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15 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1985

Role of chemotaxis in the ecology of denitrifiers

It has been recognized that the process of denitrification represents a major sequence in the nitrogen cycle. It involves the anaerobic reduction of nitrate or nitrite to nitrous oxide or elemental nitrogen. This process is responsible for significant losses of nitrogen from agricultural soils. Up to now, little attention has been paid to the ecology of the organisms responsible for denitrification. It is pointed out that chemotaxis would probably offer a strong competitive mechanism for denitrifiers, since chemotaxis would allow denitrifiers to actively reach nitrate by directed motility, rather than by random movement or diffusion of nitrate. The present investigation was initiated to examine the chemotactic responses of several denitrifiers to nitrate and nitrite. Attention is given to bacterial strains, culture media and cell preparation, chemotaxis assays, and competition experiments. It was found that several denitrifiers, including P. aeruginosa, P. fluorescens, and P. Stutzeri, were strongly attracted to NO3(-) and NO2(-).

Kennedy, M. J.↗
NASA NTRS1989

The sensory transduction pathways in bacterial chemotaxis

Bacterial chemotaxis is a useful model for investigating in molecular detail the behavioral response of cells to changes in their environment. Peritrichously flagellated bacteria such as coli and typhimurium swim by rotating helical flagella in a counterclockwise direction. If flagellar rotation is briefly reversed, the bacteria tumble and change the direction of swimming. The bacteria continuously sample the environment and use a temporal sensing mechanism to compare the present and immediate past environments. Bacteria respond to a broad range of stimuli including changes in temperature, oxygen concentration, pH and osmotic strength. Bacteria are attracted to potential sources of nutrition such as sugars and amino acids and are repelled by other chemicals. In the methylation-dependent pathways for sensory transduction and adaptation in E. coli and S. typhimurium, chemoeffectors bind to transducing proteins that span the plasma membrane. The transducing proteins are postulated to control the rate of autophosphorylation of the CheA protein, which in turn phosphorylates the CheY protein. The phospho-CheY protein binds to the switch on the flagellar motor and is the signal for clockwise rotation of the motor. Adaptation to an attractant is achieved by increasing methylation of the transducing protein until the attractant stimulus is cancelled. Responses to oxygen and certain sugars involve methylation-independent pathways in which adaption occurs without methylation of a transducing protein. Taxis toward oxygen is mediated by the electron transport system and changes in the proton motive force. Recent studies have shown that the methylation-independent pathway converges with the methylation-dependent pathway at or before the CheA protein.

Taylor, Barry L.↗
NASA NTRS1998

Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

Non-NASA Center↗
NASA NTRS1995

Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.

NASA Program Exobiology↗
NASA NTRS2015

Effects of Angular Frequency During Clinorotation on Mesenchymal Stem Cell Morphology and Migration

Background/Objectives: Ground-based microgravity simulation can reproduce the apparent effects of weightlessness in spaceflight using clinostats that continuously reorient the gravity vector on a specimen, creating a time-averaged nullification of gravity. In this work, we investigated the effects of clinorotation speed on the morphology, cytoarchitecture, and migration behavior of human mesenchymal stem cells (hMSCs). Methods: We compared cell responses at clinorotation speeds of 0, 30, 60, and 75 rpm over 8 hours in a recently developed lab-on-chip-based clinostat system. Time lapse light microscopy was used to visualize changes in cell morphology during and after cessation of clinorotation. Cytoarchitecture was assessed by actin and vinculin staining, and chemotaxis was examined using time lapse light microscopy of cells in NGF (100 ng/ml) gradients. Results: Among clinorotated groups, cell area distributions indicated a greater inhibition of cell spreading with higher angular frequency (p is less than 0.005), though average cell area at 30 rpm after 8 hours became statistically similar to control (p = 0.794). Cells at 75rpm clinorotation remained viable and were able to re-spread after clinorotation. In chemotaxis chambers clinorotation did not alter migration patterns in elongated cells, but most clinorotated cells exhibited cell retraction, which strongly compromised motility.

microgravity↗
NASA NTRS2000

Extracellular calcium elicits a chemokinetic response from monocytes in vitro and in vivo

