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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 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
NASA NTRS2002

Demonstration of four immunoassay formats using the array biosensor

The ability of a fluorescence-based array biosensor to measure and quantify the binding of an antigen to an immobilized antibody has been demonstrated using the four different immunoassay formats: direct, competitive, displacement, and sandwich. A patterned array of antibodies specific for 2,4,6-trinitrotoluene (TNT) immobilized onto the surface of a planar waveguide and used to measure signals from different antigen concentrations simultaneously. For direct, competitive, and displacement assays, which are one-step assays, measurements were obtained in real time. Dose-response curves were calculated for all four assay formats, demonstrating the array biosensor's ability to quantify the amount of antigen present in solution.

NASA Discipline Life Sciences Technologies↗
NASA NTRS1988

Evidence against the involvement of ionically bound cell wall proteins in pea epicotyl growth

Ionically bound cell wall proteins were extracted from 7 day old etiolated pea (Pisum sativum L. cv Alaska) epicotyls with 3 molar LiCl. Polyclonal antiserum was raised in rabbits against the cell wall proteins. Growth assays showed that treatment of growing region segments (5-7 millimeters) of peas with either dialyzed serum, serum globulin fraction, affinity purified immunoglobulin, or papain-cleaved antibody fragments had no effect on growth. Immunofluorescence microscopy confirmed antibody binding to cell walls and penetration of the antibodies into the tissues. Western blot analysis, immunoassay results, and affinity chromatography utilizing Sepharose-bound antibodies confirmed recognition of the protein preparation by the antibodies. Experiments employing in vitro extension as a screening measure indicated no effect upon extension by antibodies, by 50 millimolar LiCl perfusion of the apoplast or by 3 molar LiCl extraction. Addition of cell wall protein to protease pretreated segments did not restore extension nor did addition of cell wall protein to untreated segments increase extension. It is concluded that, although evidence suggests that protein is responsible for the process of extension, the class(es) of proteins which are extracted from pea cell walls with 3 molar LiCl are probably not involved in this process.

NASA Program Space Biology↗
NASA NTRS2000

Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells

We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well. Copyright 2000 Wiley-Liss, Inc.

NASA Program Fundamental Space Biology↗
NASA NTRS2001

Vascular defense responses in rice: peroxidase accumulation in xylem parenchyma cells and xylem wall thickening

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae is a vascular pathogen that elicits a defensive response through interaction with metabolically active rice cells. In leaves of 12-day-old rice seedlings, the exposed pit membrane separating the xylem lumen from the associated parenchyma cells allows contact with bacterial cells. During resistant responses, the xylem secondary walls thicken within 48 h and the pit diameter decreases, effectively reducing the area of pit membrane exposed for access by bacteria. In susceptible interactions and mock-inoculated controls, the xylem walls do not thicken within 48 h. Xylem secondary wall thickening is developmental and, in untreated 65-day-old rice plants, the size of the pit also is reduced. Activity and accumulation of a secreted cationic peroxidase, PO-C1, were previously shown to increase in xylem vessel walls and lumen. Peptide-specific antibodies and immunogold-labeling were used to demonstrate that PO-C1 is produced in the xylem parenchyma and secreted to the xylem lumen and walls. The timing of the accumulation is consistent with vessel secondary wall thickening. The PO-C1 gene is distinct but shares a high level of similarity with previously cloned pathogen-induced peroxidases in rice. PO-C1 gene expression was induced as early as 12 h during resistant interactions and peaked between 18 and 24 h after inoculation. Expression during susceptible interactions was lower than that observed in resistant interactions and was undetectable after infiltration with water, after mechanical wounding, or in mature leaves. These data are consistent with a role for vessel secondary wall thickening and peroxidase PO-C1 accumulation in the defense response in rice to X. oryzae pv. oryzae.

NASA Discipline Plant Biology↗
NASA NTRS1995

Polar distribution of annexin-like proteins during phytochrome-mediated initiation and growth of rhizoids in the ferns Dryopteris and Anemia

