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At least 73 records · Page 4

RWPV bioreactor mass transport: earth-based and in microgravity

Mass transport and mixing of perfused scalar quantities in the NASA Rotating Wall Perfused Vessel bioreactor are studied using numerical models of the flow field and scalar concentration field. Operating conditions typical of both microgravity and ground-based cell cultures are studied to determine the expected vessel performance for both flight and ground-based control experiments. Results are presented for the transport of oxygen with cell densities and consumption rates typical of colon cancer cells cultured in the RWPV. The transport and mixing characteristics are first investigated with a step change in the perfusion inlet concentration by computing the time histories of the time to exceed 10% inlet concentration. The effects of a uniform cell utilization rate are then investigated with time histories of the outlet concentration, volume average concentration, and volume fraction starved. It is found that the operating conditions used in microgravity produce results that are quite different then those for ground-based conditions. Mixing times for microgravity conditions are significantly shorter than those for ground-based operation. Increasing the differential rotation rates (microgravity) increases the mixing and transport, while increasing the mean rotation rate (ground-based) suppresses both. Increasing perfusion rates enhances mass transport for both microgravity and ground-based cases, however, for the present range of operating conditions, above 5-10 cc/min there are diminishing returns as much of the inlet fluid is transported directly to the perfusion exit. The results show that exit concentration is not a good indicator of the concentration distributions in the vessel. In microgravity conditions, the NASA RWPV bioreactor with the viscous pump has been shown to provide an environment that is well mixed. Even when operated near the theoretical minimum perfusion rates, only a small fraction of the volume provides less than the required oxygen levels. 2002 Wiley Periodicals, Inc.

Models, Biological↗

Inventions Utilizing Microfluidics and Colloidal Particles

Several related inventions pertain to families of devices that utilize microfluidics and/or colloidal particles to obtain useful physical effects. The families of devices can be summarized as follows: (1) Microfluidic pumps and/or valves wherein colloidal-size particles driven by electrical, magnetic, or optical fields serve as the principal moving parts that propel and/or direct the affected flows. (2) Devices that are similar to the aforementioned pumps and/or valves except that they are used to manipulate light instead of fluids. The colloidal particles in these devices are substantially constrained to move in a plane and are driven to spatially order them into arrays that function, variously, as waveguides, filters, or switches for optical signals. (3) Devices wherein the ultra-laminar nature of microfluidic flows is exploited to effect separation, sorting, or filtering of colloidal particles or biological cells in suspension. (4) Devices wherein a combination of confinement and applied electrical and/or optical fields forces the colloidal particles to become arranged into three-dimensional crystal lattices. Control of the colloidal crystalline structures could be exploited to control diffraction of light. (5) Microfluidic devices, incorporating fluid waveguides, wherein switching of flows among different paths would be accompanied by switching of optical signals.

Marr, David W.↗

Membrane peptides and their role in protobiological evolution

How simple membrane peptides performed such essential protocellular functions as transport of ions and organic matter across membranes separating the interior of the cell from the environment, capture and utilization of energy, and transduction of environmental signals, is a key question in protobiological evolution. On the basis of detailed, molecular-level computer simulations we explain how these peptides fold at water-membrane interfaces, insert into membranes, self-assemble into higher-order structures and acquire functions. We have investigated the interfacial behavior and folding of several peptides built of leucine and glutamine residues and have demonstrated that many of them tend to adopt ordered structures. Further, we have studied the insertion of an alpha-helical peptide containing leucine (L) and serine (S) of the form (LSLLLSL)3 into a model membrane. The transmembrane state is metastable, and approximately 15 kcal mol(-1) is required to insert the peptide into the membrane. Investigations of dimers formed by (LSLLLSL)3 and glycophorin A demonstrate how the favorable free energy of helix association can offset the unfavorable free energy of insertion, leading to self-assembly of peptide helices in the membrane. An example of a self-assembled structure is the tetrameric transmembrane pore of the influenza virus M2 protein, which is an efficient and selective voltage-gated proton channel. Our simulations explain the gating mechanism and provide guidelines how to re-engineer the channel to act as a simple proton pump. In general, emergence of integral membrane proteins appears to be quite feasible and may be easier to envision than the emergence of water-soluble proteins.

