Mariko Umeda

Single-walled carbon nanotubes acquire a specific lectin-affinity through supramolecular wrapping with lactose-appended schizophyllan.

Single-walled carbon nanotubes can be entrapped within a helical superstructure composed of schizophyllan bearing lactoside-appendages to show an excellent water-solubility as well as a specific lectin-affinity.

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.

Lactose-appended schizophyllan is a potential candidate as a hepatocyte-targeted antisense carrier.

A schizophyllan (beta-1,3-glucan) derivative carrying lactose-appendages prepared by reductive amination can form stable macromolecular complexes with polynucleotides, shows excellent affinity with a lactose-binding lectin, and effectively mediates gene transfection into hepatocytes.

Polysaccharide–polynucleotide complexes (15): thermal stability of schizophyllan (SPG)/poly(C) triple strands is controllable by α-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.

MicroRNA-200a Regulates the Development of Mandibular Condylar Cartilage

Mandibular condylar cartilage (MCC) is classified as secondary cartilage, the histologic structure of which is unique from that of primary cartilage. MicroRNA (miRNA) is a small noncoding RNA that binds to the messenger RNA (mRNA) target to repress its translation and plays an important role in cell differentiation, proliferation, and death. Microarray analysis revealed that miR-200a was characteristically expressed during embryonic development. We hypothesized that miR-200a may be involved in regulating the formation of cartilage during MCC growth. We investigated the function of miR-200a by transfecting an inhibitor or mimic into MCC organ and cell cultures. A histologic examination revealed the localized inhibitory effects of the miR-200a mimic and widespread enhancing effects of the inhibitor on chondrocytic differentiation in the MCC organ culture system. An immunohistochemical examination and gene expression analysis demonstrated that the miR-200a inhibitor enhanced chondrogenesis, while the mimic had the opposite effect by enhancing cell proliferation. Quantitative reverse transcription polymerase chain reaction analysis revealed that miR-200a downregulated the gene expression of chondrocyte markers. Moreover, transfection of the miR-200a mimic into ATDC5 cells repressed the formation of the cartilaginous matrix. These results indicate that miR-200a contributed to chondrogenesis in developing MCC by controlling proliferation and differentiation in MCC cells.

The role of miRNA-200a in the early stage of the mandibular development

Abstract Background MicroRNAs are non-coding small RNA molecules that regulate various cellular functions. In this study, we analyzed the function of microRNA-200a (miR-200a) during the development of primary and secondary cartilages in the mandible during the embryonic stage. Materials and methods Mandibular processes were excised from ICR mouse embryos. Meckel’s cartilage and the complex of condylar and angular cartilages were examined as primary and secondary cartilages, respectively. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridization were performed to examine the expression of miR-200a during normal mandibular development. Transfection of a miR-200a-mimic and -inhibitor were performed to analyze the effect of miR-200a on mandibular cartilage formation. Prior to transfection, DiI labeling was performed to detect the transfection sites and examine the effect of miR-200a on Meckel’s and condylar cartilage formation. Nine days after transfection, Alcian blue staining and quantification were performed to analyze the formation of the cartilage. Results The expression levels of miR-200a gradually increased from embryonic day 9–14 in mandibular processes. However, the expression levels of miR-200a in Meckel’s cartilage were nearly identical from embryonic day 12–16. The positive hybridization signals were observed in Meckel’s cartilage and mesenchymal condensation of mandibular condylar cartilage. Alcian blue analysis showed a significant decrease in the complex of condylar and angular cartilage formation in the lateral-anterior-miR-200a-mimic transfected samples compared to miR-200a-inhibitor-transfected and control samples. Conclusion miR-200a negatively regulates the formation of condylar cartilage in early mandibular development.