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Specific amino acid substitutions in degenerin mechano-gated channels (DEGs) of C. elegans convert these channels into constitutively active mutants that induce the degeneration of neurons where DEGs are expressed. Acid-sensing ion channel-2a (ASIC2a), a proton-gated cation channel predominantly expressed in central neurons, is a mammalian ortholog of DEGs, and it can remain unclosed to be cytotoxic once the same mutations as the DEG mutants are introduced into its gene. Here we show that heterozygous transgenic (Tg) rats expressing ASIC2a-G430F (ASIC2a

Aim: This project aims to explore a novel magnetic field (MF) health care based on the cholesterol metabolism. Methods: Mouse peritoneal macrophages foam cells were incubated in CO 2 -independent culture medium containing apoA-I and were exposed in a MF (0.4 T) using the electromagnet of EPR spectrometer. C57BL/6N mice (8 week-old) were peritoneally injected F-ara-EdU (133μg/g mouse body weight) then exposed to MF under the identical condition. Mouse plasma, cerebrospinal fluid, liver and brain were harvested on the 28 th day. Lipoprotein profiles were examined enzymatic detection followed by a gel-permeable-HPLC (Skylight Biotech Inc.). Amyloid β40 and β42 levels were determined by WAKO ELISA kit. Fixed mouse brain sections (40um) were prepared by Leica CM1900 cryostat. Alexa fluor® 488 conjugates were link to F-ara-EdU by a click chemistry method and fluor positive nucleus images were captured by a confocal super resolution SpinSR10 (Olympus, Inc.). Results: ABCA1-apoA-I mediated cellular cholesterol release was not affected or reduced by the exposure in the MF of 0.4 T. The Pcsk9 expression showed significant reduction in the hepatocytes. Reduction of Abca1 expression and increase of Scarb1 were obtained non significantly. The newly generated hippocampal dentate gyrus cells in the control mice group and the MF exposed group were 1.6±1.17, and 0.47±0.62 cells per dentate gyrus section, respectively (P=0.003) despite bulk of new cells in olfactory bulbs in the both groups. Discussions: No substantial increase in cellular cholesterol export was observed by MF irradiation of mouse peritoneal macrophage cells. In addition, the lipoprotein profile showed a decrease in HDL cholesterol even after 28 days. As a result of gene expression in the liver, a decrease in ABCA1 expression and an increase in SR-BI expression inferred reduction of HDL. The number of new cells detected in the granular zone and the subgranular zone in the dentate gyrus of the hippocampus was significantly reduced in the mice subjected to the MF treatment. In the future, it is necessary to investigate in detail whether these newborn cells are neuron or glial cells.

Background Little is known on the role of transient receptor potential ankyrin 1 (TRPA1) in fibroblast—myofibroblast transition (FMT) that can lead to airway remodeling which is a major problem for severe asthma and fibrosis. Thus, this study investigated the effect of TRPA1 modulators on transforming growth factor beta 1(TGF-β1) -treated lung fibroblasts. Methods MRC-5 cells were preincubated with TGF-β1 for 24 h. TRPA1 agonist or antagonist were added and further incubated for 24 h. The changes in TRPA1 and alpha-smooth muscle actin (α-SMA) expressions by stimuli were evaluated using qRT-PCR, western blot and immunohistochemical analyses. Statistical significance was determined by using one- or two-way ANOVA, followed by Bonferroni’s post hoc analysis for comparison of multiple groups and paired 2-tailed Student’s t-test between 2 groups. Results MRC-5 cells treated by TGF-β1 significantly upregulated α-SMA mRNA expressions (P P P P P P P P Conclusion We found that AITC exerts protective effect on TGF-β1-induced α-SMA induction by activating ERK1/2 MAPK and NRF2/HO-1 pathways in lung fibroblasts. It also overcomes corticosteroids insensitivity in TGF-β1-induced α-SMA induction. TRPA1 antagonist modulates the suppressive effect, but not prevent it. AITC and TRPA1 antagonist may be therapeutic agents in treating chronic respiratory diseases.

Neuronal migration is a critical step for the development of neuronal circuits in the brain. Immature new neurons (neuroblasts) generated in the postnatal ventricular-subventricular zone (V-SVZ) show a remarkable potential to migrate for a long distance at a high speed in the postnatal mammalian brain, and are thus a powerful model to analyze the molecular and cellular mechanisms of neuronal migration. Here we describe a methodology for in vitro time-lapse imaging of the primary cilium and its related structures in migrating V-SVZ-derived neuroblasts using confocal or superresolution laser-scanning microscopy. The V-SVZ tissues are dissected from postnatal day 0-1 (P0-1) mouse brains and dissociated into single cells by trypsinization and gentle pipetting. These cells are then transduced with a plasmid(s) encoding a gene(s) of interest, aggregated by centrifugation, and cultured for 2 days in Matrigel. Time-lapse images of migratory behaviors of cultured neuroblasts and their ciliary structures, including the ciliary membrane and basal body, are acquired by confocal or superresolution laser-scanning microscopy. This method provides information about the spatiotemporal dynamics of neuroblasts' morphology and ciliary structures, and is widely applicable to various types of migrating neuronal and nonneuronal cells in various species.

