The cytoskeleton, apoptosis, and gene expression in T lymphocytes and other mammalian cells exposed to altered gravity.
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Animals↗
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Ag+ alters cell growth, neurite extension, cardiomyocyte beating, and fertilized egg constriction.
BACKGROUND: The Russian Space Agency uses electrochemically generated silver ions (Ag+) to purify drinking water for their space station, Mir, and their portion of the International Space Station. U.S. EPA guidelines allow 10.6 micromol x L(-1) Ag+ in human drinking water for up to 10 d. Studies correlate Ag+ exposure with tissue dysfunction in humans, rats, and mice, and with altered ion transport, skeletal muscle contraction, and embryonic cell constriction in other animal cells. Ag+ effects on cell shape change-related functions have not been assessed. METHODS: Immortalized embryonic human intestinal epithelial cells, freshly explanted embryonic avian nerve cells and cardiomyocytes, and marine fertilized eggs were grown in vitro in medium containing AgNO3. RESULTS: Intestinal cells detach from the substratum and viable cell number decreases by 5-6 d at 5 micromol x L(-1) AgNO3, and faster at higher concentrations. Microtubules appear unaltered in adherent cells. Detached cells are nonviable. Neurite outgrowth and glial cell migration from dorsal root ganglia are inhibited by 3 d at 15 micromol x L(-1) AgNO3 or greater. Contractions stop temporarily in most cardiomyocytes by 5 min at 5 micromol x L(-1) AgNO3 or more, but some cardiomyocytes beat 3 times faster than normal at 7.5-20 micromol x L(-1) AgNO3. Picomolar Ag+ increases marine egg polar lobe constriction within an hour, even in the absence of microtubules. CONCLUSION: Ag+ alters animal cell growth and shape changes by a MT-independent mechanism. This is the first report of Ag+ effects on vertebrate neurite outgrowth, glial cell migration, or cardiomyocyte beat rate.
Animals↗
Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells.
Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.
Blotting, Western↗
Regulation of heat shock protein message in Jurkat cells cultured under serum-starved and gravity-altered conditions.
Although our understanding of effects of space flight on human physiology has advanced significantly over the past four decades, the potential contribution of stress at the cellular and gene regulation level is not characterized. The objective of this ground-based study was to evaluate stress gene regulation in cells exposed to altered gravity and environmentally suboptimal conditions. We designed primers to detect message for both the constitutive and inducible forms of the heat shock protein, HSP-70. Applying the reverse transcriptase-polymerase chain reaction (RT-PCR), we probed for HSP-70 message in human acute T-cell leukemia cells, Jurkat, subjected to three types of environmental stressors: (1) altered gravity achieved by centrifugation (hypergravity) and randomization of the gravity vector in rotating bioreactors, (2) serum starvation by culture in medium containing 0.05% serum, and (3) temperature elevation (42 degrees C). Temperature elevation, as the positive control, significantly increased HSP-70 message, while centrifugation and culture in rotating bioreactors did not upregulate heat shock gene expression. We found a fourfold increase in heat shock message in serum-starved cells. Message for the housekeeping genes, actin and cyclophilin, were constant and comparable to unstressed controls for all treatments. We conclude that gravitational perturbations incurred by centrifugal forces, exceeding those characteristic of a Space Shuttle launch (3g), and culture in rotating bioreactors do not upregulate HSP-70 gene expression. In addition, we found RT-PCR useful for evaluating stress in cultured cells.
Aerospace Medicine↗
Regulatory coding of lymphoid lineage choice by hematopoietic transcription factors.
During lymphopoiesis, precursor cells negotiate a complex regulatory space, defined by the levels of several competing and cross-regulating transcription factors, before arriving at stable states of commitment to the B-, T- and NK-specific developmental programs. Recent perturbation experiments provide evidence that this space has three major axes, corresponding to the PU.1 versus GATA-1 balance, the intensity of Notch signaling through the CSL pathway, and the ratio of E-box transcription factors to their Id protein antagonists.
Animals↗
How can plants tell which way is up?
