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At least 289 records · Page 16
All sourcesNASA NTRSDOE OSTI
NASA NTRS1998

Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

Love, Felisha D.↗
NASA NTRS1996

Transport proteins of the plant plasma membrane

Recently developed molecular and genetic approaches have enabled the identification and functional characterization of novel genes encoding ion channels, ion carriers, and water channels of the plant plasma membrane.

Non-NASA Center↗
NASA NTRS1980

Glucose metabolism in different regions of the rat brain under hypokinetic stress influence

Glucose metabolism in rats kept under long term hypokinetic stress was studied in 7 brain regions. Determination was made of the regional levels of glucose, lactate, glutamate, glutamine, aspartate, gamma-aminobutyrate and the incorporation of C-14 from plasma glucose into these metabolites, in glycogen and protein. From the content and activity data the regional glucose flux was approximated quantitatively. Under normal conditions the activity gradient cortex and frontal pole cerebellum, thalamus and mesencephalon, hypothalamus and pons and medulla is identical with that of the regional blood supply (measured with I131 serum albumin as the blood marker). Within the first days of immobilization a functional hypoxia occurred in all brain regions and the utilization of cycle amino acids for protein synthesis was strongly diminished. After the first week of stress the capillary volumes of all regions increased, aerobic glucose metabolism was enhanced (factors 1.3 - 2.0) and the incorporation of glucose C-14 via cycle amino acids into protein was considerably potentiated. The metabolic parameters normalized between the 7th and 11th week of stress. Blood supply and metabolic rate increased most in the hypothalamus.

Konitzer, K.↗
NASA NTRS1974

Studies of hypokinesia in animals to solve urgent problems of space biology and medicine

The effects of hypokinesia on animals were studied by observing: (1) hormonal and mediator balance of the body; (2) gas exchange and tissue respiration; (3) protein content in skeletal muscles; (4) structure of skeletal muscles; and (5) function of skeletal muscles. Sharp limitation of motor activity causes interconnected processes of a dystropic and pathological character expressed as a reduction in the force of various muscle group with disturbance of velocity properties and motor coordination due to disturbances in the control link of the neuromuscular system.

Baranski, S.↗
NASA NTRS1978

Polyglutaraldehyde - A new reagent for coupling proteins to microspheres and for labeling cell-surface receptors

Glutaraldehyde polymerized in basic aqueous solutions was found to react with low molecular weight amines, immunoglobulins and hemoglobin. The polyglutaraldehyde was covalently bound to hydrophilic microspheres. The rate of addition of proteins to the polyglutaraldehyde-derivatized microspheres was investigated spectrophotometrically as a function of pH and temperature. The reaction of polyglutaraldehyde was found to be faster than that of the monomer. The findings led to successful labeling of human lymphocyte subpopulations.

Rembaum, A.↗
NASA NTRS1983

Life in motion, in motion!

A 120 day limited mobility experiment with young male rats and its results, including retarded growth and degenerative changes in the cardiac muscle, are described. A 120 day strict bedrest experiment with 10 human volunteers and its results are described and discussed. Early subjective complaints, subsequent adaptation and eventual progressive changes in excitability and reactivity, reduction in functional capability of the cerebral cortex, and disturbances in water-salt, protein and fat metabolism, including development of precursors of atherosclerosis, as well as poor results of the orthostatic test after 4 months, are presented. These results are explained as applied to sedentary workers and recommendations are given for such persons to exercise in the morning, at work and in the evening in order to prevent hypokinesis and its physical, mental and physiological effects.

Kovalenko, Y. A.↗
NASA NTRS1998

Peculiarities of Crystallization of the Restriction Endonuclease EcoRII

Nucleases interfere with most standard molecular biology procedures. We have purified and crystallized the restriction endonuclease EcoRII, which belongs to the type II of restriction- modification enzyme, to study the protein crystallization process using a "non standard" macromolecule. A procedure for the purification of EcoRII was developed and 99% pure protein as determined by SDS PAGE electrophoresis obtained. Light scattering experiments were performed to assist in screening protein suitable crystallization conditions. The second virial coefficient was determined as a function of precipitating salt concentration, using sodium chloride, ammonium sulfate, and sodium sulfate. Small (maximum size approximately 0.2 mm) well shaped crystals have been obtained. Larger poorly formed crystals (ca 0.5 mm) have also been obtained, but we have been unable to mount them for diff-raction analysis due to their extreme fragility. Crystallization experiments with PEG have shown that using this precipitant, the best crystals are obtained from slightly over-saturated solutions. Use of higher precipitant concentration leads to dendritic crystal formation. EcoRII is difficult to solubilize and meticulous attention must be paid to the presence of reducing agents.

