BR Signaling Research Articles

Administration of GABA B receptor positive allosteric modulators inhibit the expression of previously established methamphetamine-induced conditioned place preference

Little is known about the role of GABA B receptors (GABA BRs) in the maintenance of memories associated with using abused substances. We have embarked on a series of studies designed to determine if enhancing the efficacy of GABA-occupied GABA BRs with positive allosteric modulators (PAMs) can negate previously established conditioned place preference (CPP) induced by methamphetamine. In the current study, we evaluated the effects of acute administration of GABA BR PAMs, GS39783 and CGP7930. We determined that post-conditioning treatments with these PAMs, administered in the home cage, blocked the subsequent expression of methamphetamine-induced CPP. These data indicate that selectively augmenting GABA-occupied GABA BR signaling is sufficient to reduce memory maintenance and/or the salience of contextual cues previously associated with methamphetamine.

Soluble graphene through edge-selective functionalization

Thermally expanded graphite was functionalized with 4-bromophenyl addends using the in situ diazonium formation procedure, and after mild sonication treatment in N,N′-dimethylformamide, thin graphene layers were exfoliated from the bulk graphite. These chemically-assisted exfoliated graphene (CEG) sheets had higher solubility than pristine graphene without any stabilizer additive. More than 70% of these soluble flakes had less than 5 layers. Energy filtered transmission electron microscopy (EFTEM) elemental mapping provided evidence of the edge-selective diazonium functionalization with graphene. A majority of the Br signals came from the edges of the CEG indicating that the basal planes were not highly functionalized. The CEG was also characterized by X-ray photoelectron spectroscopy, atomic force microscopy, Raman spectroscopy, and transmission electron microscopy.

73. Involvement of BR signaling in the regulation of organ size by SHORT GRAIN 1

73. Involvement of BR signaling in the regulation of organ size by SHORT GRAIN 1

Synchrotron X-Ray Fluorescent Imaging and Spectroscopy Studies of the Role of Copper in the Stem Cell Niche Architecture of Adult Neural Stem Cells

Improvements in sensitivity and spatial resolution of X-ray fluorescent (XRF) imaging and spectroscopy allowed us to study the distribution of metals in brain tissues. We discovered the specific Cu enrichment in cells in the subventricular zone (SVZ) of the lateral ventricle. This area in the brain contains adult neural stem cells (NSCs). NSCs niche architecture enables to continuously generate functional neurons in specific brain regions throughout life. Knowledge about the mechanisms controlling NSCs self-renewal, proliferation and differentiation is of critical importance for future therapeutic interventions of major brain disorders.XRF-imaging with sub-cellular (200 nm) resolution on rat brains demonstrated that sub-population of cells in the SVZ builds up sub-cellular Cu accumulations. The Cu concentration inside these structures is as high as 50 mM. It is well established that SVZ has four types of cells: ependymal cells, type B progenitors, type C transit amplifying cells and type A migrating neuroblasts. Imaging the Br signal in brains of BrdU treated rats, we found that actively dividing cells are largely depleted of Cu accumulations. However, cells surrounding actively dividing cells, which are assigned to type B progenitors, demonstrate significant Cu accumulations. Cu K-edge micro-XANES demonstrated that Cu is in Cu(I) form with and spectrum has shape characteristic of a Cu(I)-thiolate multimetallic cluster. The Cu co-localizes with increased sulfur signal which allows quantitation of the Cu/S content.Copper is known to play an important role in the brain's development and function. However, the role of Cu in the viability and control of the NSCs is presently unknown. Our study is a first attempt to look at the role of Cu in mechanisms controlling the NSCs.

