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NASA NTRS1998

Helicity, membrane incorporation, orientation and thermal stability of the large conductance mechanosensitive ion channel from E. coli

In this report, we present structural studies on the large conductance mechanosensitive ion channel (MscL) from E. coli in detergent micelles and lipid vesicles. Both transmission Fourier transform infrared spectroscopy and circular dichroism (CD) spectra indicate that the protein is highly helical in detergents as well as liposomes. The secondary structure of the proteins was shown to be highly resistant towards denaturation (25-95 degrees C) based on an ellipticity thermal profile. Amide H+/D+ exchange was shown to be extensive (ca. 66%), implying that two thirds of the protein are water accessible. MscL, reconstituted in oriented lipid bilayers, was shown to possess a net bilayer orientation using dichroic ratios measured by attenuated total-reflection Fourier transform infrared spectroscopy. Here, we present and discuss this initial set of structural data on this new family of ion-channel proteins.

NASA Discipline Cell Biology↗

NASA NTRS1997

Kinetics, Ca2+ dependence, and biophysical properties of integrin-mediated mechanical modulation of transmitter release from frog motor nerve terminals

Neurotransmitter release from frog motor nerve terminals is strongly modulated by change in muscle length. Over the physiological range, there is an approximately 10% increase in spontaneous and evoked release per 1% muscle stretch. Because many muscle fibers do not receive suprathreshold synaptic inputs at rest length, this stretch-induced enhancement of release constitutes a strong peripheral amplifier of the spinal stretch reflex. The stretch modulation of release is inhibited by peptides that block integrin binding of natural ligands. The modulation varies linearly with length, with a delay of no more than approximately 1-2 msec and is maintained constant at the new length. Moreover, the stretch modulation persists in a zero Ca2+ Ringer and, hence, is not dependent on Ca2+ influx through stretch activated channels. Eliminating transmembrane Ca2+ gradients and buffering intraterminal Ca2+ to approximately normal resting levels does not eliminate the modulation, suggesting that it is not the result of release of Ca2+ from internal stores. Finally, changes in temperature have no detectable effect on the kinetics of stretch-induced changes in endplate potential (EPP) amplitude or miniature EPP (mEPP) frequency. We conclude, therefore, that stretch does not act via second messenger pathways or a chemical modification of molecules involved in the release pathway. Instead, there is direct mechanical modulation of release. We postulate that tension on integrins in the presynaptic membrane is transduced mechanically into changes in the position or conformation of one or more molecules involved in neurotransmitter release, altering sensitivity to Ca2+ or the equilibrium for a critical reaction leading to vesicle fusion.

Non-NASA Center↗

NASA NTRS1996

Distinct transcripts are recognized by sense and antisense riboprobes for a member of the murine HSP70 gene family, HSP70.2, in various reproductive tissues

The expression of hsp70.2, an hsp70 gene family member, originally characterized by its high levels of expression in germ cells in the adult mouse testis, was detected in several other reproductive tissues, including epididymis, prostate, and seminal vesicles, as well as in extraembryonic tissues of mid-gestation fetuses. In addition, hybridization with RNA probes transcribed in the sense orientation surprisingly indicated the presence of slightly larger "antisense" transcripts in several tissues. The levels of antisense transcripts varied among the tissues, with the highest signal detected in the prostate and no signal being detectable in the testis. Consistent with these results, in situ hybridization analysis clearly localized the sense-orientation transcripts to pachytene spermatocytes, while no antisense-orientation transcripts were observed in adjacent sections of the same tubules. Our findings have thus shown that although hsp70.2 was expressed abundantly and in a highly stage-specific manner in the male germ line, it was also expressed in other murine tissues. Furthermore, we have made the surprising observation of antisense transcription of the hsp70.2 gene in several mouse tissues, revealing another level of complexity in the regulation and function of heat shock proteins.

