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At least 1,639 records · Page 91

Mining a human transcriptome database for chemical modulators of NRF2

Nuclear factor erythroid-2 related factor 2 (NRF2) encoded by the NFE2L2 gene is a transcription factor critical for protecting cells from chemically-induced oxidative stress. We developed computational procedures to identify chemical modulators of NRF2 in a large database of human microarray data. A gene expression biomarker was built from statistically-filtered gene lists derived from microarray experiments in primary human hepatocytes and cancer cell lines exposed to NRF2-activating chemicals (oltipraz, sulforaphane, CDDOIm) or in which the NRF2 suppressor Keap1 was knocked down by siRNA. Directionally consistent biomarker genes were further filtered for those dependent on NRF2 using a microarray dataset from cells after NFE2L2 siRNA knockdown. The resulting 143-gene biomarker was evaluated as a predictive tool using the correlation-based Running Fisher algorithm. Using 59 gene expression comparisons from chemically-treated cells with known NRF2 activating potential, the biomarker gave a balanced accuracy of 93%. The biomarker was comprised of many well-known NRF2 target genes (AKR1B10, AKR1C1, NQO1, TXNRD1, SRXN1, GCLC, GCLM), 69% of which were found to be bound directly by NRF2 using ChIP-Seq. NRF2 activity was assessed across ~9840 microarray comparisons from ~1460 studies examining the effects of ~2260 chemicals in human cell lines. A total of 260 and 43 chemicals were found to activate or suppress NRF2, respectively, most of which have not been previously reported to modulate NRF2 activity. Using a NRF2-responsive reporter gene in HepG2 cells, we confirmed the activity of a set of chemicals predicted using the biomarker. The biomarker will be useful for future gene expression screening studies of environmentally-relevant chemicals.

59 BASIC BIOLOGICAL SCIENCES↗

Longitudinal analysis of host protein serum signatures of treatment and recovery in pulmonary tuberculosis

A better understanding of treatment progression and recovery in pulmonary tuberculosis (TB) infectious disease is crucial. This study analyzed longitudinal serum samples from pulmonary TB patients undergoing interventional treatment to identify surrogate markers for TB-related outcomes. Serum that was collected at baseline and 8, 17, 26, and 52 weeks from 30 TB patients experiencing durable cure were evaluated and compared using a sensitive LC-MS/MS proteomic platform for the detection and quantification of differential host protein signatures relative to timepoint. The global proteome signature was analyzed for statistical differences across the time course and between disease severity and treatment groups. A total of 676 proteins showed differential expression in the serum over these timepoints relative to baseline. Comparisons to understand serum protein dynamics at 8 weeks, treatment endpoints at 17 and 26 weeks, and post-treatment at 52 weeks were performed. The largest protein abundance changes were observed at 8 weeks as the initial effects of antibiotic treatment strongly impacted inflammatory and immune modulated responses. However, the largest number of proteome changes was observed at the end of treatment time points 17 and 26 weeks respectively. Post-treatment 52-week results showed an abatement of differential proteome signatures from end of treatment, though interestingly those proteins uniquely significant at post-treatment were almost exclusively downregulated. Patients were additionally stratified based upon disease severity and compared across all timepoints, identifying 461 discriminating proteome signatures. These proteome signatures collapsed into discrete expression profiles with distinct pathways across immune activation and signaling, hemostasis, and metabolism annotations. Insulin-like growth factor (IGF) and Integrin signaling maintained a severity signature through 52 weeks, implying an intrinsic disease severity signature well into the post-treatment timeframe. Previous proteome studies have primarily focused on the 8-week timepoint in relation to culture conversion status. While this study confirms previous observations, it also highlights some differences. The inclusion of additional end of treatment and post-treatment time points offers a more comprehensive assessment of treatment progression within the serum proteome. Examining the expression dynamics at these later time periods will help in the investigation of relapse patients and has provided indicative markers of response and recovery.