Many people think of plants as essentially sessile organisms that do not actively respond to their environment. What could be further from the truth! In fact, plants are capable of a variety of movements, including the dramatic nastic responses (such as Venus fly trap closure) and the less sensational tropisms. These latter movements are directed growth responses to some type of external stimulus such as gravity (gravitropism, formerly known as geotropism) or light (phototropism). This paper describes some interesting exercises that are derived from recent work, including research that has led to experiments performed on two Space Shuttle missions in 1997 (Kiss et al. 1998). The study of tropisms can be a useful way to introduce students to plant biology in high school and introductory college courses. In our experience, students are fascinated by plant movements when they are presented in lectures and find laboratory experiences on this topic quite engaging. Laboratory work on plant tropisms can also be used to introduce important concepts in science such as hypothesis testing, quantitative analysis, and the use of statistics. The laboratory exercises described in this paper involve the higher plant Arabidopsis thaliana, which has become an important organism in molecular biology research and is the focus of an international plant genome project. Based on the material presented here, a number of plant gravitropism laboratory exercises with Arabidopsis that are simple in terms of equipment/materials and procedures can be developed. These exercises are robust in that they work well even in the hands of introductory students, and they can be expanded according to the individual instructor's needs. This paper describes two exercises that have been performed by beginning college students, and these exercises can easily be performed in biology classes in most high school settings.
Arabidopsis↗
Changing the axis changes the perspective.
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Animals↗
Expression and function of FGF10 in mammalian inner ear development.
We have investigated the expression of FGF10 during ear development and the effect of an FGF10 null mutation on ear development. Our in situ hybridization data reveal expression of FGF10 in all three canal crista sensory epithelia and the cochlea anlage as well as all sensory neurons at embryonic day 11.5 (E11.5). Older embryos (E18.5) displayed strong graded expression in all sensory epithelia. FGF10 null mutants show complete agenesis of the posterior canal crista and the posterior canal. The posterior canal sensory neurons form initially and project rather normally by E11.5, but they disappear within 2 days. FGF10 null mutants have no posterior canal system at E18.5. In addition, these mutants have deformations of the anterior and horizontal cristae, reduced formation of the anterior and horizontal canals, as well as altered position of the remaining sensory epithelia with respect to the utricle. Hair cells form but some have defects in their cilia formation. No defects were detected in the organ of Corti at the cellular level. Together these data suggest that FGF10 plays a major role in ear morphogenesis. Most of these data are consistent with earlier findings on a null mutation in FGFR2b, one of FGF10's main receptors.
Animals↗
Fluid shear stress stimulates prostaglandin and nitric oxide release in bone marrow-derived preosteoclast-like cells.
Bone is a porous tissue that is continuously perfused by interstitial fluid. Fluid flow, driven by both vascular pressure and mechanical loading, may generate significant shear stresses through the canaliculi as well as along the bone lining at the endosteal surface. Both osteoblasts and osteocytes produce signaling factors such as prostaglandins and nitric in response to fluid shear stress (FSS); however, these humoral agents appear to have more profound affects on osteoclast activity at the endosteal surface. We hypothesized that osteoclasts and preosteoclasts may also be mechanosensitive and that osteoclast-mediated autocrine signaling may be important in bone remodeling. In this study, we investigated the effect of FSS on nitric oxide (NO), prostaglandin E(2) (PGE(2)), and prostacyclin (PGI(2)) release by neonatal rat bone marrow-derived preosteoclast-like cells. These cells were tartrate-resistant acid phosphatase (TRAP) positive, weakly nonspecific esterase (NSE) positive, and capable of fusing into calcitonin-responsive, bone-resorbing, multinucleated cells. Bone marrow-derived preosteoclast-like cells exposed for 6 h to a well-defined FSS of 16 dynes/cm(2) produced NO at a rate of 7.5 nmol/mg protein/h, which was 10-fold that of static controls. This response was completely abolished by 100 microM N(G)-amino-L-arginine (L-NAA). Flow also stimulated PGE(2) production (3.9 microg/mg protein/h) and PGI(2) production (220 pg/mg protein/h). L-NAA attenuated flow-induced PGE(2) production by 30%, suggesting that NO may partially modulate PGE(2) production. This is the first report demonstrating that marrow derived cells are sensitive to FSS and that autocrine signaling in these cells may play an important role in load-induced remodeling and signal transduction in bone.
Animals↗
Plant lignans and health: cancer chemoprevention and biotechnological opportunities.
