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Chang, D.

Publications and source records attributed to Chang, D..

18 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS2016

Fluid Shifts

NASA is focusing on long-duration missions on the International Space Station (ISS) and future exploration-class missions beyond low-Earth orbit. Visual acuity changes observed after short-duration missions were largely transient, but more than 50% of ISS astronauts experienced more profound, chronic changes with objective structural and functional findings such as papilledema and choroidal folds. Globe flattening, optic nerve sheath dilation, and optic nerve tortuosity also are apparent. This pattern is referred to as the visual impairment and intracranial pressure (VIIP) syndrome. VIIP signs and symptoms, as well as postflight lumbar puncture data, suggest that elevated intracranial pressure (ICP) may be associated with the spaceflight-induced cephalad fluid shifts, but this hypothesis has not been tested. The purpose of this study is to characterize fluid distribution and compartmentalization associated with long-duration spaceflight, and to correlate these findings with vision changes and other elements of the VIIP syndrome. We also seek to determine whether the magnitude of fluid shifts during spaceflight, as well as the VIIP-related effects of those shifts, is predicted by the crewmember's preflight conditions and responses to acute hemodynamic manipulations (such as head-down tilt). Lastly, we will evaluate the patterns of fluid distribution in ISS astronauts during acute reversal of fluid shifts through application of lower body negative pressure (LBNP) interventions to characterize and explain general and individual responses. METHODS: We will examine a variety of physiologic variables in 10 long-duration ISS crewmembers using the test conditions and timeline presented in the Figure below. Measures include: (1) fluid compartmentalization (total body water by D2O, extracellular fluid by NaBr, intracellular fluid by calculation, plasma volume by CO rebreathe, interstitial fluid by calculation); (2) forehead/eyelids, tibia, calcaneus tissue thickness (by ultrasound); (3) vascular dimensions by ultrasound (jugular veins, cerebral and carotid arteries, vertebral arteries and veins, portal vein); (4) vascular dynamics by MRI (head/neck blood flow, cerebrospinal fluid pulsatility); (5) ocular measures (optical coherence tomography, intraocular pressure, 2-dimensional ultrasound including optic nerve sheath diameter, globe flattening, and retina-choroid thickness, Doppler ultrasound of ophthalmic and retinal arteries, and veins); (6) cardiac variables by ultrasound (inferior vena cava, tricuspid flow and tissue Doppler, pulmonic valve, stroke volume, right heart dimensions and function, four-chamber views); and (7) ICP measures (tympanic membrane displacement, distortion-product otoacoustic emissions, and ICP calculated by MRI). On the ground, acute head-down tilt will induce cephalad fluid shifts, whereas LBNP will oppose these shifts. Controlled Mueller maneuvers will manipulate cardiovascular variables. Through interventions applied before, during, and after flight, we intend to fully evaluate the relationship between fluid shifts and the VIIP syndrome.

Stenger, M.↗
NASA NTRS2015

Fluid Shifts

INTRODUCTION: Mechanisms responsible for the ocular structural and functional changes that characterize the visual impairment and intracranial pressure (ICP) syndrome (VIIP) are unclear, but hypothesized to be secondary to the cephalad fluid shift experienced in spaceflight. This study will relate the fluid distribution and compartmentalization associated with long-duration spaceflight with VIIP symptoms. We also seek to determine whether the magnitude of fluid shifts during spaceflight, as well as the VIIP-related effects of those shifts, can be predicted preflight with acute hemodynamic manipulations, and also if lower body negative pressure (LBNP) can reverse the VIIP effects. METHODS: Physiologic variables will be examined pre-, in- and post-flight in 10 International Space Station crewmembers including: fluid compartmentalization (D2O and NaBr dilution); interstitial tissue thickness (ultrasound); vascular dimensions and dynamics (ultrasound and MRI (including cerebrospinal fluid pulsatility)); ocular measures (optical coherence tomography, intraocular pressure, ultrasound); and ICP measures (tympanic membrane displacement, otoacoustic emissions). Pre- and post-flight measures will be assessed while upright, supine and during 15 deg head-down tilt (HDT). In-flight measures will occur early and late during 6 or 12 month missions. LBNP will be evaluated as a countermeasure during HDT and during spaceflight. RESULTS: The first two crewmembers are in the preflight testing phase. Preliminary results characterize the acute fluid shifts experienced from upright, to supine and HDT postures (increased stroke volume, jugular dimensions and measures of ICP) which are reversed with 25 millimeters Hg LBNP. DISCUSSION: Initial results indicate that acute cephalad fluid shifts may be related to VIIP symptoms, but also may be reversible by LBNP. The effect of a chronic fluid shift has yet to be evaluated. Learning Objectives: Current spaceflight VIIP research is described, including novel hardware and countermeasures.

