Modulation of Intracellular Cyclic AMP Levels by Different Human Dopamine D4 Receptor Variants

Abstract: To investigate whether polymorphic forms of the human dopamine D4 receptor have different functional characteristics, we have stably expressed cDNAs of the D4.2, D4.4, and D4.7 isoforms in several cell lines. Chinese hamster ovary CHO-K1 cell lines expressing D4 receptor variants displayed pharmacological profiles that were in close agreement with previous data from transiently expressed D4 receptors in COS-7 cells. Dopamine stimulation of the D4 receptors resulted in a concentration-dependent inhibition of the forskolin-stimulated cyclic AMP (cAMP) levels. The potency of dopamine to inhibit cAMP formation was about twofold reduced for D4.7 (EC₅₀ of ∼37 n M ) compared with the D4.2 and D4.4 variants (EC₅₀ of ∼16 n M ). Antagonists block the dopamine-mediated inhibition of cAMP formation with a rank order of potency of emonapride > haloperidol = clozapine ≫ raclopride. There was no obvious correlation between the efficacy of inhibition of forskolin-stimulated cAMP levels and the D4 subtypes. Dopamine could completely reverse prostaglandin E₂-stimulated cAMP levels for all three D4 receptor variants. Deletion of the repeat sequence does not affect functional activity of the receptor. The data presented indicate that the polymorphic repeat sequence causes only small changes in the ability of the D4 receptor to block cAMP production in CHO cells.     

Biodegradation of 4-nitrophenol by indigenous microbial populations in Everglades soils

The Everglades in South Florida are a unique ecological system. As a result of the widespread use of pesticides and herbicides in agricultural areas upstream from these wetlands, there is a serious potential for pollution problems in the Everglades. The purpose of this study was to evaluate the ability of indigenous microbial populations to degrade xenobiotic organic compounds introduced by agricultural and other activities. Such biodegradation may facilitate the remediation of contaminated soils and water in the Everglades. The model compound selected in this study is 4-nitrophenol, a chemical commonly used in the manufacture of pesticides. The mineralization of 4-nitrophenol at various concentrations was studied in soils collected from the Everglades. At concentrations of 10 and 100 microg/g soil, considerable mineralization occurred within a week. At a higher concentration, i.e., 10 mg/g soil, however, no mineralization of 4-nitrophenol occurred over a 4-month period; such a high concentration apparently produced an inhibitory effect. The rate and extent of 4-nitrophenol mineralization was enhanced on inoculation with previously isolated nitrophenol-degrading microorganisms. The maximum mineralization extent measured, however, was less than 30% suggesting conversion to biomass and/or unidentified intermediate products. These results indicate the potential for natural mechanisms to mitigate the adverse effects of xenobiotic pollutants in a complex system such as the Everglades.     

Effects of nonionic surfactants on the solubilization and mineralization of phenanthrene in soil-water systems

The solubilization and mineralization of (14)C-phenanthrene in soil-water systems was examined with several commercially available surface-active agents, viz., an alkyl ethoxylate C(12)E(4); two alkylphenol ethoxylate surfactants: C(8)PE(9.5) and C(9)PE(10.5); two sorbitan ethoxylate surfactants: the sorbitan monolaurate (Tween 20) and the sorbitan monooleate (Tween 80); two pairs of nonionic ethoxylate surfactant mixtures: C(12)E(4)/C(12)E(23) at a 1:1 ratio, and C(12-15)E(3)/C(12-15)E(9) at a 1:3 ratio; and two surfactants possessing relatively high critical micelle concentration (CMC) values and low aggregation numbers: CHAPS and octyglucoside. Surface tension experiments were performed to evaluate surfactant sorption onto soil and the surfactant doses required to attain the CMC in the soil-water systems. Surfactant solubilization of (14)C-phenanthrene commenced with the onset of micellization. The addition of surface-active agents was observed not to be beneficial to the microbial mineralization of phenanthrene in the soil-water systems and, for supra-CMC surfactant doses, phenanthrene mineralization was completely inhibited for all the surfactants tested. A comparison of solubilization, surface tension, and mineralization data confirms that the inhibitory effect on microbial degradation of phenanthrene is related to the CMC of the surfactant in the presence of soil. Additional tests demonstrated the recovery of mineralization upon dilution of surfactant concentration to sub-CMC levels, and a relatively high exit rate for phenanthrene from micelles. These tests suggest that the inhibitory effect is probably related to a reversible physiological surfactant micelle-bacteria interaction, possibly through partial complexing or release of membrane material with disrupting membrane lamellar structure. This study indicates that nonionic surfactant solubilization of sorbed hydrophobic organic compounds from soil may not be beneficial for the concomitant enhancement of soil bioremediation. Additional work is needed to address physicochemical processes for bioavailability enhancement, and effects of solubilizing agents on microorganisms for remediation and treatment of hydrophobic organic compounds and nonaqueous phase liquids. (c) 1992 John Wiley & Sons Inc.     

