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Yoshio Kato

Publications and source records attributed to Yoshio Kato.

At least 19 recordsLinked to original sources
PubMed2006

Sequence-specific interference by small RNAs derived from adenovirus VAI RNA.

A virus-associated RNA (VAI) of adenoviruses is a cytoplasmic non-coding RNA and it plays an important role for viral replication in infected cells. VAI RNA transcripts, produced by RNA polymerase III (pol III), form tightly structured stems, which confer resistance to cellular defense systems. We demonstrate here that small RNAs of approximately 22 nucleotides are produced from a terminal stem region but not from an apical stem of VAI RNA. We determined the processing sites of VAI RNA by S1 nuclease mapping and further confirmed that the processed small RNA can act as small interfering RNAs (siRNAs) or as microRNAs (miRNAs) in transient transfection assays and during viral infection. Our data demonstrate that non-coding RNAs synthesized by pol III can be substrates for Dicer, and diced small RNAs might regulate cellular phenomena as siRNAs and miRNAs.

Adenoviridae↗
PubMed2006

Prognostic value of FDG-PET in patients with ovarian carcinoma following surgical treatment.

OBJECTIVE: [corrected] To determine the prognostic value of FDG-PET after surgical resection in patients with ovarian carcinoma, we compared the results of FDG-PET and serum CA-125 level and prognosis of patients. METHODS: Eighteen patients underwent a total of 32 FDG-PET examinations following surgery for ovarian carcinoma from October 2001 to December 2002 at our hospital (median follow-up period, 31 months). Age of the patients at the time of the initial FDG-PET examination ranged from 31 to 73 years (mean 52 years) and the period from surgery to the initial FDG-PET examination ranged from 5 to 109 months (mean 30 months). Serum CA-125 levels were determined on the occasion of each FDG-PET examination. Recurrent tumors were treated with surgery in 5 cases, radiotherapy in 2 cases, and chemotherapy in 9 cases. RESULTS: The initial FDG-PET examinations revealed that 13 cases had positive and 5 cases had negative findings, which included 2 false positive cases. The survival rate for all patients at 1 year and 2 years after the initial examination was 82% and 63%, respectively. Two-year survival rates in patients with positive and negative FDG-PET findings were 51% and 83%, respectively, and the difference was not statistically significant (p = 0.19). Furthermore, 4 patients with normal CA-125 levels and 14 patients with elevated CA-125 levels showed 2-year survival rates of 100% and 51%, respectively, and they were not significantly different (p = 0.11). For all 32 examinations, the 2-year survival rates for patients with normal CA-125 levels (100%) were significantly higher (p = 0.025) than that for patients with elevated CA-125 levels (47%), however there was no significant difference (p = 0.20) between FDG-PET positive cases (53%) and negative cases (83%). CONCLUSION The prognosis of patients with positive FDG-PET findings was less favorable than that of patients with negative findings. However, over the mean extended observation period of about 2.5 years, no significant difference in the prognosis of patients was observed between the two groups. The results of the present study indicate that elevated serum CA-125 levels may be more useful for evaluating the prognosis of ovarian cancer during the post-operative follow-up than FDG-PET findings.

Female↗
PubMed2006

Correlation of structure and activity of short hairpin RNA.

To explore the relationship between the structure of the precursor of short hairpin RNA (shRNA) and the activity of the shRNA-expression vector, 32 kinds of vectors, which expressed shRNAs with different flanking sequences, were tested. Addition of the poly(A) enhanced the activity of shRNA driven by U6 promoter. The activity of poly (A) tagged U6 promoter driven shRNA was lower than that of the CMV promoter driven constructs, in spite of the identical sequence of both transcripts. The result indicated that it is not only the structure but also the cascade of the transcripts that affects the RNAi activity.

Base Sequence↗
PubMed2006

Detection of the mature, but not precursor, RNA using a fluorescent DNA probe.

Fluorescently labelled oligonucleotide probes have been widely used in biotechnology and diagnostic field. We improved the molecular beacon probe to detect the mature, but not precursor, RNA selectively. Based on the principle that a kind of fluorophore can be quenched by adjacent guanine base, therefore, the noise fluorescence caused by the false targets can be avoided. As an example, we designed the probe for the selective detection of a mature microRNA (miRNA). We demonstrate that the probe detects mature miRNA with high specificity. Since the probe itself does not fluoresce but becomes fluorescent upon hybridization to the mature target RNA, it provides a quick and selective way for detection of various forms of RNAs.