Stenger, M. B.↗
NASA NTRS1996

A Comparison of the Physiology and Mechanics of Exercise in LBNP and Upright Gait

Bone, muscular strength, aerobic capacity, and normal fluid pressure gradients within the body are lost during bed rest and spaceflight. Lower Body Negative Pressure (LBNP) exercise may create musculoskeletal and cardiovascular strains equal to a greater than those experienced on Earth and elucidate some of the mechanisms for maintaining bone integrity. LBNP exercise simulates gravity during supine posture by using negative pressure to pull subjects inward against a treadmill generating footward forces and increasing transmural pressures. Footward forces are generated which equal the product of the pressure differential and the cross-sectional area of the LBNP waist seal. Subjects lie supine within the chamber with their legs suspended from one another via cuffs, bungee cords, and pulleys, such that each leg acts as a counterweight to the other leg during the gait cycle. The subjects then walk or run on a treadmill which is positioned vertically within the chamber. Supine orientation allows only footward force production due to the negative pressure within the chamber. The purpose of this study was to determine if the kinematics, kinetics, and metabolic rate during supine walking and slow running on a vertical treadmill within LBNP are similar to those on a treadmill in 1-g environment in an upright posture.

Boda, W. L.↗
NASA NTRS1994

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

Non-NASA Center↗
NASA NTRS1994

Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

NASA Discipline Developmental Biology↗
NASA NTRS1994

Advanced Mico-Gn&C Technology for Low-Cost Planetary

The new NASA paradigm calls for more frequent, low-cost, small spacecraft missions capable of returning high-value planetary science. This challenge also extends to the rapid insertion of advanced technologies across all spacecraft subsystems as an enabling tool for building these highly capable miniaturized science platforms in the spirit of.

planetary spacecraft tracking gravity-field mappin↗
NASA NTRS1993

Characterization of the DNA binding properties of polyomavirus capsid protein

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

NASA Discipline Cell Biology↗
NASA NTRS1993

Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

Non-NASA Center↗
NASA NTRS1993

Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals

The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.

NASA Discipline Number 93-10↗
NASA NTRS1992

Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2

A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.

Non-NASA Center↗
NASA NTRS1992

The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1

A nuclear localization signal (NLS) has been identified in the N-terminal (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic "subtractive" study using the constructs pSG5VP1 (full-length VP1) and pSG5 delta 5'VP1 (truncated VP1, lacking amino acids Ala1-Cys11). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an "additive" approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys3-Arg-Lys5) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.

Non-NASA Center↗
NASA NTRS1992

Early events of polyoma infection: adsorption, penetration and nuclear transport

Polyoma virions have different attachment proteins which are responsible for hemagglutination of erythrocytes and attachment to cultured mouse kidney cells (MKC). Virion binding studies demonstrated that MKC possess specific (productive infection) and nonspecific (nonproductive) receptors. Empty polyoma capsids have hemagglutination activity and bind to non-specific MKC receptors, but they are not capable of competing for specific virion cell receptors or preventing productive infection. Isoelectric focusing of the virion major capsid protein, VP1, separated this protein into six species (A through F). These species had identical amino acid sequences, but differed in degree of modification (phosphorylation, acetylation, sulfation and hydroxylation). Evidence based upon precipitation with specific antisera supports the view that VP1 species E is required for specific adsorption and that D and F are required for hemagglutination. The virion attachment domain has been localized to an 18 kilodalton fragment of the C-terminal region of VP1. Monopinocytotic vesicles containing 125I-labeled polyoma virions were isolated from infected MKC. A crosslinker was used to bind the MKC cell receptor(s) covalently to VP1 attachment protein, and a new 120 kilodalton band was identified by SDS-PAGE. An anti-idiotype antibody prepared against a neutralizing polyoma monoclonal antiody was used to identify a putative 50 kilodalton receptor protein from a detergent extract of MKC, as well as from MKC membrane preparation.

NASA Discipline Developmental Biology↗
NASA NTRS1977

Sound-tube measurements of the relaxation frequency of moist nitrogen

At audible frequencies, the vibrational relaxation of nitrogen contributes significantly to the absorption of sound in still air. The accurate measurement of the humidity dependence of the relaxation frequency of nitrogen, as yet, has eluded careful measurement due to the difficulty in measuring small absorption at low frequencies. Recently, equipment has been constructed for measuring sound absorption in air as a function of humidity over the frequency range from 4 to 100 kHz. In the experiment described here, the temperature and humidity range of this equipment has been extended so that it can be used to study the relaxation absorption in nitrogen at temperatures from 311 K to 418 K. The results indicate that, over this temperature range, the frequency of maximum absorption in moist nitrogen, f, can be given by f/P = 260 x h, Hz/atm, where h is the percent mole fraction of water, and P is the pressure in atm. To the accuracy of the measurements reported here, f is independent of temperature over the range of temperatures at which the measurements were made.

Chang, D.↗