Loss-of-function mutation of c-Ret causes cerebellar hypoplasia in mice with Hirschsprung disease and Down's syndrome.

The c-RET proto-oncogene encodes a receptor-tyrosine kinase. Loss-of-function mutations of RET have been shown to be associated with Hirschsprung disease and Down's syndrome (HSCR-DS) in humans. DS is known to involve cerebellar hypoplasia, which is characterized by reduced cerebellar size. Despite the fact that c-Ret has been shown to be associated with HSCR-DS in humans and to be expressed in Purkinje cells (PCs) in experimental animals, there is limited information about the role of activity of c-Ret/c-RET kinase in cerebellar hypoplasia. We found that a loss-of-function mutation of c-Ret Y1062 in PCs causes cerebellar hypoplasia in c-Ret mutant mice. Wild-type mice had increased phosphorylation of c-Ret in PCs during postnatal development, while c-Ret mutant mice had postnatal hypoplasia of the cerebellum with immature neurite outgrowth in PCs and granule cells (GCs). c-Ret mutant mice also showed decreased numbers of glial fibers and mitogenic sonic hedgehog (Shh)-positive vesicles in the external germinal layer of PCs. c-Ret-mediated cerebellar hypoplasia was rescued by subcutaneous injection of a smoothened agonist (SAG) as well as by reduced expression of Patched1, a negative regulator for Shh. Our results suggest that the loss-of-function mutation of c-Ret Y1062 results in the development of cerebellar hypoplasia via impairment of the Shh-mediated development of GCs and glial fibers in mice with HSCR-DS.

Hair graying with aging in mice carrying oncogenic RET.

Hair graying is a representative sign of aging in animals and humans. However, the mechanism for hair graying with aging remains largely unknown. In this study, we found that the microscopic appearance of hair follicles without melanocyte stem cells (MSCs) and descendant melanocytes as well as macroscopic appearances of hair graying in RET-transgenic mice carrying RET oncogene (RET-mice) are in accordance with previously reported results for hair graying in humans. Therefore, RET-mice could be a novel model mouse line for age-related hair graying. We further showed hair graying with aging in RET-mice associated with RET-mediated acceleration of hair cycles, increase of senescent follicular keratinocyte stem cells (KSCs), and decreased expression levels of endothelin-1 (ET-1) in bulges, decreased endothelin receptor B (Ednrb) expression in MSCs, resulting in a decreased number of follicular MSCs. We then showed that hair graying in RET-mice was accelerated by congenitally decreased Ednrb expression in MSCs in heterozygously Ednrb-deleted RET-mice [Ednrb(+/-);RET-mice]. We finally partially confirmed common mechanisms of hair graying with aging in mice and humans. Taken together, our results suggest that age-related dysfunction between ET-1 in follicular KSCs and endothelin receptor B (Ednrb) in follicular MSCs via cumulative hair cycles is correlated with hair graying with aging.

Peptides containing the MXXCW motif inhibit oncogenic RET kinase activity with a novel mechanism of action.

REarranged during Transition (RET) is a tyrosine kinase associated with the development of several malignancies. Identification of RET kinase inhibitors promises valuable therapeutic tools for the intervention of RET-driven tumors. Most currently available tyrosine kinase inhibitors target the ATP binding site, but there are several drawbacks of these ATP-competitive drugs. Therefore, there is a need to develop new kinase inhibitors with alternative mechanisms of action. We have previously reported that a conserved cysteine in the MXXCW motif of RET is crucial to the disulfide-bonded dimerization-linked activation of RET kinases. Reagents which bind to this cysteine may inhibit the activity of RET kinases through disulfide-bond mediated dimerization. Here, we examine the potential of MXXCW motif-containing peptides as candidate kinase inhibitors. We demonstrate that MXXCW motif-containing peptides bind to RET in a redox-sensitive manner and block enzymatic activity, causing inhibition of the RET-dependent activity of extracellular signal-regulated kinases and effectively reducing the malignant potential of RET-papillary thyroid carcinoma-1 (PTC)-expressing cells. These motif-containing peptides were also found to be effective against the drug resistant mutant of RET. The inhibition of RET kinase activity by these peptides resulted in suppression of RET-PTC-1-mediated cancer growth. The great potency of these cysteine targeted peptides could indicate promising approaches for novel molecular-targeted therapies for RET-associated cancers.

