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Masami Mizu

Publications and source records attributed to Masami Mizu.

At least 19 recordsLinked to original sources
PubMed2005

CpG DNA/zymosan complex to enhance cytokine secretion owing to the cocktail effect.

Zymosan, classified among beta-(1-->3)-d-glucans, is produced from the cell wall of yeast and well known to induce proinflammatory cytokines when ingested by immune cells. We found that zymosan forms a complex with immunostimulatory CpG DNA, where both zymosan and CpG DNA can induce cytokine secretion according to the different mechanisms (i.e., recognized by different receptors). The complex activated macrophages and induced cytokine secretion, more efficiently than separate administration of zymosan or CpG DNA. Microscopic observation showed that this increment of the cytokine secretion can be explained by the fact that zymosan and zymosan/CpG DNA complex are up-taken more than naked CpG DNA. Additionally, existence of two different immunostimulants in the same cells may enhance the immunoresponse. This report presents a new strategy to construct a delivering vehicle for CpG DNA and to enhance its activity with the 'cocktail effect' of the two immunostimulants.

Animals↗
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

Galactose-PEG dual conjugation of beta-(1-->3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake.

Antisense oligonucleotides (AS ODNs) are applied to silence a particular gene, and this approach is one of the potential gene therapies. However, naked oligonucleotides are easy to be degraded or absorbed in biological condition. Therefore, we need a carrier to deliver AS ODNs. This paper presents galactose moieties that were conjugated to the side chain of SPG to enhance cellular ingestion through endocytosis mediated by asialoglycoprotein receptor specifically located on parenchymal liver cells. We introduced galactose with two types of chemical bonds; amide and amine, and the amine connection showed lower ingestion and more toxicity than the amide one. Since PEG was known to induce endocytosis escape, we combined PEG and galactose aiming to provide both cellular up-take and subsequent endocytosis escape. We designed lactose or galactose moieties to attach to the end of the PEG chain that connects to the SPG side chain. When the PEG had the molecular weight of 5000-6000, the antisense effect reached the maximum. We believe that this new type of galactose and PEG dual conjugation broaden the horizon in antisense delivery.

Biocompatible Materials↗
PubMed2005

Parallel vs. anti-parallel orientation in a curdlan/oligo(dA) complex as estimated by a FRET technique.

We already found that beta-1,3-glucan polysaccharides form polymeric complexes with certain polynucleotides, but the parallel vs. anti-parallel orientation in those complexes had remained unsolved. In this paper, this controversial problem has been discussed for curdlan/oligo(dA) complexes utilizing two different energy transfer techniques. The first system consists of a combination of fluorescein-labeled curdlan and 3'-(or 5'-)tetramethyl-rhodamine (TAMRA)-labeled oligo(dA). The second system utilizes gold nanoparticles: that is, two curdlan chains were linked by a disulfide bond and after complexation with oligo(dA), the complex was immobilized on gold nanoparticles. In this system, TAMRA was attached to the 3'(or 5') end of oligo(dA) and the gold particle acted as a fluorescence quencher (energy acceptor). These experiments have led us to conclude that in the curdlan/oligo(dA) complex, parallel orientation is more favourable than anti-parallel orientation. These findings have enabled us to envision a clearer image for the complexation mode between beta-1,3-glucan polysaccharides and polynucleotides.

Fluorescein↗
PubMed2005

PEG-appended beta-(1-->3)-D-glucan schizophyllan to deliver antisense-oligonucleotides with avoiding lysosomal degradation.

Schizophyllan is a natural beta-(1-->3)-d-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO). As we already reported, when a homo-polynucleotide [e.g., poly(dA) or poly(C)] is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. One of the potential applications for this novel complex is an antisense-oligonucleotide (AS ODN) carrier. The present paper describes a modification technique that enabled us to introduce PEG only to the side chain of schizophyllan. This technique consisted of periodate oxidation of the glucose side chain and subsequent reaction between methoxypolyethylene glycol amine and the formyl terminate, followed by reduction with NaBH4. Subsequently, we made a complex from PEG-appended schizophyllan and an AS ODN sequence, and carried out an in vitro antisense assay, administrating the AS ODN complex to depress A375 c-myb mRNA of A375 melanoma cell lines. The PEG-SPG/AS ODN complex showed more enhanced antisnese effect than naked AS ODN dose, i.e., the same level as that of RGD-appended SPG. Here, the RGD system has been shown one on the most effective AS ODN carrier (Science 261 (1993) 1004-1012). When we added nigericin to the assay system, the antisense effect was not affected in the PEG-SPG system, on the other hand, it was almost eliminated in the RGD system. Nigericin is well known to interrupt transport from endosome to lysosome. Therefore, the difference between the PEG and RGD complexes indicates that, in the PEG system, AS ODN was able to escape from lysosomal degradation. The present work has thus proposed a new strategy to delivery AS ODN using schizophyllan as a new carrier.

