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At least 559 records · Page 31

Nonenzymatic, template-directed ligation of oligoribonucleotides is highly regioselective for the formation of 3'-5' phosphodiester bonds

We have found that nonenzymatic, template-directed ligation reactions of oligoribonucleotides display high selectivity for the formation of 3'-5' rather than 2'-5' phosphodiester bonds. Formation of the 3'-5'-linked product is favored regardless of the metal ion catalyst or the leaving group, and for several different ligation junction sequences. The degree of selectivity depends on the leaving group: the ratio of 3'-5'- to 2'-5'-linked products was 10-15:1 when the 5'-phosphate was activated as the imidazolide, and 60-80:1 when the 5'-phosphate was activated by the formation of a 5'-triphosphate. Comparison of oligonucleotide ligation reactions with previously characterized single nucleotide primer extension reactions suggests that the strong preference for 3'-5'-linkages in oligonucleotide ligation is primarily due to occurence of ligation within the context of an extended Watston-Crick duplex. The ability of RNA to correctly self-assemble by template-directed ligation is an intrinsic consequence of its chemical structure and need not be imposed by an external catalyst (i.e., an enzyme polymerase); RNA therefore provides a reasonable structural basis for a self-replicating system in a prebiological world.

Non-NASA Center↗

An algorithm for converting a virtual-bond chain into a complete polypeptide backbone chain

A systematic analysis is presented of the algorithm for converting a virtual-bond chain, defined by the coordinates of the alpha-carbons of a given protein, into a complete polypeptide backbone. An alternative algorithm, based upon the same set of geometric parameters used in the Purisima-Scheraga algorithm but with a different "linkage map" of the algorithmic procedures, is proposed. The global virtual-bond chain geometric constraints are more easily separable from the loal peptide geometric and energetic constraints derived from, for example, the Ramachandran criterion, within the framework of this approach.

NASA Discipline Exobiology↗

Transformations in Air Transportation Systems For the 21st Century

Globally, our transportation systems face increasingly discomforting realities: certain of the legacy air and ground infrastructures of the 20th century will not satisfy our 21st century mobility needs. The consequence of inaction is diminished quality of life and economic opportunity for those nations unable to transform from the 20th to 21st century systems. Clearly, new thinking is required regarding business models that cater to consumers value of time, airspace architectures that enable those new business models, and technology strategies for innovating at the system-of-networks level. This lecture proposes a structured way of thinking about transformation from the legacy systems of the 20th century toward new systems for the 21st century. The comparison and contrast between the legacy systems of the 20th century and the transformed systems of the 21st century provides insights into the structure of transformation of air transportation. Where the legacy systems tend to be analog (versus digital), centralized (versus distributed), and scheduled (versus on-demand) for example, transformed 21st century systems become capable of scalability through technological, business, and policy innovations. Where air mobility in our legacy systems of the 20th century brought economic opportunity and quality of life to large service markets, transformed air mobility of the 21st century becomes more equitable available to ever-thinner and widely distributed populations. Several technological developments in the traditional aircraft disciplines as well as in communication, navigation, surveillance and information systems create new foundations for 21st thinking about air transportation. One of the technological developments of importance arises from complexity science and modern network theory. Scale-free (i.e., scalable) networks represent a promising concept space for modeling airspace system architectures, and for assessing network performance in terms of robustness, resilience, and other metrics. The lecture offers an air transportation system topology and a scale-free network linkage graphic as framework for transportation system innovation. Successful outcomes of innovation in air transportation could lay the foundations for new paradigms for aircraft and their operating capabilities, air transportation system topologies, and airspace architectures and procedural concepts. These new paradigms could support scalable alternatives for the expansion of future air mobility to more consumers in more parts of the world.

