Synthesis of cyclic ß-1,6-oligosaccharides from glucosamine monomers by electrochemical polyglycosylation.

The synthesis of protected precursors of cyclic ß-1,6-oligoglucosamines from thioglycosides as monomers is performed by electrochemical polyglycosylation. The monomer with a 2,3-oxazolidinone protecting group afforded the cyclic disaccharide exclusively. Cyclic oligosaccharides up to the trisaccharide were obtained using the monomer with a 2-azido-2-deoxy group.

Two-Dimensional Living Supramolecular Polymerization: Improvement in Edge Roughness of Supramolecular Nanosheets by Using a Dummy Monomer.

Supramolecular polymers are formed through nucleation (i. e., initiation) and polymerization processes, and kinetic control over the nucleation process has recently led to the realization of living supramolecular polymerization. Changing the viewpoint, herein we focus on controlling the polymerization process, which we expect to pave the way to further developments in controlled supramolecular polymerization. In our previous study, two-dimensional living supramolecular polymerization was used to produce supramolecular nanosheets with a controlled area; however, these had rough edges. In this study, the growth of the nanosheets was controlled by using a 'dummy' monomer to produce supramolecular nanosheets with smoothed edges.

Electrochemical synthesis of the protected cyclic (1,3;1,6)-ß-glucan dodecasaccharide.

Automated electrochemical assembly is an electrochemical method to synthesise middle-sized molecules, including linear oligosaccharides, and some linear oligosaccharides can be electrochemically converted into the corresponding cyclic oligosaccharides effectively. In this study, the target cyclic oligosaccharide is a protected cyclic (1,3;1,6)-ß-glucan dodecasaccharide, which consists of two types of glucose trisaccharides with ß-(1,3)- and ß-(1,6)-glycosidic linkages. The formation of the protected cyclic dodecasaccharide was confirmed by the electrochemical one-pot dimerisation-cyclisation of the semi-circular hexasaccharide. The yield of the protected cyclic dodecasaccharide was improved by using a stepwise synthesis via the linear dodecasaccharide.

Synthesis of protected precursors of chitin oligosaccharides by electrochemical polyglycosylation of thioglycosides.

The synthesis of protected precursors of chitin oligosaccharides by electrochemical polyglycosylation of thioglycosides as monomer is described. Oligosaccharides up to the hexasaccharide were synthesized under optimized reaction conditions. Further, a modified method enabled the synthesis of oligosaccharides up to the octasaccharide by repeating electrolysis with additional monomers. The mechanism of the electrochemical polyglycosylation is also discussed, based on the oxidation potential of the monomer and oligosaccharides.

Glycan characteristics of human heart constituent cells maintaining organ function: relatively stable glycan profiles in cellular senescence.

Cell surface glycoproteins, which are good indicators of cellular types and biological function; are suited for cell evaluation. Tissue remodeling using various cells is a key feature of regenerative therapy. For artificial heart remodeling, a mixture of heart constituent cells has been investigated for organ assembly, however, the cellular characteristics remain unclear. In this study, the glycan profiles of human cardiomyocytes (HCMs), human cardiac fibroblasts (HCFs), and human vascular endothelial cells (ECs) were analyzed using evanescent-field lectin microarray analysis, a tool of glycan profiling, to clarify the required cellular characteristics. We found that ECs had more "α1-2fucose" and "core α1-6fucose" residues than other cells, and that "α2-6sialic acid" residue was more abundant in ECs and HCMs than in HCFs. HCFs showed higher abundance of "ß-galactose" and "ß-N-acetylgalactosamine" residues on N-glycan and O-glycan, respectively, compared to other cells. Interestingly, cardiac glycan profiles were insignificantly changed with cellular senescence. The residues identified in this study may participate in organ maintenance by contributing to the preservation of glycan components. Therefore, future studies should investigate the roles of glycans in optimal tissue remodeling since identifying cellular characteristics is important for the development of regenerative therapies.

