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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 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
NASA NTRS2002

Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants

Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca2+-transport and storage pathways that include Ca2+ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca2+ binding characteristics of the C-domain of calreticulin to selectively increase Ca2+ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20-50 moles of Ca2+ per mole of protein and has been shown to be the major site of Ca2+ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9-35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca2+ stores, and that this Ca2+ reserve can be used by the plant in times of stress.

NASA Discipline Plant Biology↗
NASA NTRS2003

Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

NASA Discipline Musculoskeletal↗
NASA NTRS1997

Activation of the adenylyl cyclase/cyclic AMP/protein kinase A pathway in endothelial cells exposed to cyclic strain

The aim of this study was to assess the involvement of the adenylyl cyclase/cyclic AMP/protein kinase A pathway (AC) in endothelial cells (EC) exposed to different levels of mechanical strain. Bovine aortic EC were seeded to confluence on flexible membrane-bottom wells. The membranes were deformed with either 150 mm Hg (average 10% strain) or 37.5 mm Hg (average 6% strain) vacuum at 60 cycles per minute (0.5 s strain; 0.5 s relaxation) for 0-60 min. The results demonstrate that at 10% average strain (but not 6% average strain) there was a 1.5- to 2.2-fold increase in AC, cAMP, and PKA activity by 15 min when compared to unstretched controls. Further studies revealed an increase in cAMP response element binding protein in EC subjected to the 10% average strain (but not 6% average strain). These data support the hypothesis that cyclic strain activates the AC/cAMP/PKA signal transduction pathway in EC which may occur by exceeding a strain threshold and suggest that cyclic strain may stimulate the expression of genes containing cAMP-responsive promoter elements.

Non-NASA Center↗
NASA NTRS1996

The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy

Insulin-like growth factor-1 (IGF-1) is known to have anabolic effects on skeletal muscle cells. This study examined the time course of muscle hypertrophy and associated IGF-1 peptide and mRNA expression. Data were collected at 3, 7, 14, and 28 days after surgical removal of synergistic muscles of both normal and hypophysectomized (HX) animals. Overloading increased the plantaris (Plant) mass, myofiber size, and protein-to-body weight ratio in both groups (normal and HX; P < 0.05). Muscle IGF-1 peptide levels peaked at 3 (normal) and 7 (HX) days of overloading with maximum 4.1-fold (normal) and 6.2-fold (HX) increases. Increases in muscle IGF-1 preceded the hypertrophic response. Total DNA content of the overloaded Plant increased in both groups. There was a strong positive relationship between IGF-1 peptide and DNA content in the overloaded Plant from both groups. These results indicate that 1) the muscles from rats with both normal and severely depressed systemic levels of IGF-1 respond to functional overload with an increase in local IGF-1 expression and 2) this elevated IGF-1 may be contributing to the hypertrophy response, possibly via the mobilization of satellite cells to provide increases in muscle DNA.

NASA Discipline Musculoskeletal↗
NASA NTRS2004

Phosphate binding protein as the biorecognition element in a biosensor for phosphate

This work explores the potential use of a member of the periplasmic family of binding proteins, the phosphate binding protein (PBP), as the biorecognition element in a sensing scheme for the detection of inorganic phosphate (Pi). The selectivity of this protein originates from its natural role which, in Escherichia coli, is to serve as the initial receptor for the highly specific translocation of Pi to the cytoplasm. The single polypeptide chain of PBP is folded into two similar domains connected by three short peptide linkages that serve as a hinge. The Pi binding site is located deep within the cleft between the two domains. In the presence of the ligand, the two globular domains engulf the former in a hinge-like manner. The resultant conformational change constitutes the basis of the sensor development. A mutant of PBP (MPBP), where an alanine was replaced by a cysteine residue, was prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The mutant was expressed, from plasmid pSD501, in the periplasmic space of E. coli and purified in a single chromatographic step on a perfusion anion-exchange column. Site-specific labeling was achieved by attaching the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), to the protein through the sulfhydryl group of the cysteine moiety. Steady-state fluorescence studies of the MPBP-MDCC conjugate showed a change in the intensity of the signal upon addition of Pi. Calibration curves for Pi were constructed by relating the intensity of the fluorescence signal with the amount of analyte present in the sample. The sensing system was first developed and optimized on a spectrofluorometer using ml volumes of sample. It was then adapted to be used on a microtiter plate arrangement with microliter sample volumes. The system's versatility was finally proven by developing a fiber optic fluorescence-based sensor for monitoring Pi. In all three cases the detection limits for the analyte were in the sub-microMolar range. It was also demonstrated that the sensing system was selective for phosphate over other structurally-similar anions, paving the way for the design and development of a new family of biosensors utilizing the specific binding properties of periplasmic proteins. c2003 Elsevier B.V. All rights reserved.

