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At least 199 records · Page 11

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NASA NTRS1973

Effects of oxygen-augmented atmosphere on the immune response.

Antibovine serum albumin antibody and nonspecific protein production was evaluated in female rabbits (11-14.5 kg) housed in special cages ventilated with 20% or 40% oxygen at normal barometric pressure. Animals exposed to 40% oxygen do not show normal steady increase of serum antibody. Instead, their titers show a pattern of undershoot, overshoot, undershoot, and finally equilibration at a subnormal level; they have a depressed popliteal node polysome level and have an abnormally low proportion of membrane-bound polysomes. They also show reduced capability of popliteal nodes to synthesize protein (as expected from the reduced number of polysomes). However, the ratio of newly-synthesized specific antibody: nonspecific protein remains normal.

Guttman, H. N.↗

NASA NTRS1983

Denervation and reinnervation of skeletal muscle

A review is presented of the physiological and biochemical changes that occur in mammalian skeletal muscle after denervation and reinnervation. These changes are compared with those observed after altered motor function. Also considered is the nature of the trophic influence by which nerves control muscle properties. Topics examined include the membrane and contractile properties of denervated and reinnervated muscle; the cholinergic proteins, such as choline acetyltransferase, acetylcholinesterase, and the acetylcholine receptor; and glucose-6-phosphate dehydrogenase.

Mayer, R. F.↗

NASA NTRS2003

Discrimination of Single Base Pair Differences Among Individual DNA Molecules Using a Nanopore

The protein toxin alpha-hemolysin form nanometer scale channels across lipid membranes. Our lab uses a single channel in an artificial lipid bilayer in a patch clamp device to capture and examine individual DNA molecules. This nanopore detector used with a support vector machine (SVM) can analyze DNA hairpin molecules on the millisecond time scale. We distinguish duplex stem length, base pair mismatches, loop length, and single base pair differences. The residual current fluxes also reveal structural molecular dynamics elements. DNA end-fraying (terminal base pair dissociation) can be observed as near full blockades, or spikes, in current. This technique can be used to investigate other biological processes dependent on DNA end-fraying, such as the processing of HIV DNA by HIV integrase.

Vercoutere, Wenonah↗

NASA NTRS2017

Small Ion Channel Linking Molecular Simulations and Electrophysiology

Ion channels are pore-forming protein assemblies that mediate the transport of small ions across cell membranes. Otherwise, membrane bilayers would be almost impermeable to ions incapable to traverse the low dielectric constant, hydrophobic membrane core. Ion channels are ubiquitous to all life forms. In humans and other higher organisms they play the central role in conducting nerve impulses, cardiac functions, muscle contraction and apoptosis. On the other extreme of biological complexity, viral ion channels (viroporins) influence many stages of the virus infection cycle either through regulating virus replication, such as entry, assembly and release or modulating the electrochemical balance in the subcellular compartments of host cells. Ion channels were crucial components of protocells. Their emergence facilitated adaptation of nascent life to different environmental conditions. The earliest ion channels must have been much simpler than most of their modern ancestors. Viral channels are among only a few naturally occurring models to study the structure, function and evolution of primordial channels. Experimental studies of these properties are difficult and often unreliable. In principle, computational methods, and molecular dynamics (MD) simulations in particular, can aid in providing information about both the structure and the function of ion channels. However, MD suffers from its own problems, such as inability to access sufficiently long time scales or limited accuracy of force fields. It is, therefore, essential to determine the reliability of MD simulations. We propose to do so on the basis of two criteria. One is channel stability on time scales that extend for several microseconds or longer. The other is the ability to reproduce the measured ionic conductance as a function of applied voltage. If both the stability and the calculated ionic conductance are satisfactory it will greatly increase our confidence that the structure and the function of a channel are described sufficiently accurately. To our knowledge, long time scale stability (approx.10 micro-sec) and the correct electrophysiology have been shown so far for only one channel - the synthetic LS3 hexamer). In this presentation, this approach will be discussed in application to two viral channels - Vpu, encoded by the HIV-1 genome and p7 of hepatitis C.

