Browsing by Subject "Kinetics"

Now showing 1 - 20 of 59
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    A beta-adrenergic receptor kinase-like enzyme is involved in olfactory signal termination.
    (Proc Natl Acad Sci U S A, 1993-02-15) Schleicher, S; Boekhoff, I; Arriza, J; Lefkowitz, RJ; Breer, H
    We have previously shown that second-messenger-dependent kinases (cAMP-dependent kinase, protein kinase C) in the olfactory system are essential in terminating second-messenger signaling in response to odorants. We now document that subtype 2 of the beta-adrenergic receptor kinase (beta ARK) is also involved in this process. By using subtype-specific antibodies to beta ARK-1 and beta ARK-2, we show that beta ARK-2 is preferentially expressed in the olfactory epithelium in contrast to findings in most other tissues. Heparin, an inhibitor of beta ARK, as well as anti-beta ARK-2 antibodies, (i) completely prevents the rapid decline of second-messenger signals (desensitization) that follows odorant stimulation and (ii) strongly inhibits odorant-induced phosphorylation of olfactory ciliary proteins. In contrast, beta ARK-1 antibodies are without effect. Inhibitors of protein kinase A and protein kinase C also block odorant-induced desensitization and phosphorylation. These data suggest that a sequential interplay of second-messenger-dependent and receptor-specific kinases is functionally involved in olfactory desensitization.
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    A Comprehensive Study of Guanosine-5'-triphosphate Hydrolysis by the Bacterial Cell Division Protein FtsZ
    (2018) Salsburg, Andrew

    The bacterial protein FtsZ plays a vital role in cytokinesis in prokaryotes as it polymerizes to form an FtsZ ring (Z ring) at the division septum midcell. FtsZ exhibits a GTP hydrolysis activity and attempts have been made to model the kinetics of this process. There is a major discrepancy, however, over the concentration of GTP needed for activity. The dissociation constant (KD) between wild-type FtsZ protein and GTP was measured to be 30 nM using isothermal titration calorimetry. In contrast, several research groups have reported that GTP hydrolysis required GTP concentrations in the millimolar range. They used Michaelis-Menten kinetics to model the GTP concentration dependence and obtained an apparent binding constant (Km) in the range of 300-1,000 µM GTP. Km and KD are not identical measures of binding given that they differ by the kinetic constant governing catalysis, kcat, but we suggest that a five order of magnitude difference between the values is unprecedented and this was a problem that needed investigating.

    My overall goal in this work has been to perform a comprehensive in vitro study of the FtsZ GTP hydrolysis activity using an enzyme-coupled regenerating system. With this system rates of GTP hydrolysis by FtsZ are obtained spectrophotometrically. I have confirmed for wild-type FtsZ that GTP hydrolysis rates show little to no dependence on GTP concentrations in the range of 50-3,000 µM, contradicting the high values of Km reported in some previous studies. Since we have failed to reproduce the high Km with three different preparations of FtsZ protein, we cannot propose a definitive mechanism for the previous results.

    I also measured the GTP hydrolysis of several mutants of FtsZ: E238A, L169R, and FtsZ84 (G105S). I investigated each of these mutants to see if they had a high apparent Km. FtsZ84 had a low overall hydrolysis rate, but did show a large increase in hydrolysis rates when GTP was increased from 50-3,000 µM. We hypothesize that a lower affinity for GTP is not a Michaelis-Menten Km, but likely a reflection of a weak binding of GTP by FtsZ84, giving KD in the millimolar range.

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    A kinesin motor in a force-producing conformation.
    (BMC Struct Biol, 2010-07-05) Heuston, Elisabeth; Bronner, C Eric; Kull, F Jon; Endow, Sharyn A
    BACKGROUND: Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states. RESULTS: Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central beta-sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1. CONCLUSIONS: Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor.
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    A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes.
    (J Gen Physiol, 1988-02) Marchetti, C; Premont, RT; Brown, AM
    Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.
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    Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1 and induce beta-chemokines.