NASA Program Fundamental Space Biology↗
NASA NTRS2000

Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.

NASA Discipline Plant Biology↗
NASA NTRS2002

Alteration of gene expression profiles in skeletal muscle of rats exposed to microgravity during a spaceflight

To clarify the mechanism of skeletal muscle wasting during spaceflights, we investigated whether intramuscular gene expression profiles are affected, by using DNA microarray methods. Male rats sent on the 17-day NASA STS-90 Neurolab spaceflight were sacrificed 24 hours after return to earth (MG group). Ground control rats were maintained for 17 days in flight-simulated cages (CS group). Spaceflight induced a 19% and 23% loss of tibialis anterior and gastrocnemius muscle mass, respectively, as compared to ground controls. Muscle RNA was analyzed by the Clontech Atlas DNA expression array in four rats, with two MG/ CS pairs for the tibialis anterior, and one pair for the gastrocnemius. Alterations in gene expression were verified for selected genes by reverse-transcription PCR. In both muscles of MG rats, mRNAs for 12 genes were up-regulated by over 2-fold, and 38 were down-regulated compared to controls. There was inhibition of genes for cell proliferation and growth factor cascades, including cell cycle genes and signal transduction proteins, such as p21 Cip1, retinoblastoma (Rb), cyclins G1/S, -E and -D3, MAP kinase 3, MAD3, and ras related protein RAB2. These data indicate that following exposure to microgravity, there is downregulation of genes involved in regulation of muscle satellite cell replication.

NASA Program Fundamental Space Biology↗
NASA NTRS1994

Retinoic acid stimulates interstitial collagenase messenger ribonucleic acid in osteosarcoma cells

The rat osteoblastic osteosarcoma cell line UMR 106-01 secretes interstitial collagenase in response to retinoic acid (RA). The present study demonstrates by Northern blot analysis that RA causes an increase in collagenase messenger RNA (mRNA) at 6 h, which is maximal at 24 h (20.5 times basal) and declines toward basal level by 72 h. This stimulation is dose dependent, with a maximal response at 5 x 10(-7) M RA. Nuclear run-on assays show a greater than 20-fold increase in the rate of collagenase mRNA transcription between 12-24 h after RA treatment. Cycloheximide blocks RA stimulation of collagenase mRNA, demonstrating the need for de novo protein synthesis. RA not only causes an increase in collagenase secretion, but is known to decrease collagen synthesis in UMR 106-01 cells. In this study, the increase in collagenase mRNA is accompanied by a concomitant decrease in the level of alpha 1(I) procollagen mRNA, which is maximal at 24 h (70% decrease), with a return to near-control levels by 72 h. Nuclear run-on assays demonstrated that the decrease in alpha 1 (I) procollagen expression does not have a statistically significant transcriptional component. RA did not statistically decrease the stability of alpha 1 (I) procollagen mRNA (calculated t1/2 = 8.06 +/- 0.30 and 9.01 +/- 0.62 h in the presence and absence of RA, respectively). However, transcription and stability together probably contribute to the major decrease in stable alpha 1 (I) procollagen mRNA observed. Cycloheximide treatment inhibits basal level alpha 1 (I) procollagen mRNA accumulation, demonstrating the need for on-going protein synthesis to maintain basal expression of this gene.

