NR4A1 (Nur77) mediates thyrotropin-releasing hormone-induced stimulation of transcription of the thyrotropin β gene: analysis of TRH knockout mice

Thyrotropin-releasing hormone (TRH) is a major stimulator of thyrotropin-stimulating hormone (TSH) synthesis in the anterior pituitary, though precisely how TRH stimulates the TSHβ gene remains unclear. Analysis of TRH-deficient mice differing in thyroid hormone status demonstrated that TRH was critical for the basal activity and responsiveness to thyroid hormone of the TSHβ gene. cDNA microarray and K-means cluster analyses with pituitaries from wild-type mice, TRH-deficient mice and TRH-deficient mice with thyroid hormone replacement revealed that the largest and most consistent decrease in expression in the absence of TRH and on supplementation with thyroid hormone was shown by the TSHβ gene, and the NR4A1 gene belonged to the same cluster as and showed a similar expression profile to the TSHβ gene. Immunohistochemical analysis demonstrated that NR4A1 was expressed not only in ACTH- and FSH- producing cells but also in thyrotrophs and the expression was remarkably reduced in TRH-deficient pituitary. Furthermore, experiments in vitro demonstrated that incubation with TRH in GH4C1 cells increased the endogenous NR4A1 mRNA level by approximately 50-fold within one hour, and this stimulation was inhibited by inhibitors for PKC and ERK1/2. Western blot analysis confirmed that TRH increased NR4A1 expression within 2 h. A series of deletions of the promoter demonstrated that the region between bp -138 and +37 of the TSHβ gene was responsible for the TRH-induced stimulation, and Chip analysis revealed that NR4A1 was recruited to this region. Conversely, knockdown of NR4A1 by siRNA led to a significant reduction in TRH-induced TSHβ promoter activity. Furthermore, TRH stimulated NR4A1 promoter activity through the TRH receptor. These findings demonstrated that 1) TRH is a highly specific regulator of the TSHβ gene, and 2) TRH mediated induction of the TSHβ gene, at least in part by sequential stimulation of the NR4A1-TSHβ genes through a PKC and ERK1/2 pathway.     

Selenoproteins mediate T cell immunity through an antioxidant mechanism

Selenium is an essential dietary element with antioxidant roles in immune regulation, but there is little understanding of how this element acts at the molecular level in host defense and inflammatory disease. Selenium is incorporated into the amino acid selenocysteine (Sec), which in turn is inserted into selenoproteins in a manner dependent on Sec tRNA([Ser]Sec). To investigate the molecular mechanism that links selenium to T cell immunity, we generated mice with selenoprotein-less T cells by cell type-specific ablation of the Sec tRNA([Ser]Sec) gene (trsp). Herein, we show that these mutant mice exhibit decreased pools of mature T cells and a defect in T cell-dependent antibody responses. We also demonstrate that selenoprotein deficiency leads to oxidant hyperproduction in T cells and thereby suppresses T cell proliferation in response to T cell receptor stimulation. These findings offer novel insights into immune function of selenium and physiological antioxidants.     

Discovery of novel series of benzoic acid derivatives containing biphenyl ether moiety as potent and selective human beta(3)-adrenergic receptor agonists: Part IV

Identification and SAR study of novel series of beta(3)-AR agonists with benzoic acid are described. Conversion of ether linkage position of phenoxybenzoic acid derivative 2b led to compound 7b with moderate beta(3)-AR activity. Further modification in right, center and left parts of compound 7b was investigated to improve the beta(3)-AR potency and selectivity. Compounds 7g and 7k, with the bulky aliphatic-substituted group at 2-position of benzoic acid moiety, were identified as potent and selective beta(3)-AR agonists. In addition, in vivo efficacy of compounds 7g and 7k was exhibited on dog OAB model.     

Enzymatic synthesis of dimaltosyl-beta-cyclodextrin via a transglycosylation reaction using TreX, a Sulfolobus solfataricus P2 debranching enzyme

Di-O-α-maltosyl-β-cyclodextrin ((G2)₂-β-CD) was synthesized from 6-O-α-maltosyl-β-cyclodextrin (G2-β-CD) via a transglycosylation reaction catalyzed by TreX, a debranching enzyme from Sulfolobus solfataricus P2. TreX showed no activity toward glucosyl-β-CD, but a transfer product (1) was detected when the enzyme was incubated with maltosyl-β-CD, indicating specificity for a branched glucosyl chain bigger than DP2. Analysis of the structure of the transfer product (1) using MALDI-TOF/MS and isoamylase or glucoamylase treatment revealed it to be dimaltosyl-β-CD, suggesting that TreX transferred the maltosyl residue of a G2-β-CD to another molecule of G2-β-CD by forming an α-1,6-glucosidic linkage. When [¹⁴C]-maltose and maltosyl-β-CD were reacted with the enzyme, the radiogram showed no labeled dimaltosyl-β-CD; no condensation product between the two substrates was detected, indicating that the synthesis of dimaltosyl-β-CD occurred exclusively via transglycosylation of an α-1,6-glucosidic linkage. Based on the HPLC elution profile, the transfer product (1) was identified to be isomers of 6¹,6³- and 6¹,6⁴-dimaltosyl-β-CD. Inhibition studies with β-CD on the transglycosylation activity revealed that β-CD was a mixed-type inhibitor, with a K_i value of 55.6 μmol/mL. Thus, dimaltosyl-β-CD can be more efficiently synthesized by a transglycosylation reaction with TreX in the absence of β-CD. Our findings suggest that the high yield of (G2)₂-β-CD from G2-β-CD was based on both the transglycosylation action mode and elimination of the inhibitory effect of β-CD.     