A unique system that can sensitively assess the risk of chemical leukoderma by using murine tail skin.

Chemical leukoderma is a patchy hypopigmentation in the skin. Phenol derivatives such as raspberry ketone have been reported to cause the development of occupationally induced leukoderma. Recently, 2% (w/w) rhododenol, a reduced form of raspberry ketone used in a skin-lightning agent, also caused the development of leukoderma in >16,000 users, about 2% of all users, in Asian countries including Japan. However, a method for assessing the risk of leukoderma caused by 2% rhododenol has not been established despite the fact that the development of leukoderma caused by 30% rhododenol was previously shown in animal experiments. Establishment of a novel technique for risk assessment of leukoderma in humans caused by external treatment with chemicals is needed to prevent a possible future chemical disaster. This study demonstrated that external treatment with 2% rhododenol and the same concentration of raspberry ketone caused the development of leukoderma in murine tail skin without exception with significant decreases in the amount of melanin and number of melanocytes in the epidermis. Thus, a novel in vivo technique that can assess the risk of leukoderma caused by 2% rhododenol was developed. The unique technique using tail skin has the potential to prevent chemical leukoderma in the future.

Inhibition of mast cell degranulation by melanin.

Melanin is a dark naturally occurring pigment produced in nature and in many organisms. Although several reports have demonstrated applications for melanins in various therapeutic treatments, to date, no research has examined the anti-allergic effect of melanin. In this study, we for the first time found that solubilized or synthesized soluble melanin acts as a potent inhibitor of the degranulation of mast cells. We found that squid-ink-derived melanin significantly inhibited antigen-IgE-FcεRI-mediated degranulation of the mucosal mast cell line RBL-2H3. A homogenized melanin nanoparticle prepared by laser ablation also clearly suppressed antigen-induced mast cell degranulation. We also successfully solubilized synthetic melanin in a neutral biochemical buffer and found that it also significantly inhibited IgE-sensitized mast cells. The anti-degranulation activity of synthesized melanin was abolished in the melanin fraction below 50-kD molecular weight. All melanins used in this study did not exert significant cell death. Signal transduction analysis revealed that melanin suppressed antigen-triggered phosphorylation of signaling molecules as well as calcium influx. Transmission electron microscopy revealed that homogenized melanin nanoparticles partially attached to the cell surface and some nanoparticles were internalized to the cell. Flow cytometry revealed that the number of FcεRI-bound IgE molecules was decreased by melanin. Fluorescence recovery after photobleaching analysis indicated that melanin attenuated both plasma membrane and cytoplasmic fluidity, implying that melanin increased their viscosities. In vivo experiments using passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA) mouse models demonstrated that oral administration of melanin accelerated the recovery of decreased body temperature after antigen infection in PSA, and combination sensitization of IgE with melanin attenuated antigen-induced extravasation in PCA. These findings indicated that melanin exhibits preventative effects against IgE-mast cell-mediated anaphylaxis. This study provides the first evidence that homogenized melanin may be a potential therapeutic agent for diseases involving mast cells.

Arsenic level in toenails is associated with hearing loss in humans.

