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At least 343 records · Page 19

Lymphocyte trafficking and HIV infection of human lymphoid tissue in a rotating wall vessel bioreactor

The pathogenesis of HIV infection involves a complex interplay between both the infected and noninfected cells of human lymphoid tissue, the release of free viral particles, the de novo infection of cells, and the recirculatory trafficking of peripheral blood lymphocytes. To develop an in vitro model for studying these various aspects of HIV pathogenesis we have utilized blocks of surgically excised human tonsils and a rotating wall vessel (RWV) cell culture system. Here we show that (1) fragments of the surgically excised human lymphoid tissue remain viable and retain their gross cytoarchitecture for at least 3 weeks when cultured in the RWV system; (2) such lymphoid tissue gradually shows a loss of both T and B cells to the surrounding growth medium; however, this cellular migration is reversible as demonstrated by repopulation of the tissue by labeled cells from the growth medium; (3) this cellular migration may be partially or completely inhibited by embedding the blocks of lymphoid tissue in either a collagen or agarose gel matrix; these embedded tissue blocks retain most of the basic elements of a normal lymphoid cytoarchitecture; and (4) both embedded and nonembedded RWV-cultured blocks of human lymphoid tissue are capable of productive infection by HIV-1 of at least three various strains of different tropism and phenotype, as shown by an increase in both p24 antigen levels and free virus in the culture medium, and by the demonstration of HIV-1 RNA-positive cells inside the tissue identified by in situ hybridization. It is therefore reasonable to suggest that gel-embedded and nonembedded blocks of human lymphoid tissue, cocultured with a suspension of tonsillar lymphocytes in an RWV culture system, constitute a useful model for simulating normal lymphocyte recirculatory traffic and provide a new tool for testing the various aspects of HIV pathogenesis.

Tissue Culture/instrumentation/methods↗

Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway

Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.

NASA Discipline Musculoskeletal↗

Graphical User Interface (GUI) Implementation for Agent-Based Microbial Radiobiology Model

Sending human life past the Low Earth Orbit (LEO) to explore the Moon and Mars will be challenging. The Earth’s magnetic field naturally protects life from deep-space particle radiation such as Galactic Cosmic Rays (GCR) and Solar Particle Events (SPE); these will pose health risks to humans in deep space. Research has been done to investigate these effects, like BioSentinel, the first biological CubeSat to fly beyond the LEO, designed to culture yeast in a microfluidic device and record optical measurements of growth and metabolism. However, experiments can only report cell damage as bulk growth curves, while deep-space radiation causes damage that is heterogeneous among individual cells. AMMPER is an open-source, agent-based, computational model coded in Python to simulate the effects of deep-space radiation on individual yeast cells (Saccharomyces cerevisiae) to facilitate interpretation of biological radiation experiments. Version 1.0 of the code ran in a command line interface (CLI), limiting use to those familiar with modularization, object-oriented programming, and computational models. Here we present a graphical user interface (GUI) for AMMPER to increase its accessibility. GUI development included converting input points and UI files, designing an application and logo, and expanding program packages. Additionally, we added optical assistance that corresponded with simulation parameters, which included simulation type, cell type, ROS model, and radiation dosage, as well as customizable display and file exportation features. Following a pilot testing period, its structure was updated further to enhance abilities, adding increased runs, video visualization, data plotting, and an educational/tutorial component. Future work will include creating a bit installer and runtime environment for AMMPER. Ultimately, the creation of the GUI has two main goals: to facilitate the integration of computational models into the work of researchers in microbial radiobiology, and to act as an interactive and visual resource for space biology education.

yeast↗

Algal culture studies related to a closed ecological life support system

Studies on the steady-state long-term (4 month) culture of Scenedesmus obliquus algae, maintained in an annular air-lift column operated as a turbidostat, were carried out to evaluate the life-supporting possibilities of this system. Chlorophyll production and cell number as functions of the dry weight were linear at constant illumination. Productivity (measured as the product of dry weight, mg/ml, and the growth rate, ml/hr) vs. dry weight rose linearly until the cell density reached a level at which light became limiting (89 percent absorption of the photosynthetically active radiation). In the initial, linear portion of the curve, the productivity was limited by cell growth at the given light intensity. The maximum dilution rate of the system corresponded to the doubling time of 13.4 hr, about half the maximum rate, with a productivity of 80 percent of the maximum theoretical productivity. The high light utilization efficiencies were contributed by the low (10 percent of full sunlight) incident intensities.

