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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 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
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↗
DOE OSTI2025

Astrocyte FABP7 Modulates Seizure Activity-Dependent Protein Expression in Mouse Brain

Background/Objectives: Patients with epilepsy commonly experience patterns of seizures that change with sleep/wake behavior or diurnal rhythms. The cellular and molecular mechanisms that underlie these patterns in seizure activity are not well understood but may involve non-neuronal cells, such as astrocytes. Our previous studies show the critical importance of one specific astrocyte factor, the brain-type fatty acid binding protein Fabp7, in the regulation of time-of-day-dependent electroshock seizure threshold and neural activity-dependent gene expression in mice. Here, we examined whether Fabp7 influences differential seizure activity-dependent protein expression, by comparing Fabp7 knockout (KO) to wild-type (WT) mice under control conditions and after reaching the maximal electroshock seizure threshold (MEST). Methods: We analyzed the proteome in cortical–hippocampal extracts from MEST and SHAM groups of WT and KO mice using mass spectrometry (MS), followed by Gene Ontology (GO) and pathway analyses. GO and pathway analyses of all groups revealed a diverse set of up- and downregulated differentially expressed proteins (DEPs). Results: We identified 65 significant DEPs in the comparison of KO SHAM versus WT SHAM; 33 proteins were upregulated and 32 were downregulated. We found downregulation in mitochondrial-associated proteins in WT MEST compared to WT SHAM controls, including Slc1a4, Slc25a27, Cox7a2, Cox8a, Micos10, and Atp5mk. Several upregulated DEPs in the KO SHAM versus WT SHAM comparison were associated with the 20S proteasomal subunit, suggesting proteasomal activity is elevated in the absence of Fabp7 expression. We also observed 92 DEPs significantly altered in the KO MEST versus WT MEST, with 49 proteins upregulated and 43 downregulated. Conclusions: Together, these data suggest that the astrocyte Fabp7 regulation of time-of-day-mediated neural excitability is modulated by multiple cellular mechanisms, which include proteasomal pathways, independent of its role in activity-dependent gene expression.

Neural Excitability↗
DOE OSTI2024

Ribosome-inactivation by a class of widely distributed C-tail anchored membrane proteins

Ribosome hibernation is a commonly used strategy that protects ribosomes under unfavorable conditions and regulates developmental processes. Multiple ribosome-hibernation factors have been identified in all domains of life, but due to their structural diversity and the lack of a common inactivation mechanism, it is currently unknown how many different hibernation factors exist. Here, we show that the YqjD/ElaB/YgaM paralogs, initially discovered as membrane-bound ribosome binding proteins in E. coli, constitute an abundant class of ribosome-hibernating proteins, which are conserved across all proteobacteria and some other bacterial phyla. Our data demonstrate that they inhibit in vitro protein synthesis by interacting with the 50S ribosomal subunit. In vivo cross-linking combined with mass spectrometry revealed their specific interactions with proteins surrounding the ribosomal tunnel exit and even their penetration into the ribosomal tunnel. Thus, YqjD/ElaB/YgaM inhibit translation by blocking the ribosomal tunnel and thus mimic the activity of antimicrobial peptides and macrolide antibiotics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Thermodynamics of calcium binding to heparin: Implications of solvation and water structuring for polysaccharide biofunctions

Heparan sulfates are found in all animal tissues and have essential roles in living systems. This family of biomacromolecules modulates binding to calcium ions (Ca 2+ ) in low free energy reactions that influence biochemical processes from cell signaling and anticoagulant efficacy to biomineralization. Despite their ubiquity, the thermodynamic basis for how heparans and similarly functionalized biomolecules regulate Ca 2+ interactions is not yet established. Using heparosan (Control) and heparins with different positions of sulfate groups, we quantify how SO 3 − and COO − content and SO 3 − position modulate Ca 2+ binding by isothermal titration calorimetry. The free energy of all heparin-Ca2+ interactions (ΔG rxn ) is dominated by entropic contributions due to favorable water release from polar, hydrophilic groups. Heparin with both sulfate esters (O-SO 3 − ) and sulfamides (N-SO 3 − ) has the strongest binding to Ca 2+ compared to heparosan and to heparin with only O-SO 3 − groups (~3X). By linking Ca 2+ binding thermodynamics to measurements of the interfacial energy for calcite (CaCO 3 ) crystallization onto polysaccharides, we show molecule-specific differences in nucleation rate can be explained by differences in water structuring during Ca 2+ interactions. A large entropic term (-TΔS rxn ) upon Ca 2+ –polysaccharide binding correlates with high interfacial energy to CaCO 3 nucleation. Combining our measurements with literature values indicates many Ca 2+ –polysaccharide interactions have a shared thermodynamic signature. The resulting enthalpy–entropy compensation relationship suggests these interactions are generally dominated by water restructuring involving few conformational changes, distinct from Ca 2+ –protein binding. Our findings quantify the thermodynamic origins of heparin-specific interactions with Ca 2+ and demonstrate the contributions of solvation and functional group position during biomacromolecule-mediated ion regulation.