Holmes, Bruce J.↗

Detection of Cracks at Welds in Steel Tubing Using Flux Focusing Electromagnetic Probe

The inspection of weldments in critical pressure vessel joints is a major concern in the nuclear power industry. Corrosive environments can speed the fatigue process and access to the critical area is often limited. Eddy current techniques have begun to be used to help overcome these obstacles [1]. As direct contact and couplants are not required, remote areas can be inspected by simply snaking an eddy current coil into the intake tube of the vessel. The drawback of the eddy current method has been the high sensitivity to small changes in the conductivity and permeability of the test piece which are known to vary at weldments [1]. The flaw detection mechanism of the flux focusing electromagnetic probe can help alleviate these difficulties and provide a unique capability for detecting longitudinal fatigue cracks in critical tube structures. The Flux Focusing Electromagnetic Flaw Detector, originally invented for the detection of fatigue and corrosion damage in aluminum plates [2-3], has been adapted for use in testing steel tubing for longitudinal fatigue cracks. The modified design allows for the probe to be placed axisymmetrically into the tubing, inducing eddy currents in the tube wall. The pickup coil of the probe is fixed slightly below the primary windings and is rotated 90 so that its axis is normal to the tube wall. The magnetic flux of the primary coil is focused through the use of ferromagnetic material so that in the absence of fatigue damage there will be no flux linkage with the pickup coil. The presence of a longitudinal fatigue crack will cause the eddy currents induced in the tube wall to flow around the flaw and directly under the pickup coil. The magnetic field associated with these currents will then link the pickup coil and an unambiguous increase in the output voltage of the probe will be measured. The use of the flux focusing electromagnetic probe is especially suited for the detection of flaws originating at or near tube welds. The probe is shown to discriminate against signals due solely to the weld joint so that flaw signals are not hidden in the background in these locations. Experimental and finite element modeling results are presented for the flaw detection capabilities of the probe in stainless steel tubes.

Wincheski, Buzz↗

Effect of dinucleoside pyrophosphates on the oligomerization of activated mononucleotides on Na(+)-montmorillonite: reaction of 5'-phosphoro-4-(dimethylamino)pyridinium [4-(CH3)2NpypA] with A5'ppA

The oligomerization of adenosine 5'-phosphoro-4-(dimethylamino)pyridinium (4-(CH3)2-NpypA) and diadenosine 5',5'-pyrophosphate (A5'ppA) (9:1) on Na(+)-montmorillonite was studied. The oligomers were isolated and analyzed by selective enzymatic hydrolyses and the oligomeric composition and the percent of 3',5'-phosphodiester linkages present in each fraction was determined. The longest oligomers formed (11-mers) are slightly shorter than those produced in the absence of A5'ppA (12-mers). Smaller amounts of A5'ppA are incorporated into the oligomers than in the ImpA/A5'ppA reaction. The regioselectivity of 3',5'-phosphodiester bond formation is comparable to that of the oligomerization of 4-(CH3)2NpypA alone. An explanation of these data is proposed and the possible effect of dinucleoside pyrophosphate on prebiotic RNA formation is discussed.

Non-NASA Center↗

The structure of cockpit management attitudes

A revised version of the Cockpit Management Attitudes Questionnaire (CMAQ) is introduced. Factor analyses of responses from 3 different samples reveal comparable factor structure (previous attempts to factor analyze this measure had produced equivocal results). Implications for the measurement of attitudes and the assessment of attitude change are discussed. It is argued that the CMAQ will benefit both special training programs and efforts to explore attitude-performance linkages in air-transport operations.

NASA Program Space Human Factors↗

Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells

A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.

Non-NASA Center↗

Palmitoylation of caveolin-1 in endothelial cells is post-translational but irreversible