Gangliosides as Signaling Regulators in Cancer.

At the plasma membrane, gangliosides, a group of glycosphingolipids, are expressed along with glycosphingolipids, phospholipids, and cholesterol in so-called lipid rafts that interact with signaling receptors and related molecules. Most cancers present abnormalities in the intracellular signal transduction system involved in tumor growth, invasion, and metastasis. To date, the roles of gangliosides as regulators of signal transduction have been reported in several cancer types. Gangliosides can be expressed by the exogenous ganglioside addition, with their endogenous expression regulated at the enzymatic level by targeting specific glycosyltransferases. Accordingly, the relationship between changes in the composition of cell surface gangliosides and signal transduction has been investigated by controlling ganglioside expression. In cancer cells, several types of signaling molecules are positively or negatively regulated by ganglioside expression levels, promoting malignant properties. Moreover, antibodies against gangliosides have been shown to possess cytotoxic effects on ganglioside-expressing cancer cells. In the present review, we highlight the involvement of gangliosides in the regulation of cancer cell signaling, and we explore possible therapies targeting ganglioside-expressing cancer.

Cell Surface and Functional Features of Cortical Bone Stem Cells.

The newly established mouse cortical-bone-derived stem cells (mCBSCs) are unique stem cells compared to mouse mesenchymal stem cells (mMSCs). The mCBSC-treated hearts after myocardial infarction have been reported to have greater improvement in myocardial structure and functions. In this study, we examined the stemness features, cell surface glycan profiles, and paracrine functions of mCBSCs compared with mMSCs. The stemness analysis revealed that the self-renewing capacity of mCBSCs was greater than mMSCs; however, the differentiation capacity of mCBSCs was limited to the chondrogenic lineage among three types of cells (adipocyte, osteoblast, chondrocyte). The cell surface glycan profiles by lectin array analysis revealed that α2-6sialic acid is expressed at very low levels on the cell surface of mCBSCs compared with that on mMSCs. In contrast, the lactosamine (Galß1-4GlcNAc) structure, poly lactosamine- or poly N-acetylglucosamine structure, and α2-3sialic acid on both N- and O-glycans were more highly expressed in mCBSCs. Moreover, we found that mCBSCs secrete a greater amount of TGF-ß1 compared to mMSCs, and that the TGF-ß1 contributed to the self-migration of mCBSCs and activation of fibroblasts. Together, these results suggest that unique characteristics in mCBSCs compared to mMSCs may lead to advanced utility of mCBSCs for cardiac and noncardiac repair.

Characterization of the metastatic potential of the floating cell component of MIA PaCa-2, a human pancreatic cancer cell line.

In pancreatic cancer, morphologically and functionally heterogeneous cancer cells reside within the same patient. The heterogeneity is believed to promote metastasis and resistance to chemoradiotherapy. MIA PaCa-2, an established human pancreatic ductal adenocarcinoma (PDAC) cell line, contains round and spindle-shaped adherent cells, as well as, round floating cells. In this study, we aimed to assess if the floating cells might have greater metastatic potential and/or be more resistant to drug-induced apoptosis compared to adherent cells. Time-lapse analysis revealed that the two types of adherent cells transformed bilaterally, and some of the adherent, round cells converted to floating cells. Flow cytometry and electron microscopy showed that approximately 90% of the floating cells were viable. qRT-PCR analysis revealed that floating cells expressed lower levels of integrins and ATP-binding cassette (ABC) transporters than adherent cells. In contrast, except for vimentin, floating cells expressed more epithelial to mesenchymal transition markers than adherent cells. Floating cells included a larger population of G2/M-phase cells, and migration assays revealed a decreased migration ability by floating cells relative to adherent cells. A cell aggregation assay showed that the aggregative properties of the floating cells were lower than those of the adherent cells. In 3D culture, spheres derived from floating cells were more sensitive to anti-cancer drugs, including gemcitabine, 5-FU, and abraxane, than those derived from adherent cells. Expression levels of stemness markers in the spheres derived from floating cells were lower than those derived from adherent cells. Morphological characterization of human PDAC cell lines may help to clarify the series of alterations cancer cells undergo during the metastatic process and may contribute to the development of new PDAC diagnostics and more patient-specific treatments for those with PDAC.

