Biomedical subjects
Publications and source records attributed to Kazuo Tanaka.
Photostimulated hole transport through DNA duplexes immobilized on gold electrodes has been investigated. By modifying a gold electrode with a DNA duplex containing a photosensitizer, we have observed a sequence-dependent cathodic photocurrent. DNA acts as a good mediator for cathodic photocurrent when appropriate sequences are selected.
We report on the enzymatic synthesis of DNA wires by successive incorporation of artificial nucleotides. We synthesized an efficient hole-transporting nucleoside triphosphate, d(MD)ATP, and have examined the screening of polymerases and reverse transcriptases for enzymatic incorporation of d(MD)ATP into DNA. We found that KOD Dash is an effective enzyme for the enzymatic synthesis of (MD)A-containing DNA. Furthermore, the efficiency of enzymatic incorporation was remarkably improved by the addition of manganese chloride. By applying the PCR method to the synthesis of DNA containing a run of (MD)A, the (MD)A run was amplified up to 6 x 10(6) times. (MD)A runs prepared by the enzymatic method exhibited a high hole-transporting ability.
A conceptually new logic gate based on DNA has been devised. Methoxybenzodeazaadenine ((MD)A), an artificial nucleobase which we recently developed for efficient hole transport through DNA, formed stable base pairs with T and C. However, a reasonable hole-transport efficiency was observed in the reaction for the duplex containing an (MD)A/T base pair, whereas the hole transport was strongly suppressed in the reaction using a duplex where the base opposite (MD)A was replaced by C. The influence of complementary pyrimidines on the efficiency of hole transport through (MD)A was quite contrary to the selectivity observed for hole transport through G. The orthogonality of the modulation of these hole-transport properties by complementary pyrimidine bases is promising for the design of a new molecular logic gate. The logic gate system was executed by hole transport through short DNA duplexes, which consisted of the "logic gate strand", containing hole-transporting nucleobases, and the "input strand", containing pyrimidines which modulate the hole-transport efficiency of logic bases. A logic gate strand containing multiple (MD)A bases in series provided the basis for a sharp AND logic action. On the other hand, for OR logic and combinational logic, conversion of Boolean expressions to standard sum-of-product (SOP) expressions was indispensable. Three logic gate strands were designed for OR logic according to each product term in the standard SOP expression of OR logic. The hole-transport efficiency observed for the mixed sample of logic gate strands exhibited an OR logic behavior. This approach is generally applicable to the design of other complicated combinational logic circuits such as the full-adder.
We report on a new method for the detection of a base at a specific site in a DNA sequence by monitoring the fluorescence emission of fluorescein. To achieve this goal, we developed a new base-discriminating fluorescent (BDF) nucleobase, naphthodeazaadenine ((ND)A). The fluorescence spectrum of the duplex possessing a cytosine base as a complementary base of (ND)A showed a fluorescence peak at 383 nm when using an excitation wavelength of 350 nm. When the complementary base of (ND)A was one of the other bases, the fluorescence intensity was very low. The fluorescence emission spectrum of (ND)A overlapped with the fluorescence excitation spectrum of fluorescein in the wavelength range of 400-500 nm. Thus, we designed FRET-BDF probes containing (ND)A as the FRET donor and fluorescein as the acceptor. The interaction of these two fluorophores, which are separated by defined base pairs, allowed an efficient energy transfer that resulted in a dominant fluorescence emission of fluorescein at 520 nm when using an excitation wavelength of 350 nm. Fluorescence emission from FRET-BDF probes was observed only when the complementary base of (ND)A is C, thus achieving a clear distinction of a C base on the complementary DNA strand. However, the general utility of our method is limited due to the quenching of the (ND)A fluorescence by a G/C base pair flanking (ND)A.
Diffraction of an optical field by an aperture in a thick metallic screen is analyzed numerically by use of a three-dimensional volume integral equation together with a generalized conjugate residual method and fast Fourier transformation. Numerical results were validated by reciprocity and the independence of the results of the truncated discretized volume size used in numerical calculations. Near and far fields of square, circular, and triangular apertures in a thick screen are obtained numerically. Some of the numerical results obtained in the present study agree with previously reported experimental results. The surface plasmon polaritons excited on the sidewalls of the aperture can explain the basic characteristics of near-field distribution of apertures. The Bethe-Bouwkamp theory was found to be insufficient to explain the basic characteristics of the near field around the subwavelength aperture in a practical metallic screen.