Pohorille, Andrzej↗

NASA NTRS2000

Quantitative image analysis of laminin immunoreactivity in skin basement membrane irradiated with 1 GeV/nucleon iron particles

We previously reported that laminin immunoreactivity in mouse mammary epithelium is altered shortly after whole-body irradiation with 0.8 Gy from 600 MeV/nucleon iron ions but is unaffected after exposure to sparsely ionizing radiation. This observation led us to propose that the effect could be due to protein damage from the high ionization density of the ion tracks. If so, we predicted that it would be evident soon after radiation exposure in basement membranes of other tissues and would depend on ion fluence. To test this hypothesis, we used immunofluorescence, confocal laser scanning microscopy, and image segmentation techniques to quantify changes in the basement membrane of mouse skin epidermis. At 1 h after exposure to 1 GeV/nucleon iron ions with doses from 0.03 to 1.6 Gy, neither the visual appearance nor the mean pixel intensity of laminin in the basement membrane of mouse dorsal skin epidermis was altered compared to sham-irradiated tissue. This result does not support the hypothesis that particle traversal directly affects laminin protein integrity. However, the mean pixel intensity of laminin immunoreactivity was significantly decreased in epidermal basement membrane at 48 and 96 h after exposure to 0.8 Gy 1 GeV/nucleon iron ions. We confirmed this effect with two additional antibodies raised against affinity-purified laminin 1 and the E3 fragment of the long-arm of laminin 1. In contrast, collagen type IV, another component of the basement membrane, was unaffected. Our studies demonstrate quantitatively that densely ionizing radiation elicits changes in skin microenvironments distinct from those induced by sparsely ionizing radiation. Such effects may might contribute to the carcinogenic potential of densely ionizing radiation by altering cellular signaling cascades mediated by cell-extracellular matrix interactions.

Non-NASA Center↗

NASA NTRS2002

Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells

During normal and pathological conditions, endothelial cells (ECs) are subjected to locally generated reactive oxygen species, produced by themselves or by other vessel wall cells. In excess these molecules cause oxidative injury to the cell but at moderate levels they might modulate intracellular signalling pathways. We have investigated the effect of oxidative stress on the palmitoylation and trafficking of caveolin-1 in bovine aortic ECs. Exogenous H2O2 did not alter the intracellular localization of caveolin-1 in ECs. However, metabolic labelling experiments showed that H2O2 inhibited the trafficking of newly synthesized caveolin-1 to membrane raft domains. Several mechanisms potentially responsible for this inhibition were examined. Impairment of caveolin-1 synthesis by H2O2 was not responsible for diminished trafficking. Similarly, the inhibition was independent of H2O2-induced caveolin-1 phosphorylation as shown by the markedly different concentration dependences. We tested the effect of H2O2 on palmitoylation of caveolin-1 by the incorporation of [3H]palmitic acid. Exposure of ECs to H2O2 markedly inhibited the palmitoylation of caveolin-1. Comparable inhibition was observed after treatment of cells with H2O2 delivered either as a bolus or by continuous delivery with glucose and glucose oxidase. Kinetic studies showed that H2O2 did not alter the rate of caveolin-1 depalmitoylation but instead decreased the 'on-rate' of palmitoylation. Together these results show for the first time the modulation of protein palmitoylation by oxidative stress, and suggest a cellular mechanism by which stress might influence caveolin-1-dependent cell activities such as the concentration of signalling proteins and cholesterol trafficking.

NASA Discipline Cell Biology↗

NASA NTRS1990

Mechanism of transport and distribution of organic solvents in blood

Little is known about the mechanism of transport and distribution of volatile organic compounds in blood. Studies were conducted on five typical organic solvents to investigate how these compounds are transported and distributed in blood. Groups of four to five rats were exposed for 2 hr to 500 ppm of n-hexane, toluene, chloroform, methyl isobutyl ketone (MIBK), or diethyl ether vapor; 94, 66, 90, 51, or 49%, respectively, of these solvents in the blood were found in the red blood cells (RBCs). Very similar results were obtained in vitro when aqueous solutions of these solvents were added to rat blood. In vitro studies were also conducted on human blood with these solvents; 66, 43, 65, 49, or 46%, respectively, of the added solvent was taken up by the RBCs. These results indicate that RBCs from humans and rats exhibited substantial differences in affinity for the three more hydrophobic solvents studied. When solutions of these solvents were added to human plasma and RBC samples, large fractions (51-96%) of the solvents were recovered from ammonium sulfate-precipitated plasma proteins and hemoglobin. Smaller fractions were recovered from plasma water and red cell water. Less than 10% of each of the added solvents in RBC samples was found in the red cell membrane ghosts. These results indicate that RBCs play an important role in the uptake and transport of these solvents. Proteins, chiefly hemoglobin, are the major carriers of these compounds in blood. It can be inferred from the results of the present study that volatile lipophilic organic solvents are probably taken up by the hydrophobic sites of blood proteins.

