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Arabidopsis thaliana responses to mechanical stimulation do not require ETR1 or EIN2
Plants exposed to repetitive touch or wind are generally shorter and stockier than sheltered plants. These mechanostimulus-induced developmental changes are termed thigmomorphogenesis and may confer resistance to subsequent stresses. An early response of Arabidopsis thaliana to touch or wind is the up-regulation of TCH (touch) gene expression. The signal transduction pathway that leads to mechanostimulus responses is not well defined. A role for ethylene has been proposed based on the observation that mechanostimulation of plants leads to ethylene evolution and exogenous ethylene leads to thigmomorphogenetic-like changes. To determine whether ethylene has a role in plant responses to mechanostimulation, we assessed the ability of two ethylene-insensitive mutants, etr1-3 and ein2-1, to undergo thigmomorphogenesis and TCH gene up-regulation of expression. The ethylene-insensitive mutants responded to wind similarly to the wild type, with a delay in flowering, decrease in inflorescence elongation rate, shorter mature primary inflorescences, more rosette paraclades, and appropriate TCH gene expression changes. Also, wild-type and mutant Arabidopsis responded to vibrational stimulation, with an increase in hypocotyl elongation and up-regulation of TCH gene expression. We conclude that the ETR1 and EIN2 protein functions are not required for the developmental and molecular responses to mechanical stimulation.
Crystallization of bFGF-DNA Aptamer Complexes Using a Sparse Matrix Designed for Protein-Nucleic Acid Complexes
The Sparse Matrix approach for obtaining lead crystallization conditions has proven to be very fruitful for the crystallization of proteins and nucleic acids. Here we report a Sparse Matrix developed specifically for the crystallization of protein-DNA complexes. This method is rapid and economical, typically requiring 2.5 mg of complex to test 48 conditions. The method was originally developed to crystallize basic fibroblast growth factor (bFGF) complexed with DNA sequences identified through in vitro selection, or SELEX, methods. Two DNA aptamers that bind with approximately nanomolar affinity and inhibit the angiogenic properties of bFGF were selected for co-crystallization. The Sparse Matrix produced lead crystallization conditions for both bFGF-DNA complexes.
Assessment of Resistance of Bacillus Horneckiae Endospores to UV Radiation and Function of Their Extraneous Layer in Resistance
Spore-forming microbes are highly resistant to various physical and chemical conditions, which include ionizing and UV radiation, desiccation and oxidative stress, and the harsh environment of outer space or planetary surfaces. The spore's resistance might be due to their metabolically dormant state, and/or by the presence of a series of protective structures that encase the interior-most compartment, the core, which houses the spore chromosome. These spores have multiple layers surrounding the cell that are not found in vegetative cells, and some species have an outer layer of proteins and glycoproteins termed the "exosporium" or a fibrous "extraneous layer" (EL). Bacillus horneckiae is an EL-producing novel sporeformer isolated from a Phoenix spacecraft assembly clean room, and it has previously demonstrated resistance to UV radiation up to 1000 J/m(sup 2). The EL appears to bind B. horneckiae spores into large aggregations, or biofilms, and may confer some UV resistance to the spores. Multiple culturing and purification schemes were tried to achieve high purity spores because vegetative cells would skew UV resistance results. An ethanol-based purification scheme produced high purity spores. Selective removal of the EL from spores was attempted with two schemes: a chemical extraction method and physical extraction (sonication). Results from survival rates in the presence and absence of the external layer will provide a new understanding of the role of biofilms and passive resistance that may favor survival of biological systems in aggressive extra-terrestrial environments. The chemical extraction method decreased viable counts of spores and lead to an inconclusive change UV resistance relative to non-extracted spores. The physical extraction method lead to non-aggregated spores and did not alter viability; however, it produced UV resistance profiles similar to non-extracted spores. In addition to the EL-removal study, samples of B. horneckiae spores dried on aluminum coupons and exposed to increasing UV (200-400 nm range) levels (0 to 8.0 x 105 kJ/m(sup 2)) were tested for viability, which indicated that the maximum UV exposure level that still resulted in viable spores was 5.0 x 10? kJ/m(sup 2).