We have developed a novel nucleosbase, edaG, that efficiently releases various reporter units upon one-electron oxidation. The edaG-selective degradation of ODNs was achieved by various mild oxidizing agents. The oxidant-dependent molecular releasing technique is quite useful not only for DNA-based drug releasing systems but also for the detection of long-range hole transport through DNA without time-consuming analysis.
It has been reported that spontaneous or interferon (IFN)-induced hepatitis B e (HBe) seroconversion has usually been associated with the development of a stop codon in the precore region. However, the difference between lamivudine-induced seroconversion and spontaneous or IFN-induced seroconversion is not known. The aim of this study was to investigate the correlation between the evolution of the precore and core promoter mutations and lamivudine-induced seroconversion. Forty-five patients with chronic hepatitis B virus (HBV) infection who were treated with lamivudine for more than 1 year were enrolled. The nucleotide sequence of the precore and core promoter region was determined before and after treatment with lamivudine for 1 year. Among 29 patients who were hepatitis B e antigen (HBeAg)-positive before treatment, 12 (41.3%) lost HBeAg during the course of treatment for 1 year. Of these, eight patients (66.7%) still had precore wild type HBV after 1 year. After 1 year, reversion to precore wild type HBV was detected in 11 (64.7%) of 17 patients who had precore mutant HBV before treatment. Twelve (70.6%) of 17 patients who were persistently HBeAg-positive had precore wild type HBV before and after treatment for 1 year. Despite the loss of HBeAg, two thirds of the patients still had precore wild type HBV after the 1-year treatment. It is suggested that lamivudine-induced seroconversion differs from spontaneous or IFN-induced seroconversion in the change of nucleotides in the precore region. The reversion in the precore region may be caused by the difference of drug-susceptibility to lamivudine. The antiviral effect of lamivudine may be more effective in the precore mutant HBV than in the precore wild type HBV.
In 1990, we conducted an epidemiological study of 509 residents in a hepatitis C virus (HCV) hyperendemic area in Japan. The purpose of the present study was to examine the approach to therapy for liver diseases accompanied by HCV and hepatitis B virus (HBV) infections among the surviving residents in the town after 12 years. Fifty-three with HCV or HBV infections among 385 people who resided in the town were clearly analyzed in 2002. The number of persons diagnosed with the liver diseases was as follows: HCV-related asymptomatic healthy carrier (1), past history of HCV infection (15), chronic hepatitis C (22), HCV-related liver cirrhosis (6), HCV-related hepatocellular carcinoma (HCC) (5), and HBV-related asymptomatic healthy carrier (4). HCC was detected in residents who did not have periodic regular hospital checkups. Only 19% of the 53 inhabitants consulted liver medical specialists, and 75% (3/4) who received interferon therapy received treatment from a liver medical specialist. It is necessary to provide continuous medical treatment to HCV carriers, minimizing difference in treatment quality in different medical institution. An efficient HCV medical checkup and a program of subsequence health management are important problems to be solved for improved health care.
A new type of long narrow aperture in a pyramidal structure on a thick metallic screen is proposed, and optical wave scattering by this structure is simulated. This aperture structure provides high emission intensity and small spot size simultaneously through excitation of the surface plasmon polaritons on the sidewalls of the pyramidal structure. Scattering of optical waves by this structure in the thick metallic screen is solved numerically with a volume integral equation by generalized conjugate residual iteration and fast Fourier transformation. The basic characteristics of the near-field intensities of the aperture are investigated in detail.
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We report a novel method for base detection using a base-discriminating fluorescent (BDF) nucleoside. We developed BDF probes containing methoxybenzodeazaadenine MDA and methoxybenzodeazainosine MDI, which give strong fluorescence only when the base on the complementary strand is cytosine and thymine, respectively. Thus, the MDA- and MDI-containing ODNs can be used as a very effective BDF probe for the detection of single base alterations, such as SNPs and point mutations. The present method using BDF probes is a very powerful tool for SNP typing that does not require any enzymes and time-consuming steps, and can avoid hybridization errors. In addition, a combination of MDA- and MDI-containing BDF probes facilitates the T/C SNP typing of a heterozygous sample.
