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Biomedical subjects

Tetsuji Yamaoka

Publications and source records attributed to Tetsuji Yamaoka.

At least 19 recordsLinked to original sources
PubMed2006

Three-dimensional cell seeding and growth in radial-flow perfusion bioreactor for in vitro tissue reconstruction.

Radial-flow perfusion bioreactor systems have been designed and evaluated to enable direct cell seeding into a three-dimensional (3-D) porous scaffold and subsequent cell culture for in vitro tissue reconstruction. However, one of the limitations of in vitro regeneration is the tissue necrosis that occurs at the central part of the 3-D scaffold. In the present study, tubular poly-L-lactic acid (PLLA) porous scaffolds with an optimized pore size and porosity were prepared by the lyophilization method, and the effect of different perfusion conditions on cell seeding and growth were compared with those of the conventional static culture. The medium flowed radially from the lumen toward the periphery of the tubular scaffolds. It was found that cell seeding under a radial-flow perfusion condition of 1.1 mL/cm2 x min was effective, and that the optimal flow rate for cell growth was 4.0 mL/cm2 x min. At this optimal rate, the increase in seeded cells in the perfusion culture over a period of 5 days was 7.3-fold greater than that by static culture over the same period. The perfusion cell seeding resulted in a uniform distribution of cells throughout the scaffold. Subsequently, the perfusion of medium and hence the provision of nutrients and oxygen permitted growth and maintenance of the tissue throughout the scaffold. The perfusion seeding/culture system was a much more effective strategy than the conventional system in which cells are seeded under a static condition and cultured in a bioreactor such as a spinner flask.

Animals↗
PubMed2006

Real-time monitoring of antibody secretion from hybridomas on a microchip by time-resolved luminescence anisotropy analysis.

This article presents a real-time monitoring system for cellular analysis using micro total analysis systems technology. Time-resolved luminescence anisotropy analysis was adopted for real-time detection of small amounts of a target protein produced by a small number of cells. The system was tested by real-time monitoring of the antibody secretion by hybridomas. The cells were successfully cultivated in a micro-incubation chamber (240 nl) fabricated on a microchip. The quantification of the antibody was achieved using the Ru(II) complex-labeled Staphylococcus aureus protein A probe, which can bind specifically to the Fc region of the antibody. Using this system, we detected as little as 24 fmol of immunoglobulin G under physiological conditions without the bound/free separation protocol. We successfully achieved real-time and quantitative monitoring of small amounts of antibody production by approximately 200 hybridoma cells. This method could be applied to various cellular analyses using small numbers of cells.

Animals↗
PubMed2006

Various properties of polymeric carriers improved the transfection efficiency.

In order to develop novel efficient gene carriers, we have been focusing on the transcription of transgene in the nucleus among various steps in the gene transfer system. Optimal carrier properties for improving the transgene recognition by transcription factors have not been clarified so far. In the present study, we established a novel evaluation system for the intranuclear transcription efficiency of the transgene using microinjection technique. Polyplexes composed of polypeptides with different molecular weights were microinjected into the cytoplasm or nucleus of COS-1 cells, and the relationship between the carrier properties, such as molecular weight, and the intranuclear transcription efficiency was evaluated. The molecular weight (Mw) dependency of the transgene transcription in the living cells was successfully quantified, and the low Mw polymers were found not to suppress the transcription but high Mw polymers allowed almost no transcription. Interestingly, the transcription efficiencies of poly(arginine) (PR) and poly(lysine) (PK) were almost same although the PR is widely reported to be more efficient gene carrier than PK. The difference in the various properties and intracellular trafficking of PR/DNA and PK/DNA polyplexes will be discussed.

Animals↗
PubMed2005

Preparation and properties of ProNectin F-coated biodegradable hollow fibers.

