Biomedical subjects

Takahiro Kanagawa

Publications and source records attributed to Takahiro Kanagawa.

15 recordsLinked to original sources

PubMed2006

A proportional analysis method using non-kinetic real-time PCR.

Prompted by increasing interest in proportional analysis of genetic types, we developed a simple assay technique for determining the ratio of a specific target gene in the total genes that can be amplified with the same PCR primer. The key feature of this method is that the following two tasks are performed in a single-tube real-time PCR system: task 1, PCR amplification of the total genes including the target using a labeled PCR primer, with concurrent monitoring of the total copy number of the PCR product; task 2, detection of the signal of the target gene at each cycle of amplification, using a labeled nucleotide probe. In principle, the ratio of the target gene to the total genes is represented by the signal detected in 'task 2' at the cycle in which the PCR product reached a prescribed copy number (assessed by 'task 1').

DNA↗

PubMed2006

Affinity capillary electrophoresis with a DNA-nanoparticle conjugate as a new tool for genotyping.

We have developed a novel method for genotyping based on free solution affinity capillary electrophoresis. We prepared DNA-nanoparticle conjugates by mixing biotin-modified DNA and NeutrAvidin-modified polystyrene nanoparticles; this mixture was then injected into a capillary. Subsequently, we injected the fluorescent-labeled sample DNAs into the capillary, applied the voltage, increased its temperature after 7 min, and detected the fluorescence at its anodic end. This novel method was applied for genotyping human c-K-ras, and the three genotypes were definitely distinguishable with high reproducibility. This method can be easily automated, and it is useful for high-throughput gene mutation analysis.

Base Sequence↗

PubMed2005

Quantification of genetically modified soybean by quenching probe polymerase chain reaction.

Quenching probe (QProbe) polymerase chain reaction (PCR) is a simple and cost-effective real-time PCR assay in comparison with other real-time PCR assays such as the TaqMan assay. We used QProbe-PCR to quantify genetically modified (GM) soybean (Roundup Ready soybean). We designed event-specific QProbes for Le1 (soy endogenous gene) and RRS (recombinant gene), and we quantified certified reference materials containing 0.1, 0.5, 1, 2, and 5% GM soybean. The TaqMan assay was also applied to the same samples, and the results were compared. The accuracy of QProbe-PCR was similar to that of TaqMan assay. When GM soybean content was 0.5% or more, the relative standard deviations of QProbe-PCR were less than 20%. QProbe-PCR is sensitive enough to monitor labeling systems and has acceptable levels of accuracy and precision.

DNA Probes↗

PubMed2005

Novel approach to quantitative detection of specific rRNA in a microbial community, using catalytic DNA.

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.

Base Sequence↗

PubMed2005

Bacterial communities in petroleum oil in stockpiles.

Bacterial communities in crude-oil samples from Japanese oil stockpiles were investigated by 16S rRNA gene cloning, followed by denaturing gradient gel electrophoresis (DGGE) analysis. 16S rRNA genes were successfully amplified by PCR after isooctane treatment from three kinds of crude-oil sample collected at four oil stockpiles in Japan. DGGE profiles showed that bacteria related to Ochrobactrum anthropi, Burkholderia cepacia, Stenotrophomonas maltophilia, Propionibacterium acnes, and Brevundimonas diminuta were frequently detected in most crude-oil samples. The bacterial communities differed in the sampling time and layer. Among the predominant bacteria detected in the crude oil, only three species were found for bacteria isolated on agar plates and were related to Burkholderia, Stenotrophomonas, and Propionibacterium, while Ochrobactrum sp. could not be isolated although this species seemed to be the most abundant bacterium in crude oil from the DGGE profiles. Using an archaea-specific primer set, methanogens were found in crude-oil sludge but not in crude-oil samples, indicating that methanogens might be involved in sludge formation in oil stockpiles.

Archaea↗

PubMed2004

A FRET-based analysis of SNPs without fluorescent probes.

