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At least 289 records · Page 16

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NASA NTRS1997

Suppressed PHA Activation of T Lymphocytes in Simulated Microgravity Is Restored by Direct Activation of Protein Kinase C with Phorbol Ester

Various aspects of spaceflight, including microgravity, cosmic radiation, and physiological stress, may perturb immune function. We sought to understand the impact of microgravity alone on the cellular mechanisms critical to immunity. We utilized clinostatic RWV bioreactors that simulate aspects of microgravity to analyze the response of human PBMC to polyclonal activation. PHA responsiveness in the RWV was almost completely diminished. IL-2 and IFN-gamma secretion was reduced whereas IL- 1 beta and IL-6 secretion was increased, suggesting that monocytes may not be as adversely affected by simulated microgravity as T cells. Activation marker expression (CD25, CD69, CD71) was significantly reduced in RWV cultures. Furthermore, addition of exogenous IL-2 to these cultures did not restore proliferation. Reduced cell-cell and cell-substratum interactions may play a role in the loss of PHA responsiveness. However, PHA activation in Teflon culture bags that limit cell-substratum interactions did not suppress PHA activation. Furthermore, increasing cell density and, therefore, cell-cell interactions in the RWV cultures did not help restore PHA activation. However, placing PBMC within small collagen beads did partially restore PHA responsiveness. Activation of both PBMC and purified T cells with PMA and ionomycin was unaffected by RWV culture, indicating that signaling mechanisms downstream of PKC activation and calcium flux are not sensitive to simulated microgravity. Furthermore, submitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture. Thus, our data indicate that during polyclonal activation the signaling pathways upstream of PKC activation are sensitive to simulated microgravity.

Cooper, David↗

NASA NTRS1997

Influence of Zero-Shear on Yeast Development

The objective of the research was to begin evaluating the effect of zero-shear on the development of the cell wall of Saccharomyces cerevisiae employing the High Aspect Rotating-Wall Vessel (HARV) NASA bioreactor. This particular yeast has enormous potential for research as a model eukaryotic system on the International Space Station, as well as the production of food stuffs' at the future lunar colony. Because the cell wall is the barrier between the cell and the environment, its form and function as influenced by microgravity is of great importance. Morphologic studies revealed that the circularity and total area of the individual yeast cells were essentially the same in both the control and test HARV's. The growth rates were also essentially the same. In zero-shear, the yeast grew in clumps consisting of rudimentary pseudohyphae in contrast to solitary budding cells in the control. Based upon mechanical and sonic shear applied to the yeast cells, those grown in zero-shear had stronger cell walls and septa. This suggests that there are structural differences, most likely related to the chitin skeleton of the cell wall. From this research further NASA support was obtained to continue the work. Investigations will deal with gene expression and ultrastructure. These will lead to a clearer assessment of the value of S. cerevisiae eukaryotic as a model for space station research.

McGinnis, Michael R.↗

NASA NTRS1997

Tissue Culture in Microgravity

Attempts to simulate normal tissue micro-environments in vitro have been thwarted by the complexity and plasticity of the extracellular matrix, which is important in regulating cytoskeletal and nuclear matrix proteins. Gravity is one of the problems, tending to separate components that should be kept together. For space shuttle experiments, NASA engineers devised a double-walled rotating bioreactor, which is proving to be a useful tissue culture device on earth as well as in space.

Pellis, Neal R.↗

NASA NTRS1997

STS-85 Day 06 Highlights

On this sixth day of the STS-85 mission, the flight crew, Cmdr. Curtis L. Brown, Jr., Pilot Kent V. Rominger, Payload Cmdr. N. Jan Davis (Ph.D.), Mission Specialists Robert L. Curbeam, Jr. and Stephen K. Robinson (Ph.D.), and Payload Specialist Bjarni V. Tryggvason today continue their work with the Bioreactor Demonstration System designed to perform cell biology experiments under controlled conditions. Tryggvason, today continues his work with the Microgravity Vibration Isolation Mount which uses magnets to levitate a platform and protect sensitive microgravity processing experiments from vibrations.

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NASA NTRS1997

STS-85 Day 05 Highlights

On this fifth day of the STS-85 mission, the flight crew, Cmdr. Curtis L. Brown, Jr., Pilot Kent V. Rominger, Payload Cmdr. N. Jan Davis (Ph.D.), Mission Specialists Robert L. Curbeam, Jr. and Stephen K. Robinson (Ph.D.), and Payload Specialist Bjarni V. Tryggvason once again test the small robotic arm serving as a prototype for one that will fly as part of the Japanese Experiment Module on the International Space Station. Simulated orbital replacement unit detachment and reattachment will be the focus. Bob Curbeam discusses the progress of the flight with a television station in St Louis, before continuing his work with the Bioreactor Demonstration System designed to perform cell biology experiments under controlled conditions. Immediately after Curbeam's interview, Canadian Payload Specialist Bjarni Tryggvason is set to talk to elementary and high school students at a summer camp in Saskatchewan, Canada.

