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Biomedical subjects

Kazunori Ikebukuro

Publications and source records attributed to Kazunori Ikebukuro.

At least 19 recordsLinked to original sources
PubMed2007

In silico panning for a non-competitive peptide inhibitor.

BACKGROUND: Peptide ligands have tremendous therapeutic potential as efficacious drugs. Currently, more than 40 peptides are available in the market for a drug. However, since costly and time-consuming synthesis procedures represent a problem for high-throughput screening, novel procedures to reduce the time and labor involved in screening peptide ligands are required. We propose the novel approach of 'in silico panning' which consists of a two-stage screening, involving affinity selection by docking simulation and evolution of the peptide ligand using genetic algorithms (GAs). In silico panning was successfully applied to the selection of peptide inhibitor for water-soluble quinoprotein glucose dehydrogenase (PQQGDH). RESULTS: The evolution of peptide ligands for a target enzyme was achieved by combining a docking simulation with evolution of the peptide ligand using genetic algorithms (GAs), which mimic Darwinian evolution. Designation of the target area as next to the substrate-binding site of the enzyme in the docking simulation enabled the selection of a non-competitive inhibitor. In all, four rounds of selection were carried out on the computer; the distribution of the docking energy decreased gradually for each generation and improvements in the docking energy were observed over the four rounds of selection. One of the top three selected peptides with the lowest docking energy, 'SERG' showed an inhibitory effect with Ki value of 20 microM. PQQGDH activity, in terms of the Vmax value, was 3-fold lower than that of the wild-type enzyme in the presence of this peptide. The mechanism of the SERG blockage of the enzyme was identified as non-competitive inhibition. We confirmed the specific binding of the peptide, and its equilibrium dissociation constant (KD) value was calculated as 60 microM by surface plasmon resonance (SPR) analysis. CONCLUSION: We demonstrate an effective methodology of in silico panning for the selection of a non-competitive peptide inhibitor from small virtual peptide library. This study is the first to demonstrate the usefulness of in silico evolution using experimental data. Our study highlights the usefulness of this strategy for structure-based screening of enzyme inhibitors.

Acinetobacter calcoaceticus↗
PubMed2006

Analysis of the evolution of the thrombin-inhibiting DNA aptamers using a genetic algorithm.

We previously identified a thrombin-inhibiting DNA aptamer that was presumed to form a G-quartet structure with a duplex. To investigate the importance of the sequences in the duplex region and to obtain aptamers with higher inhibitory activities, we randomized the sequences of the duplex region of this aptamer and carried out selection based on inhibitory activity using a genetic algorithm. This method consisted of selection via an inhibition assay, crossover, and mutation in silico. After two cycles, we obtained ligands with greater inhibitory activities than that of the original aptamer. In addition, the duplex sequences were found to contribute to the inhibitory activities of aptamers.

Algorithms↗
PubMed2006

Characterization and application of aptamers for Taq DNA polymerase selected using an evolution-mimicking algorithm.

Using an evolution-mimicking algorithm (EMA), we have recently identified DNA aptamers that inhibit Taq DNA polymerase. In the present study, we have attempted to improve further the inhibitory activities of aptamers, as well as to characterize those aptamers with the most potent inhibitory activities. To characterize the most potent aptamer and demonstrate its applicability, the abilities to inhibit Tth DNA polymerase and to modulate specific amplification in PCR were investigated. This aptamer inhibited both Tth DNA polymerase and Taq DNA polymerase and improved the specificity of detection of a low-copy-number target gene in PCR using these DNA polymerases.

Algorithms↗
PubMed2006

Pyrroloquinoline quinone (PQQ) prevents fibril formation of alpha-synuclein.