Recruitment of macrophages to sites of cell death is critical for induction of an immunologic response. Calcium concentrations in extracellular fluids vary markedly, and are particularly high at sites of injury or infection. We hypothesized that extracellular calcium participates in modulating the immune response, perhaps acting via the seven-transmembrane calcium-sensing receptor (CaR) on mature monocytes/macrophages. We observed a dose-dependent increase in monocyte chemotaxis in response to extracellular calcium or the selective allosteric CaR activator NPS R-467. In contrast, monocytes derived from mice deficient in CaR lacked the normal chemotactic response to a calcium gradient. Notably, CaR activation of monocytes bearing the receptor synergistically augmented the transmigration response of monocytes to the chemokine MCP-1 in association with increased cell-surface expression of its cognate receptor, CCR2. Conversely, stimulation of monocytes with MCP-1 or SDF-1alpha reciprocally increased CaR expression, suggesting a dual-enhancing interaction of Ca(2+) with chemokines in recruiting inflammatory cells. Subcutaneous administration in mice of Ca(2+), MCP-1, or (more potently) the combination of Ca(2+) and MCP-1, elicited an inflammatory infiltrate consisting of monocytes/macrophages. Thus extracellular calcium functions as an ionic chemokinetic agent capable of modulating the innate immune response in vivo and in vitro by direct and indirect actions on monocytic cells. Calcium deposition may be both consequence and cause of chronic inflammatory changes at sites of injury, infection, and atherosclerosis.

Non-NASA Center↗
NASA NTRS1998

Extracellular calcium (Ca2+o)-sensing receptor in a mouse monocyte-macrophage cell line (J774): potential mediator of the actions of Ca2+o on the function of J774 cells

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone remodeling and may play a role in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for bone marrow mononuclear cells in the vicinity, leading us to investigate whether such mononuclear cells express the CaR. In this study, we used the mouse J774 cell line, which exhibits a pure monocyte-macrophage phenotype. Both immunocytochemistry and Western blot analysis, using polyclonal antisera specific for the CaR, detected CaR protein in J774 cells. The use of reverse transcriptase-polymerase chain reaction with CaR-specific primers, including a set of intron-spanning primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in J774 cells. Exposure of J774 cells to high Ca2+o (2.8 mM or more) or the polycationic CaR agonist, neomycin (100 microM), stimulated both chemotaxis and DNA synthesis in J774 cells. Therefore, taken together, our data strongly suggest that the monocyte-macrophage cell line, J774, possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney.

NASA Discipline Musculoskeletal↗
NASA NTRS1976

The effects of space flight on polymorphonuclear leukocyte response experiment MA-032

In a series of studies performed at intervals from 30 day before flight to 30 days after recovery, blood samples were obtained from the three astronauts of the Apollo Soyuz Test Project and from eight control subjects. To determine the effects of space flight on polymorphonuclear leukocytes, tests were performed on blood samples obtained as quickly as possible after splashdown and on the day following recovery. The astronauts' inhalation of propellant gases and the inception of corticosteroid therapy 1 day after recovery provided an additional opportunity to investigate the possible effects of these factors on leukocyte function. Data were obtained during each time period on the total leukocyte count, differential count, leukocyte adhesion, leukocyte migration and chemotaxis, phagocytosis, and histochemical staining for leukocyte acid and alkaline phosphatase. These observations present a variety of in vitro correlates to white blood cell function within the body. Taken together, they serve as a reasonable approximation of the effects of space flight on leukocyte function.

Martin, R. R.↗
NASA NTRS1998

Extracellular calcium (Ca2+(o))-sensing receptor in a murine bone marrow-derived stromal cell line (ST2): potential mediator of the actions of Ca2+(o) on the function of ST2 cells

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+(o)) homeostasis by mediating the actions of Ca2+(o) on parathyroid gland and kidney. Bone marrow stromal cells support the formation of osteoclasts from their progenitors as well as the growth of hematopoietic stem cells by secreting humoral factors and through cell to cell contact. Stromal cells also have the capacity to differentiate into bone-forming osteoblasts. Bone resorption by osteoclasts probably produces substantial local increases in Ca2+(o) that could provide a signal for stromal cells in the immediate vicinity, leading us to determine whether such stromal cells express the CaR. In this study, we used the murine bone marrow-derived, stromal cell line, ST2. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in ST2 cells. We also identified CaR transcripts in ST2 cells by Northern analysis using a CaR-specific probe and by RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of ST2 cells to high Ca2+(o) (4.8 mM) or to the polycationic CaR agonists, neomycin (300 microM) or gadolinium (100 microM), stimulated both chemotaxis and DNA synthesis in ST2 cells. Therefore, taken together, our data strongly suggest that the bone marrow-derived stromal cell line, ST2, possesses both CaR protein and messenger RNA that are very similar if not identical to those in parathyroid and kidney. Furthermore, as ST2 cells have the potential to differentiate into osteoblasts, the CaR in stromal cells could participate in bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local, osteoclast-mediated release of Ca2+(o) and, thereafter, initiating bone formation after their differentiation into osteoblasts.