Models, Chemical↗

Enabling Experimental Evolution: Multi-Parameter Sensor System Integration into a Culture/Stressor Biofluidics System

Experimental evolution (EE) exposes microbial communities to ecological stressors, simulating dynamics up to near-extinction events. Combined with comparative sequencing and other molecular tools, such data can inform the genetic and other biological mechanisms underlying extremophile adaptation, and other observed effects. Automating this type of experiment using biofluidics can mitigate many traditional obstacles, including delays in assay results and environment adjustment and the need for many replicates. A first-generation device for automating EE procedures, the Automated Adaptive Directed Evolution Chamber (AADEC), was developed at NASA Ames. UV-C radiation was the stressor, an LED-photodiode array measured optical density, magnetic agitation and peristaltic pump systems ensured nutrient availability, and Arduino microcontrollers provided control. Escherichia coli in LB kanamycin media was used for testing and performance verification. A manual laboratory procedure with timed exposure to UV-C was performed to typify tolerance acquisition. Approximately a 106 factor increase in survival ratio was recorded over multiple iterations. Currently, a second-generation device is being developed integrating more real-time sensors: redox potential (ORP), indicating available/consumed metabolic energy; dissolved oxygen (DO), indicating aerobic/anaerobic growth; pH, indicating metabolic products; and electrical conductivity (EC), another indicator of metabolic products. The EC sensor system was constructed and calibrated in-house and matched commercial sensors in the required range. A Raspberry Pi computer automated the electrical system, allowing real-time data acquisition. The fluidics card was made of CNC-milled polycarbonate for biocompatibility. Each sensor parameter can also be used as a selection pressure alone or in combination with others to create extreme microbial environments. As a proof of concept, this work demonstrated sensor operation in one pair of growth-sensor chambers. It can be expanded to a multi-chamber system to enable inter-culture comparisons and multi-population studies. The prior Arduino system will be ported to the RPi system. Future stressors to be added include thermal, reactive oxygen species, and varying nutrient availability.

Govinda Raj, Chinmayee↗

BioSentinel: Optimizing Growth Conditions for Improved Yeast Cell Viability After Long-Term Desiccation

NASA's BioSentinel mission is one of thirteen secondary payloads to be deployed on the Space Launch System Exploration Mission-1 (SLS EM-1). The BioSentinel nanosatellite will be sent into a heliocentric orbit beyond Low Earth Orbit (LEO), to study the effects of deep space radiation on the budding yeast, Saccharomyces cerevisiae. Ionizing radiation encountered in deep space can create damaging lesions in DNA, including double strand breaks (DSBs). Budding yeast is suitable as a biological model to study these effects, as it is eukaryotic, and can be desiccated for prolonged periods while retaining viability, thus serving as a robust analog for human cells. On the ground, yeast cells are grown in liquid medium, then loaded into the wells of microfluidic cards and air dried prior to integration into the payload. Once the spacecraft reaches its target heliocentric orbit, a mixture of growth medium and metabolic indicator dye will be pumped into the microwells at specific time points to rehydrate the cells and allow them to grow. A 3-color LED detection system will measure changes in growth and metabolism resulting from ionizing radiation exposure. BioSentinel contains a wild type control strain and a rad51 mutant that is defective for DNA damage repair. In this study, we will determine the optimal amount of time to grow diploid yeast cells in liquid culture before they are desiccated for space flight. After an extended time in stationary phase, they become more tolerant to desiccation due to stress caused by nitrogen starvation. However, excessive exposure can lead to loss of viability and to a heterogeneous cell population due to sporulation. Since viability loss during desiccation poses a risk to mission success, a stress preconditioning process during initial growth may increase long-term cell viability. To determine the growth period that improves desiccation tolerance but allows for retention of uniform radiation sensitivity, we will grow both strains in liquid medium for a varying number of days (4 to 7), desiccate the cells, and then observe changes to cell viability and ionizing radiation sensitivity over time. Supported by the Space Life Sciences Training Program at NASA Ames Research Center.

