Biomedical subjects

Hideyoshi Harashima

Publications and source records attributed to Hideyoshi Harashima.

At least 37 records · Page 2Linked to original sources

PubMed2006

Genome wide expression analysis of white blood cells and liver of pre-diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats using a cDNA microarray.

In a prior study, we reported on a significant decrease in calpain10 gene expression in white blood cells (WBC) as well as the major insulin-target tissues including liver and adipose tissue, before the onset of diabetes in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. In this study, we extended our hypothesis that some type 2 diabetes mellitus (NIDDM) susceptible genes are up/down-regulated before the onset in WBC of OLETF rats, reflecting their up/down-regulation in major insulin-target tissues, such as the liver. We tested this hypothesis using rat cDNA microarrays. The findings show that 1080 genes are up/down-regulated by more than 2-fold compared to the controls, Long-Evans Tokushima Otsuka rats, before the onset in WBC and liver under fasted or insulin administered condition. Fifty-seven of the 1080 genes were up/down-regulated in both WBC and the liver. More than half have been reported to NIDDM susceptible genes and the remainder have not been reported to be related to NIDDM. These results indicate that there some NIDDM related genes are up/down-regulated in WBC before the onset of diabetes.

Animals↗

PubMed2006

[Development of non-viral vector based on the quantitative comparison of intracellular trafficking with viral vector].

For the development of efficient gene vector, intracellular processes such as cellular uptake, endosomal release and nuclear delivery must be overcome. Viruses have also evolved and have developed sophisticated mechanisms for controlling intracellular trafficking for the efficient delivery of their genomes to nuclei in host cells for symbiosis. In the light of these mechanisms, various kinds of artificial devices have been developed to overcome the intracellular barriers. However, in the majority of studies, variation of the transfection activity before and after the modification of devices was evaluated, and intracellular trafficking remained unclear. Therefore, it is understand to recognize which of the intracellular barrier should be intensively improved to enhance the transfection activity. To clarify the rate-limited process in the current non-viral vector, we compared the intracellular trafficking between adenovirus and LipofectAMINE PLUS. As a result, we found that difference of the transfection efficiency between adenovirus and LipofectAMINE PLUS was dominantly derived from the differences on transcription activity. Therefore it is essential to consider the regulation of the intranuclear events to improve the transfection activity of artificial vector.

Active Transport, Cell Nucleus↗

PubMed2006

[Envelope-type nano device for gene delivery].

We introduced a new concept "Programmed Packaging" for the rational development of non viral gene delivery system. There are many non-viral gene delivery system, however, their utility is limited in the in vitro experiments. The major reason is considered that it is difficult to package many functional devices into a nanoparticle. The new concept which we introduce here is named as Programmed Packaging, which is composed of the strategy to targeting, molecular design of functional devices to overcome barriers, and assembly technique to package them into a nanoparticle. In this article, we explain this concept and provide newly developed gene delivery systems based on this concept.

Gene Transfer Techniques↗

PubMed2005

Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems.

To develop nonviral gene vectors that are sufficient for clinical application, it is necessary to understand why and to what extent nonviral vectors are inferior to viral vectors, which in general show a more efficient transfection activity. This study describes a systematic and quantitative comparison of the cellular uptake and subsequent intracellular distribution (e.g., endosome/lysosome, cytosol, and nucleus) of exogenous DNA transfected by viral and nonviral vectors in living cells, using a combination of TaqMan PCR and a recently developed confocal image-assisted three-dimensionally integrated quantification method. As a model, adenovirus (Ad) and Lipofectamine Plus (LFN) were used for comparison since they are highly potent and widely used viral and nonviral vectors, respectively. The findings indicate that the efficiency of cellular uptake for LFN is significantly higher than that for Ad. Once taken up by a cell, Ad exhibited comparable endosomal escape and slightly higher nuclear transfer efficiency compared with LFN. In contrast, LFN requires 3 orders of magnitude more intranuclear gene copies to exhibit a transgene expression comparable to that of the Ad, suggesting that the difference in transfection efficiency principally arises from differences in nuclear transcription efficiency and not from a difference in intracellular trafficking between Ad and LFN.

Adenoviridae↗

PubMed2005

Non-linear pharmacodynamics in a non-viral gene delivery system: positive non-linear relationship between dose and transfection efficiency.

A remarkable non-linearity was found between dose and transfection activities of non-viral gene delivery systems, such as a Lipofectamine/DNA complex and an octaarginine-modified multifunctional envelope-type nano device (R8-MEND). We measured the nuclear delivery of pDNA to distinguish the non-linearity in intracellular pharmacokinetics or pharmacodynamics after transfection with R8-MEND at different doses. A remarkable positive non-linearity was found in the pharmacodynamics when the dose was increased. Even dummy pDNA enhanced the efficiency of transcription and/or translation per pDNA in the nucleus, but empty liposomes did not. These results suggest the importance of controlled pharmacodynamics as well as the importance of intracellular pharmacokinetics for the rational design of non-viral gene delivery systems.