DNA Probes↗
PubMed2005

Linear double-stranded DNA that mimics an infective tail of virus genome to enhance transfection.

Our previous work showed that a natural beta-(1-->3)-d-glucan schizophyllan (SPG) can form a stable complex with single-stranded oligonucleotides (ssODNs). When protein transduction peptides were attached to SPG and this modified SPG was complexed with ssODNs, the resultant complex could induce cellular transfection of the bound ODNs, without producing serious cytotoxicity. However, no technique was available to transfect double-stranded DNAs (dsDNA) or plasmid DNA using SPG. This paper presents a new approach to transfect dsDNA, showing preparation and transfection efficiency for a minimal-size gene having a loop-shaped poly(dA)(80) on both ends. This poly(dA) loops of dsDNA can form a complex with SPG. An siRNA-coding dsDNA with the poly(dA) loop was complexed with Tat-attached SPG to silence luciferase expression. When LTR-Luc-HeLa cells that can express luciferase under the control of the LTR promoter were exposed to this complex, the expression of luciferase was suppressed (i.e., RNAi effect was enhanced). Cytotoxicity studies showed that the Tat-SPG complex induced much less cell death compared to polyethylenimine, indicating that the proposed method caused less harm than the conventional method. The Tat-SPG/poly(dA) looped dsDNA complex had a structure similar to the viral genome in that the dsDNA ends were able to induce transfection and protection. The present work identifies the SPG and poly(dA) looped minimum-sized gene combination as a candidate for a non-toxic gene delivery system.

Adenosine↗
PubMed2005

Functional gene-discovery systems based on libraries of hammerhead and hairpin ribozymes and short hairpin RNAs.

Abundant information about the nucleotide sequence of the human genome has become readily available and it is now necessary to develop methods for the identification of genes that are involved in important cellular, developmental and disease-related processes. Identification methods based on the activities of hammerhead and hairpin ribozymes and of short hairpin RNAs (shRNAs), whose target specificities are coupled with loss-of-function phenotypes, have received increasing attention as possible tools for the rapid identification of key genes involved in such processes. We describe here recent advances that have been made with libraries of ribozymes and shRNAs and compare the advantages of the different types of library. The use of such libraries has already revealed new details of several important physiological phenomena.

Genetic Vectors↗
PubMed2005

Novel methods for expressing RNA interference in human cells.

RNA interference (RNAi) is a conserved process in which a double-stranded RNA (dsRNA) induces sequence-specific gene silencing. Recent developments in the use of the 21-nt small interfering RNA (siRNA) have allowed the specific degradation of mRNA without induction of nonspecific effects in mammalian cells. RNAi provides a method for knocking down genes of interest and a powerful tool for studies on gene functions in various organisms. Although many vector-based siRNA expression systems have been developed for production of siRNAs in mammalian cells, many technical issues for an effective production of siRNAs still need to be resolved. In this chapter, we describe methods for construction of genetically stable and highly active siRNA expression systems and also mention some strategies to overcome serious technical problems.

Base Sequence↗
PubMed2005

Chemistry-based RNA technologies: demonstration of usefulness of libraries of ribozymes and short hairpin RNAs (shRNAs).

Mechanism of action of hammerhead ribozymes has been investigated and their intracellular activities have been improved. Based on the improved ribozymes and more recently discovered natural RNAi, we have created libraries of both ribozymes and short hairpin RNAs (shRNAs). The introduction of a library of active ribozymes or shRNAs into cells, and the subsequent screening for phenotypic changes, allows the rapid identification of gene function.

Animals↗
PubMed2004

Analysis of the conserved P9-G10.1 metal-binding motif in hammerhead ribozymes with an extra nucleotide inserted between A9 and G10.1 residues.