NASA Discipline Musculoskeletal↗
NASA NTRS2016

Impact of The Protective Renin-Angiotensin System (RAS) on The Vasoreparative Function of CD34+ CACs in Diabetic Retinopathy

Purpose: In diabetes, the impaired vasoreparative function of Circulating Angiogenic Cells (CACs) is believed to contribute to the progression of diabetic retinopathy (DR). Accumulating evidence suggests that the protective arm of renin-angiotensin system (RAS) ACE2 Angiotensin-(1-7) Mas plays an important role in restoring the function of diabetic CACs. We examined the protective RAS in CACs in diabetic individuals with different stages of retinopathy. Methods: Study subjects (n43) were recruited as controls or diabetics with either no DR, mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR). Fundus photography and fluorescein angiograms were analyzed using Vessel Generation Analysis (VESGEN) software in a cohort of subjects. CD34+ CACs were isolated from peripheral blood of diabetics and control subjects. RAS gene expressions in CACs were measured by qPCR. The vasoreparative function of CACs was assessed by migration ability toward CXCL12 using the QCM 5M 96-well chemotaxis cell migration assay. Results: ACE2 gene is a key enzyme converting the deleterious Angiotensin II to the beneficial Angiotensin-(1-7). ACE2 expression in CACs from diabetic subjects without DR was increased compared to controls, suggestive of compensation (p0.0437). The expression of Mas (Angiotensin-(1-7) receptor) in CACs was also increased in diabetics without DR, while was reduced in NPDR compared to controls (p0.0002), indicating a possible loss of compensation of the protective RAS at this stage of DR. The presence of even mild NPDR was associated with CD34+ CAC migratory dysfunction. When pretreating CACs of DR subjects with Angiotensin-(1-7), migratory ability to a chemoattractant CXCL12 was restored (p0.0008). By VESGEN analysis, an increase in small vessel density was observed in NPDR subjects when compared with the controls. Conclusions: These data suggest the protective RAS axis within diabetic CACs may help maintain their vasoreparative potential. The VESGEN analysis supports the presence of retinal repair in small vessels. The loss of the protective arm of RAS may predict the progression of DR.

diabetes↗
NASA NTRSDate not supplied

Multi-Omics Analysis of Mouse Retina Following Low Dose Radiation and/or Hindlimb Unloading

Rodent models have been used as analogs for studying the effects of spaceflight. NASA’s GeneLab provides access to omics datasets generated from spaceflight and ground-based experiments allowing for additional retrospective analysis. We used GeneLab’s GLDS-203, a dataset generated by researchers at Loma Linda University to study the impact of prolonged unloading and/or low-dose radiation on mouse retina. The purpose of this study was to understand the effect of gamma radiation and/or hindlimb unloading on mice retinas through a multi-omics analysis. In the experiment that generated the omics data, mice were irradiated with gamma-ray and/or subjected to hindlimb unloading for 21 days and multi-omics analysis was performed at 7 days, 1 month, or 4 months post exposure. In the current study, for each of the three timepoints, we compared epigenomic profiles for retinas from exposed mice against timepoint-matched controls. We identified a total of 5,271 differentially methylated loci (DML) and 321 differentially methylated regions (DMR; using a sliding window and step size of 500 bp) with methylation difference > 10% and q-value < 0.05 (sliding linear model corrected p-value) across the nine exposure groups. Highest correlation in methylation difference was seen for significant DMLs (q-value < 0.05) across different conditions at same post exposure timepoint (Figure 1).The location of DMLs and DMRs were characterized with respect to CpG islands and shores, putative promoters, gene body, and intergenic regions (Table 1). We analyzed RNA-seq counts and performed gene set enrichment analysis using differential expression results from comparing each exposure group to its timepoint-matched control group. Significant pathways (adjusted p-value <0.05) enriched in all three microgravity-only groups were related to morphogenesis of a branching epithelium, skeletal muscle cell differentiation, and response to fibroblast growth factor. Common processes across all timepoints in the radiation-only groups were retina homeostasis, synaptic vesicle exocytosis-endocytosis, and chemotaxis. In the combination groups, regulation of trans-synaptic signaling, and Rho protein signal transduction were enriched at all three timepoints. Processes related to purine nucleotide metabolism were enriched in all nine exposure groups, with activation at 1 month, and suppression at 7 days and 4 months. A total of 14 genes contained at least one DML and were differentially expressed at adjusted p-value < 0.05, including genes implicated in cataract development (Sipa1l3, Crybb3) and those involved in cytoskeletal organization (Plec, Flnb, Eef1a1). This analysis is part of a larger effort to understand the molecular mechanisms following spaceflight exposures that can help translate effects observed in animal models to human impacts.