Radmer, R.↗

Diffusion length variation and proton damage coefficients for InP/In(x)Ga(1-x)As/GaAs solar cells

Indium phosphide solar cells are more radiation resistant than gallium arsenide and silicon solar cells, and their growth by heteroepitaxy offers additional advantages leading to the development of lighter, mechanically strong and cost-effective cells. Changes in heteroepitaxial InP cell efficiency under 0.5 and 3 MeV proton irradiations are explained by the variation in the minority-carrier diffusion length. The base diffusion length versus proton fluence is calculated by simulating the cell performance. The diffusion length damage coefficient K(L) is plotted as a function of proton fluence.

Jain, R. K.↗

Properties of cirrus generating cells.

Particle size spectra were measured during 20 hr of sampling in cirrus generating cells (uncinus, stratus, spissatus, thunderstorm anvil) and the particle concentration, mean crystal length, ice water content, reflectivity factor, and precipitation rate were calculated from these spectra. Average values of the physical properties in the generating cells were obtained. Growth was found to be downward, reaching a maximum ice water content just below the base of the generating cell. The maximum ice water contents (not including the thunderstorm anvil) were found in cirrus uncinus. Liquid water was not found throughout the cirrus sampling by measurement with the Johnson-Williams hot-wire liquid-water meter; however, it is believed that liquid water is present as a transient phase.

Heymsfield, A. J.↗

Diffusion length variation in 0.5- and 3-MeV-proton-irradiated, heteroepitaxial indium phosphide solar cells

Indium phosphide (InP) solar cells are more radiation resistant than gallium arsenide (GaAs) and silicon (Si) solar cells, and their growth by heteroepitaxy offers additional advantages leading to the development of light weight, mechanically strong, and cost-effective cells. Changes in heteroepitaxial InP cell efficiency under 0.5- and 3-MeV proton irradiations have been explained by the variation in the minority-carrier diffusion length. The base diffusion length versus proton fluence was calculated by simulating the cell performance. The diffusion length damage coefficient, K(sub L), was also plotted as a function of proton fluence.

Jain, Raj K.↗

Mechanisms of Gravity-Evoked Neuronal Plasticity

The grant focuses on a gene we identified called, serum and glucocorticoid- induced kinase (SGK), during a previously funded NASA project. The abundance of SGK messenger RNA (mRNA) and protein is increased in CNS tissues from animals reared in microgravity in comparison with 1G reared animals. In the funded proposal we had three aims: 1) characterize the distribution of SGK mRNA in the developing and adult rat CNS, 2) determine if expression of enzymatically active or inactive forms of SGK in cells influenced cell morphology (neurite growth), and 2) determine if SGK is a CREB kinase - that is, a protein kinase that adds phosphate groups to the transcription factor CREB. Over the past year we have made strong progress in the two most difficult parts of the project, namely specific aims 2 and 3. In specific aim #2 we planned to express a dominant negative or a constitutively active form of SGK in PC12 cells and assay the effects on neurite growth. Several methods are available for examining the effects of a transgene on PC12 neurite growth. Relevant variables include the performance of the assay +/- serum, +/- NGF, substratum for growth, timing between transfection and assay. Over the past 8 months we have customized the assay to enable us to most readily determine the effects of transgene expression on neurite growth. We have also compared the relative utility of transfecting DNA as opposed to protein itself. We are now well positioned to study the effects of SGK on neurite growth. We have also made progress in parallel studies in primary neurons. We have made constructs which will lead to transgene expression in cultures of spinal cord neurons. Co-transfection of a reporter and the SGK constructs can now be performed.

Kalb, Robert↗

Problems and potentialities of cultured plant cells in retrospect and prospect

The past, present and expected future accomplishments and limitations of plant cell and tissue culture are reviewed. Consideration is given to the pioneering insights of Haberlandt in 1902, the development of culture techniques, and past work on cell division, cell and tissue growth and development, somatic embryogenesis, and metabolism and respiration. Current activity in culture media and technique development for plant regions, organs, tissues, cells, protoplasts, organelles and embryos, totipotency, somatic embryogenesis and clonal propagation under normal and space conditions, biochemical potentialities, and genetic engineering is surveyed. Prospects for the investigation of the induced control of somatic cell division, the division of isolated protoplasts, the improvement of haploid cell cultures, liquid cultures for somatic embryogenesis, and the genetic control of development are outlined.