15 GEOTHERMAL ENERGY↗
DOE OSTI2026

A characterization of recombinant Arabidopsis FRIABLE1 (FRB1) reveals robust rhamnogalacturonan-I rhamnosyltransferase activity and critical catalytic residues

Plant cell walls are glycan-rich extracellular matrices that fundamentally impact essential cellular processes, such as growth, adhesion, and cell shape acquisition. Understanding plant cell wall glycans requires the identification and characterization of the biosynthetic enzymes that produce these polymers. Most successful in vitro protein expression studies of plant cell wall glycosyltransferases have relied on insect, fungal/yeast, or human cell expression systems, whereas prokaryotic expression systems have been generally unsuccessful. Here, we show that Arabidopsis FRIABLE1 (FRB1)/rhamnogalacturonan-I rhamnosyltransferase 8 (RRT8) can be produced in Escherichia coli RosettaGami2 cells as N-terminal maltose-binding protein fusion proteins containing C-terminal 6X-His-tags. We also report the catalytic constants of FRB1/RRT8 with apparent K M and K cat values of 226 μM and 33 min -1 for UDP-Rhamnose and 117 μM and 28.7 min -1 for rhamnogalacturonan-I (RG-I), respectively. We examine the catalytic activities of mutated FRB1/RRT8 proteins based on an AlphaFold 3-generated FRB1/RRT8 protein structural model with a virtually docked UDP-Rha donor. Enzymatic characterization of the mutated and wildtype FRB1/RRT8 protein confirmed that mutation of predicted catalytic site amino acid residues resulted in a 20-fold reduction in RRT activity. FRB1 also robustly polymerizes RG-I in combination with RG-I galacturonosyltransferase 1. These results show how a robust E. coli expression system combined with artificial intelligence tools can be used to increase understanding of plant cell wall glycosyltransferase structure and function.

glycosyltransferase↗
NASA NTRS1991

Cloned Hemoglobin Genes Enhance Growth Of Cells

Experiments show that portable deoxyribonucleic acid (DNA) sequences incorporated into host cells make them produce hemoglobins - oxygen-binding proteins essential to function of red blood cells. Method useful in several biotechnological applications. One, enhancement of growth of cells at higher densities. Another, production of hemoglobin to enhance supplies of oxygen in cells, for use in chemical reactions requiring oxygen, as additive to serum to increase transport of oxygen, and for binding and separating oxygen from mixtures of gases.

Khosla, Chaitan↗
DOE OSTI2024

Structure-driven development of a biomimetic rare earth artificial metalloprotein

The 2011 discovery of the first rare earth–dependent enzyme in methylotrophic Methylobacterium extorquens AM1 prompted intensive research toward understanding the unique chemistry at play in these systems. This enzyme, an alcohol dehydrogenase (ADH), features a La 3+ ion closely associated with redox-active coenzyme pyrroloquinoline quinone (PQQ) and is structurally homologous to the Ca 2+ -dependent ADH from the same organism. AM1 also produces a periplasmic PQQ-binding protein, PqqT, which we have now structurally characterized to 1.46-Å resolution by X-ray diffraction. This crystal structure reveals a Lys residue hydrogen-bonded to PQQ at the site analogously occupied by a Lewis acidic cation in ADH. Accordingly, we prepared K 142 A- and K 142 D-PqqT variants to assess the relevance of this site toward metal binding. Isothermal titration calorimetry experiments and titrations monitored by UV–Vis absorption and emission spectroscopies support that K 142 D-PqqT binds tightly (Kd = 0.6 ± 0.2 μM) to La 3+ in the presence of bound PQQ and produces spectral signatures consistent with those of ADH enzymes. These spectral signatures are not observed for WT- or K 142 A-variants or upon addition of Ca 2+ to PQQ ⸦ K 142 D-PqqT. Addition of benzyl alcohol to La 3+ -bound PQQ ⸦ K 142 D-PqqT (but not Ca 2+ -bound PQQ ⸦ K 142 D-PqqT, or La 3+ -bound PQQ ⸦ WT-PqqT) produces spectroscopic changes associated with PQQ reduction, and chemical trapping experiments reveal the production of benzaldehyde, supporting ADH activity. By creating a metal binding site that mimics native ADH enzymes, we present a rare earth-dependent artificial metalloenzyme primed for future mechanistic, biocatalytic, and biosensing applications.