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NASA NTRS1997

STS-85 Day 08 Highlights

On this eighth day of the STS-85 mission, the flight crew, Cmdr. Curtis L. Brown, Jr., Pilot Kent V. Rominger, Payload Cmdr. N. Jan Davis (Ph.D.), Mission Specialists Robert L. Curbeam, Jr. and Stephen K. Robinson (Ph.D.), and Payload Specialist Bjarni V. Tryggvason entered the final portion of its flight. The new Mir 24 crew of Commander Anatoly Solovyev and Flight Engineer Pavel Vinogradov, who arrived on the station the same day Discovery was launched, bid farewell to Mir 23 Commander Vasily Tsibliev and Flight Engineer Alexander Lazutkin who are returning home after 185 days in space. The Soyuz vehicle carrying the Mir 23 crew home undocked from the station. Robinson again used the Southwest Ultraviolet Imaging System (SWUIS), a 7-inch imaging telescope that is pointed out of the orbiter's middeck hatch window, to observe the Hale-Bopp comet. Curbeam continued his work with the Bioreactor Demonstration System designed to perform cell biology experiments under controlled conditions. Tryggvason spent part of his time troubleshooting a computer hard drive system that supports the Microgravity Vibration Isolation Mount experiment.

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NASA NTRS1997

STS-85 Day 09 Highlights

On this ninth day of the STS-85 mission, the flight crew, Cmdr. Curtis L. Brown, Jr., Pilot Kent V. Rominger, Payload Cmdr. N. Jan Davis (Ph.D.), Mission Specialists Robert L. Curbeam, Jr. and Stephen K. Robinson (Ph.D.), and Payload Specialist Bjarni V. Tryggvason watch over the Manipulator Flight Demonstration (MFD) experiment while Japanese investigators again maneuvere the Small Fine Arm remotely from a control room near Mission Control. It is the final planned work with the arm during this mission. While MFD operations are ongoing, Robinson again uses the Southwest Ultraviolet Imaging System's ultraviolet imaging telescope to observe Comet Hale-Bopp and Curbeam continue his work with the Bioreactor Demonstration System designed to perform cell biology experiments under controlled conditions. Tryggvason spends his day supporting data gathering with the Microgravity Vibration Isolation Mount experiment. Before the crew's workday began, they discussed the mission's progress with reporters in the U.S. and Canada as part of the traditional crew news conference. Questions ranged from life in space for the first time space travelers to providing a report card on the more than 24 experiments being conducted throughout the mission.

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NASA NTRS1996

Altered Gravity and Early Heart Development in Culture

The macromolecules comprising the cytoskeleton and extracellular matrix of cells may be sensitive to gravitation. Since early development of organs depends on dynamic interactions across cell surfaces, altered gravity may disturb development. We investigated this possibility for heart development. Previous studies showed that the extracellular matrix glycoprotein fibronectin (Fn) is necessary for normal heart development. We cultured precardiac tissue explants in a high aspect ratio bioreactor vessel (HARV) to simulate microgravity. We observed tissue morphology, contraction, and Fn distribution by immunolocalization in HARV rotated and control (lxg) explants, cultured 18 hr. We also measured Fn amount by immunoassay. Explants in HARV were rotated at 6 rpm to achieve continuous freefall. Thirty-five of 37 control, but only 1 of 37 matched rotated explants exhibited contractions. Tissue architecture was identical. Immunolocalization of Fn showed remarkable differences which may be related to the development of contractions. The Fn staining in the HARV explants was less intense in all areas. Areas of linear staining along epithelia were present but shorter, and there was less intercellular staining in both mesenchymal tissue and myocardium. Initial immunoassay results of 5 matched pairs of explants showed a 22% reduction in total tissue Fn in the HARV rotated samples. Our results indicate that altered gravity in the HARV reduced the amount and distribution of Fn, as assessed by two independent criteria. This was correlated with a reduction in the development of contractile activity.