Arsenic (As) pollution in drinking water is a worldwide health risk for humans. We previously showed hearing loss in young people who live in areas of As-polluted drinking water and in young mice orally treated with As. In this study, we epidemiologically examined associations between As levels in toenails and hearing in 145 Bangladeshi aged 12-55 years in 2014. Levels of As in toenails, but not those in urine, were shown to be significantly correlated with hearing loss at 4 kHz [odds ratio (OR) = 4.27; 95% confidence interval (CI): 1.51, 12.05], 8 kHz (OR = 3.91; 95% CI: 1.47, 10.38) and 12 kHz (OR = 4.15; 95% CI: 1.55, 11.09) by multivariate analysis with adjustments for age, sex, smoking and BMI. Our experimental study further showed a significant association between As levels in inner ears and nails (r = 0.8113, p = 0.0014) in mice orally exposed to As, suggesting that As level in nails is a suitable index to assess As level in inner ears. Taken together, the results of our study suggest that As level in nails could be a convenient and non-invasive biomarker for As-mediated hearing loss in humans.

Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections.

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 °C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding 'tuft.' Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

A comprehensive study including monitoring, assessment of health effects and development of a remediation method for chromium pollution.

Chromium (Cr) pollution caused by wastewater from tanneries is a worldwide environmental problem. To develop a countermeasure, we performed a comprehensive study using Hazaribagh, the tannery area in Dhaka City, Bangladesh, as a model. Our environmental monitoring indicated that the soluble form of Cr, but not barium or arsenic, in Buriganga River is derived from Hazaribagh. Our chemical analysis next showed that Cr, the primary pollutant in canal water at Hazaribagh, consisted of ≤0.7 µM hexavalent Cr [Cr(VI)] and ≤1705 µM trivalent Cr [Cr(III)]. Our biological study then showed that coexposure to Cr(VI) and Cr(III) at possible ratios in canal water at Hazaribagh synergistically promotes transforming activity of human non-tumorigenic HaCaT keratinocytes with activated MEK/ERK and AKT. Our environmental engineering study finally indicated that a magnesium and iron-based hydrotalcite-like compound (MF-HT), our original depurative, can maximally adsorb 9.0 mg/g Cr(VI) and 1041 mg/g Cr(III). Our results suggested the importance of removal of Cr(III) as well as Cr(VI) by showing that Cr(III), which is generally recognized as a chemical with low toxicity, synergistically promoted carcinogenicity of a low level of Cr(VI). Therefore, we propose the use of our original high-efficient and low-cost depurative as a countermeasure to address the worldwide problem of environmental Cr pollution.

Increased expression level of Hsp70 in the inner ears of mice by exposure to low frequency noise.

Previous studies showed that people in urban areas are possibly exposed to 60-110 dB of low frequency noise (LFN) defined as noise of ≤100 Hz in their daily life. Previous studies also showed increased health risks by exposure to high levels (130-140 dB) of LFN in animals. However, little is known about the health effects of exposure to an ordinary level of LFN. We biochemically and immunohistochemically assessed the effects of exposure to inaudible LFN for mice (12 h/day of 100 Hz LFN at 95 dB for 5 days), at a level to which people are possibly exposed in daily life, on a murine inner ear by targeting 9 stress-reactive molecules. There was more than a 5-fold increased transcript level of heat shock protein 70 (Hsp70) in the whole inner ear exposed to LFN. However, the transcript levels of the other 8 stress-reactive molecules including Hsp27 and Hsp90 were comparable in LFN-exposed and unexposed murine inner ears. Only the transcript level of Cebpß among the previously reported 4 transcriptional activators for Hsp70 expression was more than 3-fold increased by LFN exposure. Hsp70 transcript expression levels in the inner ears 3 days after LFN exposure were comparable to those in unexposed inner ears. The protein level of Hsp70, but not the levels of Hsp27 and Hsp90, was also increased in the vestibule by LFN exposure. However, hearing levels as well as expression levels of Hsp70 protein in the cochleae were comparable in LFN-exposed mice and unexposed mice. Our results demonstrated that the inner ear might be one of the organs that is negatively affected by stress from inaudible LFN exposure. Moreover, LFN exposure might increase Hsp70 expression level via Cebpß in the inner ear. Thus, Hsp70 and Cebpß levels could be candidates of biomarkers for response to LFN exposure.