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Animals↗
Gene fishing: the use of a simple protocol to isolate multiple homeodomain classes from diverse invertebrate taxa.
Comparison of relevant gene sequence and functional data is central to understanding the evolution of metazoan development. The conservation of portions of regulatory genes, such as homeoboxes, allows for the design of PCR-based sequence isolation and amplification strategies. Here we describe a simple protocol that uses a degenerate primer pair to isolate a variety of homeobox-containing genes from diverse metazoan taxa. In a nonexhaustive survey, we have isolated 28 gene sequence fragments from 15 taxa, representing eight invertebrate phyla (Mollusca, Echiura, Annelida, Platyhelminth, Acoela, Ctenophora, Cnidaria, and Porifera). Based on BLAST and parsimony analyses, these gene fragments affiliate with several gene groups (PAIRED-like, HOX, and ParaHOX) and several single genes, including pancreas/duodenum homeoboxes (Pdx), empty spiracles (ems/Emx), gastrulation brain homeoboxes (Gbx), hematopoietically expressed homeoboxes (HEX), brain specific homeobox (bsh/BarH1/BarH2), NK-1 (NK-1/s59/slouch), and ladybird (Lbl/Lbe/Lbx). In several cases, these fragments represent the first reported orthologue for the phylum or superphyletic group (i.e., Lophotrochozoa).
Amino Acid Sequence↗
Gravity-directed calcium current in germinating spores of Ceratopteris richardii.
Gravity directs the early polar development in single cells of Ceratopteris richardii Brogn. It acts over a limited period of time during which it irreversibly determines the axis of the spore cell's development. A self-referencing calcium selective electrode was utilized to record the net movement of calcium across the cell membrane at different positions around the periphery of the spore during the period in which gravity orients the polarity of the spore. A movement of calcium into the cell along the bottom and out of the cell along the top was detected. This movement was specific, polarized, and strongest in a direction that opposed the vector of gravity. Treatment with nifedipine, a calcium-channel blocker, diminished the calcium current and caused the cell to lose its responsiveness to the orienting influence of gravity. Results shown suggest that calcium plays a crucial role in the ability of a single cell to respond to gravity and in the subsequent establishment of its polarity.
Calcium↗
The auxin-resistant diageotropica mutant of tomato responds to gravity via an auxin-mediated pathway.
Hypocotyls of the diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) do not elongate in response to exogenous auxin, but can respond to gravity. This appears paradoxical in light of the Cholodny-Went hypothesis, which states that shoot gravicurvature results from asymmetric stimulation of elongation by auxin. While light-grown dgt seedlings can achieve correct gravitropic reorientation, the response is slow compared to wild-type seedlings. The sensitivity of dgt seedlings to inhibition of gravicurvature by immersion in auxin or auxin-transport inhibitors is similar to that of wild-type plants, indicating that both an auxin gradient and auxin transport are required for the gravitropic response and that auxin uptake, efflux, and at least one auxin receptor are functional in dgt. Furthermore, dgt gravicurvature is the result of asymmetrically increased elongation as would be expected for an auxin-mediated response. Our results suggest differences between elongation in response to exogenous auxin (absent in dgt) and elongation in response to gravistimulation (present but attenuated in dgt) and confirm the presence of two phases during the gravitropic response, both of which are dependent on functional auxin transport.
Benzoic Acid↗
A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification.
A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.
Amino Acid Sequence↗
The Potato virus X TGBp3 protein associates with the ER network for virus cell-to-cell movement.
Potato virus X (PVX) TGBp3 is required for virus cell-to-cell movement. Cell-to-cell movement of TGBp3 was studied using biolistic bombardment of plasmids expressing GFP:TGBp3. TGBp3 moves between cells in Nicotiana benthamiana, but requires TGBp1 to move in N. tabacum leaves. In tobacco leaves GFP:TGBp3 accumulated in a pattern resembling the endoplasmic reticulum (ER). To determine if the ER network is important for GFP:TGBp3 and for PVX cell-to-cell movement, a single mutation inhibiting membrane binding of TGBp3 was introduced into GFP:TGBp3 and into PVX. This mutation disrupted movement of GFP:TGBp3 and PVX. Brefeldin A, which disrupts the ER network, also inhibited GFP:TGBp3 movement in both Nicotiana species. Two deletion mutations, that do not affect membrane binding, hindered GFP:TGBp3 and PVX cell-to-cell movement. Plasmids expressing GFP:TGBp2 and GFP:TGBp3 were bombarded to several other PVX hosts and neither protein moved between adjacent cells. In most hosts, TGBp2 or TGBp3 cannot move cell-to-cell.