Solvents/administration & dosage/metabolism/pharma↗

NASA NTRS1992

Thin-Membrane Sensor With Biochemical Switch

Modular sensor electrochemically detects chemical or biological agent, indicating presence of agent via gate-membrane-crossing ion current triggered by chemical reaction between agent and recognition protein conjugated to channel blocker. Used in such laboratory, industrial, or field applications as detection of bacterial toxins in food, military chemical agents in air, and pesticides or other contaminants in environment. Also used in biological screening for hepatitis, acquired immune-deficiency syndrome, and like.

Case, George D.↗

NASA NTRS1992

Determination of monomer concentrations in crystallizing lysozyme solutions

We have developed a non-optical technique for the study of aggregation in lysozyme and other protein solutions. By monitoring the rate at which lysozyme traverses a semipermeable membrane it was possible to quantitate the degree of aggregation in supersaturated solutions. Using this technique, we have measured the concentration of monomers and larger aggregates in under- and oversaturated lysozyme solutions, and in the presence of crystals, at pH 4.0 and 3 percent NaCl (0.1M NaAc). Comparison of these concentration profiles with (110) face growth rate data supports the theory that tetragonal lysozyme crystals grow by addition of preformed aggregates and not by monomer addition. The data suggest that a considerable population of aggregates larger than dimers are present at lysozyme concentrations above 22 mg/ml. Determination of dimer concentrations, and equilibrium constants for subsequent aggregation levels, are currently underway.

Wilson, L. J.↗

NASA NTRS1994

More About Thin-Membrane Biosensor

Report presents additional information about device described in "Thin-Membrane Sensor With Biochemical Switch" (MFS-26121). Device is modular sensor that puts out electrical signal indicative of chemical or biological agent. Signal produced as membrane-crossing ion current triggered by chemical reaction between agent and recognition protein conjugated to channel blocker. Prototype of biosensor useful in numerous laboratory, industrial, or field applications; such as to detect bacterial toxins in food, to screen for disease-producing micro-organisms, or to warn of toxins or pollutants in air.

Case, George D.↗

NASA NTRS2002

The Effect of Microgravity on the Smallest Space Travelers: Bacterial Physiology and Virulence on Earth and in Microgravity

Since the first human flights outside of Earth's gravity, crew health and well-being have been major concerns. Exposure to microgravity during spaceflight is known to affect the human immune response, possibly making the crew members more vulnerable to infectious disease. In addition, biological experiments previously flown in space have shown that bacteria grow faster in microgravity than they do on Earth. The ability of certain antibiotics to control bacterial infections may also differ greatly in microgravity. It is therefore critical to understand how spaceflight and microgravity affect bacterial virulence, which is their ability to cause disease. By utilizing spaceflight hardware provided by the European Space Agency (ESA), Dr. Barry Pyle and his team at Montana State University, Bozeman, will be performing an experiment to study the effects of microgravity on the virulence of a common soil and water bacterium, Pseudomonas aeruginosa. Importantly, these bacteria have been detected in the water supplies of previous Space Shuttle flights. The experiment will examine the effects of microgravity exposure on bacterial growth and on the bacterium's ability to form a toxin called Exotoxin A. Another goal is to evaluate the effects of microgravity on the physiology of the bacteria by analyzing their ability to respire (produce energy), by studying the condition of the plasma membrane surrounding the cell, and by determining if specific enzymes remain active. Proteins produced by the bacteria will also be assayed to see if the normal functions of the bacteria are affected. In the context of human life support in spaceflight, the results of this experiment will offer guidance in providing the highest possible water quality for the Shuttle in order to limit the risk of infection to human occupants and to minimize water system and spacecraft deterioration.

