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.     

Thermotomaculum hydrothermale gen. nov., sp. nov., a novel heterotrophic thermophile within the phylum Acidobacteria from a deep-sea hydrothermal vent chimney in the Southern Okinawa Trough

A novel heterotrophic, thermophilic bacterium, designated strain AC55^T, was isolated from a deep-sea hydrothermal vent chimney at the Hatoma Knoll in the Okinawa Trough, Japan. Cells of strain AC55^T were non-motile, long rods (2.0- to 6.8-μm long and 0.3- to 0.6-μm wide). The strain was an obligatory anaerobic heterotroph capable of fermentative growth on complex proteinaceous substances. Elemental sulfur was reduced to hydrogen sulfide but did not stimulate growth. Growth was observed between 37 and 60°C (optimum 55°C), pH 5.5 and 8.5 (optimum pH 6.6), and in the presence of 1.5–4.5% (w/v) NaCl (optimum 2.5%, w/v). Menaquinone-7 and -8 were the major respiratory quinones. The G + C content of the genomic DNA from strain AC55^T was 51.6 mol%. The 16S rRNA gene sequence analysis revealed that strain AC55^T was the first cultivated representative of Acidobacteria subdivision 10. Based on the physiological and phylogenetic features of the novel isolate, the genus name Thermotomaculum gen. nov. is proposed, with Thermotomaculum hydrothermale sp. nov. as the type species. The type strain is AC55^T (=JCM 17643^T = DSM 24660^T = NBRC 107904^T).     

Type IV pilin is glycosylated in Pseudomonas syringae pv. tabaci 6605 and is required for surface motility and virulence

Type IV pilin (PilA) is a major constituent of pilus and is required for bacterial biofilm formation, surface motility and virulence. It is known that mature PilA is produced by cleavage of the short leader sequence of the pilin precursor, followed by methylation of N-terminal phenylalanine. The molecular mass of the PilA mature protein from the tobacco bacterial pathogen Pseudomonas syringae pv. tabaci 6605 (Pta 6605) has been predicted to be 12 329 Da from its deduced amino acid sequence. Previously, we have detected PilA as an approximately 13-kDa protein by immunoblot analysis with anti-PilA-specific antibody. In addition, we found the putative oligosaccharide-transferase gene tfpO downstream of pilA. These findings suggest that PilA in Pta 6605 is glycosylated. The defective mutant of tfpO (ΔtfpO) shows reductions in pilin molecular mass, surface motility and virulence towards host tobacco plants. Thus, pilin glycan plays important roles in bacterial motility and virulence. The genetic region around pilA was compared among P. syringae pathovars. The tfpO gene exists in some strains of pathovars tabaci, syringae, lachrymans, mori, actinidiae, maculicola and P. savastanoi pv. savastanoi. However, some strains of pathovars tabaci, syringae, glycinea, tomato, aesculi and oryzae do not possess tfpO, and the existence of tfpO is independent of the classification of pathovars/strains in P. syringae. Interestingly, the PilA amino acid sequences in tfpO-possessing strains show higher homology with each other than with tfpO-nonpossessing strains. These results suggest that tfpO and pilA might co-evolve in certain specific bacterial strains.     

Claudin-4 Deficiency Results in Urothelial Hyperplasia and Lethal Hydronephrosis

Claudin (Cld)-4 is one of the dominant Clds expressed in the kidney and urinary tract, including selective segments of renal nephrons and the entire urothelium from the pelvis to the bladder. We generated Cldn4 ^−/− mice and found that these mice had increased mortality due to hydronephrosis of relatively late onset. While the renal nephrons of Cldn4 ^−/− mice showed a concomitant diminution of Cld8 expression at tight junction (TJ), accumulation of Cld3 at TJ was markedly enhanced in compensation and the overall TJ structure was unaffected. Nonetheless, Cldn4 ^−/− mice showed slightly yet significantly increased fractional excretion of Ca²⁺ and Cl⁻, suggesting a role of Cld4 in the specific reabsorption of these ions via a paracellular route. Although the urine volume tended to be increased concordantly, Cldn4 ^−/− mice were capable of concentrating urine normally on dehydration, with no evidence of diabetes insipidus. In the urothelium, the formation of TJs and uroplaques as well as the gross barrier function were also unaffected. However, intravenous pyelography analysis indicated retarded urine flow prior to hydronephrosis. Histological examination revealed diffuse hyperplasia and a thickening of pelvic and ureteral urothelial layers with markedly increased BrdU uptake in vivo. These results suggest that progressive hydronephrosis in Cldn4 ^−/− mice arises from urinary tract obstruction due to urothelial hyperplasia, and that Cld4 plays an important role in maintaining the homeostatic integrity of normal urothelium.     