59 BASIC BIOLOGICAL SCIENCES↗

Rhythmic Mechanisms Governing CAM Photosynthesis in Kalanchoe fedtschenkoi : High-Resolution Temporal Transcriptomics

Crassulacean acid metabolism (CAM) is a specialized photosynthetic pathway that enhances water-use efficiency by temporally separating nocturnal CO 2 uptake from daytime decarboxylation and carbon fixation. To uncover the regulatory mechanisms coordinating these temporal dynamics, we generated high-resolution, 48 h time-course transcriptomes for the CAM model Kalanchoe fedtschenkoi under both 12 h/12 h light/dark (LD) cycles and continuous light (LL). A rhythmicity analysis revealed that diel light cues are the dominant driver of transcript oscillations: 16,810 genes (54.3% of annotated genes) exhibited rhythmic expression only under LD, whereas just 399 genes (1.3%) remained rhythmic under LL. A smaller set of 3009 genes (9.7%) oscillated in both conditions, indicating that the intrinsic circadian clock sustains rhythmicity for a limited subset of the transcriptome. A gene co-expression network analysis revealed extensive integration between circadian clock components, core CAM pathway enzymes, and stomatal regulators, defining regulatory modules that coordinate metabolic and physiological timing. Notably, key hub genes associated with post-translational and post-transcriptional regulation, including the E3 ubiquitin ligase HUB2 and several pentatricopeptide repeat (PPR) proteins, act as central nodes in CAM-associated networks. This discovery implicates epigenetic and organellar regulation as previously unrecognized critical tiers of control in CAM. Together, our results support a regulatory model in which CAM rhythmicity is governed by both external light/dark cues and the endogenous circadian clock through multi-level control spanning transcriptional and protein-level regulation. To support community exploration, we also provide an interactive eFP (electronic Fluorescent Pictograph) browser for visualizing time-resolved gene expression profiles.

09 BIOMASS FUELS↗

Examination of Genetic Control Elements in the Phototrophic Firmicute Heliomicrobium modesticaldum

Heliomicrobium modesticaldum has been used as a model organism for the Heliobacteria, the only phototrophic family in the Firmicutes. It is a moderately thermophilic anoxygenic phototrophic bacterium that is capable of fermentative growth in the dark. The genetic manipulation of H. modesticaldum is still in its infancy. Methods to introduce genes through the use of exogenous plasmids and to delete genes from the chromosome through the use of the native CRISPR/Cas system have been developed in the last several years. To expand our genetic toolkit, it was necessary to control gene expression. In this study, we analyzed constitutive and inducible promoters developed for clostridia for their use in H. modesticaldum and further tested two reporters, adhB and lacZ, as indicators of promoter strength. Alcohol dehydrogenase (AdhB) was unsuitable as a reporter in this species due to high endogenous activity and/or low activity of the reporter, but a thermostable LacZ worked well as a reporter. A set of constitutive promoters previously reported to work in Clostridium thermocellum was found to be reliable for controlling the expression of the lacZ reporter gene in H. modesticaldum at a range of activities spanning an order of magnitude. An anhydrotetracycline-inducible promoter was created by inserting tetO operators into a strong constitutive promoter, but it was not fully repressible. The implementation of a xylose-inducible promoter resulted in complete repression of β-gal in the absence of xylose, and reliable expression tunable through the concentration of xylose added to the culture.

59 BASIC BIOLOGICAL SCIENCES↗

The potato virus X TGBp2 protein association with the endoplasmic reticulum plays a role in but is not sufficient for viral cell-to-cell movement

Potato virus X (PVX) TGBp1, TGBp2, TGBp3, and coat protein are required for virus cell-to-cell movement. Plasmids expressing GFP fused to TGBp2 were bombarded to leaf epidermal cells and GFP:TGBp2 moved cell to cell in Nicotiana benthamiana leaves but not in Nicotiana tabacum leaves. GFP:TGBp2 movement was observed in TGBp1-transgenic N. tabacum, indicating that TGBp2 requires TGBp1 to promote its movement in N. tabacum. In this study, GFP:TGBp2 was detected in a polygonal pattern that resembles the endoplasmic reticulum (ER) network. Amino acid sequence analysis revealed TGBp2 has two putative transmembrane domains. Two mutations separately introduced into the coding sequences encompassing the putative transmembrane domains within the GFP:TGBp2 plasmids and PVX genome, disrupted membrane binding of GFP:TGBp2, inhibited GFP:TGBp2 movement in N. benthamiana and TGBp1-expressing N. tabacum, and inhibited PVX movement. A third mutation, lying outside the transmembrane domains, had no effect on GFP:TGBp2 ER association or movement in N. benthamiana but inhibited GFP:TGBp2 movement in TGBp1-expressing N. tabacum and PVX movement in either Nicotiana species. Thus, ER association of TGBp2 may be required but not be sufficient for virus movement. TGBp2 likely provides an activity for PVX movement beyond ER association.