Control over the Aspect Ratio of Supramolecular Nanosheets by Molecular Design.

Recent developments in kinetically controlled supramolecular polymerization permit control of the size (i.e., length and area) of self-assembled nanostructures. However, control of molecular self-assembly at a level comparable with organic synthetic chemistry and the achievement of structural complexity at a hierarchy larger than the molecular level remain challenging. This study focuses on controlling the aspect ratio of supramolecular nanosheets. A systematic understanding of the relationship between the monomer structure and the self-assembly energy landscape has derived a new monomer capable of forming supramolecular nanosheets. With this monomer in hand, the aspect ratio of a supramolecular nanosheet is demonstrated that it can be controlled by modulating intermolecular interactions in two dimensions.

Rapamycin promotes endothelial-mesenchymal transition during stress-induced premature senescence through the activation of autophagy.

BACKGROUND: Rapamycin is known to be effective in suppressing senescence and the senescence-associated secretory phenotype (SASP). Therefore, it is highly expected to represent an anti-aging drug. Its anti-aging effect has been demonstrated at the mouse individual level. However, there are not many clinical findings with respect to its activity in humans. Here, we aimed to clarify the effect of rapamycin on human endothelial cells (ECs) as an in vitro model of human blood vessels. METHODS: Over the course of oxidative stress-induced senescence using hydrogen peroxide, we examined the effect of rapamycin on human coronary artery ECs (HCAECs). Senescence was evaluated by detecting senescence-associated ß-galactosidase (SA-ß-Gal) activity and the real-time PCR analysis of p16INK4a. Furthermore, expression levels of SASP factors were examined by real-time PCR and the expression of senescence-related antigens, such as intercellular adhesion molecule-1 (ICAM-1) and ganglioside GM1, were examined by fluorescence-activated cell sorting analysis and immunostaining. The inhibitory effect of rapamycin on mTOR signaling was examined by immunoblotting. The adhesion of leukocytes to HCAECs was evaluated by adhesion assays. Endothelial-mesenchymal transition (EndMT) induced by rapamycin treatment was evaluated by real-time PCR analysis and immunostaining for EndMT markers. Finally, we checked the activation of autophagy by immunoblotting and examined its contribution to EndMT by using a specific inhibitor. Furthermore, we examined how the activation of autophagy influences TGF-ß signaling by immunoblotting for Smad2/3 and Smad7. RESULTS: A decrease in SA-ß-Gal activity and the suppression of SASP factors were observed in HCAECs undergoing stress-induced premature senescence (SIPS) after rapamycin treatment. In contrast, ICAM-1 and ganglioside GM1 were upregulated by rapamycin treatment. In addition, leukocyte adhesion to HCAECs was promoted by this treatment. In rapamycin-treated HCAECs, morphological changes and the promotion of EndMT were also observed. Furthermore, we found that autophagy activation induced by rapamycin treatment, which led to activation of the TGF-ß pathway, contributed to EndMT induction. CONCLUSIONS: We revealed that although rapamycin functions to inhibit senescence and suppress SASP in HCAECs undergoing SIPS, EndMT is induced due to the activation of autophagy. Video abstract.

Establishment and characterization of a novel neuroendocrine carcinoma cell line derived from a human ascending colon tumor.

