R-Ras controls axon specification upstream of glycogen synthase kinase-3beta through integrin-linked kinase

The initial event in establishing a polarized neuron is the specification of a single axon. Spatially regulated glycogen synthase kinase-3beta (GSK-3beta) activity is critical for specifying axon-dendrite fate; however, the upstream signaling of GSK-3beta in the determination of neuronal polarity still remains obscure. Here, we found that, in cultured hippocampal neurons, the small GTPase R-Ras selectively localized in a single neurite of stage 2 neurons and that its activity increased after plating and peaked between stages 2 and 3. Ectopic expression of R-Ras induced global inactivation of GSK-3beta and formation of multiple axons, whereas knockdown of endogenous R-Ras by RNA interference blocked GSK-3beta inactivation and axon formation. GSK-3beta inactivation and axon formation by R-Ras required integrin-linked kinase (ILK), and subcellular localization of ILK was strictly regulated by R-Ras-mediated phosphatidylinositol 3-kinase activity. In addition, membrane targeting of ILK was sufficient to inactivate GSK-3beta and to form multiple axons. Our study demonstrates a novel role of R-Ras and ILK upstream of GSK-3beta in the regulation of neuronal polarity.     

Complementary roles of farnesoid X receptor,pregnane X receptor,and constitutive androstane receptor in protection against bile acid toxicity

The nuclear receptors, farnesoid X receptor (FXR) and pregnane X receptor (PXR), are important in maintaining bile acid homeostasis. Deletion of both FXR and PXR in vivo by cross-breeding B6;129-Fxrtm1Gonz (FXR-null) and B6;129-Pxrtm1Glaxo-Wellcome (PXR-null) mice revealed a more severe disruption of bile acid, cholesterol, and lipid homeostasis in B6;129-Fxrtm1Gonz Pxrtm1Glaxo-Wellcome (FXR-PXR double null or FPXR-null) mice fed a 1% cholic acid (CA) diet. Hepatic expression of the constitutive androstane receptor (CAR) and its target genes was induced in FXR- and FPXR-null mice fed the CA diet. To test whether up-regulation of CAR represents a means of protection against bile acid toxicity to compensate for the loss of FXR and PXR, animals were pretreated with CAR activators, phenobarbital or 1,4-bis[2-(3,5-dichlorpyridyloxy)]benzene (TCPOBOP), followed by the CA diet. A role for CAR in protection against bile acid toxicity was confirmed by a marked reduction of serum bile acid and bilirubin concentrations, with an elevation of the expression of the hepatic genes involved in bile acid and/or bilirubin metabolism and excretion (CYP2B, CYP3A, MRP2, MRP3, UGT1A, and glutathione S-transferase alpha), following pretreatment with phenobarbital or TCPOBOP. In summary, the current study demonstrates a critical and combined role of FXR and PXR in maintaining not only bile acid but also cholesterol and lipid homeostasis in vivo. Furthermore, FXR, PXR, and CAR protect against hepatic bile acid toxicity in a complementary manner, suggesting that they serve as redundant but distinct layers of defense to prevent overt hepatic damage by bile acids during cholestasis.     

Dopamine Receptor 2 Regulates L-Type Voltage-Gated Calcium Channel in Primary Cultured Mouse Midbrain Neural Network

1. Synchronous oscillation of intracellular Ca²⁺ in the central nervous system is essential for neural development. We previously reported that endogenous dopamine was involved with synchronous Ca²⁺ oscillation of primary cultured midbrain neurons, and that regulation of dopamine in synchronous oscillation was distinctly different through dopamine receptor 1 (D1R) and 2 (D2R): the action of dopamine through D1R or D2R was facilitative or suppressive, respectively, to the Ca²⁺ influx of synchronous oscillation.2. In the present study, we confirmed that the suppressive effects of D2R were mediated by the regulation of the L-type voltage-gated Ca²⁺ channel, not by the regulation of NMDA receptor on the Ca²⁺ influx in the midbrain neural network showing synchronous oscillation.3. This evidence promotes better understanding of the regulation of neural activity by endogenous dopamine in networked neurons.     

The peptide near the C terminus regulates receptor CAR nuclear translocation induced by xenochemicals in mouse liver

In response to phenobarbital (PB) and other PB-type inducers, the nuclear receptor CAR translocates to the mouse liver nucleus (T. Kawamoto et al., Mol. Cell. Biol. 19:6318-6322, 1999). To define the translocation mechanism, fluorescent protein-tagged human CAR (hCAR) was expressed in the mouse livers using the in situ DNA injection and gene delivery systems. As in the wild-type hCAR, the truncated receptor lacking the C-terminal 10 residues (i.e., AF2 domain) translocated to the nucleus, indicating that the PB-inducible translocation is AF2 independent. Deletion of the 30 C-terminal residues abolished the receptor translocation, and subsequent site-directed mutagenesis delineated the PB-inducible translocation activity of the receptor to the peptide L313GLL316AEL319. Ala mutations of Leu313, Leu316, or Leu319 abrogated the translocation of CAR in the livers, while those of Leu312 or Leu315 did not affect the nuclear translocation. The leucine-rich peptide dictates the nuclear translocation of hCAR in response to various PB-type inducers and appears to be conserved in the mouse and rat receptors.     

Volatilization of mercury under acidic conditions from mercury-polluted soil by a mercury-resistant Acidithiobacillus ferrooxidans SUG 2-2.