    (J Exp Med, 2010-04-12) Moody, MA; Liao, HX; Alam, SM; Scearce, RM; Plonk, MK; Kozink, DM; Drinker, MS; Zhang, R; Xia, SM; Sutherland, LL; Tomaras, GD; Giles, IP; Kappes, JC; Ochsenbauer Jambor, C; Edmonds, TG; Soares, M; Barbero, G; Forthal, DN; Landucci, G; Chang, C; King, SW; Kavlie, A; Denny, TN; Hwang, KK; Chen, PP; Thorpe, PE; Montefiori, DC; Haynes, BF
    Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to approximately 10 microg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1alpha and MIP-1beta. The release of these beta-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes.
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    ItemRestricted
    beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein.
    (Proc Natl Acad Sci U S A, 1998-11-24) Premont, RT; Claing, A; Vitale, N; Freeman, JL; Pitcher, JA; Patton, WA; Moss, J; Vaughan, M; Lefkowitz, RJ
    G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced beta2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating beta2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.
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    Binding of MetJ repressor to specific and nonspecific DNA and effect of S-adenosylmethionine on these interactions.
    (Biochemistry, 2010-04-20) Augustus, Anne M; Sage, Harvey; Spicer, Leonard D
    We have used analytical ultracentrifugation to characterize the binding of the methionine repressor protein, MetJ, to synthetic oligonucleotides containing zero to five specific recognition sites, called metboxes. For all lengths of DNA studied, MetJ binds more tightly to repeats of the consensus sequence than to naturally occurring metboxes, which exhibit a variable number of deviations from the consensus. Strong cooperative binding occurs only in the presence of two or more tandem metboxes, which facilitate protein-protein contacts between adjacent MetJ dimers, but weak affinity is detected even with DNA containing zero or one metbox. The affinity of MetJ for all of the DNA sequences studied is enhanced by the addition of SAM, the known cofactor for MetJ in the cell. This effect extends to oligos containing zero or one metbox, both of which bind two MetJ dimers. In the presence of a large excess concentration of metbox DNA, the effect of cooperativity is to favor populations of DNA oligos bound by two or more MetJ dimers rather than a stochastic redistribution of the repressor onto all available metboxes. These results illustrate the dynamic range of binding affinity and repressor assembly that MetJ can exhibit with DNA and the effect of the corepressor SAM on binding to both specific and nonspecific DNA.
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    cAMP stimulates transcription of the beta 2-adrenergic receptor gene in response to short-term agonist exposure.
    (Proc Natl Acad Sci U S A, 1989-07) Collins, S; Bouvier, M; Bolanowski, MA; Caron, MG; Lefkowitz, RJ
    In addition to conveying cellular responses to an effector molecule, receptors are often themselves regulated by their effectors. We have demonstrated that epinephrine modulates both the rate of transcription of the beta 2-adrenergic receptor (beta 2AR) gene and the steady-state level of beta 2AR mRNA in DDT1MF-2 cells. Short-term (30 min) exposure to epinephrine (100 nM) stimulates the rate of beta 2AR gene transcription, resulting in a 3- to 4-fold increase in steady-state beta 2AR mRNA levels. These effects are mimicked by 1 mM N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) or foskolin but not by phorbol esters. The half-life of the beta 2AR mRNA after addition of actinomycin D (46.7 +/- 10.2 min; mean +/- SEM; n = 5) remained unchanged after 30 min of epinephrine treatment (46.8 +/- 10.6 min; mean +/- SEM; n = 4), indicating that a change in transcription rate is the predominant factor responsible for the increase of beta 2AR mRNA. Whereas brief exposure to epinephrine or Bt2cAMP does not significantly affect the total number of cellular beta 2ARs (assessed by ligand binding), continued exposure results in a gradual decline in beta 2AR number to approximately 20% (epinephrine) or approximately 45% (Bt2cAMP) of the levels in control cells by 24 hr. Similar decreases in agonist-stimulated adenylyl cyclase activity are observed. This loss of receptors with prolonged agonist exposure is accompanied by a 50% reduction in beta 2AR mRNA. Transfection of the beta 2AR promoter region cloned onto a reporter gene (bacterial chloramphenicol acetyltransferase) allowed demonstration of a 2- to 4-fold induction of transcription by agents that elevate cAMP levels, such as forskolin or phosphodiesterase inhibitors. These results establish the presence of elements within the proximal promoter region of the beta 2AR gene responsible for the transcriptional enhancing activity of cAMP and demonstrate that beta 2AR gene expression is regulated by a type of feedback mechanism involving the second messenger cAMP.
