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NASA NTRS2001

Connexin45 interacts with zonula occludens-1 in osteoblastic cells

Connexin43 (Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.

Non-NASA Center↗

NASA NTRS2002

XCTK1: A Xenopus C-terminal Kinesin-like Protein

XCTK1 is 97kDa kinesin-like protein homologous to FKIF2 and KIFC3. XCTK1 is present at picomolar levels in eggs, embryos and cultured cells in a soluble high-molecular weight complex that is not associated with membranes. XCKT1 localizes to centrosomes in Xenopus A6 cells. Anti-XCTK1 antibodies also localize to spindle poles when injected into A6 cells or when added to extracts during in vitro spindle assembly reactions. XCTK1 is associated with the center of taxol-induced microtubule asters in extracts. Therefore its localization to poles is dependent on microtubule minus-ends and not on centrosomes per se. Overexpression of XCTK1 leads to centrosome destruction in cultured cells. XCTK1 was tagged at either the N- or C-terminus and transfected into Xenopus A6 cells At low expression levels, XCTK1 associated with centrosomes. At higher levels, the protein localized to insoluble cytoplasmic structures. Gamma-tubulin staining was dramatically decreased from centrosomes or altogether absent. The centrosomal SPJ antigen colocalized with XCTK1-containing structures. Upon nocodozole treatment, microtubules failed to regrow from the centrosomes indicating that overexpression of XCTK1 severely compromises centrosomal function. Current studies are aimed at determining whether XCTK1 interacts directly with centrosomal proteins and to determine the effects of XCTK1 depletion on oocyte maturation and embryogenesis.

Winfree, Seth↗

NASA NTRS1987

Controlled ecological life support systems; Proceedings of Workshop II of the 26th COSPAR Plenary Meeting, Toulouse, France, June 30-July 11, 1986

The present conference on the development status of Controlled Ecological Life Support Systems (CELSSs) discusses food production and gas exchange with the Spirulina blue-green alga, biomass recycling for greater energy efficiency in algal culture CELSSs, algal proteins for food processing in a CELSS, a CELSS with photosynthetic N2-fixing cyanobacteria, the NASA CELSS program, and vapor compression ditillation and membrane technology for water revitalization. Also discussed are a fundamental study of CELSS gas monitoring, the application of catalytic wet oxidation to CELSS, a large-scale perspective on ecosystems, Japanese CELSS research activities, the use of potatoes in bioregenerative life-support, wheat production in controlled environments, and a trickle water and feeding system in plant culture.

Macelroy, R. D.↗

NASA NTRS1998

Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.

Non-NASA Center↗

NASA NTRS1983

Calcium movements and the cellular basis of gravitropism

Evidence that calcium is a significant factor in gravitropism of plants is examined, together with the regulatory mechanism that controls calcium efflux in plant cells. Calcium has been found to accumulate on the upper surface of a plant placed in a horizontal attitude, which is followed by growth to the vertical within a half-hour. It has also been determined that the upper surface of the plant simultaneously experiences a reduced growth rate, compared to the lower surface. The substance calmodulin has been identified as the calcium-binding regulatory protein most influential in the transduction process leading to gravitropism, while chlorpromazine can inhibit its effects. The calcium is transported by ATPase located in the cell membrane. A possibility that the calcium pump is regulated by a feedback mechanism involving the presence of calmodulin is discussed.

Roux, S. J.↗

NASA NTRS1997

The Gravity of Giraffe Physiology

By virtue of its tallness and terrestrial environment, the giraffe is a uniquely sensitive African animal to investigate tissue adaptations to gravitational stress. One decade ago, we studied transcapillary fluid balance and local tissue adaptations to high cardiovascular and musculoskeletal loads in adult and fetal giraffes. Previous studies by Goetz, Pattersson, Van Citters, Warren and their colleagues revealed that arterial pressure near the giraffe heart is about twice that in humans, to provide more normal blood pressure and perfusion to the brain. Another important question is how giraffes avoid pooling of blood and tissue fluid (edema) in dependent tissue of the extremities. As monitored by radiotelemetry, the blood and tissue fluid pressures that govern transcapillary exchange vary greatly with exercise. These pressures, combined with a tight skin layer, move fluid upward against gravity. Other mechanisms that prevent edema include precapillary vasoconstriction and low permeability of capillaries to plasma proteins. Other anatomical adaptations in dependent tissues of giraffes represent developmental adjustments to high and variable gravitational forces. These include vascular wall hypertrophy, thickened capillary basement membrane and other connective tissue adaptations. Our results in giraffe suggest avenues of future gravitational research in other animals including humans.

