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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 739 records · Page 41
All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

Site-Directed Mutagenesis of IRX9, IRX9L and IRX14 Proteins Involved in Xylan Biosynthesis: Glycosyltransferase Activity Is Not Required for IRX9 Function in Arabidopsis

Xylans constitute the main non-cellulosic polysaccharide in the secondary cell walls of plants. Several genes predicted to encode glycosyltransferases are required for the synthesis of the xylan backbone even though it is a homopolymer consisting entirely of b-1,4-linked xylose residues. The putative glycosyltransferases IRX9, IRX14, and IRX10 (or the paralogs IRX9L, IRX14L, and IRX10L) are required for xylan backbone synthesis in Arabidopsis. To investigate the function of IRX9, IRX9L, and IRX14, we identified amino acid residues known to be essential for catalytic function in homologous mammalian proteins and generated modified cDNA clones encoding proteins where these residues would be mutated. The mutated gene constructs were used to transform wild-type Arabidopsis plants and the irx9 and irx14 mutants, which are deficient in xylan synthesis. The ability of the mutated proteins to complement the mutants was investigated by measuring growth, determining cell wall composition, and microscopic analysis of stem cross-sections of the transgenic plants. The six different mutated versions of IRX9 and IRX9-L were all able to complement the irx9 mutant phenotype, indicating that residues known to be essential for glycosyltransferases function in homologous proteins are not essential for the biological function of IRX9/IRX9L. Two out of three mutated IRX14 complemented the irx14 mutant, including a mutant in the predicted catalytic amino acid. A IRX14 protein mutated in the substrate-binding DxD motif did not complement the irx14 mutant. Thus, substrate binding is important for IRX14 function but catalytic activity may not be essential for the function of the protein. The data indicate that IRX9/IRX9L have an essential structural function, most likely by interacting with the IRX10/ IRX10L proteins, but do not have an essential catalytic function. Most likely IRX14 also has primarily a structural role, but it cannot be excluded that the protein has an important enzymatic activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Identification of a classical mutant in the industrial host Aspergillus niger by systems genetics: LaeA is required for citric acid production and regulates the formation of some secondary metabolites

Rapid acidification of the culture medium by the production of organic acids and the production of acid-induced proteases are key characteristics of the filamentous fungus Aspergillus niger. The D15 mutant of A. niger is non-acidifying mutant and used often for the expression of recombinant proteins in A. niger, because of its reduced production of extracellular proteases under non-acidic conditions. In this study, the D15 mutant is characterized in detail. Strongly reduced levels of citric and oxalic acid were observed in the D15 mutant both in shake flask cultures and in controlled batch cultivations. To identify the mutation in the D15 mutant, we successfully combined high-throughput sequencing (Illumina) with bulk segregant analysis. Because of the lack of a sexual cycle for A. niger, the parasexual cycle was used to generate a pool of segregants. From the 52 single nucleotide polymorphisms (SNPs) between the parental strains, three SNPs were homozygous in the genomic DNA of pool of segregants. These three SNPs mapped to all the right arm of chromosome II, indicating that this region contains the genetic locus affecting the phenotype related to acid production. Of the three SNPs, one mutation resulted in a missense mutation in the gene encoding the A. niger homologue of the A. nidulans methyltransferase gene laeA. Complementation analysis of the original mutant with the laeA gene and targeted disruption of laeA further confirmed that LaeA is involved in citric acid production in A. niger lab (N402) and citric acid production strains (ATCC 11414). Analysis of the secondary metabolite (SM) profile of the laeA mutants indicate that LaeA is required for the production of several SMs (asperrubrol, atromentin and JBIR86), but deletion of laeA also resulted in the presence of SMs (aspernigrin A/B and BMS-192548) that were not detected in the wild-type strain. The levels of ten other SMs were not strongly affected as a result of laeA deletion indicating that only a limited number of SM gene clusters are controlled by LaeA activity.

