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33 records · Page 2

Elucidating the Gravome: Quantitative Proteomic Profiling of the Response to Chronic Hypergravity in Drosophila

Altered gravity conditions, such as experienced by organisms during spaceflight, is known to cause transcriptomic and proteomic changes. We describe the proteomic changes in the whole body of adult Drosophila melanogaster (fruit fly), but focus specifically on the localized changes in the adult head in response to chronic hypergravity (3G) treatment. Canton S adult female flies (2-3 days old) were exposed to chronic hypergravity for 9 days and compared with parallel 1G controls. After hypergravity treatment, whole flies and fly heads were separated, and evaluated for quantitative comparison of the two gravity conditions using an isobaric tagging liquid chromatography-tandem mass spectrometry approach. Data revealed a total of 1948 (whole flies) and 1480 (head) proteins to be differentially present in hypergravity-treated flies. Gene Ontology analysis of head specific proteomics revealed host immune response and humoral stress proteins were significantly upregulated. Proteins related to calcium signaling, ion transport and ATPase were decreased. Enhanced expression of cuticular proteins may suggest an alteration in chitin metabolism and in chitin-based cuticle development. We therefore present a comprehensive quantitative survey of proteomic changes in response to chronic hypergravity in Drosophila, which will help elucidate the underlying molecular mechanisms associated with altered gravity environments.

Hypergravity↗

Maize Rough Endosperm6 (rgh6) Encodes A Predicted Dead-Box RNA Helicase and Affects Mirna Processing in Endosperm Development

Maize rough endosperm (rgh) mutants have defective kernels with a rough, etched, or pitted endosperm surface. Molecular genetic analysis of this mutant class has identified multiple RNA processing proteins critical to endosperm development. Here, we report on the developmental and molecular function of the rgh6 locus. The rgh6 mutant was isolated from the UniformMu transposon tagging population. Mutant kernels have reduced endosperm size and defective embryos that develop in a more apical position than typical for defective embryos. TB translocation crosses revealed that rgh6 mutant endosperm inhibits normal embryo development. Positional cloning of the rgh6 locus found that it encodes a predicted DEAD-box RNA helicase. Consistent with a predicted function for RNA processing, transient expression of a RGH6-GFP fusion protein is localized to nucleolus and nuclear speckles in Nicotiana benthamiana leaves. Rgh6 transcripts are highly expressed in endosperm epidermal cell types such as the aleurone, basal endosperm cell layer, embryo surrounding region, and endosperm adjacent to scutellum. Markers of these cell types show increased levels in rgh6 mutant kernels. Mutant endosperm tissues have increased precursor microRNA (pre-miRNA) and decreased mature miRNA relative to normal sibling endosperm, indicating that rgh6 is required for miRNA processing. The transcript levels for most miRNA target genes accumulate to a higher level in rgh6 mutant tissue. These results suggest that miRNA processing and regulation of miRNA target genes are required for normal endosperm development.

Plant Sciences↗

XCTK1: A Xenopus C-terminal Kinesin-like Protein

XCTK1 is 97kDa kinesin-like protein homologous to FKIF2 and KIFC3. XCTK1 is present at picomolar levels in eggs, embryos and cultured cells in a soluble high-molecular weight complex that is not associated with membranes. XCKT1 localizes to centrosomes in Xenopus A6 cells. Anti-XCTK1 antibodies also localize to spindle poles when injected into A6 cells or when added to extracts during in vitro spindle assembly reactions. XCTK1 is associated with the center of taxol-induced microtubule asters in extracts. Therefore its localization to poles is dependent on microtubule minus-ends and not on centrosomes per se. Overexpression of XCTK1 leads to centrosome destruction in cultured cells. XCTK1 was tagged at either the N- or C-terminus and transfected into Xenopus A6 cells At low expression levels, XCTK1 associated with centrosomes. At higher levels, the protein localized to insoluble cytoplasmic structures. Gamma-tubulin staining was dramatically decreased from centrosomes or altogether absent. The centrosomal SPJ antigen colocalized with XCTK1-containing structures. Upon nocodozole treatment, microtubules failed to regrow from the centrosomes indicating that overexpression of XCTK1 severely compromises centrosomal function. Current studies are aimed at determining whether XCTK1 interacts directly with centrosomal proteins and to determine the effects of XCTK1 depletion on oocyte maturation and embryogenesis.