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    Changes in axonally transported proteins during axon regeneration in toad retinal ganglion cells.
    (The Journal of cell biology, 1981-04) Skene, JH; Willard, M
    In an effort to understand the regulation of the transition of a mature neuron to the growth, or regenerating, state we have analyzed the composition of the axonally transported proteins in the retinal ganglion cells of the toad Bufo marinus after inducing axon regeneration by crushing the optic nerve. At increasing intervals after axotomy, we labeled the retinal ganglion cells with [35S]methionine and subsequently analyzed the labeled transported polypeptides in the crushed optic nerve by means of one- and two-dimensional electrophoretic techniques. The most significant conclusion from these experiments is that, while the transition from the mature to the regenerating state does not require a gross qualitative alteration in the composition of axonally transported proteins, the relative labeling of a small subset of rapidly transported proteins is altered dramatically (changes of more than 20-fold) and reproducibly (more than 30 animals) by axotomy. One of these growth-associated proteins (GAPs) was soluble in an aqueous buffer, while three were associated with a crude membrane fraction. The labeling of all three of the membrane-associated GAPs increased during the first 8 d after axotomy, and they continued to be labeled for at least 4 wk. The modulation of these proteins after axotomy is consistent with the possibility that they are involve in growth-specific functions and that the altered expression of a small number of genes is a crucial regulatory event in the transition of a mature neuron to a growth state. In addition to these selective changes in rapidly transported proteins, we observed the following more general metabolic correlates of the regeneration process: The total radioactive label associated with the most rapidly transported proteins (groups I and II) increased three to fourfold during the first 8 d after the nerve was crushed, while the total label associated with more slowly moving proteins (group IV) increased about 10-fold during this same period. Among these more slowly transported polypeptides, five were observed whose labeling increased much more than the average. Three of these five polypeptides resemble actin and alpha- and beta-tubulin in their electrophoretic properties.
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    Characterization of the murine BEK fibroblast growth factor (FGF) receptor: activation by three members of the FGF family and requirement for heparin.
    (Proc Natl Acad Sci U S A, 1992-04-15) Mansukhani, A; Dell'Era, P; Moscatelli, D; Kornbluth, S; Hanafusa, H; Basilico, C
    The bek gene encodes a member of the high-affinity fibroblast growth factor receptor family. The BEK/FGFR-2 receptor is a membrane-spanning tyrosine kinase with the typical features of FGF receptors. We have cloned a murine bek cDNA and expressed it in receptor-negative Chinese hamster ovary cells and in 32D myeloid cells. The BEK receptor expressed in Chinese hamster ovary cells binds acidic FGF, basic FGF, and Kaposi FGF equally well but does not bind keratinocyte growth factor or FGF-5 appreciably. Upon treatment with basic FGF or Kaposi FGF, the BEK receptor is phosphorylated and a mitogenic response is achieved. Heparan sulfate proteoglycans have been shown to play an obligate role in basic FGF binding to the high-affinity FLG receptor. Unlike the BEK-expressing Chinese hamster ovary cells, 32D cells expressing the BEK receptor require the addition of exogenous heparin in order to grow in the presence of basic FGF or Kaposi FGF. We show that the addition of heparin greatly enhances the binding of radio-labeled basic FGF to the receptor. Thus the BEK receptor, like FLG, also requires an interaction with heparan sulfate proteoglycans to facilitate binding to its ligands.