Non-NASA Center↗
NASA NTRSDate not supplied

Oxygen Deficiency in Spaceflight & its Impact on Plants’ Adaptive Changes

The goal of this study was to investigate the effects of hypoxic conditions in spaceflight. The distribution of genes involved with hypoxia in Arabidopsis thaliana and Brassica rapa were analyzed with the results from past spaceflight experiments to evaluate genes for future studies. Transcriptomes data of two different spaceflight studies of Arabidopsis thaliana from the NASA GeneLab database, GLDS-7 and GLDS-17, were compared. DNA microarrays were utilized for transcription profiling to conduct these studies. For GLDS-7, the response in spaceflight was studied with approaches that collected gene expression data. Leaves, hypocotyls, and root tissues were compared to the whole plant. For GLDS-17, seedlings and undifferentiated cultured cells were placed in the Biological Research in Canisters (BRIC), specifically BRIC-16. The genes related to hypoxia in Arabidopsis thaliana from these two studies were compared to genes in Brassica rapa with the TOAST database to evaluate similarities. When transcriptomes were analyzed for GLDS-7 and 17, genes that were considered significant had p-values ≤ 0.05 and log fold change values ≤ -1 or ≥1. Sixteen genes fulfilled the criteria. The genes related to hypoxia were alcohol dehydrogenase, elongation factor, ethylene-responsive factor, GUS, heat-shock proteins, NAP, RAP2.12, and RD20. The genes most impacted by spaceflight were heat-shock proteins. These genes were compared with Brassica rapa through Arabidopsis Ensemble Orthology from the TOAST Database. Similarities were seen in alcohol dehydrogenase, elongation factor, ethylene-responsive factor, heat-shock proteins, NAP, and RAP2.12. Overall, transcription profiling indicates that plants’ survival in spaceflight is dependent on adaptive changes with gene expression. This study also indicates that there are similarities in gene expression between Arabidopsis thaliana and Brassica rapa with comparable gene expression. Future studies could include analyzing additional species to understand which genes could be modified to ensure better yield of space crops amid hypoxic conditions.

hypoxia↗
NASA NTRS1996

Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts

Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

Non-NASA Center↗
NASA NTRS2006

Computer Modeling of Protocellular Functions: Peptide Insertion in Membranes

Lipid vesicles became the precursors to protocells by acquiring the capabilities needed to survive and reproduce. These include transport of ions, nutrients and waste products across cell walls and capture of energy and its conversion into a chemically usable form. In modem organisms these functions are carried out by membrane-bound proteins (about 30% of the genome codes for this kind of proteins). A number of properties of alpha-helical peptides suggest that their associations are excellent candidates for protobiological precursors of proteins. In particular, some simple a-helical peptides can aggregate spontaneously and form functional channels. This process can be described conceptually by a three-step thermodynamic cycle: 1 - folding of helices at the water-membrane interface, 2 - helix insertion into the lipid bilayer and 3 - specific interactions of these helices that result in functional tertiary structures. Although a crucial step, helix insertion has not been adequately studied because of the insolubility and aggregation of hydrophobic peptides. In this work, we use computer simulation methods (Molecular Dynamics) to characterize the energetics of helix insertion and we discuss its importance in an evolutionary context. Specifically, helices could self-assemble only if their interactions were sufficiently strong to compensate the unfavorable Free Energy of insertion of individual helices into membranes, providing a selection mechanism for protobiological evolution.

Rodriquez-Gomez, D.↗
NASA NTRS2010

Versatile platform for nanotechnology based on circular permutations of chaperonin protein

The present invention provides chaperonin polypeptides which are modified to include N-terminal and C-terminal ends that are relocated from the central pore region to various different positions in the polypeptide which are located on the exterior of the folded modified chaperonin polypeptide. In the modified chaperonin polypeptide, the naturally-occurring N-terminal and C-terminal ends are joined together directly or with an intervening linker peptide sequence. The relocated N-terminal or C-terminal ends can be covalently joined to, or bound with another molecule such as a nucleic acid molecule, a lipid, a carbohydrate, a second polypeptide, or a nanoparticle. The modified chaperonin polypeptides can assemble into double-ringed chaperonin structures. Further, the chaperonin structures can organize into higher order structures such as nanofilaments or nanoarrays which can be used to produce nanodevices and nanocoatings.