Non-NASA Center↗

NASA NTRS1995

Development of the otolith organs and semicircular canals in the Japanese red-bellied newt, Cynops pyrrhogaster

The sequence in which the otoliths and semicircular canals and their associated sensory epithelia appear and develop in the newt are described. Three-dimensional reconstruction of serial sections through the otic vesicle of newt embryos from stages 31 through 58 demonstrate the first appearance, relative position and growth of the otoliths. A single otolith is first seen in stage 33 embryos (approximately 9 days old); this splits into separate utricular and saccular otoliths at stage 40 (13 days). The lateral semicircular canal is the first to appear, at stage 41 (14 days). The anterior and posterior canals appear approximately one week later and the vestibular apparatus is essentially fully formed at stage 58 (approximately 5 weeks). The data reported here will serve as ground-based controls for fertilized newt eggs flown on the International Microgravity Laboratory-2 Space Shuttle flight, to investigate the influence of microgravity on the development of the gravity-sensing organs.

NASA Discipline Neuroscience↗

NASA NTRS1994

Permeation of membranes by the neutral form of amino acids and peptides: relevance to the origin of peptide translocation

The flux of amino acids and other nutrient solutes such as phosphate across lipid bilayers (liposomes) is 10(5) slower than facilitated inward transport across biological membranes. This suggest that primitive cells lacking highly evolved transport systems would have difficulty transporting sufficient nutrients for cell growth to occur. There are two possible ways by which early life may have overcome this difficulty: (1) The membranes of the earliest cellular life-forms may have been intrinsically more permeable to solutes; or (2) some transport mechanism may have been available to facilitate transbilayer movement of solutes essential for cell survival and growth prior to the evolution of membrane transport proteins. Translocation of neutral species represents one such mechanism. The neutral forms of amino acids modified by methylation (creating protonated weak bases) permeate membranes up to 10(10) times faster than charged forms. This increased permeability when coupled to a transmembrane pH gradient can result in significantly increased rates of net unidirectional transport. Such pH gradients can be generated in vesicles used to model protocells that preceded and were presumably ancestral to early forms of life. This transport mechanism may still play a role in some protein translocation processes (e.g. for certain signal sequences, toxins and thylakoid proteins) in vivo.

NASA Discipline Exobiology↗

NASA NTRS1993

Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase

A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

NASA Discipline Cell Biology↗

NASA NTRS1993

The correlated blanching of synaptic bodies and reduction in afferent firing rates caused by transmitter-depleting agents in the frog semicircular canal

Synaptic bodies (SBs) associated with rings of synaptic vesicles and well-defined, pre- and post-synaptic membrane structures are indicators of maturity in most hair cell-afferent nerve junctions. The role of the SBs remains elusive despite several experiments showing that they may be involved in storage of neurotransmitter. Our results demonstrate that SBs of the adult posterior semicircular canal (SCC) cristae hair cells become less electron dense following incubation of the SCC with the transmitter-depleting drug tetrabenazine (TBZ). Objective quantification and comparison of the densities of the SBs in untreated and TBZ-treated frog SCC demonstrated that TBZ significantly decreased the electron density of SBs. This reduction in electron density was accompanied by a reduction in firing rates of afferent fibers innervating the posterior SCC. A second transmitter-depleting drug, guanethidine, previously shown to reduce the electron density of hair cell SBs, also reduced the firing rates of afferent fibers innervating the posterior SCC. In contrast, the electron density of dense granules (DG), similar in size and shape to synaptic bodies (SB) in hair cells, did not change after incubation in TBZ, thus indicating that granules and SBs are not similar in regard to their electron density. The role of SBs in synaptic transmission and the transmitter, if any, stored in the SBs remain unknown. Nonetheless, the association of the lessening of electron density with a reduction in afferent firing rate provides impetus for the further investigation of the SB's role in neurotransmission.