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    Cloning and expression of a human kidney cDNA for an alpha 2-adrenergic receptor subtype.
    (Proc Natl Acad Sci U S A, 1988-09) Regan, JW; Kobilka, TS; Yang-Feng, TL; Caron, MG; Lefkowitz, RJ; Kobilka, BK
    An alpha 2-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet alpha 2-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet alpha 2-adrenergic receptor and is consistent with the structure of other members of the family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the alpha 2-adrenergic ligand [3H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the alpha 2B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet alpha 2-adrenergic receptor (alpha 2A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective alpha-adrenergic ligands.
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    Conformational kinetics reveals affinities of protein conformational states.
    (Proc Natl Acad Sci U S A, 2015-07-28) Daniels, Kyle G; Suo, Yang; Oas, Terrence G
    Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein's affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states.
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    Critical appraisal of four IL-6 immunoassays.
    (PLoS One, 2012) Thompson, Dana K; Huffman, Kim M; Kraus, William E; Kraus, Virginia Byers
    BACKGROUND: Interleukin-6 (IL-6) contributes to numerous inflammatory, metabolic, and physiologic pathways of disease. We evaluated four IL-6 immunoassays in order to identify a reliable assay for studies of metabolic and physical function. Serial plasma samples from intravenous glucose tolerance tests (IVGTTs), with expected rises in IL-6 concentrations, were used to test the face validity of the various assays. METHODS AND FINDINGS: IVGTTs, administered to 14 subjects, were performed with a single infusion of glucose (0.3 g/kg body mass) at time zero, a single infusion of insulin (0.025 U/kg body mass) at 20 minutes, and frequent blood collection from time zero to 180 minutes for subsequent Il-6 measurement. The performance metrics of four IL-6 detection methods were compared: Meso Scale Discovery immunoassay (MSD), an Invitrogen Luminex bead-based multiplex panel (LX), an Invitrogen Ultrasensitive Luminex bead-based singleplex assay (ULX), and R&D High Sensitivity ELISA (R&D). IL-6 concentrations measured with MSD, R&D and ULX correlated with each other (Pearson Correlation Coefficients r = 0.47-0.94, p<0.0001) but only ULX correlated (r = 0.31, p = 0.0027) with Invitrogen Luminex. MSD, R&D, and ULX, but not LX, detected increases in IL-6 in response to glucose. All plasma samples were measurable by MSD, while 35%, 1%, and 4.3% of samples were out of range when measured by LX, ULX, and R&D, respectively. Based on representative data from the MSD assay, baseline plasma IL-6 (0.90 ± 0.48 pg/mL) increased significantly as expected by 90 minutes (1.29 ± 0.59 pg/mL, p = 0.049), and continued rising through 3 hours (4.25 ± 3.67 pg/mL, p = 0.0048). CONCLUSION: This study established the face validity of IL-6 measurement by MSD, R&D, and ULX but not LX, and the superiority of MSD with respect to dynamic range. Plasma IL-6 concentrations increase in response to glucose and insulin, consistent with both an early glucose-dependent response (detectable at 1-2 hours) and a late insulin-dependent response (detectable after 2 hours).
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    Defective lymphocyte chemotaxis in beta-arrestin2- and GRK6-deficient mice.