Pyle, Barry↗

NASA NTRS2001

Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells

Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

NASA Discipline Cell Biology↗

NASA NTRS1993

The mechanosensory calcium-selective ion channel: key component of a plasmalemmal control centre?

Mechanosensory calcium-selective ion channels probably serve to detect not only mechanical stress but also electrical, thermal, and diverse chemical stimuli. Because all stimuli result in a common output, most notably a shift in second messenger calcium concentration, the channels are presumed to serve as signal integrators. Further, insofar as second messenger calcium in turn gives rise to mechanical, electrical, and diverse chemical changes, the channels are postulated to initiate regulatory feedbacks. It is proposed that the channels and the feedback loops play a wide range of roles in regulating normal plant function, as well as in mediating disturbance of normal function by environmental stressors and various pathogens. In developing evidence for the physiological performance of the channel, a model for a cluster of regulatory plasmalemmal proteins and cytoskeletal elements grouped around a set of wall-to-membrane and transmembrane linkers has proved useful. An illustration of how the model might operate is presented. It is founded on the demonstration that several xenobiotics interfere both with normal channel behaviour and with gravitropic reception. Accordingly, the first part of the illustration deals with how the channels and the control system within which they putatively operate might initiate gravitropism. Assuming that gravitropism is an asymmetric expression of growth, the activities of the channels and the plasmalemmal control system are extrapolated to account for regulation of both rate and allometry of cell expansion. Finally, it is discussed how light, hormones, redox agents and herbicides could in principle affect growth via the putative plasmalemmal control cluster or centre.

NASA Program Space Biology↗

NASA NTRS2004

Oxygen Effect on the Low Temperature Tolerance of Facultative Anaerobes from Antarctica, Alaska, and Patagonia

Psychrotolerance as an adaptation to survival in extreme environments is widespread among many of the mesophilic microorganisms. Red-ox potential, pH and salinity could significantly alter the features of ecosystems by providing liquid water at subzero temperatures. Furthermore, organisms can respond to temperature changes by several known mechanisms, including changing the conformation capacities of constitutional proteins or by the synthesis of mucopolysaccharides around the cell wall and membrane. Such protective mechanisms make it possible for cells to not only passively survive low-temperature in a state of anabiosis, but also to be capable of actively metabolizing substrates and reproducing normally. The physiological and biochemical characteristics of species as well as genetics could be remarkably changed due to -on and surviving m extreme environments. The cold shock genes for some of the studied strains of psychrotolerant facultative anaerobes already were published In this paper we present experimental data for psychrotolerant facultative anaerobes isolated from geographically different cold regions of our planet. We show the growth response on the changing of anaerobic conditions to aerobic with cultivation at subzero temperatures.

Pikuta, Elena V.↗

NASA NTRS2004

Growth of the Facultative Anaerobes from Antarctica, Alaska, and Patagonia at Low Temperatures

Psychotolerance, as an adaptation for surviving in extreme environments, is widespread among mesophilic microorganisms. Physico-chemical factors such as pressure, red-ox potential, pH and salinity could significantly alter the features of ecosystems by providing liquid water at subzero temperatures. Furthermore, organisms can respond to temperature changes by several known mechanisms, including changing the conformation capacities of constitutional proteins or by the synthesis of mucopolysaccharides around the cell wall and membrane. Such protective mechanisms make it possible for cells to not only passively survive low temperatures in a state of anabiosis, but also to be capable of actively metabolizing substrates and reproducing normally. The physiological and biochemical characteristics of the species, as well as genetics, could be remarkably changed due to adaptation and surviving in extreme environments. The cold shock genes of some of the studied strains of psychotolerant facultative anaerobes were reported previously. In this paper we present experimental data for psychotolerant, non spore-forming, facultative anaerobes isolated from geographically different cold regions of our planet. We show the growth response on changing from anaerobic conditions to aerobic with cultivation at low temperatures.