BRASSINOSTEROID UPREGULATED1, Encoding a Helix-Loop-Helix Protein, Is a Novel Gene Involved in Brassinosteroid Signaling and Controls Bending of the Lamina Joint in Rice

Brassinosteroids (BRs) are involved in many developmental processes and regulate many subsets of downstream genes throughout the plant kingdom. However, little is known about the BR signal transduction and response network in monocots. To identify novel BR-related genes in rice (Oryza sativa), we monitored the transcriptomic response of the brassinosteroid deficient1 (brd1) mutant, with a defective BR biosynthetic gene, to brassinolide treatment. Here, we describe a novel BR-induced rice gene BRASSINOSTEROID UPREGULATED1 (BU1), encoding a helix-loop-helix protein. Rice plants overexpressing BU1 (BU1:OX) showed enhanced bending of the lamina joint, increased grain size, and resistance to brassinazole, an inhibitor of BR biosynthesis. In contrast to BU1:OX, RNAi plants designed to repress both BU1 and its homologs displayed erect leaves. In addition, compared to the wild type, the induction of BU1 by exogenous brassinolide did not require de novo protein synthesis and it was weaker in a BR receptor mutant OsbriI (Oryza sativa brassinosteroid insensitive1, d61) and a rice G protein alpha subunit (RGA1) mutant d1. These results indicate that BU1 protein is a positive regulator of BR response: it controls bending of the lamina joint in rice and it is a novel primary response gene that participates in two BR signaling pathways through OsBRI1 and RGA1. Furthermore, expression analyses showed that BU1 is expressed in several organs including lamina joint, phloem, and epithelial cells in embryos. These results indicate that BU1 may participate in some other unknown processes modulated by BR in rice.

Phenotypic Analysis of Arabidopsis Mutants: Brassinosteroid Hormone Response

INTRODUCTIONBrassinosteroid (BR) is required for normal plant growth and development; most notably, BR promotes cell elongation. Thus, mutants with defects in BR biosynthesis or response have defects in cell elongation. They fail to undergo skotomorphogenesis (etiolation) in the dark and they are dwarves when grown in the light. Whether a cell elongation defect can be rescued, unaffected, or alleviated by BR treatment provides an important criterion for whether a mutant has a defect in BR biosynthesis or BR response or whether the mutant phenotype is unrelated to BR. On the other hand, mutants with increased BR signaling are less sensitive to the BR-biosynthesis inhibitor brassinazole (BRZ). The hypocotyl length of dark-grown seedlings and petiole length of light-grown plants are often measured to determine BR responsiveness; BRZ inhibition of BR biosynthesis can reduce hypocotyl and petiole elongation. Wild-type seedlings grown on 1 μM BRZ in the dark have hypocotyls approximately one-third the length of those on medium lacking BRZ. This protocol describes the measurement of BR responsiveness, with or without BRZ, in light- and dark-grown seedlings.

Multiple mechanisms modulate brassinosteroid signaling

Brassinosteroids are essential hormones for plant growth and development. Genetic studies have identified key components of the BR signaling pathway, including the cell-surface receptor kinases that perceive BR, an intracellular kinase and a phosphatase, and nuclear transcription factors. Subsequent biochemical studies have revealed many details about signaling events from BR perception at the cell surface to gene expression in the nucleus. Recent studies have identified the 14-3-3 proteins as BR signaling components and elucidated multiple mechanisms by which phosphorylation modulates the BR transcription factors. In addition, BRI1 signaling from the endosomes and BR-independent functions of BAK1 have been observed. However, a major gap still exists in the current BR signaling pathway between the receptor complex and downstream signaling events.

TomatoBRASSINOSTEROID INSENSITIVE1Is Required for Systemin-Induced Root Elongation inSolanum pimpinellifoliumbut Is Not Essential for Wound Signaling

The tomato Leu-rich repeat receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) has been implicated in both peptide (systemin) and steroid (brassinosteroid [BR]) hormone perception. In an attempt to dissect these signaling pathways, we show that transgenic expression of BRI1 can restore the dwarf phenotype of the tomato curl3 (cu3) mutation. Confirmation that BRI1 is involved in BR signaling is highlighted by the lack of BR binding to microsomal fractions made from cu3 mutants and the restoration of BR responsiveness following transformation with BRI1. In addition, wound and systemin responses in the cu3 mutants are functional, as assayed by proteinase inhibitor gene induction and rapid alkalinization of culture medium. However, we observed BRI1-dependent root elongation in response to systemin in Solanum pimpinellifolium. In addition, ethylene perception is required for normal systemin responses in roots. These data taken together suggest that cu3 is not defective in systemin-induced wound signaling and that systemin perception can occur via a non-BRI1 mechanism.