Amino Acid Sequence↗
The potato virus X TGBp2 protein association with the endoplasmic reticulum plays a role in but is not sufficient for viral cell-to-cell movement.
Potato virus X (PVX) TGBp1, TGBp2, TGBp3, and coat protein are required for virus cell-to-cell movement. Plasmids expressing GFP fused to TGBp2 were bombarded to leaf epidermal cells and GFP:TGBp2 moved cell to cell in Nicotiana benthamiana leaves but not in Nicotiana tabacum leaves. GFP:TGBp2 movement was observed in TGBp1-transgenic N. tabacum, indicating that TGBp2 requires TGBp1 to promote its movement in N. tabacum. In this study, GFP:TGBp2 was detected in a polygonal pattern that resembles the endoplasmic reticulum (ER) network. Amino acid sequence analysis revealed TGBp2 has two putative transmembrane domains. Two mutations separately introduced into the coding sequences encompassing the putative transmembrane domains within the GFP:TGBp2 plasmids and PVX genome, disrupted membrane binding of GFP:TGBp2, inhibited GFP:TGBp2 movement in N. benthamiana and TGBp1-expressing N. tabacum, and inhibited PVX movement. A third mutation, lying outside the transmembrane domains, had no effect on GFP:TGBp2 ER association or movement in N. benthamiana but inhibited GFP:TGBp2 movement in TGBp1-expressing N. tabacum and PVX movement in either Nicotiana species. Thus, ER association of TGBp2 may be required but not be sufficient for virus movement. TGBp2 likely provides an activity for PVX movement beyond ER association.
Amino Acid Sequence↗
Migration of lymphocytes on fibronectin-coated surfaces: temporal evolution of migratory parameters.
Lymphocytes typically interact with implanted biomaterials through adsorbed exogenous proteins. To provide a more complete characterization of these interactions, analysis of lymphocyte migration on adsorbed extracellular matrix proteins must accompany the commonly performed adhesion studies. We report here a comparison of the migratory and adhesion behavior of Jurkat cells (a T lymphoblastoid cell line) on tissue culture treated and untreated polystyrene surfaces coated with various concentrations of fibronectin. The average speed of cell locomotion showed a biphasic response to substrate adhesiveness for cells migrating on untreated polystyrene and a monotonic decrease for cells migrating on tissue culture-treated polystyrene. A modified approach to the persistent random walk model was implemented to determine the time dependence of cell migration parameters. The random motility coefficient showed significant increases with time when cells migrated on tissue culture-treated polystyrene surfaces, while it remained relatively constant for experiments with untreated polystyrene plates. Finally, a cell migration computer model was developed to verify our modified persistent random walk analysis. Simulation results suggest that our experimental data were consistent with temporally increasing random motility coefficients.
Adsorption↗
Pretreatment methods to improve nerve immunostaining in corneas from long-term fixed embryonic quail eyes.
Pretreatment methods were used to improve neurofilament immunostaining in corneas from embryonic day 16 Japanese quail corneas that had been stored in fixative solution for several months. A sequential combination of the following three pretreatments: brief microwave heating in saline, followed by extraction with sodium dodecyl sulfate (SDS) at 37 degrees C, followed by digestion with hyaluronidase at 37 degrees C, produced significantly increased antibody staining of corneal neurofilament proteins, compared with embryonic corneas subjected to no prior pretreatments or to single or two-step protocols. After applying the sequence of all three pretreatments, darkest nerve staining and increased numbers of fine branches were observed, together with lower background staining. Thus, the result of applying the three-step pretreatment sequence is better than that of applying any of its component single pretreatments or even combinations of any two of them. These findings therefore suggest that each of these three pretreatments causes a unique effect, beneficial to immunostaining of neurofilament proteins, and that their individual effects are independent and additive. In addition to embryonic corneas, the three-step procedure also may be useful for immunostaining of nerves in other very delicate, highly-hydrated tissues containing an abundance of extracellular matrix.
Animals↗