Hammerhead ribozymes (Rz) have catalytically important tandem G:A pairs in the core region, and we recently demonstrated that the P9-G10.1 motif (a sheared-type G:A pair with a guanine residue on the 3' side of the adenine residue) with several flanking base pairs is sufficient for capture of divalent cations, such as Mg(2+) and Cd(2+) ions that are important to maintain full activities (Tanaka et al. J. Am. Chem. Soc. 2002, 124, 4595-4601; Tanaka et al. J. Am. Chem. Soc. 2004, 126, 744-752). We also found that mutant hammerhead ribozymes that have an additional G residue inserted between A9 and G10.1 residues (the metal-binding P9-G10.1 motif) have significant catalytic activities. In this study, we demonstrate that the hammerhead ribozymes are capable of maintaining the catalytically competent structure even when the tandem, sheared-type G:A pairs were perturbed by an insertion of an additional nucleotide, whereas the chirality of the phosphorothioate at the P9 position significantly influenced the enzymatic activity for both the natural and G-inserted ribozymes.

Adenine↗
PubMed2004

Phosphorylation at 5' end of guanosine stretches inhibits dimerization of G-quadruplexes and formation of a G-quadruplex interferes with the enzymatic activities of DNA enzymes.

During an analysis of DNA enzymes by gel electrophoresis, we found that some DNA enzymes can adopt more than one conformation. The DNA enzyme Dz31 that formed more than one conformer contained a stretch of G residues. Further investigations, involving kinetic analysis and measurements of circular dichroism, indicated that this DNA enzyme and its derivatives formed G-quadruplexes. Moreover, we found that some derivative oligomers were capable of forming dimeric G-quadruplexes. We also compared the catalytic activities of Dz31 and its mutant derivatives. The present findings suggest that DNA enzymes with five or more continuous G residues are less favorable than those without G5 in the association step in the enzymatic reaction and, thus, the choice of targets that contain a continuous stretch of C residues downstream of the cleavage site should be avoided. In addition, we found that negative charge-charge repulsion disrupted the dimerization of G-quadruplexes when a phosphate group was added directly to the 5'-terminal G of oligomers with continuous guanosine residues. In the case of 5'-phosphorylated G5CTA, direct attachment of a phosphate group to the continuous G5 sequence inhibited dimerization of G-quadruplexes, at least during electrophoresis on a denaturing gel.

Circular Dichroism↗
PubMed2004

Hydrophobic interaction chromatography at low salt concentration for the capture of monoclonal antibodies.

We evaluated hydrophobic interaction chromatography (HIC) at low salt concentration for the capture of proteins from feed stocks by using monoclonal antibodies as model samples. It was indicated that the HIC at low salt concentration on critical hydrophobicity supports has a potential for capturing hydrophobic monoclonal antibodies directly from large volumes of feed stocks and recovering bound monoclonal antibodies in high yield. On the other hand, the HIC at low salt concentration did not seem so useful for the capture of weakly hydrophobic monoclonal antibodies. The recovery of weakly hydrophobic monoclonal antibodies from columns packed with critical hydrophobicity supports was not quantitative and significantly decreased as the residence time of the monoclonal antibodies in the columns became longer.

Antibodies, Monoclonal↗
PubMed2004

Effect of chromatographic conditions on resolution in high-performance ion-exchange chromatography of proteins on macroporous anion-exchange resin.

We explored chromatographic conditions to obtain high resolution in protein separations by ion-exchange chromatography (IEC) on a macroporous anion-exchange resin of 10 microm in particle diameter. We studied effects of flow-rate, gradient time (steepness of salt concentration gradient) and column length on resolution in wide ranges. It was found that very high resolutions are attainable at long gradient times with long columns. The resolution continuously became higher as the gradient time and the column length became longer except in some special cases. The dependence of resolution on gradient time was particularly great when the column was long and the gradient time for the change of 0-0.5 M NaCl was longer than 2 h. On the other hand, the effect of flow-rate on resolution was very small. Although the separations at long gradient times with long columns have not been popular in high-performance IEC and it takes several hours for one separation, such separations should be advantageous when very high resolutions are required like in proteomics research.

Anion Exchange Resins↗
PubMed2004

Functional gene discovery using hybrid ribozyme libraries.