Hargens, Alan R.↗

NASA NTRS1994

Production of RNA by a polymerase protein encapsulated within phospholipid vesicles

Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.

Non-NASA Center↗

NASA NTRS1991

Effects of murine leukemia virus env gene proteins on macrophage-mediated cytotoxicity in vitro

F5b Tumor cells were incubated with concentrated culture supernatants taken from cells resistant (F5m) or sensitive (F5b) to contact-dependent macrophage cytotoxicity. Macrophage cell line B6MP102 and murine peritoneal macrophages killed targets incubated with supernatants taken from sensitive cells but poorly killed cells incubated in supernatants isolated from resistant cells. Membranes from cells resistant to macrophage killing, F5m, were fused into F5b cells. The fused F5b cells were killed significantly less than F5b cells fused with F5b cell membranes or untreated F5b cells. The decreased killing of F5b cells corresponded to increased concentrations of gp70(a) molecules on F5b cells. Affinity purified gp70(a) was added to cytotoxicity assays but failed to inhibit macrophage cytotoxicity. P15E molecules were detectable on both F5b and F5m cells. In addition, a synthetic peptide found to exhibit the inhibitory properties of p15E was added to cytotoxicity assays. P15E synthetic peptide also did not inhibit macrophage cytotoxicity. Therefore, env gene proteins of murine leukemia virus do not appear responsible for inducing tumor cell resistance to activated macrophage contact-dependent cytotoxicity.

NASA Program NSCORT↗

NASA NTRS1986

Protobiological informatoin, bidirectional recognition and reverse translation

Emergence of protobiological information has been suggested by experiments in which heated mixtures of alpha-amino acids order themselves into a self limited array of thermal proteins. The polymers display selective catalytic, hormonal, and other activities. Interactions of varied cationic thermal proteins with polynucleotides indicate selective recognition in both directions. Reverse translation is partly a missing link in the molecular evolution flowsheet. The self ordering of amino acids serves conceptually as a deterministic evolutionary precursor of the modern coding mechanism. The possibility for the evolution of information at an early nontemplated protein stage is supported by findings of electrical signals from proteinoid microspheres prepared with no DNA/RNA in their history. The deposition of thermal copolyamino acids on lipid membranes in the Mueller-Rudin apparatus has here been found to produce electrical behavior like that evoked by bacterial EIM polypeptide. A new procedure is to make a film of membrane on the electrode; the results provide maximal repeatability. The principle of nonrandom biomacromolecular specificity identified by these studies in molecular evolution have been extrapolated to principles of evolution of advanced organisms.

Fox, S. W.↗

NASA NTRS2006

Novel Concept for LSS Based on Advanced Microalgal Biotechnologies

One of the key issues for successful human space exploration is biomedical life support in hostile space and planetary environments that otherwise cannot sustain life. Bioregenerative life support systems (LSS) are one of the options for atmospheric regeneration. To date, no bioregenerative LSS has shown capability for 100% air regeneration. Nor have these LSS been robust enough to simultaneously provide a regenerable complete food source. In contrast to microalgae, traditional plant approaches, e.g. wheat and lettuce, are lacking essential amino acids, vitamins, and micronutrients. Moreover, the rate of photosynthesis by microalgae significantly exceeds that of high plants. Nevertheless, the employment of microalgae in LSS technology was restricted, until recently, due to high water demands. Also the per person requirement of a 40L volume of microalgae in a photobioreactor, to provide daily O2 production, made an algae-based approach less attractive. By employing a vertically stacked membrane bioreactor, coupled with a solar tracker and photon-delivery system, a lightweight air revitalization system for space based applications, with minimal water requirements, can be developed. Our preliminary estimations suggest that a membrane bioreactor, 8m3 in volume, comprised of 80m2 (twenty 2m x 2m membranes, each spaced 10 cm apart), and a total of 70L of water could produce 2.7 kg of dried microalgal biomass that would supply the energy and essential amino acid requirements, as well as producing sufficient O2 for the daily needs of a 15 member crew. Research on the biochemical content of edible blue-green alga Spirulina (Arthrospira) platensis shows a wide spectrum of stable Spirulina mutants with an enhanced content of amino acids, -carotene, and phycobilliprotein c-phycocyanin. Feeding animals suffering from radiation-induced lesions, c-phycocyanin, extracted from strain 27G, led to a correction in the decrement of dehydrogenase activity and energy-rich phosphate levels, as well as improved antioxidant defense and pyruvate levels, compared to untreated animals. Experimental anemia in rats was corrected by feeding Spirulina platensis strains 198B and 27G, (with an enhanced content of methionine, phycobiliproteins and carotenoids). Spirulina was recently shown by Ananyev et al, 2005, to be an oxygenic organism with the highest level of photosystem II activity (O2 production). We propose therefore to develop a design for membrane-based photoreactors for Lunar and Mars exploration habitat LSS, for the cultivation of genetically modified strains of Spirulina to scrub CO2 and supply astronauts with O2, protein, vitamins, and immunostimulators.

