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Well-differentiated liposarcoma (WD) acquires fully malignant potential when the histological progression named dedifferentiation occurs. This progression is supposed to occur in a time-dependent manner but this is still a debated issue. Clinically, the prediction of dedifferentiation for WD is very important from the therapeutic point of view. To identify genes that are predictive of dedifferentiation and to understand the mechanism of dedifferentiation, we investigated clinical information of 50 cases and studied the gene expression profiles of 36 lipomatous tumors using cDNA microarray. The clinical study showed that the dedifferentiation did not always seem to occur in a time-dependent manner. Interestingly, from the gene expression study, unsupervised hierarchical clustering analysis of well-differentiated lesions obtained from dedifferentiated liposarcoma (DD) cases that were indistinguishable from WD pathologically showed a clearly distinct gene expression pattern from WD. Using the pattern-matching program, 1687 genes including 487 known genes were identified, which discriminated WD cases from well-differentiated lipomatous lesions obtained from DD cases. These results suggest that the dedifferentiation may arise from different types of WD that could be distinguished from gene expression profiling but could hardly be classified by the pathological studies.

We successfully developed a novel selection method for the acquisition of DNA aptamers that selectively recognize resorufin using on‐chip selection in combination with method for point mutations. This method proved efficient for selection for DNA aptamers of single‐stranded oligo‐DNAs. A genetic algorithm was applied to produce oligonucleotides for the combinatorial library. A fluorescent molecule, resorufin, was applied for the ligand selection as a target. The binding affinity of the library was analyzed by the DNA chip. This selection method of DNA ligands includes on‐chip selection and point‐mutated sequence, which the highest affinity was selected. The fluorescence intensity of the library on the DNA chip increased after three repetitions of the selection round. The average of response for the affinity test increased with each generation.

A technique for detecting Raphidophycean, a bloom-forming genus of algae, was developed using a specific DNA probe. The design of the probe was based on a sequence polymorphism within the small subunit (SSU) ribosomal RNA gene (rDNA) of this strain by using fluorescence polarization (FP) analysis and the BIAcore 2000 biosensor, which utilized surface plasmon resonance (SPR). The specific sequence in SSU rDNA for Heterosigma carterae was determined by sequence data analysis. One pair of polymerase chain reaction (PCR) probes was designed for use in making the identification. H. carterae SSU rDNA was amplified by PCR. Using a fluoroscein isothiocyanate-labeled or biotin-labeled oligonucleotide probe, the PCR-amplified rDNA was selectively detected as an FP-intensity change via FP analysis or as a resonance-unit change via SPR. Although total time for final detection after sampling was within 3 hours, specific rDNA could be detected within 10 minutes after PCR through these detection methods.

We describe a novel sensor system to detect toxic chemicals based on measurement of the RT-PCR-amplified single-stranded DNA (ssDNA) of induced mRNA in Anabaena variabilis. Detection was conducted using a flow-injection type of sensor system based on surface plasmon resonance (SPR). The oligonucleotide probe containing the complementary sequence to a part of ribonuclease P RNA B (rnpB) and cyp110 mRNA was immobilized on the sensor chip. We succeeded in detecting 10 ng of amplified ssDNA using oligonucleotide probes. Using this sensor system, we were able to detect hexadecane using transcriptomic analysis of induced mRNA in A. variabilis treated with 0.2% (vol/vol) n-hexadecane. This method allows for rapid and simple detection of chemicals.

A real-time detection technique for mRNA was developed using a specific DNA probe. The design of the probe was based on a sequence polymorphism within the expressed mRNA of Anabaena variabilis PCC 7120 by using the BIAcore 2000 biosensor, which uses surface plasmon resonance (SPR). The reverse transcription polymerase chain reaction (RT-PCR)-amplified single-stranded DNA (ssDNA), specific for ribonuclease P RNA B (rnpB) from A. variabilis, was determined by an SPR biosensor. Through these detection methods, the ssDNA amplified from rnpB could be detected within 10 min after asymmetric RT-PCR.