Caveolin-1 is a palmitoylated protein involved in assembly of signaling molecules in plasma membrane subdomains termed caveolae and in intracellular cholesterol transport. Three cysteine residues in the C terminus of caveolin-1 are subject to palmitoylation, which is not necessary for caveolar targeting of caveolin-1. Protein palmitoylation is a post-translational and reversible modification that may be regulated and that in turn may regulate conformation, membrane association, protein-protein interactions, and intracellular localization of the target protein. We have undertaken a detailed analysis of [(3)H]palmitate incorporation into caveolin-1 in aortic endothelial cells. The linkage of palmitate to caveolin-1 was hydroxylamine-sensitive and thus presumably a thioester bond. However, contrary to expectations, palmitate incorporation was blocked completely by the protein synthesis inhibitors cycloheximide and puromycin. In parallel experiments to show specificity, palmitoylation of aortic endothelial cell-specific nitric-oxide synthase was unaffected by these reagents. Inhibitors of protein trafficking, brefeldin A and monensin, blocked caveolin-1 palmitoylation, indicating that the modification was not cotranslational but rather required caveolin-1 transport from the endoplasmic reticulum and Golgi to the plasma membrane. In addition, immunophilin chaperones that form complexes with caveolin-1, i.e. FK506-binding protein 52, cyclophilin A, and cyclophilin 40, were not necessary for caveolin-1 palmitoylation because agents that bind immunophilins did not inhibit palmitoylation. Pulse-chase experiments showed that caveolin-1 palmitoylation is essentially irreversible because the release of [(3)H]palmitate was not significant even after 24 h. These results show that [(3)H]palmitate incorporation is limited to newly synthesized caveolin-1, not because incorporation only occurs during synthesis but because the continuous presence of palmitate on caveolin-1 prevents subsequent repalmitoylation.

Non-NASA Center↗

The relationship between dietary intake, exercise, energy balance and the space craft environment

Space flight is associated with the loss of skeletal muscle, principally from muscles with anti-gravity functions. Examination of data across different missions can permit a distinction to be made between true microgravity responses and what are mission-specific responses. Protein metabolism has been investigated on six missions, four short-term [Shuttle missions Space Life Sciences 1 (1991, SLSI), Space Life Sciences 2 (1993, SLS2), Deutsche-2 (1993, D2) and the Life and Microgravity Sciences (1996, LMS)] and two long-term missions (Skylab 1993 and NASA/MIR, 1996-1998). Measurements made include dietary intake (six missions), nitrogen balance (four missions), whole-body protein kinetics with [15N]glycine as the tracer (four missions) and cortisol excretion (three missions). Also available for comparison are bed rest studies with and without exercise. The purpose of this paper is to see what can be learnt about the muscle loss problem by comparing metabolic results across the six missions for which data are available and against bed rest. The analysis suggests that there is a linkage between the inability to maintain energy balance and exercise, and the connection is the decreased efficiency of removal of the metabolic by-products of exercise (heat, CO2) during space flight.

long duration↗

Context-dependent arm pointing adaptation

We sought to determine the effectiveness of head posture as a contextual cue to facilitate adaptive transitions in manual control during visuomotor distortions. Subjects performed arm pointing movements by drawing on a digitizing tablet, with targets and movement trajectories displayed in real time on a computer monitor. Adaptation was induced by presenting the trajectories in an altered gain format on the monitor. The subjects were shown visual displays of their movements that corresponded to either 0.5 or 1.5 scaling of the movements made. Subjects were assigned to three groups: the head orientation group tilted the head towards the right shoulder when drawing under a 0.5 gain of display and towards the left shoulder when drawing under a 1.5 gain of display; the target orientation group had the home and target positions rotated counterclockwise when drawing under the 0.5 gain and clockwise for the 1.5 gain; the arm posture group changed the elbow angle of the arm they were not drawing with from full flexion to full extension with 0.5 and 1.5 gain display changes. To determine if contextual cues were associated with display alternations, the gain changes were returned to the standard (1.0) display. Aftereffects were assessed to determine the efficacy of the head orientation contextual cue compared to the two control cues. The head orientation cue was effectively associated with the multiple gains. The target orientation cue also demonstrated some effectiveness while the arm posture cue did not. The results demonstrate that contextual cues can be used to switch between multiple adaptive states. These data provide support for the idea that static head orientation information is a crucial component to the arm adaptation process. These data further define the functional linkage between head posture and arm pointing movements.