Pyrroloquinoline quinone (PQQ) is a noncovalently bound cofactor in the bacterial oxidative metabolism of alcohols. PQQ also exists in plants and animals. Due to its inherent chemical feature, namely its free-radical scavenging properties, PQQ has been drawing attention from both the nutritional and the pharmacological viewpoint. alpha-Synuclein, a causative factor of Parkinson's disease (PD), has the propensity to oligomerize and form fibrils, and this tendency may play a crucial role in its toxicity. We show that PQQ prevents the amyloid fibril formation and aggregation of alpha-synuclein in vitro in a PQQ-concentration-dependent manner. Moreover, PQQ forms a conjugate with alpha-synuclein, and this PQQ-conjugated alpha-synuclein is also able to prevent alpha-synuclein amyloid fibril formation. This is the first study to demonstrate the characteristics of PQQ as an anti-amyloid fibril-forming reagent. Agents that prevent the formation of amyloid fibrils might allow a novel therapeutic approach to PD. Therefore, together with further pharmacological approaches, PQQ is a candidate for future anti-PD reagent compounds.

Amyloid↗
PubMed2006

Homogeneous DNA sensing using enzyme-inhibiting DNA aptamers.

A novel aptameric enzyme subunit (AES) which can detect target DNAs has been developed. AES is an enzyme-inhibiting aptamer bearing a target-molecule binding site which can allosterically control enzymatic activity. The thrombin-inhibiting aptamer bearing a G-quartet structure was chosen as the enzyme-inhibiting aptamer. The stem-and-loop structure, which contains a strand complementary to the target DNA, was inserted into the G-quartet structure of the thrombin-inhibiting aptamer to disrupt the G-quartet structure through the hybridization of the target DNA with the complementary strand in the AES. The disruption of the G-quartet structure led to a decrease of the inhibitory activity of the whole aptameric complex. Using this designed aptamer, we were able to detect target DNAs by measuring the thrombin activity in a homogeneous solution without bound/free separation, and the lower detection limit was 20 nM.

Aptamers, Peptide↗
PubMed2006

A screening method for DNA aptamers that bind to a specific, unidentified protein in tissue samples.

Aptamers are oligonucleotide ligands with a high affinity to, and specificity for, various target molecules and they are expected to be powerful tools for proteomic analysis. To select aptamers that bind to a specific unidentified protein in tissues for protein analysis, a screening method was developed using chicken skeletal muscle as a model. Target proteins in the target mixture were separated by electrophoresis and transferred to a membrane, and a DNA library was added onto it. The aptamers that bound to the target protein were visualized by chemiluminescence and collected by cutting out the visualized band. The specific aptamers to the target protein were selected by only one round of selection using this screening, suggesting this screening method might be useful for selecting aptamers for proteome analysis.

Animals↗
PubMed2006

Aptamer selection based on inhibitory activity using an evolution-mimicking algorithm.

In order to efficiently select aptamers that bind to and inhibit proteins, we developed a method that involves screening DNA aptamers based on their inhibitory activities using an evolution-mimicking algorithm after the pre-selection by SELEX. The value of this method was demonstrated by the identification of an inhibitor of Taq DNA polymerase in a unique single-stranded DNA library, which was expected to form a G-quartet structure. This method consists of selection via an inhibition assay, sequence shuffling, and mutation in silico. After six rounds of selection, the inhibitory activities of the aptamers had evolved significantly. This demonstrates the utility of this strategy for screening aptamers based on their inhibitory actions.

Algorithms↗
PubMed2006

Aptameric enzyme subunit for biosensing based on enzymatic activity measurement.

The aptameric enzyme subunit (AES), which is a DNA aptamer composed of an enzyme-inhibiting aptamer and a target molecule-binding aptamer, has been developed for the biosensing of target molecules. We used a thrombin-inhibiting aptamer as the aptamer that inhibits enzymatic activity. The thrombin-inhibiting aptamer folds into the G-quartet structure, which plays an important role in its inhibitory activity. As a target molecule-binding aptamer, an adenosine-binding aptamer was inserted into the G-quartet structure of the thrombin-inhibiting aptamer to enable the change of the G-quartet structure upon the recognition of adenosine. In the present study, the change in the G-quartet structure led to a change in the thrombin inhibition activity, and adenosine was successfully detected by measuring the thrombin activity in a homogeneous solution without bound/free separation. We constructed two kinds of AESs; one of the structures is universal and can be used for designing any target molecule-binding aptamer. Since the enzyme activity is measured, AESs enable the simple and high-sensitivity detection of target molecules in a homogeneous assay.