Biodegradation, Environmental↗
PubMed2004

Removal of the side-chain glucose groups from schizophyllan improves the thermal stability of the polycytidylic acid complexes under the physiological conditions.

Thermal stabilization of the complex between polycytidylic acid [poly(C)] and the modified schizophyllan (SPG) whose hydrophilic side-chain glucose groups are selectively removed utilizing mild Smith-degradation has been investigated. With the decrease in the side-chain glucose groups of schizophyllan, the complex with poly(C) can be considerably stabilized compared with unmodified SPG; for example, the T(m) value after the removal of the side-chain glucose groups from 33.3 (unmodified) to 1.0 is enhanced by 14 degrees C. In addition, the thermal stabilization effect is even operative under the physiological conditions ([NaCl] = 0.15 mol dm(-3)). This effect is exerted owing to the construction of the hydrophobic atmosphere around the complex. Although schizophyllan lost the side-chain glucose groups, it still kept the protection effect of the bound poly(C) chain against RNaseA-mediated hydrolysis as observed for unmodified schizophyllan. The assessment of the cytotoxicity for A375:human malignant melanoma, and HL60:human promyelocytic leukemia revealed that the modified schizophyllan scarcely increases the cytotoxicity. These results indicate that the present modification for schizophyllan is of great significance in a viewpoint to develop the practical gene carriers operative even under the physiological conditions.

Cell Line, Tumor↗
PubMed2004

Proposal of new modification technique for linear double-stranded DNAs using the polysaccharide schizopyllan.

A natural polysaccharide schizophyllan (SPG) has been known to form a stable complex with poly(dA). We attached a poly(dA)(80) tail to the both ends of a linear double-stranded DNA, which had been prepared from a plasmid DNA vector. The poly(dA) tailed DNA verified to form complex with SPG by gel electrophoresis and atomic force microscopy (AFM). AFM images indicated that the complexes exhibit a dumbbell-like architecture, that is, quite similar to that of adenovirus genome. The complex demonstrated excellent exonuclease resistance, probably because of the protection effect by SPG complexation.

Carbohydrate Conformation↗
PubMed2004

Schizophyllans carrying oligosaccharide appendages as potential candidates for cell-targeted antisense carrier.

Schizophyllans carrying beta-lactoside and alpha-mannoside appendages were prepared from native schizophyllan through NaIO4 oxidation followed by reductive amination using aminoethyl-beta-lactoside and alpha-mannoside, respectively. The resulting schizophyllans form stable macromolecular complexes with polynucleotides, such as poly(C) and poly(dA). Specific affinity between these macromolecular complexes and saccharide-binding proteins was demonstrated by surface plasmon resonance and agarose gel staining assays. beta-lactoside-appended schizophyllan enhanced an antisense activity in hepatocytes which express lactoside-binding proteins on their cell surfaces.

Cell Count↗
PubMed2004

A polysaccharide carrier for immunostimulatory CpG DNAs to enhance cytokine secretion.

A beta-(1 --> 3)-d-glucan schizophyllan (SPG) forms a stoichiometric complex with some polynucleotides. This communication describes our attempt to apply the SPG complex to deliver CpG DNA to endosomes to enhance cytokine secretion. To increase cellular uptake, we introduced spermine, arginine-glycine-aspartic acid tripeptide, octaarginine, or cholesterol to the SPG side chain. The chemically modified SPG showed essentially no cytotoxicity. When CpG DNA complex made therefrom was exposed to macrophages, dramatic enhancement in the cytokine secretion was observed. It increased 5-10 times from the naked dose and 100 times from the background. This performance promises that SPG can be an excellent carrier for CpG DNA.

Adjuvants, Immunologic↗
PubMed2004

Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake efficiency.