NASA Discipline Plant Biology↗

[The role of the serotonin system in the stress response of various cells]

The recombinant mouse brain serotonin receptor (5HT1c) was used to study the response of plant cells and oocytes to a stress signal activated by the serotonin-serotonin receptor interaction and associated Ca2+ flow. Based on plant expression vectors, recombinant constructs were obtained to direct production of 5HT1c fused with the green fluorescent protein in plant cells. The mRNAs for hybrid proteins were synthesized in an in vitro transcription system. The expression and function of the hybrid protein and the function of the associated ion channels were electrophysiologically studied in Xenopus laevis oocytes injected with the hybrid mRNA. The hybrid protein was functional and changed the operation of the Ca2+ channel in oocytes. To study the expression of the hybrid constructs in plant cells, the in vitro transcription product was inoculated in tobacco leaves, which then fluoresced.

Non-NASA Center↗

Transcription profiling of the early gravitropic response in Arabidopsis using high-density oligonucleotide probe microarrays

Studies of plant tropisms, the directed growth toward or away from external stimuli such as light and gravity, began more than a century ago. Yet biochemical, physiological, and especially molecular mechanisms of plant tropic responses remain for the most part unclear. We examined expression of 8,300 genes during early stages of the gravitropic response using high-density oligonucleotide probe microarrays. Approximately 1.7% of the genes represented on the array exhibited significant expression changes within the first 30 min of gravity stimulation. Among gravity-induced genes were a number of genes previously implicated to be involved in gravitropism. However, a much larger number of the identified genes have not been previously associated with gravitropism. Because reorientation of plants may also expose plants to mechanical perturbations, we also compared the effects of a gentle mechanical perturbation on mRNA levels during the gravity response. It was found that approximately 39% of apparently gravity-regulated genes were also regulated by the mechanical perturbation caused by plant reorientation. Our study revealed the induction of complex gene expression patterns as a consequence of gravitropic reorientation and points to an interplay between the gravitropic and mechanical responses and to the extreme sensitivity of plants to even very gentle mechanical perturbations.

NASA Discipline Plant Biology↗

Plants, plant pathogens, and microgravity--a deadly trio

Plants grown in spaceflight conditions are more susceptible to colonization by plant pathogens. The underlying causes for this enhanced susceptibility are not known. Possibly the formation of structural barriers and the activation of plant defense response components are impaired in spaceflight conditions. Either condition would result from altered gene expression of the plant. Because of the tools available, past studies focused on a few physiological responses or biochemical pathways. With recent advances in genomics research, new tools, including microarray technologies, are available to examine the global impact of growth in the spacecraft on the plant's gene expression profile. In ground-based studies, we have developed cDNA subtraction libraries of rice that are enriched for genes induced during pathogen infection and the defense response. Arrays of these genes are being used to dissect plant defense response pathways in a model system involving wild-type rice plants and lesion mimic mutants. The lesion mimic mutants are ideal experimental tools because they erratically develop defense response-like lesions in the absence of pathogens. The gene expression profiles from these ground-based studies will provide the molecular basis for understanding the biochemical and physiological impacts of spaceflight on plant growth, development and disease defense responses. This, in turn, will allow the development of strategies to manage plant disease for life in the space environment.

