Potency of Agonists and Competitive Antagonists on Adult- and Fetal-Type Nicotinic Acetylcholine Receptors

1. The potency of agonists and competitive antagonists on the two expressed forms of the nicotinic acetylcholine receptor (adult or junctional subtype, -AChR; fetal or extrajunctional subtype, -AChR) have not previously been compared systematically in homogeneous receptor preparations.2. Each subtype of the receptor was expressed separately in Xenopus oocytes by cytoplasmic injection of combinations of RNA transcribed in vitro. The presence of each type of receptor was confirmed by single-channel recordings. Expressing oocytes were assayed using discontinuous, single-electrode voltage clamp by measuring peak currents in response to test compounds.3. The extrajunctional subtype was more potently activated by the nicotinic agonist dimethylphenyl piperazinium iodide (DMPP) than was the junctional form. There was no statistically significant difference in potency between the two subtypes for other nicotinic agonists (nicotine, cytisine and succinylcholine). The rank order of potency for -AChR was succinylcholine>cytisine>DMPP>nicotine, and that for -AChR was DMPP>cytisine>succinylcholine>nicotine.4. Two agonists (cytisine and succinylcholine) displayed six- to eight-fold greater intrinsic activity in activating -AChR over -AChR. There was no difference between the two forms of receptor in efficacy for nicotine.5. The extrajunctional form was much more potently inhibited by the steroidal competitive antagonist pancuronium than was the junctional receptor. However, there was no significant difference in potency of inhibition by the curariform drug atracurium.6. Contrary to previous reports, there is no consistent relation between the effect of agonists and antagonists and the subtype of receptor. These data suggest that the resistance or sensitivity to these agents seen in various clinical settings are related to other cellular factors.     

Glycerol 3-phosphate acylation in microsomes of type II cells isolated from adult rat lung

Glycerol 3-phosphate acylation was studied in type II cells isolated from adult rat lung. The process was found to be largely microsomal. In the microsomes phosphatidic acid is the main product of glycerol 3-phosphate acylation. Glycerol-3-phosphate acyltransferase is rate limiting in the phosphatidic acid formation by the microsomes. Type II cell microsomes incorporate palmitoyl and oleoyl residues into phosphatidic acid at an equal rate if palmitoyl-CoA and oleoyl-CoA are added separately. However, if palmitoyl-CoA and oleoyl-CoA are added as an equimolar mixture the unsaturated fatty acyl moiety is incorporated much faster. Under the latter conditions monoenoic species constitute the most abundant products of glycerol 3-phosphate acylation. The microsomes incorporate both palmitoyl and oleoyl residues readily into both the 1- and 2-position of phosphatidic acid, even when palmitoyl-CoA and oleoyl-CoA are added together. Assuming that both phosphatidic acid phosphatase and cholinephosphotransferase do not discriminate against substrates with an unsaturated acyl moiety at the 1-position and a saturated acyl moiety at the 2-position, the last two observations indicate that a considerable percentage of phosphatidylcholine molecules synthesized de novo may have a saturated fatty acid at the 2-position and an unsaturated fatty acid at the 1-position, and that remodeling at the 1-position may be important for the formation of surfactant dipalmitoylphosphatidylcholine. They also indicate that type II cell microsomes are capable of synthesizing the dipalmitoyl species of phosphatidic acid. However, since there is a preference for the acylation of glycerol 3-phosphate with unsaturated fatty acyl residues, the percentage of dipalmitoyl species in the synthesized phosphatidic acid, and thereby the percentage of dipalmitoyl species in the phosphatidylcholine synthesized de novo, will probably depend on the relative availability of the various acyl-CoA species.     

Plant-soil interactions associated with acid,weathered soils

Plant-soil interactions in weathered soils are so complex that unqualified statements about a suitable pH for plants are risky. Conventional experimental designs and statistical methods may not be appropriate for investigating such complexities. Lime experiments using continuous function designs and observation of plant response to indigenous variability in soil pH permit detailed observations of plant-soil interactions that are frequently not detected. A graphical boundary-line approach to interpreting data can make good sense out of apparent confusion. Increasing the pH of variable-charge soils by adding lime or by indigenous means increased CEC and retarded cation leaching, but Ca solubility changed very little over the range pH 5 to 6. N fixation and yield was closely related to soil pH, soil Mn and Mn uptake by soybean. This result was clearly demonstrated regardless of numerous other limiting factors. Plant yield response curves resolved into distinct segments that corresponded with associated soil properties. Excess Al compounded by Ca deficiency is suspect in the pH range <5. Excess Mn, and Ca deficiency probably limited yields in the pH range 5.0 to 5.7. Yields were stable, and Ca and P were constant in the pH interval 5.7 to 6.0. Yields abruptly increased in the pH interval 6.0 to 6.3. This was associated with elevated Ca concentrations in soil solutions.     

Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the splicing of mRNA precursors is disrupted by a severe heat shock. Mild heat treatments prior to severe heat shock protect splicing from disruption, as was previously reported for Drosophila melanogaster. In contrast to D. melanogaster, protein synthesis during the pretreatment is not required to protect splicing in yeast cells. However, protein synthesis is required for the rapid recovery of splicing once it has been disrupted by a sudden severe heat shock. Mutations in two classes of yeast hsp genes affect the pattern of RNA splicing during the heat shock response. First, certain hsp70 mutants, which overproduce other heat shock proteins at normal temperatures, show constitutive protection of splicing at high temperatures and do not require pretreatment. Second, in hsp104 mutants, the recovery of RNA splicing after a severe heat shock is delayed compared with wild-type cells. These results indicate a greater degree of specialization in the protective functions of hsps than has previously been suspected. Some of the proteins (e.g., members of the hsp70 and hsp82 gene families) help to maintain normal cellular processes at higher temperatures. The particular function of hsp104, at least in splicing, is to facilitate recovery of the process once it has been disrupted.     