Karpove, Elizaveta↗
NASA NTRS1985

The Radioimmunoassay of Fluid and Electrolyte Hormones

The subject of the paper will be the assay of fluid/electrolyte hormones. ADH (antidiuretic hormone also referred to as vasopressin) reduces fluid loss by increasing water reabsorption by the kidney. The stimuli for its release from the pituitary are loss of blood, dehydration, or increased salt intake. Angiotensin II is the next hormone of interest. It is "generated" from a blood protein by the release of renin from the kidney. One of its functions is to stimulate the secretion of aldosterone from the adrenal gland. Release of renin is also stimulated by volume and sodium loss.

Keil, Lanny C.↗
NASA NTRS2001

Evidence for a role in growth and salt resistance of a plasma membrane H+-ATPase in the root endodermis

The plasma membrane of plant cells is energized by an electrochemical gradient produced by P-type H+-ATPases (proton pumps). These pumps are encoded by at least 12 genes in Arabidopsis. Here we provide evidence that isoform AHA4 contributes to solute transport through the root endodermis. AHA4 is expressed most strongly in the root endodermis and flowers, as suggested by promoter-GUS reporter assays. A disruption of this pump (aha4-1) was identified as a T-DNA insertion in the middle of the gene (after VFP(574)). Truncated aha4-1 transcripts accumulate to approximately 50% of the level observed for AHA4 mRNA in wild-type plants. Plants homozygous for aha4-1 (-/-) show a subtle reduction in root and shoot growth compared with wild-type plants when grown under normal conditions. However, a mutant phenotype is very clear in plants grown under salt stress (e.g., 75 or 110 mM NaCl). In leaves of mutant plants subjected to Na stress, the ratio of Na to K increased 4-5-fold. Interestingly, the aha4-1 mutation appears to be semidominant and was only partially complemented by the introduction of additional wild-type copies of AHA4. These results are consistent with the hypothesis that aha4-1 may produce a dominant negative protein or RNA that partially disrupts the activity of other pumps or functions in the root endodermal tissue, thereby compromising the function of this cell layer in controlling ion homeostasis and nutrient transport.

NASA Discipline Plant Biology↗
NASA NTRS2017

Vascular Patterning Analysis by VESGEN 2D/3D with Bioinformatics: Updates for Rodent Tissues

Fractally branching vascular systems are a complex physiological requirement shared by humans with all higher terrestrial life forms, including other vertebrates, insects, and higher land plants. Vascular trees, networks, and tree-network composites are therefore mapped and quantified by the VESsel GENeration Analysis (VESGEN) software according to weighted physiological vascular rules that include vessel connectivity, tapering and bifurcational branching. According to fluid dynamics, successful vascular transport depends upon a complex distributed system of highly regulated laminar flow. VESGEN has elucidated changes in vascular patterning resulting from inflammatory, developmental and other signaling pathways within numerous tissues of major model organisms important for Space Biology, especially for rodents. Important early stage regenerative opportunities have been identified by VESGEN vascular analysis for visual impairments in the human retina, and is currently being used for research into astronaut visual and ocular disorders associated with long duration missions. The VESGEN 2D software is a mature, automated, widely published capability for which beta testing and public release by NASA is planned for the upcoming year. Early-stage capabilities for VESGEN 3D analysis are under development for the rodent retina and intestine as prototype tissues. A prototype VESGEN 2D Bioinformatics software capability has also been developed to associate phenotypic changes in molecular expression with vascular structure and function. By new VESGEN bioinformatic innovations, expression patterns of the genetic, transcriptional, protein and other markers for regulatory molecules such as vascular endothelial growth factor (VEGF) and their receptors, often indicators of tissue oxygenation status, are co-localized with alterations in vascular pattern. Biomarkers are therefore mapped and quantified as information dimensions directly correlated with the spatial dimensions of a vascular pattern. Further important technology innovations by NASA include substantial image segmentation advances for more automated binary extraction of the grayscale vascular patterns, together with informative associated image quality assessments. Vascular mapping and quantification capabilities for the rodent retina and intestine are illustrated for VESGEN 2D, along with technology status reports on VESGEN 3D and Bioinformatic capabilities. Research partially supported by Ames Center Innovation Awards.