ProNectin F-coated biodegradable hollow fibers were newly prepared and their cytocompatibility was evaluated in vitro. Although the coating efficiency onto poly(L-lactic acid) (PLLA) and poly(lactide-co-caprolactone) [p(LA/CL)] matrices was similar, the cell adhesion properties were greatly affected by the nature of the polymer substrate. ProNectin F-coated PLLA showed about seven times higher cytocompatibility than ProNectin F-coated p(LA/CL). The single-extruded melt spinning method and the core-sheath bicomponent melt spinning method were employed to prepare PLLA hollow fibers. The effect of the spinning conditions, such as the melt draw ratio, spinneret temperature, and take-up speed, on the diameter and wall thickness of the spun fibers was studied in detail. For single-extruded melt spinning, a segmented type of spinneret was used, and the effect of the flow rate of nitrogen, which was confined in the hollow part of fibers, was studied. X-ray photographs of the drawn hollow fibers, clarified the significant molecular orientation, which was much higher than that in drawn solid PLLA fiber under identical drawing conditions. The morphology and mechanical properties of hollow fibers demonstrated an increase in the tensile strength and a decrease in the thickness of the PLLA wall with increased nitrogen flow rates and melt draw ratios for single-extruded melt spinning. These results indicate the unique characteristics of ProNectin F-coated PLLA hollow fibers, which can be successfully utilized as a biodegradable substrate.

Absorbable Implants↗
PubMed2005

Synthesis of antisense oligonucleotides containing a photocleavable protecting group on a guanine base and their photoinduced duplex formation.

An oligonucleotide containing a photocleavable protecting group at a guanine base was synthesized to induce the duplex formation by photo-irradiation. Alpha-methyl-2-nitropiperonyl (MeNP) group was used for the photocleavable protecting group at O6 position of deoxyguanosine. The oligonucleotide containing MeNP group (MeNP-ODN:5'-dTTCTG(MeNP)TCTGT-3') was synthesized by phosphoramidite method. The MeNP group was found to be removable by UV irradiation at wavelength of 365 nm for 5 min in 98% yield. UV-melting temperature (Tm value) analysis indicated that the duplex of MeNP-ODN with the complementary RNA was significantly unstable compared with the unmodified DNA/RNA duplex (deltaTm = -25 degrees C). After UV irradiation at 365 nm, the Tm value of the mixture increased to the same as that of the unmodified duplex. These results suggest that the RNA binding ability of the MeNP-ODN can be induced by photocleavage of the MeNP group.

Benzodioxoles↗
PubMed2005

Luminescence anisotropy-based detection of nucleic acids and proteins using long-lifetime Ru(II) complex as a luminescent label.

Luminescence anisotropy-based methods are powerful tool for the detection of biomolecules in homogeneous physiological media without Bound/Free separation. However, analyses based on the method are always disturbed by the autofluorescence from biological specimen. This disturbance limits the sensitivity of the method. To improve the detection limit of the method, a long-lifetime luminophore was adopted as the label. Because of the lifetime of the autofluorescence is below 10 ns, it is expected that the use of the long-lifetime luminophore enables us to avoid the disturbance by adopting time-resolved luminescence anisotropy measurement. To verify this concept, oligodeoxyribonucleotides and Staphylococcus aureus protein A were labeled with Ru(II) complex (tau approximately 500 nsec) and used for the detection of Escherichia coli 16S ribosomal RNA (16S rRNA) and immunoglobulin G (IgG), respectively. Results indicated that the methods were useful for the detection of rRNA and IgG without disturbance of the autofluorescence.

Anisotropy↗
PubMed2005

Synthesis and properties of photo-reactive antisense oligonucleotides containing 2'-O-psoralen-conjugated adenosine.

In order to selectively regulate mRNA having a point mutation, the photo-reactive antisense oligonucleotides were developed. Two types of photo-reactive oligonucleotides containing adenosine whose 2'-OH was modified with 4,5',8-trimethylpsoralen (psoralen) were synthesized (2'-Ps-oligo). One contains psoralen via a methylene linkage (2'-Ps-met), and the other via an amidomethylene linkage (2'-Ps-amd). 2'-Ps-oligos were then subjected to the photo-cross-linking reaction. 2'-Ps-met cross-linked to the complementary RNA and scarcely did to the RNA having a single mismatch base. Contrarily, 2'-Ps-amd did not cross-link to both RNA strands. These results suggest the structure of the linkage might affect the efficiency of the photo-cross-linking.

Adenosine↗
PubMed2005

The destabilization of polyplexes facilitates intranuclear transcription efficiency.