No genetic association with recurrent pregnancy loss (RPL) caused by embryonic aneuploidy has been found. Recent studies have indicated that the common genetic variant rs2305957, surrounding the PLK4 gene, contributes to mitotic-origin aneuploidy risk during human early embryo development. The decrease in meiosis-specific cohesin causes predivision of sister chromatids in the centromere and chromosome segregation errors. STAG3 is a component of cohesin and is a meiosis-specific gene. Our case-control study included 184 patients with RPL whose previous products of conception (POC) exhibited aneuploidy and 190 fertile control women without a history of miscarriage. We performed a genetic association study to examine the genotype distribution at PLK4 (rs2305957) and STAG3 in patients with RPL caused by aneuploidy compared with controls. Regarding STAG3, SNPs with a minor allele frequency (MAF) threshold > 0.05 that were predicted to be binding sites of transcription factors and that showed significant associations in expression quantitative trait locus (e-QTL) analysis were selected. No significant differences in the MAF or distribution in any model of PLK4 (rs2305957) and 5 selected tag SNPs in STAG3 were found between the patients and controls. A further genome-wide association study is needed since a combination of genetic risk alleles might be useful in predicting future age-dependent RPL caused by aneuploidy., Pregnancy: Finding genes to predict recurrent pregnancy loss Researchers have ruled out two potential genetic causes of recurrent pregnancy loss (RPL). Defined as loss of two or more pregnancies at any stage, RPL is caused by loss or gain of a chromosome (aneuploidy) in almost half of cases. Identifying genetic factors could help to predict the risk of RPL, which increases with maternal age. Hiroyuki Yoshihara at Nagoya City University in Japan and co-workers investigated whether two genes involved in chromosome distribution during cell division were associated with aneuploidy-related RPL. In a study of almost 400 women, half of whom had experienced RPL, they showed that neither gene was involved. Because multiple genetic factors could combine to cause RPL, the researchers recommend a genome-wide search. A better understanding of the genetic risk factors of RPL could help women in making fertility-related choices.

This study aimed to establish a rapid and simple method for isolating exosomes from raw bovine milk and to compare the quality of the isolated exosomes with those isolated by a standard method involving ultracentrifugation (UC).To remove caseins, which are major milk proteins consisting more than 80% of milk protein (35% in human breast milk) and hamper isolation and purification of exosomes, hydrochloride (HCl) was added to milk for isoelectric precipitation (IP). The effects of acidification on morphological features, particle size distribution, surface charge, and exosome surface proteins were analyzed by electron microscopy, tunable resistive pulse sensing (TRPS), and Western blot (WB) analysis, respectively.Electron microscopy showed that some of the exosomes isolated using IP had rough surfaces; most exosomes were successfully isolated without breakage, and their morphological features were similar to those of exosomes isolated by UC. TRPS showed that their surface charge and peaks (mode) for particle size distribution did not significantly differ between both methods. WB analysis using antibodies against the exosome surface marker proteins - milk fat globule-epidermal growth factor 8 (MFG-E8) and CD63 - revealed that the structures of exosome surface proteins were not affected by adding HCl.IP can be used to remove caseins to reduce operation time. This method will be useful for efficient isolation and purification of bovine milk exosomes and contribute to progression of research on health management of dairy cattle and drug delivery systems in human medicine, which require large amounts of milk exosomes.

SAD kinases regulate presynaptic vesicle clustering and neuronal polarization. A previous report demonstrated that Sada−/− and Sadb−/− double-mutant mice showed perinatal lethality with a severe defect in axon/dendrite differentiation, but their single mutants did not. These results indicated that they were functionally redundant. Surprisingly, we show that on a C57BL/6N background, SAD-A is essential for cortical development whereas SAD-B is dispensable. Sada−/− mice died within a few days after birth. Their cortical lamination pattern was disorganized and radial migration of cortical neurons was perturbed. Birth date analyses with BrdU and in utero electroporation using pCAG-EGFP vector showed a delayed migration of cortical neurons to the pial surface in Sada−/− mice. Time-lapse imaging of these mice confirmed slow migration velocity in the cortical plate. While the neurites of hippocampal neurons in Sada−/− mice could ultimately differentiate in culture to form axons and dendrites, the average length of their axons was shorter than that of the wild type. Thus, analysis on a different genetic background than that used initially revealed a nonredundant role for SAD-A in neuronal migration and differentiation.

Acid-sensing ion channel 4 (ASIC4) belongs to the ASIC gene family of neuronal proton-gated cation channels, and is the least understood subtype among the members. Previous studies of ASIC4 expression in the mammalian central nervous system have shown that ASIC4 is abundantly expressed in the spinal cord and in various brain regions, such as the cerebral cortex, the hippocampus, and the cerebellum. However, the detailed distribution of ASIC4 transcripts in mammalian brains still remains to be elucidated. In the present study, radioactive in situ hybridization histochemistry with an ASIC4-specific cRNA probe was performed on wild-type mouse brains, followed by X-gal staining experiments with Asic4-lacZ reporter mice Asic4tm1a(KOMP)Mbp. It was found that ASIC4 mRNAs were widely expressed throughout the wild-type brain, but preferentially concentrated in the olfactory bulb, the piriform cortex, the caudate putamen, the preoptic area, the paraventricular nucleus, the medial habenular nucleus, the pretectal area, the lateral geniculate nucleus, the amygdaloid complex, the superior colliculus, the interpeduncular nucleus, and the granule cell layer of the ventral hippocampus, and these results were in agreement with the X-gal-positive reactions observed in the mutant brain. In addition, X-gal staining combined with immunohistochemistry identified intense signals for ASIC4 transcriptional activity in most of the choline acetyltransferase (ChAT)-positive principal neurons located in the basal forebrain cholinergic nuclei. Our data provide useful information to speculate possible roles of ASIC4 in diverse brain functions.