Paavola, Chad D.↗
NASA NTRS1998

BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields

We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

Non-NASA Center↗
NASA NTRS1999

Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats

Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of (125)I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2(14k) and E3alpha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E2(14k) inhibited this increase in ubiquitination rates. Both E2(14k) and E3alpha were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3alpha (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2(14k) and E3alpha content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2(14k) and E3alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.

Non-NASA Center↗
NASA NTRS2023

Uncovering Unique Molecular Adaptations in the Arabidopsis Thaliana Cvi-0 Ecotype

This research proposal aims to investigate the unique molecular adaptations exhibited by Arabidopsis Thaliana, specifically focusing on the Cape Verde Islands (Cvi-0) ecotype, in response to microgravity conditions. The study examines data from NASA’s Open Science Data Repository and applies a multifaceted RNAseq analysis pipeline using tools in the UseGalaxy.org open platform. Through transcriptomic analysis, differential gene expression patterns were identified in Cvi-0, revealing an absence of heat shock protein (HSP) upregulation and an upregulation of Rubisco Activase (RCA) and chloroplast-related pathways. To test the hypothesis that these adaptations may contribute to Cvi-0’s increased adaptability in microgravity, a three-fold experimental design is proposed. Four experimental groups will be cultivated under simulated microgravity and ground control conditions, including Cvi-0, Col-0, and genetically modified Col-0 with silenced HSP genes, and genetically modified Col-0 with upregulated RCA gene. Growth parameters will be measured to assess plant resilience, and RNA sequencing will provide transcriptomic data for pathway analysis. Anticipated outcomes include improved markers of plant health (mass, growth, etc.) of Cvi-0 in simulated microgravity and enhanced resilience in genetically altered Col-0 variants, providing insights into potential mechanisms of adaptation. This research would bear significance for space agriculture, nutrition for extended space missions, and sustainable terrestrial crop enhancement. Moreover, the insights gained could reshape crop engineering on Earth, enhancing robustness to climate induced stresses and bolstering global food security. The proposal’s trajectory blends scientific curiosity with practical applicability, forging a path towards sustainable food production and improving human exploration beyond our planet.

GL4HS↗
NASA NTRS2001

Differential expression of neuroleukin in osseous tissues and its involvement in mineralization during osteoblast differentiation

Osteoblast differentiation is a multistep process that involves critical spatial and temporal regulation of cellular processes marked by the presence of a large number of differentially expressed molecules. To identify key functional molecules, we used differential messenger RNA (mRNA) display and compared RNA populations isolated from the defined transition phases (proliferation, matrix formation, and mineralization) of the MC3T3-E1 osteoblast-like cell line. Using this approach, a complementary DNA (cDNA) fragment was isolated and identified as neuroleukin (NLK), a multifunctional cytokine also known as autocrine motility factor (AMF), phosphoglucose isomerase (PGI; phosphohexose isomerase [PHI]), and maturation factor (MF). Northern analysis showed NLK temporal expression during MC3T3-E1 cell differentiation with a 3.5-fold increase during matrix formation and mineralization. Immunocytochemical studies revealed the presence of NLK in MC3T3-E1 cells as well as in the surrounding matrix, consistent with a secreted molecule. In contrast, the NLK receptor protein was detected primarily on the cell membrane. In subsequent studies, a high level of NLK expression was identified in osteoblasts and superficial articular chondrocytes in bone of 1-, 4-, and 8-month-old normal mice, as well as in fibroblasts, proliferating chondrocytes, and osteoblasts within a fracture callus. However, NLK was not evident in hypertrophic chondrocytes or osteocytes. In addition, treatment of MC3T3 cells with 6-phosphogluconic acid (6PGA; a NLK inhibitor) resulted in diminishing alkaline phosphatase (ALP) activity and mineralization in MC3T3-E1 cells, especially during the matrix formation stage of differentiating cells. Taken together, these data show specific expression of NLK in discrete populations of bone and cartilage cells and suggest a possible role for this secreted protein in bone development and regeneration.