Pikuta, Elena V.↗

NASA NTRS1999

Plasma membrane ordering agent pluronic F-68 (PF-68) reduces neurotransmitter uptake and release and produces learning and memory deficits in rats

A substantial body of evidence indicates that aged-related changes in the fluidity and lipid composition of the plasma membrane contribute to cellular dysfunction in humans and other mammalian species. In the CNS, reductions in neuronal plasma membrane order (PMO) (i.e., increased plasma membrane fluidity) have been attributed to age as well as the presence of the beta-amyloid peptide-25-35, known to play an important role in the neuropathology of Alzheimer's disease (AD). These PMO increases may influence neurotransmitter synthesis, receptor binding, and second messenger systems as well as signal transduction pathways. The effects of neuronal PMO on learning and memory processes have not been adequately investigated, however. Based on the hypothesis that an increase in PMO may alter a number of aspects of synaptic transmission, we investigated several neurochemical and behavioral effects of the membrane ordering agent, PF-68. In cell culture, PF-68 (nmoles/mg SDS extractable protein) reduced [3H]norepinephrine (NE) uptake into differentiated PC-12 cells as well as reduced nicotine stimulated [3H]NE release. The compound (800-2400 microg/kg, i.p., resulting in nmoles/mg SDS extractable protein in the brain) decreased step-through latencies and increased the frequencies of crossing into the unsafe side of the chamber in inhibitory avoidance training. In the Morris water maze, PF-68 increased the latencies and swim distances required to locate a hidden platform and reduced the time spent and distance swam in the previous target quadrant during transfer (probe) trials. PF-68 did not impair performance of a well-learned working memory task, the rat delayed stimulus discrimination task (DSDT), however. Studies with 14C-labeled PF-68 indicated that significant (pmoles/mg wet tissue) levels of the compound entered the brain from peripheral (i.p.) injection. No PF-68 related changes were observed in swim speeds or in visual acuity tests in water maze experiments, rotorod performance, or in tests of general locomotor activity. Furthermore, latencies to select a lever in the DSDT were not affected. These results suggest that PF-68 induced deficits in learning and memory without confounding peripheral motor, sensory, or motivational effects at the tested doses. Furthermore, none of the doses induced a conditioned taste aversion to a novel 0.1% saccharin solution indicating a lack of nausea or gastrointestinal malaise induced by the compound. The data indicate that increases in neuronal plasma membrane order may have significant effects on neurotransmitter function as well as learning and memory processes. Furthermore, compounds such as PF-68 may also offer novel tools for studying the role of neuronal PMO in mnemonic processes and changes in PMO resulting from age-related disorders such as AD.

Poloxamer/pharmacology↗

NASA NTRS1987

Experimental and theoretical investigation of the stability of stepwise pH gradients in continuous flow electrophoresis

Isoelectric focusing in the continuous flow mode can be more quickly and economically performed by admitting a stepwise pH gradient composed of simple buffers instead of uniform mixtures of synthetic carrier ampholytes. The time-consuming formation of the pH gradient by the electric field is thereby omitted. The stability of a three-step system with arginine - morpholinoethanesulfonic acid/glycylglycine - aspartic acid is analyzed theoretically by one-dimensional computer simulation as well as experimentally at various flow rates in a continuous flow apparatus. Excellent agreement between experimental and theoretical data was obtained. This metastable configuration was found to be suitable for focusing of proteins under continuous flow conditions. The influence of various combinations of electrolytes and membranes between electrophoresis chamber and electrode compartments is also discussed.

Kuhn, Reinhard↗

NASA NTRS1991

Photosynthetic reaction center complexes from heliobacteria

The goal of this project is to understand the early evolutionary development of photosynthesis by examining the properties of reaction centers isolated from certain contemporary organisms that appear to contain the simplest photosynthetic reaction centers. The major focus of this project is the family of newly discovered strictly anaerobic photosynthetic organisms known as Heliobacteria. These organisms are the only known photosynthetic organisms that are grouped with the gram-positive phylum of bacteria. The properties of these reaction centers suggest that they might be the decendants of an ancestor that also gave rise to Photosystem 1 found in oxygen-evolving photosynthetic organisms. Photoactive reaction center-core antenna complexes have been isolated from the photosynthetic bacteria Heliobacillus mobilis and Heliobacterium gestii. The absorption and fluorescence properties of membranes and reaction centers are almost identical, suggesting that a single pigment-protein complex serves as both antenna and reaction center. Experiments in progress include sequence determination of the 48,000 Mr reaction center protein, and evolutionary comparisons with other reaction center proteins.

Trost, J. T.↗