Adenosine↗
PubMed2005

Development of photocatalytic biosensor for the evaluation of biochemical oxygen demand.

The photocatalytic biosensor of flow system using semiconductor TiO2 was developed to evaluate biochemical oxygen demand (BOD) levels in river water. Photocatalysis of sample was carried out in a photoreactor with TiO2 and a 6W black-light blue fluorescent tube as light source. Sample from a photoreactor outlet was measured by an oxygen electrode with a biofilm. The sensor response of photocatalytic biosensor was between 5 and 10 min depending on concentration of biochemical in the samples. At BOD of 1 mgl-1, the sensor response increased 1.33-fold in comparison with that without photocatalysis. The degradation of tannic acid and humic acid with photocatalysis were 51.8 and 38.4%, respectively. Gum arabic and linear alkylbenzene sulfonate (LAS) were degraded a little, but gave the responses of more than double to the sensor. Free radicals yielded by photocatalysis in a photoreactor did not affect the sensor response because their lifetime is extremely short. Fairly good correlation (r=0.983) between the sensor method and the conventional method was obtained for test samples. This biosensor using photocatalytic pretreatment improved the sensitivity.

Biofilms↗
PubMed2005

A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm.

Thrombin-inhibiting DNA aptamers have already been obtained through the systematic evolution of ligands by exponential enrichment (SELEX). However, SELEX is a method that screens DNA aptamers that bind to their target molecules, and it sometimes fails to screen good inhibitors. Therefore, it is necessary to develop a method of screening DNA aptamers based on their inhibitory effects on the target molecules. We developed a novel method of detecting aptamers using an evolution-mimicking algorithm, and we applied it to the search of new aptamers which inhibit thrombin. First, we randomly designed and synthesized ten 15mer oligonucleotides presumed to form G-quartet structures, and then measured their thrombin-inhibiting activities. The aptamers showing high inhibitory activity were selected, and we shuffled and mutated those sequences in silico to generate 10 new sequences of next-generation aptamers. After repeating the cycle five times, we successfully obtained the same aptamers reported previously, and they showed high inhibitory activity. In addition, we added 8mer oligonucleotides to both the 5' and the 3' end of the selected 15mer aptamers, and then repeated the evolution in silico. After two cycles, we were able to obtain aptamers with higher inhibitory activity than that of the 15mer aptamers.

Algorithms↗
PubMed2005

Development of a novel glucose enzyme fuel cell system employing protein engineered PQQ glucose dehydrogenase.

Glucose dehydrogenase harboring pyrroloquinoline quinone as the prosthetic group (PQQGDH) from Acinetobacter calcoaceticus is an ideal enzyme for the anode of biofuel cell, because of its oxygen insensitivity and high catalytic efficiency. However, the application of PQQGDH for the bioanode is inherently limited because of its instability. Using Ser415Cys mutant whose stability was greatly improved, we constructed the biofuel cell system employing the engineered PQQGDH as the bioanode enzyme and bilirubin oxidase (BOD) as the biocathode, and compared the stability of the biofuel cell with that employing wild-type PQQGDH. The maximum power density was 17.6 microW/cm2 at an external optimal load of 200 k omega. Using Ser415Cys mutant, the lifetime of the biofuel cell system was greatly extended to 152 h, more than six times as that of the biofuel cell employing the wild-type.

Acinetobacter calcoaceticus↗
PubMed2005

Novel electrochemical sensor system for protein using the aptamers in sandwich manner.