NASA Discipline Neuroscience↗

NASA NTRS1992

Permeability of lipid bilayers to amino acids and phosphate

Permeability coefficients for amino acid classes, including neutral, polar, hydrophobic, and charged species, were measured and compared with values for other ionic solutes such as phosphate. The rates of efflux of glycine, lysine, phenylalanine, serine and tryptophan were determined after they were passively entrapped in large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine (EPC) or dimyristoylphosphatidylcholine (DMPC). The following permeability coefficients were obtained for: glycine, 5.7 x 10(-12) cm s-1 (EPC), 2.0 x 10(-11) cm s-1 (DMPC); serine, 5.5 x 10(-12) cm s-1 (EPC), 1.6 x 10(-11) cm s-1 (DMPC); lysine, 5.1 x 10(-12) cm s-1 (EPC), 1.9 x 10(-11) cm s-1 (DMPC); tryptophan, 4.1 x 10(-10) cm s-1 (EPC); and phenylalanine, 2.5 x 10(-10) cm s-1 (EPC). Decreasing lipid chain length increased permeability slightly, while variations in pH had only minor effects on the permeability coefficients of the amino acids tested. Phosphate permeability was in the range of 10(-12)-10(-13) cm s-1 depending on the pH of the medium. The values for the polar and charged amino acids were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium, which are in the range of 10(-12)-10(-13) cm s-1, depending on conditions and the lipid species used. This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. The results are relevant to the permeation of certain peptides into lipid bilayers during protein translocation and membrane biogenesis.

NASA Discipline Exobiology↗

NASA NTRS1992

Effects of spaceflight in the adductor longus muscle of rats flown in the Soviet Biosatellite COSMOS 2044. A study employing neural cell adhesion molecule (N-CAM) immunocytochemistry and conventional morphological techniques (light and electron microscopy)

The effects of spaceflight upon the "slow" muscle adductor longus were examined in rats flown in the Soviet Biosatellite COSMOS 2044. The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leukocytes and mononuclear cells. Neural cell adhesion molecule immunoreactivity (N-CAM-IR) was seen on the myofiber surface and in regenerating myofibers. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with apparent preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments. The principal electron microscopic changes of the neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles replaced by microtubules and neurofilaments, degeneration of axon terminals, vacant axonal spaces and changes suggestive of axonal sprouting. The present observations suggest that alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

NASA Experiment Number COS 2044-10↗

NASA NTRS1991

A serum factor promotes collagenase synthesis by an osteoblastic cell line

Regulation of the synthesis of collagenase was investigated in the osteoblastic cell line, UMR 106-01. The cells were stained by the avidin-biotin-complex technique for the presence of the enzyme. By this method, it was possible to identify cells producing collagenase. Synthesis, but not secretion, was found to be constitutive in these cells with the enzyme located intracellularly in cytoplasmic vesicles and the Golgi apparatus. The amount of collagenase contained within UMR cells and the number of cells synthesizing the enzyme were proportional to the concentration of fetal bovine serum in the incubating medium. When serum was withdrawn from the osteosarcoma cells, the content of collagenase decreased with time and the enzyme became undetectable by 48 h of serum depletion. The decrease in collagenase content could be completely reversed by resupplying serum to the cells. The collagenase promoting activity of serum could not be eliminated by adsorption on activated charcoal but was retained by a dialysis membrane with a 12,000 mol wt cutoff. A range of bone-seeking hormones or agents known to affect collagenase secretion was added to the medium in an attempt to mimic the effect of serum on collagenase accumulation. None of these agonists, including parathyroid hormone, could reproduce the effect of serum on these cells, although parathyroid hormone could act as a collagenase secretagogue in the presence or absence of serum. It is concluded that fetal bovine serum contains a yet unidentified factor or factors greater than 12,000 mol wt responsible for the continued synthesis of collagenase by UMR 106-01 cells.

Non-NASA Center↗

NASA NTRS1990

Cosmos 1887: morphology, histochemistry, and vasculature of the growing rat tibia

Light microscopy, electron microscopy, and enzyme histochemistry were used to study the effects of spaceflight on metaphyseal and cortical bone of the rat tibia. Cortical cross-sectional area and perimeter were not altered by a 12.5-day spaceflight in 3-month-old male rats. The endosteal osteoblast population and the vasculature near the periosteal surface in flight rats compared with ground controls showed more pronounced changes in cortical bone than in metaphyseal bone. The osteoblasts demonstrated greater numbers of transitional Golgi vesicles, possibly caused by a decreased cellular metabolic energy source, but no difference in the large Golgi saccules or the cell membrane-associated alkaline phosphatase activity. The periosteal vasculature in the diaphysis of flight rats often showed lipid accumulations within the lumen of the vessels, occasional degeneration of the vascular wall, and degeneration of osteocytes adjacent to vessels containing intraluminal deposits. These changes were not found in the metaphyseal region of flight animals. The focal vascular changes may be due to ischemia of bone or a developing fragility of the vessel walls as a result of spaceflight.

