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At least 37 records · Page 2

Compartmental Control of UDP-Araf Supply: Golgi Lumen UDP-Arabinopyranose Mutase Depletes Plant Cell Wall Araf Linkages

L-Arabinofuranose (Araf) is a major constituent of plant cell wall polysaccharides. UDP-Araf is generated from UDP-L-arabinopyranose (UDP-Arap) by a UDP-Arap mutase (UAM) on the cytosolic face of the Golgi and then imported into the Golgi lumen for utilization by arabinosyltransferases (AraTs). Yet, a substantial fraction of UDP-Arap is synthesized in the Golgi lumen by the UDP-xylose 4-epimerase (UXE), creating a topological paradox where luminal UDP-Arap must be exported to the cytosol for conversion to UDP-Araf by UAM and then re-imported into the Golgi lumen. To test the functional significance of this compartmentation, we mislocalized Arabidopsis (Arabidopsis thaliana) UAM1 to the Golgi lumen by fusing it to the UXE1 transmembrane domain. The fusion protein was present in the Golgi, and protease-protection assays on microsomes supported its luminal orientation. In wild-type and uam1 plants, Golgi-targeted UAM1 reduced total cell wall Arabinose without consistent changes in other monosaccharides. Linkage analyses showed that this decrease reflects selective loss of Araf residues, whereas Arap remain largely unchanged, consistent with reduced UDP-Araf availability for luminal AraTs. Under salt stress, expression of Golgi-targeted UAM1 in uam1 caused root-growth inhibition and maturation-zone swelling that exogenous L-Ara failed to rescue. These results support a hypothesis that the strong thermodynamic bias of the UAM reaction toward UDP-Arap requires spatial separation of UDP-Arap production from UDP-Araf formation to sustain UDP-Araf flux into Golgi arabinosylation and avoid futile intraluminal back-conversion.

59 BASIC BIOLOGICAL SCIENCES

Nitrogen metabolism in Lignifying Pinus taeda cell cultures

The primary metabolic fate of phyenylalanine, following its deamination in plants, is conscription of its carbon skeleton for lignin, suberin, flavonoid, and related metabolite formation. Since this accounts for approximately 30-40% of all organic carbon, an effective means of recycling the liberated ammonium ion must be operative. In order to establish how this occurs, the uptake and metabolism of various 15N-labeled precursors (15N-Phe, 15NH4Cl, 15N-Gln, and 15N-Glu) in lignifying Pinus taeda cell cultures was investigated, using a combination of high performance liquid chromatography, 15N NMR, and gas chromatograph-mass spectrometry analyses. It was found that the ammonium ion released during active phenylpropanoid metabolism was not made available for general amino acid/protein synthesis. Rather it was rapidly recycled back to regenerate phenylalanine, thereby providing an effective means of maintaining active phenylpropanoid metabolism with no additional nitrogen requirement. These results strongly suggest that, in lignifying cells, ammonium ion reassimilation is tightly compartmentalized.

NASA Discipline Number 40-50

Models of glycolysis: Glyceraldehyde as a source of energy and monomers for prebiotic condensation reactions

All organisms require energy in a chemical form for maintenance and growth. In contemporary life this chemical energy is obtained by the synthesis of the phosphoanhydride bonds of ATP. Among the biological processes that yield ATP, fermentation is generally considered primitive, because it operates under anaerobic conditions by substrate-level phosphorylation which does not require compartmentation by membranes. Fermentation by the glycolytic pathway, which is found in almost every living cell, is an especially attractive energy source for primitive life. Glycolysis not only produces useful chemical energy (ATP), but intermediates of this pathway are also involved in amino acid synthesis and photosynthetic carbon-fixation. It is believed that energy and substrates needed for the origin of life were provided by nonenzymatic chemical reactions that resemble the enzyme-mediated reactions of glycolysis. These nonenzymatic reactions would have provided a starting point for the evolutionary development of glycolysis.

Weber, A. L.