Steward, F. C.↗

Development of processes for the production of low cost silicon dendritic web for solar cells

High area output rates and continuous, automated growth are two key technical requirements for the growth of low-cost silicon ribbons for solar cells. By means of computer-aided furnace design, silicon dendritic web output rates as high as 27 sq cm/min have been achieved, a value in excess of that projected to meet a $0.50 per peak watt solar array manufacturing cost. The feasibility of simultaneous web growth while the melt is replenished with pelletized silicon has also been demonstrated. This step is an important precursor to the development of an automated growth system. Solar cells made on the replenished material were just as efficient as devices fabricated on typical webs grown without replenishment. Moreover, web cells made on a less-refined, pelletized polycrystalline silicon synthesized by the Battelle process yielded efficiencies up to 13% (AM1).

Duncan, C. S.↗

Development of Gravity Sensitive Plant Cells (Ceratodon) in Microgravity

Protonemata of the moss Ceratodon are tip-growing cells that grow up in the dark. This cell type is unique compared to cells in almost any other organism, since the growth of the plant cell itself is completely oriented by gravity. Thus, both the processes of gravity sensing and the gravity response occur in the same cell. Gravity sensing appears to rely upon amyloplasts (starch-filled plastids) that sediment. This sedimentation occurs in specific zones and plastid zonation is complex with respect to plastid morphology, distribution, and gravity. Microtubules restrict the extent of plastid sedimentation (i.e., they are load-bearing). Light also is important since apical cells have a phytochrome-based positive phototropism, light quality influences plastid zonation and sedimentation (photomorphogenesis), and red light suppresses gravitropism at higher but not lower light intensities. Many of these processes were examined in a 16 day spaceflight experiment, "SPM-A" space moss" or "SPAM)) on STS-87 that landed in December, 1997. The work described here involves the definition of a second flight experiment that builds upon the data and questions arising from STS-87. Effort was directed towards further definition of an experiment for the Shuttle (dubbed "SOS" for "Son of SPAM"). Our current target is STS 107 that is scheduled to fly in January 2001. This definition addressed two goals of the STS107 experiment. The goals of the current experiment were to determine whether the cytoskeleton plays a role in maintaining and generating an apical (non-random) plastid distribution in microgravity and to determine the development and extent of clockwise spiral tip-growth in microgravity.

Sack, Fred D.↗

Skeletal unloading causes resistance of osteoprogenitor cells to parathyroid hormone and to insulin-like growth factor-I

Skeletal unloading decreases bone formation and osteoblast number in vivo and decreases the number and proliferation of bone marrow osteoprogenitor (BMOp) cells in vitro. We tested the ability of parathyroid hormone (PTH) to stimulate BMOp cells in vivo by treating Sprague Dawley rats (n = 32) with intermittent PTH(1-34) (1 h/day at 8 microg/100 g of body weight), or with vehicle via osmotic minipumps during 7 days of normal weight bearing or hind limb unloading. Marrow cells were flushed from the femur and cultured at the same initial density for up to 21 days. PTH treatment of normally loaded rats caused a 2.5-fold increase in the number of BMOp cells, with similar increases in alkaline phosphatase (ALP) activity and mineralization, compared with cultures from vehicle-treated rats. PTH treatment of hind limb unloaded rats failed to stimulate BMOp cell number, ALP activity, or mineralization. Hind limb unloading had no significant effect on PTH receptor mRNA or protein levels in the tibia. Direct in vitro PTH challenge of BMOp cells isolated from normally loaded bone failed to stimulate their proliferation and inhibited their differentiation, suggesting that the in vivo anabolic effect of intermittent PTH on BMOp cells was mediated indirectly by a PTH-induced factor. We hypothesize that this factor is insulin-like growth factor-I (IGF-I), which stimulated the in vitro proliferation and differentiation of BMOp cells isolated from normally loaded bone, but not from unloaded bone. These results suggest that IGF-I mediates the ability of PTH to stimulate BMOp cell proliferation in normally loaded bone, and that BMOp cells in unloaded bone are resistant to the anabolic effect of intermittent PTH therapy due to their resistance to IGF-I.