Diphtheria toxin (DT) administration into transgenic mice that express the DT receptor (DTR) under control of specific promoters is often used for cell ablation studies in vivo. Because DTR is not expressed in mice, DT injection has been assumed to be nontoxic to cells in vivo. In this study, we demonstrated that DT application during the juvenile stage leads to hearing loss in wild-type mice. Auditory brainstem response measurement showed severe hearing loss in C57BL/6 mice administered DT during the juvenile period, and the hearing loss persisted into adulthood. However, ototoxicity did not occur when DT was applied on postnatal day 28 or later. Histological studies demonstrated that hearing loss was accompanied by significant degeneration of inner and outer hair cells (HCs), as well as spiral ganglion neurons. Scanning electron microscopy showed quick degeneration of inner HCs within 3days and gradual degeneration of outer HCs within 1week. These results demonstrated that DT has ototoxic action on C57BL/6 mice during the juvenile period, but not thereafter, and the hearing loss was due to degeneration of inner and outer HCs by unknown DT-related mechanisms.

Bovine milk extracellular vesicles (EVs) attract research interest as carriers of biologically active cargo including miRNA from donor to recipient cells to facilitate intercellular communication. Since toxicity of edible milk seems to be negligible, milk EVs are applicable to use for therapeutics in human medicine. Casein separation is an important step in obtaining pure EVs from milk, and recent studies reported that adding hydrochloric acid (HCl) and acetic acid (AA) to milk accelerates casein aggregation and precipitation to facilitate EV isolation and purification; however, the effects of acidification on EVs remain unclear. In this study, we evaluated the acidification effects on milk-derived EVs with that by standard ultracentrifugation (UC). We separated casein from milk by either UC method or treatment with HCl or AA, followed by evaluation of EVs in milk serum (whey) by transmission electron microcopy (TEM), spectrophotometry, and tunable resistive pulse sensing analysis to determine EVs morphology, protein concentration, and EVs size and concentration, respectively. Moreover, we used anti-CD9, -CD63, -CD81, -MFG-E8, -HSP70, and -Alix antibodies for the detection of EVs surface and internal marker proteins by western blot (WB). Morphological features of EVs were spherical shape and similar structure was observed in isolated EVs by TEM. However, some of the EVs isolated by HCl and AA had shown rough surface. Although protein concentration was higher in whey obtained by UC, EV concentration was significantly higher in whey following acid treatment. Moreover, although all of the targeted EVs-marker-proteins were detected by WB, HCl- or AA-treatments partially degraded CD9 and CD81. These findings indicated that acid treatment successfully separated casein from milk to allow efficient EV isolation and purification but resulted in partial degradation of EV-surface proteins. Our results suggest that following acid treatment, appropriate EV-surface-marker antibodies should be used for accurate assess the obtained EVs for downstream applications. This study describes the acidification effects on EVs isolated from bovine milk for the first time.

New neurons, referred to as neuroblasts, are continuously generated in the ventricular-subventricular zone of the brain throughout an animal's life. These neuroblasts are characterized by their unique potential for proliferation, formation of chain-like cell aggregates, and long-distance and high-speed migration through the rostral migratory stream (RMS) toward the olfactory bulb (OB), where they decelerate and differentiate into mature interneurons. The dynamic changes of ultrastructural features in postnatal-born neuroblasts during migration are not yet fully understood. Here we report the presence of a primary cilium, and its ultrastructural morphology and spatiotemporal dynamics, in migrating neuroblasts in the postnatal RMS and OB. The primary cilium was observed in migrating neuroblasts in the postnatal RMS and OB in male and female mice and zebrafish, and a male rhesus monkey. Inhibition of intraflagellar transport molecules in migrating neuroblasts impaired their ciliogenesis and rostral migration toward the OB. Serial section transmission electron microscopy revealed that each migrating neuroblast possesses either a pair of centrioles or a basal body with an immature or mature primary cilium. Using immunohistochemistry, live imaging, and serial block-face scanning electron microscopy, we demonstrate that the localization and orientation of the primary cilium are altered depending on the mitotic state, saltatory migration, and deceleration of neuroblasts. Together, our results highlight a close mutual relationship between spatiotemporal regulation of the primary cilium and efficient chain migration of neuroblasts in the postnatal brain.SIGNIFICANCE STATEMENTImmature neurons (neuroblasts) generated in the postnatal brain have a mitotic potential and migrate in chain-like cell aggregates toward the olfactory bulb. Here we report that migrating neuroblasts possess a tiny cellular protrusion called a primary cilium. Immunohistochemical studies with zebrafish, mouse, and monkey brains suggest that the presence of the primary cilium in migrating neuroblasts is evolutionarily conserved. Ciliogenesis in migrating neuroblasts in the rostral migratory stream is suppressed during mitosis and promoted after cell cycle exit. Moreover, live imaging and 3D electron microscopy revealed that ciliary localization and orientation change during saltatory movement of neuroblasts. Our results reveal highly organized dynamics in maturation and positioning of the primary cilium during neuroblast migration that underlie saltatory movement of postnatal-born neuroblasts.