Modulation of the cytosolic androgen receptor in striated muscle by sex steroids
The effects of orchiectomy (GDX) and of subsequent administration of testosterone propionate (TP) or 17(beta)-estradiol (E2) on the maximum binding (Bmax) and apparent Kd of the cytosolic androgen receptor in levator ani (LA) and skeletal muscles of adult male Sprague-Dawley rats are investigated experimentally. The results are presented in graphs and discussed. In LA, BMAX is found to rise from a control level of 2.5 fmol/mg protein to 280, 600, 478, and 133 percent of control at 12 h, 14 d, 30 d, and 44 d after GDX, respectively, while Kd increased only insignificantly (from 680 to 960 fM); Bmax is held at control levels for 6 h by cycloheximide given at GDX, is unaffected by TP given at 30 d, and is further increased (by 480 percent at 44 d) by administration of E2 at 30 d. Bmax in skeletal muscles is found to increase to 139, 212, 220, and 158 percent of control at 12 h, 14 d, 30 d, and 44 d, respectively; Bmax is returned to control at 44 d by TP at 30 d but is not affected by E2. The effect of E2 in LA is attributed to either induction of the cytosolic receptor or a decreased rate of receptor degradation.
Protein specific fluorescent microspheres for labelling a protein
Highly fluorescent, stable and biocompatible microspheres are obtained by copolymerizing an acrylic monomer containing a covalent bonding group such as hydroxyl, amine or carboxyl, for example, hydroxyethylmethacrylate, with an addition polymerizable fluorescent comonomer such as dansyl allyl amine. A lectin or antibody is bound to the covalent site to provide cell specificity. When the microspheres are added to a cell suspension the marked microspheres will specifically label a cell membrane by binding to a specific receptor site thereon. The labeled membrane can then be detected by fluorescence of the fluorescent monomer.
TNF-alpha increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k
In some inflammatory diseases, TNF-alpha is thought to stimulate muscle catabolism via an NF-kappaB-dependent process that increases ubiquitin conjugation to muscle proteins. The transcriptional mechanism of this response has not been determined. Here we studied the potential role of UbcH2, a ubiquitin carrier protein and homologue of murine E220k. We find that UbcH2 is constitutively expressed by human skeletal and cardiac muscles, murine limb muscle, and cultured myotubes. TNF-alpha stimulates UbcH2 expression in mouse limb muscles in vivo and in cultured myotubes. The UbcH2 promoter region contains a functional NF-kappaB binding site; NF-kappaB binding to this sequence is increased by TNF-alpha stimulation. A dominant negative inhibitor of NF-kappaB activation blocks both UbcH2 up-regulation and the increase in ubiquitin-conjugating activity stimulated by TNF-alpha. In extracts from TNF-alpha-treated myotubes, ubiquitin-conjugating activity is limited by UbcH2 availability; activity is inhibited by an antiserum to UbcH2 or a dominant negative mutant of UbcH2 and is enhanced by wild-type UbcH2. Thus, UbcH2 up-regulation is a novel response to TNF-alpha/NF-kappaB signaling in skeletal muscle that appears to be essential for the increased ubiquitin conjugation induced by this cytokine.
The rhodopsins: structure and function. Introduction
Nature makes use of the propensity of retinal for light-dependent double-bond isomerization in a number of systems and in a variety of ways. The common theme for light receptors based on this kind of chemistry is that (1) the retinal is bound in most cases to a small membrane protein via a protonated lysine-retinal Schiff base, (2) the absorption maximum in the visible is tuned to a suitable wavelength largely by electrostatic interaction with polar protein residues, and (3) the light-induced bond rotations and strains in the retinal set off reaction chains during which at least part of the excess free energy acquired is transferred to the protein and causes pK shifts of acidic residues and/or backbone conformational changes. The physiological consequence of the process initiated by absorption of light is either the activation of an information transfer chain (sensory and visual rhodopsins) or energy transduction which drives the electrogenic movement of ions across the membrane (ion-motive rhodopsins). Rhodopsins with these functions occur in bacteria and in higher organisms; from an evolutionary standpoint they are not related to one another. Nevertheless, all of these proteins are remarkably similar and form a distinct family.