Loss of Stemness, EMT, and Supernumerary Tooth Formation in Cebpb-/-Runx2+/- Murine Incisors.

Adult Cebpb KO mice incisors present amelogenin-positive epithelium pearls, enamel and dentin allopathic hyperplasia, fewer Sox2-positive cells in labial cervical loop epitheliums, and reduced Sox2 expression in enamel epithelial stem cells. Thus, Cebpb acts upstream of Sox2 to regulate stemness. In this study, Cebpb KO mice demonstrated cementum-like hard tissue in dental pulp, loss of polarity by ameloblasts, enamel matrix in ameloblastic layer, and increased expression of epithelial-mesenchymal transition (EMT) markers in a Cebpb knockdown mouse enamel epithelial stem cell line. Runx2 knockdown in the cell line presented a similar expression pattern. Therefore, the EMT enabled disengaged odontogenic epithelial stem cells to develop supernumerary teeth. Cebpb and Runx2 knockdown in the cell line revealed higher Biglycan and Decorin expression, and Decorin-positive staining in the periapical region, indicating their involvement in supernumerary tooth formation. Cebpb and Runx2 acted synergistically and played an important role in the formation of supernumerary teeth in adult incisors.

Arsenic levels in cutaneous appendicular organs are correlated with digitally evaluated hyperpigmented skin of the forehead but not the sole in Bangladesh residents.

There has been no report showing the effect of arsenic level on digitized skin pigmentation level, a typical diagnostic marker for arsenicosis. Correlations among history of drinking well water, arsenic levels in hair and toenails, and digitalized skin pigmentation levels (L*-value) in sunlight-exposed (forehead) and unexposed (sole) skin areas digitally evaluated by using a reflectance spectrophotometer were examined in 150 residents of Bangladesh. Univariate analysis showed that arsenic levels in hair and toenails of subjects with a history of drinking well water were 10.6-fold and 7.1-fold higher, respectively, than those in subjects without a history of drinking well water. The mean L*-value of foreheads, but not that of soles, in subjects with a history of drinking well water was 1.15-fold lower (more pigmented) than that in subjects without a history of drinking well water. Significant correlations were found between duration of drinking well water and arsenic concentrations in hair (r=0.63; P<0.01) and toenails (r=0.60; P<0.01). Multivariate analysis showed that the arsenic levels in hair and toenails and the duration of drinking well water were strongly correlated with the digitized pigmented level of the forehead but not that of the sole. An increase in the duration of drinking well water may increase hyperpigmentation in the forehead, but not that in the sole, through an increased arsenic level in the human body as shown in cutaneous appendicular organs (hair and toenails).

Oral exposure to arsenic causes hearing loss in young people aged 12-29 years and in young mice.

There is no information on the association between oral exposure to arsenic (As) and hearing loss in humans or mice. In this combined epidemiological study and experimental study, the association of oral exposure to As with hearing loss in people aged 12-29 years and young mice was examined. Subjects in the exposure group (n = 48), who were drinking tube well water contaminated with As, showed significantly higher risks of hearing loss at 4 kHz [odds ratio (OR) = 7.60; 95% confidence interval (CI): 1.56, 57.88], 8 kHz (OR = 5.00; 95% CI: 1.48, 18.90) and 12 kHz (OR = 8.72; 95% CI: 2.09, 47.77) than did subjects in the control group (n = 29). We next performed an experiment in which young mice were exposed to As via drinking water at 22.5 mg/L, which is a much greater concentration than that in human studies. The exposure group showed hearing loss and accumulation of As in inner ears. Ex vivo exposure of the organ of Corti from mice exposed to As significantly decreased the number of auditory neurons and fibers. Thus, our combined study showed that oral exposure to As caused hearing loss in young people and young mice.

