Probe walking : development of novel probes for DNA fingerprinting

Thesis--University of Tsukuba, D.M.S.(A), no. 786, 1990. 3. 23Offprint. Originally published in: Human genetics, v. 83, pp. 223-226, 1989Joint authors: Shogo Misawa and Shintaroh UedaIncludes supplementary treatise

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

Is antipsychotic polypharmacy associated with metabolic syndrome even after adjustment for lifestyle effects?: a cross-sectional study

BACKGROUND: Although the validity and safety of antipsychotic polypharmacy remains unclear, it is commonplace in the treatment of schizophrenia. This study aimed to investigate the degree that antipsychotic polypharmacy contributed to metabolic syndrome in outpatients with schizophrenia, after adjustment for the effects of lifestyle. METHODS: A cross-sectional survey was carried out between April 2007 and October 2007 at Yamanashi Prefectural KITA hospital in Japan. 334 patients consented to this cross-sectional study. We measured the components consisting metabolic syndrome, and interviewed the participants about their lifestyle. We classified metabolic syndrome into four groups according to the severity of metabolic disturbance: the metabolic syndrome; the pre-metabolic syndrome; the visceral fat obesity; and the normal group. We used multinomial logistic regression models to assess the association of metabolic syndrome with antipsychotic polypharmacy, adjusting for lifestyle. RESULTS: Seventy-four (22.2%) patients were in the metabolic syndrome group, 61 (18.3%) patients were in the pre-metabolic syndrome group, and 41 (12.3%) patients were in visceral fat obesity group. Antipsychotic polypharmacy was present in 167 (50.0%) patients. In multinomial logistic regression analyses, antipsychotic polypharmacy was significantly associated with the pre-metabolic syndrome group (adjusted odds ratio [AOR], 2.348; 95% confidence interval [CI], 1.181-4.668), but not with the metabolic syndrome group (AOR, 1.269; 95%CI, 0.679-2.371). CONCLUSIONS: These results suggest that antipsychotic polypharmacy, compared with monotherapy, may be independently associated with an increased risk of having pre-metabolic syndrome, even after adjusting for patients' lifestyle characteristics. As metabolic syndrome is associated with an increased risk of cardiovascular mortality, further studies are needed to clarify the validity and safety of antipsychotic polypharmacy

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

5-Caffeoylquinic Acid Ameliorates Cognitive Decline and Reduces Aβ Deposition by Modulating Aβ Clearance Pathways in APP/PS2 Transgenic Mice

The accumulation of amyloid β (Aβ) in the brain is a major pathological feature of Alzheimer’s disease (AD). In our previous study, we demonstrated that coffee polyphenols (CPP) prevent cognitive dysfunction and Aβ deposition in the brain of an APP/PS2 transgenic mouse AD model. The underlying mechanisms, however, remain to be elucidated. Here, we investigated the effects of the chronic administration of 5-caffeoylquinic acid (5-CQA), the most abundant component of CPP, on cognitive dysfunction in APP/PS2 mice to identify the role of CPP in Aβ elimination. Relative to the untreated controls, the mice fed a 5-CQA-supplemented diet showed significant improvements in their cognitive function assessed by Y-maze and novel object recognition tests. Histochemical analysis revealed that 5-CQA substantially reduced Aβ plaque formation and neuronal loss in the hippocampi. Moreover, 5-CQA upregulated the gene encoding low-density lipoprotein receptor-related protein 1, an Aβ efflux receptor, and normalized the perivascular localization of aquaporin 4, which facilitates Aβ clearance along the paravascular pathway. These results suggest that 5-CQA reduces Aβ deposition in the brain by modulating the Aβ clearance pathways and ameliorating cognitive decline and neuronal loss in APP/PS2 mice. Thus, 5-CQA may be effective in preventing cognitive dysfunction in AD