Biomedical subjects

Yoji Yamamoto

Publications and source records attributed to Yoji Yamamoto.

At least 19 recordsLinked to original sources

PubMed2006

Chemical-reaction-based site-selective DNA cutter for PCR-free gene manipulation.

An artificial restriction DNA cutter (ARCUT), recently developed by the authors, was used to construct a fusion protein. The gene of WW-domain-containing oxidoreductase (WWOX) was cut by ARCUT just before its stop codon, and ligated to fuse the gene of enhanced green fluorescent protein (EGFP). The reading frames of two genes were adjusted to coincide each other. Throughout the manipulation, no PCR was employed. The fluorescent fusion protein was successfully expressed in mammalian cells, and showed entirely different subcellular localization from EGFP itself. Apparently, the DNA was kept completely intact during the manipulation. The man-made tool ARCUT has promising features for future biotechnology and molecular biology.

Animals↗

PubMed2006

Highly efficient strand invasion by peptide nucleic acid bearing optically pure lysine residues in its backbone.

Chiral PNA monomers (PNA = peptide nucleic acid), in which nucleobases are attached to N-(aminoethyl)-D-lysine, were introduced to PNAs bearing pseudo-complementary nucleobases (2,6-diaminopurine and 2-thiouracil). When these highly cationic PNAs targeted double-stranded DNA, they invaded there much more efficiently than conventional pseudo-complementary PNAs composed of achiral PNA monomers. Although introduction of N-(aminoethyl)-D-lysine backbone was effective for promotion of strand invasion, L-isomer never promote it. Simple incorporation of lysine groups to the termini of PNA was also ineffective, indicating that introduction of positive charges into PNA backbone is important. Even highly G-C rich sequence, which conventional pseudo-complementary PNAs never invade, was successfully targeted based on this strategy.

2-Aminopurine↗

PubMed2006

Highly active artificial restriction enzyme composed of Ce(IV)/EDTA and PNA bearing phosphate group--relationship between the promotion by phosphate and the structure of invasion complex.

Recently, we developed artificial restriction DNA cutter (ARCUT) composed of pseudo-complementary peptide nucleic acid (pcPNA) and Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). Here we promoted the site-selective hydrolysis by attaching phosphate groups to the pcPNAs. The promotion by the phosphates increased with decreasing length of the gap-like site. Furthermore, the scission was successful even when phosphate groups were introduced to 0 base-gap system.

Cerium↗

PubMed2006

Site-selective hydrolysis of huge DNA by artificial restriction DNA cutter.

Genomic DNAs of lambda phage (48.5 kbp) and E. coli (4.6 Mbp) were successfully cleaved at the target site by artificial restriction DNA cutter (ARCUT), which we recently developed by combining Ce(IV)/EDTA and PNA (peptide nucleic acid). PNA satisfactorily invaded the target site in such huge DNAs and activate there for Ce(IV)/EDTA-induced scission.

Bacteriophage lambda↗

PubMed2006

Site-selective RNA scission by PNA-Lu(III) hybrid system.

By using two PNA (PNA = peptide nucleic acid) additives, one-base gap structure was formed in RNA. Upon treatment of this gap-forming RNA substrate with Lu(III) ion, the phosphodiester linkage at 5'-side of the unpaired nucleotide was mainly cleaved. Promising feature of PNA as a sequence recognizing moiety of man-made RNA cutters was evidenced.

Hydrolysis↗

PubMed2005

Oligonucleotide bearing ethylenediamine-N,N,N'-Triacetates for gap-selective DNA hydrolysis by Ce4+/EDTA.

With the use of two oligonucleotides bearing ethylenediamine-N,N,N'-triacetate groups as additives, gap sites were formed at predetermined sites in substrate DNA. Upon treating these systems with a Ce(4+)/EDTA complex at pH 7.0 and 37 degrees C, the phosphodiester linkages at the gap site were selectively hydrolyzed. The DNA scission was greatly promoted by the introduction of ethylenediaminetriacetate groups, and the scission efficiency increased as the number of these groups increased. Even a one-base gap was successfully hydrolyzed when three ethylenediaminetriacetate groups were placed consecutively at both edges of the gap, although the scission was minimal in the absence of these groups. The site-selective scission could be also achieved at higher temperatures without any significant loss of site-selectivity.

Base Sequence↗

PubMed2005

Genetic recombination without using either restriction enzyme or PCR.

We recently prepared artificial restriction DNA cutters (ARCUT) for site-selective scission of double-stranded DNA by combining Ce(IV)/EDTA complex with a pair of pseudo-complementary peptide nucleic acids (pcPNAs). Here we report an improved method for genetic recombination using ARCUT. The key point is to treat the scission fragments with nuclease S1 (a single-stranded DNA specific enzyme) and form blunt ends. By this procedure, these scission fragments are efficiently ligated with foreign DNA fragments having blunt ends, providing desired recombinant DNA in high yields. Neither restriction enzyme nor PCR amplification is required.