Wiens, Darrell J.↗

NASA NTRS1998

In-Vitro Induced Immunosuppression in a Rotary Cell Culture System

The function of the innate immune system is to provide a first-line of defense against infectious organisms, via control of bacterial and viral growth using antigen nonspecific means. These nonspecific immune effectors include macrophages and Natural Killing (NK) cells, and certain cytokines elicited in response to "super antigens" on the infectious agents. This innate system usually keeps most infectious agents from rapidly growing while the adaptive immune system is generating a specific response complete with immunologic memory. Compelling evidence suggests that space flight results in various immunosuppressive effects, including reduced innate and adaptive immune responses. We were particularly concerned with reduced NK activity at landing, and have asked whether the microgravity component of space flight could be responsible for the previously observed NK defect. We have conclusively demonstrated that simulated microgravity as provided by the Synthecon bioreactors does not inhibit the NK function nor the IL-2 activation of lymphokine-activated killing (LAK). Interleukin-2 is the key cytokine responsible for activation of NK cells to express LAK, as well as to support differentiation of lymphocytes during adaptive immune responses. Therefore, we have disproved our original hypothesis based on poor NK in many of the astronauts upon landing.

Grimm, Elizabeth A.↗

NASA NTRS1999

Reactions and Surface Transformations of a Bone-Bioactive Material in a Simulated Microgravity Environment

A comprehensive program to investigate the expeditious in vitro formation of three-dimensional bone-like tissue is currently underway at the University of Pennsylvania. The study reported here forms a part of that program. Three-dimensional bone-like tissue structures may be grown under the simulated microgravity conditions of NASA designed Rotating Wall Bioreactor Vessels (RWV's). Such tissue growth will have wide clinical applications. In addition, an understanding of the fundamental changes that occur to bone cells under simulated microgravity would yield important information that will help in preventing or minimizing astronaut bone loss, a major health issue with travel or stay in space over long periods of time. The growth of three-dimensional bone-like tissue structures in RWV's is facilitated by the use of microcarriers which provide structural support. If the microcarrier material additionally promotes bone cell growth, then it is particularly advantageous to employ such microcarriers. We have found that reactive, bone-bioactive glass (BBG) is an attractive candidate for use as microcarrier material. Specifically, it has been found that BBG containing Ca- and P- oxides upregulates osteoprogenitor cells to osteoblasts. This effect on cells is preceded by BBG reactions in solution which result in the formation of a Ca-P surface layer. This surface further transforms to a bone-like mineral (i.e., carbonated crystalline hydroxyapatite (c-HA)). At normal gravity, time-dependent, immersion-induced BBG reactions and transformations are greatly affected both by variations in the composition of the milieu in which the glass is immersed and on the immersion conditions. However, the nature of BBG reactions and phase transformations under the simulated microgravity conditions of RWV's are unknown, and must be understood in order to successfully use BBG as microcarrier material in RWV'S. In this paper, we report some of our recent findings in this regard using experimental and numerical methods. BBG composition 45S5, the most reactive among known bone-bioactive glasses, was chosen for the study. BBG 45S5 behavior in physiological solutions was tested in simulated microgravity and compared with that at normal gravity. On the basis of our numerical study, we have chosen the BBG granule size to be in the range 40-70 microns, and a RWV rotational speed of 10 rpm. Our numerical study has shown that these parameters enable the microcarrier to remain suspended in the medium without experiencing collisions with the wall of the vessel. Immersion-induced changes in the solution composition and the material surface were analyzed after immersion.

Radin, S.↗

NASA NTRS1999

A Study of Aberrant Glycosylation in Simulated Microgravity Using Laser Induced AutoFluorescence and Flow Cytometry

A number of pathologies and cellular dysfunctions including neoplasms have been correlated with autofluorescence. The complications of aging and diabetes have been associated with the accumulation of non-enzymatic glycosylations of tissue macromolecules. These products are known as the Advanced Glycosylated End Products (AGEs). A physical property associated with AGEs is the emission of 570 mn or 630 nm light energy (autofluorescence) following the absorption of 448 mm energy associated with the argon laser. This investigation sought to assess the induction of argon-laser induced autofluorescence in a variety of in vitro culture systems. Different fluorescence intensities distinguished tumor lines from normal cell populations. Laser-stimulated autofluorescence discriminated primary cultures of lymphocytes grown in the presence of excess glucose as opposed to normal glucose concentrations. The effects of deglycosylating agents upon laser-induced autofluorescence were also assessed. The studies included studies of cell cycle analysis using Propidium Iodide stained DNA of cells grown in simulated microgravity using NASA Bioreactor Vessels in media of normal and elevated glucose concentrations.