Cui, Kaixin↗

Investigation of evolution-related aspects of bacterial rhodopsins

We have investigated evolution-related aspects of bacterial rhodopsins, the unique retinal-based energy transducing systems of halophilic archae. The approach was to describe both structural and functional aspects: the structure by sequencing genes to explore which regions are conserved, and the function by comparing proton and chloride transport in the closely related systems, bacteriorhodopsin and halorhodopsin, respectively. In the latter, we have made a good start toward the ultimate goal of separating the attributes of the general principles of retinal-based ionic pumps from those of the specific ion specificities, by determining the thermodynamics of the internal steps of the protein-mediated active transport process, as well as some of the intraprotein ion-transfer steps. Our present emphasis is on continuing to acquire the tools for studying what distinguishes proton transport from chloride transport. We consider it important, therefore, that we have been able to provide firm mathematical grounds for the kinetics analyses which underlies these studies. Our molecular biological studies have received a great boost from the expression vector for the bop gene based on a halobacterial plasmid, that we recently developed.

Source record↗

Performance of the CELSS Antarctic Analog Project (CAAP) Crop Production System

Regenerative life support systems potentially offer a level of self-sufficiency and a concomitant decrease in logistics and associated costs in support of space exploration and habitation missions. Current state-of-the-art in plant based, regenerative life support requires resources in excess of resource allocations proposed for candidate mission scenarios. Feasibility thresholds have been identified for candidate exploration missions. The goal of this paper is to review recent advances in performance achieved in the CELSS Antarctic Analog Project (CAAP) in light of likely resource constraints. A prototype CAAP crop production chamber has been constructed and operated at the Ames Research Center. The chamber includes a number of unique hardware and software components focused on attempts to increase production efficiency, increase energy efficiency, and control the flow of energy and mass through the system to achieve enhanced performance efficiency. Both single crop, batch production, and continuous cultivation of mixed crops Product ion scenarios have been completed. The crop productivity as well as engineering performance of the chamber will be described. For each scenario, energy required and partitioned for lighting, cooling, pumps, fans, etc. is quantified. Crop production and the resulting lighting efficiency and energy conversion efficiencies are presented. In the mixed-crop scenario, with up to 25 different crops under cultivation, 17 sq m of crop area provided a mean of 515 g edible biomass per day (83% of the approximately 620 g required for one person). Lighting efficiency (moles on photons kWh-1) approached 4 and the conversion efficiency of light energy to biomass was greatly enhanced compared with conventional growing systems. Engineering and biological performance achieved place plant-based life support systems at the threshold of feasibility.

Bubenheim, David L.↗

Applications of aerospace technology in biology and medicine

Utilization of National Aeronautics and Space Administration (NASA) technology in medicine is discussed. The objective is best obtained by stimulation of the introduction of new or improved commercially available medical products incorporating aerospace technology. A bipolar donor/recipient model of medical technology transfer is presented to provide a basis for the team's methodology. That methodology is designed to: (1) identify medical problems and NASA technology that, in combination, constitute opportunities for successful medical products; (2) obtain the early participation of industry in the transfer process; and (3) obtain acceptance by the medical community of new medical products based on NASA technology. Two commercial transfers were completed: the Stowaway, a lightweight wheelchair that provides mobility for the disabled and elderly in the cabin of commercial aircraft, and Micromed, a portable medication infusion pump for the reliable, continuous infusion of medications such as heparin or insulin. The marketing and manufacturing factors critical to the commercialization of the lightweight walker incorporating composite materials were studied. Progress was made in the development and commercialization of each of the 18 currently active projects.