We have been focusing on the last step of transfection, that is, the intranuclear recognition of polyplexes by transcription factors. In this work, we studied about the relationship between the intracellular destabilization of polyplexes and the transcription efficiency. Polypeptides containing specific sequences which are digested by intracellular proprotein convertase (PC), furin (Fur-polypeptide) were synthesized as novel carriers. When Fur-polypeptide/plasmid DNA polyplexes were incubated with furin in the cell-free transcription/translation system, the sequence specific expression was observed as time. Additionally, Fur-polypeptides/plasmid DNA polyplexes led to site-specific effective transgene expression even in COS-1 cells. These results indicate that shortened carriers lose their abilities to form polyplexes in response to furin activity in the cells, and it seems to enhance the destabilization of polyplexes, the recognition of transgene in polyplexes by transcription factors, and the expression efficiency.

Animals↗
PubMed2005

Evaluation of dynamic features of Escherichia coli 16S ribosomal RNA in homogeneous physiological solution.

There is no methodology for the estimation of the dynamic features of large-molecular-weight RNAs in homogeneous physiological media. In this report, a luminescence anisotropy-based method using a long-lifetime luminescent oligonucleotide probe for the estimation of the dynamic features of large-molecular-weight RNA is described. As a luminescent probe, Ru(II) complex-labeled oligonucleotides, which have a complementary sequence to the single-stranded regions of Escherichia coli 16S rRNA, were synthesized. After the hybridization of the probe to single-stranded regions of 16S rRNA, the segmental motions of the regions were evaluated by time-resolved luminescence anisotropy analysis. In 16S rRNA, the L2 site (323-332 nt) was found to be the most flexible among the seven sites chosen. From a comparison between the hybridization kinetics of oligonucleotides to these single-stranded regions and the rotational correlation times, it was suggested that the flexibility of the single-stranded region was closely correlated with the hybridization kinetics. Furthermore, results of the luminescence lifetime measurement and luminescence quenching experiments suggested that the highly flexible region was located on the surface of the 16S rRNA and that the less flexible region was located in the depths of 16S rRNA.

Body Fluids↗
PubMed2004

An enhanced site-specific transcription efficiency of DNA/polypeptide-vector polyplexes.

A novel gene delivery system based on the enhanced release of plasmid DNA from polyplexes in response to intracellular proprotein convertase (PC), furin, that cleaves the carboxyl side of Arg-X-Arg/Lys-Arg, was successfully established. Polypeptides that have this recognition sequence were synthesized by the Fmoc solid phase method and evaluated as gene vectors. The fragmented polypeptides were found to lose the abilities to form polyplexes with plasmid DNA. When the Fur-polypeptide/plasmid DNA polyplexes were treated with furin in vitro, their disassembly and the release of plasmid DNA was observed in agarose gel electrophoresis analysis. The Fur-polypeptide showed higher transgene expression in COS-1 cells, while the control polypeptides which do not have the recognition sites did not. These results indicate that the cationic nature of the polypeptides is not sufficient for gene carrier and this intracellular signal-responsive gene transfer system is the useful strategy to enhance the transcription efficiency of the delivered plasmid DNA.

Amino Acid Sequence↗
PubMed2004

Transcriptome analysis using fluorescence-labeled oligonucleotide.

In our previous studies, the fluorescence intensity at 480 nm of the bispyrene-labeled 2'-O-methyl-oligoribonucleotide (OMUpy2) was drastically enhanced only when it was hybridized with its complementary RNA. In this study, we demonstrated the potential of the OMUpy2 for transcriptome analysis in the cell free system and in the living cell. For the analysis in the cell free system, the micro-chamber was designed and used to evaluate the detection limit of the target RNA. The detection limit of oligo-RNA was approximately 15 fmol. For the analysis of the living cells, C-OMUpy2 was introduced to the cervical carcinoma cells, C4II, and the fluorescence from C-OMUpy2 was monitored. The fluorescence from the cells began to be observed 5 min after the serum stimulation, and the fluorescence was decreased after 20 min. These results seemed to be consistent with the results from the RT-PCR analysis and suggested that OMUpy2 could be a useful tool for the transcriptome analysis.

Cell Line, Tumor↗
PubMed2004

Synthesis and properties of fluorescent-labeled oligonucleotides containing amide-linked dinucleoside with pyrene.

