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Results for “NASA Discipline Cell Biology”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1994

Hypergravity signal transduction and gene expression in cultured mammalian cells

A number of studies have been conducted during space flight and with clinostats and centrifuges, suggesting that gravity effects the proliferation and differentiation of mammalian cells in vitro. However, little is known about the mechanisms by which mammalian cells respond to changes in gravitational stress. This paper summarizes studies designed to clarify the effects of hypergravity on the cultured human HeLa cells and to investigate the mechanism of hypergravity signal transduction in these cells.

NASA Center JSC↗
NASA NTRS2005

Homozygous diploid deletion strains of Saccharomyces cerevisiae that determine lag phase and dehydration tolerance

This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which > 70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.

NASA Discipline Cell Biology↗
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 NTRS1994

Comparative assessment of the polypeptide profiles from lateral and primary roots of Phaseolus vulgaris L

In Phaseolus vulgaris, primary roots show gravitational sensitivity soon after emerging from the seed. In contrast, lateral roots are agravitropic during early development, and become gravitropic after several cm growth. Primary and lateral root tissues were examined by polyacrylamide gel electrophoresis, coupled with western blotting techniques, to compare proteins which may contribute to the acquisition of gravitational sensitivity. Root tips and zones of cell elongation were compared for each root type, using immunological probes for calmodulin, alpha-actin, alpha-tubulin, and proteins of the plastid envelope. Lateral roots contained qualitatively less calmodulin, and showed a slightly different pattern of actin-related epitope proteins, than did primary root tissues, suggesting that polypeptide differences may contribute to the gravitational sensitivity which these root types express.

Non-NASA Center↗
NASA NTRS1993

Cytoskeleton-amyloplast interactions in sweet clover

The distribution of organelles within columella cells of sweet clover was examined by transmission electron microscopy following growth under static or clinorotating conditions. A developmentally conditioned polarity was observed, with a proximal location of the nucleus and a distal accumulation of the endoplasmic reticulum. This polarity was insensitive to clinorotation. In contrast, clinorotation altered the location of amyloplasts. Application of cytoskeletal poisons (colchicine, cytochalasin D, taxol, and phalloidin), especially during clinorotation, had interesting effects on the maintenance of columella cell polarity, with a profound effect on the extent, location, and structure of the endoplasmic reticulum. The site of cytoskeletal interactions with sedimenting amyloplasts is thought to be the amyloplast envelope. An envelope fraction, having over 17 polypeptides, was isolated using immobilized antibody technology, and will provide a means of assessing the role of specific peptides in cytoskeleton/amyloplast interactions.

Non-NASA Center↗
NASA NTRS1996

Effects of microgravity on osteoblast growth activation

Space flight is an environmental condition where astronauts can lose up to 19% of weight-bearing bone during long duration missions. We used the MC3T3-E1 osteoblast to investigate bone cell growth in microgravity (10(-6) to 10(-9)g). Osteoblasts were launched on the STS-56 shuttle flight in a quiescent state with 0.5% fetal calf serum (FCS) medium and growth activation was initiated by adding fresh medium with 10% FCS during microgravity exposure. Four days after serum activation, the cells were fixed before return to normal Earth gravity. Ground controls were treated in parallel with the flight samples in identical equipment. On landing, cell number, cell cytoskeleton, glucose utilization, and prostaglandin synthesis in flight (n = 4) and ground controls (n = 4) were examined. The flown osteoblasts grew slowly in microgravity with total cell number significantly reduced (55 +/- 6 vs 141 +/- 8 cells per microscopic field). The cytoskeleton of the flight osteoblasts had a reduced number of stress fibers and a unique abnormal morphology. Nuclei in the ground controls were large and round with punctate Hoechst staining of the DNA nucleosomes. The flight nuclei were 30% smaller than the controls (P < 0.0001) and oblong in shape, with fewer punctate areas. Due to their reduced numbers, the cells activated in microgravity used significantly less glucose than ground controls (80.2 +/- 0.7 vs 50.3 +/- 3.7 mg of glucose/dl remaining in the medium) and had reduced prostaglandin E2 (PGE2) synthesis when compared to controls (57.3 +/- 17 vs 138.3 +/- 41 pmol/ml). Cell viability was normal since, on a per-cell basis, glucose use and prostaglandin synthesis were comparable for flight and ground samples. Taken together, these data suggest that growth activation in microgravity results in reduced growth, causing reduced glucose utilization and reduced prostaglandin synthesis, with significantly altered actin cytoskeleton in osteoblasts.

