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NASA NTRS1990

The mGA1.0: A common LISP implementation of a messy genetic algorithm

Genetic algorithms (GAs) are finding increased application in difficult search, optimization, and machine learning problems in science and engineering. Increasing demands are being placed on algorithm performance, and the remaining challenges of genetic algorithm theory and practice are becoming increasingly unavoidable. Perhaps the most difficult of these challenges is the so-called linkage problem. Messy GAs were created to overcome the linkage problem of simple genetic algorithms by combining variable-length strings, gene expression, messy operators, and a nonhomogeneous phasing of evolutionary processing. Results on a number of difficult deceptive test functions are encouraging with the mGA always finding global optima in a polynomial number of function evaluations. Theoretical and empirical studies are continuing, and a first version of a messy GA is ready for testing by others. A Common LISP implementation called mGA1.0 is documented and related to the basic principles and operators developed by Goldberg et. al. (1989, 1990). Although the code was prepared with care, it is not a general-purpose code, only a research version. Important data structures and global variations are described. Thereafter brief function descriptions are given, and sample input data are presented together with sample program output. A source listing with comments is also included.

Goldberg, David E.↗
NASA NTRS1991

Three Hierarchies in Skeletal Muscle Fibre Classification Allotype, Isotype and Phenotype

Immunocytochemical analyses using specific anti-myosin antibodies of mammalian muscle fibers during regeneration, development, and after denervation have revealed two distinct myogenic components determining fiber phenotype. The jaw-closing muscles of the cat contain superfast fibers which express a unique myosin not found in limb muscles. When superfast muscle is transplanted into a limb muscle bed, regenerating myotubes synthesize superfast myosin independent of innervation. Reinnervation by the nerve to a fast muscle leads to the expression of superfast and not fast myosin, while reinnervation by the nerve to a slow muscle leads to the expression of a slow myosin. When limb muscle is transplanted into the jaw muscle bed, only limb myosins are synthesized. Thus jaw and limb muscles belong to distinct allotypes, each with a unique range of phenotype options, the expressions of which may be modulated by the nerve. Primary and secondary myotubes in developing jaw and limb muscles are observed to belong to different categories characterized by different patterns of myosin gene expression. By taking into consideration the pattern of myosins synthesized and the changes in fiber size after denervation, 3 types of primary (fast, slow, and intermediate) fibers can be distinguished in rat fast limb muscles. All primaries synthesize slow myosin soon after their formation, but this is withdrawn in fast and intermediate primaries at different times. After neonatal denervation, slow and intermediate primaries express slow primaries hypertrophy with other fibers atrophy. In the mature rat, the number of slow fibers in the EDL is less than the number of slow primaries. Upon denervation, hypertrophic slow fibers matching the number and topographic distribution of slow primaries appear, suggesting that a subpopulation of the slow primaries acquire the fast phenotype during adult life, but reveal their original identity as slow primaries in response to denervation by hypertrophying and synthesizing slow myosin. It is proposed that within each muscle allotype, the various isotypes of primary and secondary fibers are myogenically determined, and are derived from different lineage of myoblasts.

Hoh, Joseph F. Y.↗
NASA NTRS1990

A study of murine bone marrow cells cultured in bioreactors which create an environment which simulated microgravity