Rapid radiation degradation in the XPS analysis of antibacterial coatings of brominated furanones

AbstractBrominated furanones are promising antibacterial compounds, both in solution and immobilized on solid surfaces, and there is interest in their utilization as protective coatings on biomedical devices to combat device‐centered infections. The rational design and optimization of antibacterial furanone coatings requires detailed spectroscopic surface characterization of the coatings produced and correlation of surface analysis data with antibacterial performance. However, as reported in this study, the extreme sensitivity of brominated furanones to radiation damage in the course of XPS analysis greatly complicates characterization. Our data show that rapid degradation occurs, as shown by the emergence of a signal assignable to Br− ion in the Br 3d spectrum. This Br− is water soluble as shown by washing experiments, whereas the CBr signal does not become reduced during washing, consistent with covalent interfacial immobilization of the furanone molecules. Furanone molecules were immobilized onto two different hydrogel interlayers (polyacrylic acid and polyethylene oxide (PEO)), and XPS analyses show that the interlayers did not suffer significant radiation degradation; the damage is limited to the furanone molecules. The results of this study may assist in XPS analyses of other brominated compounds on surfaces; the possibility of Br− ions deriving from XPS analysis–damage must be investigated before assigning their generation to side reactions in the coating or surface‐modification procedure. Copyright © 2006 John Wiley & Sons, Ltd.

A brassinolide‐suppressed rice MADS‐box transcription factor, OsMDP1, has a negative regulatory role in BR signaling

The MADS-box transcription factor-encoding genes are expressed mainly during plant reproductive development, where they play important roles in controlling floral organ initiation and identity. Few previous reports have investigated the functions of MADS-box transcription factors expressed in vegetative tissues. Here we describe the functional characterization of a rice AG-like MADS-box protein, OsMDP1 (Oryza sativa MADS-domain-containing protein 1). A partial cDNA encoding a MADS-box domain was identified via high-throughput screening of rice brassinolide-regulated genes, and the full-length cDNA was subsequently isolated via screening of a cDNA library constructed from rice materials at tillering stage. Expression pattern analyses indicated that OsMDP1 is transcribed mainly in vegetative tissues, including the mature leaf, coleoptile, root-elongation zone, culm internode, and especially the joint region between the leaf blade and sheath. Further studies revealed that transcription of OsMDP1 is stimulated by darkness and suppressed by brassinolide treatment. OsMDP1 deficiency resulted in shortened primary roots, elongated coleoptiles and enhanced lamina joint inclinations. Moreover, transgenic plants showed hypersensitivities to exogenous brassinolide in terms of lamina joint inclination and coleoptile elongation. OsMDP1 deficiency resulted in enhanced expression of OsXTR1, which encodes xyloglucan endotransglycosylase, the cell-wall loosening enzyme necessary for cell elongation, and modulated expressions of multiple genes involved in cell signalling and gene transcription, indicating the key negative regulatory role of OsMDP1 in BR signalling.