Parsons-Wingerter, P.↗
NASA NTRSDate not supplied

Genetic Determinants of Microbial Survival in Space

Space flight agencies envision a future for humankind beyond Earth, including missions back to the Moon and to Mars in the coming decades. Sending humans into space inevitably includes their microbiomes as well, leading to trillions of bacteria being shed in their living areas. These bacteria shape the lives of their hosts as well as their environment; thus, it is crucial to understand the adaptations of these microbial spacefarers in spaceflight conditions. We aimed to elucidate the genetic determinants of microbial survival in space using a pan-genome analysis of 12 genera cultured from the International Space Station (ISS) from 2017 to 2018. Analysis was performed on each of the genera individually with terrestrial analogs to identify the core and accessory genomes of the spaceflight and terrestrial strains. We then compared the flight and terrestrial core and accessory genomes for each genera using a Bray-Curtis index and visualized the resulting dissimilarity using an Non-Metric Dimensional Scaling plot. The core proteins available in only the spaceflight organisms were then manually characterized for function and genomic location. In every core genome comparison in each genus, there was significant dissimilarity in the core of the spaceflight organisms when compared to the terrestrial organisms. This trend was present in some of the accessory genomes, but was not ubiquitous. Functional analysis of the core content of the ISS genomes showed the majority of genes unique to the core were clustered by location. These gene clusters suggested a set of genetic determinants confer survival in spacecraft-built environments, notably through the uptake of extracellular DNA such as bacteriophage and plasmids. The clear difference between spaceflight and terrestrial microorganisms shows that spaceflight conditions are selective, which has long term implications for their human hosts and environments.

MoBE↗
NASA NTRS2020

Shedding Light on Microbial Dark Matter with A Universal Language of Life

The majority of microbial genomes have yet to be cultured, and most proteins predicted from microbial genomes or sequenced from the environment cannot be functionally annotated. As a result, current computational approaches to describe microbial systems rely on incomplete reference databases that cannot adequately capture the full functional diversity of the microbial tree of life, limiting our ability to model high-level features of biological sequences. The scientific community needs a means to capture the functionally and evolutionarily relevant features underlying biology, independent of our incomplete reference databases. Such a model can form the basis for transfer learning tasks, enabling downstream applications in environmental microbiology, medicine, and bioengineering. Here we present LookingGlass, a deep learning model capturing a “universal language of life”. LookingGlass encodes contextually-aware, functionally and evolutionarily relevant representations of short DNA reads, distinguishing reads of disparate function, homology, and environmental origin. We demonstrate the ability of LookingGlass to be fine-tuned to perform a range of diverse tasks: to identify novel oxidoreductases, to predict enzyme optimal temperature, and to recognize the reading frames of DNA sequence fragments. LookingGlass is the first contextually-aware, general purpose pre-trained “biological language” representation model for short-read DNA sequences. LookingGlass enables functionally relevant representations of otherwise unknown and unannotated sequences, shedding light on the microbial dark matter that dominates life on Earth.

A Hoarfrost↗
NASA NTRS2000

TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice

The transforming-growth-factor-beta-activated kinase TAK1 is a member of the mitogen-activated protein kinase kinase kinase family, which couples extracellular stimuli to gene transcription. The in vivo function of TAK1 is not understood. Here, we investigated the potential involvement of TAK1 in cardiac hypertrophy. In adult mouse myocardium, TAK1 kinase activity was upregulated 7 days after aortic banding, a mechanical load that induces hypertrophy and expression of transforming growth factor beta. An activating mutation of TAK1 expressed in myocardium of transgenic mice was sufficient to produce p38 mitogen-activated protein kinase phosphorylation in vivo, cardiac hypertrophy, interstitial fibrosis, severe myocardial dysfunction, 'fetal' gene induction, apoptosis and early lethality. Thus, TAK1 activity is induced as a delayed response to mechanical stress, and can suffice to elicit myocardial hypertrophy and fulminant heart failure.