Effect of electrical stimulation of the LES on LES pressure in a canine model

Gastric electrical stimulation modulates lower esophageal sphincter pressure (LESP). High-frequency neural stimulation (NES) can induce gut smooth muscle contractions. To determine whether lower esophageal sphincter (LES) electrical stimulation (ES) can affect LESP, bipolar electrodes were implanted in the LES of four dogs. Esophageal manometry during sham or ES was performed randomly on separate days. Four stimuli were used: 1) low-frequency: 350-ms pulses at 6 cycles/min; 2) high-frequency-1: 1-ms pulses at 50 Hz; 3) high-frequency-2: 1-ms pulses at 20 Hz; and 4) NES: 20-ms bipolar pulses at 50 Hz. Recordings were obtained postprandially. Tests consisted of three 20-min periods: baseline, stimulation/sham, and poststimulation. The effect of NES was tested under anesthesia and following IV administration of l-NAME and atropine. Area under the curve (AUC) and LESP were compared among the three periods, by ANOVA and t-test, P < 0.05. Data are shown as means +/- SD. We found that low-frequency stimulation caused a sustained increase in LESP: 32.1 +/- 12.9 (prestimulation) vs. 43.2 +/- 18.0 (stimulation) vs. 50.1 +/- 23.8 (poststimulation), P < 0.05. AUC significantly increased during and after stimulation. There were no significant changes with other types of ES. With NES, LESP initially rose and then decreased below baseline (LES relaxation). During NES, N(G)-nitro-l-arginine methyl ester increased both resting LESP and the initial rise in LESP and markedly diminished the relaxation. Atropine lowered resting LESP and abolished the initial rise in LESP. In conclusion, low frequency ES of the LES increases LESP in conscious dogs. NES has dual effect on LESP: an initial stimulation, cholinergically mediated, followed by relaxation mediated by nitric oxide.     

CHMetal(II) axial interaction in planar complexes (metal = Cu, Pd) and implications for possible environmental effects of alkyl groups at biological copper sites

Intramolecular M(II)H–C interactions (M(II)=Cu(II), Pd(II)) involving a side chain alkyl group of planar d⁸ and d⁹ metal complexes of the N-alkyl (R) derivatives of N,N-bis(2-pyridylmethyl)amine with an N₃Cl donor set were established by structural and spectroscopic methods. The methyl group from the branched alkyl group (R = 2,2-dimethylpropyl and 2-methylbutyl) axially interacts with the metal ion with the MC and MH distances of 3.056(3)–3.352(9) and 2.317(1)–2.606(1) Å, respectively, and the M–H–C angles of 122.4–162.3°. The Cu(II) complexes showing the interaction have a higher redox potential as compared with those without it, and the ¹H NMR signals of the interacting methyl group in Pd(II) complexes shifted downfield relative to the ligand signals. Dependence of the downshift values on the dielectric constants of the solvents used indicated that the M(II)H–C interaction is mainly electrostatic in nature and may be regarded as a weak hydrogen bond. Implications for possible environmental effects of the leucine alkyl group at the type 1 Cu site of fungal laccase are also discussed.     

Migration of mesenchymal cell fated to blastema is necessary for fish fin regeneration

Urodeles and fish have higher regeneration ability in a variety of tissues and organs than do other vertebrate species including mammals. Though many studies have aimed at identifying the cellular and molecular basis for regeneration, relatively little is known about the detailed cellular behaviors and involved molecular basis. In the present study, a small molecule inhibitor was used to analyzed the role of phosphoinositide 3-kinase (PI3K) signaling during regeneration. We showed that the inhibitor disrupted the formation of blastema including the expression of characteristic genes. The failure of blastema formation was due to the impaired migration of mesenchymal cells to the distal prospective blastema region, although it had a little affect on cell cycle activation in mesenchymal cells. Moreover, we found that the epidermal remodeling including cell proliferation, distal cell migration and Akt phosphorylation was also affected by the inhibitor, implying a possible involvement of epidermis for proper formation of blastema. From these data, we propose a model in which distinct signals that direct the cell cycle activation, mesenchymal cell migration and epidermal remodeling coordinate together to accomplish the correct blastema formation and regeneration.     

Clathrin dependent endocytosis of E-cadherin is regulated by the Arf6GAP isoform SMAP1

E-cadherin is a central component of the adherens junction in epithelial cells and continuously undergoes endocytosis via clathrin-coated vesicles and/or caveolae depending on the cell type. In this study, we examined the role of SMAP1, a clathrin-interacting GTPase-activating protein (GAP) for the ADP-ribosylation factor 6 (Arf6) GTPase, in E-cadherin endocytosis. Mardin-Darby canine kidney (MDCK) epithelial cells were used as a model, and SMAP1 localized in the cytoplasm and along the adherens junction where E-cadherin was present. Next, activity of SMAP1 was compared with that of other Arf6GAPs (and/or an effector of Arf6-GTP), namely GIT1 and AMAP2/DDEF2. Overexpression of SMAP1 but not GIT1 nor AMAP2/DDEF2 strongly inhibited basal, as well as phorbolester-induced, internalization of E-cadherin. Notably, AMAP2/DDEF2 rather enhanced the caveolae-mediated incorporation of a membrane protein other than E-cadherin. Thus, in MDCK cells, E-cadherin appeared to be endocytosed solely through SMAP1-regulated clathrin-coated vesicles. Furthermore, MDCK cells overexpressing SMAP1 showed a reduced degree of cell migration compared to untransfected cells, as assessed by wound healing and Transwell assays, and this reduction in migration appeared to be due to the accumulation of E-cadherin at the adherens junction in cells overexpressing SMAP1. Collectively, SMAP1 likely represents a key Arf6GAP in clathrin dependent endocytosis of E-cadherin in MDCK cells. This activity of SMAP1 in E-cadherin turnover may be involved in epithelial organization and/or epithelial-mesenchymal transition.