NASA Discipline Plant Biology↗

Dirigent proteins and dirigent sites in lignifying tissues

Tissue-specific dirigent protein gene expression and associated dirigent (site) localization were examined in various organs of Forsythia intermedia using tissue printing, in situ mRNA hybridization and immunolabeling techniques, respectively. Dirigent protein gene expression was primarily noted in the undifferentiated cambial regions of stem sections, whereas dirigent protein sites were detected mainly in the vascular cambium and ray parenchyma cell initials. Immunolocalization also revealed cross-reactivity with particular regions of the lignified cell walls, these being coincident with the known sites of initiation of lignin deposition. These latter regions are considered to harbor contiguous arrays of dirigent (monomer binding) sites for initiation of lignin biopolymer assembly. Dirigent protein mRNA expression was also localized in the vascular regions of roots and petioles, whereas in leaves the dirigent sites were primarily associated with the palisade layers and the vascular bundle. That is, dirigent protein mediated lignan biosynthesis was initiated primarily in the cambium and ray cell initial regions of stems as well as in the leaf palisade layers, this being in accordance with the occurrence of the lignans for defense purposes. Within lignified secondary xylem cell walls, however, dirigent sites were primarily localized in the S(1) sublayer and compound middle lamella, these being coincident with previously established sites for initiation of macromolecular lignin biosynthesis. Once initiation occurs, lignification is proposed to continue through template polymerization.

NASA Discipline Plant Biology↗

Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system

The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin. Copyright 2000 Academic Press.

Non-NASA Center↗

atonal regulates neurite arborization but does not act as a proneural gene in the Drosophila brain

Drosophila atonal (ato) is the proneural gene of the chordotonal organs (CHOs) in the peripheral nervous system (PNS) and the larval and adult photoreceptor organs. Here, we show that ato is expressed at multiple stages during the development of a lineage of central brain neurons that innervate the optic lobes and are required for eclosion. A novel fate mapping approach shows that ato is expressed in the embryonic precursors of these neurons and that its expression is reactivated in third instar larvae (L3). In contrast to its function in the PNS, ato does not act as a proneural gene in the embryonic brain. Instead, ato performs a novel function, regulating arborization during larval and pupal development by interacting with Notch.

NASA Discipline Developmental Biology↗

Localization of the human 64kD autoantigen D1 to myofibrils in a subset of extraocular muscle fibers

PURPOSE. To evaluate the tissue-specific expression pattern of the 64kD human autoantigen D1, a tropomodulin-related protein that may be involved in thyroid-associated ophthalmopathy. METHODS. Recombinant 64kD human autoantigen D1 was generated in a bacterial expression system and used to immunize rabbits. Specific antibodies were affinity-purified and used for Western blots on normal and hyperthyroid rat and rabbit tissue, and immunofluorescence localization on cryosections of rat tissue. RESULTS. Anti-64kD human autoantigen D1 antibodies recognize specifically a approximately 70kD polypeptide in western blots of extraocular muscle, sternothyroid muscle, and smooth muscle. Immunofluorescence staining demonstrates that the 64kD human autoantigen D1 localizes to myofibrils in slow fibers from rat extraocular and sternothyroid muscle. The level of this protein is not altered in extraocular muscles from hyperthyroid rabbits. CONCLUSIONS. The 64kD human autoantigen D1 is expressed in slow fibers of extraocular and sternothyroid muscles as a component of myofibrils, and is not upregulated in conditions of hyperthyroidism.

Proteins/genetics/immunology/metabolism↗

Rapid and systemic accumulation of chloroplast mRNA-binding protein transcripts after flame stimulus in tomato

It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5-15 min) in transcript accumulation, a decline to basal levels (15-45 min), and then a second, more prolonged increase (60-90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding.

NASA Discipline Plant Biology↗

Model system for plant cell biology: GFP imaging in living onion epidermal cells

The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

NASA Discipline Plant Biology↗

CCAAT/enhancer-binding protein delta is a critical regulator of insulin-like growth factor-I gene transcription in osteoblasts

