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Articles by K. Itoh
Total Records ( 4 ) for K. Itoh
  F Moriyasu and K. Itoh

OBJECTIVE. The purpose of this study was to assess the efficacy and safety of contrast-enhanced ultrasound performed with perflubutane microbubbles in comparison with unenhanced ultrasound and dynamic CT in the characterization of focal liver lesions during the vascular phase of imaging and in the detection of lesions during the Kupffer phase.

SUBJECTS AND METHODS. A total of 196 patients were enrolled at 15 centers in Japan. Vascular phase images were obtained before contrast injection until 1 minute after injection. Kupffer phase images were obtained 10 minutes after injection. Dual-phase CT was performed as determined by standard clinical practice at each center. Unenhanced ultrasound, contrast-enhanced ultrasound, and CT images were read by blinded reviewers, and the results they reached regarding characterization and detection were compared with reference standard findings made by onsite investigators. The safety observation period was 72 hours after contrast administration.

RESULTS. Among the 190 patients included in the characterization analysis, the accuracy of contrast-enhanced ultrasound (88.9%) was significantly greater than that of unenhanced ultrasound (68.4%) and dynamic CT (80.5%) (p < 0.001 and p = 0.008). Among the 191 patients in the detection analysis, the efficacy of contrast-enhanced ultrasound in detection of lesions was significantly higher than that of unenhanced ultrasound and dynamic CT (p < 0.001 and p = 0.008), predominantly because more metastatic lesions were detected (both p < 0.001). In particular, contrast-enhanced ultrasound was superior to dynamic CT in the detection of metastatic lesions measuring 1 cm or smaller. The incidence of adverse events was 49.2% and that of adverse drug reactions was 10.4%. All adverse drug reactions were mild.

CONCLUSION. Compared with unenhanced ultrasound and dynamic CT, contrast-enhanced ultrasound with perflubutane microbubbles improved diagnostic efficacy in both characterization and detection of focal liver lesions with no serious adverse drug reactions.

  R.A.A. Lelono , S. Tachibana and K. Itoh
  Gardenia jasminoides E. (Rubiaceae) methanol extracts showed the highest level of antifungal activity against Pleurotus ostreatus, a wood-rotting fungus, compared to five other methanol plants extracts; [Thuja orientalis L. (Cupressaceae), Datura innoxia (Solanaceae), Ligustrum japonicum T. (Oleaceae), Juniperus chinensis var. procumbens (Cupressaceae) and Mallotus japonica M. (Euphorbiaceae)] and selected for further analysis. Two antifungal compounds were isolated from n-butanol and ethyl acetate solubles in the methanol extracts of Gardenia jasminoides leaves and stems by bioassay-guided fractionation, using Pleurotus ostreatus. The antifungal compounds found for the first time in Gardenia jasminoides against Pleurotus ostreatus were identified as genipin and geniposide based on instrumental analyses. Both also had potent inhibitory effects on two plant pathogenic fungi; Fusarium oxysporum and Corynespora cassiicola.
  M Ohshima , K Inoue , H Hayashi , D Tsuji , M Mizugaki and K. Itoh

DNA methylation is involved in many diseases such as cancer and autoimmunity. We generated recombinant single-chain Fv (scFv) antibodies against 5-methyl-2'-deoxycytidine (m5dCyd) using phage display technology and a hyperimmunized mouse, and the scFv of most interest were contructed as fusion proteins with green fluorescent protein obtained from Aequorea coerulescens GFP (AcGFP). Using RNA isolated from mouse spleens, we constructed a scFv library consisting of light chains. The scFv library was selected against m5Cyd-BSA and enriched through four rounds of panning. The scFv library was concentrated about 390-fold and an individual clone was reacted with m5Cyd-BSA. Two scFvs with high reactivity for m5Cyd-BSA termed 1–2 and 1–12 were produced. Furthermore, methylated DNA-binding activities of the scFvs were confirmed using an indirect immunofluorescence assay. Additionally, N- and C-terminal scFv 1–2 fusion with AcGFP were constructed, and we observed the N-terminal AcGFP exhibited much higher fluorescence intensity than the C-terminal fusions. The AcGFP-scFv 1–2 modified N-terminus of scFv with AcGFP had high fluorescence intensity, but the scFv 1-2-AcGFP modified C-terminus of scFv with AcGFP had low fluorescence intensity. The cross-reactivity of AcGFP-scFv 1–2 was similar to scFv 1–2, and thus, AcGFP-scFv 1–2 could be used in a direct immunofluorescence assay. The scFv fusion proteins may be useful for the detection and quantification of cellular methylated DNA in various specimens.

  M Ohshima , T Tadakuma , H Hayashi , K Inoue and K. Itoh

We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m5dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly4-Ser)3. The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m5dCyd and weakly with 5-methylcytidine (m5Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Although the specificities of scFv and MoAb were almost identical, the sensitivity of the scFv (IC50 0.054 µg/ml) was ~80 times higher than that of the parent MoAb (IC50 4.27 µg/ml), determined by inhibition ELISA. As a biochemical application of this scFv, we quantified the m5dCyd content of genomic DNA by enzymatic hydrolysis using inhibition ELISA. The cancer cell lines HeLa, HeLa S3 and MDA-MB-453 contained ~1% of the methylated DNA in total genomic DNA, as did peripheral blood cell genomic DNA from healthy volunteers, but HT29 and T-47D showed hypomethylation compared with the HeLa, HeLa S3 and MDA-MB-453 cell lines. The scFv generated here may be applicable to the assessment of cellular DNA methylation levels and is more sensitive than the MoAb.

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