Biomedical subjects

Hirotada Fujii

Publications and source records attributed to Hirotada Fujii.

9 recordsLinked to original sources

PubMed2006

Development of an ESR/MR dual-imaging system as a tool to detect bioradicals.

A system combining electron spin resonance imaging (ESRI) with another imaging modality capable of enabling visualization of the distribution of bioradicals on an anatomical map of the specimens would be a superior biomedical imaging system. We describe the development of an ESR/MR dual-imaging system with one permanent magnet and the biomedical applications of this system. The magnetic circuit developed for the ESR/MR dual-imaging system consisted of the permanent magnet made of Fe-Nd-B, pole pieces, and poke. The permanent magnet was installed on the MR side only, and the ESR side was made of pole pieces only. The magnetic field was adjusted to 0.5T at MR and to 0.042T at ESR. The overall dimensions of the magnet developed for the ESR/MR imaging system were 460 (W)x440 (D)x460 (H) mm, and it weighed 220 kg. The distance of each center for the magnet for ESR and MR imaging could be set as close as 200 mm. The entire ESR/MR imaging system can be installed in a common laboratory without magnetic shielding. MR images of plants (myoga) and small animals (mice and rats) were successfully acquired with or without ESR operation. ESR spectra of nitroxyl spin probes were also measured, even with MRI operation. ESR signals of triarylmethyl derivatives with narrow line-width (0.026 mT) were observed in living mice while MRI was operating. The ESR/MR imaging dual functions work properly with no electric or magnetic interference. The ESR/MR dual images demonstrate that this system enables visualization of the distribution of bioradicals on the anatomical map of the object.

Animals↗

PubMed2004

Binding of FAD to cytochrome b558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production.

The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47(phox), p67(phox), p40(phox), and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b(558) (cyt b(558)) with different amounts of FAD. A remarkably high activity, over 100 micromol/s/micromol heme, was obtained in the oxidase with purified cyt b(558), ternary complex (p47-p67-p40(phox)), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant K(d) of FAD in cyt b(558) of reconstituted oxidase was estimated as nearly 1 nm. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b(558) increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b(558) recovered electron flow from NADPH to heme. From both K(d) values of FAD and the amount of incorporated FAD in cyt b(558) of reconstituted oxidase, in combination with spectra, we propose the model in which the K(d) values of FAD in cyt b(558) is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.

Animals↗

PubMed2004

Detection of bioradicals by in vivo L-band electron spin resonance spectrometry.

The applications of in vivo electron paramagnetic resonance (EPR) or electron spin resonance (ESR) spectroscopy have been impressive over a relatively short period despite the many obstacles which had to be overcome, such as dielectric absorption and biodestruction. The loop-gap resonators and modified loop coil systems have emerged as the most suitable resonators for in vivo EPR experiments. This paper briefly discusses instrumental aspects as a prelude to several examples related to the in vivo monitoring and detection of bioradicals. Recent progress in detection of bioradicals induced by drugs or chemicals is discussed with regard to nitrosocompounds, nitric oxide and metals in vivo. A clinical EPR application is also discussed.

Animals↗

PubMed2004

Decoupling of automatic control systems in a continuous-wave electron paramagnetic resonance spectrometer for biomedical applications.

This article describes a systematic approach to decoupling automatic tuning control (ATC) and automatic matching control (AMC) systems in continuous-wave (CW) electron paramagnetic resonance (EPR) spectroscopy for animal experiments. This technique enables us to improve the stability of CW-EPR spectroscopy even if the animal is moving during the data acquisition of EPR spectra. The control systems are formulated to allow the behavior of interference between them to be investigated, since they are generally coupled due to the characteristics of the microwave resonator. The stability of the entire control system in a 1.1 GHz CW-EPR spectrometer is evaluated with the generalized Nyquist stability criterion. We compare the EPR spectra of a triarylmethyl (TAM) radical that is dosed in anesthetized mice in terms of the signal-to-noise ratio (SNR) to test the precompensator for decoupling the ATC and AMC systems. The experimental findings suggest that the present technique is useful for improving the SNR of EPR spectra in animal experiments. The SNR of the measured EPR spectra was improved by about 50% with the precompensator.

Algorithms↗

PubMed2004

In vivo spin trapping of nitric oxide.

The measurement of nitric oxide (NO) in biological samples has normally required destructive chemical techniques. The ability to detect NO non-invasively in living animals or excised organs has great potential using specialized electron paramagnetic resonance (EPR) methods. Although NO is paramagnetic, it cannot be observed directly unless it is complexed with ferrous iron-dithiocarbamate ligand spin trap complexes. Despite the minimally invasive nature of the technique, highly sensitive localized concentrations of NO may be observed ("trapped") in vivo by both L-band EPR and magnetic resonance imaging.

Animals↗

PubMed2004

A constitutive NADPH oxidase-like system containing gp91phox homologs in human keratinocytes.