Science & Technology - Other Topics↗
DOE OSTI2024

Lanthanide binding peptide surfactants at air–aqueous interfaces for interfacial separation of rare earth elements

Rare earth elements (REEs) are critical materials to modern technologies. They are obtained by selective separation from mining feedstocks consisting of mixtures of their trivalent cation. We are developing an all-aqueous, bioinspired, interfacial separation using peptides as amphiphilic molecular extractants. Lanthanide binding tags (LBTs) are amphiphilic peptide sequences based on the EF-hand metal binding loops of calcium-binding proteins which complex selectively REEs. We study LBTs optimized for coordination to Tb 3+ using luminescence spectroscopy, surface tensiometry, X-ray reflectivity, and X-ray fluorescence near total reflection, and find that these LBTs capture Tb 3+ in bulk and adsorb the complex to the interface. Molecular dynamics show that the binding pocket remains intact upon adsorption. We find that, if the net negative charge on the peptide results in a negatively charged complex, excess cations are recruited to the interface by nonselective Coulombic interactions that compromise selective REE capture. If, however, the net negative charge on the peptide is −3, resulting in a neutral complex, a 1:1 surface ratio of cation to peptide is achieved. Surface adsorption of the neutral peptide complexes from an equimolar mixture of Tb 3+ and La 3+ demonstrates a switchable platform dictated by bulk and interfacial effects. The adsorption layer becomes enriched in the favored Tb 3+ when the bulk peptide is saturated, but selective to La 3+ for undersaturation due to a higher surface activity of the La 3+ complex.

Ortuno Macias, Luis E. (ORCID:0000000284342192)↗
NASA NTRS1997

Activation of G proteins mediates flow-induced prostaglandin E2 production in osteoblasts

Interstitial fluid flow may play a role in load-induced bone remodeling. Previously, we have shown that fluid flow stimulates osteoblast production of cAMP inositol trisphosphate (IP3), and PGE2. Flow-induced increases in cAMP and IP3 were shown to be a result of PG production. Thus, PGE2 production appears to be an important component in fluid flow induced signal transduction. In the present study, we investigated the mechanism of flow-induced PGE2 synthesis. Flow-induced a 20-fold increase in PGE2 production in osteoblasts. Increases were also observed with ALF4-(10mM) (98-fold), an activator of guanidine nucleotide-binding proteins (G proteins), and calcium ionophore A23187 (2 microM) (100-fold) in stationary cells. We then investigated whether flow stimulation is mediated by G proteins and increases in intracellular calcium. Flow-induced PGE2 production was inhibited by the G protein inhibitors GDP beta S (100 microM) and pertussis toxin (1 microgram/ml) by 83% and 72%, respectively. Chelation of extracellular calcium by EGTA (2 mM) and intracellular calcium by quin-2/AM (30 microM) blocked flow stimulation by 87% and 67%, respectively. These results suggest that G proteins and calcium play an important role in mediating mechanochemical signal transduction in osteoblasts.

NASA Discipline Cell Biology↗
DOE OSTI2024

The protein structurome of Orthornavirae and its dark matter

Metatranscriptomics is uncovering more and more diverse families of viruses with RNA genomes comprising the viral kingdom Orthornavirae in the realm Riboviria. Thorough protein annotation and comparison are essential to get insights into the functions of viral proteins and virus evolution. In addition to sequence- and hmm profile-based methods, protein structure comparison adds a powerful tool to uncover protein functions and relationships. We constructed an Orthornavirae “structurome” consisting of already annotated as well as unannotated (“dark matter”) proteins and domains encoded in viral genomes. We used protein structure modeling and similarity searches to illuminate the remaining dark matter in hundreds of thousands of orthornavirus genomes. The vast majority of the dark matter domains showed either “generic” folds, such as single α-helices, or no high confidence structure predictions. Nevertheless, a variety of lineage-specific globular domains that were new either to orthornaviruses in general or to particular virus families were identified within the proteomic dark matter of orthornaviruses, including several predicted nucleic acid-binding domains and nucleases. In addition, we identified a case of exaptation of a cellular nucleoside monophosphate kinase as an RNA-binding protein in several virus families. Notwithstanding the continuing discovery of numerous orthornaviruses, it appears that all the protein domains conserved in large groups of viruses have already been identified. The rest of the viral proteome seems to be dominated by poorly structured domains including intrinsically disordered ones that likely mediate specific virus-host interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