Oligonucleotides containing 2'-pyrene modified uridine (Upy) exhibit the increase of the pyrene monomer emission when hybridize with the complementary RNA. In order to examine the effect of restricted C3'-endoconformation of the Upy residue for the enhancement of the pyrene monomer emission, an oligodeoxyribonucleotide containing amide-linked dinucleoside with a pyrene group at the sugar residue (UapyODN) was synthesized by use of 3'-carboxymethyl-5'-O-dimethoxytrityl-2'-O-(1-pyrenylmethyl)-3'-deoxyuridine. NMR analysis indicated that the furanose ring of the uridine derivative was fixed to C3'-endo conformation. The UapyODN showed increase of fluorescence intensity to 7-fold at 375 nm when hybridized with complementary oligoribonucleotide. This result suggests that the UapyODN can detect the target RNA.

Amides↗
PubMed2003

Detection of acceptor sites for antisense oligonucleotides on native folded RNA by fluorescence spectroscopy.

Antisense strategy has high potential for curing diseases and studying gene functions by suppressing the translation step. For the strategy, it is essential to detect acceptor sites of antisense molecules on mRNA under physiological conditions. We propose a new analytical method for the detection of acceptor sites of antisense molecules with high sensitivity. 2'-O-Methyloligoribonucleotide containing 2'-O-(1-pyrenylmethyl)uridine (OMUpy) was chosen as the fluorescence probe. The fluorescence intensity due to the pyrene in single-stranded OMUpy was scarcely observed. When OMUpy was hybridized with the complementary oligoRNA, the fluorescence intensity at 375 nm was remarkably increased. It was found that the increase was derived from the localization of the pyrene by the measurements of time-resolved fluorescence spectroscopy, CD and UV absorption spectra. These results suggest that the change of the fluorescence intensity of OMUpy can be a useful index to monitor hybridization. In this study, we chose Escherichia coli. 16S-rRNA as the model RNA and chose seven regions for probing by OMUpy based on the reported secondary structure of 16S-rRNA. The fluorescence intensity of an equimolar mixture of OMUpy with 16S-rRNA varied depending on the sequence. In particular, the increment in the system of OMUpy-8, which can hybridize with region 887-896 nt of 16S-rRNA, was most significant among the systems. These results indicated that the site targeted by OMUpy-8 was exposed to regulatory molecules, and suggest that the method presented here is useful to design antisense molecules.

Base Sequence↗
PubMed2003

Synthesis of antisense oligonucleotides containing photocleavable protecting groups on the thymine bases and their photoinduced duplex formation.

Oligonucleotides containing photocleavable protecting groups at thymine bases were synthesized to induce the duplex formation by photo-irradiation. 6-Nitroveratryloxycarbonyl (NVOC) group was used for the photocleavable protecting group at N3 position of thymidine. An oligonucleotide containing NVOC groups (NVOC-ODN2:5'-dATG CAC CAT(NVOC) TCT(NVOC)GTC TGT-3') was synthesized by phosphoramidite method. The NVOC groups were found to be removable by UV irradiation at wavelength of 365 nm for 5 h. UV-melting temperature (Tm value) analysis indicated that the duplex of NVOC-ODN2 with the complementary RNA was significantly unstable compared with the unmodified DNA/RNA duplex (delta Tm=-13 degrees C). After UV irradiation at 365 nm, the Tm value of the mixture increased to the almost same as that of the unmodified duplex. These results suggest that the RNA binding ability of the NVOC-ODN2 can be induced by photocleavage of the NVOC groups.

Base Sequence↗
PubMed2003

Detection of acceptor sites for antisense oligonucleotides on native folded RNA by fluorescence-labeled oligonucleotide.

Pyrene-labeled 2'-O-methyloligoribonucleotide (OMUpy) and 5'-Ru(II) complex labeled oligodeoxyribonucleotie (Ru-probe) were prepared as the fluorescence probes to detect acceptor sites of antisense molecules on native folded RNA under the physiological condition. OMUpy showed the remarkable increase of the fluorescence intensity (334-fold at 375 nm) only when hybridized with complementary oligoRNA. When OMUpy was applied to E. coli 16S-rRNA, the fluorescence intensities were increased in a sequence specific manner. The rotational correlation time of Ru-probe complementary to 16S-rRNA were largely dependent on the sequence of Ru-probe. These results suggest that the accessibility of the antisense molecules for RNA can be evaluated by those fluorescent probes.

Fluorescent Dyes↗