Animals↗

PubMed2005

High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression.

The mechanism of the arginine-rich peptide-mediated cellular uptake is currently a controversial issue. Several factors, including the type of peptide, the nature of the cargo, and the linker between them, appear to affect uptake. One of the less studied factors, which may affect the uptake mechanism, is the effect of peptide density on the surface of the cargo. Here, we examined the mechanism of cellular uptake and intracellular trafficking of liposomes modified with different densities of the octaarginine (R8) peptide. Liposomes modified with a low R8 density were taken up mainly through clathrin-mediated endocytosis, leading to extensive lysosomal degradation, whereas those modified with a high R8 density were taken up mainly through macropinocytosis and were less subject to lysosomal degradation. Furthermore, the high density R8-liposomes were able to stimulate the macropinocytosis-mediated uptake of other particles. When plasmid DNA was condensed and encapsulated in R8-liposomes, the levels of gene expression were three orders of magnitude higher for the high density liposomes. The enhanced gene expression by the high density R8-liposomes was highly impaired by blocking uptake through macropinocytosis. The different extents of gene expression from different densities of the R8 peptide on the liposomes could be explained principally by the existence of an intracellular trafficking route, but not by the uptake amount, of internalized liposomes. These results show that the density of the R8 peptide on liposomes determines the uptake mechanism and that this is directly linked to intracellular trafficking, resulting in different levels of gene expression.

Animals↗

PubMed2005

Factors affecting SFHR gene correction efficiency with single-stranded DNA fragment.

A 606-nt single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, improves the gene correction efficiency by 12-fold as compared with a PCR fragment, which is the conventional type of fragment used in the small fragment homologous replacement method [H. Tsuchiya, H. Harashima, H. Kamiya, Increased SFHR gene correction efficiency with sense single-stranded DNA, J. Gene Med. 7 (2005) 486-493]. To reveal the characteristic features of this gene correction with the ss DNA fragment, the effects on the gene correction in CHO-K1 cells of the chain length, 5'-phosphate, adenine methylation, and transcription were studied. Moreover, the possibility that the ss DNA fragment is integrated into the target DNA was examined with a radioactively labeled ss DNA fragment. The presence of methylated adenine, but not the 5'-phosphate, enhanced the gene correction efficiency, and the optimal length of the ss DNA fragment (approximately 600 nt) was determined. Transcription of the target gene did not affect the gene correction efficiency. In addition, the target DNA recovered from the transfected CHO-K1 cells was radioactive. The results obtained in this study indicate that length and adenine methylation were important factors affecting the gene correction efficiency, and that the ss DNA fragment was integrated into the double-stranded target DNA.

Adenine↗

PubMed2005

Mitochondrial delivery of mastoparan with transferrin liposomes equipped with a pH-sensitive fusogenic peptide for selective cancer therapy.

Mastoparan (MP), a potent facilitator of mitochondrial permeability transition (PT), could be used as an antitumor agent, if it were encapsulated in a tumor selective delivery system. We recently developed transferrin-modified liposomes (Tf-L) with a pH-sensitive fusogenic peptide (GALA), which delivers an encapsulated fluorescent marker into cytosol efficiently. In this study, we encapsulated MP into Tf-L with GALA for the selective delivery to mitochondria of tumor cells. The MP showed potent PT activity at concentrations above 25 microM in a homogenate of K 562 cells as well as in isolated mitochondria in the presence of phosphate. Tf-L equipped with cholesteryl GALA can release encapsulated sulforhodamine B, while Tf-L failed, as evidenced by confocal laser scanning microscopy. The MP, which was delivered with Tf-L with GALA, released cytochrome c (cyt c) from mitochondria to the cytosol, while free MP released cyt c not only to the cytosol but also extracellulary. These results demonstrate the utility of MP in Tf-L with GALA for cancer therapy.

Antineoplastic Agents↗

PubMed2005

A multifunctional envelope-type nano device for novel gene delivery of siRNA plasmids.

