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Kazuo Sakurai

Publications and source records attributed to Kazuo Sakurai.

At least 37 records · Page 2Linked to original sources
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

Beta-1,3-glucan (schizophyllan) can act as a one-dimensional host for creation of novel poly(aniline) nanofiber structures.

We here demonstrate the creation of novel poly(aniline) (PANI) nanofiber structures by a polymer wrapping method using schizophyllan (SPG). Mannose-modified SPG can also wrap PANIs to give nanofibers having a lectin affinity. This interaction is applicable to designing novel PANI/protein composites. The results establish that SPG can act as a novel "host" to assemble PANIs into one-dimensional superstructures. [reaction: see text]

Aniline Compounds↗
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

Rod-like architecture and helicity of the poly(C)/schizophyllan complex observed by AFM and SEM.

Microscopic studies of the complex between poly(C) and schizophyllan (SPG), employing both AFM and SEM, revealed that the complex takes the same rod-like architecture on the mica surface as those of the renatured SPG and the original triple helix of SPG, indicating that the complex also has a helical structure. The SEM observations showed the helical pattern on the rod surface, only when the sample was metal shadowed. The pitch evaluated from the image is comparable with that obtained from crystallographic data. The ability to visualize the helical structure can be explained from the hypothesis that the platinum grains may assemble on the sample using the molecular surface of the SPG (or complex) as the template.

Carbohydrate Conformation↗
PubMed2004

Low Mw sulfated curdlan with improved water solubility forms macromolecular complexes with polycytidylic acid.

The water solubility of curdlan was enhanced by partial sulfation at O-6. Circular dichroism measurements suggest that the sulfated curdlan with the degree of substitution (DS) from 0 to 8.7 mol% forms macromolecular complexes with polycytidylic acid (poly(C)). Although the thermal stability of the complexes decreased with increase in DS, this could be overlapped by addition of NaCl in the concentration above that of serum. The results clearly indicate that the drawback arising from the electrostatic repulsion between the anionic charges can be partially compensated by the presence of salt. Furthermore, the polynucleotide chain complexed with the sulfated curdlan was protected from the enzymatic hydrolysis, corroborating the assumption that the sulfated curdlan has an ability to bind poly(C).

Anions↗
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

Proposal of a new hydrogen-bonding form to maintain curdlan triple helix.

Curdlan and other beta-1,3-D-glucans form right-handed triple helices, and it has been believed that the intermolecular H-bond is present at the center of the helix to maintain the structure. In this H-bond model, three secondary OH groups form an inequilateral hexagonal shape perpendicular to the helix axis. This hexagonal form seems to be characteristic for beta-1,3-D-glucans and is widely accepted. We carried out MOPAC and ab initio calculations for the curdlan helix, and we propose a new intermolecular H-bonding model. In our model, the H-bonds are formed between the O2-atoms on different x-y planes along the curdlan helix, hence the H-bonds are not perpendicular to the helix axis. The new H-bonds are connected along the helix, traversing three curdlan chains to make a left-handed helix. Therefore, the H-bonding array leads to a reverse helix of the main chain. According to our MOPAC calculation, this model is more stable than the previous one. We believe that the continuous H-bonding array is stabilized by cooperative phenomena in the polymeric system.

Carbohydrate Conformation↗
PubMed2004

Composite lymphoma arising in the parotid gland: a case report.

A case of composite lymphoma (CL) arisen in the parotid gland in a 58-year-old male is reported. Histologically, most of the tumor region was follicular lymphoma (FL), grade 2. However complicating classical Hodgkin lymphoma (CHL) was noted inside the lesion. There have been a few reports of composite lymphoma, complicated by multiple histologic types in the same organ. Extra-nodal cases are particularly rare. This is a very significant case with regard to differences between B cell-derived non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL) and its histological origin.

Antineoplastic Combined Chemotherapy Protocols↗
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 (15): thermal stability of schizophyllan (SPG)/poly(C) triple strands is controllable by alpha-amino acid modification.

Schizophyllan (SPG), a beta-1,3-glucan polysaccharide which is known to form macromolecular complexes with certain polynucleotides, was modified by a reductive amination method with alpha-amino acids (Arg, Lys, and Ser). The thermal stability of the complexes as estimated by T(m) was enhanced in SPG-Arg and SPG-Lys conjugates which have pI values higher than the pH of the medium (8.0). The T(m) shift increased with the increase in the percentage of alpha-amino acid introduced and the highest T(m) values attained were 64 degrees C for SPG-Arg conjugate and 62 degrees C for SPG-Lys conjugate, which are higher by 13 and 11 degrees C, respectively, than those of the unmodified SPG+poly(C) complex. In the SPG-Ser conjugate with a pI lower than the medium pH (8.0), the T(m) values decreased with an increase in the percentage of Ser. Formation of the macromolecular complex was no longer detected above 13.2% Ser. The findings indicate that the T(m) values are easily controllable by the type and percentage of the introduced alpha-amino acids. We believe, therefore, that the present conjugates, consisting of naturally originated SPG and alpha-amino acids, provide an important lead for developing nontoxic artificial vectors and to control the affinity with polynucleotides in response to medium pH and temperature.

Amino Acids↗