Aspergillus niger↗
DOE OSTI2025

Electronic Interactions Between the Receptor-Binding Domain of Omicron Variants and Angiotensin-Converting Enzyme 2: A Novel Amino Acid–Amino Acid Bond Pair Concept

SARS-CoV-2 remains a severe threat to worldwide public health, particularly as the virus continues to evolve and diversify into variants of concern (VOCs). Among these VOCs, Omicron variants exhibit unique phenotypic traits, such as immune evasion, transmissibility, and severity, due to numerous spike protein mutations and the rapid subvariant evolution. These Omicron subvariants have more than 15 mutations in the receptor-binding domain (RBD), a region of the SARS-CoV-2 spike protein that is important for recognition and binding with the angiotensin-converting enzyme 2 (ACE2) human receptor. To address the impact of these high numbers of Omicron mutations on the binding process, we have developed a novel method to precisely quantify amino acid interactions via the amino acid–amino acid bond pair (AABP). We applied this concept to investigate the interface interactions of the RBD–ACE2 complex in four Omicron Variants (BA.1, BA.2, BA.5, and XBB.1.16) with its Wild Type counterpart. Based on the AABP analysis, we have identified all the sites that are affected by mutation and have provided evidence that unmutated sites are also impacted by mutation. We have calculated that the binding between RBD and ACE2 is strongest in OV BA.1, followed by OV BA.2, WT, OV BA.5, and OV XBB.1.16. We also present the partial charge values for all 311 residues across these five models. Our analysis provides a detailed understanding of changes caused by mutation in each Omicron interface complex.

Biochemistry & Molecular Biology↗
DOE OSTI2017

Deconstruction of the Ras switching cycle through saturation mutagenesis

Ras proteins are highly conserved signaling molecules that exhibit regulated, nucleotide-dependent switching between active and inactive states. The high conservation of Ras requires mechanistic explanation, especially given the general mutational tolerance of proteins. Here, we use deep mutational scanning, biochemical analysis and molecular simulations to understand constraints on Ras sequence. Ras exhibits global sensitivity to mutation when regulated by a GTPase activating protein and a nucleotide exchange factor. Removing the regulators shifts the distribution of mutational effects to be largely neutral, and reveals hotspots of activating mutations in residues that restrain Ras dynamics and promote the inactive state. Evolutionary analysis, combined with structural and mutational data, argue that Ras has co-evolved with its regulators in the vertebrate lineage. Overall, our results show that sequence conservation in Ras depends strongly on the biochemical network in which it operates, providing a framework for understanding the origin of global selection pressures on proteins.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1993

Genetic effects of HZE and cosmic radiation (L-9)

The purpose of our experiment is to detect mutations in Drosophila possibly induced by space radiation during the SL-J mission, so that we will be able to obtain basic information about 'the genetic (mutational) risk of space radiation' which can be used to estimate human risk of cancer induction by space flight. As an example of somatic mutation, we will analyze morphological changes in hair growing on the surface of the wing of an adult fly. A piece of wing consists of about 30 thousand wing cells and in the wild type Drosophila a long single piece of hair is growing on the surface of each wing cell. When Drosophila is exposed to radiation as its early stage of development, such as embryonic stage or larval (maggot) stage, some mutations will appear in the wing hair of the adult fly with a certain low frequency, depending on the radiation dose. Among the mutations, the most frequent one is a change in the number of hairs per cell, that is, usually three or more hairs are coming out from a single wing cell. In the actual SL-J flight, we will install thousands of Drosophila larvae (maggots) into the Space Shuttle Discovery and expose them to space radiation during the 7-day mission. Immediately after the re-entry to the ground, these larvae are expected to develop (emerge) into adult flies. Then the wings will be fixed by ethylalcohol and permanent samples will be prepared. Finally, we will analyze the wing samples microscopically in order to detect mutations.