The incidence of rare neuroendocrine tumors (NET) is rapidly increasing. Neuroendocrine carcinoma (NEC) is a NET with poorly differentiated histological features, high proliferative properties and associated poor prognoses. As these carcinomas are so rare and, thus, affect only a small number of patients allowing for few cell lines to be derived from patient biopsies, the histological, immunohistochemical, and clinical characteristics associated with colorectal NEC and NEC in other organs have yet to be clearly defined. Herein, we describe the establishment of a novel NEC cell line (SS-2) derived from a tumor resection of the ascending colon from a 59-year-old Japanese woman. The histological, electron microscopic and immunohistochemical features of chromogranin A (CgA) as well as confirmation of synaptophysin positivity in this tumor were typical of those commonly observed in surgically resected colorectal NEC. Further, the Ki-67 labeling index of the resected tumor was >20% and, thus, the tumor was diagnosed as an NEC of the ascending colon. The SS-2 cell line maintained characteristic features to those of the resected tumor, which were further retained following implantation into subcutaneous tissues of nude mice. Additionally, when SS-2 cells were seeded into ultra-low attachment plates, they formed spheres that expressed higher levels of the cancer stem cell (CSC) marker CD133 compared to SS-2 cells cultured under adherent conditions. SS-2 cells may, therefore, contribute to the current knowledge on midgut NEC biological function while providing a novel platform for examining the effects of colorectal NEC drugs, including CSC.

Fetal bovine serum enlarges the size of human pancreatic cancer spheres accompanied by an increase in the expression of cancer stem cell markers.

Pancreatic ductal adenocarcinoma (PDAC) is a major histological type of pancreatic cancer and remains one of the most lethal cancers with a high mortality rate owing to its aggressive growth, high metastatic rate, and recurrence. Recent studies on cancer stem cells (CSCs) have suggested that the aggressive growth, high metastatic rate, and recurrence might be caused by the ability of CSCs to self-renew, differentiate, and drive tumorigenesis. Thus, CSCs are expected to be a therapeutic target for PDAC. Sphere forming assay of cancer cells, including PDAC cells, is commonly performed using epidermal growth factor and fibroblast growth factor-2 containing serum-free medium to identify and isolate the enriched CSCs. Recently, we observed that PDAC spheres cultured in fetal bovine serum containing medium are morphologically similar to spheres cultured in the growth factor containing medium. In this study, we cultured two PDAC cell lines, PANC-1 and PK-1, in growth factor containing serum-free medium or serum containing medium, and compared the morphology of the spheres formed in detail by electron microscopy and examined the expression of major CSC marker genes. Both the PDAC cells formed larger spheres in the serum containing medium than in the growth factor containing medium. PK-1 cells formed more morphologically differentiated spheres, with peripheral flat lining cells, in the serum containing medium. Expression levels of most of the CSC markers were higher in the spheres of the two PDAC cells in both the culture mediums than in the cells cultured under adherent conditions. The expression levels of CSC markers in PDAC spheres cultured in the growth factor containing medium were not necessarily higher than that in the spheres cultured in the serum containing medium. These findings suggest that sphere forming assay using serum containing medium, by which large PDAC spheres with enriched CSCs are formed, may be useful for deciphering the characteristics of CSCs and for developing anti-CSC therapies for PDAC.

Vascular Diseases and Gangliosides.

Vascular diseases, such as myocardial infarction and cerebral infarction, are most commonly caused by atherosclerosis, one of the leading causes of death worldwide. Risk factors for atherosclerosis include lifestyle and aging. It has been reported that lifespan could be extended in mice by targeting senescent cells, which led to the suppression of aging-related diseases, such as vascular diseases. However, the molecular mechanisms underlying the contribution of aging to vascular diseases are still not well understood. Several types of cells, such as vascular (endothelial cell), vascular-associated (smooth muscle cell and fibroblast) and inflammatory cells, are involved in plaque formation, plaque rupture and thrombus formation, which result in atherosclerosis. Gangliosides, a group of glycosphingolipids, are expressed on the surface of vascular, vascular-associated and inflammatory cells, where they play functional roles. Clarifying the role of gangliosides in atherosclerosis and their relationship with aging is fundamental to develop novel prevention and treatment methods for vascular diseases based on targeting gangliosides. In this review, we highlight the involvement and possible contribution of gangliosides to vascular diseases and further discuss their relationship with aging.