Novel electrochemical detection system for protein in sandwich manner using the aptamers was developed. Two different aptamers, which recognize different positions of thrombin, were chosen to construct sandwich type sensing system for protein, and one was immobilized onto the gold electrode for capturing thrombin onto the electrode and the other was used for detection. To obtain the signal, the aptamer for detection was labeled with pyrroquinoline quinone glucose dehydrogenase ((PQQ)GDH), and the electrical current, generated from glucose addition after the formation of the complex of thrombin, gold immobilized aptamer and the (PQQ)GDH labeled aptamer on the electrode, was measured. The increase of the electric current generated by (PQQ)GDH was observed in dependent manner of the concentration of thrombin added, and more than 10nM thrombin was detected selectively. The batch type protein sensing system was constructed using the two different aptamers sandwiching thrombin and it showed linear response to the increase of the thrombin concentration in the range of 40-100 nM.

Biosensing Techniques↗
PubMed2005

Screening of DNA aptamers against multiple proteins in tissue.

We report the development of a novel screening method for DNA aptamers against multiple proteins in the target tissue. Although a purified single protein is generally used in screening of aptamers at present, such a simultaneous selection would be very advantageous in efficiency and selectivity. We first carried out the screening in situ and it was suggested that aptamers against particular target proteins were enriched. We also developed another novel screening method named Aptamer Blotting based on protein separation by PAGE and its visualization with fluorescein labeled aptamers and it was demonstrated that this method allowed the simultaneous selection of specific aptamers against multiple proteins.

Animals↗
PubMed2005

Development of a novel sensing probe using DNA aptamer inhibiting enzymatic activity.

A novel sensing probe for adenosine has been developed using the thrombin-inhibiting DNA aptamer fused with the adenosine-binding DNA aptamer. The adenosine aptamer was inserted into the G-quartet structure of the thrombin aptamer to change its inhibitory activity to thrombin upon binding of the adenosine aptamer part to adenosine. This designed aptamer had lower inhibitory activity than that of the original thrombin aptamer, and it was increased in the presence of adenosine. CD spectra of this designed aptamer indicated that its structure was changed by its binding to adenosine. Using this designed aptamer, we were able to detect adenosine by measuring thrombin activity in homogeneous assay.

Adenosine↗
PubMed2004

Single nucleotide polymorphism typing on DNA array with hydrophobic surface fabricated by plasma-polymerization technique.

A DNA array has been fabricated on glass substrates, which enables high-throughput analysis of single-base mismatches. In this work, microfabrication-compatible plasma-polymerization (PP) method was used for immobilizing probe DNAs to study the hybridization behavior by changing surface properties. The immobilization matrix consisting of 35 A of PP layer, applied additionally on the streptavidin absorbed hexamethyldisiloxane (HMDS)-PP layer, was constructed on the substrates to anchor biotinylated DNA probes onto the surface. The hydrophobic immobilization matrix was considered to enhance hybridization accuracy and efficiency, compared with its hydrophilic acetonitrile-PP layers. The oligonucleotide arrays fabricated on HMDS-PP surface were shown to be effective in detection of single nucleotide polymorphisms (SNPs) of ApoE gene.

Apolipoproteins E↗
PubMed2004

Development of a novel DNA sensing system using DNA aptamer inhibited enzymatic activity 1.

A novel DNA detection system has been developed using the DNA aptamer inhibiting thrombin activity. The complementary strand to the target DNA was inserted into the loop region of the G-quartet structure of this adapter aiming at the disruption of it by the hybridization of the target DNA with its complementary strand in the aptamer. Using this modified aptamer, we were able to detect the target DNA by measuring thrombin activity.

Aptamers, Nucleotide↗
PubMed2004

Development of a novel DNA sensing system using DNA aptamer that inhibits enzymatic activity 2.

We have developed novel DNA sensor system using the DNA aptamers for thrombin. We modified original thrombin aptamer by addition of the target DNA complementary strands to the 5' end or 3' end of aptamer. Inhibitory activity of this modified aptamer increased in the presence of target DNA. We were able to detect the target DNA by measuring thrombin activity using this aptamer without the separation of bound and free separation.

Aptamers, Nucleotide↗