Bass, B.↗

Model study of ATP and ADP buffering, transport of Ca(2+) and Mg(2+), and regulation of ion pumps in ventricular myocyte

We extended the model of the ventricular myocyte by Winslow et al. (Circ. Res 1999, 84:571-586) by incorporating equations for Ca(2+) and Mg(2+) buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na(+)-K(+) pump, sarcolemmal and sarcoplasmic Ca(2+) pumps). The results indicate that, under normal conditions, Ca(2+) binding by low-affinity ATP and diffusion of CaATP may affect the amplitude and time course of intracellular Ca(2+) signals. The model also suggests that a fall in ATP/ADP ratio significantly reduces sarcoplasmic Ca(2+) content, increases diastolic Ca(2+), lowers systolic Ca(2+), increases Ca(2+) influx through L-type channels, and decreases the efficiency of the Na(+)/Ca(2+) exchanger in extruding Ca(2+) during periodic voltage-clamp stimulation. The analysis suggests that the most important reason for these changes during metabolic inhibition is the down-regulation of the sarcoplasmic Ca(2+)-ATPase pump by reduced diastolic MgATP levels. High Ca(2+) concentrations developed near the membrane might have a greater influence on Mg(2+), ATP, and ADP concentrations than that of the lower Ca(2+) concentrations in the bulk myoplasm. The model predictions are in general agreement with experimental observations measured under normal and pathological conditions.

NASA Discipline Cardiopulmonary↗

Subtropical Gyre Variability Observed by Ocean Color Satellites

The subtropical gyres of the world are extensive, coherent regions that occupy about 40% of the surface of the earth. Once thought to be homogeneous and static habitats, there is increasing evidence that mid-latitude gyres exhibit substantial physical and biological variability on a variety of time scales. While biological productivity within these oligotrophic regions may be relatively small, their immense size makes their total contribution significant. Global distributions of dynamic height derived from satellite altimeter data, and chlorophyll concentration derived from satellite ocean color data, show that the dynamic center of the gyres, the region of maximum dynamic height where the thermocline is deepest, does not coincide with the region of minimum chlorophyll concentration. The physical and biological processes by which this distribution of ocean properties is maintained, and the spatial and temporal scales of variability associated with these processes, are analyzed using global surface chlorophyll-a concentrations, sea surface height, sea surface temperature and surface winds from operational satellite and meteorological sources, and hydrographic data from climatologies and individual surveys. Seasonal and interannual variability in the areal extent of the subtropical gyres are examined using 8 months (November 1996 - June 1997) of OCTS and nearly 5 years (September 1997 - June 02) of SeaWiFS ocean color data and are interpreted in the context of climate variability and measured changes in other ocean properties (i.e., wind forcing, surface currents, Ekman pumping, and vertical mixing). The North Pacific and North Atlantic gyres are observed to be shrinking over this period, while the South Pacific, South Atlantic, and South Indian Ocean gyres appear to be expanding.

McClain, Charles R.↗

The phytoplankton bloom in the northwestern Arabian Sea during the southwest monsoon of 1979

The present study investigates the biological variability of the northwestern Arabian Sea during the 1979 southwest monsoon by the synthesis of satellite ocean color remote sensing with an analysis of in situ hydrographic and meteorological data sets and the results of wind-driven modeling of upper-ocean circulation. The phytoplankton bloom peaked during August-September, extended from the Oman coast to about 65 deg E, and lagged behind the development of open-sea upwelling by at least 1 mo. The pigment distributions, hydrographic data, and model results all suggest that the boom was driven by spatially distinct upward nutrient fluxes to the euphotic zone forced by the physical processes of coastal upwelling and offshore Ekman pumping. Coastal upwelling was evident from May through September, yielded the most extreme concentrations of phytoplankton biomass, and, along the Arabian coast, was limited to the continental shelf in the promotion of high concentrations of phytoplankton.