Gangliosides Contribute to Vascular Insulin Resistance.

Insulin in physiological concentrations is important to maintain vascular function. Moreover, vascular insulin resistance contributes to vascular impairment. In the elderly, other factors including hypertension, dyslipidemia, and chronic inflammation amplify senescence of vascular endothelial and smooth muscle cells. In turn, senescence increases the risk for vascular-related diseases such as arteriosclerosis, diabetes, and Alzheimer's disease. Recently, it was found that GM1 ganglioside, one of the glycolipids localized on the cell membrane, mediates vascular insulin resistance by promoting senescence and/or inflammatory stimulation. First, it was shown that increased GM1 levels associated with aging/senescence contribute to insulin resistance in human aortic endothelial cells (HAECs). Second, the expression levels of gangliosides were monitored in HAECs treated with different concentrations of tumor necrosis factor-alpha (TNFα) for different time intervals to mimic in vivo acute or chronic inflammatory conditions. Third, the levels of insulin signaling-related molecules were monitored in HAECs after TNFα treatment with or without inhibitors of ganglioside synthesis. In this review, we summarize the molecular mechanisms of insulin resistance in aged/senescent and TNFα-stimulated endothelial cells mediated by gangliosides and highlight the possible roles of gangliosides in vascular insulin resistance-related diseases.

Reduced expression of the H19 long non-coding RNA inhibits pancreatic cancer metastasis.

H19 is an oncofetal RNA expressed in the developing embryo as well as in bladder, breast, gastric, pancreatic, hepatocellular, and prostate cancers. Recent studies have shown that H19 enhances cancer invasion and metastasis; however, its roles in cancer remain controversial. In the current study, H19 exhibited the second largest increase (82.4-fold) and represented the only non-protein coding gene among 11 genes identified that were elevated over 10-fold in lung-metastasis-derived pancreatic cancer cells compared with their parental cells using a mouse metastatic model. Subsequently, we further clarified the roles of H19 in pancreatic cancer growth and metastasis using in vitro and in vivo techniques. In situ hybridization showed that H19 was detected in 23 of 139 invasive ductal carcinomas (17%), and that H19 expression positively correlated with higher histological grades (P < 0.0001). Overexpression of H19 in PANC-1 pancreatic cancer cells induced higher motilities, whereas H19 inhibition using shRNA and siRNA showed opposite results; however, cell growth rates were not impacted. Intravenous injection of H19 shRNA vector-transfected PANC-1 cells yielded marked inhibition of metastasis in the liver and lungs of immunodeficient mice. These findings suggest that H19 has important roles in pancreatic cancer metastasis, and that inhibition of H19 represents a novel candidate for pancreatic cancer therapy.

Stemness and anti-cancer drug resistance in ATP-binding cassette subfamily G member 2 highly expressed pancreatic cancer is induced in 3D culture conditions.

The expression of ATP-binding cassette subfamily G member 2 (ABCG2) is related to tumorigenic cancer stem cells (CSC) in several cancers. However, the effects of ABCG2 on CSC-related malignant characteristics in pancreatic ductal adenocarcinoma (PDAC) are not well elucidated. In this study, we compared the characteristics of low (ABCG2-) and high (ABCG2+)-ABCG2-expressing PDAC cells after cell sorting. In adherent culture condition, human PDAC cells, PANC-1, contained approximately 10% ABCG2+ cell populations, and ABCG2+ cells displayed more and longer microvilli compared with ABCG2- cells. Unexpectedly, ABCG2+ cells did not show significant drug resistance against fluorouracil, gemcitabine and vincristine, and ABCG2- cells exhibited higher sphere formation ability and stemness marker expression than those of ABCG2+ cells. Cell growth and motility was greater in ABCG2- cells compared with ABCG2+ cells. In contrast, epithelial-mesenchymal transition ability between ABCG2- and ABCG2+ cells was comparable. In 3D culture conditions, spheres derived from ABCG2- cells generated a large number of ABCG2+ cells, and the expression levels of stemness markers in these spheres were higher than spheres from ABCG2+ cells. Furthermore, spheres containing large populations of ABCG2+ cells exhibited high resistance against anti-cancer drugs presumably depending on ABCG2. ABCG2+ cells in PDAC in adherent culture are not correlated with stemness and malignant behaviors, but ABCG2+ cells derived from ABCG2- cells after sphere formation have stemness characteristics and anti-cancer drug resistance. These findings suggest that ABCG2- cells generate ABCG2+ cells and the malignant potential of ABCG2+ cells in PDAC varies depending on their environments.