    (Proc Natl Acad Sci U S A, 2002-05-28) Fong, Alan M; Premont, Richard T; Richardson, Ricardo M; Yu, Yen-Rei A; Lefkowitz, Robert J; Patel, Dhavalkumar D
    Lymphocyte chemotaxis is a complex process by which cells move within tissues and across barriers such as vascular endothelium and is usually stimulated by chemokines such as stromal cell-derived factor-1 (CXCL12) acting via G protein-coupled receptors. Because members of this receptor family are regulated ("desensitized") by G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and beta-arrestin binding, we examined signaling and chemotactic responses in splenocytes derived from knockout mice deficient in various beta-arrestins and GRKs, with the expectation that these responses might be enhanced. Knockouts of beta-arrestin2, GRK5, and GRK6 were examined because all three proteins are expressed at high levels in purified mouse CD3+ T and B220+ B splenocytes. CXCL12 stimulation of membrane GTPase activity was unaffected in splenocytes derived from GRK5-deficient mice but was increased in splenocytes from the beta-arrestin2- and GRK6-deficient animals. Surprisingly, however, both T and B cells from beta-arrestin2-deficient animals and T cells from GRK6-deficient animals were strikingly impaired in their ability to respond to CXCL12 both in transwell migration assays and in transendothelial migration assays. Chemotactic responses of lymphocytes from GRK5-deficient mice were unaffected. Thus, these results indicate that beta-arrestin2 and GRK6 actually play positive regulatory roles in mediating the chemotactic responses of T and B lymphocytes to CXCL12.
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    Design, selection, and development of cyclic peptide ligands for human erythropoietin.
    (Journal of chromatography. A, 2017-06) Kish, William S; Sachi, Hiroyuki; Naik, Amith D; Roach, Matthew K; Bobay, Benjamin G; Blackburn, Robert K; Menegatti, Stefano; Carbonell, Ruben G
    This work presents the selection and characterization of erythropoietin (EPO)-binding cyclic peptide ligands. The sequences were selected by screening a focused library of cyclic depsipeptides cyclo[(Nα-Ac)Dap(A)-X1-X6-AE], whose structure and amino acid compositions were tailored to mimic the EPO receptor. The sequences identified through library screening were synthesized on chromatographic resin and characterized via binding-and-elution studies against EPO to select a pool of candidate ligands. Sequences with higher hydrophobicity consistently showed stronger binding to EPO, with the exception of FSLLSH, which was noted for its lower hydrophobicity and high EPO binding. Mutagenesis studies performed on FSLLSH with natural and non-natural amino acid substitutions led to the identification of critical EPO-binding determinants, and the discovery of new peptide ligands. In particular, histidine-scanning mutagenesis performed on three lead sequences yielded the discovery of variants whose EPO-binding is more pH-sensitive, which facilitates EPO recovery. Selected ligands were studied to correlate the elution yield to the salinity of the binding buffer and the elution pH. Elution yields were consistently higher when EPO binding was performed at low ionic strength. The crystal structures of lead cyclic peptides were docked in silico against EPO to estimate the binding affinity in solution. Isotherm adsorption studies performed on FSLLSH indicated that the cyclic version of the ligand (KD=0.46μM) has a higher affinity for EPO than its corresponding linear variant (KD=1.44μM). Collectively, these studies set the stage for use of the cyclic peptide ligands as EPO purification and detection tools.
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    Electrosprayed core-shell microspheres for protein delivery.
    (Chem Commun (Camb), 2010-07-14) Wu, Yiquan; Liao, I-Chien; Kennedy, Scott J; Du, Jinzhi; Wang, Jun; Leong, Kam W; Clark, Robert L
    This communication describes a single-step electrospraying technique that generates core-shell microspheres (CSMs) with encapsulated protein as the core and an amphiphilic biodegradable polymer as the shell. The protein release profiles of the electrosprayed CSMs showed steady release kinetics over 3 weeks without a significant initial burst.
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    Emergence of limit-periodic order in tiling models.
    (Phys Rev E Stat Nonlin Soft Matter Phys, 2014-07) Marcoux, Catherine; Byington, Travis W; Qian, Zongjin; Charbonneau, Patrick; Socolar, Joshua ES
    A two-dimensional (2D) lattice model defined on a triangular lattice with nearest- and next-nearest-neighbor interactions based on the Taylor-Socolar monotile is known to have a limit-periodic ground state. The system reaches that state during a slow quench through an infinite sequence of phase transitions. We study the model as a function of the strength of the next-nearest-neighbor interactions and introduce closely related 3D models with only nearest-neighbor interactions that exhibit limit-periodic phases. For models with no next-nearest-neighbor interactions of the Taylor-Socolar type, there is a large degenerate class of ground states, including crystalline patterns and limit-periodic ones, but a slow quench still yields the limit-periodic state. For the Taylor-Socolar lattic model, we present calculations of the diffraction pattern for a particular decoration of the tile that permits exact expressions for the amplitudes and identify domain walls that slow the relaxation times in the ordered phases. For one of the 3D models, we show that the phase transitions are first order, with equilibrium structures that can be more complex than in the 2D case, and we include a proof of aperiodicity for a geometrically simple tile with only nearest-neighbor matching rules.