Previous research indicated that mouse bone marrow cells could be grown in conditions of simulated microgravity. This environment was created in rotating bioreactor vessels. On three attempts mouse cells were grown successfully in the vessels. The cells reached a stage where the concentrations were doubling daily. Phenotypic analysis using a panel of monoclonal antibodies indicated that the cell were hematopoietic pluripotent stem cells. One unsuccessful attempt was made to reestablish the immune system in immunocompromised mice using these cells. Since last summer, several unsuccessful attempts were made to duplicate these results. It was determined by electron microscopy that the cells successfully grown in 1989 contained virus particles. It was suggested that these virally parasitized cells had been immortalized. The work of this summer is a continuation of efforts to grow mouse bone marrow in these vessels. A number of variations of the protocol were introduced. Certified pathogen free mice were used in the repeat experiments. In some attempts the medium of last summer was used; in others Dexture Culture Medium containing Iscove's Medium supplemented with 20 percent horse serum and 10-6 M hydrocortisone. Efforts this summer were directed solely to repeating the work of last summer. Plans were made for investigations if stem cells were isolated. Immortalization of the undifferentiated stem cell would be attempted by transfection with an oncogenic vector. Selective differentiation would be induced in the stem cell line by growing it with known growth factors and immune response modulators. Interest is in identifying any surface antigens unique to stem cells that would help in their characterization. Another goal was to search for markers on stem cells that would distinguish them from stem cells committed to a particular lineage. If the undifferentiated hematopoietic stem cell was obtained, the pathways that would terminally convert it to myeloid, lyphoid, erythroid, or other cell lines would be studied. Transfection with a known gene would be attempted and then conversion to a terminally identifiable cell.

Lawless, Brother Desales↗
NASA NTRS1991

Exploration of RNA structure spaces

In order to understand the structure of real structure spaces, we are studying the 5S rRNA structure space experimentally. A plasmid containing a synthetic 5S rRNA gene, two rRNA promoters, and transcription terminators has been assembled. Assays are conducted to determine if the foreign 5S rRNA is expressed, and to see whether or not it is incorporated into ribosomes. Evolutionary competition is used to determine the relative fitness of strains containing the foreign 5S rRNA and a control 5S rRNA. By using site directed mutagenesis, a number of mutants can be made in order to study the boundaries of the structure space and how sharply defined they are. By making similar studies in the vicinity of structure space, it will be possible to determine how homogeneous the 5S rRNA structure space is. Useable experimental protocols have been developed, and a number of mutants have already been studied. Initial results suggest an explanation of why single stranded regions of the RNA are less subject to mutation than double stranded regions.

Fox, G. E.↗
NASA NTRS1991

Functional characteristics of the calcium modulated proteins seen from an evolutionary perspective

We have constructed dendrograms relating 173 EF-hand proteins of known amino acid sequence. We aligned all of these proteins by their EF-hand domains, omitting interdomain regions. Initial dendrograms were computed by minimum mutation distance methods. Using these as starting points, we determined the best dendrogram by the method of maximum parsimony, scored by minimum mutation distance. We identified 14 distinct subfamilies as well as 6 unique proteins that are perhaps the sole representatives of other subfamilies. This information is given in tabular form. Within subfamilies one can easily align interdomain regions. The resulting dendrograms are very similar to those computed using domains only. Dendrograms constructed using pairs of domains show general congruence. However, there are enough exceptions to caution against an overly simple scheme in which one pair of gene duplications leads from one domain precurser to a four domain prototype from which all other forms evolved. The ability to bind calcium was lost and acquired several times during evolution. The distribution of introns does not conform to the dendrogram based on amino acid sequences. The rates of evolution appear to be much slower within subfamilies, especially within calmodulin, than those prior to the definition of subfamily.

Kretsinger, R. H.↗
NASA NTRS1991

Symbiosis and the origin of eukaryotic motility

Ongoing work to test the hypothesis of the origin of eukaryotic cell organelles by microbial symbioses is discussed. Because of the widespread acceptance of the serial endosymbiotic theory (SET) of the origin of plastids and mitochondria, the idea of the symbiotic origin of the centrioles and axonemes for spirochete bacteria motility symbiosis was tested. Intracellular microtubular systems are purported to derive from symbiotic associations between ancestral eukaryotic cells and motile bacteria. Four lines of approach to this problem are being pursued: (1) cloning the gene of a tubulin-like protein discovered in Spirocheata bajacaliforniesis; (2) seeking axoneme proteins in spirochets by antibody cross-reaction; (3) attempting to cultivate larger, free-living spirochetes; and (4) studying in detail spirochetes (e.g., Cristispira) symbiotic with marine animals. Other aspects of the investigation are presented.