Clinical Trial↗

Analysis of cell mechanics in single vinculin-deficient cells using a magnetic tweezer

A magnetic tweezer was constructed to apply controlled tensional forces (10 pN to greater than 1 nN) to transmembrane receptors via bound ligand-coated microbeadswhile optically measuring lateral bead displacements within individual cells. Use of this system with wild-type F9 embryonic carcinoma cells and cells from a vinculin knockout mouse F9 Vin (-/-) revealed much larger differences in the stiffness of the transmembrane integrin linkages to the cytoskeleton than previously reported using related techniques that measured average mechanical properties of large cell populations. The mechanical properties measured varied widely among cells, exhibiting an approximately log-normal distribution. The median lateral bead displacement was 2-fold larger in F9 Vin (-/-) cells compared to wild-type cells whereas the arithmetic mean displacement only increased by 37%. We conclude that vinculin serves a greater mechanical role in cells than previously reported and that this magnetic tweezer device may be useful for probing the molecular basis of cell mechanics within single cells. Copyright 2000 Academic Press.

Non-NASA Center↗

Neocortical malformation as consequence of nonadaptive regulation of neuronogenetic sequence

Variations in the structure of the neocortex induced by single gene mutations may be extreme or subtle. They differ from variations in neocortical structure encountered across and within species in that these "normal" structural variations are adaptive (both structurally and behaviorally), whereas those associated with disorders of development are not. Here we propose that they also differ in principle in that they represent disruptions of molecular mechanisms that are not normally regulatory to variations in the histogenetic sequence. We propose an algorithm for the operation of the neuronogenetic sequence in relation to the overall neocortical histogenetic sequence and highlight the restriction point of the G1 phase of the cell cycle as the master regulatory control point for normal coordinate structural variation across species and importantly within species. From considerations based on the anatomic evidence from neocortical malformation in humans, we illustrate in principle how this overall sequence appears to be disrupted by molecular biological linkages operating principally outside the control mechanisms responsible for the normal structural variation of the neocortex. MRDD Research Reviews 6:22-33, 2000. Copyright 2000 Wiley-Liss, Inc.

Review↗

Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism

The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.

NASA Program Biomedical Research and Countermeasur↗

A complex ligase ribozyme evolved in vitro from a group I ribozyme domain

Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains. The component domains of a protein can associate in alternative combinations to form molecules with different functions. These observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains. To test this hypothesis, an in vitro evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool of 10(16) different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3',5'-phosphodiester linkage between adjacent template-bound oligonucleotides, one bearing a 3' hydroxyl and the other a 5' triphosphate. Ligation occurs in the context of a Watson-Crick duplex, with a catalytic rate of 0.26 min(-1) under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common structural domains while exhibiting distinct catalytic functions.

NASA Discipline Exobiology↗

Stoichiometry of the large conductance bacterial mechanosensitive channel of E. coli. A biochemical study

MscL, a 15 kDa transmembrane protein, is the only component involved in the formation of a 3 nS channel in the inner membrane of Escherichia coli that opens in response to mechanical or osmotic stress. While previous data had suggested that the functional MscL complex might be a hexamer, a recent crystallographic study of the MscL homologue from M. tuberculosis reveals a pentameric structure. The present work further examines the stoichiometry of the E. coli MscL using a variety of biochemical approaches. Detergent-purified 6His-MscL in solution and MscL in the membrane could be chemically crosslinked with the products displaying ladderlike patterns on SDS gels. Three crosslinking agents (EDC, DMS, and DMA) used at saturating concentrations invariably generated pentamers as the largest product. DSS produced additional bands corresponding to larger complexes although the pentamer band appeared to be the predominant product at high levels of crosslinker. It is not clear whether these extra bands reflect a difference in the crosslinking chemistry of DSS or whether its spacer arm is the longest of those used, or a combination of both facts. For the detergent-solubilized 6His-MscL both sedimentation equilibrium and gel chromatography showed the presence of multiple species. Thus the longer spacer arm could permit both intra- and intercomplex linkages. Nonetheless, the patterns obtained with all agents are consistent with and strongly suggest a pentameric organization for the MscL channel. Expression of MscL as genetically engineered double or triple subunit tandems yields low numbers of functional channels as compared to expressed monomers. The double-tandem assemblies must have an even number of subunits and crosslinking in the membrane confirmed hexamerization. Gel chromatography clearly demonstrated that the channels formed from the double tandems were larger than those formed from WT MscL, consistent with the native channel being pentameric. The observation that both double and triple tandems form channels of normal conductance implies that the pentameric assembly is to some degree independent of the number of subunit repeats in the polypeptide precursor. The channel is thus a pentameric core with the 'extra' subunits left out of the functional complex. From sedimentation equilibrium and size-exclusion chromatography, we also conclude that MscL complexes are not in a dynamic equilibrium with monomers, but are pre-assembled; and thus, their gating properties must result from changes in the conformation of the entire complex induced by the mechanical stress.

