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Takanori Kubo

Publications and source records attributed to Takanori Kubo.

18 recordsLinked to original sources
PubMed2006

Dual-labeled telomere sensing probes for quantification of telomerase activity assay.

The present study describes an empirically discovered phenomenon that might be useful for development of a sensitive and rapid methodology for quantification of telomerase activity assay with simple data acquisition and possibility for calculation of telomerase product in absolute units. The method is based on the design and application of two single-stranded telomere sensing probes consisting of dual-labeled 16-mer oligonucleotides (fluorescent Cy3/Cy3-labeled and non-fluorescent IowaBlack/BHQ-labeled) that can simultaneously hybridize on the primary product of the telomerase reaction.

Base Sequence↗
PubMed2005

Controlled intracellular localization and enhanced antisense effect of oligonucleotides by chemical conjugation.

Oligonucleotides can be covalently linked to peptides composed of any sequence of amino acids by solid phase fragment condensation. The peptides incorporated into the conjugates include nuclear localizing signals (NLS), nuclear export signals (NES), membrane fusion domain of some viral proteins and some designed peptides with amphipathic character. Evaluation of biological properties of DNA-peptide conjugates indicated that (a) the conjugates could bind to target RNA and dsDNA with increased affinity, (b) the conjugates were more resistant to cellular nuclease degradation, (c) the conjugate-RNA hybrids could activate RNase H as effectively as native oligonucleotides, (d) the conjugates with fusion peptides showed largely enhanced cellular uptake, (e) the conjugates with NLS could be predominantly delivered into the cell nucleus, (f) the conjugates with NES could be localized in the cytoplasm. As a result, antisense oligonucleotides conjugated with NLS could inhibit human telomerase in human leukemia cells much more strongly than phosphorothioate oligonucleotides.

Amino Acid Sequence↗
PubMed2005

Efficient cleavage of RNA, enhanced cellular uptake, and controlled intracellular localization of conjugate DNAzymes.

Conjugate DNAzymes with polyamines and peptides were successfully prepared by solid phase fragment condensation (SPFC) and showed up to 4.2 times higher catalytic efficiency (k(cat)/K(m)) and enhanced tolerance against DNase 1digestion. To be pointed out, intracellular localization of DNAzymes could be controlled by conjugated with naturally occurring signal peptides responsible for nuclear cytoplasmic transport of proteins.

Base Sequence↗
PubMed2005

Suppression of bcr/abl chimeric gene by conjugate DNA enzymes in human cells.

Conjugate DNAzymes with polyamines and peptides were successfully prepared by solid phase fragment condensation (SPFC) and showed up to 4.2 times higher catalytic efficiency (kcat/Km). Intracellular localization of DNAzymes could be controlled by conjugated with naturally occurring signal peptides which are responsible for nuclear cytoplasmic transport of proteins. Suppression of bcr/abl chimeric gene on Philadelphia chromosome by conjugate DNA enzymes were largely enhanced in human leukemia cells.

Catalysis↗
PubMed2005

Highly sensitive inhibition of hTERT mRNA expression and telomerase activity by DNA-signal-peptide conjugates.

In the present study, we investigated the antisense properties of conjugate oligonucleotides (ODNs) inhibiting human telomerase activity. Conjugate oligonucleotides assembled with signal peptides, artificially designed peptides, amines and sugars were synthesized by solid phase fragment condensation (SPFC) in sufficient yields. Conjugate ODNs showed a high resistance to nuclease degradation and sufficient binding affinity to target RNA, comparatively rapid and sufficient intracellular delivery and specific localization controlled by signal peptides (nuclear localization signals, NLS; nuclear export signals, NES). ODN-NLS conjugates demonstrated high antisense inhibitory effects against human telomerase activity into the nucleus (e. g, phosphorothioate conjugate inhibited the telomerase activity over 95%), whereas ODN-NES conjugates inhibited target mRNA expression into the cytoplasm.

Biological Transport↗
PubMed2004

Atypical protein-kinase Czeta, but neither conventional Ca2+ -dependent protein-kinase C isoenzymes nor Ca2+ -calmodulin, participates in regulation of telomerase activity in Burkitt's lymphoma cells.