Manganese-Mediated Decrease in Levels of c-RET and Tyrosine Hydroxylase Expression In Vitro.

Previous studies showed that overexposure to manganese causes parkinsonism, a disorder of dopaminergic neurons. Previous studies also showed that activity of c-RET kinase controls dopamine production through regulation of tyrosine hydroxylase (TH) expression, suggesting the involvement of c-RET in the development of parkinsonism. To our knowledge, however, there is no report showing a correlation between manganese-mediated parkinsonism and c-RET. In this study, we examined the effect of manganese on the expression and/or activation levels of c-RET and TH in human TH-expressing cells (TGW cells). We first found that treatment with 30 and 100 µM manganese resulted in reduction of c-RET transcript level and degradation of c-RET protein through promotion of ubiquitination. We then examined the biological significance of manganese-mediated decrease of c-RET protein expression. Decreased TH expression with decreased c-RET kinase activity was observed in c-RET protein-depleted TGW cells by treatment with manganese (30 µM) as well as by c-RET siRNA transfection. Since TH protein has been shown to be involved in the dopamine-producing pathway in previous studies, our results indicate the possibility that manganese-mediated reduction of TH expression and phosphorylation via decreased expression of c-RET protein in neural cells is involved in parkinsonism induced by manganese.

Arsenic-mediated hyperpigmentation in skin via NF-kappa B/endothelin-1 signaling in an originally developed hairless mouse model.

Chronic exposure to arsenic is associated with various diseases in humans. Skin hyperpigmentation is the most sensitive objective symptom for patients with arsenicosis. However, there is very limited information about the mechanism of arsenic-mediated skin hyperpigmentation in vivo. In this study, hairless homozygous mice (Hr/Hr-mice) that drank water containing 3 and 30 µM arsenic for 2 months developed skin hyperpigmentation with increased levels of arsenic and number of melanocytes in the skin. Since it is possible for humans to be exposed to 3 µM of arsenic in well drinking water, our results suggest that the Hr/Hr-mice could be a novel model sensitively reflecting arsenic-mediated skin hyperpigmentation. We then analyzed the mechanism of arsenic-mediated skin hyperpigmentation. The epidermis of Hr/Hr-mice and human HaCaT skin keratinocytes exposed to arsenic for 2 and 4 months, respectively, showed 5.4-21.5-fold increased levels of endothelin-1 (ET-1) expression via NF-kappa B activation. Coexposure of primary normal human epithelial melanocytes to arsenic and ET-1 activated their proliferation and melanin synthesis with increased levels of MITF-M and ET-1 receptor expression. Our results suggest that interaction between keratinocytes and melanocytes in the skin through ET-1 and its receptor contributes to arsenic-mediated skin pigmentation, a hallmark of arsenicosis.

Limited effectiveness of household sand filters for removal of arsenic from well water in North Vietnam.

Since well water utilized for domestic purposes in the Red River Delta of North Vietnam has been reported to be polluted by arsenic, barium, iron, and manganese, household sand filters consisting of various components are used. Information regarding the effectiveness of various sand filters for removal of the four toxic elements in well water is limited. In this study, arsenic levels in 13/20 of well water samples and 1/7 of tap water samples exceeded World Health Organization (WHO) health-based guideline value for drinking water. Moreover, 2/20, 6/20, and 4/20 of well water samples had levels exceeding the present and previous guideline levels for barium, iron, and manganese, respectively. Levels of iron and manganese, but not arsenic, in well water treated by sand filters were lower than those in untreated water, although previous studies showed that sand filters removed all of those elements from water. A low ratio of iron/arsenic in well water may not be sufficient for efficient removal of arsenic from household sand filters. The levels of barium in well water treated by sand filters, especially a filter composed of sand and charcoal, were significantly lower than those in untreated water. Thus, we demonstrated characteristics of sand filters in North Vietnam.

Manganese-mediated acceleration of age-related hearing loss in mice.