NASA Discipline Cell Biology↗

Heavy-ion radiobiology: new approaches to delineate mechanisms underlying enhanced biological effectiveness

Shortly after the discovery of polonium and radium by Marie Curie and her husband and colleague, Pierre Curie, it was learned that exposure to these alpha-particle emitters produced deleterious biological effects. The mechanisms underlying the increased biological effectiveness of densely ionizing radiations, including alpha particles, neutrons and highly energetic heavy charged particles, remain an active area of investigation. In this paper, we review recent advances in several areas of the radiobiology of these densely ionizing radiations, also known as heavy ions. Advances are described in the areas of DNA damage and repair, chromosome aberrations, mutagenesis, neoplastic transformation in vitro, genomic instability, normal tissue radiobiology and carcinogenesis in vivo. We focus on technical innovations, including novel applications of pulsed-field gel electrophoresis, fluorescence in situ hybridization (FISH), linkage analysis, and studies of gene expression and protein expression. We also highlight the use of new cellular and animal systems, including those with defined DNA repair deficiencies, as well as epithelial cell model systems to assess neoplastic transformation both in vitro and in vivo. The studies reviewed herein have had a substantial impact on our understanding of the genotoxic effects of heavy ions as well as their distinct effects on tissue homeostasis. The use of these radiations in cancer therapy is also discussed. The use of both heavy-ion and proton therapy is on the upswing in several centers around the world, due to their unique energy deposition characteristics that enhance the therapeutic effect and help reduce damage to normal tissue.

Review, Academic↗

Virtual Observatories: Are We Virtually There Yet?

Virtual observatories are tools for simplifying access to and use of astronomical data from an increasing number of data sources of rapidly growing volume. Now that a variety of virtual observatory development efforts are under way around the world, a cursory review of the efforts outside solar physics, and an only slightly more detailed consideration of those within, demonstrates a commonality of conceptual model if not of approach or application. The linkages among virtual observatories optimized for different scientific communities present an interesting challenge to the designers: should virtual observatories be designed for the most expert users? For the least? For everyone? It is too early to provide definitive answers, but examination of current efforts does offer some clues.

Gurman, J. B.↗

Oxidative acylation using thioacids

Several important prebiotic reactions, including the coupling of amino acids into polypeptides by the formation of amide linkages, involve acylation. Theae reactions present a challenge to the understanding of prebiotic synthesis. Condensation reactions relying on dehydrating agents are either inefficient in aqueous solution or require strongly acidic conditions and high temperatures. Activated amino acids such as thioester derivatives have therefore been suggested as likely substrates for prebiotic peptide synthesis. Here we propose a closely related route to amide bond formation involving oxidative acylation by thioacids. We find that phenylalanine, leucine and phenylphosphate are acylated efficiently in aqueous solution by thioacetic acid and an oxidizing agent. From a prebiotic point of view, oxidative acylation has the advantage of proceeding efficiently in solution and under mild conditions. We anticipate that oxidative acylation should prove to be a general method for activating carboxylic acids, including amino acids.

Non-NASA Center↗