NASA Discipline Cardiopulmonary↗
NASA NTRS1993

Evaluation of ovostatin and ovostatin assay

Ovostatin is a 780,000 MW protein, originally isolated from chicken egg white, which is active as a protease inhibitor. Structural studies indicate that the protein is a tetramer of identical subunits of 165,000 MW which can be separated upon reduction with beta- mercaptoethanol. Chicken ovostatin is an inhibitor of metalloproteases such as collagenase and thermolysin, and of acid proteases such as pepsin and rennin. Ovostatin isolated from duck eggs and from crocodile eggs appears to be similar to chicken egg ovostatin, but with significant differences in structure and function. Duck ovostatin contains a reactive thiol ester which is not found in the chicken protein, and duck and crocodile ovostatin inhibit serine protease such as trypsin and chymotrypsin, while chicken ovostatin does not. Electron microscopy of ovostatin indicates that two subunits associated near the middle of each polypeptide to form a dimer with four arms. Two of these dimers then associate to produce a tetramer with eight arms, with the protease binding site near the center of the molecule. Upon binding of the protease, a conformational change causes all eight arms to curl toward the center of the molecule, effectively trapping the protease and sterically hindering access of the substrates to its active site. The structural organization and mechanism of action proposed for ovostatin are nearly identical to that proposed for alpha(sub 2)- macroglobulin, a serum protease inhibitor which may play an important role in regulation of proteases in animal tissues. Although the general arrangement of subunits appears to be the same for all ovostatins studied, some differences have been observed, with chicken ovostatin more closely resembling reptilian ovostatin than the duck protein. This is a surprising result, given the evolutionary relatedness of chickens and ducks. It is possible that the differences in structures may be due to deformed subunit arrangements which occur during the processing and fixing necessary for electron microscopy. Examination of the native structure of these proteins using X-ray crystallography would help clarify these discrepancies.

Moriarity, Debra M.↗
NASA NTRS1997

Calmodulin-Dependent Protein Kinase mediates Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

A number of basic cellular functions, e.g., electrolyte concentration cell growth rate, glucose utilization, bone formation, response to growth stimulation and exocytosis are modified by microgravity or during spaceflight. Studies with intact animal during spaceflights have found lipid accumulations within the lumen of the vasculature and degeneration of the vascular wall. Capillary alterations with extensive endothelial invaginations were also seen. Hemodynamic studies have shown that there is a redistribution of blood from the lower extremities to the upper part of the body; this will alter vascular permeability, resulting in leakage into surrounding tissues. These studies indicate that changes in gravity will affect a number of physiological systems, including the vasculature. However, few studies have addressed the effect of microgravity on vascular cell function and metabolism. A major problem with ground based studies is that achieving a true microgravity hand, environment for prolonged period is not possible. On the other increasing gravity (i.e., hypergravity) is easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell limes (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies suggest that hypergravity will alter the behavior of most cells. Several investigators have shown that hypergravity affects the expression of the early response genes (c-fos and c-myc) and the activation of several protein kinases (PK's) in cells (10,11). In this study we investigated whether hypergravity alters the expression of f-actin by aortic endothelial cells, and the possible role of protein kinases (calmodulin(II)-dependent and PKA) as mediators of these effects.

Love, Felisha D.↗
NASA NTRS1998

Co- and/or post-translational modifications are critical for TCH4 XET activity

TCH4 encodes a xyloglucan endotransglycosylase (XET) of Arabidopsis thaliana. XETs endolytically cleave and religate xyloglucan polymers; xyloglucan is one of the primary structural components of the plant cell wall. Therefore, XET function may affect cell shape and plant morphogenesis. To gain insight into the biochemical function of TCH4, we defined structural requirements for optimal XET activity. Recombinant baculoviruses were designed to produce distinct forms of TCH4. TCH4 protein engineered to be synthesized in the cytosol and thus lack normal co- and post-translational modifications is virtually inactive. TCH4 proteins, with and without a polyhistidine tag, that harbor an intact N-terminus are directed to the secretory pathway. Thus, as predicted, the N-terminal region of TCH4 functions as a signal peptide. TCH4 is shown to have at least one disulfide bond as monitored by a mobility shift in SDS-PAGE in the presence of dithiothreitol (DTT). This disulfide bond(s) is essential for full XET activity. TCH4 is glycosylated in vivo; glycosidases that remove N-linked glycosylation eliminated 98% of the XET activity. Thus, co- and/or post-translational modifications are critical for optimal TCH4 XET activity. Furthermore, using site-specific mutagenesis, we demonstrated that the first glutamate residue of the conserved DEIDFEFL motif (E97) is essential for activity. A change to glutamine at this position resulted in an inactive protein; a change to aspartic acid caused protein mislocalization. These data support the hypothesis that, in analogy to Bacillus beta-glucanases, this region may be the active site of XET enzymes.