NASA Program Flight↗
NASA NTRS1988

Dynamic cell culture system: a new cell cultivation instrument for biological experiments in space

The prototype of a miniaturized cell cultivation instrument for animal cell culture experiments aboard Spacelab is presented (Dynamic cell culture system: DCCS). The cell chamber is completely filled and has a working volume of 200 microliters. Medium exchange is achieved with a self-powered osmotic pump (flowrate 1 microliter h-1). The reservoir volume of culture medium is 230 microliters. The system is neither mechanically stirred nor equipped with sensors. Hamster kidney (Hak) cells growing on Cytodex 3 microcarriers were used to test the biological performance of the DCCS. Growth characteristics in the DCCS, as judged by maximal cell density, glucose consumption, lactic acid secretion and pH, were similar to those in cell culture tubes.

NASA Program Flight↗
NASA NTRS1989

Bioprocessing under microgravity--an introduction

The main biological processes presently considered for applications in space laboratories are: (i) bioseparation, (ii) cell cultivation and (iii) cell electrofusion. All three technologies were discussed by experts in the field at the 1988-COSPAR Meeting in Helsinki.

NASA Discipline Cell Biology↗
NASA NTRS1985

Separation of mouse testis cells on a Celsep (TM) apparatus and their usefulness as a source of high molecular weight DNA or RNA

The use of a self-contained unit-gravity cell separation apparatus for separation of populations of mouse testicular cells is described. The apparatus, a Celsep (TM), maximizes the unit area over which sedimentation occurs, reduces the amount of separation medium employed, and is quite reproducible. Cells thus isolated have been good sources for isolation of DNA, and notably, high molecular weight RNA.

NASA Discipline Cell Biology↗
NASA NTRS1995

Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix

This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In contrast, cell spreading could be completely inhibited by combining suboptimal doses of cytochalasin D and nocodazole, suggesting that intact microtubules can stabilize cell form when the microfilament lattice is partially compromised. The physiological relevance of the cytoskeleton and cell shape in hepatocyte physiology was highlighted by the finding that a short exposure (6 hour) of cells to nocodazole resulted in production of smaller cells 42 hours later that exhibited enhanced production of a liver-specific product (albumin). These data demonstrate that spreading and flattening of the entire cell body is not driven directly by net polymerization of either microfilaments or microtubules.(ABSTRACT TRUNCATED AT 400 WORDS).

NASA Discipline Cell Biology↗
NASA NTRS1996

Cytoplasm-to-myonucleus ratios following microgravity

The cytoplasmic volume-to-myonucleus ratio in the tibialis anterior and gastrocnemius muscles of juvenile rats after 5.4 days of microgravity was studied. Three groups of rats (n = 8 each) were used. The experimental group (space rats) was flown aboard the space shuttle Discovery (NASA, STS-48), while two ground-based groups, one hindlimb suspended (suspended rats), one non-suspended (control), served as controls. Single fibre analysis revealed a significant decrease in cross-sectional area (microns2) in the gastrocnemius for both the space and the suspended rats; in the tibialis anterior only the suspended rats showed a significant decrease. Myonuclei counts (myonuclei per mm) in both the tibialis anterior and gastrocnemius were significantly increased in the space rats but not in the suspended rats. The mean myonuclear volume (individual nuclei: microns3) in tibialis anterior fibres from the space rats, and in gastrocnemius fibres from both the space and the suspended rats, was significantly lower than that in the respective control group. Estimation of the total myonuclear volume (microns3 per.mm), however, revealed no significant differences between the three groups in either the tibialis anterior or gastrocnemius. The described changes in the cross-sectional area and myonuclei numbers resulted in significant decreases in the cytoplasmic volume-to-myonucleus ratio (microns3 x 10(3)) in both muscles and for both space and suspended rats (tibialis anterior; 15.6 +/- 0.6 (space), 17.2 +/- 1.0 (suspended), 20.8 +/- 0.9 (control): gastrocnemius; 13.4 +/- 0.4 (space) and 14.9 +/- 1.1 (suspended) versus 18.1 +/- 1.1 (control)). These results indicate that even short periods of unweighting due to microgravity or limb suspension result in changes in skeletal muscle fibres which lead to significant decreases in the cytoplasmic volume-to-myonucleus ratio.