PURPOSE: To clarify the role of the pathways dependent on protein-kinase C (PK-C) and Ca2+/calmodulin (CaM) in the regulation of telomerase activity in Burkitt's lymphoma cells. METHODS: Burkitt's lymphoma cells (Raji and Daudi) were treated with the PK-C inhibitor, bisindolylmaleimide (BIM), or the CaM inhibitor, trifluoperazine (TFPZ), in a dose-dependent manner and in a time-dependent manner. The activities of PK-C isoenzymes were analyzed fluorimetrically using POLARIS assay kits. CaM-kinase II activity was analyzed radiographically, using CaMK-II immunoprecipitation kinase assay kits. Telomerase activity was detected by a conventional telomeric repeat amplification protocol and Stretch PCR. The level of catalytic subunit of telomerase (hTERT) in drug-treated and nontreated cells was analyzed by flow cytometry using anti-hTERT antibody labeled with ZenonAlexa Fluor-488 IgG. Apoptosis was estimated in terms of phosphatidylserine exposure on the cell surface and DNA fragmentation. RESULTS: It was found that BIM inhibited telomerase activity and this process preceded apoptosis. The subsequent addition of exogenous PK-C (mixture of isoenzymes) to the cell lysates restored telomerase activity if incubation of cells with BIM was up to 24 h. Using PK-C isoenzymes, it was established that atypical PK-Czeta, but not conventional Ca2+ -dependent PK-Calpha, PK-Cbeta or PK-Cgamma, is responsible for the reactivation of telomerase in BIM-treated cells. BIM also showed a well-expressed cytotoxicity against intact leukemia cells. In contrast, the CaM inhibitor TFPZ showed the same cytotoxic effect without any influence on telomerase activity during incubation for 24 h with leukemia cells. After incubation for 48 h, TFPZ markedly suppressed telomerase activity. However, the effect followed apoptosis and appeared to be a result of cell death. The addition of exogenous CaMK-II to the cell lysates obtained from TFPZ-treated cells did not reactivate telomerase. CONCLUSION: The present study confirmed the participation of atypical PK-Czeta, but not conventional Ca2+ -dependent PK-C isoenzymes (alpha, beta, gamma) nor the Ca2+/CaM-dependent pathway, in the regulation of telomerase activity in Burkitt's lymphoma cells.

Apoptosis↗
PubMed2004

Antisense inhibition of Bcr-Abl/c-Abl synthesis promotes telomerase activity and upregulates tankyrase in human leukemia cells.

Clinical studies in chronic myelogenous leukemia demonstrate that the overexpression of Bcr-Abl tyrosine kinase is usually accompanied by relatively low telomerase activity in the chronic phase, which reverts to a high activity in blast crisis. The present study was designed to investigate the cross-talk between both enzymes, using Bcr-Abl-positive K-562 and Bcr-Abl-negative Jurkat cell lines, treated with antisense oligodeoxyribonucleotides (ODNs) against Bcr-Abl/c-Abl mRNA. The decreased amount and enzyme activity of Bcr-Abl/c-Abl provoked telomerase activation in both cell lines. After short-term treatment with anti-Bcr-Abl/c-Abl ODNs (6 days), no variations in hTERT and phospho-hTERT were detected. The decreased amount of Bcr-Abl/c-Abl was accompanied by: alterations in telomeric associated proteins-overexpression of tankyrase and decreased amount of TRF1/Tin2, cell growth arrest of K-562 cells, reaching a plateau after 6 days treatment, and increased proliferating activity of Jurkat cells. No changes in telomere length were detected after short-term treatment. In contrast, after long-term treatment with anti-Bcr-Abl/c-Abl ODNs (36 days), a significant elongation of telomeres and enhancement of hTERT were established, accompanied by an increased proliferating activity of both cell lines. These data provide evidence that the inhibition of Bcr-Abl or c-Abl synthesis keeps a potential to restore or induce cell proliferation through telomere lengthening control and telomerase activation.

Base Sequence↗
PubMed2004

Control of intracellular delivery of oligonucleotides by signal peptides and genetic expression in human cells.

In the present study, membrane permeability and intracellular localization of oligonucleotide (ODN) conjugated with naturally occurring functional peptides or designed peptide were investigated, as well as antisense properties of them to inhibit of telomerase activities. All conjugate antisense ODNs showed higher membrane permeability and nuclease resistance than natural ODN. Intracellular localization of ODN could be precisely controlled by conjugation with functional peptides. Conjugate antisense ODNs indicated thousand fold higher inhibitory effects than natural ODN in cellular extract and 95% suppression in human leukemia cells.

Base Sequence↗
PubMed2004

Precisely controlled intracellular delivery of DNA-peptide conjugates.