Ikenaga, Mituo↗
NASA NTRSDate not supplied

Determining Chlamydomonas reinhardtii resistance to ionizing radiation at a genome-wide scale

Spaceflight is a complex mixture of abiotic stresses that causes a myriad of cellular responses including increased genome-wide mutation load. The impact of spaceflight on mutation load is not well understood for most organisms and is especially critical to characterize for candidate microorganisms that could be part of biological life support systems. Prior research in our laboratory revealed an unexpectedly high level of mutation during spaceflight in the eukaryotic green algae, C. reinhardtii. Based on the spectrum of base changes observed, we concluded that the mutations were generated through a molecular mechanism unique to the spaceflight environment. Spaceflight is a complex set of stresses, and we are using an Earth-based analog to test ionizing radiation as a potential causative variable for the unique mutation spectrum observed in spaceflight. C. reinhardtii was grown exposed to chronic radiation from a Cesium-137 source. Four biological replicate cultures of wild-type cells were grown in gas permeable plastic tissue culture bags in liquid media following the design used on the International Space Station (ISS) during the previous space algae experiment. Radiation levels for this experiment approximate ionizing radiation observed on the moon, in low Earth orbit (LEO), and on Earth. Cells from each replicate were passaged to inoculate the next cycle of growth, then samples were taken from each culture to characterize cell morphology, quantify biomass, and extract DNA. Whole genome sequencing from each culture will be used to identify the frequency and types of mutations in each radiation level. In growth cycles 3 and 4, there was a significant increase in biomass accumulation under lunar radiation level compared to LEO or Earth radiation levels. These results such that ionizing radiation affected growth of C. reinhardtii.

Microbiology↗
NASA NTRSDate not supplied

Determining Chlamydomonas Reinhardtii Resistance to Ionizing Radiation at A Genome-Wide Scale

Spaceflight is a complex mixture of abiotic stresses that causes a myriad of cellular responses including increased genome-wide mutation load. The impact of spaceflight on mutation load is not well understood for most organisms and is especially critical to characterize for candidate microorganisms that could be part of biological life support systems. Prior research in our laboratory revealed an unexpectedly high level of mutation during spaceflight in the eukaryotic green algae, C. reinhardtii. Based on the spectrum of base changes observed, we concluded that the mutations were generated through a molecular mechanism unique to the spaceflight environment. Spaceflight is a complex set of stresses, and we are using an Earth-based analog to test ionizing radiation as a potential causative variable for the unique mutation spectrum observed in spaceflight. C. reinhardtii was grown exposed to chronic radiation from a Cesium-137 source. Four biological replicate cultures of wild-type cells were grown in gas permeable plastic tissue culture bags in liquid media following the design used on the International Space Station (ISS) during the previous space algae experiment. Radiation levels for this experiment approximate ionizing radiation observed on the moon, in low Earth orbit (LEO), and on Earth. Cells from each replicate were passaged to inoculate the next cycle of growth, then samples were taken from each culture to characterize cell morphology, quantify biomass, and extract DNA. Whole genome sequencing from each culture will be used to identify the frequency and types of mutations in each radiation level. In growth cycles 3 and 4, there was a significant increase in biomass accumulation under lunar radiation level compared to LEO or Earth radiation levels. These results show that ionizing radiation affected growth of C. reinhardtii.

Hope Hersh↗
NASA NTRSDate not supplied

Benchmarking Computational Tools for Calling SNPs and Indels in Complex Microbial Populations

The NASA BioNutrients missions seek to understand the suitability of microorganisms for bioproduction during space flight. One topic of interest is the stability of microbial genomes during long-term ambient storage and subsequent rehydration and growth. To address these questions, samples from 8 species were flown to ISS for 5 years of desiccated storage at ambient temperature (Stasis Packs) and 2 species were packaged along with powdered media inside a bioreactor system to allow hydration and growth in microgravity (Production Packs). For both systems, Whole Genome Sequencing (WGS) of the DNA extracted from the returned samples and paired ground controls will be conducted to identify changes in genome stability due to time, storage conditions and growth in space. Across the technical replicates, ground controls, 10 timepoints, and multiple experimental conditions, ~300 samples have been selected for initial analysis with WGS sequencing to 100x coverage. A flexible and resource efficient mutation calling pipeline is needed to process this large dataset and allow for comparisons between species. Many bioinformatics tools for calling Indels and Single Nucleotide Variants (SNVs) are designed for use with pure isolates, where true variations from the reference genome are expected to dominate the reads aligning to the location of mutation. In contrast, DNA from the Stasis Pack (SP) samples was collected directly after recovery from desiccated storage and the Production Pack (PP) samples were collected after fermentation. In this context, reads with mutations are expected to be less frequent than reads that align with the reference genome, as each sample will include multiple lines of cells. Thus, BioNutrients samples are expected to be similar to samples from cancer cell or “pooled” sequencing approaches. In preparation for the analysis of the BioNutrients samples, we have tested three mutation calling tools (GATK for Microbes, BreSeq and DiscoSNP) designed for complex samples. A challenge of validating mutation identification pipelines is a lack of “Ground Truth” datasets, especially for complex samples. To compare these three tools, we sought to identify mutations in pre-existing WGS data collected from populations of Chlamydomonas reinhardtii that were exposed to UV mutagenesis and growth in LEO as part of the Space Algae-1 mission. Here we present a summary of these tools against the analysis originally conducted using the CRISP tool. Critical metrics are compared such as runtime, the number of SNPs, the number and size of Indels, and patterns of transversion and transitions identified by each tool are reported. By sharing these benchmarking results collected in support of the BioNutrients mission, we aim to guide others seeking to identify SNVs in similarly complex microbial samples.