In non-phagocytic cells, superoxide has been implicated in physiological and pathological cellular functions in the skin and mucosa, such as, host defense, mitogenic responses, and malignant conversion. Here, we identify a constitutively expressed heme-flavoprotein NADPH oxidase (Nox) system as a source of superoxide in human skin (HaCaT) and gingival mucosal (GM16) keratinocyte cell lines. Western blot analysis showed that both cell lines expressed the phagocyte oxidase (phox) cytosolic proteins Rac1, p40phox, and p67phox. With respect to the catalytic flavoheme protein subunit, HaCaT membranes, which expressed p22phox, showed an absorbance peak at 558 nm indicative of a b-type cytochrome. At mRNA levels, both GM16 and HaCaT cells expressed gp91phox homologs Nox1, Nox2, and Nox4, however, HaCaT cells expressed very low levels of Nox1 mRNA. At protein levels, Nox1 was readily detected in HaCaT but was nearly undetectable in GM16 cells. Consistently, Nox activity of HaCaT membranes was demonstrated by electron paramagnetic resonance spin-trapping and cytochrome c reduction, and the activity was sensitive to the flavoprotein inhibitor diphenylene iodonium. V(max) values were 20-fold lower than those reported for phagocytic oxidase. In conclusion, keratinocytes expressed a Nox distinct from the phox isoform of phagocytes providing molecular evidence for a source of superoxide that may regulate cell proliferation and host defense in skin and oral mucosa.

Cell Line↗

PubMed2003

Effects of high gravity on amphibian development.

In order to clarify the possible effects of high gravity environments on eggs and developing embryos, Rana rugosa and Xenopus laevis fertilized eggs and early embryos were raised in 2 G, 5 G, 7 G and 10 G up to the hatched tadpole stage. The results showed that: (1) High gravity significantly retarded the development of eggs and embryos beginning treatment before the blastula stage and induced various abnormalities, including two heads and microcephally suggesting that high gravity is apt to disrupt the animal-vegital axis. On the other hand, embryos beginning treatment after the gastrula stage showed a striking increase in the number of normal-appearing feeding tadpoles. (2) Autopsy revealed that brains, notochords and muscles were reduced in development and differentiation for embryos and tadpoles developed in high gravity. (3) It seems likely that the system for hydrogen peroxide detoxification develops abnormally in high gravity-treated embryos and tadpoles, which probably results in oxidative stress, leading to considerable cell damage.

Animals↗

PubMed2002

Consequences of nitric oxide generation in epileptic-seizure rodent models as studied by in vivo EPR.

The role of nitric oxide (NO) in epileptogenesis was studied in pentylenetetrazole (PTZ)-treated animals using in vivo and ex vivo EPR spectroscopy. NO generation was measured directly in the brain of a PTZ-induced mouse in vivo by an L-band EPR spectrometer. An elevation in NO production in the brain was observed during convulsions, and more NO was generated in the tonic seizure vs. the clonic seizure. NO content in several brain tissues (including the cerebral cortex (CR), cerebellum (CL), olfactory bulb (OB), hippocampus (HI), and hypothalamus (HT)) of PTZ-doped rats was analyzed quantitatively ex vivo by X-band EPR. To test the involvement of NO in seizure development, pharmacological analyses were performed using the NO synthase (NOS) inhibitors N(G)-nitro-L-arginine (L-NNA), N(G)-monomethyl-L-arginine (L-NMMA), and 3-bromo-7-nitroindazole (3Br-7NI). All of these inhibitors suppressed the convulsions, holding them at the clonic level, and prevented development of a tonic convulsion in rats doped with up to 80 mg/kg PTZ. 3Br-7NI completely inhibited NO production, but L-NNA and L-NMMA showed only 70% inhibition of NO production in PTZ-doped rats. In order to examine the contributions of NO in convulsions, rats were treated with anticonvulsants (phenytoin and diazepam) before PTZ treatment. Both drugs completely suppressed tonic convulsion in PTZ-doped rats at doses up to 80 mg/kg, but NO levels were similar to those detected in a clonic convulsion. These results support the notion that NO does not directly induce a clonic convulsion, but may be generated as a consequence of onset of seizure.

Analysis of Variance↗

PubMed2002

In vivo fate of superparamagnetic iron oxides during sepsis.

The enzymatic generation of nitric oxide (NO) in vivo has been reported to be modulated by ions, such as copper and iron. Superparamagnetic iron oxide (SPIO) or ferumoxides is a liver-specific magnetic resonance contrast agent that is taken up by the Kupffer cells, where NO is generated by inducible nitric oxide synthase (iNOS). Thus, it is important to evaluate SPIO in vivo under conditions, such as infectious disease, where significant amounts of NO are generated by iNOS. In this study, we monitored the pharmacokinetics of SPIO in the liver of septic-shock mice and rats. A significant decrease in the ferric iron EPR signal was observed during NO generation in septic-shock mice compared with control mice doped with only SPIO. These results were also confirmed in a model reaction system consisting of SPIO and the NO donor, S-nitroso-N-acetyl DL penicillamine (SNAP). We compared NO generation quantitatively in the liver of the septic-shock rats, either in the presence or absence of SPIO, and found that the presence of SPIO did not affect the NO-generating activity of NOS expressed in the liver. T2-weighted MR images of an agarose gel phantom containing different SPIO to NO donor (SNAP) ratios clearly demonstrated that the contrast enhancement by SPIO decreased with increasing NO at constant SPIO levels. The reduced contrast is most probably due to the reduction of ferric to ferrous irons, resulting in a decrease in paramagnetic relaxation of water protons. These results show that SPIO can be a versatile NO-sensitive indicator, especially employing MRI as a powerful tool to 'visualize' sites of NO generation.

Animals↗