NASA Discipline Cell Biology↗
NASA NTRS2003

Conservation of Endo16 expression in sea urchins despite evolutionary divergence in both cis and trans-acting components of transcriptional regulation

Evolutionary changes in transcriptional regulation undoubtedly play an important role in creating morphological diversity. However, there is little information about the evolutionary dynamics of cis-regulatory sequences. This study examines the functional consequence of evolutionary changes in the Endo16 promoter of sea urchins. The Endo16 gene encodes a large extracellular protein that is expressed in the endoderm and may play a role in cell adhesion. Its promoter has been characterized in exceptional detail in the purple sea urchin, Strongylocentrotus purpuratus. We have characterized the structure and function of the Endo16 promoter from a second sea urchin species, Lytechinus variegatus. The Endo16 promoter sequences have evolved in a strongly mosaic manner since these species diverged approximately 35 million years ago: the most proximal region (module A) is conserved, but the remaining modules (B-G) are unalignable. Despite extensive divergence in promoter sequences, the pattern of Endo16 transcription is largely conserved during embryonic and larval development. Transient expression assays demonstrate that 2.2 kb of upstream sequence in either species is sufficient to drive GFP reporter expression that correctly mimics this pattern of Endo16 transcription. Reciprocal cross-species transient expression assays imply that changes have also evolved in the set of transcription factors that interact with the Endo16 promoter. Taken together, these results suggest that stabilizing selection on the transcriptional output may have operated to maintain a similar pattern of Endo16 expression in S. purpuratus and L. variegatus, despite dramatic divergence in promoter sequence and mechanisms of transcriptional regulation.

NASA Discipline Evolutionary Biology↗
NASA NTRS2010

The Earliest Ion Channels in Protocellular Membranes

Cellular membranes with their hydrophobic interior are virtually impermeable to ions. Bulk of ion transport through them is enabled through ion channels. Ion channels of contemporary cells are complex protein molecules which span the membrane creating a cylindrical pore filled with water. Protocells, which are widely regarded as precursors to modern cells, had similarly impermeable membranes, but the set of proteins in their disposal was much simpler and more limited. We have been, therefore, exploring an idea that the first ion channels in protocellular membranes were formed by much smaller peptide molecules that could spontaneously selfassemble into short-lived cylindrical bundles in a membrane. Earlier studies have shown that a group of peptides known as peptaibols is capable of forming ion channels in lipid bilayers when they are exposed to an electric field. Peptaibols are small, non-genetically encoded peptides produced by some fungi as a part of their system of defense against bacteria. They are usually only 14-20 residues long, which is just enough to span the membrane. Their sequence is characterized by the presence of non-standard amino acids which, interestingly, are also expected to have existed on the early earth. In particular, the presence of 2-aminoisobutyric acid (AIB) gives peptaibols strong helix forming propensities. Association of the helices inside membranes leads to the formation of cylindrical bundles, typically containing 4 to 10 monomers. Although peptaibols are excellent candidates for models of the earliest ion channels their structures, which are stabilized only by van der Waals forces and occasional hydrogen bonds between neighboring helices, are not very stable. Although it might properly reflect protobiological reality, it is also a major obstacle in studying channel behavior. For this reason we focused on two members of the peptaibol family, trichotoxin and antiamoebin, which are characterized by a single conductance level. This indicates that their structures are unique and stable. In addition, it is also believed that the trichotoxin channel displays some selectivity between potassium and chloride ions. This makes trichotoxin and antiamoebin ideal models of the earliest ion channels that could provide insight into the origins of ion conductance and selectivity. In the absence of crystal structure of the trichotoxin and antiamoebin channels, we propose their molecular models based on experimentally determined number of monomers forming the bundles. We use molecular dynamics simulations to validate the models in terms of their conductance and selectivity. On the basis of our simulations we show that the emergence of channels built of small, alpha-helical peptides was protobiologically plausible and did not require highly specific amino acid sequences, which is a convenient evolutionary trait. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. To this end, we will discuss how the amino acid sequence and structure of primitive channels give rise to the phenomena of ionic conductance and selectivity across the earliest cell walls, which were essential functions for the emergence and early evolution of protocells. Furthermore, we will argue that even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

Mijajlovic, Milan↗