Biomedical subjects
Publications and source records attributed to Hiroaki Ozaki.
Triphosphate of a thymidine analogue bearing acridone was prepared and incorporated as a substrate for PCR using KOD Dash DNA polymerase forming a new fluorescent-labeled DNA that is useful as a DNA probe.
We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.
Concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) may affect its degradation kinetics in advanced oxidation systems, and combinations of two or more systems can be more effective for its mineralization at low concentration levels. Degradations and mineralizations of 0.045mM 2,4-D using O(3), O(3)/UV, UV/TiO(2) and O(3)/UV/TiO(2) systems were compared, and influence of reaction temperature on the mineralization in O(3)/UV/TiO(2) system was investigated. 2,4-D degradations by O(3), O(3)/UV and UV/TiO(2) systems were similar to the results of earlier investigations with higher 2,4-D concentrations. The degradations and total organic carbon (TOC) removals in the four systems were well described by the first-order reaction kinetics. The degradation and removal were greatly enhanced in O(3)/UV/TiO(2) system, and further enhancements were observed with larger O(3) supplies. The enhancements were attributed to hydroxyl radical (()OH) generation from more than one reaction pathway. The degradation and removal in O(3)/UV/TiO(2) system were very efficient with reaction temperature fixed at 20 degrees C. It was suspected that reaction temperature might have influenced ()OH generation in the system, which needs further attention.
Beer bran was found to effectively adsorb several organic compounds, such as dichloromethane, chloroform, trichloroethylene, benzene, pretilachlor, and esprocarb. Equilibrium adsorption isotherms conformed to the Freundlich isotherm (log-log linear). Adsorption of these organic compounds by beer bran was observed in the pH range of 1-11. At equilibrium, the adsorption efficiency of beer bran for benzene, chloroform, and dichiloromethane was higher than that of activated carbon. The removal of these organic compounds by beer bran was attributed to the uptake by intracellular particles called spherosomes. The object of this work was to investigate several adsorbents for the effective removal of organic compounds from wastewater.
Fluorescent DNA probes for L-argininamide were developed by a combination of DNA aptamers and fluorophore-quencher pairs. These molecules were synthesized by a combination of pre- and post-synthetic modification methods. The fluorescence-labeled aptamer could detect L-argininamide specifically. The binding affinities were defined by the binding affinity of the original aptamer to indicate that the end labeling of the aptamer did not influence the affinities.
A rapid infiltration (RI) system using granulated materials was developed for wastewater and river water treatment. Granules were processed by optimized procedures from soil and waste materials with high sorption of recalcitrant dissolved organic matter (DOM) and phosphorus. Granule performance was examined by laboratory column and pilot-scale tests. During the first year of the pilot test, removal of phosphorus was removed from livestock-farm effluent at the hydraulic loading rate of 2 m/day was 50-70% and that of refractory DOM was 10-30%.
Triphosphate of a thymidine analogue bearing acridone was prepared from a modified thymidine nucleotide with a terminal amino group at C5 position and an acridone derivative as a new fluorecsent-tagged nucleotide. The acridone-tagged nucleotide was incorporated into DNA enzymatically during PCR using KOD Dash DNA polymerase. Further, we introduced the acridone derivative into an amino-modified DNA chemically by post-synthetic modification, and investigated their fluorescence properties and hybridization ability. The new fluorescent-labeled DNA bearing acridone will be useful as a DNA probe.
We synthesized modified 2'-deoxyuridine triphosphates bearing amino acids at the C5 position and investigated their substrate properties for KOD Dash DNA polymerase during polymerase chain reaction (PCR). PCR using C5-modified dUTP having an amino acyl group (arginyl, histidyl, lysyl, phenylalanyl, tryptophanyl, leucyl, prolyl, glutaminyl, seryl, O-benzyl seryl or threonyl group) gave the corresponding full-length PCR products in good yield. Although dUTP analogues bearing aspartyl, glutamyl or cysteinyl were found to be poor substrates for PCR catalyzed by KOD Dash DNA polymerase, optimization of the reaction conditions resulted in substantial generation of full-length product. In the case of reaction using dUTP analogue having a cysteinyl group, addition of a reducing agent improved the reaction yield. Thus, PCRs using KOD Dash DNA polymerase together with amino acyl dUTP provide convenient and efficient preparation of various modified DNA libraries with potential protein-like activities.
A series of C5 substituted analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate were synthesized and investigated their substrate properties for thermostable DNA polymerases during polymerase chain reaction (PCR). The PCR assays indicated that the relative yield of the full-length product is dependent on whether the substitution is on dU or dC, and on the nature of the polymerase.
A binder that is specific for a target will be applicable to detection of a variety of molecules such as bioactive substances, environmental contaminants and so on. Nucleic acid aptamers obtained by in vitro selection method are interesting and promising binders. To enhance the ability of nucleic acid aptamers, we prepared a combinatorial modified DNA library containing arginyl uracil base by using arginyl dUTP, then attempted screening of glutamic acid-binding aptamers from the library by in vitro selection method.
1,2-Bis(1,10-phenanthrolin-2-yl)ethylenediamine (1) displays a unique long-life time excimer fluorescence at 528 nm by binding with calf thymus DNA, while its monomer fluorescence at 400 nm shows a decrease upon binding with DNA.