Nutrition and dietary habits contribute to the onset and progression of sensorineural hearing loss (SNHL). Fructo-oligosaccharides (FOS) are non-digestible oligosaccharides and are known as prebiotics, which enhance short-chain fatty acid (SCFA) production and antioxidant activity. Although a substantial number of studies have shown that FOS play a role in the prevention of lifestyle-related diseases as prebiotics, little is known about the effects on the inner ear. The purpose of this study is to investigate the effect of FOS on gene expression and spiral ganglion neuron (SGN) protection in the inner ear of DBA/2 J mice, which is a model for early-onset progressive hearing loss. DBA/2 J mice were fed either control diet or FOS diet contained 10% (w/w) of FOS for 8 weeks. Analysis of mice fed the FOS diet revealed a change in intestinal flora including an inversion of the ratio of Bacteroidetes and Firmicutes, which was followed by a significant increase in SCFAs in the cecum and a decrease in an oxidative stress marker in the serum. In the inner ear, gene expression of neurotrophin, brain-derived neurotrophic factor (BDNF), its receptor, tyrosine kinase receptor b (Trkb), and the SCFA receptor, free fatty acid receptor 3 (FFAR3), were increased by FOS. In addition, the survival rate of SGNs in the inner ear was maintained in FOS-fed mice. Altogether, these results suggest that a compositional variation of the intestinal flora due to a prebiotic effect may be involved in the progression of SNHL.

Background: Plasma HDL particle is one of the essential player for maintaining cholesterol homeostasis in mammal. Even though cholesterol can be ubiquitously biosynthesized upon requirement, cholesterol in diet is efficiently used to maintain cholesterol homeostasis which implicates existence of sensing system for cholesterol in diet. The study of taste preference on cholesterol was not well explored. In this project we examined taste preference in mice using a novel method called dual diet test (DDT) system. Methods: Chow pellets of 1.2% cholesterol or 0.02% Acesulfame potassium (AmK, calorie-free sweet molecule) were prepared based on standard mouse chow, CE-2(0.2% cholesterol, Crea Japan). The mouse chow tray was divided into three sections. Water nozzle was set in the center section. Each chow was weighed then set ether right or left section. One session was composed by 1-3 days and remaining chow was weighed to calculate consumption of each chow diet. The diet position was switched randomly at each session. The operation was performed between ZT9-ZT10 when mice were less active in a day. To avoid social isolation stress, 2-5 littermates were placed per cage. Results: C56B/6 wild type mice and LCAT null mice significantly preferred 1.2% cholesterol containing chow compared to control chow during 6 sessions by 18-28 weeks old littermates (n=16, n=8, respectively). These mice also significantly preferred AmK-chow compared to the control chow. ABCA1 null and ABCA1 heterozygote mice exhibited significance on the preference of 1.2% cholesterol in the first 3 sessions but not for the later sessions. Repeating the experiment after interval period improved their cholesterol preference in ABCA1 null mice. No gender difference was observed. Preferences to AmK-chow were significant in all trials. Conclusions: The preference in higher cholesterol or sweet in diet were detected at this novel DDT system, significantly. In ABCA1 null or heterozygote mice, long-term preferences to high cholesterol diet were abolished perhaps due to their lack of focus on cholesterol-rich chow of which less reward to these mice. In this experiment, mice synchronized in consuming higher cholesterol containing diet indicating mice are sensing cholesterol as a preferred tastant.

We elucidated the mechanism of spermatogonial stem cell disturbance of cryptorchidism and investigated the expression of miRNAs and their target genes in undescended testes.Using microarray analysis we compared total miRNA expression in unilateral undescended testes with that in contralateral descended and normal testes in a rat model of cryptorchidism. The model was derived by administering flutamide to pregnant Sprague Dawley® rats. We identified mRNA targets of miRNAs by bioinformatic analysis, followed by in situ hybridization and immunohistochemistry to localize candidate miRNAs and mRNAs, respectively. We also investigated whether miRNAs could inhibit target protein expression in vitro.Microarray analysis and subsequent quantitative reverse transcriptase-polymerase chain reaction showed that only miR-135a was expressed at a lower level in undescended testes. We identified its target as FoxO1, which is essential for stem cell maintenance. miR-135a and FoxO1 localized to spermatogonial stem cells. FoxO1 localized to the spermatogonial stem cell nucleus less frequently in undescended testes, indicating that the activity of FoxO1, which acts as a transcription factor, is altered in undescended testes. Finally, miR-135a transfection into spermatogonia in vitro resulted in down-regulation of FoxO1 expression.In cryptorchid testes there is a decreased number of spermatogonial stem cells in which FoxO1 is activated, indicating that failure of spermatogonial stem cell maintenance results in spermatogenesis alteration. We also noted interaction between miR-135a and FoxO1, and propose that miR-135a contributes to spermatogonial stem cell maintenance through modulation of FoxO1 activity.

Aripiprazole has made a significant contribution to the treatment of schizophrenia and related disorders. It has improved its safety and tolerability profiles, and these effects have been attributed to its pharmacological profile at the serotonin 5-HT and dopamine D(2) receptors. To discover compounds that have a similar pharmacological profile, we introduced a generic single-cell-based calcium imaging assay that standardizes the readouts from various assays used in previous studies on aripiprazole. In the present assay, the efficacy and potency of known ligands of serotonin 5-HT(1A), 5-HT(2A), 5-HT(2C), 5-HT(7) and dopamine D(2L) receptors were comparable to those found in previous studies using a variety of readouts. The developed assay was also able to reproduce the partial agonist activity, the low intrinsic activity and the selective activation of aripiprazole at the dopamine D(2L) receptors. Under identical experimental conditions, geissoschizine methyl ether (GM), a plant indole alkaloid, behaved as a partial agonist at the serotonin 5-HT(1A) receptor, a partial agonist/antagonist at the dopamine D(2L) receptor and an antagonist at the serotonin 5-HT(2A), 5-HT(2C) and 5-HT(7) receptors. Interestingly, GM showed a relatively low intrinsic activity and evoked a partial activation response in a subset of cells expressing the dopamine D(2L) receptor; both of these effects were similarly observed for aripiprazole. Although GM is far less potent at the dopamine receptor than aripiprazole at dopamine D(2L) receptors (EC(50)=4.4 μM for GM vs. EC(50)=56 nM for aripiprazole), GM and GM derivatives may comprise a new set of candidates for atypical antipsychotics.