Biology↗
DOE OSTI2016

Genetic characterization of an adapted pandemic 2009 H1N1 influenza virus that reveals improved replication rates in human lung epithelial cells

The 2009 influenza pandemic originated from a swine-origin H1N1 virus, which, although less pathogenic than anticipated, may acquire additional virulence-associated mutations in the future. To estimate the potential risk, we sequentially passaged the isolate A/Hamburg/04/2009 in A549 human lung epithelial cells. After passage 6, we observed a 100-fold increased replication rate. High-throughput sequencing of viral gene segments identified five dominant mutations, whose contribution to the enhanced growth was analyzed by reverse genetics. The increased replication rate was pinpointed to two mutations within the hemagglutinin (HA) gene segment (HA{sub 1} D130E, HA{sub 2} I91L), near the receptor binding site and the stem domain. The adapted virus also replicated more efficiently in mice in vivo. Enhanced replication rate correlated with increased fusion pH of the HA protein and a decrease in receptor affinity. Our data might be relevant for surveillance of pre-pandemic strains and development of high titer cell culture strains for vaccine production. - Highlights: • We observed a spontaneous mutation of a 2009-pandemic H1N1 influenza virus in vitro. • The adaptation led to a 100-fold rise in replication rate in human A549 cells. • Adaptation was caused by two mutations in the HA gene segment. • Adaptation correlates with increased fusion pH and decreased receptor affinity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Proteogenomic strategies for identification of aberrant cancer peptides using large-scale Next Generation Sequencing data

Cancer is driven by the acquisition of somatic DNA lesions. Distinguishing the early driver mutations from subsequent passenger mutations is key to molecular sub-typing of cancers, and the discovery of novel biomarkers. The availability of genomics technologies (mainly wholegenome and exome sequencing, and transcript sampling via RNA-seq, collectively referred to as NGS) have fueled recent studies on somatic mutation discovery. However, the vision is challenged by the complexity, redundancy, and errors in genomic data, and the difficulty of investigating the proteome using only genomic approaches. Recently, combination of proteomic and genomic technologies are increasingly employed. However, the complexity and redundancy of NGS data remains a challenge for proteogenomics, and various trade-offs must be made to allow for the searches to take place. This paperprovides a discussion of two such trade-offs, relating to large database search, and FDR calculations, and their implication to cancer proteogenomics. Moreover, it extends and develops the idea of a unified genomic variant database that can be searched by any mass spectrometry sample. A total of 879 BAM files downloaded from TCGA repository were used to create a 4.34 GB unified FASTA database which contained 2,787,062 novel splice junctions, 38,464 deletions, 1105 insertions, and 182,302 substitutions. Proteomic data from a single ovarian carcinoma sample (439,858 spectra) was searched against the database. By applying the most conservative FDR measure, we have identified 524 novel peptides and 65,578 known peptides at 1% FDR threshold. The novel peptides include interesting examples of doubly mutated peptides, frame-shifts, and non-sample-recruited mutations, which emphasize the strength of our approach.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Characterization of amino acid residues within the N-terminal region of Ubc9 that play a role in Ubc9 nuclear localization