Lawless, B. DeSales↗

NASA NTRS1998

Develop Recovery Systems for Separations of Salts from Process Streams for use in Advanced Life Support System

The main objectives of this project were the development of a four-compartment electrolytic cell using high selective membranes to remove nitrate from crop residue leachate and convert it to nitric acid, and the development of an six compartment electrodialysis cell to remove selectively sodium from urine wastes. The recovery of both plant inedible biomass and human wastes nutrients to sustain a biomass production system are important aspects in the development of a controlled ecological life support system (CELSS) to provide the basic human needs required for life support during long term space missions. A four-compartment electrolytic cell has been proposed to remove selectively nitrate from crop residue and to convert it to nitric acid, which is actually used in the NASA-KSC Controlled Ecological Life Support System to control the pH of the aerobic bioreactors and biomass production chamber. Human activities in a closed system require large amount of air, water and minerals to sustain life and also generate wastes. Before using human wastes as nutrients, these must be treated to reduce organic content and to remove some minerals which have adverse effects on plant growth. Of all the minerals present in human urine, sodium chloride (NACl) is the only one that can not be used as nutrient for most plants. Human activities also requires sodium chloride as part of the diet. Therefore, technology to remove and recover sodium chloride from wastes is highly desirable. A six-compartment electrodialysis cell using high selective membranes has been proposed to remove and recover NaCl from human urine.

Colon, Guillermo↗

NASA NTRS1999

Training Under-Represented Students in Biological Research at Fisk University

The objectives of our training and research project in biology at Fisk are to motivate and train our African-American undergraduate and graduate students by (a) teaching the basic principles and applications of different biological, biochemical and biophysical research techniques; (b) providing a "hands on experience" with laboratory instrumentation (c) requiring the students to participate in the proposed research project entitled "Cyanobacterial Bioreactors for Oxygen and Ammonia Production under "CELSS" Conditions" to gain confidence in independently conducting experiments and (d) providing training in scientific data collection and presentation to peers in scientific conferences or meetings.

Gunasekaran, Muthukumaran↗

NASA NTRS1998

Analysis of proteins involved in biodegradation of crop biomass

The biodegradation of crop biomass for re-use in crop production is part of the bioregenerative life support concept proposed by the National Aeronautics and Space Administration (NASA) for long duration, manned space exploration. The current research was conducted in the laboratory to evaluate the use of electrophoretic analysis as a means of rapidly assaying for constitutive and induced proteins associated with the bacterial degradation of crop residue. The proteins involved in crop biomass biodegradation are either constitutive or induced. As a result, effluent and cultures were examined to investigate the potential of using electrophoretic techniques as a means of monitoring the biodegradation process. Protein concentration for optimum banding patterns was determined using the Bio-Rad Protein Assay kit. Four bacterial soil isolates were obtained from the G.W. Carver research Farm at Tuskegee University and used in the decomposition of components of plant biomass. The culture, WDSt3A was inoculated into 500 mL of either Tryptic Soy Broth or Nutrient Broth. Incubation, with shaking of each flask was for 96 hours at 30 C. The cultures consistently gave unique banding patterns under denaturing protein electrophoresis conditions, The associated extracellular enzymes also yielded characteristic banding patterns over a 14-day period, when native electrophoresis techniques were used to examine effluent from batch culture bioreactors. The current study evaluated sample preparation and staining protocols to determine the ease of use, reproducibility and reliability, as well as the potential for automation.

Crawford, Kamau↗

NASA NTRS1999

Loss of Signal Transduction and Inhibition of Lymphocyte Locomotion in a Ground-Based Model of Microgravity

Inflammatory adherence to, and locomotion through the interstitium is an important component of the immune response. Conditions such as true gravity (TG) and modeled microgravity (MMG) severely inhibit lymphocyte locomotion in vitro through gelled Type I collagen (Pellis et al., 1994, 1997). We used the rotating-wall vessel bioreactor (RWV) as a prototype for modeled microgravity. After observing that lymphocyte locomotion was severely affected in modeled microgravity, we found that polyclonal activation of lymphocytes before exposure to modeled microgravity reversed the locomotion inhibition. Phorbol myristate acetate (PMA) treatment of normal peripheral blood lymphocytes, after exposure to modeled microgravity, restored lymphocyte locomotion by 84%. Calcium ionophore had no effect on modeled microgravity-exposed lymphocytes. Therefore, the signal pathways involving calcium may not be affected by modeled microgravity. However, direct activation of Protein Kinase C (PKC) with PMA was effective in restoring locomotion in modeled microgravity almost comparable to normal levels in lymphocytes cultured in static T flasks. Thus, events either at the level of PKC or upstream are affected by modeled microgravity. Treatment of lymphocytes with mitomycin C prior to exposure to modeled microgravity, followed by PMA, restored locomotion to the same extent as nonmitomycin C-treated lymphocytes exposed to modeled microgravity (80-85%). Therefore 1) new DNA synthesis is not necessary to restore locomotion and 2) traditional activation and locomotion share common pathways up to PKC. Thereafter the signals diverge. Furthermore PMA added immediately before or after initiation of modeled microgravity prevents the loss of lymphocyte locomotion.