To reveal the mechanism of urinary urgency evoked by cold sensation, we investigated the convergence of the primary sensory afferents of the skin and bladder. Dichotomizing afferents of L6-S1 dorsal root ganglion neurons that innervate the skin and bladder was constantly observed with retrograde neuron tracers in rats. In-situ hybridization revealed that approximately 8.0% of the double-labelled cells expressed transient receptor potential channel melastatin member 8 (TRPM8) transcripts in the dorsal root ganglions. Cold and menthol stimuli to the skin generated bladder nerve responses conducted through dichotomizing axons, which significantly decreased in the presence of the TRPM8 blocker BCTC. Taken together, TRPM8-expressing sensory neurons with dichotomizing axons projecting to the skin and bladder may be responsible for the urinary urgency evoked by cold sensation.

Transient receptor potential vanilloid 3 (TRPV3), a member of the calcium-permeable thermosensitive TRP (thermoTRP) subfamily of receptors, is an important cutaneous sensor that detects thermal and chemical stimuli. TRPV3 is activated by innocuous warm temperature stimuli (>33 degrees C) and a variety of physiologically active substances. While the corneal epithelium is known to respond to such stimuli, it is unknown whether TRPV3 is involved in this phenomenon. We show here that TRPV3 mRNA and protein are abundantly expressed in the epithelial cells of human and mouse cornea. Carvacrol, an agonist of TRPV3, elevated cytosolic Ca2+ concentration in both primary mouse corneal epithelial cells and cultured human corneal epithelial cells (HCE-T cells). The response to carvacrol was inhibited by ruthenium red, a TRPV channel antagonist. Moreover, repetitive agonist stimulation sensitized the response with gradually increasing amplitude, suggesting that the TRPV3 in the cornea has similar physiological and pharmacological characteristics to that in skin keratinocytes. Finally, a wound healing assay revealed that appropriate calcium ion influx via activated TRPV3 in corneal epithelial cells accelerated their proliferation. Thus, functional TRPV3 is present in corneal epithelial cells and may play a role not only in thermosensation, but also in the regulation of cell proliferation.

The thermo-transient receptor potential (thermoTRP) subfamily is composed of channels that are important in nociception and thermo-sensing. Here, we show a selective expression of TRPV3 channel in the distal colon throughout the gastrointestinal tract. Expression analyses clearly revealed that TRPV3 mRNA and proteins were expressed in the superficial epithelial cells of the distal colon, but not in those of the stomach, duodenum or proximal colon. In a subset of primary epithelial cells cultured from the distal colon, carvacrol, an agonist for TRPV3, elevated cytosolic Ca(2+)concentration in a concentration-dependent manner. This response was inhibited by ruthenium red, a TRPV channel antagonist. Organotypic culture supported that the carvacrol-responsive cells were present in superficial epithelial cells. Moreover, application of carvacrol evoked ATP release in primary colonic epithelial cells. We conclude that TRPV3 is present in absorptive cells in the distal colon and may be involved in a variety of cellular functions.

Conclusion: Immunohistochemical detection of Reelin in granular cells and disabled-1 in cochlear nucleus suggests a possible Reelin signaling pathway in mature rat cochlear nucleus. Materials and Methods: Six-week-old Wister rats were used throughout this study. The expression of reelin and disabled-1 were studied by using in situ hybridization technique and immunohistochemistry. Results: Reelin mRNA expression was observed in granular cell layer of dorsal cochlear nucleus. Immunohistochemistry using anti-reelin monoclonal antibodies confirmed reelin expression in granule cells at protein level. We also examined disabled-1 expression in cochlear nucleus and observed positive immunoreactivity in both ventricular and dorsal cochlear nucleus. In the dorsal cochlear nucleus, fusiform and cartwheel cells were labeled. In the ventricular cochlear nucleus, relatively large cells were labeled with anti-disabled-1 polyclonal antibody but the subtypes of disabled-1 positive cells could not be identified.

The transient receptor potential melastatin subfamily (TRPM), which is a mammalian homologue of cell death-regulated genes in Caenorhabditis elegans and Drosophila, has potential roles in the process of the cell cycle and regulation of Ca2+signaling. Among this subfamily, TRPM8 (also known as Trp-p8) is a Ca2+-permeable channel that was originally identified as a prostate-specific gene upregulated in tumors. Here we showed that the TRPM8 channel was expressed in human melanoma G-361 cells, and activation of the channel produced sustainable Ca2+influx. The application of menthol, an agonist for TRPM8 channel, elevated cytosolic Ca2+concentration in a concentration-dependent manner with an EC50value of 286 μM in melanoma cells. Menthol-induced responses were significantly abolished by the removal of external Ca2+. Moreover, inward currents at a holding potential of −60 mV in melanoma cells were markedly potentiated by the addition of 300 μM menthol. The most striking finding was that the viability of melanoma cells was dose-dependently depressed in the presence of menthol. These results reveal that a functional TRPM8 protein is expressed in human melanoma cells to involve the mechanism underlying tumor progression via the Ca2+handling pathway, providing us with a novel target of drug development for malignant melanoma.