Margulis, L.↗
NASA NTRS1992

The neurochemical basis of photic entrainment of the circadian pacemaker

Circadian rhythmicity in mammals is controlled by the action of a light-entrainable hypothalamus, in association with two cell clusters known as the supra chiasmatic nuclei (SCN). In the absence of temporal environmental clues, this pacemaker continues to measure time by an endogenous mechanism (clock), driving biochemical, physiological, and behavioral rhythms that reflect the natural period of the pacemaker oscillation. This endogenous period usually differs slightly from 24 hours (i.e., circadian). When mammals are maintained under a 24 hour light-dark (LD) cycle, the pacemaker becomes entrained such that the period of the pacemaker oscillation matches that of the LD cycle. Potentially entraining photic information is conveyed to the SCN via a direct retinal projection, the retinohypothalamic tract (RHT). RHT neurotransmission is thought to be mediated by the release of excitatory amino acids (EAA) in the SCN. In support of this hypothesis, recent experiments using nocturnal rodents have shown that EAA antagonists block the effects of light on pacemaker-driven behavioral rhythms, and attenuate light induced gene expression in SCN cells. An understanding of the neurochemical basis of the photic entrainment process would facilitate the development of pharmacological strategies for maintaining synchrony among shift workers in environments, such as the Space Station, which provide unreliable or conflicting temporal photic clues.

Rea, Michael A.↗
NASA NTRS1991

Use of T7 RNA polymerase to direct expression of outer Surface Protein A (OspA) from the Lyme disease Spirochete, Borrelia burgdorferi

The OspA gene from a North American strain of the Lyme disease Spirochete, Borrelia burgdorferi, was cloned under the control of transciption and translation signals from bacteriophage T7. Full-length OspA protein, a 273 amino acid (31kD) lipoprotein, is expressed poorly in Escherichia coli and is associated with the insoluble membrane fraction. In contrast, a truncated form of OspA lacking the amino-terminal signal sequence which normally would direct localization of the protein to the outer membrane is expressed at very high levels (less than or equal to 100 mg/liter) and is soluble. The truncated protein was purified to homogeneity and is being tested to see if it will be useful as an immunogen in a vaccine against Lyme disease. Circular dichroism and fluorescence spectroscopy was used to characterize the secondary structure and study conformational changes in the protein. Studies underway with other surface proteins from B burgdorferi and a related spirochete, B. hermsii, which causes relapsing fever, leads us to conclude that a strategy similar to that used to express the truncated OspA can provide a facile method for producing variations of Borrelia lipoproteins which are highly expressed in E. coli and soluble without exposure to detergents.

Dunn, John J.↗
NASA NTRS1992

Microgravitational effects on chromosome behavior (7-IML-1)

The effects of the two major space-related conditions, microgravity and radiation, on the maintenance and transmission of genetic information have been partially documented in many organisms. Specifically, microgravity acts at the chromosomal level, primarily on the structure and segregation of chromosomes, in producing major abberations such as deletions, breaks, nondisjunction, and chromosome loss, and to a lesser degree, cosmic radiation appears to affect the genic level, producing point mutations and DNA damage. To distinguish between the effects from microgravity and from radiation, it is necessary to monitor both mitotic and meiotic genetic damage in the same organism. The yeast Saccharomyces cerevisiae is used to monitor at high resolution the frequency of chromosome loss, nondisjunction, intergenic recombination, and gene mutation in mitotic and meiotic cells, to a degree impossible in other organisms. Because the yeast chromosomes are small, sensitive measurements can be made that can be extrapolated to higher organisms and man. The objectives of the research are: (1) to quantitate the effects of microgravity and its synergism with cosmic radiation on chromosomal integrity and transmission during mitosis and meiosis; (2) to discriminate between chromosomal processes sensitive to microgravity and/or radiation during mitosis and meiosis; and (3) to relate these findings to anomalous mitotic mating type switching and ascosporogenesis following meiosis.