Brock, John C.↗

Sample Processor for Life on Icy Worlds (SPLIce): Design and Test Results

We report the design, development, and testing of the Sample Processor for Life on Icy Worlds (SPLIce) system, a microfluidic sample processor to enable autonomous detection of signatures of life and measurements of habitability parameters in Ocean Worlds. This monolithic fluid processing-and-handling system (Figure 1; mass 0.5 kg) retrieves a 50-L-volume sample and prepares it to supply a suite of detection instruments, each with unique preparation needs. SPLIce has potential applications in orbiter missions that sample ocean plumes, such as found in Saturns icy moon Enceladus, or landed missions on the surface of icy satellites, such as Jupiters moon Europa. Answering the question Are we alone in the universe? is captivating and exceptionally challenging. Even general criteria that define life very broadly include a significant role for water [1,2]. Searches for extinct or extant life therefore prioritize locations of abundant water whether in ancient (Mars), or present (Europa and Enceladus) times. Only two previous planetary missions had onboard fluid processing: the Viking Biology Experiments [3] and Phoenixs Wet Chemistry Laboratory (WCL) [4]. SPLIce differs crucially from those systems, including its capability to process and distribute L-volume samples and the integration autonomous control of a wide range of fluidic functions, including: 1) retrieval of fluid samples from an evacuated sample chamber; 2) onboard multi-year storage of dehydrated reagents; 3) integrated pressure, pH, and conductivity measurement; 4) filtration and retention of insoluble particles for microscopy; 5) dilution or vacuum-driven concentration of samples to accommodate instrument working ranges; 6) removal of gas bubbles from sample aliquots; 7) unidirectional flow (check valves); 8) active flow-path selection (solenoid-actuated valves); 9) metered pumping in 100 nL volume increments. The SPLIce manifold, made of three thermally fused layers of precision-machined cyclo-olefin polymer, supports all fluidic components (Figure 1) and integrated microchannels (125 x 250 m). Fluid is pumped by a stepper-motor-driven pump (Lee Co.). The functionality of the integrated MEMS pressure sensor (Honeywell) and passive check valves (Figure 2) were tested in conjunction with our newly designed integral bubble traps (Figure 3) and hydrophobic membrane-based concentrator (Figure 4). The concentrator (initially tested as a standalone component) demonstrated 5-fold vacuum-evaporative concentration. Polyethylene fused bead beds (PEFBBs; 50 porosity) store drylyophilized buffers, calibrants, and fluorescent dyes, and also promote mixing of sample with calibrant, dye, or H2O. Software-controlled automated tests demonstrated successful 1) fluid delivery to each component 2) valve and pump synchronization 3) sample aliquot delivery to instrument interface ports, and 4) rehydration of vacuum-dried fluorescent dye. In Figure 5, fluorescein on PEFBBs was rehydrated for 15 min using a pump-delivered water aliquot; it is displaced as H2O enters the bottom of the channel and pushes the dye into a check valve. Ultimately, SPLIce will fluorescently label amino acids in the sample for microchip-based electrophoretic (MCE) chiral separation and detection to seek and quantify key organic bio-signatures [5]; it will also deliver sample to a microfluidic version of WCL (mWCL) to measure soluble ions and redox-active species.

Life detection↗

Automated Fluidics Device for Extraction and Quantification of miRNA Biomarkers From Blood