A multifunctional envelope-type nano device (MEND) for use in the delivery of siRNA expression plasmids is described. The plasmid DNA encoding anti-luciferase short interfering RNA (siRNA) was condensed by poly-L-lysine (PLL) and packaged into the MEND. The silencing effect of the MEND(PLL) showed a 96% inhibition of luciferase activities in a co-transfection study. The silencing effect was maintained at more than 60%, even under the 100-fold diluted conditions. In the luciferase transformed cells, however, the MEND(PLL) showed no significant silencing effect (10%), indicating heterogeneity in transfection by the MEND(PLL). To solve this problem, the DNA condensing agents were optimized by comparing PLL, stearyl octaarginine (STR-R8) and protamine (Prot). No difference in silencing effect (95-97%) was found among these MENDs in a co-transfection study. However, the MEND(Prot) showed a 70% silencing effect in the transformed cells. These results suggest that the MEND(Prot) has less heterogeneity in transfection, while the MEND(PLL) and the MEND(STR-R8) have large heterogeneities. These results demonstrate that MEND(Prot) is a promising gene delivery system for siRNA expression plasmids with less heterogeneity associated with the transfection.

Animals↗

PubMed2005

Pharmacokinetics and brain uptake of lactoferrin in rats.

Lactoferrin (Lf) is an iron-binding glycoprotein belonging to the transferrin (Tf) family. Lf was reported to cross the blood brain barrier (BBB) via receptor-mediated transcytosis in an in vitro model of the BBB. In the present study, we compared the in vivo brain uptake of Lf with that of OX26, an anti-Tf receptor antibody, and Tf. These three proteins were radiolabeled with 125I and administered to rats by i.v. injection. We found that Lf was more rapidly eliminated from the blood compared with OX26 and Tf (The half-life of Lf was approximately 8 and 6 times shorter than that of OX26 and Tf, respectively; the area under the blood concentration-time curve of Lf was approximately 15 and 17 times smaller than that of OX26 and Tf, respectively), and mainly accumulated in the liver, spleen, and kidney. Markedly high brain uptake was observed for Lf relative to Tf and OX26. Lf might be useful as a ligand for facilitating drug delivery into the brain.

Animals↗

PubMed2005

Kinetic analysis of protein production after DNA transfection.

The production of an exogenous protein by the transfection of a plasmid DNA encoding the protein was kinetically analyzed, to determine the efficiency of the transfection. Cultured NIH3T3 or HeLa cells, and the luciferase protein were used as a model system in this experiment. The findings indicate that at least a 8x10(4)- and 4x10(3)-fold molar amounts of luciferase protein was produced from one copy of the plasmid DNA molecule in NIH3T3 and HeLa cells, respectively. The rate of elimination of luciferase activity upon DNA transfection was smaller than that for the luciferase protein itself (k(el) for DNA transfection<k(el) for the luciferase protein), suggesting that a decrease in intranuclear active DNA was the main determinant of the elimination rate in this case. A preliminary pharmacokinetic model is proposed, based on the results obtained.

Animals↗

PubMed2005

RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured mammalian cells.

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.

Animals↗

PubMed2005

New NTP analogs: the synthesis of 4'-thioUTP and 4'-thioCTP and their utility for SELEX.

The synthesis of the triphosphates of 4'-thiouridine and 4'-thiocytidine, 4'-thioUTP (7; thioUTP) and 4'-thioCTP (8; thioCTP), and their utility for SELEX (systematic evolution of ligands by exponential enrichment) is described. The new nucleoside triphosphate (NTP) analogs 7 and 8 were prepared from appropriately protected 4'-thiouridine and -cytidine derivatives using the one-pot method reported by J. Ludwig and F. Eckstein [(1989) J. Org. Chem., 54, 631-635]. Because SELEX requires both in vitro transcription and reverse transcription, we examined the ability of 7 and 8 for SELEX by focusing on the two steps. Incorporation of 7 and 8 by T7 RNA polymerase to give 4'-thioRNA (thioRNA) proceeded well and was superior to those of the two sets of frequently used modified NTP analogs for SELEX (2'-NH2dUTP and 2'-NH2dCTP; 2'-FdUTP and 2'-FdCTP), when an adequate leader sequence of DNA template was selected. We revealed that a leader sequence of about +15 of DNA template is important for the effective incorporation of modified NTP analogs by T7 RNA polymerase. In addition, reverse transcription of the resulting thioRNA into the complementary DNA in the presence of 2'-deoxynucleoside triphosphates (dNTPs) also proceeded smoothly and precisely. The stability of the thioRNA toward RNase A was 50 times greater than that of the corresponding natural RNA. With these successful results in hand, we attempted the selection of thioRNA aptamers to human alpha-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity (K(d) = 4.7 nM).

Base Sequence↗

PubMed2005

Amino acid residues involved in substrate recognition of the Escherichia coli Orf135 protein.