Fluorescence resonance energy transfer (FRET) is a simple procedure for detecting specific DNA sequences, and is therefore used in many fields. However, the cost is relatively high, because FRET-based methods usually require fluorescent probes. We have designed a cost-effective way of using FRET, and developed a novel approach for the genotyping of single nucleotide polymorphisms (SNPs) and allele frequency estimation. The key feature of this method is that it uses a DNA-binding fluorogenic molecule, SYBR Green I, as an energy donor for FRET. In this method, single base extension is performed with dideoxynucleotides labeled with an orange dye and a red dye in the presence of SYBR Green I. The dyes incorporated into the extended products accept energy from SYBR Green I and emit fluorescence. We have validated the method with ten SNPs, which were successfully discriminated by end-point measurements of orange and red fluorescence intensity in a microplate fluorescence reader. Using a mixture of homozygous samples, we also confirmed the potential of this method for estimation of allele frequency. Application of this strategy to large-scale studies will reduce the time and cost of genotyping a vast number of SNPs.

Benzothiazoles↗

PubMed2004

A new approach to SNP genotyping with fluorescently labeled mononucleotides.

Fluorescence resonance energy transfer (FRET) is one of the most powerful and promising tools for single nucleotide polymorphism (SNP) genotyping. However, the present methods using FRET require expensive reagents such as fluorescently labeled oligonucleotides. Here, we describe a novel and cost-effective method for SNP genotyping using FRET. The technique is based on allele-specific primer extension using mononucleotides labeled with a green dye and a red dye. When the target DNA contains the sequence complementary to the primer, extension of the primer incorporates the green and red dye-labeled nucleotides into the strand, and red fluorescence is emitted by FRET. In contrast, when the 3' end nucleotide of the primer is not complementary to the target DNA, there is no extension of the primer, or FRET signal. Therefore, discrimination among genotypes is achieved by measuring the intensity of red fluorescence after the extension reaction. We have validated this method with 11 SNPs, which were successfully determined by end-point measurements of fluorescence intensity. The new strategy is simple and cost-effective, because all steps of the preparation consist of simple additions of solutions and incubation, and the dye-labeled mononucleotides are applicable to all SNP analyses. This method will be suitable for large-scale genotyping.

Alleles↗

PubMed2004

Changes in phosphorus removing performance and bacterial community structure in an enhanced biological phosphorus removal reactor.

A lab-scale-enhanced biological phosphorus removal (EBPR) reactor was operated for 204 days to investigate the correlation between phosphorus removing performance and bacterial community structure. The phosphorus removing performance was good from day 1 to 92 and from day 172 to 204. However, the removal activity was in a deteriorated state from day 93 to 171. From day 69 (2 weeks before the beginning of the deterioration) to 118 (2 weeks after the beginning of the deterioration), sludge P content decreased. The amounts of ubiquinone-8 and menaquinone-8 (H(4)) decreased during this period while the amount of ubiquinone-10 increased. The comparison of these changes and the general attribution of each quinone to the bacterial phylogenetic groups suggested that beta proteobacteria and Actinobacteria contributed to EBPR positively, and that alpha proteobacteria were related to this EBPR deterioration. Glycogen accumulating organisms (GAOs) are considered to detrimentally affect EBPR ability by outcompeting the phosphorus accumulating organisms by using aerobically synthesized glycogen as the energy source to assimilate organic substrates anaerobically to form polyhydroxyalkanoates. However, in this research, there was nearly no substrate uptake during the anaerobic period at the middle of the deteriorated performance period. This suggests that the deterioration observed in this research does not agree with the GAOs inhibition model. In this research, the excess P release at the anaerobic period was concluded to cause the deterioration.

Actinobacteria↗

PubMed2004

Reevaluation and reduction of a PCR bias caused by reannealing of templates.

We reevaluated the bias toward a 1:1 ratio of products in multitemplate PCR used in ecological studies and showed that the template reannealing at the annealing step would not cause the bias; however, the preferential homoduplex formation during temperature decrease from denaturation to annealing step would cause the bias.

Actinomycetales↗

PubMed2003

Bias and artifacts in multitemplate polymerase chain reactions (PCR).

Polymerase chain reaction (PCR) is often used for the amplification of a mixture of homologous genes. PCR bias and artifact formation can occur in multitemplate PCR, and provide incorrect information on the abundance and diversity of genes. PCR bias and artifact formation occur at a higher rate during the last few cycles of the reaction, and therefore can be avoided by stopping the PCR earlier.

Journal Article↗

PubMed2003

Flow cytometric sorting, phylogenetic analysis and in situ detection of Oscillospira guillermondii, a large, morphologically conspicuous but uncultured ruminal bacterium.