Synthesis of highly fluorescent nitrogen and phosphorus doped carbon dots for the detection of Fe3+ ions in cancer cells

Highly fluorescent nitrogen and phosphorus‐doped carbon dots with a quantum yield 59% have been successfully synthesized from citric acid and di‐ammonium hydrogen phosphate by single step hydrothermal method. The synthesized carbon dots have high solubility as well as stability in aqueous medium. The as‐obtained carbon dots are well monodispersed with particle sizes 1.5–4 nm. Owing to a good tunable fluorescence property and biocompatibility, the carbon dots were applied for intercellular sensing of Fe³⁺ ions as well as cancer cell imaging. Copyright © 2015 John Wiley & Sons, Ltd.     

Induction of Krüppel‐like factor 2 reduces K/BxN serum‐induced arthritis

Krüppel‐like factor 2 (KLF2) critically regulates activation and function of monocyte, which plays important pathogenic role in progressive joint destruction in rheumatoid arthritis (RA). It is yet to be established the molecular basis of KLF2‐mediated regulation of monocytes in RA pathogenesis. Herein, we show that a class of compound, HDAC inhibitors (HDACi) induced KLF2 expression in monocytes both in vitro and in vivo. KLF2 level was also elevated in tissues, such as bone marrow, spleen and thymus in mice after infusion of HDACi. Importantly, HDACi significantly reduced osteoclastic differentiation of monocytes with the up‐regulation of KLF2 and concomitant down‐regulation of matrixmetalloproteinases both in the expression level as well as in the protein level. In addition, HDACi reduced K/BxN serum‐induced arthritic inflammation and joint destruction in mice in a dose‐dependent manner. Finally, co‐immunoprecipitation and overexpression studies confirmed that KLF2 directly interacts with HDAC4 molecule in cells. These findings provide mechanistic evidence of KLF2‐mediated regulation of K/BxN serum‐induced arthritic inflammation.     

Dynamics of Membrane-Bound G12V-KRAS from Simulations and Single-Molecule FRET in Native Nanodiscs

Recent studies have shown that the small GTPase KRAS adopts multiple orientations with respect to the plane of anionic model membranes, whereby either the three C-terminal helices or the three N-terminal β-strands of the catalytic domain face the membrane. This has functional implications because, in the latter, the membrane occludes the effector-interacting surface. However, it remained unclear how membrane reorientation occurs and, critically, whether it occurs in the cell in which KRAS operates as a molecular switch in signaling pathways. Herein, using data from a 20 μs-long atomistic molecular dynamics simulation of the oncogenic G12V-KRAS mutant in a phosphatidylcholine/phosphatidylserine bilayer, we first show that internal conformational fluctuations of flexible regions in KRAS result in three distinct membrane orientations. We then show, using single-molecule fluorescence resonance energy transfer measurements in native lipid nanodiscs derived from baby hamster kidney cells, that G12V-KRAS samples three conformational states that correspond to the predicted orientations. The combined results suggest that relatively small energy barriers separate orientation states and that signaling-competent conformations dominate the overall population.     

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans

Dopamine-modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine-mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1-like dopamine receptor gene (dop-1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop-1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild-type copy of the gene. The dop-1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop-1 gene in mechanosensory neurons using the mec-7 promoter rescues the mechanosensory deficit in dop-1 mutant animals. The tyrosine hydroxylase-deficient C. elegans mutant (cat-2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.