Precisely controlled intracellular delivery of oligonucleotides was achieved by conjugation with signal peptides and amphiphilic designed peptides. Localization in the cellular nucleus was accelerated by nuclear localizing signals derived from naturally occurring viral proteins and arginine rich designed peptides, and cytoplasmic localization was enhanced by nuclear export signals and membrane fusion peptides.

Amino Acid Sequence↗
PubMed2003

Synthesis of DNA-peptide conjugates by solid-phase fragment condensation.

[reaction: see text] DNA-peptide conjugates were synthesized by condensing partially protected peptide fragments and oligonucleotides on a CPG support using diisocyanatoalkane as a linker. After cleavage and deprotection with aqueous ammonia, pure products were obtained by single RPHPLC purification in satisfactory yields and identified by a MALDI-TOF MS spectrometer. This method allows one to prepare DNA-peptide conjugates with any components and sequences of DNA and peptides.

Amino Acid Sequence↗
PubMed2003

Conjugate DNAzymes.

Conjugate DNAzymes were synthesized by solid phase fragment condensation and their biological properties were characterized. They have increased affinity to target RNA, enhanced stability against DNase 1 digestion and comparable or higher RNA cleaving activity compared with native and also phosphorothioate DNA zymes. It was also demonstrated that conjugate DNAzymes could inhibit BCR-ABL tyrosine kinase in cellular lysis of human leukemia cell line. Consequently DNAzymes can be expected to act effectively in cellular system and also in vivo system.

Base Sequence↗
PubMed2003

Antisense effects of DNA-peptide conjugates.

Antisense conjugate DNA covalently bound to functional amines, sugars or peptides such as nuclear localization signals (NLS), nuclear export signals (NES) and artificial amphiphilic alpha-helical and beta-sheet peptides were synthesized by solid phase fragment condensation (SPFC). Inhibitory effects on human telomerase of synthesized antisense conjugate DNAs were evaluated by TRAP assay. Antisense conjugate DNAs showed several times higher inhibitory effects on telomerase activity compared to native antisense DNA in cell lysis solutions. Antisense conjugate DNAs showed comparable or higher as same as inhibitory activity compared with antisense phosphorothioate DNA (S-DNA). Interestingly, antisense conjugate DNA showed much higher inhibition against telomerase activity with native antisense DNA or antisense phosphorothioate DNA in cellular system. We observed enhanced cellular uptake and positively controlled intracellular localization of conjugate DNAs by use of confocal fluorescent microscope.

Amino Acid Sequence↗
PubMed2003

Control of intracellular delivery and inhibition of genetic expression by DNA-peptide conjugates.

Various types of DNA-peptide conjugates were synthesized by solid phase fragment condensation (SPFC). DNA-LNS (nuclear localizing signal) peptide conjugate was proved to be delivered and localized into cellular nucleus and exhibited higher antisense inhibitory effect against telomerase than antisense phosphorothioate DNA. In contrast, DNAzymes conjugated with NES (nuclear export signal) peptide was shown to be taken up and localized in cytoplasm. Inhibitory effect of the conjugate DNAzyme against BCR-ABL tyrosine kinase was evaluated to be more significant than the native DNAzyme.

Amino Acid Sequence↗
PubMedDate not supplied

DNA conjugates as novel functional oligonucleotides.

Oligodeoxynucleotides with RNA cleavage activity 1) were conjugated with amines and peptides by solid phase fragment condensation (SPFC). It was found that 29 mer DNA enzyme conjugated with spermine at its 5'-end showed higher affinity to the target RNA sequence and 40 times higher activity of cleavage than native DNA enzyme. It is also to be noted that conjugate DNA enzymes showed increased resistance against nuclease digestion.

Base Sequence↗
PubMedDate not supplied

Synthesis of DNA conjugates by solid phase fragment condensation.

Development of a novel method for the synthesis of DNA conjugates is described. Oligonucleotides were successfully conjugated with a variety of functional molecules on a solid phase (Solid Phase Fragment Condensation) using an amino, a hydroxyl, a thiol, and a carboxyl group. DNA-peptide conjugate was obtained as a pure from by a single RPHPLC purification approximately in 20% yield. Moreover, it was demonstrated that the present method was effective for the preparation of conjugate molecules, DNA-sugar, DNA-polyamine, DNA-lipid and so on. The study to create new intelligent DNAs by accumulation various biofunctions on the molecule by SPFC is now in progress in our laboratory.

Base Sequence↗