As the sole E2 enzyme for SUMOylation, Ubc9 is predominantly nuclear. However, the underlying mechanisms of Ubc9 nuclear localization are still not well understood. Here we show that RNAi-depletion of Imp13, an importin known to mediate Ubc9 nuclear import, reduces both Ubc9 nuclear accumulation and global SUMOylation. Furthermore, Ubc9-R13A or Ubc9-H20D mutation previously shown to interrupt the interaction of Ubc9 with nucleus-enriched SUMOs reduces the nuclear enrichment of Ubc9, suggesting that the interaction of Ubc9 with the nuclear SUMOs may enhance Ubc9 nuclear retention. Moreover, Ubc9-R17E mutation, which is known to disrupt the interaction of Ubc9 with both SUMOs and Imp13, causes a greater decrease in Ubc9 nuclear accumulation than Ubc9-R13A or Ubc9-H20D mutation. Lastly, Ubc9-K74A/S89D mutations that perturb the interaction of Ubc9 with nucleus-enriched SUMOylation-consensus motifs has no effect on Ubc9 nuclear localization. Altogether, our results have elucidated that the amino acid residues within the N-terminal region of Ubc9 play a pivotal role in regulation of Ubc9 nuclear localization. - Highlights: • Imp13-mediated nuclear import of Ubc9 is critical for global SUMOylation. • Ubc9 mutations disrupting Ubc9-SUMO interaction decrease Ubc9 nuclear accumulation. • N-terminal amino acid residues of Ubc9 are critical for Ubc9 nuclear enrichment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

JAK2V617F influences epigenomic changes in myeloproliferative neoplasms

Highlights: • Ectopically expressed JAK2V617F in Ba/F3 cells reduced the binding specificity of pSTAT3- and EZH2-associated targets. • JAK2V617F can induce downstream epigenomic modifications. • The methylation changes in the JAK2-STAT3 target loci were clustered differently from the control and other mutation genes. Negative valine (V) to phenylalanine (F) switch at the Janus kinase (JAK2) 617 codon (V617F) is the dominant driver mutation in patients with myeloproliferative neoplasms (MPNs). JAK2V617F was proved to be sufficient for cell transformation; however, independent mutations might influence the following epigenomic modifications. To assess the JAK2V617F-induced downstream epigenomic changes without interferences, we profiled the epigenomic changes in ectopically expressed JAK2V617F in Ba/F3 cells. Antibodies against phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and enhancer of zeste homolog 2 (EZH2) were used for chromatin-immunoprecipitation sequencing (ChIP-seq) to detect the downstream epigenomic targets in the JAK2–STAT3 signaling pathway. To confirm the JAK2V617F-induced epigenetic changes in vivo, DNA methylation changes in the target loci in patients with MPNs were detected through methylation-specific polymerase chain reaction and were clustered against the changes within controls. We found that ectopically expressed JAK2V617F in Ba/F3 cells reduced the binding specificity; it was associated with cis-regulatory elements and recognized DNA motifs in both pSTAT3-downstream and EZH2-associated targets. Overlapping target loci between the control and JAK2V617F were FOXH1, HOXC9, and SRF) were clustered independently from the control locus (L1TD1) and other mutation genes (HMGA2 and Lin28A) in the analyzed MPN samples. Therefore, JAK2V617F influences target binding in both pSTAT3 and EZH2. Without mutations in epigenetic regulators, JAK2V617F can induce downstream epigenomic modifications. Thus, epigenetic changes in JAK2 downstream targets might be trackable in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Tissue-specific in vivo genetic toxicity of nine polycyclic aromatic hydrocarbons assessed using the Muta™Mouse transgenic rodent assay