Flow cytometric sorting based on its large cell size enabled an enriched fraction of Oscillospira guillermondii cells to be obtained from the rumen contents of a sheep. Phylogenetic analysis based on cloned 16S rDNA sequences indicated that the bacterium is a member of the low-G+C Gram-positive bacterial cluster. Sporobacter termitidis and Papillibacter cinnamivorans were the most closely related known species, with sequence similarities of only 86.3-88.1 %. Fluorescently labelled 16S rRNA-targeted oligonucleotide probes specific for Oscillospira were designed and applied to the rumen sample from which the enriched fraction was obtained. The probes hybridized specifically with the large, morphologically conspicuous Oscillospira cells.

Animals↗

PubMed2003

Extracellular acidic polysaccharide production by a two-membered bacterial coculture.

A two-membered coculture of strains KYM-7 and KYM-8, identified as Cellulomonas cellulans and Agrobacterium tumefaciens, respectively, produced a large amount of an extracellular polysaccharide, designated APK-78, from starch. Each strain in pure culture produced only very little amount of polysaccharide from starch; the coexistence of the two strains from the early stage of cultivation was indispensable for a large amount of polysaccharide to be produced. The polysaccharide APK-78 was acidic and composed of glucose, galactose, succinic acid, and pyruvic acid with a molar ratio of 8.1:1.0:1.7:1.0, indicating that it is a succinoglycan type of polysaccharide.

Agrobacterium tumefaciens↗

PubMed2002

Characterization of filamentous Eikelboom type 021N bacteria and description of Thiothrix disciformis sp. nov. and Thiothrix flexilis sp. nov.

The phenotypic and genotypic characteristics of 15 strains of Eikelboom type 021 N bacteria isolated from wastewater treatment plants were investigated. The strains shared many characters with Thiothrix species. However, the Eikelboom type 021N bacteria had only 88.3-92.0% 16S rDNA sequence similarity to members of the Thiothrix nivea group, including T. nivea, 'Thiothrix ramosa', Thiothrix unzii and Thiothrix fructosivorans, and were differentiated from them in sugar utilization and other properties, suggesting that the Eikelboom type 021N bacteria belong to species distinct from the T. nivea group. The 15 Eikelboom type 021N bacteria that were investigated were divided into three distinct groups (I to III) on the basis of their genotypic and phenotypic characteristics. The creation of two novel species is proposed, Thiothrix disciformis sp. nov. for the group I strains (type strain B3-1T = JCM 11364T = DSM 14473T) and Thiothrix flexilis sp. nov. for the group III strains (type strain EJ2M-BT = JCM 11135T = DSM 14609T). Thiothrix eikelboomii AP3T was included in group II and shared many characters with the other group II strains. The inclusion of all group II strains within the species T. eikelboomii is proposed,together with emendation of the description of T. eikelboomii.

Bacterial Proteins↗

PubMed2002

Identification of a gene essential for sheathed structure formation in Sphaerotilus natans, a filamentous sheathed bacterium.

Sphaerotilus natans, a filamentous bacterium that causes bulking in activated sludge processes, can assume two distinct morphologies, depending on the substrate concentration for growth; in substrate-rich media it grows as single rod-shaped cells, whereas in substrate-limited media it grows as filaments. To identify genes responsible for sheath formation, we carried out transposon Tn5 mutagenesis. Of the approximately 20,000 mutants obtained, 7 did not form sheathed structures. Sequencing of the Tn5-flanking regions showed that five of the seven Tn5 insertions converged at the same open reading frame, designated sthA. The deduced amino acids encoded by sthA were found to be homologous to glycosyltransferase, which is known to be involved in linking sugars to lipid carriers during bacterial exopolysaccharide biosynthesis. Disruption of the gene of the wild-type strain by inserting a kanamycin resistance gene cassette also resulted in sheathless growth under either type of nutrient condition. These findings indicate that sthA is a crucial component responsible for sheath formation.

Bacterial Proteins↗

PubMed2002

Photosynthetic apparatus in Roseateles depolymerans 61A is transcriptionally induced by carbon limitation.

Production of a photosynthetic apparatus in Roseateles depolymerans 61A, a recently discovered freshwater beta-Proteobacterium showing characteristics of aerobic phototrophic bacteria, was observed when the cells were subjected to a sudden decrease in carbon sources (e.g., when cells grown with 0.1 to 0.4% Casamino Acids were diluted or transferred into medium containing or=0.2% O(2)), and was reduced in the presence of light. Transcription of the R. depolymerans puf operon is considered to be controlled by changes in carbon nutrients in addition to oxygen tension and light intensity.

Aerobiosis↗