Acid-sensing ion channels (ASICs) are strong candidates for mammalian mechanoreceptors. We investigated whether mouse acid-sensing ion channel-1b (ASIC1b) is sensitive to mechanical stimuli using oocyte electrophysiology, because ASIC1b is located in the mechanosensory stereocilia of cochlear hair cells. Hypotonic stimuli that induced membrane stretch of oocytes evoked no significant current in ASIC1b-expressing oocytes at pH 7.5. However, acid (pH 4.0 or 5.0)-evoked currents in the oocytes were substantially enhanced by the hypotonicity, showing mechanosensitivity of ASIC1b and possible mechanogating of the channel in the presence of other components. Interestingly, the ASIC1b channel was permeable to K(+) (a principal charge carrier for cochlear sensory transduction) and the affinity of the channel for amiloride (IC(50) (inhibition constant)=approximately 48.3 microM) was quite similar to that described for the mouse hair cell mechanotransducer current. Taken together, these data raise the possibility that ASIC1b participates in cochlear mechanoelectrical transduction.

Malignant melanoma is the most deadly form of skin cancer and its incidence is steadily increasing worldwide. The plasma membrane in melanoma cells possesses a variety of ion channels, so its profile is thought to lead to a novel target for medical treatment for malignant melanoma. Here we showed that human melanoma G-361 cells expressed the epithelial Na(+) channel delta subunit (ENaC delta), which is largely unknown in physiological and pathological functions in non-neuronal tissues. Expression analyses at the level of mRNA clearly revealed that ENaC delta transcript was strongly expressed in human melanoma cells using reverse transcription-polymerase chain reaction and cell-based in situ hybridization techniques. Other ENaC subunits (alpha, beta, and gamma) were also distributed in human melanoma cells. In addition, human melanoma cells possessed an abundant expression of ENaC delta protein by immunocytochemistry. These results provide an attractive target for drug development of malignant melanoma.

P2X7 receptor is a member of the P2X family of ATP-gated ion channels. The cellular localization of P2X7 receptors in the central nervous system remains controversial because immunohistochemical staining patterns are inconsistent among antibodies. Here we examined the precise distribution of P2X7 mRNAs in the rat brain using isotopic in situ hybridization. P2X7-positive glial-like small cells were sporadically scattered in almost all areas of the brain. P2X7-positive glial-like small cells were also observed in nerve fiber tracts such as the anterior commissure, corpus callosum (CC), optic tract, and internal capsule. P2X7-positive neurons were found in the anterior olfactory nucleus, cerebral cortex, piriform cortex (Pir), lateral septal nucleus (LS), hippocampal pyramidal cell layers of CA1, CA3, CA4, pontine nuclei, external cuneate nucleus, and medial vestibular nucleus. P2X7 hybridization signals were also observed in the motor neurons of the trigeminal motor nucleus, facial nucleus, hypoglossal nucleus, and the anterior horn of the spinal cord. P2X7 mRNA was expressed in the ependymal cells around the olfactory ventricle, lateral ventricles (LV), third ventricle (3V), cerebral aqueduct (Aq), fourth ventricle (4V), and central canal. The P2X7 hybridization signal was also very strong in the area postrema (AP). The double staining experiments demonstrate that neurons, oligodendrocytes, and microglia expressed P2X7 receptor mRNAs. These findings suggest that P2X7 receptors may play a variety of roles in a wide range of cell types in the brain.

Basal cells in the nasal epithelium (olfactory and airway epithelia) are stem/progenitor cells that are capable of dividing, renewing and differentiating into specialized cells. These stem cells can sense their biophysical microenvironment, but the underlying mechanism of this process remains unknown. Here, we demonstrate the prominent expression of the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca2+-permeable channel that is known to act as a sensor for hypo-osmotic and mechanical stresses, in the basal cells of the mouse nasal epithelium. TRPV4 mRNA was expressed in the basal portions of the prenatal mouse nasal epithelium, and this expression continued into adult mice. The TRPV4 protein was also detected in the basal layers of the nasal epithelium in wild-type but not in TRPV4-knockout (TRPV4-KO) mice. The TRPV4-positive immunoreactions largely overlapped with those of keratin 14 (K14), a marker of basal cells, in the airway epithelium, and they partially overlapped with those of K14 in the olfactory epithelium. Ca2+ imaging analysis revealed that hypo-osmotic stimulation and 4α-phorbol 12,13 didecanoate (4α-PDD), both of which are TRPV4 agonists, caused an increase in the cytosolic Ca2+ concentration in a subset of primary epithelial cells cultured from the upper parts of the nasal epithelium of the wild-type mice. This response was barely noticeable in cells from similar parts of the epithelium in TRPV4-KO mice. Finally, there was no significant difference in BrdU-labeled proliferation between the olfactory epithelia of wild-type and TRPV4-KO mice under normal conditions. Thus, TRPV4 channels are functionally expressed in basal cells throughout the nasal epithelium and may act as sensors for the development and injury-induced regeneration of basal stem cells., Highlights • TRPV4 is expressed in basal stem cells of the nasal airway and olfactory epithelium. • TRPV4 expression appears in the nasal epithelium during the late prenatal stages. • TRPV4 activation causes an increase in cytosolic Ca2+ concentration. • TRPV4 may be involved in a variety of cellular functions in progenitor/stem cells.