Although the calcium requirement of phytochrome-mediated fern spore germination and early rhizoid growth is well established, the calcium-binding proteins that serve as transducers for these responses are not known. Here we report the presence of annexin-like proteins in germinating spores of Dryopteris filix-mas (L.) Schott and Anemia phyllitidis (L.) Sw. and evidence that they may be important participants in early photomorphogenic changes in gametophytes. Immunolocalization and immunoblot assays of these proteins were carried out using polyclonal antibodies raised either against a 35-kDa annexin-like protein from pea or against anchorin CII from chicken. Western-blot analysis showed that crude protein extracts obtained from both species after red-light treatment contained two cross-reactive protein bands with molecular weights around 70 kDa. These proteins were annexin-like in that they bound to a phosphatidylserine affinity column in a calcium-dependent fashion. Using this column, two protein bands around 70 kDa, i.e. 67 and 73 kDa, were partially purified together with proteins at 36 kDa and a doublet at 54 kDa. Proteins of these latter molecular weights are suggested to be members of the annexin family, but no cross-reactivity could be found between these and the two antibodies used in our investigations. Immunodetectable levels of these proteins were observed only after light-mediated induction of spore germination. Imaging of the immuno-localization patterns observed with both antibodies showed that the annexin-like proteins are concentrated at the extreme tips of the rhizoids in D. filix-mas and A. phyllitidis during rhizoid initiation and all stages of elongation. We suggest that these proteins may play a major role in the tip-oriented exocytosis events that are critical for the initiation and growth of fern rhizoids.

NASA Discipline Number 40-50↗
NASA NTRS2001

Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells

A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.

Non-NASA Center↗
NASA NTRS1993

The 'tubulin-like' S1 protein of Spirochaeta is a member of the hsp65 stress protein family

A 65-kDa protein (called S1) from Spirochaeta bajacaliforniensis was identified as 'tubulin-like' because it cross-reacted with at least four different antisera raised against tubulin and was isolated, with a co-polymerizing 45-kDa protein, by warm-cold cycling procedures used to purify tubulin from mammalian brain. Furthermore, at least three genera of non-cultivable symbiotic spirochetes (Pillotina, Diplocalyx, and Hollandina) that contain conspicuous 24-nm cytoplasmic tubules displayed a strong fluorescence in situ when treated with polyclonal antisera raised against tubulin. Here we summarize results that lead to the conclusion that this 65-kDa protein has no homology to tubulin. S1 is an hsp65 stress protein homologue. Hsp65 is a highly immunogenic family of hsp60 proteins which includes the 65-kDa antigens of Mycobacterium tuberculosis (an active component of Freund's complete adjuvant), Borrelia, Treponema, Chlamydia, Legionella, and Salmonella. The hsp60s, also known as chaperonins, include E. coli GroEL, mitochondrial and chloroplast chaperonins, the pea aphid 'symbionin' and many other proteins involved in protein folding and the stress response.

NASA Discipline Exobiology↗
NASA NTRS1995

Some growth factors stimulate cultured adult rabbit ventricular myocyte hypertrophy in the absence of mechanical loading

Cultured adult rabbit cardiac myocytes treated with recombinant growth factors display enhanced rates of protein accumulation (ie, growth) in response to insulin and insulin-like growth factors (IGFs), but epidermal growth factor, acidic or basic fibroblast growth factor, and platelet-derived growth factor failed to increase contractile protein synthesis or growth of the heart cells. Insulin and IGF-1 increased growth rates by stimulating anabolic while simultaneously inhibiting catabolic pathways, whereas IGF-2 elevated growth modestly by apparently inhibiting lysosomal proteolysis. Neutralizing antibodies directed against either IGF-1 or IGF-2 or IGF binding protein 3 blocked protein accumulation. A monoclonal antibody directed against the IGF-1 receptor also inhibited changes in protein turnover provoked by recombinant human IGF-1 but not IGF-2. Of the other growth factors tested, only transforming growth factor-beta 1 increased the fractional rate of myosin heavy chain (MHC) synthesis, with beta-MHC synthesis being elevated and alpha-MHC synthesis being suppressed. However, the other growth factors were able to modestly stimulate the rate of DNA synthesis in this preparation. Bromodeoxyuridine labeling revealed that these growth factors increased DNA synthesis in myocytes and nonmyocytes alike, but the heart cells displayed neither karyokinesis or cytokinesis. In contrast, cocultures of cardiac myocytes and nonmyocytes and nonmyocyte-conditioned culture medium failed to enhance the rate of cardiac MHC synthesis or its accumulation, implying that quiescent heart cells do not respond to "conditioning" by cardiac nonmyocytes. These findings demonstrated that insulin and the IGFs promote passively loaded cultured adult rabbit heart cells to hypertrophy but suggest that other growth factors tested may be limited in this regard.