Schizophyllan is a natural beta-(1-->3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when some homo-phosphodiester polynucleotide (for example, poly(dA) or poly(C)) is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. Furthermore, we have already demonstrated that one of the potential applications of this novel complex is an antisense-oligonucleotide (AS ODN) carrier. This work describes a versatile and universal modification technique which enables us to introduce various functional groups only to the side chain of schizophyllan. This technique consists of periodate oxidation of the glucose side chain (it does not react with the main chain because of the absence of the 1,2-diol group in beta-(1-->3)-glucan) and subsequent introduction of the functional groups into the formyl terminate. In the present work, the introduced functional groups were spermine, octa-arginine (R8), arginine-glycine-aspartic acid tripeptide (RGD) and some amino or alpha-amino acid compounds. Using these compounds, we made the complexes and carried out an in vitro antisense assay for them, administrating a phosphorothioate AS ODN to the melanoma A375 or leukemia HL-60 cell lines to depress their c-myb mRNA. When we used the R8 or RGD modified schizophyllan as the antisense carrier, the antisense effect was most enhanced among others. Their superiority can be ascribed to enhancement of endocytosis due to these functional peptides. Furthermore, the cytotoxicity for these two modified schizophyllans was negligibly as small as the natural (unmodified) schizophyllan. One of the peculiar features of our system is that the complex (i.e., carrier+AS ODN) is charged negatively in total, which is different from the conventional systems. The present work has thus clarified that schizophyllan can act as a new potential candidate for AS ODN carriers.

Animals↗
PubMed2004

Polysaccharide/polynucleotide complexes. Part 6: complementary-strand-induced release of single-stranded DNA bound in the schizophyllan complex.

Spectroscopic properties of single-stranded DNA/schizophyllan ternary complexes (ss-DNA2s-SPG), induced by addition of either complementary or noncomplementary strands, have been investigated. The addition of the complementary strands to ss-DNA2s-SPG induced the quick release of the bound ss-DNA to the complementary strands (both DNA and RNA), whereas the ternary complex was unaffected upon addition of noncomplementary strands. Our experiments imply that SPG has complexation properties indispensable to the gene carriers. As far as we know, there is no report on exploitation of such nonviral gene carriers that can accomplish an intelligent release of the bound ss-DNA toward the complementary strands. We believe, therefore, that SPG, a natural and neutral polysaccharide, has a great potential to become a new ss-DNA carrier.

DNA, Complementary↗
PubMed2004

First observation by fluorescence polarization of complexation between mRNA and the natural polysaccharide schizophyllan.

Schizophyllan is a natural beta-(1-->3)-D-glucan that exists as a triple helix in H(2)O and as a single chain in dimethylsulfoxide (DMSO) or basic solution (pH >13). As we have already reported, when a homo-polynucleotide (e.g., poly(dA), poly(A), or poly(C)) is added to a schizophyllan/DMSO solution, and, subsequently, DMSO is exchanged for H(2)O, the single chain of schizophyllan forms a complex with the polynucleotide. Since eukaryotic mRNAs have poly(A) tails, we hypothesized that schizophyllan can bind to mRNA by interacting with this tail. However, we have not yet observed complexation between schizophyllan and mRNA after exchanging DMSO for H(2)O. In this report, we show that the complexation can be accelerated when the solution pH is changed from 13 to 7-8 in the presence of schizophyllan and polynucleotides. By this approach, we found that schizophyllan forms a complex with a yeast mRNA.

Adjuvants, Immunologic↗
PubMed2004

Protection of polynucleotides against nuclease-mediated hydrolysis by complexation with schizophyllan.

Schizophyllan is a beta-(1-->3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when some homo-polynucleotide (for example, poly(dA) or poly(C)) is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan (s-SPG) forms a complex with the polynucleotide. The present work demonstrates that the polynucleotide bound in the complex is more stable to nuclease-mediated hydrolysis than the polynucleotide itself (i.e., naked polynucleotide), using high-performance liquid chromatography and ultraviolet absorbance technique. A kinetic study for the hydrolysis clarified that the simple Michaelis-Menten relation is held and the maximum velocity for the complex is one-sixth as small as that of the naked polynucleotide. This low hydrolysis rate for the complex suggests that s-SPG is applicable to a carrier for antisense oligonucleotides.

Biocompatible Materials↗
PubMed2004

Antisense oligonucleotides bound in the polysaccharide complex and the enhanced antisense effect due to the low hydrolysis.