Bruschi, Carlo↗
NASA NTRS1992

Advanced Manufacturing and Value-added Products from US Agriculture

An objective of the US Department of Agriculture (USDA) Agriculture Research Service (ARS) is to develop technology leading to a broad portfolio of value-added marketable products. Modern scientific disciplines such as chemical engineering are brought into play to develop processes for converting bulk commodities into high-margin products. To accomplish this, the extremely sophisticated processing devices which form the basis of modern biotechnology, namely, genes and enzymes, can be tailored to perform the required functions. The USDA/ARS is a leader in the development of intelligent processing equipment (IPE) for agriculture in the broadest sense. Applications of IPE are found in the production, processing, grading, and marketing aspects of agriculture. Various biotechnology applications of IPE are discussed.

Villet, Ruxton H.↗
NASA NTRS1990

Rapid increase of inositol 1,4,5-trisphosphate in the HeLa cells after hypergravity exposure

The IP3 level in HeLa cells has been elevated through the application in hypergravity in a time-dependent manner. The data obtained for the hydrolytic products of PIP2, IP3, and DG are noted to modulate c-myc gene expression. It is also established that the cAMP accumulation by the IBMX in hypergravity-exposed cells was suppressed relative to the control. In light of IP3 increase and cAMP decrease results, a single GTP-binding protein may play a role in the hypergravity signal transduction of HeLa cells by stimulating PLC while inhibiting adenylate cyclase.

Kumei, Yasuhiro↗
NASA NTRS1992

Microbiological concerns and methodological approaches related to bacterial water quality in spaceflight

A number of microbiological issues are of critical importance to crew health and system performance in spacecraft water systems. This presentation reviews an army of these concerns which include factors that influence water treatment and disinfection in spaceflight such as biofilm formation and the physiological responses of bacteria in clean water systems. Factors associated with spaceflight like aerosol formation under conditions of microgravity are also discussed within the context of airborne infections such as Legionellosis. Finally, a spectrum of analytical approaches is reviewed to provide an evaluation of methodological alternatives that have been suggested or used to detect microorganisms of interest in water systems. These range from classical approaches employing colony formation on specific microbiological growth media to direct (i.e. microscopic) and indirect (e.g. electrochemical) methods as well as the use of molecular approaches and gene probes. These techniques are critically evaluated for their potential utility in determining microbiological water quality through the detection of microorganisms under the influence of ambient environmental stress inherent in spaceflight water systems.

Pyle, Barry H.↗
NASA NTRS1993

Toxicity study of dimethylethoxysilane (DMSES), the waterproofing agent for the Orbiter heat protective system

DMES, a volatile liquid, is used by NASA to waterproof the Orbiter thermal protective system. During waterproofing operations at the Oribter Processing Facility at KSC, workers could be exposed to DMES vapor. To assess the toxicity of DMES, acute and subchronic (2-week and 13-week) inhalation studies were conducted with rats. Studies were also conducted to assess the potential of DMES. Inhalation exposure concentrations ranged from 40 ppm to 4000 ppm. No mortality was observed during the studies. Exposures to 2100 ppm produced narcosis and ataxia. Post-exposure recovery from these CNS effects was rapid (less than 1 hr). These effects were concentration-dependent and relatively independent of exposure length. Exposure to 3000 ppm for 2 weeks (5 h/d, 5 d/wk) produced testicular toxicity. The 13-week study yielded similar results. Results from the genotoxicity assays (in vivo/in vitro unscheduled DNA synthesis in rat primary heptaocytes, chromosomal aberrations in rat bone marrow cells; reverse gene mutation in Salmonella typhimurium; and forward mutation in Chinese hamster culture cells) were negative. These studies indicated that DMES is mildly to moderately toxic but not a multagen.