Sundaresan, Alamelu↗

NASA NTRS1999

PKC Isoform Expression in Modeled Microgravity

Our previous studies showed that modeled (MMG) and true (USA Space Shuttle Missions STS-54 and STS-56) microgravity (MG) inhibit human lymphocyte locomotion, Modeled MG also suppressed polyclonal and antigen-specific lymphocyte activation. Activation of PKC by phorbol myristate acetate (PMA) restored the microgravity-inhibited lymphocyte locomotion as well as activation by phytohaemagglutinin (PHA), whereas calcium ionophore (ionomycin) was unable to restore these functions. Based on these results we hypothesized that MG-induced changes in lymphocyte functions are caused by a fundamental defect in signal transduction mechanism. This defect may be localized either at the PKC level or upstream of PKC, most likely, at the cell membrane level. In this study we examined the expression of PKC isoforms alpha, epsilon and delta in PBMC cultured in rotating wall vessel bioreactor, developed at NASA JSC, which models microgravity by sustaining cells in continuous free fall. The assessment of the isoforms was performed by FACS analysis following cell permeabilization. A decrease in the expression of isoforms epsilon and delta, but not isoform a, was observed in PBMC cultured in microgravity conditions. These data suggest that MMG might selectively affect the expression of Ca2+ independent isoforms of PKC Molecular analysis confirm selective suppression of Ca2+ independent isoforms of PKC.

Risin, Diana↗

NASA NTRS1999

Gravitational Effects on Signal Transduction

An understanding of the mechanisms by which individual cells perceive gravity and how these cells transduce and respond to gravitational stimuli is critical for the development of long-term manned space flight experiments. We now propose to use a well-characterized model erythroid cell system and to investigate gravitational perturbations of its erythropoietin (Epo) signaling pathway and gene regulation. Cells will be grown at 1-G and in simulated microgravity in the NASA Rotating Wall Vessel bioreactor (RWV). Cell growth and differentiation, the Epo-receptor, the protein kinase C pathway to the c-myc gene, and the protein phosphatase pathway to the c-myb gene will be studied and evaluated as reporters of gravitational stimuli. The results of these experiments will have impact on the problems of 1) gravitational sensing by individual cells, and 2) the anemia of space flight. This ground-based study also will serve as a Space Station Development Study in gravitational effects on intracellular signal transduction.

Sytkowski, Arthur J.↗

NASA NTRS1997

Endothelial Cell Morphology and Migration are Altered by Changes in Gravitational Fields

Many of the physiological changes of the cardiovascular system during space flight may originate from the dysfunction of basic biological mechanisms caused by microgravity. The weightlessness affects the system when blood and other fluids move to the upper body causing the heart to enlarge to handle the increased blood flow to the upper extremities and decrease circulating volume. Increase arterial pressure triggers baroreceptors which signal the brain to adjust heart rate. Hemodynarnic studies indicate that the microgravity-induced headward fluid redistribution results in various cardiovascular changes such as; alteration of vascular permeability resulting in lipid accumulation in the lumen of the vasculature and degeneration of the the vascular wall, capillary alteration with extensive endothelial invagination. Achieving a true microgravity environment in ground based studies for prolonged periods is virtually impossible. The application of vector-averaged gravity to mammalian cells using horizontal clinostat produces alterations of cellular behavior similar to those observed in microgravity. Similarly, the low shear, horizontally rotating bioreactor (originally designed by NASA) also duplicates several properties of microgravity. Additionally, increasing gravity, i.e., hypcrgravity is easily achieved. Hypergravity has been found to increase the proliferation of several different cell lines (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. The effect of altered gravity on cells maybe similar to those of other physical forces, i.e. shear stress. Previous studies examining laminar flow and shear stress on endothelial cells found that the cells elongate, orient with the direction of flow, and reorganize their F-actin structure, with concomitant increase in cell stiffness. These studies suggest that alterations in the gravity environment will change the behavior of most cells, including vascular cells. However, few studies have been directed at assessing the effect of altered gravitational field on vascular cell fiction and metabolism, Using image analysis we examined how bovine aortic endothelial cells altered their morphological characteristics and their response to a denudation injury when cells were subjected to simulated microgravity and hypergravity.

Melhado, Caroline↗