NASA Discipline Musculoskeletal↗
NASA NTRS1995

Human osteoblast-like cells respond to mechanical strain with increased bone matrix protein production independent of hormonal regulation

Exposure of osteosarcoma cell lines to chronic intermittent strain increases the activity of mechano-sensitive cation (SA-cat) channels. The impact of mechano-transduction on osteoblast function has not been well studied. We analyzed the expression and production of bone matrix proteins in human osteoblast-like osteosarcoma cells, OHS-4, in response to chronic intermittent mechanical strain. The OHS-4 cells exhibit type I collagen production, 1,25-Dihydroxyvitamin D-inducible osteocalcin, and mineralization of the extracellular matrix. The matrix protein message level was determined from total RNA isolated from cells exposed to 1-4 days of chronic intermittent strain. Northern analysis for type I collagen indicated that strain increased collagen message after 48 h. Immunofluorescent labeling of type I collagen demonstrated that secretion was also enhanced with mechanical strain. Osteopontin message levels were increased several-fold by the application of mechanical load in the absence of vitamin D, and the two stimuli together produced an additive effect. Osteocalcin secretion was also increased with cyclic strain. Osteocalcin levels were not detectable in vitamin D-untreated control cells. However, after 4 days of induced load, significant levels of osteocalcin were observed in the medium. With vitamin D present, osteocalcin levels were 4 times higher in the medium of strained cells compared to nonstrained controls. We conclude that mechanical strain of osteoblast-like cells is sufficient to increase the transcription and secretion of matrix proteins via mechano-transduction without hormonal induction.

NASA Discipline Number 40-40↗
NASA NTRS2000

Molecular Simulations in Astrobiology

One of the main goals of astrobiology is to understand the origin of cellular life. The most direct approach to this problem is to construct laboratory models of protocells. Such efforts, currently underway in the NASA Astrobiology Program, are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures that are capable of performing protocellular functions. Many of these functions, such as importing nutrients, capturing energy and responding to changes in the environment, are carried out by proteins bound to membranes. We use computer simulations to address the following questions about these proteins: (1) How do small proteins self-organize into ordered structures at water-membrane interfaces and insert into membranes? (2) How do peptides form membrane-spanning structures (e.g. channels)? (3) By what mechanisms do such structures perform their functions? The simulations are performed using the molecular dynamics method. In this method, Newton's equations of motion for each atom in the system are solved iteratively. At each time step, the forces exerted on each atom by the remaining atoms are evaluated by dividing them into two parts. Short-range forces are calculated in real space while long-range forces are evaluated in reciprocal space, using a particle-mesh algorithm which is of order O(NInN). With a time step of 2 femtoseconds, problems occurring on multi-nanosecond time scales (10(exp 6)-10(exp 8) time steps) are accessible. To address a broader range of problems, simulations need to be extended by three orders of magnitude, which requires algorithmic improvements and codes scalable to a large number of processors. Work in this direction is in progress. Two series of simulations are discussed. In one series, it is shown that nonpolar peptides, disordered in water, translocate to the nonpolar interior of the membrane and fold into helical structures (see Figure). Once in the membrane, the peptides exhibit orientational flexibility with changing conditions, which may have provided a mechanism of transmitting signals between the protocell and its environment. In another series of simulations, the mechanism by which a simple protein channel efficiently mediates proton transport across membranes was investigated. This process is a key step in cellular bioenergetics. In the channel under study, proton transport is gated by four histidines that occlude the channel pore. The simulations identify the mechanisms by which protons move through the gate.

Pohorille, Andrew↗