The Cav3.2 isoform of the T-type calcium channel is expressed in primary sensory neurons of the dorsal root ganglion (DRG), and these channels contribute to nociceptive and neuropathic pain in rats. However, there are conflicting reports on the roles of these channels in pain processing in rats and mice. In addition, the function of T-type channels in persistent inflammatory hyperalgesia is poorly understood. We performed behavioral and comprehensive histochemical analyses to characterize Cav3.2-expressing DRG neurons and examined the regulation of T-type channels in DRGs from C57BL/6 mice with carrageenan-induced inflammatory hyperalgesia. We show that approximately 20% of mouse DRG neurons express Cav3.2 mRNA and protein. The size of the majority of Cav3.2-positive DRG neurons (69 ± 8%) ranged from 300 to 700 μm2 in cross-sectional area and 20 to 30 μm in estimated diameter. These channels co-localized with either neurofilament-H (NF-H) or peripherin. The peripherin-positive cells also overlapped with neurons that were positive for isolectin B4 (IB4) and calcitonin gene-related peptide (CGRP) but were distinct from transient receptor potential vanilloid 1 (TRPV1)-positive neurons during normal mouse states. In mice with carrageenan-induced inflammatory hyperalgesia, Cav3.2 channels, but not Cav3.1 or Cav3.3 channels, were upregulated in ipsilateral DRG neurons during the sub-acute phase. The increased Cav3.2 expression partially resulted from an increased number of Cav3.2-immunoreactive neurons; this increase in number was particularly significant for TRPV1-positive neurons. Finally, preceding and periodic intraplantar treatment with the T-type calcium channel blockers mibefradil and NNC 55-0396 markedly reduced and reversed mechanical hyperalgesia during the acute and sub-acute phases, respectively, in mice. These data suggest that Cav3.2 T-type channels participate in the development of inflammatory hyperalgesia, and this channel might play an even greater role in the sub-acute phase of inflammatory pain due to increased co-localization with TRPV1 receptors compared with that in the normal state.

Peripheral inflammation leads to ipsilateral and contralateral mechanical hyperalgesia. The transient receptor potential channel vanilloid type 1 (TRPV1), a nonselective cation channel expressed in mammalian primary sensory neurons and the spinal cord, may be involved in peripheral inflammation, but there is no consensus on the role of this channel in inflammation-induced mechanical hyperalgesia. Here, we examined the role of TRPV1 channels in carrageenan-induced mechanical hyperalgesia using wild-type and TRPV1-knockout (KO) mice and compared the results with those obtained in mice peripherally administered capsazepine, a TRPV1 antagonist, or capsaicin, a TRPV1 agonist. In the TRPV1-KO mice, ipsilateral mechanical hyperalgesia was significantly reduced during the acute phase (10-60 min), and the contralateral mechanical hyperalgesia nearly disappeared during both the acute and subacute phases. Blocking peripheral TRPV1 using capsazepine before carrageenan administration resulted in similar effects as those observed in the TRPV1-KO mice, except that it was less effective against contralateral mechanical hyperalgesia during the subacute phase. In contrast, capsaicin remarkably decreased ipsilateral and contralateral mechanical hyperalgesia throughout both phases, but this analgesic effect was not observed in the TRPV1-KO mice. Thus, TRPV1 channels could be involved in the development of both ipsilateral and contralateral mechanical hyperalgesia after inflammation. Peripheral TRPV1 could participate in acute hyperalgesia, whereas central TRPV1 may participate in subacute secondary hyperalgesia. Capsaicin potentially acts on both primary and secondary hyperalgesia in a TRPV1-dependent manner.

Taste receptor cells detect gustatory stimuli using a complex arrangement of ion channels, G protein-coupled receptors, and signaling cascades. Sour and salty tastes are detected by ion channels in the rat. Using a combination of homology screening and functional expression approaches, we screened a rat circumvallate papilla cDNA library and identified acid-sensing ion channel-2a (ASIC2a) and ASIC2b as candidates for the rat sour-sensing channels. In situ hybridization and reverse transcription-polymerase chain reaction experiments revealed that ASIC2a and ASIC2b transcripts were localized in taste bud cells. Immunohistochemistry and immunoprecipitation also revealed that both subunits were expressed in a subset of taste cells and that some of the cells expressed ASIC2a/ASIC2b heteromeric assemblies. Electrophysiological studies demonstrated that stimulation of acetic acid produced larger ASIC2 currents than did hydrochloric acid at the same pH. ASIC2a/ ASIC2b channels generated maximal inward currents at pH

The epithelial Na(+) channel (ENaC) regulates Na(+) homeostasis in cells and across epithelia. Four homologous ENaC subunits (alpha, beta, gamma, and delta) have been isolated in mammals. Combination of alpha-, beta-, and gamma-subunits or delta-, beta-, and gamma-subunits forms fully functional channels. Amiloride is a well known blocker of the ENaC family that inhibits both channel complexes. However, no specific antagonists are currently known that distinguish them. Here, we show that Evans blue, a diagnostic aid for the measurement of blood volume and vascular permeability, inhibits the activity of the delta-subunit expressed in Xenopus oocytes. The inward currents at a holding potential of -60 mV in human ENaCdeltabetagamma-expressing oocytes were inhibited by the application of Evans blue in a concentration-dependent manner with an IC(50) value of 143 muM. Evans blue markedly inhibited the delta-subunit current but did not block the alpha-subunit current. In conclusion, Evans blue is the first known delta-subunit-specific antagonist of ENaC. This finding provides us with a key compound for elucidating the physiological and pathological functions of ENaCdelta in humans and for drug development in the ENaC family.