NASA Discipline Cell Biology↗
NASA NTRS1996

Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

NASA Discipline Cell Biology↗
NASA NTRS1992

Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte

During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.

Non-NASA Center↗
NASA NTRS1992

Development and use of domain-specific antibodies in a characterization of the large subunits of soybean photosystem 1

The molecular architecture of the soybean photosystem 1 reaction center complex was examined using a combination of surface labeling and immunological methodology on isolated thylakoid membranes. Synthetic peptides (12 to 14 amino acids in length) were prepared which correspond to the N-terminal regions of the 83 and 82.4 kDa subunits of photosystem 1 (the PsaA and PsaB proteins, respectively). Similarly, a synthetic peptide was prepared corresponding to the C-terminal region of the PsaB subunit. These peptides were conjugated to a carrier protein, and were used for the production of polyclonal antibodies in rabbits. The resulting sera could distinguish between the PsaA and PsaB photosystem 1 subunits by Western blot analysis, and could identify appropriate size classes of cyanogen bromide cleavage fragments as predicted from the primary sequences of these two subunits. When soybean thylakoid membranes were surface-labeled with N-hydroxysuccinimidobiotin, several subunits of the complete photosystem 1 lipid/protein complex incorporated label. These included the light harvesting chlorophyll proteins of photosystem 1, and peptides thought to aid in the docking of ferredoxin to the complex during photosynthetic electron transport. However, the PsaA and PsaB subunits showed very little biotinylation. When these subunits were examined for the domains to which biotin did attach, most of the observed label was associated with the N-terminal domain of the PsaA subunit, as identified using a domain-specific polyclonal antisera.

NASA Program NSCORT↗
NASA NTRS1994

Effect of hexylene glycol-altered microtubule distributions on cytokinesis and polar lobe formation in fertilized eggs of Ilyanassa obsoleta

Some effects of gravity on early morphogenesis are correlated with microtubule locations within cells. During first cleavage in Ilyanassa obsoleta embryos, a transitory polar lobe constriction forms and then relaxes, allowing the polar lobe to merge with one daughter cell. If the polar lobe is equally divided or removed, morphogenesis is severely disrupted. To examine microtuble locations during early Ilyanassa development, eggs were fixed and stained for polymerized alpha-tubulin during first cleavage. The mitotic apparatus assembles at the animal pole. The cleavage furrow forms between the asters, constricting to a stabilized intercellular bridge encircling midbody-bound microtubules, whereas the polar lobe constriction forms below and parallel to the spindle, constricting to a transitory intercellular bridge encircling no detectable microtubules. At metaphase an alpha-tubulin epitope is distributed throughout the spindle, whereas a beta-tubulin epitope is present predominantly in the asters. Incubation in hexylene glycol, a drug that increases microtubule polymerization, during mitosis causes the polar lobe constriction to tighten around polymerized alpha-tubulin and remain stably constricted. If hexylene glycol is removed, alpha-tubulin staining disappears from the polar lobe constriction, which relaxes, whereas microtubules remain in the cleavage furrow, which remains constricted. These observations suggest that asymmetric distribution of microtubules affects early Ilyanassa cleavage patterns, and that continued presence of microtubules extending through an intercellular bridge is important for stabilization of the bridge constriction prior to completion of cytokinesis. These data provide the basis for further analysis of the role of microtubules in possible microgravity disruptions of Ilyanassa development.

NASA Discipline Cell Biology↗