DNA is a promising conductive biopolymer. However, there are problems that need to be solved to realize real DNA wires. These include the low efficiency of hole transport and the serious oxidative damage that can occur during hole transport. We have demonstrated a protocol for the design of a DNA wire that can effectively mediate hole transport that is not adversely affected by oxidation during hole transport through the DNA duplex. We have synthesized a stable and effective DNA wire by incorporating a designer nucleobase, benzodeazaadenine derivatives, which have lower oxidation potentials and wider stacking areas but are not decomposed during hole transport.
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BACKGROUNDS AND AIM: Clarithromycin (CAM)-resistant Helicobacter pylori sometimes offers serious problems with eradication by antibiotics. The aim of this study was to determine whether a probiotic can be an alternative therapy in CAM-resistant Hp infection. METHODS: The effects of Lactobacillus gasseri (strain OLL2716) on the growth of CAM-susceptible and CAM-resistant H. pylori and interleukin (IL)-8 production provoked by these strains were examined by in vitro experiments. Moreover, mice were infected with these CAM-susceptible or CAM-resistant H. pylori, and were treated with CAM or L. gasseri. RESULTS: In vitro experiments demonstrated that L. gasseri inhibited the growth of H. pylori and suppressed H. pylori-associated IL-8 production. Such effects were noted in CAM-resistant and CAM-susceptible H. pylori. Similarly, in an in vivo model of H. pylori infection, H. pylori colonization was significantly decreased by L. gasseri. CONCLUSION: Therefore, L. gasseri was found to act as a probiotic in CAM-resistant H. pylori infection.
DNA is known as a good hole carrier, and the application for a molecular wire has been studied. The efficiency of hole transfer was estimated as band intensity of phosphorimagery after electrophoresis in polyacrylamide gel. For the application of long-range hole transport through DNA to gene diagnosis and electrochemical technology, the hole transport without complicated and unwieldy analyzing processes is strongly desired. Herein, we developed novel nucleosides, which release various function units by oxidation. We synthesized novel nucleosides, which were designed based on the chemistry of an efficient hole trapping nucleoside 8-methoxy G.
New fluorescent nucleosides, methoxybenzodeazaadenine ((MD)A) and methoxybenzodeazainosine ((MD)I) which can sharply distinguish between C and T bases, respectably. The hybridization of an ODN probe containing (MD)A and (MD)I with a target DNA facilitates the judgment with the fluorescence spectra of the type of pyrimidine bases located at a specific site on the target DNA. The (MD)A- and (MD)I containing ODN are very effective probes for pyrimidine SNP typing.
The ODNs containing (MD)A, which effectively mediate hole transport, were enzymatically ligated, and photo-induced hole transport reaction proceeded efficiently through a ligated long duplex. The ligated duplex showed high hole transport efficiency.
OBJECTIVES: During treatment of chronic hepatitis B with lamivudine, changes in the level of hepatitis B virus (HBV) DNA were investigated using a real-time polymerase chain reaction (PCR) method with a detection limit of 1.7 log copies/ml (50 copies/ml) to clarify its clinical significance, particularly the association between HBV DNA levels and the emergence of tyrosine-methionine-aspartate-aspartate (YMDD) mutants. METHODS: Twenty-four patients who had received lamivudine therapy for >1 yr were studied. HBV DNA levels were determined using transcription-mediated amplification for sera with >3.7 log genome equivalents/ml, the Roche Monitor kit for sera with >/=2.6 log copies/ml, and real-time PCR for sera with < 2.6 log copies/ml (the detection limit was 1.7 log copies/ml). Patients were classified into three groups according to the minimal HBV DNA level attained during lamivudine therapy: the <1.7 log copies/ml group (eight patients), the 1.7-2.5 log copies/ml group (five patients), and the >/=2.6 log copies/ml group (11 patients). RESULTS: Pretreatment HBV DNA levels were significantly lower in the <1.7 copies/ml group than in the other two groups (p < 0.05). Neither the emergence of YMDD mutants nor a virological breakthrough of serum HBV DNA was observed in any of the eight patients in the <1.7 copies/ml group. In contrast, in the 1.7-2.5 copies/ml and >/=2.6 copies/ml groups, virological breakthroughs resulting from the emergence of YMDD mutants were observed in two of five patients and in all 11 patients, respectively (p < 0.001). Virological breakthroughs were observed at a mean of 49.6 +/- 18.4 wk in 11 the patients in the >/=2.6 copies/ml group and at wk 107 and 115 in two patients in the 1.7-2.5 copies/ml group. CONCLUSIONS: The real-time PCR method is useful for predicting the emergence of YMDD mutants and the estimated time of their emergence.