Volatilization of mercury under acidic conditions from soil polluted with mercuric chloride (1.5 mg Hg/kg soil) was studied with resting cells of a mercury-resistant strain, Acidithiobacillus ferrooxidans SUG 2-2. When resting cells of SUG 2-2 (0.01 mg of protein) were incubated for 10 d at 30 degrees C in 20 ml of 1.6 mM sulfuric acid (pH 2.5) with ferrous sulfate (3%) and mercury-polluted soil (1 g), which contained 7.5 nmol of Hg, approximately 4.1 nmol of mercury was volatilized, indicating that 54% of the total mercury in the soil was volatilized. The amount of mercury volatilized from the soil was dependent on the concentration of Fe2+ added to the medium. When elemental sulfur, sodium tetrathionate, and pyrite were used as an electron donor for the mercury reduction, 16, 2.4 and 0.84%, respectively, of the total mercury added to the solution were volatilized. The optimum pH and temperature for mercury volatilization were 2.5 and 30 degrees C. Approximately 92% of the total mercury in a salt solution (pH 2.5) with resting cells of SUG 2-2 (0.01 mg of protein), ferrous sulfate (3%) and mercury-polluted soil (1 g) was volatilized by further addition of both resting cells and Fe2+ and by incubating for 30 d at 30 degrees C.     

Substrate gating confers steroid specificity to estrogen sulfotransferase.

Estrogen sulfotransferase (EST) exhibits a high substrate specificity and catalytic efficiency toward estrogens such as estradiol (E2) but insignificant ability to sulfate hydroxysteroids such as dehydroepiandrosterone (DHEA). To provide the structural basis for this estrogen specificity, we mutated amino acid residues that constitute the substrate-binding site of EST. Among these mutants, only Tyr-81 decreased E2 and increased DHEA sulfotransferase activities. Substitution for Tyr-81 by smaller hydrophobic residues increased K(m(E2)) for E2 activity, whereas the k(cat(E2)) remained relatively constant. The Y81L mutant exhibited the same DHEA activity as wild-type hydroxysteroid sulfotransferase, for which K(m(DHEA)) remained relatively constant, and k(cat(DHEA)) was markedly increased. The side chain of Tyr-81 is directed at the A-ring of the E2 molecule in the substrate-binding pocket of EST, constituting a steric gate with Phe-142 sandwiching E2 from the opposite side. The present mutagenesis study indicates that the 3beta-hydroxyl group of the DHEA molecule is excluded from the catalytic site of EST through steric hindrance of Tyr-81 with the C-19 methyl group of DHEA. Thus, this stricture-like gating caused by steric hindrance appears to be a structural principle for conferring estrogen specificity to EST.     

Proofreading of a mutagenic nucleotide, N4-aminodeoxycytidylic acid, by Escherichia coli DNA polymerase I

N4-Aminodeoxycytidine triphosphate, a putative metabolite of N4-aminocytidine which is a potent mutagen, is incorporated, in vitro, into polynucleotides in place of dCTP and at a much lesser extent, but significantly, in place of dTTP by E. coli DNA polymerase I large fragment. The activity of the polymerase to proofread this unnatural nucleotide has now been investigated. The results indicate that the 3'-5' exonuclease in the polymerase recognizes N4-aminocytosine as an incorrect base when N4-aminocytosine is incorporated opposite adenine but the enzyme cannot distinguish N4-aminocytosine from cytosine when it is incorporated opposite guanine.     

Iruka Eliminates Dysfunctional Argonaute by Selective Ubiquitination of Its Empty State

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MARCKS dephosphorylation is involved in bradykinin-induced neurite outgrowth in neuroblastoma SH-SY5Y cells

Bradykinin (BK) plays a major role in producing peripheral sensitization in response to peripheral inflammation and in pain transmission in the central nerve system (CNS). Because BK activates protein kinase C (PKC) through phospholipase C (PLC)-β and myristoylated alanine-rich C kinase substrate (MARCKS) has been found to be a substrate of PKC, we explored the possibility that BK could induce MARCKS phosphorylation and regulate its function. BK stimulation induced transient MARCKS phosphorylation on Ser159 with a peak at 1 min in human neuroblastoma SH-SY5Y cells. By contrast, PKC activation by the phorbol ester phorbol 12,13-dibutyrate (PDBu) elicited MARCKS phosphorylation which lasted more than 10 min. Western blotting analyses and glutathione S-transferase (GST) pull-down analyses showed that the phosphorylation by BK was the result of activation of the PKC-dependent RhoA/Rho-associated coiled-coil kinase (ROCK) pathway. Protein phosphatase (PP) 2A inhibitors calyculin A and fostriecin inhibited the dephosphorylation of MARCKS after BK-induced phosphorylation. Moreover, immunoprecipitation analyses showed that PP2A interacts with MARCKS. These results indicated that PP2A is the dominant PP of MARCKS after BK stimulation. We established SH-SY5Y cell lines expressing wild-type MARCKS and unphosphorylatable MARCKS, and cell morphology changes after cell stimulation were studied. PDBu induced lamellipodia formation on the neuroblastoma cell line SH-SY5Y and the morphology was sustained, whereas BK induced neurite outgrowth of the cells via lamellipodia-like actin accumulation that depended on transient MARCKS phosphorylation. Thus these findings show a novel BK signal cascade-that is, BK promotes neurite outgrowth through transient MARCKS phosphorylation involving the PKC-dependent RhoA/ROCK pathway and PP2A in a neuroblastoma cell line.