When a day makes a difference. Interpreting data from endoplasmic reticulum-targeted green fluorescent protein fusions in cells grown in suspension culture
No abstract available
Documentation of angiotensin II receptors in glomerular epithelial cells
Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial cells participate in angiotensin II-mediated control of the glomerular filtration barrier.
Structural Studies of Human Pyruvate Dehydrogenase
Human pyruvate dehydrogenase (E1) catalyzes the irreversible decarboxylation of pyruvate in the presence of Mg(2+) and thiamin pyrophosphate (TPP) followed by the rate-limiting reductive acetylation of the lipoyl moiety linked to dihydrolipoamide acetyltransferase. The three-dimensional structure of human E1 is elucidated using the methods of macromolecular X-ray crystallography. The structure is an alpha, alpha', beta and beta' tetramer with the protein units being in the tetrahedral arrangement. Each 361-residue alpha-subunit and 329-residue beta-subunit is composed of a beta-sheet core surrounded by alpha-helical domains. Each subunit is in extensive contact with all the three subunits involving TPP and magnesium cofactors, and potassium ions. The two binding sites for TPP are at the alpha-beta' and alpha'-beta interfaces, each involving a magnesium ion and Phe6l, His63, Tyr89, and Met200 from the alpha-subunit (or alpha'-subunit), and Met81 Phe85, His128 from the beta-subunit (or beta'-subunit). K+ ions are nestled between two beta-sheets and the end of an alpha-helix in each beta-subunit, where they are coordinated by four carbonyl oxygen groups from Ile12, Ala160, Asp163, and Asnl65, and a water molecule. The catalytic C2 carbon of thiazolium ring in this structure forms a 3.2 A contact with a water molecule involved in a series of H-bonds with other water molecules, and indirectly with amino acids including those involved in the catalysis and regulation of the enzyme.
Beta-Adrenergic Receptor Expression in Muscle Cells
beta-adrenergic receptor (bAR) agonists presumably exert their physiological action on skeletal muscle cells through the bAR. Since the signal generated by the bAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of bAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 uM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the bAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 uM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in (beta)AR population, with a maximum increase of approximately 50% at 10 uM. This increase in (beta)AR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of (beta)AR population. Clenbuterol and isoproterenol gave similar effects on bAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 UM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.
Beta-Adrenergic Receptor Population is Up-Regulated in Chicken Skeletal Muscle Cells Treated with Forskolin
Skeletal muscle hypertrophy is promoted by in vivo administration of beta-adrenergic receptor (betaAR) agonists. These compounds presumably exert their physiological action through the betaAR, and alterations in the population of betaAR could potentially change the ability of the cell to respond to the betaAR agonists. Since the intracellular chemical signal generated by the betaAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of functional betaAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 microM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the betaAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 microM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in betaAR population, with a maximum increase of approximately 50% at 10 microM. This increase in PAR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of betaAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 microM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.
The origin of nuclear receptor proteins: a single precursor distinct from other transcription factors
No abstract available
Evidence for vestibular regulation of autonomic functions in a mouse genetic model
Physiological responses to changes in the gravitational field and body position, as well as symptoms of patients with anxiety-related disorders, have indicated an interrelationship between vestibular function and stress responses. However, the relative significance of cochlear and vestibular information in autonomic regulation remains unresolved because of the difficulties in distinguishing the relative contributions of other proprioceptive and interoceptive inputs, including vagal and somatic information. To investigate the role of cochlear and vestibular function in central and physiological responses, we have examined the effects of increased gravity in wild-type mice and mice lacking the POU homeodomain transcription factor Brn-3.1 (Brn-3bPou4f3). The only known phenotype of the Brn-3.1(-/-) mouse is related to hearing and balance functions, owing to the failure of cochlear and vestibular hair cells to differentiate properly. Here, we show that normal physiological responses to increased gravity (2G exposure), such as a dramatic drop in body temperature and concomitant circadian adjustment, were completely absent in Brn-3.1(-/-) mice. In line with the lack of autonomic responses, the massive increase in neuronal activity after 2G exposure normally detected in wild-type mice was virtually abolished in Brn-3.1(-/-) mice. Our results suggest that cochlear and vestibular hair cells are the primary regulators of autonomic responses to altered gravity and provide genetic evidence that these cells are sufficient to alter neural activity in regions involved in autonomic and neuroendocrine control.
