記述言語：英語  

To explore Cu(II) ion coordination by His(186) in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP(C).

DOI： 10.1016/j.bbrc.2010.03.003

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記述言語：英語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.48.S23_1

CiNii Research

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記述言語：英語  

We analyzed the pH-induced mobility changes in moPrP(C) alpha-helix and beta-sheets by cysteine-scanning site-directed spin labeling (SDSL) with ESR. Nine amino acid residues of alpha-helix1 (H1, codon 143-151), four amino acid residues of beta-sheet1 (S1, codon 127-130), and four amino acid residues of beta-sheet2 (S2, codon 160-163) were substituted for by cysteine residues. These recombinant mouse PrP(C) (moPrP(C)) mutants were reacted with a methane thiosulfonate sulfhydryl-specific spin labeling reagent (MTSSL). The 1/deltaH of the central (14N hyperfine) component (M(I) = 0) in the ESR spectrum of spin-labeled moPrP(C) was measured as a mobility parameter of nitroxide residues (R1). The mobilities of E145R1 and Y149R1 at pH 7.4, which was identified as a tertiary contact site by a previous NMR study of moPrP, were lower than those of D143R1, R147R1, and R150R1 reported on the helix surface. Thus, the mobility in the H1 region in the neutral solution was observed with the periodicity associated with a helical structure. On the other hand, the values in the S2 region, known to be located in the buried side, were lower than those in the S1 region located in the surface side. These results indicated that the mobility parameter of the nitroxide label was well correlated with the 3D structure of moPrP. Furthermore, the present study clearly demonstrated three pH-sensitive sites in moPrP, i.e., (1) the N-terminal tertiary contact site of H1, (2) the C-terminal end of H1, and (3) the S2 region. In particular, among these pH-sensitive sites, the N-terminal tertiary contact region of H1 was found to be the most pH-sensitive one and was easily converted to a flexible structure by a slight decrease of pH in the solution. These data provided molecular evidence to explain the cellular mechanism for conversion from PrP(C) to PrP(Sc) in acidic organelles such as the endosome.

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記述言語：英語  

Although enhancement of ultrasound-induced cell killing by photodynamic reagents has been shown, the sonochemical mechanism in detail is still not clear. Here, comparison between sonodynamic effect and photodynamic effect with photosensitizers at a concentration of 10 microM on free radical formation and cell killing was made. When electron paramagnetic-resonance spectroscopy (EPR) was used to detect 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TAN) after photo-irradiation or sonication with 2,2,6,6-tetramethyl-4-piperidone (TMPD), the order of TAN formation in the photo-irradiated samples was as follows: rhodamine 6G (R6) > sulforhodamine B (SR) > hematoporphyrin (Hp) > rhodamine 123 (R123) > rose bengal (RB)>erythrosine B (Er) = 0; although there was time-dependent TAN formation when the samples were sonicated, no significant difference among these agents were observed. All these agents suppressed ultrasound-induced OH radical formation detected by EPR-spin trapping. Sensitizer-derived free radicals were markedly observed in SR, RB and Er, while trace level of radicals derived from R6 and R123 were observed. Enhancement of ultrasound-induced decrease of survival in human lymphoma U937 cells was observed at 1.5 W/cm(2) (less than inertial cavitation threshold) for R6, R123, SR and Er, and at 2.3 W/cm(2) for R6, R123, Er, RB and SR. On the other hand, photo-induced decrease of survival was observed for R6, Hp and RB at the same concentration (10 microM). These comparative results suggest that (1) (1)O(2) is not involved in the enhancement of ultrasound-induced loss of cell survival, (2) OH radicals and sensitizer-derived free radicals do not take part in the enhancement, and (3) the mechanism is mainly due to certain mechanical stress such as augmentation of physical disruption of cellular membrane by sensitizers in the close vicinity of cells and/or cavitation bubbles.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: The present study was undertaken to reconfirm heme oxygenase-1 (HO-1) induction by ultrasound, and elucidate the mechanism by which this occurs. METHODS: After exposure of human lymphoma U937 cells to 1 MHz continuous ultrasound (US), gene profiling by using cDNA microarray analysis, cell viability by using the trypan blue dye exclusion test, mRNA expression by using real-time quantitative polymerase chain reaction, and protein expression by using Western blotting were examined. As an indicator of cavitation, hydroxyl radical formation was studied by using electron paramagnetic resonance-spin trapping. RESULTS: The cDNA microarray analysis reconfirmed HO-1 induction in human lymphoma U937 cells after exposure to US, and further identified one upregulated and two downregulated genes. When U937 cells were exposed to US for 1 min, HO-1 induction, as examined by real-time quantitative polymerase chain reaction and Western blotting, was observed at intensities higher than the cavitational threshold. When a potent antioxidant, N-acetyl-L-cysteine, was added to the culture medium before or after sonication, the induction was attenuated, indicating that reactive oxygen species are involved in HO-1 induction. A decrease in mitochondrial membrane potential and generation of superoxide anion radicals were also observed in the cells exposed to US. CONCLUSION: We used a cDNA microarray system to confirm upregulation of the HO-1 gene and to discover new genes that respond to ultrasonic cavitation. Increased intracellular oxidative stress secondary to the sonomechanical effects arising from ultrasonic cavitation is suggested to be the mechanism of enhancement of HO-1 expression.