Microgravity changes in heart structure and cyclic-AMP metabolism

The effects of microgravity on cardiac ultrastructure and cyclic AMP metabolism in tissues of rats flown on Spacelab 3 are reported. Light and electron microscope studies of cell structure, measurements of low and high Km phosphodiesterase activity, cyclic AMP-dependent protein kinase activity, and regulatory subunit compartmentation show significant deviations in flight animals when compared to ground controls. The results indicate that some changes have occurred in cellular responses associated with catecholamine receptor interactions and intracellular signal processing.

Philpott, D. E.

Design and development of a 5 kV isolated solid state switch

This paper describes the design and fabrication of a 5000 volt isolated hybrid switch developed by Hughes Aircraft Company under contract to NASA/Lewis. Hughes did the packaging design and NASA did the circuit design. This unique microcircuit is intended for use as a shorting switch for large extraterrestrial solar cell arrays. The packaging design for the 5 kV isolated hybrid switch is different from most hybrid microcircuits in that it utilizes a compartmentalized plastic case (a portion of which is encapsulated), is not hermetic, and is designed for high voltage operation.

Holbrook, R. J.

Cellular and Network Mechanisms Underlying Information Processing in a Simple Sensory System

Realistic, biophysically-based compartmental models were constructed of several primary sensory interneurons in the cricket cercal sensory system. A dynamic atlas of the afferent input to these cells was used to set spatio-temporal parameters for the simulated stimulus-dependent synaptic inputs. We examined the roles of dendritic morphology, passive membrane properties, and active conductances on the frequency tuning of the neurons. The sensitivity of narrow-band low pass interneurons could be explained entirely by the electronic structure of the dendritic arbors and the dynamic sensitivity of the SIZ. The dynamic characteristics of interneurons with higher frequency sensitivity required models with voltage-dependent dendritic conductances.

Jacobs, Gwen

Design and development of a 5 kV isolated solid state switch

A unique microcircuit intended for use as a shorting switch for large extraterrestrial solar cell arrays is described. The packaging design for the 5 kV isolated hybrid switch is different from most hybrid microcircuits in that it utilizes a compartmentalized plastic case (a portion of which is encapsulated), is not hermetic, and is designed for high voltage operation.

Holbrook, R. J.

Cyclic AMP-receptor proteins in heart muscle of rats flown on Cosmos 1887

The cellular compartmentalization of the cyclic AMP-receptor proteins in heart ventricular tissue obtained from rats flown on the Cosmos 1887 is determined. Photoaffinity labeling of soluble and particular cell fractions with a (32P)-8-azido analog of cyclic AMP is followed by electrophoretic separation of the proteins and by autoradiographic identification of the labeled isoforms of cAPK R subunits. It is shown that RII in the particulate subcellular fraction was significantly decreased in heart cells from rats in the flight group when compared to controls. Protein banding patterns in both the cytoplasmic fraction and in a fraction enriched in chromatin-bound proteins exhibited some variability in tissues of individual animals, but showed no changes that could be directly attributed to flight conditions. No significant change was apparent in the distribution of RI or RII cyclic AMP binding in the soluble fractions. It is inferred that the cardiac cell integrity or its protein content is not compromised under flight conditions.

Mednieks, Maija I.

The decay of HIV under anti-retroviral therapy is biphasic even in humanized mice with just T cells

HIV-1 plasma viral load decays in a biphasic manner during antiretroviral therapy (ART). It was hypothesized that this is due to infection of different cell types, namely CD4+ T cells and macrophages. We studied this possibility directly by modeling the decay of HIV-1 in humanized mice. We utilized previously published data from humanized T-cell only mice (TOM) and myeloid-only mice (MOM) infected with HIV-1 and treated with a potent ART regimen. Viral load decay dynamics were modeled using either a single or a biexponential decay fitted using nonlinear mixed effects techniques. Fits were compared using the corrected Bayesian information criterion (BICc). In TOM, the biphasic model was significantly better than a single-phase decay model (ΔBICc ≈ 16) despite additional parameters. In MOM, the biphasic decay was statistically better, but there was substantial uncertainty because the virus goes below detection very fast. The first-phase half-life was consistent between groups (1.2 days in MOM and 1.3 days in TOM) and similar to the half-life estimated in human infection. The second-phase decay in these mice was minimal likely due to low initial viral loads. Additional analyses with mice containing both CD4+ T cells and macrophages or X4-tropic virus-infected MOM mice confirmed the biphasic pattern, demonstrating the robustness of this result. The biphasic decline in HIV-1 occurs, even with only CD4+ T cells, refuting the hypothesis that distinct cell populations (CD4+ T cells and macrophages) drive each decay phase. These findings support an alternative model in which the observed dynamics arise from intrinsic properties of the viral infection lifecycle rather than from cellular compartmentalization.