NASA Discipline Musculoskeletal↗

Terrestrial nitrogen cycles: Some unanswered questions

Nitrogen is generally considered to be the element which most often limits the growth of plants in both natural and agricultural ecosystems. It regulates plant growth because photosynthetic rates are strongly dependent on the concentration of nitrogen in leaves, and because relatively large mounts of protein are required for cell division and growth. Yet nitrogen is abundant in the biosphere - the well-mixed pool in the atmosphere is considered inexhaustible compared to biotic demand, and the amount of already fixed organic nitrogen in soils far exceeds annual plant uptake in terrestrial ecosystems. In regions where natural vegetation is not nitrogen limited, continuous cultivation induces nitrogen deficiency. Nitrogen loss from cultivated lands is more rapid than that of other elements, and nitrogen fertilization is generally required to maintain crop yield under any continuous system. The pervasiveness of nitrogen deficiency in many natural and most managed sites is discussed.

Vitousek, P.↗

Macrophage cell lines derived from major histocompatibility complex II-negative mice

Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

NASA Discipline Musculoskeletal↗

Experiments with suspended cells on the Space Shuttle

Spaceflight experiments since 1981 have demonstrated that certain cell functions are altered by micro-g. Biophysical models suggest that cell membranes and organelles should not be affected directly by gravity, however, the chemical microenvironment surrounding the cell and molecular transport could be altered by reduced gravity. Most experiments have used suspended live cells in small chambers without stirring or medium exchange. Flight results include increased attachment of anchorage-dependent human cells to collagen coated microcarriers, reduced secretion of growth hormone from pituitary cells, decreased mitogenic response of lymphocytes, increased Interferon-alpha by lymphocytes, increased Interleukin-1 and Tumor Necrosis Factor secretion by macrophages. Related experiments on cells immediately postflight and on procaryotic cells have shown significant changes in secretory capacity, cell proliferation, differentiation and development. Postulated mechanism include altered cell-cell interactions, altered calcium ion transport, effects on cell cytoskeleton, transport of transmitters and interactions with receptors. The discussion includes use of new molecular methods, considerations for cell environmental control and a preview of several experiments planned for the Shuttle and Spacelab flights to study the basic effects of microgravity on cellular physiology and potential interactions of spaceflight with radiation damage and cellular repair mechanisms.

Review↗

Studying Functions of All Yeast Genes Simultaneously

A method of studying the functions of all the genes of a given species of microorganism simultaneously has been developed in experiments on Saccharomyces cerevisiae (commonly known as baker's or brewer's yeast). It is already known that many yeast genes perform functions similar to those of corresponding human genes; therefore, by facilitating understanding of yeast genes, the method may ultimately also contribute to the knowledge needed to treat some diseases in humans. Because of the complexity of the method and the highly specialized nature of the underlying knowledge, it is possible to give only a brief and sketchy summary here. The method involves the use of unique synthetic deoxyribonucleic acid (DNA) sequences that are denoted as DNA bar codes because of their utility as molecular labels. The method also involves the disruption of gene functions through deletion of genes. Saccharomyces cerevisiae is a particularly powerful experimental system in that multiple deletion strains easily can be pooled for parallel growth assays. Individual deletion strains recently have been created for 5,918 open reading frames, representing nearly all of the estimated 6,000 genetic loci of Saccharomyces cerevisiae. Tagging of each deletion strain with one or two unique 20-nucleotide sequences enables identification of genes affected by specific growth conditions, without prior knowledge of gene functions. Hybridization of bar-code DNA to oligonucleotide arrays can be used to measure the growth rate of each strain over several cell-division generations. The growth rate thus measured serves as an index of the fitness of the strain.

Stolc, Viktor↗

Electrophoretic separation of kidney and pituitary cells on STS-8

Specific secretory cells were separated from suspensions of cultured primary human embryonic cells and rat pituitary cells in microgravity conditions, with an objective of isolating the subfractions of kidney cells that produce the largest amount of urakinase, and the subfractions of rat pituitary cells that secrete growth hormones (GH), prolactin (PRL), and other hormones. It is inferred from the experimental observations that the surface charge distributions of the GH-containing cells differ from those of the PRL-containing cells, which is explained by the presence of secretory products on the surface of pituitary cells. For kidney cells, the electrophoretic mobility distributions in flight experiments were spread more than the ground controls.

Morrison, D. R.↗