DNA Restriction Enzymes↗

PubMed2005

Rapid site-selective hydrolysis of double-stranded DNA by use of Ce(IV)/EDTA and PNA bearing phosphate group.

In order to hydrolyze double-stranded DNA efficiently at the target site, two pseudo-complementary peptide nucleic acids (pcPNAs) bearing phosphate group were combined with Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). The phosphate groups as metal-binding ligands were placed near the target site, and concentrated the Ce(IV) complex thereto. As the result, the site-selective hydrolysis was notably promoted, compared with the scission by the cutters involving unmodified pcPNAs.

Cerium↗

PubMed2005

Construction of chimera protein by using artificial restriction DNA cutter.

We have already developed artificial restriction DNA cutter (ARCUT), which can hydrolyze double-stranded DNA site-selectively, by using Ce(IV)/EDTA in combination with two pseudo-complementary peptide nucleic acids (pcPNAs). Here, ARCUT was used to prepare a chimera protein. The gene for WW-domain containing oxidoreductase (WWOX) was clipped off by ARCUT just before its stop codon, and ligated with the gene for enhanced green fluorescent protein (EGFP). Conventional PCR for insertion of restriction enzyme site is never required.

Animals↗

PubMed2005

Gene manipulation using artificial restriction DNA cutter.

Artificial restriction DNA cutter (ARCUT), which we developed recently, was used for manipulation of plasmid DNA. PCR product was inserted into pBR322 plasmid vector using ARCUT, and E. coli cells were transfected with this recombinant plasmid DNA. Successful growth of cells shows that the recombination proceeds without any unexpected mutations. Furthermore, this artificial system was applied to PCR-free construction of chimera protein.

Cerium↗

PubMed2004

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA.

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson-Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37 degrees C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.

Base Sequence↗

PubMed2004

Combination of S1 nuclease and PNA for site-selective hydrolysis of double-stranded DNA. Comparison with the site-selective hydrolysis using Ce(IV)/EDTA.

The potential of the combination of SI nuclease and pseudo-complementary PNA (pcPNA) for site-selective scission of double-stranded DNA has been investigated. Through strand invasion of two pcPNAs, single-stranded portions were formed in both strands of substrate DNA. In the initial stage of the enzymatic digestion, two scission fragments were obtained due to the hydrolysis at these two gap-like sites. On prolonged reactions, however, these products (as well as the substrate DNA) were further digested to smaller fragments. Under the conditions employed here, only Ce(IV)/EDTA is available for the preparation of desired fragments from double-stranded DNA.

Cerium↗

PubMed2004

Manipulation of double-stranded DNA by artificial restriction enzyme composed of Ce(IV)/EDTA and PNA.

Through the invasion of pseudo-complementary PNA (pePNA) to double-stranded DNA, gap-like structures were formed at predetermined sites in both strands of PBR322 plasmid DNA. These gap-like sites were selectively hydrolyzed by Ce(IV)/EDTA complex, and two designed fragments were obtained. Furthermore, the scission fragment by this artificial restriction enzyme was successfully ligated with foreign DNA.

Cerium↗

PubMed2003

Oligoamine-acridine conjugates for promotion of gap-selective DNA hydrolysis by Ce(IV)/EDTA complex.

Oligoamines (spermidine, dipropylenetriamine and propylenediamine) were covalently attached to acridine via a hexamethylene linker. These oligoamine-acridine conjugates were efficiently bound to gap sites in substrate DNA, and promoted the DNA hydrolysis by a homogeneous Ce(IV)/ethylenediamine-N,N,N',N'-tetraacetate (EDTA) complex at these sites. In contrast, the hydrolysis of the double-stranded portion in the DNA was little affected by these conjugates, although they were strongly bound thereto by the intercalation of their acridine moieties. As a result, the gap site was selectively and efficiently hydrolyzed by combining the Ce(IV)/EDTA complex with the oligoamine--acridine conjugate. Either the oligoamine or the acridine was only poorly active for the purpose, substantiating the essential role of cooperation between them. The promotion of gap-selective DNA hydrolysis by the conjugates has been ascribed to electrostatic stabilization of a negatively charged transition state by their positive charges.

Acridines↗

PubMed2003

PNA for one-base differentiating protection of DNA from nuclease and its use for SNPs detection.