XPS characterization of the surface immobilization of antibacterial furanones

Brominated furanones have attracted recent interest as antibacterial compounds. To utilize them as protective coatings in biomedical device applications, they must be covalently immobilized onto solid surfaces; however, interfacial coupling protocols developed for other biomolecules are not applicable to furanones. An azide reaction scheme has enabled covalent immobilization onto fluorinated ethylene propylene copolymer but its chemistry is less predictable, requiring detailed characterization by XPS and tapping mode AFM after each step of the immobilization sequence. XPS curve fitting resolved components in the C 1s, N 1s and Br 3d regions. Angle dependent XPS was used to assess the depth distributions and layer thicknesses. The results indicated successful covalent immobilization of furanones; however, side reactions occurred. In addition to the expected C Br, a contribution from bromine ion (Br −) was detected, indicating that photo-degradation of furanones took place during UV illumination, and this reaction was found to increase with illumination time. The Br − was removed by washing with water, whereas the C Br signal from immobilized furanone remained. Spectroscopic characterization will assist in elucidating the structure of furanone coatings, understanding their mode of action when covalently immobilized on surfaces, and rationally designing and optimizing an effective antibacterial coating for biomedical applications.

Kinetics of the Reactions of the CHBr2and CHBr2O2Radicals with O2and NO

When bromoform (CHBr3) is photolyzed at 266 or 303 nm in the presence of O2 and NO, the formation of secondary Br atoms is observed. By following the rate of growth of this secondary Br atom signal as a function of conditions, rate constants have been determined for the reactions CHBr2 + O2, CHBr2 + NO (both pressure-dependent), and CHBr2O2 + NO (k(2a) = (1.74 +/- 0.16) x 10(-11) cm3 molecule(-1) s(-1) at 23 degrees C). By measuring the amplitude of the secondary Br signal compared to the primary Br formed in the initial photolysis, it is established that the CHBr2O radical spontaneously decomposes to form CHBrO + Br at least 90%, and probably 100%, of the time, in agreement with previous work and with recent ab initio calculations. A survey of four other polybrominated methanes, CH2Br2, CHClBr2, CF2Br2, and CBr4, shows that they all generate secondary Br atoms when photolyzed at 266 nm in the presence of O2 and NO, suggesting that their reaction sequences are similar to that of bromoform.

Arabidopsis RAV1 is down-regulated by brassinosteroid and may act as a negative regulator during plant development.

RAV1 is a novel DNA-binding protein with two distinct DNA-binding domains unique in higher plants, but its role in plant growth and development remains unknown. Using cDNA array, we found that transcription of RAV1 is down-regulated by epibrassinolide (epiBL) in Arabidopsis suspension cells. RNA gel blot analysis revealed that epiBL-regulated RAV1 transcription involves neither protein phosphorylation/dephosphorylation nor newly synthesized protein, and does not require the functional BRI1, suggesting that this regulation might be through a new BR signaling pathway. Overexpressing RAV1 in Arabidopsis results in a retardation of lateral root and rosette leaf development, and the underexpression causes an earlier flowering phenotype, implying that RAV1 may function as a negative regulatory component of growth and development.

Brassinosteroid signal transduction: An emerging picture

Steroid hormones play essential roles in animal growth and development. Steroid signaling in animal system is focused on the direct gene regulation response mediated by its nuclear receptors. Recently, steroid hormones are also found in plants. Identification of BRI1 — a critical component of the plasmamembrane steroid receptor complex, and the related signal transduction pathway mediated by the membrane receptor have revealed an elementary picture of BR signaling from the cell surface perception to the activation of BRresponsive nuclear genes.

Three redundant brassinosteroid early response genes encode putative bHLH transcription factors required for normal growth.

Brassinosteroids (BRs) are a class of polyhydroxylated steroids that are important regulators of plant growth and development. We have identified three closely related basic helix-loop-helix (bHLH) transcription factors, BEE1, BEE2, and BEE3, as products of early response genes required for full BR response. Comparison of the phenotypes of plants that overexpress BEE1 with bee1 bee2 bee3 triple-knockout mutant plants suggests that BEE1, BEE2, and BEE3 are functionally redundant positive regulators of BR signaling. Expression of BEE1, BEE2, and BEE3 is also regulated by other hormones, notably abscisic acid (ABA), a known antagonist of BR signaling. Reduced ABA response in plants overexpressing BEE1 suggests that BEE proteins may function as signaling intermediates in multiple pathways.

