A Novel Diagnostic Method for Thyroid Follicular Tumors Based on Immunofluorescence Analysis of p53-Binding Protein 1 Expression: Detection of Genomic Instability

The preoperative diagnosis of thyroid follicular carcinomas (FCs) by fine-needle aspiration cytology is almost impossible. It was previously demonstrated that p53-binding protein 1 (53BP1) expression, based on immunofluorescence (IF),can serve as a valuable biomarker to estimate the malignant potential of various cancers. 53BP1 belongs to a class of DNA damage response molecules that rapidly localize to the site of DNA double-strand breaks,forming nuclear foci (NF). This study aimed to elucidate the utility of 53BP1 NF expression as a biomarker to differentiate follicular tumors (FTs). Methods: Associations between 53BP1 expression based on IF and histological types of FTs were analyzed using 27 follicular adenomas (FAs), 28 minimally invasive FCs, and 14 widely invasive FCs. Furthermore, the study clarified the relationship between 53BP1 NF and copy number aberrations (CNAs) based on array comparative genomic hybridization, a hallmark of genomic instability (GIN). Results: This study demonstrates differences in 53BP1 NF expression between FA and FC. The incidence of 53BP1 at NF significantly increased with FT progression in the following order: normal follicle < FA < minimally invasive FCs< widely invasive FCs. In contrast, no significant differences were observed in CNAs among the FT samples. Furthermore, there was no significant correlation between CNAs and 53BP1 at NF in FTs. Thus, based on a comparison of these two indicators of GIN, 53BP1 NF (by IF) was better able to estimate the malignancy of FTs compared to CNA (by array comparative genomic hybridization). Interestingly, IF revealed a heterogenous distribution of 53BP1 NF,which occurred more frequently in the invasive or subcapsular area than in the center of the tumor, suggesting intratumoral heterogeneity of GIN in FTs. Conclusions: It is proposed that IF analysis of 53BP1 expression could be a novel diagnostic method to estimate the malignant potential of FTs. Because 53BP1 NF reflect DNA double-strand breaks, it is hypothesized that the incidence of 53BP1 at NF can represent the level of GIN in tumor cells. IF analysis of 53BP1 expression will not only be　an auxiliary histologic technique to diagnose FTs accurately, but also a novel technique for preoperative diagnosis　using fine-needle aspiration cytology

歯周組織破壊におけるプロスタグランディンの役割に関する実験病理学的研究

広島大学(Hiroshima University)博士(歯学)Dentistrydoctora

Heterologous Expression of Pharaonis Halorhodopsin in Xenopus laevis Oocytes and Electrophysiological Characterization of Its Light-Driven Cl- Pump Activity

Natronomonas pharaonis halorhodopsin (pHR) is an archaeal rhodopsin functioning as an inward-directed, light-driven Cl– pump. To characterize the electrophysiological features of the Cl– pump activity of pHR, we expressed pHR in Xenopus laevis oocytes and analyzed its photoinduced Cl– pump activity using the two-electrode voltage-clamp technique. Photoinduced outward currents were observed only in the presence of Cl–, Br–, I–, , and SCN–, but not in control oocytes, indicating that photoinduced anion currents were mediated by pHR. The relationship between photoinduced Cl– current via pHR and the light intensity was linear, demonstrating that transport of Cl– is driven by a single-photon reaction and that the steady-state current is proportional to the excited pHR molecule. The current-voltage relationship for pHR-mediated photoinduced currents was also linear between –150 mV and +50 mV. The slope of the line describing the current-voltage relationship increased as the number of the excited pHR molecules was increased by the light intensity. The reversal potential (VR) for Cl– as the substrate for the anion pump activity of pHR was about –400 mV. The value for VR was independent of light intensity, meaning that the VR reflects the intrinsic value of the excited pHR molecule. The value of VR changed significantly for the R123K mutant of pHR. We also show that the Cl– pump activity of pHR can generate a substantial negative membrane potential, indicating that pHR is a very potent Cl– pump. We have also analyzed the kinetics of voltage-dependent Cl– pump activity as well as that of the photocycle. Based on these data, a kinetic model for voltage-dependent Cl– transport via pHR is presented

Interaction of the Halobacterial Transducer to a Halorhodopsin Mutant Engineered so as to Bind the Transducer : Cl- Circulation within the Extracellular Channel

An alkali-halophilic archaeum, Natronomonas pharaonis, contains two rhodopsins that are halorhodopsin (phR), a light-driven inward Cl- pump and phoborhodopsin (ppR), the receptor of negative phototaxis functioning by forming a signaling complexes with a transducer, pHtrII. Previously, we reported that the phR double mutant, P240T/F250YphR, can bind with pHtrII [Y. Sudo et al. (2006) J. Mol. Biol. 357, 1274-1282]. This mutant itself can transport Cl-, while the net transport was stopped upon formation of the complex. The flash photolysis data were analyzed by a scheme in which phR ⇒ P1 → P2 → P3 → P4 → phR. The P3 of the wild-type and the double mutant contained two components, X- and O-intermediates. After the complex formation, however, the P3 of the double mutant lacked the X-intermediate. These observations imply that the X-intermediate (probably the N-intermediate) is the state having Cl- in the cytoplasmic binding site and that the complex undergoes an extracellular Cl- circulation due to the inhibition of formation of the X-intermediate