Amino acid-specific ADP-ribosylation. Evidence for two distinct NAD:arginine ADP-ribosyltransferases in turkey erythrocytes

An NAD:arginine mono-ADP-ribosyltransferase has been purified 270,000-fold from turkey erythrocytes through a five-step chromatographic procedure to apparent electrophoretic homogeneity. Molecular weight determinations of the enzyme by sodium dodecyl sulfate-gel electrophoresis, Ultrogel AcA 54, and TSK 3000 SW gel filtration were consistent with Mr = 32,000. The purified enzyme utilized arginine, other low molecular weight guanidino compounds, and various proteins as ADP-ribose acceptors. The Km values for NAD and arginine methyl ester were 36 and 3,000 microM, respectively. Unlike another transferase purified from turkey erythrocytes (Moss, J., and Stanley, S.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4809-4812), this enzyme was not activated by chaotropic salts or micromolar concentrations of histone. Thus, two distinct soluble ADP-ribosyltransferases may be present in turkey erythrocytes.     

Lack of Complete Exposure Pathways for Metals in Natural and FGD Gypsum

Use of wallboard made from synthetic gypsum generated via flue-gas desulfurization (FGD) by coal-fired power plants (FGD gypsum) raised questions concerning the potential for exposure to residual trace metals. Because gypsum is widely used (e.g., in wallboard), any issue with metals could have far-reaching implications. A conceptual site model evaluated potential human health exposure pathways for metals in gypsum, through consideration of data for 21 metals including samples of natural (mined) gypsum, and of FGD gypsum. Because there are no screening values for gypsum, comparisons were made to background soil concentrations and to risk-based concentrations for metals in soil termed preliminary remedial action goals (PRGs), which assume more frequent and prolonged contact with particulate soil than would be likely for gypsum, and thus provide a health protective means for evaluation of exposure. Additional screenings evaluated occupational exposure and agricultural use of gypsum. Maximum metal concentrations in natural and FGD gypsum samples were either consistent with background concentrations or much lower than PRGs for residential or agricultural soil, or workplace air, and thus exposure pathways for these media were considered incomplete. Separate analyses of mercury volatilization were conducted, and this pathway was also found to be incomplete.     

Isolation of the two components of Condylactis toxin.

The two toxic components from the sea anemone Condylactis gigantea have been isolated and purified using gel filtration, ion-exchange chromatography, and preparative isoelectric focusing. The molecular weight of both components is estimated by gel filtration to be 4500. The isoelectric points of the two toxins were determined to be 4.8 and 5.8 with LD₅₀ values of 19 and 58 μg/kg, respectively, when injected into Armadillidium vulgare, a terrestrial crustacean. Low-level labeling with ¹²⁵I using lactoperoxidase was effected for the purpose of estimating homogeneity.     

Live cell imaging of Gs and the beta2-adrenergic receptor demonstrates that both alphas and beta1gamma7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the beta2-adrenergic receptor

To visualize and investigate the regulation of the localization patterns of Gs and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. alphas-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of alphas, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of alphas. Functional fluorescent beta1gamma7 dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to beta1 (YFP-N-beta1) and a carboxyl-terminal YFP fragment to gamma7 (YFP-C-gamma7). When expressed together, YFP-N-beta1 and YFP-C-gamma7 produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing alphas-CFP, YFP-N-beta1/YFP-C-gamma7, and the beta2-adrenergic receptor (beta2AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, alphas-CFP and YFP-N-beta1/YFP-C-gamma7 stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with beta2AR-labeled vesicles. Moreover, internalization of beta2AR-GFP, but not alphas-CFP or YFP-N-beta1/YFP-C-gamma7, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the Gs subunits and beta2AR utilize different internalization mechanisms. Subsequent trafficking of the Gs subunits and beta2AR also differed in that vesicles labeled with the Gs subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, co-localization of alphas and beta1gamma7 on these endosomes may indicate a means of recycling specific alphasbetagamma combinations to the plasma membrane.     

Visualization of G protein betagamma dimers using bimolecular fluorescence complementation demonstrates roles for both beta and gamma in subcellular targeting

To investigate the role of subcellular localization in regulating the specificity of G protein betagamma signaling, we have applied the strategy of bimolecular fluorescence complementation (BiFC) to visualize betagamma dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to beta and a carboxyl-terminal yellow fluorescent protein fragment to gamma. When expressed together, these two proteins produced a fluorescent signal in human embryonic kidney 293 cells that was not obtained with either subunit alone. Fluorescence was dependent on betagamma assembly in that it was not obtained using beta2 and gamma1, which do not form a functional dimer. In addition to assembly, BiFC betagamma complexes were functional as demonstrated by more specific plasma membrane labeling than was obtained with individually tagged fluorescent beta and gamma subunits and by their abilities to potentiate activation of adenylyl cyclase by alpha(s) in COS-7 cells. To investigate isoform-dependent targeting specificity, the localization patterns of dimers formed by pair-wise combinations of three different beta subunits with three different gamma subunits were compared. BiFC betagamma complexes containing either beta1 or beta2 localized to the plasma membrane, whereas those containing beta5 accumulated in the cytosol or on intracellular membranes. These results indicate that the beta subunit can direct trafficking of the gamma subunit. Taken together with previous observations, these results show that the G protein alpha, beta, and gamma subunits all play roles in targeting each other. This method of specifically visualizing betagamma dimers will have many applications in sorting out roles for particular betagamma complexes in a wide variety of cell types.