DOI： 10.1007/s10396-005-0066-7

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記述言語：英語  

The structure of the mouse prion (moPrP) was studied using site-directed spin-labeling electron spin resonance (SDSL-ESR). Since a previous NMR study by Hornemanna et al., [Hornemanna, Korthb, Oeschb, Rieka, Widera, Wüthricha, Glockshubera, Recombinant full-length murine prion protein, mPrP (23-231): purification and spectroscopic characterization, FEBS Lett. 413 (1997) 277-281] has indicated that N96, D143, and T189 in moPrP are localized in a Cu(2+) binding region, Helix1 and Helix2, respectively, three recombinant moPrP mutations (N96C, D143C, and T189C) were expressed in an Escherichia coli system, and then refolded by dialysis under low pH and purified by reverse-phase HPLC. By using the preparation, we succeeded in preserving a target cystein residue without alteration of the alpha-helix structure of moPrP and were able to apply SDSL-ESR with a methane thiosulfonate spin label to the full-length prion protein. The rotational correlation times (tau) of 1.1, 3.3, and 4.8ns were evaluated from the X-band ESR spectra at pH 7.4 and 20 degrees C for N96R1, D143R1, and T189R1, respectively. tau reflects the fact that the Cu(2+) binding region is more flexible than Helix1 or Helix2. ESR spectra recorded at various temperatures revealed two phases together with a transition point at around 20 degrees C in D143R1 and T189R1, but not in N96R1. With the variation of pH from 4.0 to 7.8, ESR spectra of T189R1 at 20 degrees C showed a gradual increase of tau from 2.9 to 4.8ns. On the other hand, the pH-dependent conformational changes in N96R1 and D143R1 were negligible. These results indicated that T189 located in Helix2 possessed a structure sensitive to physiological pH changes; simultaneously, N96 in the Cu(2+) binding region and D143 in Helix1 were conserved.

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記述言語：英語  

To clarify the role of the Golgi apparatus in photodynamic therapy-induced apoptosis, its signaling pathway was studied after photodynamic treatment of human cervix carcinoma cell line HeLa, in which a photosensitizer, 2,4,5,7-tetrabromorhodamine 123 bromide (TBR), was incorporated into the Golgi apparatus. Laser scanning microscopic analysis of TBR-loaded HeLa cells confirmed that TBR was exclusively located in the Golgi apparatus. HeLa cells incubated with TBR for 1 h were then exposed to visible light using an Xe lamp. Light of wavelength below 670 nm was eliminated with a filter. Morphological observation of nuclei stained with Hoechst 33342 revealed that apoptosis of cells was induced by exposure to light. Electron spin resonance spectrometry showed that light-exposed TBR produced both singlet oxygen (1O2) and superoxide anion (O2-). Apoptosis induction by TBR was inhibited by pyrrolidine dithiocarbamate, an O2- scavenger, but not by NaN3, a quencher of 1O2. Furthermore, TBR-induced apoptosis was inhibited by aurintricarboxylic acid and ZnCl2, which are known as inhibitors of deoxyribonuclease (DNase) gamma, and (acetoxymethyl)-1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, a chelator of Ca2+, but not by acetyl Asp-Glu-Val-Asp-aldehyde, an inhibitor of caspase-3. These results suggested that O2- was responsible for TBR-induced apoptosis, and Ca(2+)-dependent and caspase-3-independent nuclease such as DNase gamma played an important role in apoptotic signaling triggered by Golgi dysfunction.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Web of Science

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/ja00010a054

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/nar/18.5.1217

Scopus

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1269/jrr.31.156

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/0009-2797(89)90032-X

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/bi00451a013

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1269/jrr.29.172

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1269/jrr.29.246

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記述言語：日本語  

J-GLOBAL

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J-GLOBAL

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記述言語：日本語  

J-GLOBAL

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記述言語：日本語  

J-GLOBAL

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記述言語：日本語   出版者・発行元：日本生物物理学会  

DOI： 10.2142/biophys.45.S38_3

CiNii Research

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記述言語：日本語  

J-GLOBAL

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J-GLOBAL

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