Extracellular calcium (Ca2+o)-sensing receptor in a mouse monocyte-macrophage cell line (J774): potential mediator of the actions of Ca2+o on the function of J774 cells
The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone remodeling and may play a role in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for bone marrow mononuclear cells in the vicinity, leading us to investigate whether such mononuclear cells express the CaR. In this study, we used the mouse J774 cell line, which exhibits a pure monocyte-macrophage phenotype. Both immunocytochemistry and Western blot analysis, using polyclonal antisera specific for the CaR, detected CaR protein in J774 cells. The use of reverse transcriptase-polymerase chain reaction with CaR-specific primers, including a set of intron-spanning primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in J774 cells. Exposure of J774 cells to high Ca2+o (2.8 mM or more) or the polycationic CaR agonist, neomycin (100 microM), stimulated both chemotaxis and DNA synthesis in J774 cells. Therefore, taken together, our data strongly suggest that the monocyte-macrophage cell line, J774, possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney.
Development of Thermostable Carbonic Anhydrases Using Structure-Guided Recombination for Use in CO2 Removal Systems on Spacecraft
Carbon capture and storage has been a research area of great interest in recent years as a method for mitigation of CO2 emissions, due to the effects of climate change. The development of technologies for the efficient capture of CO2 are also of great interest for human spaceflight applications. One of the most promising technologies in this area is CO2 scrubbing using liquid amines, unfortunately, liquid amines with low heats of desorption tend to have slow CO2 binding kinetics. One potential solution to this problem is to use the enzyme carbonic anhydrase (CA) to enhance the kinetics of CO2 binding to liquid amines, allowing the overall process to be more energy efficient. Interest in using carbonic anhydrase as a biocatalyst has led to a number of efforts to improve the thermostability and solvent tolerance of several distinct carbonic anhydrase enzymes. In the work described here, we screened through a diverse set of natural carbonic anhydrases to identify candidates for protein engineering aimed at increased stability and activity in various liquid amines. We then used SCHEMA structure-guided recombination to develop a set of chimeric carbonic anhydrases with high thermostability and activity. These chimeras were used as the starting points for further protein engineering work targeting activity in liquid amine systems. Our ultimate goal is to test the engineered enzymes in a liquid amine system for cabin air revitalization on ISS or other spacecraft.
Calcium-Dependent Protein Kinase Genes in Corn Roots
Two cDNAs encoding Ca-2(+) - Dependent Protein Kinases (CDPKs), Corn Root Protein Kinase 1 and 2 (CRPK 1, CRPK 2) were isolated from the root tip library of corn (Zea mays L., cv. Merit) and their nucleotide sequences were determined. Deduced amino acid sequences of both the clones have features characteristic of plant CDPKS, including all 11 conserved serine/threonine kinase subdomains, a junction domain and a calmodulin-like domain with four Ca-2(+), -binding sites. Northern analysis revealed that CRPKI mRNA is preferentially expressed in roots, especially in the root tip; whereas, the expression of CRPK2 mRNA was very low in all the tissues tested. In situ hybridization experiments revealed that CRPKI mRNA is highly expressed in the root apex, as compared to other parts of the root. Partially purified CDPK from the root tip phosphorylates syntide-2, a common peptide substrate for plant CDPKs, and the phosphorylation was stimulated 7-fold by the addition of Ca-2(+). Our results show that two CDPK isoforms are expressed in corn roots and they may be involved in the Ca-2(+)-dependent signal transduction process.