A two-channel fluorometric sensor system for the detection of the water bloom phytoplankton Microcystis aeruginosa has been developed. Excitation wavelengths of 620 nm and 440 nm were used; the former for detecting the cyanobacteria themselves, and the latter for subtracting the interference due to eukaryotic algae present in the sample. The fluorescence of the cyanobacteria and eukaryotic algae was measured at 645 nm and 680 nm, respectively. Applied to real samples from Lake Kasumigaura, Japan, after ultrasonication, this fluorometric method is capable of the rapid determination of phycocyanin. We used this sensor system for monitoring surface water in Lake Kasumigaura in 9 months in laboratory experiments. Moreover, an automatic device based on this system was used for the continuous in situ monitoring of phycocyanin in lakewater. As expected, higher levels of phycocyanin due to cyanobacteria were observed, compared to chlorophyll a due to eukaryotic algae in summer.

A two-channel fluorometric sensor system for the detection of the waterbloom phytoplankton Microcystis aeruginosa has been developed. Excitation wavelengths of 620 and 440 nm were used, the former for detecting the cyanobacteria themselves, and the latter for subtracting the interference due to eukaryotic algae present in the sample. The fluorescence of the cyanobacteria and eukaryotic algae was measured at 645 and 680 nm, respectively. A linear relationship between phycocyanin fluorescence was established for the species examined in the range of 10−4 to 100 g/ml−1 chlorophyll a. Furthermore, upon ultrasonic treatment of a colony of cyanobacteria, the relationship between fluorescence intensity and cell concentration was improved. Applied to real samples in Lake Kasumigaura, Japan, after ultrasonication, this fluorometric method could detect waterbloom. This system is capable of the rapid determination of phycocyanin; the time required for one measurement cycle is approximately 25 min including sonication and rinsing for 5 min. The detection limit of this method is suitable for detecting phycocyanin in the early stage of waterbloom formation. Furthermore, a device based on this system was used for the continuous in situ monitoring of phycocyanin in lake water. © 2000 John Wiley & Sons, Inc. Field Analyt Chem Technol 4: 53–61, 2000

Two chemiluminescent flow-injection analysis systems for the detection of the red tide Phytoplankton Heterosigma carterae (formerly known as Heterosigma akashiwo ) have been developed. In one system, the superoxide (O − 2 ) released by H. carterae reacts with 2-methyl-6-( p -methoxyphenyl)-3,7-dihydroimidazo[1,2- a ]-pyrazin-3-one (MCLA), a superoxide specific probe. In the other system, the hydrogen peroxide released by H. carterae reacts with luminol catalyzed by Arthromyces ramosus peroxidase (ARP). The chemiluminescence is detected by a photomultiplier tube. This system is capable of the rapid determination of H. carterae ; the time required for one measurement cycle is ca. 2 min using MCLA-dependant luminescence or 1 min in luminol/ARP luminescence. A linear response was observed from 10 2 to 10 5 cells ml −1 H. carterae. Several other species of phytoplankton gave no response using this system. The detection limit of this method is suitable for detecting H. carterae in the early stage of red tide formation.

A chemiluminescent flow-injection analysis (FIA) system for the detection of the red tide phytoplankton Chattonella antiqua has been developed based on a Cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1, 2-α]-pyrazin-3-one (MCLA), which strongly emits light at 465 nm in the presence of superoxide. The system consisted of a two reagent feeding stream, a sample injector, a joint for mixing MCLA and sample, a chemiluminescence (CL) reaction cell, a CL detector and a recorder unit. The response time is approximately 1 min for one measurement cycle. The FIA system has an optimum pH of 10.7. The calibration curves for C. antiqua displayed linearity from 2 × 103 to 2 × 104 cells ml−1. When applied to the measurement of C. antiqua, the sensitivity obtained using the FIA system is approximately 10 times higher than that of the cytochrome c method. The FIA system is a rapid practical method for the detection of C. antiqua.

Transcriptome analysis revealed close relationship between solid-state cultivation and the transcriptional regulation of the genes on the non-syntenic blocks (NSBs), which were characterized by the comparison of Aspergillus oryzae genome with those of Aspergillus fumigatus and Aspergillus nidulans. Average expression ratio of the genes on NSBs in solid-state cultivation was significantly higher than that on the syntenic blocks (SBs). Of the induced genes, the genes relating to metabolism, which are highly enriched on NSBs, most contributed to the NSB-specific induction. The analysis using the SB- and NSB-genes that had sequence similarity between the two blocks significantly decreased the difference of average expression ratios between the two blocks. In spite of remarkably high averaged expression ratio of the NSB genes encoding extracellular enzymes, no induction of PKS and NRPS genes on NSBs were observed in solid-state cultivations. These results strongly suggest that the genes on NSBs play an important role on solid-state fermentation.