Functional expression of the Aspergillus flavus PKS–NRPS hybrid CpaA involved in the biosynthesis of cyclopiazonic acid

α-Cyclopiazonic acid (CPA) is an indole tetramic acid mycotoxin. Based on our identification of the polyketide synthase–nonribosomal peptide synthase (PKS–NRPS) hybrid gene cpaA involved in cyclopiazonic acid biosynthesis in Aspergillus fungi, we carried out heterologous expression of Aspergillus flavus cpaA under α-amylase promoter in Aspergillus oryzae and identified its sole product to be the CPA biosynthetic intermediate cyclo-acetoacetyl-l-tryptophan (cAATrp). This result rationalized that the PKS–NRPS hybrid enzyme CpaA catalyzes condensation of the diketide acetoacetyl-ACP formed by the PKS module and l-Trp activated by the NRPS module. This CpaA expression system provides us an ideal platform for PKS–NRPS functional analysis, such as adenylation domain selectivity and product releasing mechanism.     

Brain-Derived Neurotrophic Factor and Interleukin-6 Levels in the Serum and Cerebrospinal Fluid of Children with Viral Infection-Induced Encephalopathy

We investigated changes in the brain-derived neurotrophic factor (BDNF) and interleukin (IL)-6 levels in pediatric patients with central nervous system (CNS) infections, particularly viral infection-induced encephalopathy. Over a 5-year study period, 24 children hospitalized with encephalopathy were grouped based on their acute encephalopathy type (the excitotoxicity, cytokine storm, and metabolic error types). Children without CNS infections served as controls. In serum and cerebrospinal fluid (CSF) samples, BDNF and IL-6 levels were increased in all encephalopathy groups, and significant increases were noted in the influenza-associated and cytokine storm encephalopathy groups. Children with sequelae showed higher BDNF and IL-6 levels than those without sequelae. In pediatric patients, changes in serum and CSF BDNF and IL-6 levels may serve as a prognostic index of CNS infections, particularly for the diagnosis of encephalopathy and differentiation of encephalopathy types.     

Homologous recombination-mediated knock-in targeting of the MET1a gene for a maintenance DNA methyltransferase reproducibly reveals dosage-dependent spatiotemporal gene expression in rice

Although homologous recombination-promoted knock-in targeting to monitor the expression of a gene by fusing a reporter gene with its promoter is routine practice in mice, gene targeting to modify endogenous genes in flowering plants remains in its infancy. In the knock-in targeting, the junction sequence between a reporter gene and an endogenous target promoter can be designed properly, and transgenic plants carrying an identical and desired knock-in allele can be repeatedly obtained. By employing a reproducible gene-targeting procedure with positive–negative selection in rice, we were able to obtain fertile transgenic knock-in plants with the promoterless GUS reporter gene encoding β-glucuronidase fused with the endogenous promoter of MET1a , one of two rice MET1 genes encoding a maintenance DNA methyltransferase. All of the primary (T₀) transgenic knock-in plants obtained were found to carry only one copy of GUS , with the anticipated structure in the heterozygous condition, and no ectopic events associated with gene targeting could be detected. We showed the reproducible, dosage-dependent and spatiotemporal expression of GUS in the selfed progenies of independently isolated knock-in targeted plants. The results in knock-in targeted plants contrast sharply with the results in transgenic plants with the MET1a promoter -fused GUS reporter gene integrated randomly in the genome: clear interindividual variation of GUS expression was observed among independently obtained plants bearing the randomly integrated transgenes. As our homologous recombination-mediated gene-targeting strategy with positive–negative selection is, in principle, applicable to modify any endogenous gene, knock-in targeting would facilitate basic and applied plant research.     

Anxiolytic effects of short- and long-term administration of cacao mass on rat elevated T-maze test

We demonstrated the effects of short- and long-term administration of cacao mass on anxiety in the elevated T-maze test, which is an animal model of anxiety. In the first study, we administered cacao mass (100 mg/100 g body weight) per os and immediately performed the elevated T-maze test. Short-term cacao mass significantly abolished delayed avoidance latency compared with the control but did not change escape latency. This result suggested that cacao mass administration reduced conditional fear-relating behavior. Short-term cacao mass administration did not affect the concentration of brain monoamines, emotion-related neurotransmitters such as norepinephrine, serotonin and dopamine, in the rat brain. In the next study, we fed a cacao mass-containing diet to rats for 2 weeks and performed the elevated T-maze test. Contrary to short-term administration, chronic consumption of cacao mass tended to increase avoidance latency and did not change escape latency. Brain serotonin concentration and its turnover were enhanced by chronic consumption of cacao mass. These results suggested that chronic consumption of cacao did not affect fear-related behavior but was involved in brain monoamine metabolism. In conclusion, we suggest that short-term cacao mass consumption showed an anxiolytic effect but chronic consumption did not.