Prachi Kothiyal↗
NASA NTRS2002

Loss of signal transduction and inhibition of lymphocyte locomotion in a ground-based model of microgravity

Inflammatory adherence to, and locomotion through the interstitium is an important component of the immune response. Conditions such as microgravity and modeled microgravity (MMG) severely inhibit lymphocyte locomotion in vitro through gelled type I collagen. We used the NASA rotating wall vessel bioreactor or slow-turning lateral vessel as a prototype for MMG in ground-based experiments. Previous experiments from our laboratory revealed that when lymphocytes (human peripheral blood mononuclear cells [PBMCs]) were first activated with phytohemaglutinin followed by exposure to MMG, locomotory capacity was not affected. In the present study, MMG inhibits lymphocyte locomotion in a manner similar to that observed in microgravity. Phorbol myristate acetate (PMA) treatment of PBMCs restored lost locomotory capacity by a maximum of 87%. Augmentation of cellular calcium flux with ionomycin had no restorative effect. Treatment of lymphocytes with mitomycin C prior to exposure to MMG, followed by PMA, restored locomotion to the same extent as when nonmitomycin C-treated lymphocytes were exposed to MMG (80-87%), suggesting that deoxyribonucleic acid replication is not essential for the restoration of locomotion. Thus, direct activation of protein kinase C (PKC) with PMA was effective in restoring locomotion in MMG comparable to the normal levels seen in Ig cultures. Therefore, in MMG, lymphocyte calcium signaling pathways were functional, with defects occurring at either the level of PKC or upstream of PKC.

NASA Discipline Cell Biology↗
NASA NTRS1999

Leukocyte subsets and neutrophil function after short-term spaceflight

Changes in leukocyte subpopulations and function after spaceflight have been observed but the mechanisms underlying these changes are not well defined. This study investigated the effects of short-term spaceflight (8-15 days) on circulating leukocyte subsets, stress hormones, immunoglobulin levels, and neutrophil function. At landing, a 1.5-fold increase in neutrophils was observed compared with preflight values; lymphocytes were slightly decreased, whereas the results were variable for monocytes. No significant changes were observed in plasma levels of immunoglobulins, cortisol, or adrenocorticotropic hormone. In contrast, urinary epinephrine, norepinephrine, and cortisol were significantly elevated at landing. Band neutrophils were observed in 9 of 16 astronauts. Neutrophil chemotactic assays showed a 10-fold decrease in the optimal dose response after landing. Neutrophil adhesion to endothelial cells was increased both before and after spaceflight. At landing, the expression of MAC-1 was significantly decreased while L-selectin was significantly increased. These functional alterations may be of clinical significance on long-duration space missions.

short duration↗
NASA NTRS1995

The development of concentration gradients in a suspension of chemotactic bacteria

When a suspension of bacterial cells of the species Bacillus subtilis is placed in a chamber with its upper surface open to the atmosphere complex bioconvection patterns are observed. These arise because the cells: (1) are denser than water; and (2) usually swim upwards, so that the density of an initially uniform suspension becomes greater at the top than the bottom. When the vertical density gradient becomes large enough, an overturning instability occurs which ultimately evolves into the observed patterns. The reason that the cells swim upwards is that they are aerotactic, i.e., they swim up gradients of oxygen, and they consume oxygen. These properties are incorporated in conservation equations for the cell (N) and oxygen (C) concentrations, and these are solved in the pre-instability phase of development when N and C depend only on the vertical coordinate and time. Numerical results are obtained for both shallow- and deep-layer chambers, which are intrinsically different and require different mathematical and numerical treatments. It is found that, for both shallow and deep chambers, a thin boundary layer, densely packed with cells, forms near the surface. Beneath this layer the suspension becomes severely depleted of cells. Furthermore, in the deep chamber cases, a discontinuity in the cell concentration arises between this cell-depleted region and a cell-rich region further below, where no significant oxygen concentration gradients develop before the oxygen is fully consumed. The results obtained from the model are in good qualitative agreement with the experimental observations.

NASA Discipline Cell Biology↗