Hybrid ribozymes that couple the cleavage activity of hammerhead ribozymes with the unwinding activity of RNA helicases are powerful tools in the study of cell genetics and pharmaceutical drug development. They are useful for targeting a specific gene as well as screening functional genes to show phenotypic alterations. By randomizing the binding arms within the ribozymes, we can create a library of ribozymes that are capable of cleaving any mRNA. After introducing the library into cells and recovering the sequence information of ribozymes that alter the cell phenotype of interest, genes that are responsible for the specific phenotype can be identified. This chapter describes a method known as the gene-discovery system to identify novel functional genes related to a specific phenotype. Our gene-discovery system should be a powerful tool for post-genome research.

Base Sequence↗
PubMed2003

Effect of chromatographic conditions on resolution in high-performance ion-exchange chromatography of proteins on nonporous support.

We explored chromatographic conditions to obtain high resolution in protein separations by ion-exchange chromatography (IEC) on a nonporous anion-exchange resin of 2.5 microm in particle diameter. We studied the effects of gradient time (steepness of salt concentration gradient), flow-rate and column length on resolution in much wider ranges than had been studied before. It was found that two distinct conditions exist that provide high resolution. The first is a condition which has widely been employed in current high-performance IEC, namely, a combination of short gradient time, high flow-rate and comparatively short column. Separation times are usually 5-30 min, and even more rapid (1-2 min) separations are possible. The second is the condition which has rarely been employed in high-performance IEC. It is a combination of long gradient time, low flow-rate and long column. Although it takes several hours for one separation, very high resolution is attainable.

Chromatography, High Pressure Liquid↗
PubMed2003

Characterization of p-aminobenzamidine-based sorbent and its use for high-performance affinity chromatography of trypsin-like proteases.

An affinity sorbent, hydrophilic polymer-based carrier of different pore size (Toyopearl) with immobilized p-aminobenzamidine (ABA), has been prepared. Its basic properties and some applications for protein purification were studied. ABA, which is a synthetic inhibitor for trypsin-like proteases, was covalently immobilized to Toyopearl by reductive amination. The ligand density and binding capacity for porcine trypsin varied depending on the pore size of Toyopearl. The maximum binding capacity of the immobilized p-aminobenzamidine Toyopearl (ABA-Toyopearl) for trypsin was more than 40 mg/ml gel. ABA-Toyopearl thus obtained was very stable below pH 8 and was successfully used for high-performance affinity chromatography of trypsin-like proteases such as trypsin, thrombin, tissue-type plasminogen activator or urokinase in a single step at 25 degrees C.

Benzamidines↗
PubMed2003

Hydrophobicity gradient columns for the separation of trypsin inhibitor by hydrophobic interaction chromatography at low salt concentration.

We investigated hydrophobicity gradient columns composed of two columns packed with supports of different hydrophobicities in order to save time in protein separation by hydrophobic interaction chromatography at low salt concentration using a crude sample of trypsin inhibitor as a model sample. One of the two hydrophobicity gradient columns was packed with a support whose hydrophobicity was critically controlled for target protein (trypsin inhibitor) and the other was packed with a support which was less hydrophobic than the critically controlled hydrophobicity support. It was found that the hydrophobicity gradient columns are useful to separate samples containing impurities of a wide range of hydrophobicities within a reasonable time.

Chromatography, Liquid↗
PubMed2003

Expression of siRNA from a single transcript that includes multiple ribozymes in mammalian cells.

RNA interference (RNAi) has been developed recently as a powerful tool for silencing of mRNAs in various organisms. In mammalian cells, the introduction of small interfering RNAs (siRNAs) can inhibit gene expression in a sequence-specific manner without induction of the nonspecific degradation that is activated by long double-stranded RNA (dsRNA) (>30 nt). Here, we report a method for generating siRNAs in mammalian cells using a self-cleaving ribozyme-expressing vector. Four ribozymes within transcripts that were expressed under the control of a cytomegalovirus (CMV) or tRNAVal promoter excised, in cis, specific sense and antisense sequences from primary transcripts and generated siRNAs in HeLa cells. The siRNAs generated by the ribozymes were able to decrease the expression of a firefly gene for luciferase. These results suggest that polymerase II (pol II) systems, particularly in view of the availability of many potential tissue-specific promoters, and pol III systems, in which siRNAs are generated by a trimming-ribozyme (TRz) system as described here, should be useful in efforts to suppress the expression of specific genes.

5' Untranslated Regions↗