Liganded Thyroid Hormone Receptor Inhibits Phorbol 12-O-Tetradecanoate-13-Acetate-Induced Enhancer Activity via Firefly Luciferase cDNA

Thyroid hormone receptor (TR) belongs to the nuclear hormone receptor (NHR) superfamily and regulates the transcription of its target genes in a thyroid hormone (T3)-dependent manner. While the detail of transcriptional activation by T3 (positive regulation) has been clarified, the mechanism of T3-dependent repression (negative regulation) remains to be determined. In addition to naturally occurring negative regulations typically found for the thyrotropin β gene, T3-bound TR (T3/TR) is known to cause artificial negative regulation in reporter assays with cultured cells. For example, T3/TR inhibits the transcriptional activity of the reporter plasmids harboring AP-1 site derived from pUC/pBR322-related plasmid (pUC/AP-1). Artificial negative regulation has also been suggested in the reporter assay with firefly luciferase (FFL) gene. However, identification of the DNA sequence of the FFL gene using deletion analysis was not performed because negative regulation was evaluated by measuring the enzymatic activity of FFL protein. Thus, there remains the possibility that the inhibition by T3 is mediated via a DNA sequence other than FFL cDNA, for instance, pUC/AP-1 site in plasmid backbone. To investigate the function of FFL cDNA as a transcriptional regulatory sequence, we generated pBL-FFL-CAT5 by ligating FFL cDNA in the 5' upstream region to heterologous thymidine kinase promoter in pBL-CAT5, a chloramphenicol acetyl transferase (CAT)-based reporter gene, which lacks pUC/AP-1 site. In kidney-derived CV1 and choriocarcinoma-derived JEG3 cells, pBL-FFL-CAT5, but not pBL-CAT5, was strongly activated by a protein kinase C activator, phorbol 12-O-tetradecanoate-13-acetate (TPA). TPA-induced activity of pBL-FFL-CAT5 was negatively regulated by T3/TR. Mutation of nt. 626/640 in FFL cDNA attenuated the TPA-induced activation and concomitantly abolished the T3-dependent repression. Our data demonstrate that FFL cDNA sequence mediates the TPA-induced transcriptional activity, which is inhibited by T3/TR

GATA2 Mediates Thyrotropin-Releasing Hormone-Induced Transcriptional Activation of the Thyrotropin β Gene

Thyrotropin-releasing hormone (TRH) activates not only the secretion of thyrotropin (TSH) but also the transcription of TSHβ and α-glycoprotein (αGSU) subunit genes. TSHβ expression is maintained by two transcription factors, Pit1 and GATA2, and is negatively regulated by thyroid hormone (T3). Our prior studies suggest that the main activator of the TSHβ gene is GATA2, not Pit1 or unliganded T3 receptor (TR). In previous studies on the mechanism of TRH-induced activation of the TSHβ gene, the involvements of Pit1 and TR have been investigated, but the role of GATA2 has not been clarified. Using kidney-derived CV1 cells and pituitary-derived GH3 and TαT1 cells, we demonstrate here that TRH signaling enhances GATA2-dependent activation of the TSHβ promoter and that TRH-induced activity is abolished by amino acid substitution in the GATA2-Zn finger domain or mutation of GATA-responsive element in the TSHβ gene. In CV1 cells transfected with TRH receptor expression plasmid, GATA2-dependent transactivation of αGSU and endothelin-1 promoters was enhanced by TRH. In the gel shift assay, TRH signal potentiated the DNA-binding capacity of GATA2. While inhibition by T3 is dominant over TRH-induced activation, unliganded TR or the putative negative T3-responsive element are not required for TRH-induced stimulation. Studies using GH3 cells showed that TRH-induced activity of the TSHβ promoter depends on protein kinase C but not the mitogen-activated protein kinase, suggesting that the signaling pathway is different from that in the prolactin gene. These results indicate that GATA2 is the principal mediator of the TRH signaling pathway in TSHβ expression

Extended posterior approach for huge popliteal aneurysm extended to superficial femoral artery

Objectives: Generally, popliteal artery aneurysms have been addressed surgically by a medial, posterior, or lateral approach. We have designed a new posterior approach that exposes the superficial femoral artery and entire popliteal artery without dividing any muscles in a just prone position. Methods and results: A 72-year old man with huge popliteal aneurysm extended to superficial femoral artery was admitted to our hospital. Surgery was performed due to a high risk of rupture. An S-shaped skin incision was made in the popliteal fossa. We could not expose the proximal side of the giant aneurysm proximal to the foramen of the adductor magnus. We extended the skin incision to the proximal and exfoliated the medial side of semitendinosus muscle. We could expose the superficial femoral artery in this approach like in a medial approach. We could perform the interposition of great saphenous vein. Conclusion: The advantages of this approach allowed for entire exposure of the popliteal aneurysm in the same patient’s position when we perform aneurysmectomy and bypass. It is possible for this approach to provide easy access to the superficial femoral artery proximal to the adductor hiatus and distal below-knee popliteal artery including the tibioperoneal trunk

超音波加温による動脈塞栓後の肝腫瘍内部のリピオドール集積検出能に関する生体内での検討

Computed tomography (CT) is the standard method to evaluate Lipiodol deposition after transarterial embolization (TAE) for a long period. However, iodine but not Lipiodol can be observed on CT. A minimally invasive other method to detect Lipiodol has been needed to evaluate accurate evaluation after procedure. The purpose of this study was to evaluate the efficacy of using the rate of change in sound velocity caused by ultrasonic heating to reflect Lipiodol accumulation after TAE in a rat liver tumor model. We analyzed the association of this developed technique with CT images and histological findings. Eight rats bearing N1S1 cells were prepared. After confirmation of tumor development in a rat liver, Lipiodol was injected via the hepatic artery. Seven days after TAE, CT scan and sound velocity changes caused by ultrasonic heating were measured, and then the rats were sacrificed. An ultrasonic pulse-echo method was used to measure the sound velocity. The temperature coefficient of the sound velocity in each treated tumor was evaluated and compared with the mean CT value and the histological Lipiodol accumulation ratio. Pearson's correlation coefficients were calculated to assess the correlation between the measured values. The correlation coefficient (r) of the mean CT value and histological Lipiodol accumulation ratio was 0.835 (p = 0.010), which was considered statistically significant. Also, those of the temperature coefficient of the sound velocity and the histological Lipiodol accumulation ratio were statistically significant (r = 0.804; p = 0.016). To our knowledge, this is the first study that reported the efficacy of ultrasonic heating to detect Lipiodol accumulation in rat liver tumors after TAE. Our results suggest that the rate of change in sound velocity caused by ultrasonic heating can be used to evaluate Lipiodol accumulation in liver tumors after TAE, and thus could represent an alternative to CT in this application. This new innovative technique is easy to treat and less invasive in terms of avoiding radiation compared with CT.博士（医学）・甲第836号・令和4年3月15日© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)