By the combination of peptide nucleic acid (PNA) with single-stranded DNA specific nucleases, alteration of a single base to another in DNA has been detected with high accuracy. Only the DNAs in DNA/PNA duplexes involving a mismatch are efficiently hydrolyzed by these enzymes, whereas fully matching sequences are kept intact. This difference is visually scored by adding 3,3'-diethylthiadicarbocyanine, which changes its color from blue to purple upon binding to DNA/PNA duplexes. These findings are applied to the convenient and straightforward detection of single nucleotide polymorphisms (SNPs). When the target site in the sample DNA is completely complementary with the PNA, a notable amount of DNA/PNA duplex remains and thus the solution exhibits purple color. In the presence of even one mismatch between PNA and DNA, however, the DNA is completely digested by the enzyme and therefore the dye shows its intrinsic blue color. The SNPs in the apolipoprotein E gene of human DNA have been successfully genotyped by this method.

Apolipoproteins E↗

PubMed2003

Reaction of poly(acrylamide-co-vinylamine) with tresyl-PEG in the presence of PC12 cells.

Tresylation of an amine containing polymer film in the presence of PC12 cells did not result in a significant loss of cell viability, at least as assessed by trypan blue exclusion or MTT assay. PC12 cells were cultured atop reactive poly(acrylamide-co-vinyl amine) films or tissue culture polystyrene and exposed for 2 h to tresylated polyethylene glycol (TPEG) or unreactive hydrolyzed TPEG in 0.1M TES (N-tris hydroxymethyl-2-aminoethane sulfonic acid). The loss in trypan blue viability was limited ( approximately 80% retained), provided the TPEG concentration was 10 micromol/g or less. Similarly when microencapsulated PC12 cells (in a non-reactive polyacrylate hydrogel) were exposed to TPEG (10 micromol/g in 0.1M TES) the loss of MTT activity was small. The loss of vaibility was attributed to the toxicity of the tresyl leaving group and not the reaction itself. Thus, it may be possible to surface modify cell containing microcapsules, at least under limited conditions, in order to improve their biocompatibility without compromising the viability of the enclosed cells. This should lead to the development of new (reactive) polymers for microencapsulation since biocompatibility need not be a design consideration in the first instance.

Amines↗

PubMed2003

Simultaneous detection of multiple single nucleotide polymorphism by single-strand-specific nuclease and PNA probe.

The combination of PNA (peptide nucleic acid) and single-strand-specific nuclease have been used for detection of single nucleotide polymorphisms (SNPs). When DNA is perfectly complementary to PNA, it is protected from digestion by the nuclease. If there exists a single-base mismatch between them, however, the DNA is completely digested. These differences are visualized by using 3,3'-diethylthiadicarbocyanine (DiSc2(5)), which changes its color from blue to purple upon binding to PNA/DNA hybrids. In terms of this methodology, homozygous and heterozygous SNPs in apoE gene have been successfully analyzed. Furthermore, the multiplex SNPs are simultaneously genotyped. This technique provides a simple, straightforward, facile, and visual genetic screening, with no need for expensive and complicated equipment.

Base Sequence↗

PubMed2002

Application of fluidized hot-melt granulation (FHMG) for the preparation of granules for tableting; properties of granules and tablets prepared by FHMG.

The objective of this study was to investigate the properties of granules and tablets prepared by a novel Fluidized Hot-Melt Granulation (FHMG) technique. Macrogol 6000 (polyethylene glycol 6000, PEG 6000), macrogol 20000 (polyethylene glycol 20000, PEG 20000), and glyceryl monostearate (GMS) were used as binders with different levels of viscosity and water solubility. The properties of both granules and tablets were compared with those obtained using the Standard Tablet Formulation (STF, lactose/corn starch/hydroxypropylcellulose/ magnesium stearate: 66/30/3.5/0.5) for fluidized-bed granulation, which is widely used for wet granulation. To obtain suitable flowability as granules for tabletting, the content of the melting material should be approximately 10 w/w%. The rate of increase in the mean diameter of the granules during FHMG was affected by both the melting temperature and the viscosity of the melting material used in the granules. The compression properties of granules prepared by FHMG were also investigated, demonstrating that these granules had a high pressure transmittance. The hardness and the disintegration time of tablets obtained from granules prepared by FHMG were influenced by the properties of the melting material, such as its compaction behavior, solubility, and wettability. No significant differences of hardness were observed when compared to STF tablets. Tablets prepared from FHMG granules disintegrated within 15 min, whereas the STF tablets showed faster disintegration. It was also demonstrated that the hardness and disintegration time of tablets prepared from FHMG granules were not affected by the tablet porosity. Therefore, tablets with a constant quality may be obtainable under a wide range of compression forces. The results of this study suggested that FHMG is a useful method of preparing granules for tableting without using any solvents or water.

Drug Compounding↗