NASA Discipline Cell Biology↗
NASA NTRS1986

Amphibian egg cytoplasm response to altered g-forces and gravity orientation

Elucidation of dorsal/ventral polarity and primary embryonic axis development in amphibian embryos requires an understanding of cytoplasmic rearrangements in fertile eggs at the biophysical, physiological, and biochemical levels. Evidence is presented that amphibian egg cytoplasmic components are compartmentalized. The effects of altered orientation to the gravitational vector (i.e., egg inversion) and alterations in gravity force ranging from hypergravity (centrifugation) to simulated microgravity (i.e., horizontal clinostat rotation) on cytoplasmic compartment rearrangements are reviewed. The behavior of yolk compartments as well as a newly defined (with monoclonal antibody) nonyolk cytoplasmic compartment, in inverted eggs and in eggs rotated on horizontal clinostats at their buoyant density, is discussed.

Neff, A. W.↗
NASA NTRS1995

Cellular localization of the Ca2+ binding TCH3 protein of Arabidopsis

TCH3 is an Arabidopsis touch (TCH) gene isolated as a result of its strong and rapid upregulation in response to mechanical stimuli, such as touch and wind. TCH3 encodes an unusual calcium ion-binding protein that is closely related to calmodulin but has the potential to bind six calcium ions. Here it is shown that TCH3 shows a restricted pattern of accumulation during Arabidopsis vegetative development. These data provide insight into the endogenous signals that may regulate TCH3 expression and the sites of TCH3 action. TCH3 is abundant in the shoot apical meristem, vascular tissue, the root columella and pericycle cells that give rise to lateral roots. In addition, TCH3 accumulation in cells of developing shoots and roots closely correlates with the process of cellular expansion. Following wind stimulation, TCH3 becomes more abundant in specific regions including the branchpoints of leaf primordia and stipules, pith parenchyma, and the vascular tissue. The consequences of TCH3 upregulation by wind are therefore spatially restricted and TCH3 may function at these sites to modify cell or tissue characteristics following mechanical stimulation. Because TCH3 accumulates specifically in cells and tissues that are thought to be under the influence of auxin, auxin levels may regulate TCH3 expression during development. TCH3 is upregulated in response to low levels of exogenous indole-3-acetic acid (IAA), but not by inactive auxin-related compounds. These results suggest that TCH3 protein may play roles in mediating physiological responses to auxin and mechanical environmental stimuli.

NASA Discipline Plant Biology↗
NASA NTRS2006

A Preliminary Analysis of Calcifying Particles in the Serum and Prostates of Patients with Prostatic Inflammation

Chronic diseases of the prostate such as benign prostatic hyperplasia (BPH) & chronic pelvic pain syndrome (CPPS) have associated findings of chronic inflammation, despite a lack of causal relationship. Numerous attempts to define an infectious agent responsible for the clinical findings have been inconsistent. The possibility of an infectious agent, that has not been uncovered with routine culturing methods, forms the basis for this study. Serum from 940 healthy Finnish men were compared with serum from 40 Crohn's, 40 path dx prostatitis, & 40 with path dx carcinoma, using an enzyme-linked immunosorbant assay (ELISA), to detect antigens specific to Nanobacteria(NB) utilizing monoclonal antibodies (Ab) 5/3 and 8D10. This ELISA has not been validated for detecting NB-associated with clinical prostatic disease, yet cross-reactivity with other bacterial species is low. Immunohistochemistry was performed on de-paraffinized prostatic tissue slides, de-calcified with EDTA and stained with the DAKO Catalyzed Signal Amplification kit, employing 8D10 as the primary (target/antigen-detecting) Ab. The mean (plus or minus SD) & median concentrations of NB antigen (U/50 L) were 379.59 (plus or minus 219.28) & 640.00 for patients with prostatitis (BPH) vs 3.31 (plus or minus 3.55) & 2.94 for prostate adenocarcinoma, 1.88 (plus or minus 2.94) & 0.80 for Crohn's disease, & 7.43 (plus or minus 25.57) & 0.00 for patients with no clinical prostatic disease. Unpaired t-tests revealed statistically significant differences between the prostatitis (BPH) sera & each of the other groups with p less than 0.005, but no differences between the other groups themselves. Preliminary studies with immunohistochemistry & 3-D confocal microscopy reveal 16/24 tissue sections + for NB Ag in BPH vs. only 2/22 tissue sections with prostate cancer. The preliminary findings of this serum screening study suggest that NB antigen may be commonly found in the serum of patients with the pathological diagnosis of prostatitis. Preliminary immunohistologic studies, suggest that NB may be found within the gland itself at a higher rate in patients with BPH relative to patients with adenocarcinoma, however confirmatory studies with a more specific ELISA technique, primary cultures, & with larger numbers of patients in a prospective design are required to determine if 1) NB are a causative organism for clinical hyperplastic and inflammatory disease, & if 2) serological testing can be used to discriminate patients with nanobacterial-associated prostatic disease.

Jones, Jeffrey A.↗
NASA NTRS1998

Acid-growth response and alpha-expansins in suspension cultures of bright yellow 2 tobacco

The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of alpha-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple alpha-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.

Non-NASA Center↗
NASA NTRS1999

Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity

The well-defined osteoblast line, MC3T3-E1 was used to examine fibronectin (FN) mRNA levels, protein synthesis, and extracellular FN matrix accumulation after growth activation in spaceflight. These osteoblasts produce FN extracellular matrix (ECM) known to regulate adhesion, differentiation, and function in adherent cells. Changes in bone ECM and osteoblast cell shape occur in spaceflight. To determine whether altered FN matrix is a factor in causing these changes in spaceflight, quiescent osteoblasts were launched into microgravity and were then sera activated with and without a 1-gravity field. Synthesis of FN mRNA, protein, and matrix were measured after activation in microgravity. FN mRNA synthesis is significantly reduced in microgravity (0-G) when compared to ground (GR) osteoblasts flown in a centrifuge simulating earth's gravity (1-G) field 2.5 h after activation. However, 27.5 h after activation there were no significant differences in mRNA synthesis. A small but significant reduction of FN protein was found in the 0-G samples 2.5 h after activation. Total FN protein 27.5 h after activation showed no significant difference between any of the gravity conditions, however, there was a fourfold increase in absolute amount of protein synthesized during the incubation. Using immunofluorescence, we found no significant differences in the amount or in the orientation of the FN matrix after 27.5 h in microgravity. These results demonstrate that FN is made by sera-activated osteoblasts even during exposure to microgravity. These data also suggest that after a total period of 43 h of spaceflight FN transcription, translation, or altered matrix assembly is not responsible for the altered cell shape or altered matrix formation of osteoblasts.

Flight Experiment↗
NASA NTRS2006

In vitro Catecholamine Exposure Produces Variable Effects on the B7 Costimulatory Pathway in Human Monocytic Cells

Catecholamines have been associated with immunomodulation of the adaptive immune system towards a Th2 response in vitro. We therefore examined the role of in vitro epinephrine (EPI) and norepinephrine (NE) exposure on the B7 costimulatory expression of antigen presenting cells (APC) from human monocytic cell lines and human peripheral blood mononuclear cells (PBMC). THP1 monocytic cells and CD14+ cells from normal human PBMC were stimulated with lipopolysaccharide (LPS) and incubated with physiologic stress levels (10(exp -6) - 10(exp -8)M) of EPI or NE for 24 hours. Cells were subsequently stained with CD80 FITC, CD86 PE, and CD14 PC5 antibodies and analyzed by flow cytometry for changes in fluorescence and mean fluorescence intensity (MFI). Exposure of THP1 to EPI in vitro at concentrations of 10(exp -6), 10(exp -7) and 10(exp -8)M significantly decreased mean CD80 from 42 plus or minus 0.7% to 11 plus or minus 0.44%, 19.1 plus or minus 2.0%, and 30.7 plus or minus 2.1% expression, respectively (p less than 0.01). In addition, CD86 expression increased with EPI at 10(exp -6), 10(exp -7) and 10(exp -8) M from 9.2 plus or minus 0.52% to 41 plus or minus 3.8%, 26.4 plus or minus 1.9%, and 15.74 plus or minus 1.8% expression, respectively (p less than 0.01). Similar results for mean CD80 and CD86 percent expression were observed for CD14+ cells from PBMC with a sample size of N = 6 and for NE when substituted for EPI. The data show that in vitro exposure to catecholamines significantly decreases %CD86 expression and significantly increases %CD86 expression in THP1 cells and human CD14+ APC. Previous studies have suggested an association between increased CD86 expression and TH2 activity. Thus, these data suggest that immunomodulation by catecholamines results in part by the variable effects of the B7 costimulatory pathway in APC.

Salicru, A. N.↗
NASA NTRS2006

Comprehensive Astronaut Immune Assessment Following a Short-Duration Space Flight

Immune system dysregulation has been demonstrated to occur during spaceflight and has the potential to cause serious health risks to crewmembers participating in exploration class missions. As a part of an ongoing NASA flight experiment assessing viral immunity (DSO-500), a generalized immune assessment was performed on 3 crewmembers who participated in the recent STS-114 Space Shuttle mission. The following assays were performed: (1) comprehensive immunophenotype analysis; (2) T cell function/intracellular cytokine profiles; (4) secreted Th1/Th2 cytokine profiles via cytometric bead array. Immunophenotype analysis included a leukocyte differential, lymphocyte subsets, T cell subsets, cytotoxic/effector CD8+ T cells, memory/naive T cell subsets and constitutively activated T cells. Study timepoints were L-180, L-65, L-10, R+0, R+3 and R+14. Detailed data are presented in the poster text. As expected from a limited number of human subjects, data tended to vary with respect to most parameters. Specific post-flight alterations were as follows (subject number in parentheses): Granulocytosis (2/3), reduced NK cells (3/3), elevated CD4/CD8 ratio (3/3), general CD8+ phenotype shift to a less differentiated phenotype (3/3), elevated levels of memory CD4+ T cells (3/3), loss of L-selectin on T cell subsets (3/3), increased levels of activated T cells (2/3), reduced IL-2 producing T cell subsets (3/3), levels of IFNg producing T cells were unchanged. CD8+ T cell expression of the CD69 activation markers following whole blood stimulation with SEA+SEB were dramatically reduced postflight (3/3), whereas other T cell function assessments were largely unchanged. Cytometric bead array assessment of secreted T cell cytokines was performed, following whole blood stimulation with either CD3/CD28 antibodies or PMA+ionomycin for 48 hours. Specific cytokines assessed were IFNg, TNFa, IL-2, IL-4, IL-5, IL-10. Following CD3/CD28 stimulation, all three crewmembers had a mission-associated reduction in the levels of secreted IFNg. One crewmember had a post-flight inversion in the IFNg/IL-10 ratio postflight, which trended back to baseline by R+14. Detailed cytokine data are presented in the poster text. This testing regimen was designed to correlate immunophenotype changes (thought to correspond to specific in-vivo immune responses or pathogenesis), against altered leukocyte function and cytokine profiles. In-flight studies are required to determine if post-flight alterations are reflective of the in-flight condition, or are a response to landing and readaptation.

Crucian, Brian↗
NASA NTRS1992

Evidence of thyroxine formation following iodine administration in Sprague-Dawley rats

Iodine (I2) has been proposed to be used as a water disinfectant on the manned space station. Previous work has shown that subchronic administration of I2 to Sprague-Dawley rats in drinking water significantly increases plasma thyroxine/triiodothyronine (T4/T3) levels. This is not observed with iodide (I-) treatment. The present study addresses the possibility that I2 reacts with deiodinated T4 metabolites in the gastrointestinal tract to resynthesize T4. Incubation of diiodothyronine (T2), T3, or reverse T3 with I2 in phosphate-buffered saline resulted in the formation of T4 as measured by radioimmunoassay. Washes from the initial segments of the small intestine of the rat show that substrates are present that react with I2 to produce T4. Single oral doses of I2 to rats produced significant dose-related increases in serum T4 and decreases in T3 concentrations after 2 h. Administration of an equivalent dose of I- did not alter significantly plasma T4 concentrations. Higher concentrations of a radioactive substance that bound a T4-specific antibody are present in plasma of animals treated with 125I2 compared to 125I-. These data support the hypothesis that I2 reacts with metabolites of thyroid hormone in the gastrointestinal tract to resynthesize T4 and elevate its levels in blood.

NASA Discipline Environmental Health↗
NASA NTRS2001

Receptor-based differences in human aortic smooth muscle cell membrane stiffness

Cells respond to mechanical stimuli with diverse molecular responses. The nature of the sensory mechanism involved in mechanotransduction is not known, but integrins may play an important role. The integrins are linked to both the cytoskeleton and extracellular matrix, suggesting that probing cells via integrins should yield different mechanical properties than probing cells via non-cytoskeleton-associated receptors. To test the hypothesis that the mechanical properties of a cell are dependent on the receptor on which the stress is applied, human aortic smooth muscle cells were plated, and magnetic beads, targeted either to the integrins via fibronectin or to the transferrin receptor by use of an IgG antibody, were attached to the cell surface. The resistance of the cell to deformation ("stiffness") was estimated by oscillating the magnetic beads at 1 Hz by use of single-pole magnetic tweezers at 2 different magnitudes. The ratio of bead displacements at different magnitudes was used to explore the mechanical properties of the cells. Cells stressed via the integrins required approximately 10-fold more force to obtain the same bead displacements as the cells stressed via the transferrin receptors. Cells stressed via integrins showed stiffening behavior as the force was increased, whereas this stiffening was significantly less for cells stressed via the transferrin receptor (P<0.001). Mechanical characteristics of vascular smooth muscle cells depend on the receptor by which the stress is applied, with integrin-based linkages demonstrating cell-stiffening behavior.

Non-NASA Center↗
NASA NTRS1999

Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate

Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.

Non-NASA Center↗