CsrA-mediated regulation of a virulence switch in Acinetobacter baumannii

CsrA is an RNA binding protein that functions as a global regulator in bacteria. We demonstrate that, in Acinetobacter baumannii, CsrA acts as a positive regulator of the switch from virulent (VIR-O) to avirulent (AV-T) subpopulations. This regulation is mediated, in part, by CsrA interfering with Rho-dependent termination in the mRNA leader region of the ABUW_1645 gene, encoding the primary TetR-type transcriptional regulator that drives cells from the VIR-O to the AV-T state. We demonstrate that CsrA directly binds to the ABUW_1645 mRNA leader region and interferes with Rho binding. We identify three small RNAs (sRNAs) designated CsrB, CsrC, and CsrD that bind to CsrA and inhibit its activity. Individual overexpression of each sRNA greatly decreased the rate of VIR-O to AV-T switching. Individual deletions of each sRNA increased the frequency of VIR-O to AV-T switching, with loss of CsrB giving the highest increase at 2.4-fold. The expression of each sRNA was strongly dependent on the GacA response regulator and the expression of each sRNA was higher in VIR-O cells than in AV-T variants. Furthermore, this regulation provides a mechanism for the differential control of CsrA activity between VIR-O and AV-T variants.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2004

The Conservation of Structure and Mechanism of Catalytic Action in a Family of Thiamin Pyrophosphate (TPP)-dependent Enzymes

Thiamin pyrophosphate (TPP)-dependent enzymes are a divergent family of TPP and metal ion binding proteins that perform a wide range of functions with the common decarboxylation steps of a -(O=)C-C(OH)- fragment of alpha-ketoacids and alpha- hydroxyaldehydes. To determine how structure and catalytic action are conserved in the context of large sequence differences existing within this family of enzymes, we have carried out an analysis of TPP-dependent enzymes of known structures. The common structure of TPP-dependent enzymes is formed at the interface of four alpha/beta domains from at least two subunits, which provide for two metal and TPP-binding sites. Residues around these catalytic sites are conserved for functional purpose, while those further away from TPP are conserved for structural reasons. Together they provide a network of contacts required for flip-flop catalytic action within TPP-dependent enzymes. Thus our analysis defines a TPP-action motif that is proposed for annotating TPP-dependent enzymes for advancing functional proteomics.

Dominiak, P.↗
NASA NTRS2002

Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 microm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.

NASA Program Fundamental Space Biology↗
NASA NTRS1994

Concentration and temperature effects on ovostatin activity

Light scattering experiments performed at Mississippi State University using MSFC ovostatin preparations indicated that at low ovostatin concentrations, below 0.2 mg/ml, the protein was dissociating from a tetramer into dimers. Since the proposed mechanism of action involved the tetrameric form of the protein, we hypothesized that perhaps under the conditions of our assays at various O/T ratios the ovostatin was becoming dissociated into an inactive dimer. To examine this possibility we assayed the ovostatin activity as a function of ovostatin concentration and of temperature of the assay. Data are presented that show the results of these assays at 23 C, 30 C, 37 C and 42 C respectively. The data are highly suggestive that there is a decrease in ovostatin activity as the concentration of the protein falls below 0.06 mg/ml. This may not be of any physiological importance, however, since the concentration of ovostatin in the egg is about 0.5 mg/ml. Curiously, the dissociation of the tetramer into dimers does not show a significant temperature dependence as would be expected for an equilibrium reaction. Whether this is in fact the case, or whether the differences are so small as to not be discerned from the current data remains to be seen. Another aspect to consider is that in the egg the primary role of the ovostatin may or may not be as a protease inhibitor. Although the inhibition of collagenase by ovostatin may be an important aspect of embryogenesis, it is also possible that it functions as a binding protein for some substance. In this regard, all ovostatin preparations from MSFC have shown an approximately 88,000 MW protein associated with the ovostatin. The identity of this protein is not currently known and may be the subject of future studies.