Radiation Assessment DuRing Exposure And long-Duration Spaceflight (RADREADS) demonstrates space-compatible point-of-care technology for quantitative biological monitoring of blood miRNA biomarkers in response to long-term low dose radiation exposure. This individualized monitoring approach will inform targeted treatment strategies to maximize medical resource utilization by accounting for individual susceptibility to radiation-related illnesses. As human spaceflight progresses beyond Earth’s magnetic shielding, radiation exposure poses a significant risk to astronaut health and safety. Extended operation in this environment comes with an increased risk of radiation exposure, leading to higher risks of radiation sickness, cancer, central nervous system effects, and degenerative diseases. While conventional physical dosimetry techniques capture radiation dose, individualistic susceptibility to radiation damage is varied. Multiple characteristics, including age, body weight, sex, genetics, and immune status, have been found to influence radiosensitivity (Liu et al. 2011, and Bouffler 2016). This differential response necessitates individualized monitoring and targeted treatment strategies to maximize medical resource utilization; however, a practical diagnostic platform for quantifying long-term, low dose radiation-induced tissue damage does not currently exist. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression by mediating the degradation of messenger RNA. The levels of particular miRNAs are influenced by biological processes such as inflammation and serve as biomarkers for a variety of conditions including cancer (Singh et al. 2017). MicroRNAs are found in various bodily fluids and are amenable to collection via liquid biopsies, providing a minimally invasive and easily quantifiable readout for a variety of radiosensitive reporters. A preliminary signature of 15 spaceflight sensitive miRNA has been identified in rodent and human studies, including miR-21-5p, miR-24-3p, miR-92a-3p, miR-17-5p, miR-16a-3p, miR-34a-3p, and miR-223-3p. These targets generally increased expression with radiation dose and linear energy transfer, though variation between individuals is not yet described. Current gaps in the field include a lack of understanding of longitudinal biological responses to long-term, low dose radiation exposure and the absence of space-compatible point-of-care technology for quantitative biological monitoring. In this body of work, we aim to develop an automated bleed-to-read system to process whole blood for the detection of miRNA biomarkers in order to monitor individualistic responses to radiation exposure. This will be achieved via separating serum (or plasma) from whole blood, followed by extraction, amplification, and quantification of the miRNA using a RT-qPCR reaction. Previously, the WetLab-2 hardware enabled execution of a RT-qPCR reaction aboard ISS; however, it is a manual system that requires crew manipulation and bulky components (Parra et al. 2017). To address these issues, automated fluid handling hardware was developed for each stage of sample preparation. Extraction of total RNA is achieved by sequentially pumping reagents through an off-the-shelf nucleic acid binding column (miRNeasy Serum/Plasma Advanced Kit, Qiagen). This approach eliminates several manual pipetting and centrifuging steps and limits the use of toxic chemicals commonly found in other sample processing techniques. The resulting elution will then be automatically dispensed for RT-qPCR analysis using a compact rotary qPCR (Mic qPCR Cycler, Bio Molecular Systems) that will improve spaceflight compatibility by removing bubbles from the detection region, another challenge highlighted by WetLab-2 (Parra et al. 2017). Efforts are also being made to simplify the RT-qPCR reaction to a 1-step air-dryable mix to improve long-term reagent stability at room temperature and reduce system complexity. By automating the RT-qPCR processes via microfluidic manipulation, RADREADS will reduce crewmember hands-on time and enable the personalized detection of radiation-induced tissue damage during long duration missions. Minimally invasive, longitudinal monitoring of individual’s response to radiation exposure will inform how the physiological system responds to long-term low dose space radiation and enables development of targeted countermeasures by the medical team. Ultimately, this portable technology will require minimal technical expertise and can also be used to monitor miRNA biomarkers associated with other diseases.

Tristen Head↗

The monitoring system for vibratory disturbance detection in microgravity environment aboard the international space station

Scientists in the Office of Life and Microgravity Sciences and Applications within the Microgravity Research Division oversee studies in important physical, chemical, and biological processes in microgravity environment. Research is conducted in microgravity environment because of the beneficial results that come about for experiments. When research is done in normal gravity, scientists are limited to results that are affected by the gravity of Earth. Microgravity provides an environment where solid, liquid, and gas can be observed in a natural state of free fall and where many different variables are eliminated. One challenge that NASA faces is that space flight opportunities need to be used effectively and efficiently in order to ensure that some of the most scientifically promising research is conducted. Different vibratory sources are continually active aboard the International Space Station (ISS). Some of the vibratory sources include crew exercise, experiment setup, machinery startup (life support fans, pumps, freezer/compressor, centrifuge), thruster firings, and some unknown events. The Space Acceleration Measurement System (SAMs), which acts as the hardware and carefully positioned aboard the ISS, along with the Microgravity Environment Monitoring System MEMS), which acts as the software and is located here at NASA Glenn, are used to detect these vibratory sources aboard the ISS and recognize them as disturbances. The various vibratory disturbances can sometimes be harmful to the scientists different research projects. Some vibratory disturbances are recognized by the MEMS's database and some are not. Mainly, the unknown events that occur aboard the International Space Station are the ones of major concern. To better aid in the research experiments, the unknown events are identified and verified as unknown events. Features, such as frequency, acceleration level, time and date of recognition of the new patterns are stored in an Excel database. My task is to carefully synthesize frequency and acceleration patterns of unknown events within the Excel database into a new file to determine whether or not certain information that is received i s considered a real vibratory source. Once considered as a vibratory source, further analysis is carried out. The resulting information is used to retrain the MEMS to recognize them as known patterns. These different vibratory disturbances are being constantly monitored to observe if, in any way, the disturbances have an effect on the microgravity environment that research experiments are exposed to. If the disturbance has little or no effect on the experiments, then research is continued. However, if the disturbance is harmful to the experiment, scientists act accordingly by either minimizing the source or terminating the research and neither NASA's time nor money is wasted.

