During the last decade, a novel mechanism of protein release has been recognized that involves small (30-100 nm) membrane vesicles termed exosomes. [1][2][3] Exosomal vesicles are secreted following the fusion of multivesicular late endosomes with the plasma membrane. While the range of exosomal proteins depends on cell type, these vesicles commonly carry cell-surface proteins and cytoskeletal proteins. Several physiologic roles have been assigned to exosomes, including the expulsion of obsolete membrane constituents, exchange of cellular material and intercellular communication. Exosome production has been observed in a variety of cell types in vitro, including reticulocytes, 4) cytotoxic T lymphocytes, 5)B lymphocytes, 6) dendritic cells 7) and neoplastic intestinal epithelial cells.8) Recent studies have reported that such vesicles are present in some physiological fluids, such as bronchoalveolar lavage 9) or urine. 10) However, there is still little evidence of whether exosomes are produced in vivo.Saliva is considered to provide the first line of oral cavity defense against bacterial and viral attack. Human whole saliva contains a potent mixture of diverse components such as mucin, immunoglobulin A (IgA), proline-rich proteins and defensins, which are produced in three major paired salivary glands (parotid, submadibular and sublingual) and several minor glands. Although comprehensive proteome analyses of whole saliva have been reported recently, 12,13) the protein constituents of saliva are not fully understood.The membrane-associated serine protease dipeptidyl peptidase IV (DPP IV), which is identical to the lymphocyte surface glycoprotein CD26, cleaves dipeptides from the N-terminus of peptides with a proline or alanine residue in the penultimate position.14) CD26/DPP IV is highly expressed on fibroblasts, epithelial and endothelial cells, and specific leukocyte subsets. The extracellular protease domain of CD26/DPP IV, which is produced by proteolytic cleavage of the membrane-bound form of CD26/DPP IV, also exists in a soluble form in plasma.15) Recently, it was reported that DPP IV is released from intestinal epithelial cells into the extracellular milieu as a constituent of exosome-like vesicles. 8)More recently, our previous report revealed that DPP IV is released into snake venom in an unprocessed form, 16) suggesting that the DPP IV is associated with membrane. In addition, we have found exosome-like vesicles that carry DPP IV in snake venom.17) DPP IV activity has been found in human saliva, 18) but the mechanism of its release has not been elucidated. Snake venom is highly modified saliva that is produced by special glands of certain species of snakes. Therefore, we hypothesized that exosome-like vesicles carrying DPP IV could be present in human saliva. We demonstrate for the first time a population of vesicles in human whole saliva similar in size to the previously described exosomes. We found that these vesicles contain DPP IV, galectin-3 and IgA. MATERIALS AND METHODS MaterialsGly-Pro-4-methyl...
Small non-coding RNAs, such as microRNAs (miRNAs), are involved in diverse processes, including organ development and tissue differentiation. Exosomes are small membrane vesicles (30-100 nm in diameter) produced by numerous cells. Recently, exosomes have been shown to contain miRNAs. However, the small RNAs contained in exosomes are not fully characterized. In a previous study, we found at least two types of salivary exosome that are different in size and have different proteomes. Studies of salivary exosomal small RNAs are limited to miRNAs. In this study, we examined small RNA transcriptomes using next generation sequencing technology to elucidate a full transcriptome set of small RNAs expressed in the two types of salivary exosomes and in whole saliva (WS). Many types of small RNA, such as miRNA, piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA) and other small RNAs are contained in salivary exosomes and WS. Among these small RNAs we identified novel miRNA candidates.
Shiga toxin (Stx) is a major virulence factor in infection with Stx-producing Escherichia coli (STEC). We developed a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), which is a receptor for Stx, and identified Gb3 polymers with highly clustered trisaccharides as Stx adsorbents functioning in the gut. The Gb3 polymers specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins. Oral administration of the Gb3 polymers protected mice after administration of a fatal dose of E. coli O157:H7, even when the polymers were administered after the infection had been established. In these mice, the serum level of Stx was markedly reduced and fatal brain damage was substantially suppressed, which suggests that the Gb3 polymers entrap Stx in the gut and prevent its entrance into the circulation. These results indicate that the Gb3 polymers can be used as oral therapeutic agents that function in the gut against STEC infections.
Exosomes are derived from various sources, including primary and cultured cell lines and body fluids. It is now evident that they are important for communication between cells. They have, therefore, been proposed as potential carriers to deliver drugs to specific sites. In this study, we examined stability of exosomes derived from human saliva. Exosomes were stored at 4°C for up to 20 months and their membrane integrity assessed. Several exosomal markers, such as dipeptidyl peptidase IV (DPP IV; membrane marker) and programmed cell death 6-interacting protein (Alix, lumen marker), were retained intact after 20 months storage at 4°C. Moreover, intact exosomes could be isolated from whole saliva that had been stored at 4°C. Membrane disruption with detergents such as Triton X-100 and Nonidet P-40 caused partial solubilization of DPP IV and release of Alix into the supernatant. In contrast, sodium dodecyl sulfate treatment caused a complete disruption of the membrane. In addition, membrane stability was maintained after freezing and thawing. These results indicated that human saliva-derived exosomes are stable, maintaining their membrane integrity over a long storage period.
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