59 BASIC BIOLOGICAL SCIENCES

Coupling Between Metabolism and Compartmentalization: Vesicle Growth in the Presence of Dipeptides

A fundamental unresolved question in studies on the origin of life is: how different, ubiquitous protocellular functions begun to work in concert setting the stage for Darwinian evolution of nascent life? From this perspective, of particular significance is coupling between growth of protocellular compartments and the encapsulated, primordial metabolism, which is one of the focal topics of the current ISSOL meeting. Specifically, growth and division of cells facilitated by the products of a metabolic reaction would confer an evolutionary advantage on protocells encapsulating this reaction, as their population would increase at the expense of other protocells. Along these lines, Adamala and Szostak have recently demonstrated that a dipeptide captured inside fatty acid vesicles catalyzes the formation of other dipeptides from activated monomers. Some of the newly synthesized dipeptides, in turn, are capable to promote competitive growth of vesicles in the presence of fatty acid micelles. As vesicles become larger, they adapt filamentous shape, which has been shown to promote their division. On the basis of computer simulations, we provide a molecularly detailed explanation of this process and draw conclusions about its generality.

Wei, C.

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity

We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe 2+ utilizing O 2 or H 2 O 2 as oxidant, and the resultant Fe 3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases.

59 BASIC BIOLOGICAL SCIENCES

Compartmentalization in proteinoid microspheres

Proteinoid microspheres with stable internal compartments and internal structure are made from acidic proteinoid and basic proteinoid with calcium. The populations of microspheres are characterized by a wide diversity of structure. A model of primitive intracellular communication is suggested by the observed movement of internal particles between compartments of a multicompartmentalized unit. Differential response to pH change and to temperature change has been demonstrated within one population and suggests one mode of adaptive selection among primordial cell populations.

Brooke, S.

Novel Confocal Microscopic and Flow Cytometric Based Assays to Visualize and Detect the (Beta)2-Adrenergic Receptor in Human Lymphocyte and Mononuclear Cell Populations

The data show that immunophenotyping of leukocyte populations with (beta)2AR is possible with the commercially available Ab, although the FC assay is limited to the IST as a result of the Ab binding site to the intracellular C-terminus of the 2AR. The FC assay has applications for measuring alterations in total (beta)2AR in human leukocyte populations as changes in fluorescence. In addition, CM confirms that both surface and intracellular compartments stain positively for the (beta)2AR and can be used for qualitative assays that screen for changes in receptor compartmentalization and localization.

Salicru, A. N.

Design of a robot-automated flat plate/reflection geometry x-ray diffraction setup for accelerated materials discovery and structural screening

Here, we report the design, construction, and automation of a flat plate sample loading, alignment, and data acquisition system for X-ray diffraction measurements in reflection geometry implemented at the Stanford Synchrotron Radiation Lightsource. The system is built onto a single platform, enabling facile transferability, and is compartmentalized into sample storage, sample transfer, and sample position/alignment segments. The core feature of this system is a six-axis robotic arm that offers a large range of highly reproducible and programable movements. The degrees of freedom of the robot arm enable adaptability in which movements can be modified to fit various beamline environments and sample configurations. Samples are housed on 3D printed sample mounts, which are arranged onto a 6 × 2 array of sample cassettes capable of holding 7 samples. Using sample mounts designed for solid oxide electrolysis button cells (SOECs), the maximum tray capacity is 84 samples, which can be aligned and run in ~ 24 hours with long exposure scans. The sample array is additionally capable of accommodating a range of sample sizes and geometries due to the rapid 3D printed fabrication. The components of the setup will be described in detail and performance will be demonstrated with a set of representative SOEC and XRD standard samples. Opportunities for future developments and integration with the automated setup are summarized.

08 HYDROGEN

Disruption of histone acetylation homeostasis reveals multilayered chromatin regulation for transcriptional resiliency

Background Epigenetic modifications, nucleosome occupancy, and three-dimensional chromatin architecture collectively create a multi-layered, highly interactive regulatory system for controlling genomic functionality. Dysregulation of epigenetic processes leads to a plethora of abnormalities including disease states. Therapies focused on epigenetic modulation can alter gene expression to correct dysfunction, though the perpetuation of these states and the relationships among chromatin regulatory layers is not well understood. Results Here, we investigated global and local chromatin structural and functional responses after acute histone deacetylase inhibitor treatment (suberoylanilide hydroxamic acid) in lung cancer cells across time. Treatment substantially increased global histone acetylation resulting in a pervasive but not distinctive signature. The spread of acetylation did not significantly impact global chromatin accessibility, and nucleosome remodeling largely occurred at finer scales in functionally relevant genomic regions. Indeed, both H 3 K 4 trimethylation, a mark of active transcription, and gene expression changes were altered in a controlled locus-specific manner, suggesting aberrant acetylation indirectly leads to balanced and bidirectional gene expression profiles from tighter regulation of other chromatin features. HDACi treatment induced (13%) genomic rearrangement in chromatin compartmentalization and moderate weakening of topologically associating domains. Conclusions Continuous wavelet analysis of these features demonstrates that scale-dependent, locus-specific factors influence the relationship between chromatin architecture and functional output, suggesting that regulation of transcription and nucleosome remodeling is not entirely (nor linearly) dependent upon large scale compartment exchange. Structural and functional responses are most pronounced early after treatment with partial persistence of differential local chromatin features and expression later in time; this highlights the plasticity of chromatin regulation, which may have implications for the efficacy of epigenetic treatments. These results demonstrate the effectiveness of multi-layered regulation of transcription: in resilient systems, disruption of one chromatin feature does not distort the regulation of other features in supporting a transcriptional program that allows for survival.

59 BASIC BIOLOGICAL SCIENCES

Modeling with uncertainty quantification reveals the essentials of a non-canonical algal carbon-concentrating mechanism

The thermoacidophilic red alga Cyanidioschyzon merolae survives its challenging environment likely in part by operating a carbon-concentrating mechanism (CCM). Here, we demonstrated that C. merolae 's cellular affinity for CO 2 is stronger than the affinity of its rubisco for CO 2 . This finding provided additional evidence that C. merolae operates a CCM while lacking the structures and functions characteristic of CCMs in other organisms. To test how such a CCM could function, we created a mathematical compartmental model of a simple CCM, distinct from those we have seen previously described in detail. The results of our modeling supported the feasibility of this proposed minimal and non-canonical CCM in C. merolae . To facilitate the robust modeling of this process, we measured and incorporated physiological and enzymatic parameters into the model. Additionally, we trained a surrogate machine-learning model to emulate the mechanistic model and characterized the effects of model parameters on key outputs. This parameter exploration enabled us to identify model features that influenced whether the model met the experimentally derived criteria for functional carbon concentration and efficient energy usage. Such parameters included cytosolic pH, bicarbonate pumping cost and kinetics, cell radius, carboxylation velocity, number of thylakoid membranes, and CO 2 membrane permeability. Our exploration thus suggested that a non-canonical CCM could exist in C. merolae and illuminated the essential features generally necessary for CCMs to function.

Steensma, Anne K. [Michigan State Univ., East Lans

Fluid Shifts: Otoacoustical Emission Changes in Response to Posture and Lower Body Negative Pressure

INTRODUCTION: The purpose of the NASA Fluid Shifts Study is to characterize fluid distribution and compartmentalization associated with long-duration spaceflight and to correlate these findings with vision changes and other elements of the visual impairment and intracranial pressure (VIIP) syndrome. VIIP signs and symptoms, as well as postflight lumbar puncture data, suggest that elevated intracranial pressure (ICP) may be associated with spaceflight-induced cephalad fluid shifts, but this hypothesis has not been tested. Due to the invasive nature of direct measures of ICP, a noninvasive technique of monitoring ICP is desired for use during spaceflight. The phase angle and amplitude of otoacoustic emissions (OAEs) have been shown to be sensitive to posture change and ICP (1, 2), therefore use of OAEs is an attractive option. OAEs are low-level sounds produced by the sensory cells of the cochlea in response to auditory stimulation. These sounds travel peripherally from the cochlea, through the oval window, to the ear canal where they can be recorded. OAE transmission is sensitive to changes in the stiffness of the oval window, occurring as a result of changes in cochlear pressure. Increased stiffness of the oval window largely affects the transmission of sound from the cochlea at frequencies between 800 Hz and 1600 Hz. OAEs can be self-recorded in the laboratory or on the ISS using a handheld device. Our primary objectives regarding OAE measures in this experiment were to 1) validate this method during preflight testing of each crewmember (while sitting, supine and in head-down tilt position), and 2) determine if OAE measures (and presumably ICP) are responsive to lower body negative pressure and to spaceflight. METHODS: Distortion-product otoacoustic emissions (DPOAEs) and transient evoked otoacoustic emissions (TEOAEs) were recorded preflight using the Otoport Advance OAE system (Otodynamics Ltd., Hatfield, UK). Data were collected in four conditions (seated, supine, 15 degrees head down tilt (HDT), and 15 degrees HDT with lower body negative pressure (LBNP)) to produce a range of ICP in each subject and test the susceptibility of OAEs to LBNP. LBNP was induced using the Russian Chibis suit to produce the same fit and pressures that would be experienced inflight during Chibis LBNP trials. Similar trials have occurred inflight on the ISS. A comparative analysis of preflight and inflight phase measurements and magnitudes was completed in both broad and narrow band frequency ranges. RESULTS: TEOAE data demonstrated notable phase shifts from 859-1640 Hz when the seated baseline condition is compared to supine, HDT, and HDT plus Chibis conditions. Changes were particularly pronounced at low frequencies and were consistent with the expected ICP changes. Preflight DPOAE magnitude data revealed changes consistent with increased ICP in two conditions at 1414 Hz, where a magnitude change (relative to the seated condition) was seen in the HDT position and in HDT plus Chibis. DISCUSSION: OAEs revealed systematic changes in phase and magnitude throughout all test conditions (including use of Chibis LBNP) that were consistent with ICP changes. Results indicate that OAEs may provide a rapid noninvasive means of monitoring ICP changes. The first two subjects are projected to complete inflight testing on the ISS in early 2016, with the full complement of 10 subjects scheduled to be complete in 2018.

Melgoza, R.

An Energetic Concept of Habitability for the Deep Subsurface

Universally, life must be characterized by a characteristic level of order and complexity. In the most general sense, habitability could then be defined as the set of factors required to allow the creation and maintenance of molecular complexity. These factors are: chemical raw materials; energy with which to assemble those materials into complex molecules and sustain the resultant state of complexity; a solvent that allows the interaction of complex molecules, promotes tertiary structure, and permits compartmentalization; and environmental conditions that permit the assembly and maintenance of complex molecules. On Earth, these general requirements correspond to the major biogenic elements C, H, O, N, P, S; chemical or light energy; the solvent water; and specific ranges of temperature, pH, radiation, ionic strength, and so forth, which have thus far been determined on and exclusively empirical basis. Importantly, while the complete absence of any of these factors ensures uninhabitable conditions, the mere presence of all four does not guarantee habitability. In each case - even that of water - it is a question of degree. This question can be couched in quantitative terms by considering the impact of each of these factors on cellular energy balance. More "extreme" conditions (e.g., high temperature, high or low pH, etc.), lower water activity, and low concentrations of nutrients incur or have potential to be addressed by increased investment of energy on the part of the cell. This must be balanced by energy conservation in the cell, noting that biochemical, mass transport, and abiotic chemical limitations intervene between environmental energy availability and biological energy capture. Similarly, lower boundary conditions are emplaced on useful environmental energy yields by the "quantized" nature of biological energy conservation, and upper boundary conditions are emplaced by energy levels or fluxes that are destructive with respect to complexity. This energetic framework, with boundary conditions supplied by the specifics of the biochemistry in question, offers a generalized, yet quantitative means of assessing the habitability of any system with respect to complex life.

Hoehler, Tori M.