Differential coupling of G protein alpha subunits to seven-helix receptors expressed in Xenopus oocytes

Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist- induced, Ca(2+)-activated Cl- currents that were measured using a two- electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX- sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor- activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase

Regions of beta 2 and beta 4 responsible for differences between the steady state dose-response relationships of the alpha 3 beta 2 and alpha 3 beta 4 neuronal nicotinic receptors

We constructed chimeras of the rat beta 2 and beta 4 neuronal nicotinic subunits to locate the regions that contribute to differences between the acetylcholine (ACh) dose-response relationships of the alpha 3 beta 2 and alpha 3 beta 4 receptors. Expressed in Xenopus oocytes, the alpha 3 beta 2 receptor displays an EC50 for ACh approximately 20-fold less than the EC50 of the alpha 3 beta 4 receptor. The apparent Hill slope (n(app)) of alpha 3 beta 2 is near one whereas the alpha 3 beta 4 receptor displays an n(app) near two. Substitutions within the first 120 residues convert the EC50 for ACh from one wild-type value to the other. Exchanging just beta 2:104-120 for the corresponding region of beta 4 shifts the EC50 of ACh dose-response relationship in the expected direction but does not completely convert the EC50 of the dose- response relationship from one wild-type value to the other. However, substitutions in the beta 2:104-120 region do account for the relative sensitivity of the alpha 3 beta 2 receptor to cytisine, tetramethylammonium, and ACh. The expression of beta 4-like (strong) cooperativity requires an extensive region of beta 4 (beta 4:1-301). Relatively short beta 2 substitutions (beta 2:104-120) can reduce cooperativity to beta 2-like values. The results suggest that amino acids within the first 120 residues of beta 2 and the corresponding region of beta 4 contribute to an agonist binding site that bridges the alpha and beta subunits in neuronal nicotinic receptors

What Does an Exemplary Middle School Mathematics Teacher Look Like? The Use of a Professional Development Rubric

A School University Research Network (SURN) committee composed of current mathematics teachers, central office math supervisors, building administrators, mathematicians, and mathematics educators researched numerous sources regarding best practices in mathematics instruction. The resulting professional development rubric synthesizes their findings and can serve a professional development role by providing teachers and administrators with a tool to develop clarity and consensus on best mathematics instructional practices, and how these practices are implemented in the classroom. It is also being used as a tool for cooperating teachers in their supervision of student teachers and as a reflective method for self-evaluation

Random Mutagenesis of G protein ɑ Subunit G_oɑ. Mutations altering nucleotide binding

Nucleotide binding properties of the G protein ɑ subunit G_oɑ were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [^(35)S]GTPyS (guanosine 5'-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified G_oɑ cDNA. Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts. A number of novel mutations reducing the affinity of G_oɑ for GTPyS or Mg^(2+) were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21^(ras) proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTPyS binding but retained the capacity to interact with the βy subunit complex as determined by pertussis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild-type G_oɑ increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl^- response, suggesting that this mutation resulted in a dominant negative phenotype

Spatial and Temporal Dynamics of Dissolved Oxygen Concentrations and Bioactivity in the Hyporheic Zone

Dissolved oxygen (DO) concentrations and consumption rates are primary indicators of heterotrophic respiration and redox conditions in the hyporheic zone (HZ). Due to the complexity of hyporheic flow and interactions between hyporheic hydraulics and the biogeochemical processes, a detailed, mechanistic, and predictive understanding of the biogeochemical activity in the HZ has not yet been developed. Previous studies of microbial activity in the HZ have treated the metabolic DO consumption rate constant (KDO) as a temporally fixed and spatially homogeneous property that is determined primarily by the concentration of bioavailable carbon. These studies have generally treated bioactivity as temporally steady state, failing to capture the temporal dynamics of a changeable system. We demonstrate that hyporheic hydraulics controls rate constants in a hyporheic system that is relatively abundant in bioavailable carbon, such that KDO is a linear function of the local downwelling flux. We further demonstrate that, for triangular dunes, the downwelling velocities are lognormally distributed, as are the KDO values. By comparing measured and modeled DO profiles, we demonstrate that treating KDO as a function of the downwelling flux yields a significant improvement in the accuracy of predicted DO profiles. Additionally, our results demonstrate the temporal effect of carbon consumption on microbial respiration rates

Photorespiration: metabolic pathways and their role in stress protection

Photorespiration results from the oxygenase reaction catalysed by ribulose-1,5-bisphosphate carboxylase/ oxygenase. In this reaction glycollate-2-phosphate is produced and subsequently metabolized in the photorespiratory pathway to form the Calvin cycle intermediate glycerate-3-phosphate. During this metabolic process, CO2 and NH3 are produced and ATP and reducing equivalents are consumed, thus making photorespiration a wasteful process. However, precisely because of this ine¤ciency, photorespiration could serve as an energy sink preventing the overreduction of the photosynthetic electron transport chain and photoinhibition, especially under stress conditions that lead to reduced rates of photosynthetic CO2 assimilation. Furthermore, photorespiration provides metabolites for other metabolic processes, e.g. glycine for the synthesis of glutathione, which is also involved in stress protection. In this review, we describe the use of photorespiratory mutants to study the control and regulation of photorespiratory pathways. In addition, we discuss the possible role of photorespiration under stress conditions, such as drought, high salt concentrations and high light intensities encountered by alpine plants

Phase equilibrium modelling of the amphibolite to granulite facies transition in metabasic rocks (Ivrea Zone, NW Italy)

The development of thermodynamic models for tonalitic melt and the updated clinopyroxene and amphibole models now allow the use of phase equilibrium modelling to estimate P–T conditions and melt production for anatectic mafic and intermediate rock types at high‐temperature conditions. The Permian mid‐lower crustal section of the Ivrea Zone preserves a metamorphic field gradient from mid amphibolite facies to granulite facies, and thus records the onset of partial melting in metabasic rocks. Interlayered metabasic and metapelitic rocks allows the direct comparison of P–T estimates and partial melting between both rock types with the same metamorphic evolution. Pseudosections for metabasic compositions calculated in the Na2O–CaO–K2O–FeO–MgO–Al2O3–SiO2–H2O–TiO2–O (NCKFMASHTO) system are presented and compared with those of metapelitic rocks calculated with consistent endmember data and a–x models. The results presented in this study show that P–T conditions obtained by phase equilibria modelling of both metabasic and metapelitic rocks give consistent results within uncertainties, allowing integration of results obtained for both rock types. In combination, the calculations for both metabasic and metapelitic rocks allows an updated and more precisely constrained metamorphic field gradient for Val Strona di Omegna to be defined. The new field gradient has a slightly lower dP/dT which is in better agreement with the onset of crustal thinning of the Adriatic margin during the Permian inferred in recent studies

Phase Fluctuations and Pseudogap Properties: Influence of Nonmagnetic Impurities

The presence of nonmagnetic impurities in a 2D ``bad metal'' depresses the superconducting Berezinskii-Kosterlitz-Thouless transition temperature, while leaving the pairing energy scale unchanged. Thus the region of the pseudogap non-superconducting phase, where the modulus of the order parameter is non-zero but its phase is random, and which opens at the pairing temperature is substantially bigger than for the clean system. This supports the premise that fluctuations in the phase of the order parameter can in principle describe the pseudogap phenomena in high-$T_c$ materials over a rather wide range of temperatures and carrier densities. The temperature dependence of the bare superfluid density is also discussed.Comment: 11 pages, LaTeX, 1 EPS figure; final version to appear in Low.Temp.Phy