Synthesis and Characterization of a Novel Glycolipid with Glucosylglycerate as a Hydrophile Showing Protective Effects on Heat-induced Protein Denaturation.

A novel glycolipid featuring a glucosylglycerate moiety as a polar head group was synthesized in two steps from sucrose, glycerate, and N-dodecylamine. Glucosylglyceric acid was formed from sucrose and glyceric acid using sucrose synthase as a catalyst, followed by condensation with N-dodecylamine using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as a condensing agent. A white solid compound was recovered with a yield of 21% after purification by hydrophobic column chromatography. The structure and purity of the isolated compound, identified as N-dodecyl glucosylglyceric acid amide (aGGA), were confirmed by 1H and 13C nuclear magnetic resonance and liquid chromatography-electrospray ionization-mass spectrometry. aGGA was soluble in several polar solvents, including acetone, dimethyl formamide, and short chain alcohols. The dissolution of aGGA in water reduced the surface tension to 27.8 mN m-1 at a critical micellar concentration of 1.57 × 10-4 M. In addition, the presence of aGGA at concentrations as low at 0.68 mM protected egg white from heat-induced denaturation. These results suggest that aGGA could be useful as a protein-protecting surfactant.

Dendritic cell inhibitory receptor 4 (DCIR4) is preferentially expressed on inflammatory and patrolling monocytes.

Dendritic cell inhibitory receptor 4 (DCIR4, Clec4a1) is a lectin receptor and a member of mouse dendritic cell immunoreceptor family. Due to the lack of antibodies against DCIR4, expression of DCIR4 protein remains unknown. In this study, we established a specific monoclonal antibody against DCIR4 and investigated the expression of DCIR4 among immune cells. We found that DCIR4 was expressed on non-granulocytic subsets of CD11b+ cells in various immune organs including bone marrow, peripheral blood, spleen, skin-associated lymph nodes and mesenteric lymph nodes. DCIR4+ CD11b+ cells were dichotomized into DCIR4High and DCIR4Low cells distinguished by different levels of DCIR4 expression. By screening a panel of cell surface markers for expression, we found that in bone marrow, blood and spleen the DCIR4Low and the DCIR4High cells were Ly-6C+ CD43Low CD11c- inflammatory- monocytes and Ly-6C- CD43HIgh CD11c+ patrolling monocytes, respectively. Using in vitro differentiation system, we also found that differentiation of Ly-6C+ monocytes into dendritic cells greatly diminished expression of DCIR4, while that into macrophages did not significantly affect DCIR4 expression. The establishment of the anti-DCIR4 antibody enables clearer definition of monocytes and provides a novel tool to investigate biology of monocytes and their progenies.

Ubiquitous versus restricted expression of the two mouse dendritic cell C-type lectin receptors, DCIR1 and DCAR2, among myeloid cells.

Dendritic cell inhibitory receptor 1 (DCIR1, also known as DCIR and Clec4a2) and dendritic cell activating receptor 2 (DCAR2, also known as DCAR and Clec4b1) are mouse lectin receptors expressed on antigen presenting cells. They have structurally similar C-type lectin domains, of which amino acid sequences show 90.5% identity, and commercially available antibodies against them cross-react each other. Here we have established novel antibodies against DCIR1 and DCAR2 that can unambiguously discriminate DCIR1 and DCAR2 and examined their distribution among various immune cells. While DCIR1 was ubiquitously expressed on myeloid cells, including conventional DCs (cDCs), macrophages, neutrophils and eosinophils, in various immune organs, significant expression of DCAR2 was detected only on subpopulations of cDCs from bone marrow and skin-draining lymph nodes. Interestingly, in FITC-painted mice, DCAR2 was expressed on all of the FITC(+) cDCs, which had migrated from the skin after FITC painting, suggesting that DCAR2 can be a marker of migratory cDCs in skin-draining lymph nodes. Our findings provide a basis to investigate in vivo function of DCIR1 and DCAR2.

Immune response to bacteria in seminiferous epithelium.

The luminal part of the seminiferous epithelium, a tissue compartment protected by the blood-testis barrier, has been considered a site of immune privilege. However, there are reports describing the production of anti-microbial peptides and the expression of Toll-like receptors in cells present in the seminiferous epithelium, evoking the possibility that this tissue compartment is immunologically active at least with regard to the innate immune response. To test this, we injected Escherichia coli into seminiferous tubules of live mice and examined the fate of bacteria, the production of chemokines and inflammatory cytokines, and the infiltration of neutrophils. The bacteria actively propagated and reached a maximal level in a day, but started to decrease after 5 days and completely disappeared in 2 months. The expression of macrophage inflammatory protein-2 and tumor necrosis factor-alpha became evident in macrophages present in the interstitial compartment of testes as early as 1-3 h after the inoculation of bacteria. Neutrophils first accumulated in the interstitial space at 9-12 h and entered the tubules after a day. On the other hand, impairment of spermatogenesis was observed a day after bacteria injection and seemed unrecoverable even after the bacteria were eliminated. By contrast, bacteria injected into the interstitial compartment were more rapidly cleared with no damage in the seminiferous epithelium. These results suggest the existence of immunity against invading microbes in the seminiferous epithelium although its effectiveness in maintaining tissue homeostasis remains equivocal.

Perturbation of spermatogenesis by androgen antagonists directly injected into seminiferous tubules of live mice.

Natural and artificial substances present in the environment can affect our health. Testicular toxicants in particular are troublesome, because they disturb gonadal function of males. Translocation of substances into the seminiferous epithelium where sperm production proceeds is restricted due to the blood-testis barrier, but this permeability barrier temporarily disappears under physiological and sub-physiological conditions. This means that any substance could enter the seminiferous epithelium and disturb sperm production. To reduce the risk posed by such toxins, it is important to accurately determine which substances possess the toxicity. However, existing assay systems are not satisfactory in terms of both accuracy and sensitivity. Here, we report the establishment of such a system. We injected the androgen antagonists, flutamide and vinclozolin, directly into seminiferous tubules of live mice, which had been treated with busulfan for a temporal arrest of spermatogenesis, and the testes were histologically examined to see the effect of the injected materials on spermatogenesis that was in the process of recovery. The injection of either substance brought about a severe impairment of spermatogenesis at an amount over a million times smaller than that used in the previous assay systems where animals are administered with test substances outside of the testis. In contrast, these androgen antagonists at the same doses showed lesser effects when intratubularly or intraperitoneally administered into mice that had not been pretreated with busulfan. We propose that the method adopted in this study is a novel assay system to identify potential testicular toxicants.