Test batteries to screen chemicals for mutagenic hazard include several endpoints regarded as effective for detecting genotoxic carcinogens. Traditional in vivo methods primarily examine clastogenic endpoints in haematopoietic tissues. Although this approach is effective for identifying systemically distributed clastogens, some mutagens may not induce clastogenic effects; moreover, genotoxic effects may be restricted to the site of contact and/or related tissues. An OECD test guideline for transgenic rodent (TGR) gene mutation assays was released in 2011, and the TGR assays permit assessment of mutagenicity in any tissue. This study assessed the responses of two genotoxicity endpoints following sub-chronic oral exposures of male Muta™Mouse to 9 carcinogenic polycyclic aromatic hydrocarbons (PAHs). Clastogenicity was assessed via induction of micronuclei in peripheral blood, and mutagenicity via induction of lacZ transgene mutations in bone marrow, glandular stomach, small intestine, liver, and lung. Additionally, the presence of bulky PAH-DNA adducts was examined. Five of the 9 PAHs elicited positive results across all endpoints in at least one tissue, and no PAHs were negative or equivocal across all endpoints. All PAHs were positive for lacZ mutations in at least one tissue (sensitivity = 100%), and for 8 PAHs, one or more initial sites of chemical contact (i.e., glandular stomach, liver, small intestine) yielded a greater response than bone marrow. Five PAHs were positive in the micronucleus assay (sensitivity = 56%). Furthermore, all PAHs produced DNA adducts in at least one tissue. The results demonstrate the utility of the TGR assay for mutagenicity assessment, especially for compounds that may not be systemically distributed. - Highlights: • The Muta™Mouse is a reliable tool for in vivo mutagenicity assessment of PAHs. • All 9 PAHs induced lacZ transgene mutations in small intestine. • Only 5 of 9 PAHs induced lacZ mutations and micronuclei in haematopoietic tissue. • Tissue-specific results are likely related to metabolism, repair, and proliferation. • For oral exposures, it is important to examine effects at the site-of-contact.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. Furthermore, these mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. When using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Structural insights into Noonan/LEOPARD syndrome-related mutants of protein-tyrosine phosphatase SHP2 ( PTPN11 )

Background: The ubiquitous non-receptor protein tyrosine phosphatase SHP2 (encoded by PTPN11) plays a key role in RAS/ERK signaling downstream of most, if not all growth factors, cytokines and integrins, although its major substrates remain controversial. Mutations in PTPN11 lead to several distinct human diseases. Germ-line PTPN11 mutations cause about 50% of Noonan Syndrome (NS), which is among the most common autosomal dominant disorders. LEOPARD Syndrome (LS) is an acronym for its major syndromic manifestations: multiple Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of genitalia, Retardation of growth, and sensorineural Deafness. Frequently, LS patients have hypertrophic cardiomyopathy, and they might also have an increased risk of neuroblastoma (NS) and acute myeloid leukemia (AML). Consistent with the distinct pathogenesis of NS and LS, different types of PTPN11 mutations cause these disorders. Results: Although multiple studies have reported the biochemical and biological consequences of NS- and LS-associated PTPN11 mutations, their structural consequences have not been analyzed fully. Here we report the crystal structures of WT SHP2 and five NS/LS-associated SHP2 mutants. These findings enable direct structural comparisons of the local conformational changes caused by each mutation. Conclusions: Our structural analysis agrees with, and provides additional mechanistic insight into, the previously reported catalytic properties of these mutants. The results of our research provide new information regarding the structure-function relationship of this medically important target, and should serve as a solid foundation for structure-based drug discovery programs.

Leopard Syndrome↗
NASA NTRS2001

Physical and biological studies with protons and HZE particles in a NASA supported research center in radiation health