Despite the fact that manganese (Mn) is known to be a neurotoxic element relevant to age-related disorders, the risk of oral exposure to Mn for age-related hearing loss remains unclear. In this study, we orally exposed wild-type young adult mice to Mn (Mn-exposed WT-mice) at 1.65 and 16.50 mg/L for 4 weeks. Mn-exposed WT-mice showed acceleration of age-related hearing loss. Mn-exposed WT-mice had neurodegeneration of spiral ganglion neurons (SGNs) with increased number of lipofuscin granules. Mn-exposed WT-mice also had increased hypoxia-inducible factor-1 alpha (Hif-1α) protein with less hydroxylation at proline 564 and decreased c-Ret protein in SGNs. Mn-mediated acceleration of age-related hearing loss involving neurodegeneration of SGNs was rescued in RET-transgenic mice carrying constitutively activated RET. Thus, oral exposure to Mn accelerates age-related hearing loss in mice with Ret-mediated neurodegeneration of SGNs.

Noninvasive, label-free, three-dimensional imaging of melanoma with confocal photothermal microscopy: Differentiate malignant melanoma from benign tumor tissue.

Skin cancer is one of the most common cancers. Melanoma accounts for less than 2% of skin cancer cases but causes a large majority of skin cancer deaths. Early detection of malignant melanoma remains the key factor in saving lives. However, the melanoma diagnosis is still clinically challenging. Here, we developed a confocal photothermal microscope for noninvasive, label-free, three-dimensional imaging of melanoma. The axial resolution of confocal photothermal microscope is ~3 times higher than that of commonly used photothermal microscope. Three-dimensional microscopic distribution of melanin in pigmented lesions of mouse skin is obtained directly with this setup. Classic morphometric and fractal analysis of sixteen 3D images (eight for benign melanoma and eight for malignant) showed a capability of pathology of melanoma: melanin density and size become larger during the melanoma growth, and the melanin distribution also becomes more chaotic and unregulated. The results suggested new options for monitoring the melanoma growth and also for the melanoma diagnosis.

Prevention of allergic rhinitis by ginger and the molecular basis of immunosuppression by 6-gingerol through T cell inactivation.

The incidence of allergies has recently been increasing worldwide. Immunoglobulin E (IgE)-mediated hypersensitivity is central to the pathogenesis of asthma, hay fever and other allergic diseases. Ginger (Zingiber officinale Roscoe) and its extracts have been valued for their medical properties including antinausea, antiinflammation, antipyresis and analgesia properties. In this study, we investigated the antiallergic effects of ginger and 6-gingerol, a major compound of ginger, using a mouse allergy model and primary/cell line culture system. In mice with ovalbumin (OVA)-induced allergic rhinitis, oral administration of 2% ginger diet reduced the severity of sneezing and nasal rubbing by nasal sensitization of OVA and suppressed infiltration of mast cells in nasal mucosa and secretion of OVA-specific IgE in serum. 6-Gingerol inhibited the expression of not only Th2 cytokines but also Th1 cytokines in OVA-sensitized spleen cells. Accordingly, 6-gingerol suppressed in vitro differentiation of both Th1 cells and Th2 cells from naïve T cells. In addition, 6-gingerol suppressed both superantigen staphylococcal enterotoxin B (SEB)- and anti-CD3-induced T cell proliferation. 6-Gingerol also abrogated PMA plus ionomycin- and SEB-induced IL-2 production in T cells, suggesting that 6-gingerol affected T cell receptor-mediated signal transduction rather than the antigen-presentation process. Indeed, 6-gingerol inhibited the phosphorylation of MAP kinases, calcium release and nuclear localization of c-fos and NF-κB by PMA and ionomycin stimulation. Thus, our results demonstrate that 6-gingerol suppresses cytokine production for T cell activation and proliferation, thereby not causing B cell and mast cell activation and resulting in prevention or alleviation of allergic rhinitis symptoms.