An oligodeoxyribonucleotide (ODN) containing three C5-substituted arabinofuranosyluracils was synthesized by the post-synthetic modification method from the ODN containing three C5-substituted 2,2'-anhydrouridines. The stability of the modified ODN/DNA duplex was lower than that of the corresponding normal duplex but that of the modified ODN/RNA duplex showed little change. The modified ODN could induce RNase H activity and was resistant against nuclease.
We produced a novel cationic-charged modified DNA aptamer for sialyllactose that is a ubiquitous component of the cell surface responsible for the infection of several viruses by using the magnetic-particle-based SELEX method. After 13 rounds of selection we selected 22 clones as sialyllactose-binding DNA aptamers composed of several modified thymidines. The DNA aptamers could form a three-way junction structure that likely forms a binding site for siallyllactose. The three-way junction structure contains several modified thymidines bearing a positively-charged amino group at the C5 position, which could enhance the binding ability for silalyllactose which has a negatively-charged carboxyl group. The dissociation constant of the aptamer that showed the strongest sialyllactose-binding ability among the clones of the aptamers was 4.9 microM.
PURPOSE: To determine whether ephrinB2 plays a role in ocular angiogenesis, we investigated the expression of ephrinB2 and EphB receptors on retinal fibroproliferative membranes. DESIGN: Experimental study of the expression of ephrinB2 and EphB receptors within fibroproliferative membranes in patients with ocular angiogenic diseases collected during vitrectomy. METHODS: Fibroproliferative membranes were obtained at the time of vitrectomy from 20 patients with proliferative diabetic retinopathy (PDR) and from 40 patients who had stage 5 retinopathy of prematurity. Specimens were investigated with immunohistochemistry using polyclonal antibodies directed against ephrinB2 and the EphB2, EphB3, and EphB4 receptors. Immunoreactivity for von Willebrand factor (factor VIII) and alpha-smooth muscle actin (alpha-SMA) was also determined to confirm the identity of the target vascular endothelial cells. RESULTS: Positive staining for ephrinB2 was observed on fibroproliferative membranes that were obtained from patients with PDR (65.0%) and retinopathy of prematurity (25.0%). Specifically, ephrinB2 was found to be present on endothelial cells, as confirmed by its colocalization with factor VIII and alpha-SMA staining. EphB2 and EphB3 expression was observed on fibroproliferative membranes that were harvested from patients with PDR (EphB2, 90.0%; EphB3, 70.0%) and retinopathy of prematurity (EphB2, 35.0%; EphB3, 45.0%). However, EphB4 expression was not observed in any of the membranes derived from patients with PDR or retinopathy of prematurity. The rate of ephrinB2 expression in patients with PDR was significantly higher than that seen in patients with retinopathy of prematurity, which probably reflected differences in the vascular density of their fibroproliferative membranes. CONCLUSION: These data suggest that the ephrinB2-EphB2/B3 system may play an important role in ocular angiogenesis.
The modified ODN's bearing C5-substituted 2'-deoxyuridine derivative were synthesized by a post-synthetic modification with an unsymmetrical triamine. The effect of the C5-substituent on the duplex formation with complementary DNA or RNA differed with the position of an imino group in the linker-arms.
Labeled oligodeoxyribonucleotide bearing fluorescent dyes at both ends and aptamer sequence for adenosine 5'-monophosphate (AMP) was synthesized. Fluorescence spectra of labeled aptamer were not so much different between with and without AMP. This result suggests the binding of AMP didn't cause the global structural change to the aptamer. Therefore, we used short complementary DNA (SCD) as an assistant DNA, which is an unmodified 11mer and have a complementary sequence of 5'-region of the labeled aptamer. In the presence of SCD, the fluorescence intensities decrease with increasing the concentration of AMP compared with a change in absence of SCD.
We attempted to utilize in vitro selection techniques to develop modified DNA aptamers that are specific for DNA secondary structure including a single-mismatch base pair. For the selection, two kinds of random modified DNA library were prepared by an enzymatic method using modified 2'-deoxynucleoside 5'-triphosphates and KOD Dash DNA polymerase: one library consists of modified DNA including amino functionality and another consists of modified DNA including both hydroxypropynyl and guanidium functionalities.
Nocardial infections in an immunocompromised host have been increasingly reported. Nocardial brain abscess, the most common presentation of nocardiosis in the central nervous system, is associated with a high mortality rate because of its delayed diagnosis and its unresponsiveness to the usual antibiotic therapy. We report four patients who experienced a long-term cure of nocardial brain abscess due to treatment by a combination of surgery and postoperative antibiotic therapy; 1 man and 3 women, ages ranging from 43 to 67 years old. Two patients were associated with systemic lupus erythematosus and two with autoimmune hemolytic anemia. Patients underwent surgical aspiration and drainage of brain abscess. Nocardia was identified from the aspirated specimen and postoperative antibiotic therapy for 5-6 weeks was performed using effective antibiotic agents; sulfamethoxazole/trimethoprim (ST), imipenem/cilastatin and minocycline (MINO) in Case 1, ST and MINO in Case 2, erythromycin in Case 3, and panipenem/betamipron and cefotaxime in Case 4. Case 3 and Case 4 with multilobulated brain abscess underwent total excision of the brain abscess. All patients showed successful cure of nocardial brain abscess with no recurrence for the period of 1-8 years. The combination of surgery and postoperative antibiotic therapy provides a good prognosis for nocardial brain abscess.