Boron Clusters Alter the Membrane Permeability of Dicationic Fluorescent DNA-Staining Dyes.

Membrane-permeable fluorescent dyes that stain DNA are useful reagents for microscopic imaging, as they can be introduced into living cells to label DNA. However, the number of these dyes, such as Hoechst 33342, is limited. Here, we show that the icosahedral dodecaborate B12Br122-, a superchaotropic carrier that delivers different types of molecules into cells, functions as an excellent carrier for membrane-impermeable fluorescent dyes. Propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI), dicationic membrane-impermeable fluorescent dyes that stain DNA, can permeate cell membranes in the presence of boron clusters. Methyl green (MG), a dicationic dye used in the histological and fluorescent staining of DNA, permeated cell membranes in the presence of boron clusters. In contrast, monocationic membrane-permeable fluorescent dyes, such as acridine orange and pyronin Y, exhibited reduced fluorescence in cells in the presence of boron clusters. Boron clusters do not quench dicationic fluorescent dyes in water in vitro but have quenching effects on monocationic fluorescent dyes. We have demonstrated that the addition of B12Br122- to impermeable dicationic fluorescent DNA-staining dyes, such as DAPI, PI, and MG, which have been widely used for numerous years, imparts membrane permeability to introduce these dyes into living cells.

Both variants of A1CF and BAZ1B genes are associated with gout susceptibility: a replication study and meta-analysis in a Japanese population.

Gout is a common type of acute arthritis that results from elevated serum uric acid (SUA) levels. Recent genome-wide association studies (GWASs) have revealed several novel single nucleotide polymorphism (SNPs) associated with SUA levels. Of these, rs10821905 of A1CF and rs1178977 of BAZ1B showed the greatest and the second greatest significant effect size for increasing SUA level in the Japanese population, but their association with gout is not clear. We examined their association with gout using 1411 clinically-defined Japanese gout patients and 1285 controls, and meta-analyzed our previous gout GWAS data to investigate any association with gout. Replication studies revealed both SNPs to be significantly associated with gout (P = 0.0366, odds ratio [OR] with 95% confidence interval [CI]: 1.30 [1.02-1.68] for rs10821905 of A1CF, P = 6.49 × 10-3, OR with 95% CI: 1.29 [1.07-1.55] for rs1178977 of BAZ1B). Meta-analysis also revealed a significant association with gout in both SNPs (Pmeta = 3.16 × 10-4, OR with 95% CI: 1.39 [1.17-1.66] for rs10821905 of A1CF, Pmeta = 7.28 × 10-5, OR with 95% CI 1.32 [1.15-1.51] for rs1178977 of BAZ1B). This study shows the first known association between SNPs of A1CF, BAZ1B and clinically-defined gout cases in Japanese. Our results also suggest a shared physiological/pathophysiological background between several populations, including Japanese, for both SUA increase and gout susceptibility. Our findings will not only assist the elucidation of the pathophysiology of gout and hyperuricemia, but also suggest new molecular targets.

Heterogeneous distribution of doublecortin-expressing cells surrounding the rostral migratory stream in the juvenile mouse.

In the postnatal mammalian brain, neural stem cells of the ventricular-subventricular zone continue to generate doublecortin (Dcx)-expressing immature neurons. Throughout life, these immature neurons migrate to the olfactory bulb through the rostral migratory stream (RMS). In this study, we investigated the distribution of these putative immature neurons using enhanced green fluorescent protein (EGFP) expression in the area surrounding the RMS of the juvenile Dcx-EGFP mice. Through the combined use of an optical clearing reagent (a 2,2'-thiodiethanol solution) and two-photon microscopy, we visualized three-dimensionally the EGFP-positive cells in the entire RMS and its surroundings. The resulting wide-field and high-definition images along with computational image processing methods developed in this study were used to comprehensively determine the position of the EGFP-positive cells. Our findings revealed that the EGFP-positive cells were heterogeneously distributed in the area surrounding the RMS. In addition, the orientation patterns of the leading process of these cells, which displayed the morphology of migrating immature neurons, differed depending on their location. These novel results provide highly precise morphological information for immature neurons and suggest that a portion of immature neurons may be detached from the RMS and migrate in various directions.

A rapid optical clearing protocol using 2,2'-thiodiethanol for microscopic observation of fixed mouse brain.

Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2'-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-µm-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.

The extract of Japanese soybean, Kurosengoku activates the production of IL-12 and IFN-γ by DC or NK1.1(+) cells in a TLR4- and TLR2-dependent manner.

During the search for immuno-improving foods, we found that a variety of the Japanese soybean, Glycine max cv. Kurosengoku (Kurosengoku), which activated Type-1 immunity in a Toll-like receptor (TLR)4- and TLR2-dependent manner. Namely, the extract of Kurosengoku first caused production of IL-12 from DC and sequentially induced IFN-γ production by NK1.1(+) NK cells and NKT cells. The IFN-γ production was significantly blocked by neutralizing mAb against IL-12 or TLR4- and TLR2-deficient condition, indicating that TLR4- and TLR2-dependent activation of DC to produce IL-12 was essential for the production of IFN-γ from spleen cells by Kurosengoku. Moreover, the extract of Kurosengoku also enhanced production of IFN-γ from human PBMC by co-stimulation with anti-CD3 mAb in a TLR2- and TLR4-dependent manner. Thus, our findings strongly suggest that Kurosengoku might a novel immuno-improving food, which would be a useful tool for preventing the tip of immune balance in developed countries.

Toll-like receptor-dependent IL-12 production by dendritic cells is required for activation of natural killer cell-mediated Type-1 immunity induced by Chrysanthemum coronarium L.

Type-1 immunity has an essential role for our host defenses against cancer and outer pathogens such as bacteria and virus. We demonstrated here that the edible plant extract of Chrysanthemum coronarium L. (C. coronarium) remarkably activates Type-1 immunity in a Toll-like receptor (TLR)2-, TLR4-, and TLR9-dependent manner. In the present experiments, the extract of C. coronarium significantly induces interferon (IFN)-γ production by mouse spleen cells. In addition, the IFN-γ production by spleen cells was completely blocked by the addition of anti-Interleukin (IL)-12 monoclonal antibodies. We confirmed that NK1.1(+) natural killer (NK) cells, NKT cells, and CD11c(+) dendritic cells (DC) were immediately activated after the stimulation with the extract of C. coronarium and the IFN-γ production was abolished in NK1.1(+) cell-depleted spleen cells. The stimulation with the extract of C. coronarium caused DC maturation involving with up-regulations of surface expression levels of MHC class I, MHC class II, CD40, and CD86 as well as induction of IL-12 production. The IFN-γ production induced by the extract was significantly reduced in the spleen cells depleted CD11c(+) cells. Furthermore, the IFN-γ production after the stimulation was strongly reduced in TLR4- and partially in TLR2- and TLR9-deficient spleen cells. Thus, we demonstrated the cellular mechanism for the activation of Type-1 immunity via NK cells, NKT cells, and DC by the extract of C. coronarium. These findings strongly suggest that C. coronarium would be a promising immuno-improving adjuvant, which might be useful for prevention of infectious, cancer, and allergic diseases through the activation of Type-1 immunity.