Winfree, Seth↗

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

Non-NASA Center↗

Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells

Homologous recombinational repair of DNA double-strand breaks and crosslinks in human cells is likely to require Rad51 and the five Rad51 paralogs (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2 and Rad51D/Rad51L3), as has been shown in chicken and rodent cells. Previously, we reported on the interactions among these proteins using baculovirus and two- and three-hybrid yeast systems. To test for interactions involving XRCC3 and Rad51C, stable human cell lines have been isolated that express (His)6-tagged versions of XRCC3 or Rad51C. Ni2+-binding experiments demonstrate that XRCC3 and Rad51C interact in human cells. In addition, we find that Rad51C, but not XRCC3, interacts directly or indirectly with Rad51B, Rad51D and XRCC2. These results argue that there are at least two complexes of Rad51 paralogs in human cells (Rad51C-XRCC3 and Rad51B-Rad51C-Rad51D-XRCC2), both containing Rad51C. Moreover, Rad51 is not found in these complexes. X-ray treatment did not alter either the level of any Rad51 paralog or the observed interactions between paralogs. However, the endogenous level of Rad51C is moderately elevated in the XRCC3-overexpressing cell line, suggesting that dimerization between these proteins might help stabilize Rad51C.

Non-NASA Center↗

Insertional Mutagenesis for Genes involved in Otic/Vestibular Development and Function in Xenopus Tropicalis

Sensitivity to gravity is essential for spatial orientation. Consequently, the gravity receptor system is one of the phylogenetically oldest sensory systems, and the special adaptations that enhance sensitivity to gravity are highly conserved. The main goal of this project is to use Xenopus (frog) to identify genes expressed during vestibular and auditory development. These studies will lead a better understanding of the molecular mechanisms involved in vestibular and auditory development and function. We are using a gene-trap approach in Xenopus tropicalis with the green fluorescent protein (GFP) gene as the transgene reporter. GFP expression occurs only when the GFP gene is correctly integrated in actively transcribed genes. Using the GFP as a tag we can easily identify and clone the mutated gene. In addition, we can study the function of the mutated gene by analyzing the defects generated by insertion of the GFP transgene. To date we have tissue specific GFP expression in X. tropicalis including expression in ear, neural tube, kidney, muscle, eyes and nose. Our transgenic animals will soon reach maturity so that we can outcross them and analyze their progeny. Our next goal is to isolate RNA from our transgenics and clone the tagged genes using RACE-PCR. Currently we are optimizing the RACE-PCR method using transgenics with crystallin GFP expression.

Torrejon, Marcela↗

Compaction agent clarification of microbial lysates

Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

NASA Discipline Environmental Health↗

Preliminary Design of a Downstream Processing System for Protein Production in Space

Biomanufacturing is a promising technology to convert in situ resources into essential products including enzymes, therapeutics, biopolymers and other chemicals required to support deep-space missions that may not be easily supplied or produced by alternative means. In addition to the biomass production operations, vital down-stream steps including biomass harvesting/concentration, cell lysis, protein capture and purification are needed to produce an application-ready product. Commercially available terrestrial processes commonly require complex, heavy equipment and highly trained operators, which are not practical in deep space environments. In this work, we aim to identify approaches required to produce an intracellular, His-tagged recombinant enzyme using E. coli at 1 L production scales within the constraints of a deep-space mission as a model use-case scenario. Based on extensive literature review and commercially available products, we identified candidate technologies and products that could be integrated for deep space biomanufacturing. Different preliminary designs were then compared in terms of total system impacts on up-mass, processing time, and consumables required. Our analysis indicated that a biomass concentrator would significantly reduce the processing time and consumables required for the overall system without a large increase in the total mass. We also identified viable technologies for other steps such as cell lysis and protein purification. Predictions from our trade study will be validated in the laboratory by testing the most promising products with the results used to optimize the design. This research will help transfer technology that is well developed on Earth to a space-ready format to produce biological products from a wide variety of microorganisms that can support deep-space missions.

Biomanufacturing↗

Biomaterials Out of Thin Air: In Situ, On-Demand Printing of Advanced Biocomposites: A New Materials Design and Production Technique Using 3D-Printed Arrays of Bioengineered Cells

