Biomedical subjects

Kazuo Sakurai

Publications and source records attributed to Kazuo Sakurai.

45 records · Page 3Linked to original sources

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↗

PubMed2002

Sclerosing mucoepidermoid carcinoma with eosinophilia of the salivary glands.

We encountered two cases of low malignant mucoepidermoid carcinoma with scanty cellular atypism which originated in the parotid or submandibular gland and was characterized by marked fibrosis and eosinophilic infiltration within tumor tissue despite the predominance of the squamous component. Here we report these two cases and provide a review of the literature. We believe that clinically these two tumors with stromal fibrosis and eosinophilic infiltration have a low malignant potential, although histological examination revealed a scanty mucus-producing epithelial component. Therefore, we consider this type of tumor as a new subtype of mucoepidermoid carcinoma. A low-malignant mucoepidermoid carcinoma with stromal fibrosis and eosinophilic infiltration, as described in these two cases, may be misdiagnosed as a highly malignant mucoepidermoid carcinoma or squamous cell carcinoma because of its histologically scanty mucus-producing epithelial component. The objective of this study was to clarify their differences and to discuss the rendering of an accurate histological diagnosis, the degree of malignancy in relation to prognosis prediction, and the choice of therapy. In addition, we propose regarding this type of tumor as a new subtype of mucoepidermoid carcinoma.

Adult↗

PubMedDate not supplied

Long-term expression with a cationic polymer derived from a natural polysaccharide: schizophyllan.

Among the various synthetic gene carriers based on biomaterials, cationic polymers with polysaccharide backbones have long been studied as nonviral vectors due to their low immunogenicity and high water solubility. Schizophyllan, a beta-(1,3)-glucan, is one of the various polysaccharides that are clinically administered. Furthermore, its safety in the human body has already been confirmed. Various functional groups can be selectively introduced into the side chain, not into the main chain of schizophyllan. Therefore, we have synthesized various oligoamine conjugates from schizophyllan. It was confirmed that their in vitro transfection efficiencies are superior to that of polyethylenimine by adjusting the molecular weight and the degree of amination of cationic schizophyllan. While it was possible to reduce cytotoxicity by adjusting the amount of DNA complex per cell, as seen with poly-L-lysine, polyethylenimine, and chitosan, PEGylation was the most effective means of reducing toxicity. Furthermore, using cationic schizophyllan carriers, it was also possible to express a reporter protein for a long period of time due to a long residence time of plasmid DNA in cells.

Animals↗

PubMedDate not supplied

Transition from a normal to inverted cylinder for an amidine-bearing lipid/pDNA complex and its excellent transfection.

A cationic lipid (TRX) having an amidine headgroup was synthesized, and a lipoplex (i.e., plasmid DNA+lipid complex) was prepared from the mixture of TRX and two other neutral colipids. Small-angle X-ray scattering showed that the addition of DNA induced a structural transition from normal to inverted hexagonally packed cylinders. Transmission electron microscopy showed a threadlike micelle was transformed to a spherical micelle about 50-200 nm in diameter by adding DNA. A combination of these results leads to the conclusion that the complexed DNA is hexagonally packed (or condensed) into spherical aggregates. The size and morphology are believed suitable for endocytosis uptake or vesicle fusion. The complex made from pDNA (pEGFP-C1) and TRX was transfected to Hep G2. Flow cytometry and confocal microscopy showed that the present system expressed green fluorescence protein (GFP) more than a conventional transfection reagent. Additionally, TRX was less cytotoxic than other transfection reagents. This paper presents the attractive possibility of the amidine group for a transfection device.

Amidines↗

PubMedDate not supplied

Synthesis and in vitro characterization of antigen-conjugated polysaccharide as a CpG DNA carrier.

Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) are known as an immune adjuvant. CpG DNAs coupled with a particular antigen enabling both CpG DNA and antigen delivery to the same antigen-presenting cell have been shown to be more effective. Based on our previous finding that beta-(1-->3)-D-glucan schizophyllan (SPG) can be used as a CpG DNA carrier, here we present the synthesis of an antigen-conjugated SPG and the characterization of the conjugate. Ovalbumin (OVA, 43 kDa) was used as a model antigen, and two OVA were conjugated to one SPG molecule (M(w) = 150,000), denoted by OVA-SPG. Circular dichroism and gel electrophoresis showed that OVA-SPG could form a complex with a (dA)(40)-tailed CpG DNA at the 3' end (1,668-(dA)(40)). When OVA-SPG was added to macrophages (J774.A1), the amount of the ingested OVA-SPG was increased compared with that of OVA itself, suggesting that Dectin-1 (proinflammatory nonopsonic receptor for beta-glucans) is involved to ingest OVA-SPG. Furthermore, the complex of the conjugate and DNA was co-localized in the same vesicles, implying that OVA (antigen) and CpG DNA (adjuvant) were ingested into the cell at the same time. This paper shows that OVA-SPG can be used as a CpG DNA carrier to induce antigen-specific immune responses.

Adjuvants, Immunologic↗

PubMedDate not supplied

Polysaccharide-polynucleotide complexes. Part 32. Structural analysis of the curdlan/poly(cytidylic acid) complex with semiempirical molecular orbital calculations.

Natural Curdlan adopts a right-handed 6(1) triple helix, in which the constituting glucan chains are underpinned with each other by the intermolecular hydrogen bonds. Curdlan can form a stoichiometric complex with polynucleotides [e.g., poly(cytidylic acid), poly(C)]. In this paper, we carried out the MOPAC (semiempirical molecular-orbital package) calculation to examine the molecular structure of the Curdlan/poly(C) complex. The calculation exhibited that two types of hydrogen bonds are formed between the Curdlan and the poly(C); the third nitrogen (N3) in cytosine forms a hydrogen bond with the second OH of one Curdlan chain, and the proton of N4 is interacting with the O2 of another Curdlan chain. In our model, the helix diameter of poly(C) is expanded from 11.0 to 15.3 A upon complexation. Despite such large conformational changes, the 6(1) helix structure of poly(C) was maintained even after the complexation. This fact is complementary to the experimental fact that the complexation does not change the band shape of the circular dichroism of poly(C). The chain length dependence of the reaction enthalpy indicated that the complexation becomes thermodynamically more favorable with the chain length increasing. This feature is also consistent with the experimental data.

Carbohydrate Conformation↗

PubMedDate not supplied

Separation technique for messenger RNAs by use of schizophyllan/poly(A) tail complexation.

Schizophyllan (SPG) is one of the water soluble beta-1,3-glucans and has a peculiar molecular recognition capability, namely, the single stranded SPG (s-SPG) can form a stoichiometric complex with certain polynucleotides such as poly(C) and poly(A), although it cannot bind poly(G) and poly(dC) at all. In this paper, we prepared an s-SPG-appended column and made an attempt to separate polynucleotides on the bases of this molecular recognition capability. The s-SPG-appended column trapped only such RNAs that could form the complex with s-SPG but eluted other RNAs which did not form the complex. Encouraged by the results in the model system, we extended the s-SPG-appended column into separation of native messenger RNAs (mRNAs) from a RNA mixture (total RNA) obtained from yeast. Since eukaryotic mRNAs have a poly(A) tail with 150-300 bases, we supposed that the tails would be trapped by the s-SPG-appended column. The results indicate that mRNAs were separated from total RNA in good yield and with high purity. It should be emphasized that this is the first device to separate natural mRNAs without using a dA/dT Watson-Crick-type interaction.

Carbohydrate Sequence↗