Non-NASA Center↗

NASA NTRS2004

Kinetics of organic transformations under mild aqueous conditions: implications for the origin of life and its metabolism

The rates of thermal transformation of organic molecules containing carbon, hydrogen, and oxygen were systematically examined in order to identify the kinetic constraints that governed origin-of-life organic chemistry under mild aqueous conditions. Arrhenius plots of the kinetic data were used to estimate the reaction of half-lifes at 50 degrees C. This survey showed that hydrocarbons and organic substances containing a single oxygenated group were kinetically the most stable; whereas organic substances containing two oxygenated groups in which one group was an alpha- or beta-positioned carbonyl group were the most reactive. Compounds with an alpha- or beta-positioned carbonyl group (aldehyde or ketone) had rates of reaction that were up to 10(24)-times faster than rates of similar molecules lacking the carbonyl group. This survey of organic reactivity, together with estimates of the molecular containment properties of lipid vesicles and liquid spherules, indicates that an origins process in a small domain that used C,H,O-intermediates had to be catalytic and use the most reactive organic molecules to prevent escape of its reaction intermediates.

Biogenesis↗

NASA NTRS2005

Organics in Meteorites, False Biomarkers, and the Search for Life in the Solar System

Bernstein, Todd↗

NASA NTRS2005

The Role of Radiation in Making Meteoritic Organics and False Biomarkers

The tons of organic material that come to the Earth every day on meteorites and dust particles probably helped to make the Earth habitable, and possibly played a role in the origin of life. These compounds."include amino acids, nucleo bases, vesicle forming fatty acids, and many others. Lab experiments suggest that low temperature radiation chemistry could account for many of these chemicals. If we find evidence of Life elsewhere in the Solar System it will probably be in form of chemical biomarkers, quintessentially biological molecules that indicate the presence of micro-organisms. While most people think of the molecules above as good biomarkers, they are not.

Bernstein, Max↗

NASA NTRS2004

Detection of microlesions induced by heavy ions using liposomes filled with fluorescent dye

In cells irradiation by heavy ions has been hypothesized to produce microlesions, regions of local damage. In cell membranes this damage is thought to manifest itself in the form of holes. The primary evidence for microlesions comes from morphological studies of cell membranes, but this evidence is still controversial, especially since holes also have been observed in membranes of normal, nonirradiated, cells. However, it is possible that damage not associated with histologically discernable disruptions may still occur. In order to resolve this issue, we developed a system for detecting microlesions based on liposomes filled with fluorescent dye. We hypothesized that if microlesions form in these liposomes as the result of irradiation, then the entrapped dye will leak out into the surrounding medium in a measurable way. Polypropylene vials containing suspensions of vesicles composed of either dipalmitoyl phosphatidylcholine, or a combination of egg phosphatidylcholine and cholesterol were irradiated at the Brookhaven National Laboratory using 56Fe ions at 1 GeV/amu. In several cases we obtained a significant loss of the entrapped dye above the background level. Our results suggest that holes may form in liposomes as the result of heavy ion irradiation, and that these holes are large enough to allow leakage of cell internal contents that are at least as large as a 1 nm diameter calcein molecule. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