The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes mutagenic 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H(2)O(2)-induced mutation frequencies. To identify the amino acid residues that interact with these nucleotides, the Glu-33, Arg-72, Arg-77, and Asp-118 residues of Orf135, which are candidates for residues interacting with the base, were substituted, and the enzymatic activities of these mutant proteins were examined. The mutant proteins with a substitution at the 33rd, 72nd, and 118th amino acid residues displayed activities affected to various degrees for each substrate, suggesting the involvement of these residues in substrate binding. On the other hand, the mutant protein with a substitution at the 77th Arg residue had activitiy similar to that of the wild-type protein, excluding the possibility that this Arg side chain is involved in base recognition. In addition, the expression of some Orf135 mutants in orf135(-) E. coli reduced the level of formation of rpoB mutants elicited by H(2)O(2). These results reveal the residues involved in the substrate binding of the E. coli Orf135 protein.

Amino Acid Substitution↗

PubMed2005

Evaluation of nuclear transfer and transcription of plasmid DNA condensed with protamine by microinjection: the use of a nuclear transfer score.

In the present study, the nuclear delivery of a green fluorescence protein (GFP)-encoding pDNA condensed by protamine was investigated in terms of trans-gene expression after cytoplasmic (E(cyt)) and nuclear (E(nuc)) microinjection. To compare the nuclear transfer process, a novel parameter; the nuclear transfer (NT) score was introduced. The E(cyt) value for protamine/pDNA particles increased in a charge ratio-dependent manner. The calculated NT score showed that this increase results from an enhancement in nuclear transfer efficiency, which was also quantitatively confirmed by a recently developed confocal image-assisted three-dimensionally integrated quantification (CIDIQ) method. Moreover, E(nuc) for protamine/pDNA particles was significantly higher than that for poly-L-lysine/pDNA particles, suggesting that pDNA, when condensed with protamine, is more accessible to intra-nuclear transcription. Collectively, protamine is an excellent DNA condenser, with bi-functional advantages: improvement in nuclear delivery and efficient intra-nuclear transcription.

Cell Nucleus↗

PubMed2005

Construction of a multifunctional envelope-type nano device by a SUV*-fusion method.

A novel assembly method "SUV*-fusion method" was developed for the construction of a small and homogenous multifunctional envelope-type nano device (MEND) by utilizing a detergent-rich small unilamellar vesicle (SUV*). The method consists of three steps: (1) DNA condensation with a polycation, (2) electrostatic interaction of the SUV* with the DNA/polycation complex (DPC) and (3) lipid coating of DPC by SUV* fusion via removal of the detergent. We confirmed the construction of the MEND by sucrose density gradient centrifugation, and isolated the MEND only from the boundary between 25% and 40% sucrose. The isolated MEND had a small diameter (155 nm), was negatively charged (-24 mV), and encapsulated 30% of the total DNA. The MEND was formed by only SUV*, not by a lipid/detergent micelle. This confirms that a small and homogenous MEND can be constructed by the SUV*-fusion method. Furthermore, we confirmed that a transferrin-modified MEND could deliver a gene into a cell through receptor-mediated endocytosis. Consequently, we report on the successful construction of a small and homogenous MEND by a novel SUV*-fusion method.

Cell Line, Tumor↗

PubMed2005

Cytokine induction by a bacterial DNA-specific modified base.

Unmethylated CpG dinucleotides in DNA contribute to a rapid inflammatory response in mammals. Here we show that N(6)-methyladenine (N(6)-MeA), a bacterium-specific modified base, also causes cytokine production. An oligodeoxyribonucleotide (ODN) containing N(6)-MeA induced cytokines when injected into mice. Co-injection of N(6)-MeA and CpG ODNs enhanced cytokines 2- to 3-fold, as compared with the injection of a CpG ODN alone. Plasmid DNA containing N(6)-MeA, complexed with cationic lipids, induced IL-12. These results indicate that the bacterium-specific base, in addition to the unmethylated CpG motif, triggers the mammalian immune response, and suggest that N(6)-MeA-containing DNA could be useful for cellular immunotherapy and DNA vaccine.

Adenine↗

PubMed2005

Dual hydrolysis of diphosphate and triphosphate derivatives of oxidized deoxyadenosine by Orf17 (NtpA), a MutT-type enzyme.

To determine whether the Orf17 (NtpA) protein of Escherichia coli, a MutT-type enzyme, functions as a hydrolyzing enzyme for a damaged deoxyribonucleotide, we purified the recombinant Orf17 protein and incubated it with oxidized deoxyribonucleotides. Of the deoxyribonucleoside 5'-triphosphates tested, 8-hydroxy-2'-deoxyadenosine 5'-triphosphate was hydrolyzed by this protein. Unexpectedly, the Orf17 protein degraded 8-hydroxy-2'-deoxyadenosine 5'-diphosphate 2.3-fold more efficiently than the corresponding triphosphate. Thus, this protein is the first MutT-type enzyme that hydrolyzes both the triphosphate and diphosphate derivatives of a deoxyribonucleoside, with similar efficiencies. These results suggest that the Orf17 protein may be involved in the hydrolysis of oxidized dATP and dADP.

Cloning, Molecular↗