Laster, Rachel M.↗

Progress toward an explicit mechanistic model for the light-driven pump, bacteriorhodopsin

Recent crystallographic information about the structure of bacteriorhodopsin and some of its photointermediates, together with a large amount of spectroscopic and mutational data, suggest a mechanistic model for how this protein couples light energy to the translocation of protons across the membrane. Now nearing completion, this detailed molecular model will describe the nature of the steric and electrostatic conflicts at the photoisomerized retinal, as well as the means by which it induces proton transfers in the two half-channels leading to the two membrane surfaces, thereby causing unidirectional, uphill transport.

Review, Tutorial↗

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

Non-NASA Center↗

Physical processes affecting availability of dissolved silicate for diatom production in the Arabian Sea

A passive tracer to represent dissolved silicate concentrations, with biologically realistic uptake kinetics, is successfully incorporated into a three-dimensional, eddy-resolving, ocean circulation model of the Indian Ocean. Hypotheses are tested to evaluate physical processes which potentially affect the availability of silicate for diatom production in the Arabian Sea. An alternative mechanism is offered to the idea that open ocean upwelling is primarily responsible for the high, vertical nutrient flux and consequent large-scale phytoplankton bloom in the northwestern Arabian Sea during the southwest monsoon. Model results show that dissolved silicate in surface waters available for uptake by diatoms is primarily influenced by the intensity of nearshore upwelling from soutwest monsoonal wind forcing and by the offshore advective transport of surface waters. The upwelling, which in the model occurs within 200 +/- 50 km of the coast, appears to be a result of a combination of coastal upwelling, Elkman pumping, and divergence of the coastal flow as it turns offshore. Localized intensifications of silicate concentrations appear to be hydrodynamically driven and geographically correlated to coastal topographic features. The absence of diatoms in sediments of the eastern Arabian Basin is consistent with modeled distributional patterns of dissolved silicate resulting from limited westward advection of upwelled coastal waters from the western continental margin of India and rapid uptake of available silicate by diatoms. Concentrations of modeled silicate become sufficiently low to become unavailable for diatom production in the eastern Arabian Sea, a region between 61 deg E and 70 deg E at 8 deg N on the south, with the east and west boundaries converging on the north at approximately 67 deg E, 20 deg N.

Young, David K.↗

NMR studies on Na+ transport in Synechococcus PCC 6311

The freshwater cyanobacterium Synechococcus PCC 6311 is able to adapt to grow after sudden exposure to salt (NaCl) stress. We have investigated the mechanism of Na+ transport in these cells during adaptation to high salinity. Na+ influx under dark aerobic conditions occurred independently of delta pH or delta psi across the cytoplasmic membrane, ATPase activity, and respiratory electron transport. These findings are consistent with the existence of Na+/monovalent anion cotransport or simultaneous Na+/H+ +anion/OH- exchange. Na+ influx was dependent on Cl-, Br-, NO3-, or NO2-. No Na+ uptake occurred after addition of NaI, NaHCO3, or Na2SO4. Na+ extrusion was absolutely dependent on delta pH and on an ATPase activity and/or on respiratory electron transport. This indicates that Na+ extrusion via Na+/H+ exchange is driven by primary H+ pumps in the cytoplasmic membrane. Cells grown for 4 days in 0.5 m NaCl medium, "salt-grown cells," differ from control cells by a lower maximum velocity of Na+ influx and by lower steady-state ratios of [Na+]in/[Na+]out. These results indicate that cells grown in high-salt medium increase their capacity to extrude Na+. During salt adaptation Na+ extrusion driven by respiratory electron transport increased from about 15 to 50%.

Non-NASA Center↗