BIN2, a new brassinosteroid-insensitive locus in Arabidopsis.

Brassinosteroids (BRs) play important roles throughout plant development. Although many genes have been identified that are involved in BR biosynthesis, genetic approaches in Arabidopsis have led to the identification of only one gene, BRI1, that encodes a membrane receptor for BRs. To expand our knowledge of the molecular mechanism(s) of plant steroid signaling, we analyzed many dwarf and semidwarf mutants collected from our previous genetic screens and identified a semidwarf mutant that showed little response to exogenous BR treatments. Genetic analysis of the bin2 (BR-INSENSITIVE 2) mutant indicated that the BR-insensitive dwarf phenotype was due to a semidominant mutation in the BIN2 gene that mapped to the middle of chromosome IV between the markers CH42 and AG. A direct screening for similar semidwarf mutants resulted in the identification of a second allele of the BIN2 gene. Despite some novel phenotypes observed with the bin2/+ mutants, the homozygous bin2 mutants were almost identical to the well-characterized bri1 mutants that are defective in BR perception. In addition to the BR-insensitive dwarf phenotype, bin2 mutants exhibited BR insensitivity when assayed for root growth inhibition and feedback inhibition of CPD gene expression. Furthermore, bin2 mutants displayed an abscisic acid-hypersensitive phenotype that is shared by the bri1 and BR-deficient mutants. A gene dosage experiment using triploid plants suggested that the bin2 phenotypes were likely caused by either neomorphic or hypermorphic gain-of-function mutations in the BIN2 gene. Thus, the two bin2 mutations define a novel genetic locus whose gene product might play a role in BR signaling.

Brassinosteroid-Insensitive-1 Is a Ubiquitously Expressed Leucine-Rich Repeat Receptor Serine/Threonine Kinase

Brassinosteroid (BR) mutants of Arabidopsis have pleiotropic phenotypes and provide evidence that BRs function throughout the life of the plant from seedling development to senescence. Screens for BR signaling mutants identified one locus, BRI1, which encodes a protein with homology to leucine-rich repeat receptor serine (Ser)/threonine (Thr) kinases. Twenty-seven alleles of this putative BR receptor have been isolated to date, and we present here the identification of the molecular lesions of 14 recessive alleles that represent five new mutations. BR-insensitive-1 (BRI1) is expressed at high levels in the meristem, root, shoot, and hypocotyl of seedlings and at lower levels later in development. Confocal microscopy analysis of full-length BRI1 fused to green fluorescent protein indicates that BRI1 is localized in the plasma membrane, and an in vitro kinase assay indicates that BRI1 is a functional Ser/Thr kinase. Among the bri1 mutants identified are mutants in the kinase domain, and we demonstrate that one of these mutations severely impairs BRI1 kinase activity. Therefore, we conclude that BRI1 is a ubiquitously expressed leucine-rich repeat receptor that plays a role in BR signaling through Ser/Thr phosphorylation.

79Br solid state NMR studies of surfaces, point defects and dislocations in silver halides

Pure and mixed silver halide crystals form the basis of chemical photography. We have used 79Br solid state NMR at 4.7 T to characterize the structure and dynamics of defects and surface effects that may be involved in determining the photographic properties of these materials. The influence of free and bound vacancies on bromine spin-lattice relaxation in doped AgBr single crystals and microcrystals was studied and interpreted within the context of the space charge model. Microcrystals subjected to compressive strain have a higher dislocation density as reflected in linewidths and “missing” Br signal intensity. For crystals under one micron in size, the integrated Br signal intensity is a function of crystallise size. We have established that the electric field gradient deviates from cubic symmetry within 200 Å of the crystal surface.

Nuclear quadrupole resonance in hafnium and zirconium tetrabromide

79 Br and 81Br Quadrupole resonance studies of solid HfBr4 and ZrBr4 support the contention that these compounds are isostructural with ZrCl4, containing both terminal and bridging Br atoms; the bridging Br signals have an anomalous temperature coefficient.