Brown, I.↗

NASA NTRS1999

Avian Blood-Vessel Formation in Space

Based on previous studies, we hypothesized that the developmental anomalies observed in the past might be related to or caused by delayed or improper vascular development. The objective of our research is to test the hypothesis that exposure to microgravity during space flight cause delayed or improper vascular development during embryogenesis. The effects of microgravity on the time course and extent of avian blood-vessel formation are assessed using two models, one for angiogenesis and one for vasculogenesis. The methodological approach is dictated by the constraints of the tissue preservation method used in space. Thus, both in the chorioallantoic membrane (CAM) and in the adrenal, we will evaluate microscopically the vascular architecture and immunostain endothelial cells with specific antibodies (anti- vWF and QH1). The extent of ECM protein deposition will be assessed by immunohistochemistry and correlated with the degree of vascularization, using computer-based image analysis. Also, the cellular source for ECM proteins will be assessed by in situ hybridization.

Lelkes, Peter I.↗

NASA NTRS2002

The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth

To investigate how roots respond to directional cues, we characterized a T-DNA-tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.

NASA Discipline Plant Biology↗

NASA NTRS1999

Rapid tachyphylaxis to hemodynamic effects of PACAP-27 after inhibition of nitric oxide synthesis

The vasodilator effects of pituitary adenylate cyclase-activating polypeptide (PACAP)-27 are subject to tachyphylaxis in rats treated with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). We examined whether this tachyphylaxis could be prevented by administration of the putative endothelium-derived nitrosyl factor S-nitroso-L-cysteine (L-SNC) and whether L-SNC may exert its effects via increases in cGMP levels in vascular smooth muscle. Five doses of PACAP-27 (2 nmol/kg iv) produced pronounced vasodilator responses in saline-treated rats. These responses were not subject to tachyphylaxis. The first injection of PACAP-27 (2 nmol/kg iv) in L-NAME-treated (50 micromol/kg iv) rats produced vasodilator responses similar to those in saline-treated rats, whereas subsequent injections produced progressively smaller responses. The injection of L-SNC (1,200 nmol/kg iv) before each injection of PACAP-27 prevented tachyphylaxis to the Gs protein-coupled receptor agonist in L-NAME-treated rats, whereas equihypotensive doses of the NO donor sodium nitroprusside (100 micrograms/kg iv) did not. The injection of the membrane-permeant cGMP analog 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-CPT-cGMP; 30 micromol/kg iv) to L-NAME-treated rats restored resting hemodynamic values to pre-L-NAME levels but did not prevent the development of tachyphylaxis to PACAP-27. These results suggest that nitrosyl factors prevent the development of tachyphylaxis to the hemodynamic actions of PACAP-27. These nitrosyl factors may act independently of their ability to generate cGMP in vascular smooth muscle.

NASA Discipline Regulatory Physiology↗

NASA NTRS2012

Quality Control Method for a Micro-Nano-Channel Microfabricated Device

A variety of silicon-fabricated devices is used in medical applications such as drug and cell delivery, and DNA and protein separation and analysis. When a fluidic device inlet is connected to a compressed gas reservoir, and the outlet is at a lower pressure, a gas flow occurs through the membrane toward the outside. The method relies on the measurement of the gas pressure over the elapsed time inside the upstream and downstream environments. By knowing the volume of the upstream reservoir, the gas flow rate through the membrane over the pressure drop can be calculated. This quality control method consists of measuring the gas flow through a device and comparing the results with a standard curve, which can be obtained by testing standard devices. Standard devices can be selected through a variety of techniques, both destructive and nondestructive, such as SEM, AFM, and standard particle filtration.

Grattoni, Alessandro↗

NASA NTRS1993

Monoclonal Antibody Testing for Cancer Metastasis

Malignant cells are characterized by the ability to invade surrounding normal tissues. Tumor invasion is abetted by proteolytic enzymes that have been correlated with recurrent disease and metastasis. These enzymes are involved in a cascade of proteolytic interactions with other enzymes and inhibitors which allow cancer cells to dissolve surrounding extracellular matrix, thereby enabling the cells to rapidly invade adjacent tissues and migrate to metastatic sites distant from the primary tumor. Among these proteases are the plasminogen activators (PA), collagenase IV, faminase, and in some cases cathepsin D, which together mediate key steps in the invasion process of metastasis. Cells which have the selective advantage for invasion and metastasis are those capable of regulating their proteolytic activity and proliferation. Cells in the process of invasion would be probably down-regulated for proliferation, but subsequent to attachment and adhesion at a distant site, would then be in a proliferative mode, up-regulating DNA replication. Urokinase (uPA) can be present in the tissues in several molecular forms. The inactive proenzyme is a single chain protein (scuPA) that is cleaved at Lys. 158 to form the double chain, high molecular weight active form (HMW-uPA) of 54 kD. A low molecular weight form (LMW-uPA) can also be produced by cleavage of the HMW-U PA at Lys. 135 - Lys. 136 giving a 35 kD active enzyme. Recently, it has been shown that the HMW active form of urokinase, bound to the tumor cell membrane, is responsible for the local lysis of the extracellular matrix, hence the tissue invasion mechanism for metastasis (Andreasen et al, 19861. Receptor- (membrane) bound uPA is twice as efficient (catalytically) as free fluid-phase uPA. Tho unbound uPA and the LMW form is not responsible for most of the local dissolution of extracellular matrix in the immediate vicinity of the metastatic tumor cell. High levels of urokinase (greater than 3.49 ng/mg of total protein) extracted from breast tumor tissues have recently been shown, together with plasminogen activator inhibitor 1 (PAI-1), to be a good prognostic indicator for high risk of recurrence and shorter patient survival times. In this project, we have attempted to develop immunocytochemical methodologies for the clinical assessment of the expression of urokinase plasminogen activator, which has been implicated to be important for initial steps in tumor invasion, and to relate it to cell proliferation and DNA replication at the single-cell level.

Source record↗

NASA NTRS1989

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.

NASA Discipline Number 61-10↗

NASA NTRS2003

Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

Non-NASA Center↗

NASA NTRS1998

Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat)

Alteration in cytoskeletal organization appears to underlie mechanisms of gravity sensitivity in space-flown cells. Human T lymphoblastoid cells (Jurkat) were flown on the Space Shuttle to test the hypothesis that growth responsiveness is associated with microtubule anomalies and mediated by apoptosis. Cell growth was stimulated in microgravity by increasing serum concentration. After 4 and 48 h, cells filtered from medium were fixed with formalin. Post-flight, confocal microscopy revealed diffuse, shortened microtubules extending from poorly defined microtubule organizing centers (MTOCs). In comparable ground controls, discrete microtubule filaments radiated from organized MTOCs and branched toward the cell membrane. At 4 h, 30% of flown, compared to 17% of ground, cells showed DNA condensation characteristic of apoptosis. Time-dependent increase of the apoptosis-associated Fas/ APO-1 protein in static flown, but not the in-flight 1 g centrifuged or ground controls, confirmed microgravity-associated apoptosis. By 48 h, ground cultures had increased by 40%. Flown populations did not increase, though some cells were cycling and actively metabolizing glucose. We conclude that cytoskeletal alteration, growth retardation, and metabolic changes in space-flown lymphocytes are concomitant with increased apoptosis and time-dependent elevation of Fas/APO-1 protein. We suggest that reduced growth response in lymphocytes during spaceflight is linked to apoptosis.

STS-76 Shuttle Project↗