Insulin-like growth factor-I (IGF-I) plays a major role in promoting skeletal growth by stimulating bone cell replication and differentiation. Prostaglandin E2 and other agents that induce cAMP production enhance IGF-I gene transcription in cultured rat osteoblasts through a DNA element termed HS3D, located in the proximal part of the major rat IGF-I promoter. We previously determined that CCAAT/enhancer-binding protein delta (C/EBPdelta) is the key cAMP-stimulated regulator of IGF-I transcription in these cells and showed that it transactivates the rat IGF-I promoter through the HS3D site. We now have defined the physical-chemical properties and functional consequences of the interactions between C/EBPdelta and HS3D. C/EBPdelta, expressed in COS-7 cells or purified as a recombinant protein from Escherichia coli, bound to HS3D with an affinity at least equivalent to that of the albumin D-site, a known high affinity C/EBP binding sequence, and both DNA elements competed equally for C/EBPdelta. C/EBPdelta bound to HS3D as a dimer, with protein-DNA contact points located on guanine residues on both DNA strands within and just adjacent to the core C/EBP half-site, GCAAT, as determined by methylation interference footprinting. C/EBPdelta also formed protein-protein dimers in the absence of interactions with its DNA binding site, as indicated by results of glutaraldehyde cross-linking studies. As established by competition gel-mobility shift experiments, the conserved HS3D sequence from rat, human, and chicken also bound C/EBPdelta with similar affinity. We also found that prostaglandin E2-induced expression of reporter genes containing human IGF-I promoter 1 or four tandem copies of the human HS3D element fused to a minimal promoter and show that these effects were enhanced by a co-transfected C/EBPdelta expression plasmid. Taken together, our results provide evidence that C/EBPdelta is a critical activator of IGF-I gene transcription in osteoblasts and potentially in other cell types and species.

NASA Discipline Musculoskeletal↗

Analysis of xylem formation in pine by cDNA sequencing

Secondary xylem (wood) formation is likely to involve some genes expressed rarely or not at all in herbaceous plants. Moreover, environmental and developmental stimuli influence secondary xylem differentiation, producing morphological and chemical changes in wood. To increase our understanding of xylem formation, and to provide material for comparative analysis of gymnosperm and angiosperm sequences, ESTs were obtained from immature xylem of loblolly pine (Pinus taeda L.). A total of 1,097 single-pass sequences were obtained from 5' ends of cDNAs made from gravistimulated tissue from bent trees. Cluster analysis detected 107 groups of similar sequences, ranging in size from 2 to 20 sequences. A total of 361 sequences fell into these groups, whereas 736 sequences were unique. About 55% of the pine EST sequences show similarity to previously described sequences in public databases. About 10% of the recognized genes encode factors involved in cell wall formation. Sequences similar to cell wall proteins, most known lignin biosynthetic enzymes, and several enzymes of carbohydrate metabolism were found. A number of putative regulatory proteins also are represented. Expression patterns of several of these genes were studied in various tissues and organs of pine. Sequencing novel genes expressed during xylem formation will provide a powerful means of identifying mechanisms controlling this important differentiation pathway.

Non-NASA Center↗

Arabidopsis TCH4, regulated by hormones and the environment, encodes a xyloglucan endotransglycosylase

Adaptation of plants to environmental conditions requires that sensing of external stimuli be linked to mechanisms of morphogenesis. The Arabidopsis TCH (for touch) genes are rapidly upregulated in expression in response to environmental stimuli, but a connection between this molecular response and developmental alterations has not been established. We identified TCH4 as a xyloglucan endotransglycosylase by sequence similarity and enzyme activity. Xyloglucan endotransglycosylases most likely modify cell walls, a fundamental determinant of plant form. We determined that TCH4 expression is regulated by auxin and brassinosteroids, by environmental stimuli, and during development, by a 1-kb region. Expression was restricted to expanding tissues and organs that undergo cell wall modification. Regulation of genes encoding cell wall-modifying enzymes, such as TCH4, may underlie plant morphogenetic responses to the environment.

NASA Discipline Plant Biology↗

Sequence, molecular properties, and chromosomal mapping of mouse lumican

PURPOSE. Lumican is a major proteoglycan of vertebrate cornea. This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization. METHODS. Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe. Tissue expression and size of lumican mRNA were determined using Northern hybridization. Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican. Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross. RESULTS. Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins. The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation. A 1.9-kb lumican mRNA is present in cornea and several other tissues. Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea. Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo. The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10. The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology. CONCLUSIONS. This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene.

NASA Discipline Cell Biology↗