Non-NASA Center↗

NASA NTRS2004

EARLY IMPACT MELTING AND SPACE EXPOSURE HISTORY OF THE PAT91501 LCHONDRITE

Collisions probably occurred frequently in the early history of the asteroid belt. Their effects, which should be recorded in meteorites, must have included heating and melting along with shock alteration of mineral textures. Some non-chondritic meteorite types e.g., eucrites and IIE and IAB irons - do indeed give evidence of extensive impact heating more than 3.4 Gyr ago. The ordinary chondrites, in contrast, show little evidence of early impact heating. The Ar-Ar and Rb-Sr ages of ordinary chondrites that experienced intense shock are for the most part relatively young, many less than 1.5 Gyr. The numerous L-chondrites with Ar- Ar ages clustering near 0.5 Gy are a well-known example. One of them, the 105-kg Chico Lchondrite, shows the effects of unusually intense heating. It is approximately 60% impact melt and likely formed as a dyke beneath a large crater when the L-chondrite parent body underwent a very large impact approximately 0.5 Gyr ago. In rare instances, older shock dates are indicated for ordinary chondrites. Dixon et al show early impact resetting of Ar-Ar ages of a few LL-chondrites including MIL 99301 at 4.23 0.03 Gyr, but in none of these stones did shock lead to extensive melting. As of 2003, searches for chondritic melts attributable to early shock had turned up only the Shaw L-chondrite, which has an Ar-Ar age of approximately 4.42 Gyr. PAT91501 is an 8.55-kg L-chondrite containing vesicles and metal-troilite nodules. It is a unique, near-total impact melt, unshocked, depleted in siderophile and chalcophile elements, and contains only approximately 10% relic chondritic material. The authors conclude that PAT91501 crystallized rapidly and from a much more homogeneous melt than did Shaw. They suggest that PAT resembles Chico and likely formed as an impact melt vein within an impact crater. To define the history of PAT, we have determined its Ar-39-Ar-40 age and measured several radioactive and stable nuclides produced during its space exposure to cosmic rays.

Bogard, Donald D.↗

NASA NTRS2005

Ice photochemistry as a source of amino acids and other organic molecules in meteorites, and implications for the origin of life and the search for life in the Solar System

The tons of extraterrestrial organic material that come to the Earth every day probably helped to made the Earth habitable, and possibly played a role in the origin of life. At the astrochemistry lab (http://www.astrochem.orq) we investigate the formation and distribution of organic molecules in space and consider the impact such molecules may have on the habitability of planets and the search for life in the Solar System. The organic compounds in meteorites include amino acids, aromatics of various sorts including purine and pyrimidine bases, and fatty acids that form bi-layer vesicles. The origin of many of these species remains mysterious, but in recent years we and others have performed experiments that suggest low temperature radiation chemistry could account for the presence and deuterium enrichment of many of these molecules. . I will present our laboratory experiments that show the viability of low temperature radiation chemistry as a source of organic molecules such as;amino acids (Nature, 2002, 416, 401-403), amphiphiles (Astrobiology, 2003, 2, 371, Proc. Nat. Acad. Sci. 2001, 98, 815), quinones (Science, 1999, 283, 1135) and other functionalized aromatic compounds (Meteoritics, 2001, 36, 351 ; Astrophysical Journal., 2003, 582, L25), some of which were invoked as potential biomarkers in the Alan Hills 84001 Martian meteorite. Understanding how components of proteins and DNA could form in sterile space environments is also of relevance to our search for life elsewhere in the Solar System, the great task now ahead of NASA. If we find evidence of Life elsewhere in the Solar System it will probably be in form of chemical biomarkers, quintessentially biological molecules that indicate the presence of micro-organisms. While most people think of molecules such as amino acids, and nucleo-bases as good candidate biomarkers, these molecules are produced non-biotically in space and are expected to be present on the surface of other planets even in the absence of Life. Understanding the range of non-biological organic molecules which could act as false biomarkers in space is a prerequisite for any reasonable search for Life on other worlds.

Bernstein, Max↗

NASA NTRS2006

Computer Modeling of Protocellular Functions: Peptide Insertion in Membranes

Lipid vesicles became the precursors to protocells by acquiring the capabilities needed to survive and reproduce. These include transport of ions, nutrients and waste products across cell walls and capture of energy and its conversion into a chemically usable form. In modem organisms these functions are carried out by membrane-bound proteins (about 30% of the genome codes for this kind of proteins). A number of properties of alpha-helical peptides suggest that their associations are excellent candidates for protobiological precursors of proteins. In particular, some simple a-helical peptides can aggregate spontaneously and form functional channels. This process can be described conceptually by a three-step thermodynamic cycle: 1 - folding of helices at the water-membrane interface, 2 - helix insertion into the lipid bilayer and 3 - specific interactions of these helices that result in functional tertiary structures. Although a crucial step, helix insertion has not been adequately studied because of the insolubility and aggregation of hydrophobic peptides. In this work, we use computer simulation methods (Molecular Dynamics) to characterize the energetics of helix insertion and we discuss its importance in an evolutionary context. Specifically, helices could self-assemble only if their interactions were sufficiently strong to compensate the unfavorable Free Energy of insertion of individual helices into membranes, providing a selection mechanism for protobiological evolution.

Rodriquez-Gomez, D.↗