Moriarity, Debra M.↗
DOE OSTI2024

Steroid responsiveness in alcohol-associated hepatitis is linked to glucocorticoid metabolism, mitochondrial repair, and heat shock proteins

Alcohol-associated hepatitis (AH) is one of the clinical presentations of alcohol-associated liver disease. AH has poor prognosis, and corticosteroids remain the mainstay of drug therapy. However, ~40% of patients do not respond to this treatment, and the mechanisms underlying the altered response to corticosteroids are not understood. The current study aimed to identify changes in hepatic protein expression associated with responsiveness to corticosteroids and prognosis in patients with AH. Patients with AH were enrolled based on the National Institute on Alcohol Abuse and Alcoholism inclusion criteria for acute AH and further confirmed by a diagnostic liver biopsy. Proteomic analysis was conducted on liver samples acquired from patients with AH grouped as nonresponders (AH-NR, n = 7) and responders (AH-R, n = 14) to corticosteroids, and nonalcohol-associated liver disease controls (n = 10). The definition of responders was based on the clinical prognostic model, the Lille Score, where a score < 0.45 classified patients as AH-R and a score > 0.45 as AH-NR. Primary outcomes used to assess steroid response were Lille Score (eg, improved liver function) and survival at 24 weeks. Reduced levels of the glucocorticoid receptor and its transcriptional co-activator, glucocorticoid modulatory element-binding protein 2, were observed in the hepatic proteome of AH-NR versus AH-R. The corticosteroid metabolizing enzyme, 11-beta-hydroxysteroid dehydrogenase 1, was increased in AH-NR versus AH-R along with elevated mitochondrial DNA repair enzymes, while several proteins of the heat shock pathway were reduced. Analysis of differentially expressed proteins in AH-NR who survived 24 weeks relative to AH-NR nonsurvivors revealed several protein expression changes, including increased levels of acute phase proteins, elevated coagulation factors, and reduced mast cell markers. This study identified hepatic proteomic changes that may predict responsiveness to corticosteroids and mortality in patients with AH.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

De novo design of miniprotein antagonists of cytokine storm inducers

Cytokine release syndrome (CRS), commonly known as cytokine storm, is an acute systemic inflammatory response that is a significant global health threat. Interleukin-6 (IL-6) and interleukin-1 (IL-1) are key pro-inflammatory cytokines involved in CRS and are hence critical therapeutic targets. Current antagonists, such as tocilizumab and anakinra, target IL-6R/IL-1R but have limitations due to their long half-life and systemic anti-inflammatory effects, making them less suitable for acute or localized treatments. Here we present the de novo design of small protein antagonists that prevent IL-1 and IL-6 from interacting with their receptors to activate signaling. The designed proteins bind to the IL-6R, GP130 (an IL-6 co-receptor), and IL-1R1 receptor subunits with binding affinities in the picomolar to low-nanomolar range. X-ray crystallography studies reveal that the structures of these antagonists closely match their computational design models. In a human cardiac organoid disease model, the IL-1R antagonists demonstrated protective effects against inflammation and cardiac damage induced by IL-1β. These minibinders show promise for administration via subcutaneous injection or intranasal/inhaled routes to mitigate acute cytokine storm effects.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1995

Lipopolysaccharide modulation of a CD14-like molecule on porcine alveolar macrophages

Cluster of differentiation antigen 14 (CD14) functions as a receptor for lipopolysaccharide (LPS) LPS-binding protein (LBP) complexes. Because LPS has varying effects on CD14 expression in vitro, we evaluated CD14 expression in response to LPS with a fully differentiated macrophage phenotype, the alveolar macrophage. By using flow microfluorometric analysis and a radioimmunoassay with an anti-human CD14 monoclonal antibody (My4) that cross-reacts with porcine CD14, we found that macrophages stimulated with LPS for 24 h exhibited a two- to fivefold increase in CD14-like antigen compared with unstimulated cells. At low concentrations of LPS, up-regulation of the CD14-like antigen was dependent on serum; at higher concentrations of LPS, serum was not required. In the absence of serum a 10-fold higher dose of LPS (10 ng/ml) was required to increase CD14-like expression. In addition, LPS-induced CD14-like up-regulation correlated with secretion of tumor necrosis factor-alpha, regardless of serum concentration. Blockade with My4 antibody significantly inhibited LPS-induced tumor necrosis factor-alpha secretion at 1 ng/ml of LPS. However, inhibition decreased as we increased the LPS concentration, suggesting the existence of CD14-independent pathways of macrophage activation in response to LPS. Alternatively, My4 may have a lower affinity for the porcine CD14 antigen than LPS, which may have only partially blocked the LPS-LBP binding site at high concentrations of LPS. Therefore, these data suggest that LPS activation of porcine alveolar macrophages for 24 h increased CD14-like receptor expression. The degree of CD14-like up-regulation was related to LPS concentration, however, activation did not require the presence of serum at high concentrations of LPS.

Non-NASA Center↗
NASA NTRS2001

The Ca(2+) status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants

To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.

NASA Discipline Plant Biology↗