Lam, Chiu-Wing↗
NASA NTRS1993

Microgravity research in plant biological systems: Realizing the potential of molecular biology

The sole all-pervasive feature of the environment that has helped shape, through evolution, all life on Earth is gravity. The near weightlessness of the Space Station Freedom space environment allows gravitational effects to be essentially uncoupled, thus providing an unprecedented opportunity to manipulate, systematically dissect, study, and exploit the role of gravity in the growth and development of all life forms. New and exciting opportunities are now available to utilize molecular biological and biochemical approaches to study the effects of microgravity on living organisms. By careful experimentation, we can determine how gravity perception occurs, how the resulting signals are produced and transduced, and how or if tissue-specific differences in gene expression occur. Microgravity research can provide unique new approaches to further our basic understanding of development and metabolic processes of cells and organisms, and to further the application of this new knowledge for the betterment of humankind.

Lewis, Norman G.↗
NASA NTRS1994

Charged-particle mutagenesis 2. Mutagenic effects of high energy charged particles in normal human fibroblasts

The biological effects of high Linear Energy Transfer (LET) charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 sq micrometer and 0.09 to 5.56 x 10(exp -3) sq micrometer respectively. The maximum values were obtained by Fe-56 with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(exp -5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

Chen, D. J.↗
NASA NTRS1994

Kinetics model for initiation and promotion for describing tumor prevalence from HZE radiation

A kinetics model for cellular repair and misrepair for multiple radiation-induced lesions (mutation-inactivation) is coupled to a two-mutation model of initiation and promotion in tissue to provide a parametric description of tumor prevalence in the Harderian gland in a mouse. Dose-response curves are described for gamma-rays and relativistic ions. The effects of nuclear fragmentation are also considered for high-energy proton and alpha particle exposures The model described provides a parametric description of age-dependent cancer induction for a wide range of radiation fields. We also consider the two hypotheses that radiation acts either solely as an initiator or as both initiator and promoter and make model calculations for fractionation exposures from gamma-rays and relativistic Fe ions. For fractionated Fe exposures, an inverse dose-rate effect is provided by a promotion hypothesis using a mutation rate for promotion typical of single-gene mutations.

Cucinotta, Francis A.↗
NASA NTRS1993

The 1993 Gordon Research Conference on Chronobiology

The study of biological timekeeping is now at a particularly fertile stage, encompassing multiple levels of biological organization, recruiting a wide array of disciplines and methodologies and uniting a host of investigators. This report summarizes a research conference on Chronobiology. Some of the topics focused on transcriptional and translational mechanisms of circadian rhythmicity, with discussions of putative 'clock genes' in cyanobacteria, algae, fungi, fruitflies, and hamsters. Cellular analysis, with emphasis on photoreceptors in frogs, neurons in molluscs, and testis in moths was addressed. New methods for investigating the circadian clock in the suprachiasmatic nucleus were introduced.

Schwartz, William J.↗
NASA NTRS1994

Heavy ion induced mutations in mammalian cells: Cross sections and molecular analysis

Our investigations of heavy ion-induced mutations in mammalian cells, which had been begun a few years ago, were systematically continued. For the first time, it was possible to cover a large LET range with a few kinds of ions. To do this, both UNILAC and SIS were used to yield comparable data for a large energy range. This is a necessary condition for a comprehensive description of the influence of such ion parameters as energy and LET. In these experiments, the induced resistance against the poison 6-thioguanin (6-TG), which is linked to the HPRT locus on the genome, is being used as mutation system. In addition to the mutation-induction cross-section measurements, the molecular changes of the DNA are being investigated by means of Multiplex PCR ('Polymerase Chain Reaction') gene amplification. From these experiments we expect further elucidation of the mutation-inducing mechanisms composing the biological action of heavy-ion radiation.

Stoll, U.↗