Direct Observation and Manipulation of Supramolecular Polymerization by High-Speed Atomic Force Microscopy.

Despite recent advances in mechanistic understanding and controlled-synthesis methodologies regarding synthetic supramolecular assemblies, it has remained challenging to capture the molecular-level phenomena in real time, thus hindering further progress in this research field. In this study, we applied high-speed atomic-force microscopy (AFM), which has extraordinary spatiotemporal resolution (1 nm and sub-100 ms), to capture dynamic events occurring during synthetic molecular self-assembly. High-speed AFM permitted the visualization of unique dynamic behavior, such as seeded growth and self-repair in real time. Furthermore, scanning-probe AFM permitted the site-specific manipulation and functionalization of a molecular self-assembly. This powerful combination of bottom-up and top-down approaches at the molecular level should enable targeted syntheses of unprecedented functional nanoarchitectures.

Total synthesis of TMG-chitotriomycin based on an automated electrochemical assembly of a disaccharide building block.

The total synthesis of TMG-chitotriomycin using an automated electrochemical synthesizer for the assembly of carbohydrate building blocks is demonstrated. We have successfully prepared a precursor of TMG-chitotriomycin, which is a structurally-pure tetrasaccharide with typical protecting groups, through the methodology of automated electrochemical solution-phase synthesis developed by us. The synthesis of structurally well-defined TMG-chitotriomycin has been accomplished in 10-steps from a disaccharide building block.

Ganglioside GM1 Contributes to the State of Insulin Resistance in Senescent Human Arterial Endothelial Cells.

Vascular endothelial cells (ECs) play central roles in physiologically important functions of blood vessels and contribute to the maintenance of vascular integrity. Therefore, it is considered that the impairment of EC functions leads to the development of vascular diseases. However, the molecular mechanisms of the EC dysfunctions that accompany senescence and aging have not yet been clarified. The carbohydrate antigens carried by glycoconjugates (e.g. glycoproteins, glycosphingolipids, and proteoglycans) mainly present on the cell surface serve not only as marker molecules but also as functional molecules. In this study, we have investigated the abundance and functional roles of glycosphingolipids in human ECs during senescence and aging. Among glycosphingolipids, ganglioside GM1 was highly expressed in abundance on the surface of replicatively and prematurely senescent ECs and also of ECs derived from an elderly subject. Insulin signaling, which regulates important functions of ECs, is impaired in senescent and aged ECs. Actually, by down-regulating GM1 on senescent ECs and overloading exogenous GM1 onto non-senescent ECs, we showed that an increased abundance of GM1 functionally contributes to the impairment of insulin signaling in ECs. Taken together, these findings provide the first evidence that GM1 increases in abundance on the cell surface of ECs under the conditions of cellular senescence and aging and causes insulin resistance in ECs. GM1 may be an attractive target for the detection, prevention, and therapy of insulin resistance and related vascular diseases, particularly in older people.

Phenotype-based clustering of glycosylation-related genes by RNAi-mediated gene silencing.

Glycan structures are synthesized by a series of reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide-O-xylosyltransferase, ß1,4-galactosyltransferase I, ß1,3-galactosyltransferase II, and ß1,3-glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N-linked, O-linked, Notch-related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N-linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes.