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Results for “In vivo assays”

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

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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1999

Radiation-Induced Cytogenetic Damage as a Predictor of Cancer Risk for Protons and Fe Ions

We have successfully completed the series of experiments planned for year 1 and the first part of year 2 measuring the induction of chromosome aberrations induced in multiple cell types by three model space radiations: Fe-ions, protons and photons. Most of these data have now been compiled and a significant part subjected to detailed data analyses, although continuing data analysis is an important part of our current and future efforts. These analyses are directed toward defining the patterns of chromosomal damage induction by the three radiations and the extent to which such patterns are dependent on the type of cell irradiated. Our studies show significant differences, both quantitatively and qualitatively, between response of different cell types to these radiations however there is an overall pattern that characterizes each type of radiation in most cell lines. Thus our data identifies general dose-response patterns for each radiation for induction of multiple types of chromosomal aberrations but also identifies significant differences in response between some cell types. Specifically, we observe significant resistance for induction of aberrations in rat mammary epithelial cells when they are irradiated in vivo and assayed in vitro. Further, we have observed some remarkable differences in susceptibility to certain radiation-induced aberrations in cells whose genome has been modulated for two cancer- relevant genes, TP53 and CDKNIA. This data, if confirmed, may represent the first evidence of gene-specific differences in cellular metabolism of damage induced by densely-ionizing radiation that confers substantial sensitivity to protons compared to photons.

Williams, Jerry R.↗
NASA NTRS2003

The evolution of transcriptional regulation in eukaryotes

Gene expression is central to the genotype-phenotype relationship in all organisms, and it is an important component of the genetic basis for evolutionary change in diverse aspects of phenotype. However, the evolution of transcriptional regulation remains understudied and poorly understood. Here we review the evolutionary dynamics of promoter, or cis-regulatory, sequences and the evolutionary mechanisms that shape them. Existing evidence indicates that populations harbor extensive genetic variation in promoter sequences, that a substantial fraction of this variation has consequences for both biochemical and organismal phenotype, and that some of this functional variation is sorted by selection. As with protein-coding sequences, rates and patterns of promoter sequence evolution differ considerably among loci and among clades for reasons that are not well understood. Studying the evolution of transcriptional regulation poses empirical and conceptual challenges beyond those typically encountered in analyses of coding sequence evolution: promoter organization is much less regular than that of coding sequences, and sequences required for the transcription of each locus reside at multiple other loci in the genome. Because of the strong context-dependence of transcriptional regulation, sequence inspection alone provides limited information about promoter function. Understanding the functional consequences of sequence differences among promoters generally requires biochemical and in vivo functional assays. Despite these challenges, important insights have already been gained into the evolution of transcriptional regulation, and the pace of discovery is accelerating.

Review, Academic↗
NASA NTRS2000

Cardiac tissue enriched factors serum response factor and GATA-4 are mutual coregulators

Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal alpha-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.

NASA Discipline Musculoskeletal↗
NASA NTRS1999

The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

NASA Discipline Plant Biology↗
NASA NTRS2017

Bone and Cartilage Degeneration in Mice Following Long-Duration Spaceflight: The Role of Bone Marrow Stem Cells

The detrimental effects of mechanical unloading in microgravity, including the musculo-skeletal system, are well documented. However, the effects of mechanical unloading on joint health and the interaction between bone and cartilage specifically, are less well known. Our ongoing studies with the mouse bone model have identified the failure of normal stem cell-based tissue regeneration, in addition to tissue degeneration, as a significant concern for long-duration spaceflight, especially in the mesenchymal and hematopoietic tissue lineages. Furthermore, we have identified the cell cycle arrest molecule, CDKN1ap21, as specifically up-regulated during spaceflight exposure and localized to osteoprecursors on the bone surface and chondroprogenitors in articular cartilage that are both required for normal tissue regeneration. The 30-day BionM1 and 37-day Rodent Research 1 (RR1) missions enabled the possibility of studying these effects in long-duration microgravity experiments. We hypothesized that the inhibition of stem cell-based tissue regeneration in short-duration spaceflight would continue during long-duration spaceflight resulting in significant tissue alterations and we specifically studied the hip joint (pelvis and proximal femur) to elucidate these effects. To test this hypothesis we analyzed bone and bone marrow stem cells using techniques including high-resolution Microcomputed Tomography (MicroCT), in-vivo differentiation and migration assays, and whole transcriptome expression profiling. We found that exposure to spaceflight for 30 days results in a significant decrease in bone volume fraction (-31), trabecular thickness (-14) and trabecular number (-20). Similar decrements in bone volume fraction (-27), trabecular number (-13) and trabecular thickness (-17) were found in female mice exposed to 37 days spaceflight. Furthermore, high-resolution MicroCT and immunohistochemical analysis of spaceflight tissues revealed a severe disruption of the epiphyseal boundary, resulting in endochondral ossification of the femoral head and perforation of articular cartilage by bone. This suggests that spaceflight in microgravity may cause rapid induction of an aging-like phenotype with signs of osteoarthritic disease in the hip joint. Microarray analysis also revealed that the top pathways altered during spaceflight include activation of matrix metalloproteinases, oxidative stress signaling and inflammation in both whole bone tissue and isolated bone marrow stem cells. In conclusion, the observed inhibition of stem cell-based tissue regeneration persists during long-duration spaceflight. Furthermore, spaceflight mice exhibit disruption of the epiphyseal boundary and endochondral ossification of the femoral head, and an inhibition of stem cell based tissue regeneration, which, taken together, may indicate onset of an accelerated aging phenotype with signs of osteoarthritic disease.

Cartilage↗
NASA NTRS1988

Conformation-dependent recognition of a protein by T-lymphocytes: apomyoglobin-specific T-cell clone recognizes conformational changes between apomyoglobin and myoglobin

A T-cell clone specific to apomyoglobin was generated. It was prepared from a T-cell culture obtained by in vitro driving of lymph node cells with apomyoglobin from SJL mice that have been primed in vivo with apomyoglobin. In proliferative assays, the T-cell clone responded to apomyoglobin but did not recognize native myoglobin or any of the synthetic peptides corresponding to the six T sites of myoglobin. The demonstration that a T-cell clone can be isolated, whose specificity is directed entirely to apomyoglobin and not to its counterpart myoglobin, with an identical amino acid composition, indicates the importance of the three-dimensional structure in the presentation of the protein to T cells.

Apoproteins/immunology↗
NASA NTRS2017

Developmental Research in Space: Predicting Adult Neurobehavioral Phenotypes via Metabolomic Imaging

As human habitation and eventual colonization of space becomes an inevitable reality, there is a necessity to understand how organisms develop over the life span in the space environment. Microgravity, altered CO2, radiation and psychological stress are some of the key factors that could affect mammalian reproduction and development in space, however there is a paucity of information on this topic. Here we combine early (neonatal) in vivo spectroscopic imaging with an adult emotionality assay following a common obstetric complication (prenatal asphyxia) likely to occur during gestation in space. The neural metabolome is sensitive to alteration by degenerative changes and developmental disorders, thus we hypothesized that that early neonatal neurometabolite profiles can predict adult response to novelty. Late gestation fetal rats were exposed to moderate asphyxia by occluding the blood supply feeding one of the rats pair uterine horns for 15min. Blood supply to the opposite horn was not occluded (within-litter cesarean control). Further comparisons were made with vaginal (natural) birth controls. In one-week old neonates, we measured neurometabolites in three brain areas (i.e., striatum, prefrontal cortex, and hippocampus). Adult perinatally-asphyxiated offspring exhibited greater anxiety-like behavioral phenotypes (as measured the composite neurobehavioral assay involving open field activity, responses to novel object, quantification of fecal droppings, and resident-intruder tests of social behavior). Further, early neurometabolite profiles predicted adult responses. Non-invasive MRS screening of mammalian offspring is likely to advance ground-based space analogue studies informing mammalian reproduction in space, and achieving high-priority multigenerational research that will enable studies of the first truly space-developed mammals.

Schorn, Julia M.↗
NASA NTRS2017

Developmental Research in Space: Predicting Adult Neurobehavioral Phenotypes via Metabolomic Imaging

As human habitation and eventual colonization of space becomes an inevitable reality, there is a necessity to understand how organisms develop over the life span in the space environment. Microgravity, altered CO2, radiation and psychological stress are some of the key factors that could affect mammalian reproduction and development in space, however there is a paucity of information on this topic. Here we combine early (neonatal) in vivo spectroscopic imaging with an adult emotionality assay following a common obstetric complication (prenatal asphyxia) likely to occur during gestation in space. The neural metabolome is sensitive to alteration by degenerative changes and developmental disorders, thus we hypothesized that that early neonatal neurometabolite profiles can predict adult response to novelty. Late gestation fetal rats were exposed to moderate asphyxia by occluding the blood supply feeding one of the rats pair uterine horns for 15min. Blood supply to the opposite horn was not occluded (within-litter cesarean control). Further comparisons were made with vaginal (natural) birth controls. In one-week old neonates, we measured neurometabolites in three brain areas (i.e., striatum, prefrontal cortex, and hippocampus). Adult perinatally-asphyxiated offspring exhibited greater anxiety-like behavioral phenotypes (as measured the composite neurobehavioral assay involving open field activity, responses to novel object, quantification of fecal droppings, and resident-intruder tests of social behavior). Further, early neurometabolite profiles predicted adult responses. Non-invasive MRS screening of mammalian offspring is likely to advance ground-based space analogue studies informing mammalian reproduction in space, and achieving high-priority.

rodent development spacefligh↗
NASA NTRS2013

From Space to the Patient: A New Cytokine Release Assay to Monitor the Immune Status of HIV Infected Patients and Sepsis Patients

Monitoring of humans either in the healthy men under extreme environmental stress like space flight, in human immunodeficiency virus (HIV) infected patients or in sepsis is of critical importance with regard to the timing of adequate therapeutic (counter-)measures. The in vivo skin delayed-type hypersensitivity test (DTH) served for many years as a tool to evaluate cell mediated immunity. However, this standardised in vivo test was removed from the market in 2002 due to the risk of antigen stabilization. To the best of our knowledge an alternative test as monitoring tool to determine cell mediated immunity is not available so far. For this purpose we tested a new alternative assay using elements of the skin DTH which is based on an ex vivo cytokine release from whole blood and asked if it is suitable and applicable to monitor immune changes in HIV infected patients and in patients with septic shock.

Kaufmann, I.↗
NASA NTRS1993

Monoclonal antibodies directed against surface molecules of multicell spheroids

The objective of this project is to generate a library of monoclonal antibodies (MAb's) to surface molecules involved in the cell-cell interactions of mammalian cells grown as multicell spheroids (MCS). MCS are highly organized 3-dimensional multicellular structures which exhibit many characteristics in vivo tissues not found in conventional monolayer or suspension culture. They also provide a functional assay for surface adhesion molecules. In brief, MCS combine the relevance of organized tissues with the accuracy of in vitro methodology. Further, one can manipulate these MCS experimentally to discern important information about their biology.

Martinez, Andrew O.↗
NASA NTRS1997

Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

Jackson, Jacquelyn↗
NASA NTRS2002

Basal glycogenolysis in mouse skeletal muscle: in vitro model predicts in vivo fluxes

A previously published mammalian kinetic model of skeletal muscle glycogenolysis, consisting of literature in vitro parameters, was modified by substituting mouse specific Vmax values. The model demonstrates that glycogen breakdown to lactate is under ATPase control. Our criteria to test whether in vitro parameters could reproduce in vivo dynamics was the ability of the model to fit phosphocreatine (PCr) and inorganic phosphate (Pi) dynamic NMR data from ischemic basal mouse hindlimbs and predict biochemically-assayed lactate concentrations. Fitting was accomplished by optimizing four parameters--the ATPase rate coefficient, fraction of activated glycogen phosphorylase, and the equilibrium constants of creatine kinase and adenylate kinase (due to the absence of pH in the model). The optimized parameter values were physiologically reasonable, the resultant model fit the [PCr] and [Pi] timecourses well, and the model predicted the final measured lactate concentration. This result demonstrates that additional features of in vivo enzyme binding are not necessary for quantitative description of glycogenolytic dynamics.

Non-NASA Center↗
NASA NTRS1985

Microgravity associated changes in pituitary growth hormone (GH) cells prepared from rats flown on Space Lab 3

The effect of microgravity on the release of pituitary growth hormone (GH) in rats is studied. The pituitary glands from six adult rats exposed to microgravity are analyzed for in vitro and in vivo changes in pituitary growth hormone cells. The GH cell functions in the somatotrophs of flight rats are compared to a control group. The two assay procedures employed in the experiment are described. It is observed that intracellular levels of GH are two to three times greater in the flight rats than in the control group; however, the amount of GH released from the somatotrophs is 1.11 + or - 0.4 micrograms for the flight rats and 1.85 + or - 1.3 micrograms for the control rats.

Hymer, W. C.↗
NASA NTRS1987

In vivo synthesis of phosphatidylcholine in rat brain via the phospholipid methylation pathway

The in vivo synthesis of brain phosphatidylcholine (PC) by the methylation of phosphatidylethanolamine (PE) was examined. (H-3)methyl)methionine was infused i.c.v., by indwelling cannula, and brain samples were taken 0.5-18 h thereafter and assayed for (H-3)PC, as well as for its biosynthetic intermediates (H-3)phosphatidyl monomethylethanolamine ((H-3)PMME) and (H-3)phosphatidyl dimethylethanolamine ((H-3)PDME), and for (H-3)lysophosphatidylcholine ((H-3)LPC) and S-(H-3)adenosylmethionine ((H-3)SAM). Most of the (H-3)PC (79-94 percent) was present ipsilateral to the infusion site; indicating that the radioactivity in the (H-3)PC was primarily of intracerebral origin, and not taken up from the blood. Moreover, only very low levels of (H-3)PC were attained in brains of animals receiving (H-3)methionine i.p. and these levels were symmetrically distributed. (H-3)PMME and (H-3)PDME turned over with apparent half-lives of 2.2 h and 2.4 h. In contrast, the accumulation of brain (H-3)PC was biphasic, suggesting the existence of two pools, the more labile of which turned over rapidly (t(sub 1/2) = 5 h) and was formed for as long as (H-3)PMME and (H-3)PDME are present in the brain, and another, which was distinguishable only at 18 h after the (H-3)methionine infusion. (The latter pool may have been synthesized from (H-3)choline that was released via the hydrolysis of some of the brain (H-3)PC previously formed by the methylation of PE.) Subcellular fractionation of brain tissue obtained after in vivo labelling with (H-3)methionine revealed that mitochondrial PC had the highest specific radioactivity (dpm per micromol total lipid phosphorus), and myelin the least. These observations affirm that rat brain does synthesize PC in vivo by methylating PE, and the technique provides an experimental system which may be useful for examining the physiological regulation of this process.

Lakher, Michael↗
NASA NTRS1994

Monoclonal antibodies directed against surface molecules of multicell spheroids

The objective of this project is to generate a library of monoclonal antibodies (MAbs) directed against surface molecules of tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, 3-dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture. Therefore MCS make better in vitro model systems to study the interactions of mammalian cells, and provide a functional assay for surface adhesion molecules. This project also involves investigations of cell-cell interactions in a gravity-based environment. It will provide a base of scientific information necessary to expand the focus of the project in future years to microgravity and hypergravity-based environments. This project also has the potential to yield important materials (e.g., cellular products) which may prove useful in the diagnosis and/or treatment of certain human diseases. Moreover, this project supports the training of both undergraduate and graduate students; thus, it will assist in developing a pool of future scientists with research experience in an area (gravitational biology) of interest to NASA.

Martinez, Andrew O.↗
NASA NTRS1993

Monoclonal antibodies directed against surface molecules of multicell spheroids

The objective of this project is to generate a library of monoclonal antibodies (MAbs) to surface molecules of mammalian tumor and transformed cells grown as multicell spheroids (MCS). These MCS are highly organized, three dimensional multicellular structures which exhibit many characteristics of in vivo organized tissues not found in conventional monolayer or suspension culture; therefore, MCS make better in vitro model systems to study the interactions of mammalian cells. Additionally, they provide a functional assay for surface adhesion molecules.

Martinez, Andrew O.↗
NASA NTRS1994

Computerized in vitro test for chemical toxicity based on tetrahymena swimming patterns

An apparatus and method for rapidly determining chemical toxicity was evaluated. The toxicity monitor includes an automated scoring of how motile biological cells (Tetrahymena pyriformis) slow down or otherwise change their swimming patterns in a hostile chemical environment. The device, called the Motility Assay Apparatus (MAA) is tested for 30 second determination of chemical toxicity in 20 aqueous samples containing trace organics and salts. With equal or better detection limits, results compare favorably to in vivo animal tests of eye irritancy, in addition to agreeing for all chemicals with previous manual evaluations of single cell motility.

Noever, David A.↗
NASA NTRS1994

Computerized In Vitro Test for Chemical Toxicity Based on Tetrahymena Swimming Patterns

An apparatus and a method for rapidly determining chemical toxicity have been evaluated as an alternative to the rabbit eye initancy test (Draize). The toxicity monitor includes an automated scoring of how motile biological cells (Tetrahymena pyriformis) slow down or otherwise change their swimming patterns in a hostile chemical environment. The method, called the motility assay (MA), is tested for 30 s to determine the chemical toxicity in 20 aqueous samples containing trace organics and salts. With equal or better detection limits, results compare favorably to in vivo animal tests of eye irritancy.

Noever, David A.↗