Schizophyllan is a beta-(1-->3)-D-glucan and can form a novel complex with some single-chains of DNAs. As the preceding paper revealed, the polynucleotide bound in the complex is more stable to nuclease-mediated hydrolysis than the polynucleotide itself (i.e., naked polynucleotide). This paper examined possibility to apply this complex to an antisense DNA carrier, using an in vitro (cell-free) transcription/translation assay. In this assay, we used a plasmid DNA coding a green fluorescence protein (GFP) and an antisense DNA designed to hybridize the ribosome-binding site in the GFP-coded mRNA. When the antisense DNA was administered as the complex, a lower GFP expression efficiency (or higher antisense effect) is observed over naked DNA. This is because the antisense DNA in the complex is protected from the attack of deoxyribonuclease. When exonuclease I, which specifically hydrolyzes single DNA chains, was present in the GEP assay system, the antisense effect was not changed for the complex while being weakened in the naked antisense DNA system. These results imply that the exonuclease I cannot hydrolyze the antisense DNA in the complex, while it can hydrolyze naked DNA to reduce its antisense effect.

Biocompatible Materials↗
PubMed2003

Polysaccharide-polynucleotide complexes. Part 7. Hydrogen-ion and salt concentration dependence of complexation between schizophyllan and single-stranded homo RNAs.

Schizophyllan belongs to a beta-1,3-D-glucan family, which exists as a random coil in dimethyl sulfoxide (DMSO) and as a triple helix in water, respectively. The schizophyllan single chain forms a complex with single-stranded homo RNAs in water/DMSO mixed solvents. Using circular dichroism, we studied the complexation and its stability as a function of apparent pH (pH(*)) in a mixed solvent system and as a function of the salt concentration. The complex is formed in the pH(*) range 6.5-10, and dissociated in the pH(*) range 4-6. Both poly(A) and poly(C) adopt a double strand in the pH(*) range 4-6 and a single strand in the pH(*) range 6.5-10. Therefore, the conformational change of each polynucleotide is responsible for dissociation/association of the complex, i.e., the single strand of the polynucleotides can form complexes, whereas the double one cannot. This result indicates that hydrogen bonding and similarity of the helix parameters are essential for the complex formation. The melting temperature of the complex reaches the maximum around 0.05 M of NaCl and KCl, and the value of the maximum temperature depends on the cation species.

Circular Dichroism↗
PubMed2003

Microanalysis of nucleic acids using the limulus G test.

The limulus G test has been used as a quantitative analysis of (1-->3)-beta-D-glucans, including schizophyllan (SPG) and curdlan. The present work extended the limulus G test to detect polynucleotide/SPG complexes. The complex showed an extremely sensitive response to the test, compared with SPG itself. The minimum concentration of the complex to show the response is almost 10-times as small as that of SPG itself, indicating the possibility to detect (1-->3)-beta-D-glucans or/and polynucleotides on the pico gram/ml scale.

Glucans↗
PubMed2002

Polysaccharide-polynucleotide complexes VIII. Cation-induced complex formation between polyuridylic acid and schizophyllan.

Schizophyllan belongs to the beta-1,3-D-glucan family, and dissolves as a single chain in dimethyl sulfoxide (DMSO) and as a triple helix in water, respectively. It is already known that when we prepare a mixture of poly(C) and schizophyllan in DMSO and subsequently exchange the solvent to water, poly(C) and schizophyllan can form a complex. When we applied this procedure to the poly(U) system, we could not induce the complex formation between poly(U) and schizophyllan at all. However, we found that addition of some alkaline cations is necessary to induce the complexation between poly(U) and schizophyllan. The complex stability strongly depends on both the cation species and the salt concentration. The complexation is sensitively reflected in the CD spectrum. The magnitude of the spectral change is followed by the order; Rb(+) >> K(+) > Na(+) ? Cs(+). This cation order in the stability is confirmed by the fluorescence polarization measurements. (23)Na-NMR spectroscopy reveals that the product system is stabilized by addition of the cations, suggesting that the OH group in schizophyllan and the phosphate anion in poly(U) synergistically form a specific ligand system for the cations. To the best of our knowledge, such a cation-induced specific interaction between saccharides and polynucleotides has not been reported, and may provide a new clue to understand the biological role of beta-1,3-D-glucans.

Circular Dichroism↗