We have completed the proof of concept described in our Phase I proposal, a two-material array of nonstructural proteins. We created an implementation of each step in our technology concept and demonstrated its critical functionality. The biological chassis and printing hardware we created as part of this work can be re-used for future work by inserting a material coding region upstream of the fluorescent tag. Overall, we showed that our technology concept is sound. The mission benefit analyses, as described in our Phase I proposal, are complete and contained in this report. These calculations show that our technology can save hundreds of kilograms of upmass for a potential planetary human habit construction mission: the mass per habitat module can be reduced by approximately one third if the biomaterials are manufactured on Earth and included in the mission upmass, and the full 240 kg per module can be saved if the materials are derived entirely from in situ resources. Mass savings between these two extremes is expected for an actual mission, depending on the level of in situ resource extraction technology. We have shown that continued advancement of this technology concept for use in a space mission environment is justified. Our survey of future development pathways proved extremely informative in light of the lessons learned from our proof of concept work and mission scenario analyses. For example, we were able for the first time to distinguish between the levels of functionality provided by production of structural proteins, other polymers such as polysaccharides, and true organic-inorganic composites such as bone and mineralized shell. This new information represents a significant advance in formulating specific applications, and key enabling technologies, for our proposed concept. We surveyed potential collaborations with other projects and synergies with enabling technologies that are developing. We have received requests for collaboration from other institutions, including labs at Stanford University and Drexel University. We have also received visits from industry, including Organovo, a tissue engineering company, and Autodesk, a major 3D and materials design software company. Finally, we have been in touch with the team behind the 2013 NIAC Phase ll 'Super Ball Bot-Structures for Planetary Landing and Exploration' and are planning to develop our biomaterial printing technology with the goal of enabling tensegrity-based rovers such as theirs to use lighter, more robust materials. A smooth transition from TRL 2 to TRL 3 assumes that the implementations of the technology concept which demonstrate critical functionality are also pathways for future development; while this is the case for most hardware or software projects, the multidisciplinary nature of our project, particularly the biological aspect of it, means that this is not always true. For example, as part of this work we showed that although there are large number of known genetic parts that correspond to non-structural materials, this is not true for sequences for structural organic proteins, let alone biominerals. These realizations allowed us to further subdivide our concept into more detailed development areas, some of which are clearly established at TRL 3, others of which were newly identified sub-technologies moved from TRL 1 to TRL 2. Similarly, although a single feasibility /benefit analysis is sufficient for advancement from TRL 2 to TRL 3, not all potential benefits to a technology concept as broad in scope as ours are apparent at TRL 2. Both our future pathways survey and our proof of concept work highlighted that the true mass savings potential of our technology concept cannot be quantified without modification of existing materials modelling tools to take into account the possibility of positional materials properties customization. Therefore, we have simultaneously both advanced one potential set of applications of our technology concept from TRL 2 to TRL 3 and also identified a previously unknown set of applications and advanced it from TRL 1 to TRL 2. Overall, we have moved the original formulation of our concept forward from TRL 2 to TRL 3, and the expanded formulation of it presented in this document has been advanced from a combination of TRL 1 and early 1RL 2 to an overall late TRL 2. We have also identified the key areas necessary for both short-term and long-term advancement, and made recommendations for specific future work in the most promising directions. With future work on a 1-2 year timeframe to continue advancement to overall TRL 3, we will be well positioned to begin work on a specific space mission technology insertion path.

Biology↗

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.↗

The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth

To investigate how roots respond to directional cues, we characterized a T-DNA-tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.

NASA Discipline Plant Biology↗

Analysis and expression of the alpha-expansin and beta-expansin gene families in maize

Expansins comprise a multigene family of proteins in maize (Zea mays). We isolated and characterized 13 different maize expansin cDNAs, five of which are alpha-expansins and eight of which are beta-expansins. This paper presents an analysis of these 13 expansins, as well as an expression analysis by northern blotting with materials from young and mature maize plants. Some expansins were expressed in restricted regions, such as the beta-expansins ExpB1 (specifically expressed in maize pollen) and ExpB4 (expressed principally in young husks). Other expansins such as alpha-expansin Exp1 and beta-expansin ExpB2 were expressed in several organs. The expression of yet a third group was not detected in the selected organs and tissues. An analysis of expansin sequences from the maize expressed sequence tag collection is also presented. Our results indicate that expansin genes may have general, overlapping expression in some instances, whereas in other cases the expression may be highly specific and limited to a single organ or cell type. In contrast to the situation in Arabidopsis, beta-expansins in maize seem to be more numerous and more highly expressed than are alpha-expansins. The results support the concept that beta-expansins multiplied and evolved special functions in the grasses.

NASA Program Fundamental Space Biology↗

The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses. Copyright 1999 Academic Press.

NASA Discipline Radiation Health↗

Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

NASA Discipline Cardiopulmonary↗

Leiomodins: larger members of the tropomodulin (Tmod) gene family

The 64-kDa autoantigen D1 or 1D, first identified as a potential autoantigen in Graves' disease, is similar to the tropomodulin (Tmod) family of actin filament pointed end-capping proteins. A novel gene with significant similarity to the 64-kDa human autoantigen D1 has been cloned from both humans and mice, and the genomic sequences of both genes have been identified. These genes form a subfamily closely related to the Tmods and are here named the Leiomodins (Lmods). Both Lmod genes display a conserved intron-exon structure, as do three Tmod genes, but the intron-exon structure of the Lmods and the Tmods is divergent. mRNA expression analysis indicates that the gene formerly known as the 64-kDa autoantigen D1 is most highly expressed in a variety of human tissues that contain smooth muscle, earning it the name smooth muscle Leiomodin (SM-Lmod; HGMW-approved symbol LMOD1). Transcripts encoding the novel Lmod gene are present exclusively in fetal and adult heart and adult skeletal muscle, and it is here named cardiac Leiomodin (C-Lmod; HGMW-approved symbol LMOD2). Human C-Lmod is located near the hypertrophic cardiomyopathy locus CMH6 on human chromosome 7q3, potentially implicating it in this disease. Our data demonstrate that the Lmods are evolutionarily related and display tissue-specific patterns of expression distinct from, but overlapping with, the expression of Tmod isoforms. Copyright 2001 Academic Press.

Carrier Proteins/biosynthesis/genetics↗