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    Estimation of in-canopy ammonia sources and sinks in a fertilized Zea mays field.
    (Environ Sci Technol, 2010-03-01) Bash, JO; Walker, JT; Katul, GG; Jones, MR; Nemitz, E; Robarg, WP
    An analytical model was developed to describe in-canopy vertical distribution of ammonia (NH(3)) sources and sinks and vertical fluxes in a fertilized agricultural setting using measured in-canopy mean NH(3) concentration and wind speed profiles. This model was applied to quantify in-canopy air-surface exchange rates and above-canopy NH(3) fluxes in a fertilized corn (Zea mays) field. Modeled air-canopy NH(3) fluxes agreed well with independent above-canopy flux estimates. Based on the model results, the urea fertilized soil surface was a consistent source of NH(3) one month following the fertilizer application, whereas the vegetation canopy was typically a net NH(3) sink with the lower portion of the canopy being a constant sink. The model results suggested that the canopy was a sink for some 70% of the estimated soil NH(3) emissions. A logical conclusion is that parametrization of within-canopy processes in air quality models are necessary to explore the impact of agricultural field level management practices on regional air quality. Moreover, there are agronomic and environmental benefits to timing liquid fertilizer applications as close to canopy closure as possible. Finally, given the large within-canopy mean NH(3) concentration gradients in such agricultural settings, a discussion about the suitability of the proposed model is also presented.
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    G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.
    (Proc Natl Acad Sci U S A, 1995-09-26) Touhara, K; Hawes, BE; van Biesen, T; Lefkowitz, RJ
    The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.
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    G-protein-coupled receptor genes as protooncogenes: constitutively activating mutation of the alpha 1B-adrenergic receptor enhances mitogenesis and tumorigenicity.
    (Proc Natl Acad Sci U S A, 1991-12-15) Allen, LF; Lefkowitz, RJ; Caron, MG; Cotecchia, S
    The alpha 1B-adrenergic receptor (alpha 1B-ADR) is a member of the G-protein-coupled family of transmembrane receptors. When transfected into Rat-1 and NIH 3T3 fibroblasts, this receptor induces focus formation in an agonist-dependent manner. Focus-derived, transformed fibroblasts exhibit high levels of functional alpha 1B-ADR expression, demonstrate a catecholamine-induced enhancement in the rate of cellular proliferation, and are tumorigenic when injected into nude mice. Induction of neoplastic transformation by the alpha 1B-ADR, therefore, identifies this normal cellular gene as a protooncogene. Mutational alteration of this receptor can lead to activation of this protooncogene, resulting in an enhanced ability of agonist to induce focus formation with a decreased latency and quantitative increase in transformed foci. In contrast to cells expressing the wild-type alpha 1B-ADR, focus formation in "oncomutant"-expressing cell lines appears constitutively activated with the generation of foci in unstimulated cells. Further, these cell lines exhibit near-maximal rates of proliferation even in the absence of catecholamine supplementation. They also demonstrate an enhanced ability for tumor generation in nude mice with a decreased period of latency compared with cells expressing the wild-type receptor. Thus, the alpha 1B-ADR gene can, when overexpressed and activated, function as an oncogene inducing neoplastic transformation. Mutational alteration of this receptor gene can result in the activation of this protooncogene, enhancing its oncogenic potential. These findings suggest that analogous spontaneously occurring mutations in this class of receptor proteins could play a key role in the induction or progression of neoplastic transformation and atherosclerosis.