NASA has established and supports a specialized center for research and training (NSCORT) to specifically address the potential deleterious effects of HZE particles on human health. The NSCORT in radiation health is a joint effort between Lawrence Berkeley National Laboratory (LBNL) and Colorado State University (CSU). The overall scope of research encompasses a broad range of subjects from microdosimetric studies to cellular and tissue responses to initial damage produced by highly energetic protons and heavy charged particles of the type found in galactic cosmic rays (GCR) spectrum. The objectives of the microdosimetry studies are to determine the response of Tissue Equivalent Proportional Counter (TEPC) to cosmic rays using ground based accelerators. This includes evaluation of energy loss due to the escape of high-energy delta rays and increased energy deposition due to the enhanced delta ray production in the wall of the detector. In this report major results are presented for 56Fe at 1000, 740, 600 and 400 MeV/nucleon. An assessment of DNA repair and early development of related chromosomal changes is extremely important to our overall understanding of enhanced biological effectiveness of high LET particle radiation. Results are presented with respect to the fidelity of the rejoining of double strand breaks and the implications of misrejoining. The relationship between molecular and cytogenetic measurements is presented by studying damage processing in highly heterochromatic supernumerary (correction of sypernumerary) X chromosomes and the active X-chromosome. One of the important consequences of cell's inability to handle DNA damage can be evaluated through mutation studies. Part of our goal is the assessment of potential radioprotectors to reduce the mutation yield following HZE exposures, and some promising results are presented on one compound. A second goal is the integration of DNA repair and mutation studies. Results are presented on a direct comparison of initial double strand breaks induction, the time course and fidelity of double strand break rejoining, cell killing and mutation induction in the same human model system. In order to understand the carcinogenic potential of protons and HZE particles, the role of damaged microenvironment in this process must be understood. In this project it has been postulated that radiation affects the microenvironment, which then modifies cell interactions in a manner conducive to neoplastic progression. Both TGF-beta and FGF-2 are important components of microenvironment. A recent result on the assessment of the role of FGF-2 and its cross-talk with TGF-beta as a function of radiation quality is presented. Theoretical modeling has so far played a central role in analyzing and integrating experimental data on repair and mutation studies and predicting new phenomena. The integrated NSCORT program also provides a broad training experience for students and postdoctoral fellows in space radiation health.

NASA Discipline Radiation Health↗
NASA NTRS1994

An initiation-promotion model of tumour prevalence from high-charge and energy radiations

A repair/misrepair kinetic model for multiple radiation-induced lesions (mutation inactivation) is coupled to a two-mutation model of initiation-promotion in tissue to provide a parametric description of tumour prevalence in the mouse Harderian gland from high-energy and charge radiations. Track-structure effects are considered using an action-cross section model. Dose-response curves are described for gamma rays and relativistic ions, and good agreement with experiment is found. 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. Radiosensitivity parameters found in the model for an initiation mutation (sigma 0 = 7.6 x 10(-10) cm2 and D0 = 148.0 Gy) are somewhat different than previously observed for neoplastic transformation of C3H10T1/2 cell cultures (sigma 0 = 0.7 x 10(-10) cm2 and D0 = 117.0 Gy). We consider the two hypotheses that radiation acts 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 with an increase of 30% or more, dependent on the dose level and fractionation schedule, using a mutation rate for promotion similar to that of single-gene mutations.

NASA Discipline Radiation Health↗
NASA NTRS2001

Different mechanisms of radiation-induced loss of heterozygosity in two human lymphoid cell lines from a single donor

Allelic loss is an important mutational mechanism in human carcinogenesis. Loss of heterozygosity (LOH) at an autosomal locus is one outcome of the repair of DNA double-strand breaks (DSBs) and can occur by deletion or by mitotic recombination. We report that mitotic recombination between homologous chromosomes occurred in human lymphoid cells exposed to densely ionizing radiation. We used cells derived from the same donor that express either normal TP53 (TK6 cells) or homozygous mutant TP53 (WTK1 cells) to assess the influence of TP53 on radiation-induced mutagenesis. Expression of mutant TP53 (Met 237 Ile) was associated with a small increase in mutation frequencies at the hemizygous HPRT (hypoxanthine phosphoribosyl transferase) locus, but the mutation spectra were unaffected at this locus. In contrast, WTK1 cells (mutant TP53) were 30-fold more susceptible than TK6 cells (wild-type TP53) to radiation-induced mutagenesis at the TK1 (thymidine kinase) locus. Gene dosage analysis combined with microsatellite marker analysis showed that the increase in TK1 mutagenesis in WTK1 cells could be attributed, in part, to mitotic recombination. The microsatellite marker analysis over a 64-cM region on chromosome 17q indicated that the recombinational events could initiate at different positions between the TK1 locus and the centromere. Virtually all of the recombinational LOH events extended beyond the TK1 locus to the most telomeric marker. In general, longer LOH tracts were observed in mutants from WTK1 cells than in mutants from TK6 cells. Taken together, the results demonstrate that the incidence of radi-ation-induced mutations is dependent on the genetic background of the cell at risk, on the locus examined, and on the mechanisms for mutation available at the locus of interest.

NASA Discipline Radiation Health↗