of both trace minerals and carbohydrates sources in order to fit their nutrient requirements. The microorganisms formed individual colonies on solid media enriched with tryptone, yeast extract and sodium chloride. Cells morphology was studied by optical and electronic microscopy. Rodshape morphologies were observed in most cases. Total bacterial genomic DNA was isolated from 50 ml late-exponential phase culture by using the CTAB miniprep protocol. The 16S rRNA genes were amplified by PCR using both Bacteria- and Archaeauniversal primer sets: 27f and 1492r, 21f and 1492r respectively. Sequences of 16S rRNA gene were determined and initially compared with reference sequences contained in the EMBL nucleotide sequence database by using the BLAST program and were subsequently aligned with 16S rRNA reference sequences in the ARB package (http://www.mikro.biologie.tu-muenchen.de). Aligned sequences were inserted within a stable phylogenetic tree by using the ARB parsimony tool. In this work we report the morphology and phylogenetic characterization of two isolates belonged to Laguna Blanca sediments.

Demergasso, C.↗

NASA NTRS2004

Radiation Risk Assessment of the Individual Astronaut: A Complement to Radiation Interests at the NIH

Predicting human risks following exposure to space radiation is uncertain in part because of unpredictable distribution of high-LET and low-dose-derived damage amongst cells in tissues, unknown synergistic effects of microgravity upon gene- and protein-expression, and inadequately modeled processing of radiation-induced damage within cells to produce rare and late-appearing malignant cancers. Furthermore, estimation of risks of radiogenic outcome within small numbers of astronauts is not possible using classic epidemiologic study. It therefore seems useful to develop strategies of risk-assessment based upon large datasets acquired from correlated biological models useful for resolving radiogenic risk-assessment for irradiated individuals. In this regard, it is suggested that sensitive 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 biomolecular risk of malignant transformation be developed in order to resolve these NASA-specific challenges. Multiparametric cellular biodosimeters could be developed using analyses of gene-expression and protein-expression whereby large datasets of cellular response to radiation-induced damage are analyzed for markers predictive for acute response as well as cancer-risk. A new paradigm is accordingly addressed wherein genomic and proteomic datasets are registered and interrogated in order to provide statistically significant dose-dependent risk estimation in individual astronauts. This evaluation of the individual for assessment of radiogenic outcomes connects to NIH program in that such a paradigm also supports assignment of a given patient to a specific therapy, the diagnosis of response of that patient to therapy, and the prediction of risks accumulated by that patient during therapy - such as risks incurred by scatter and neutrons produced during high-energy Intensity-Modulated Radiation Therapy. Value of assessment of radiogenic outcome for individuals exposed to radiation is suggested to be common to both NASA and NIH.

Richmond, Robert C.↗

NASA NTRS2003

Cell Science and Cell Biology Research at MSFC: Summary

The common theme of these research programs is that they investigate regulation of gene expression in cells, and ultimately gene expression is controlled by the macromolecular interactions between regulatory proteins and DNA. The NASA Critical Path Roadmap identifies Muscle Alterations and Atrophy and Radiation Effects as Very Serious Risks and Severe Risks, respectively, in long term space flights. The specific problem addressed by Dr. Young's research ("Skeletal Muscle Atrophy and Muscle Cell Signaling") is that skeletal muscle loss in space cannot be prevented by vigorous exercise. Aerobic skeletal muscles (i.e., red muscles) undergo the most extensive atrophy during long-term space flight. Of the many different potential avenues for preventing muscle atrophy, Dr. Young has chosen to study the beta-adrenergic receptor (betaAR) pathway. The reason for this choice is that a family of compounds called betaAR agonists will preferentially cause an increase in muscle mass of aerobic muscles (i.e., red muscle) in animals, potentially providing a specific pharmacological solution to muscle loss in microgravity. In addition, muscle atrophy is a widespread medical problem in neuromuscular diseases, spinal cord injury, lack of exercise, aging, and any disease requiring prolonged bedridden status. Skeletal muscle cells in cell culture are utilized as a model system to study this problem. Dr. Richmond's research ("Radiation & Cancer Biology of Mammary Cells in Culture") is directed toward developing a laboratory model for use in risk assessment of cancer caused by space radiation. This research is unique because a human model will be developed utilizing human mammary cells that are highly susceptible to tumor development. This approach is preferential over using animal cells because of problems in comparing radiation-induced cancers between humans and animals.

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