The amiloride-sensitive epithelial Na+ channel (ENaC) regulates Na+ homeostasis into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as alpha, beta, gamma, and delta. The chemical agents acting on ENaC are, however, largely unknown, except for amiloride and benzamil as ENaC inhibitors. In particular, there are no agonists currently known that are selective for ENaCdelta, which is mainly expressed in the brain. Here we demonstrate that capsazepine, a competitive antagonist for transient receptor potential vanilloid subfamily 1, potentiates the activity of human ENaCdeltabetagamma (hENaCdeltabetagamma) heteromultimer expressed in Xenopus oocytes. The inward currents at a holding potential of -60 mV in hENaCdeltabetagamma-expressing oocytes were markedly enhanced by the application of capsazepine (or =1 microM), and the capsazepine-induced current was mostly abolished by the addition of 100 microM amiloride. The stimulatory effects of capsazepine on the inward current were concentration-dependent with an EC50 value of 8 microM. Neither the application of other vanilloid compounds (capsaicin, resiniferatoxin, and olvanil) nor a structurally related compound (dopamine) modulated the inward current. Although hENaCdelta homomer was also significantly activated by capsazepine, unexpectedly, capsazepine had no effect on hENaCalpha and caused a slight decrease on the hENaCalphabetagamma current. In conclusion, capsazepine acts on ENaCdelta and acts together with protons. Other vanilloids tested do not have any effect. These findings identify capsazepine as the first known chemical activator of ENaCdelta.

The amiloride-sensitive epithelial Na(+) channel (ENaC) controls Na(+) transport into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as alpha, beta, gamma, and delta. ENaCdelta can associate with beta and gamma subunits and generate a constitutive current that is 2 orders of magnitude larger than that of homomeric ENaCdelta. However, the distribution pattern of ENaCdelta is not consistent with that of the beta and gamma subunits. ENaCdelta is expressed mainly in the brain in contrast to beta and gamma subunits, which are expressed in non-neuronal tissues. To explain this discrepancy, we searched for novel functional properties of homomeric ENaCdelta and investigated the detailed tissue distribution in humans. When human ENaCdelta was expressed in Xenopus oocytes and Chinese hamster ovary cells, a reduction of extracellular pH activated this channel (half-maximal pH for an activation of 5.0), and the acid-induced current was abolished by amiloride. The most striking finding was that the desensitization of the acid-evoked current was much slower (by approximately 10% 120 s later), dissociating from the kinetics of acid-sensing ion channels in the degenerin/epithelial Na(+) channel family, which were rapidly desensitized during acidification. RNA dot-blot analyses showed that ENaCdelta mRNA was widely distributed throughout the brain and was also expressed in the heart, kidney, and pancreas in humans. Northern blotting confirmed that ENaCdelta was expressed in the cerebellum and the hippocampus. In conclusion, human ENaCdelta activity is regulated by protons, indicating that it may contribute to the pH sensation and/or pH regulation in the human brain.

Many painful inflammatory and ischemic conditions such as rheumatoid arthritis, cardiac ischemia and exhausted skeletal muscles are accompanied by local tissue acidosis. In such acidotic states, extracellular protons provoke the pain by opening excitatory cation channels in nociceptors. From recent molecular investigations into sensory neurons, two important candidate genes (or gene families) encoding proton-activated cation channels - acid-sensing ion channel and transient receptor potential vanilloid receptor subtype-1 have emerged. Since both channels can be major pharmacological targets for pain therapy and therefore, for the development and screening of new analgesics, it is of great importance to confirm their involvement in acid-evoked pain in humans and to know their relative contribution to the nociception. The authors' recent psychophysical studies focused on these points and the data is presented and discussed in this review.

Acid-sensing ion channel-2a (ASIC2a) is an amiloride-blockable proton-gated cation channel, probably contributing to sour-taste detection in rat taste cells. To isolate another subtype of the sour-taste receptor, we screened a rat circumvallate papilla cDNA library and identified ASIC2b, an N-terminal splice variant of ASIC2a. Reverse transcription-PCR analyses confirmed the expression of ASIC2b transcripts in the circumvallate papilla and, moreover, demonstrated its expression in the foliate and fungiform papillae. Immunohistochemical analyses revealed that ASIC2b, as well as ASIC2a, was expressed in a subpopulation of taste cells in the circumvallate, foliate, and fungiform papillae, and some of the cells displayed both ASIC2a and ASIC2b immunoreactivities. Subsequent coimmunoprecipitation studies with circumvallate papillae extracts indicated that ASIC2b associated with ASIC2a to form assemblies and, together with our immunohistochemical findings, strongly suggested that both ASIC2 subunits formed heteromeric channels in taste cells in the circumvallate, foliate, and fungiform papillae. Oocyte electrophysiology demonstrated that the ASIC2a/ASIC2b channel generated maximal inward currents at a pH ofor =2.0, which is in agreement with the in vivo pH sensitivity of rat taste cells, and that the amiloride sensitivity of the heteromer decreased with decreasing pH and was almost completely abolished at a pH of 2.0. These findings provide persuasive explanations for the amiloride insensitivity of acid-induced responses of rat taste cells.

Pungent sensation of hot peppers is thought to be mediated by vanilloid receptor subtype-1 (VR1), which can be activated by capsaicin, but there is little information regarding its histological localization in the tongue. We examined the immunohistochemical distribution of VR1 in the rat tongue. Intensely labeled VR1-immunoreactive (VR1-IR) fibers were concentrated in the circumvallate, foliate, and fungiform papillae, while sparse VR1-IR fibers were scattered throughout the tongue. VR1-positive taste-bud cells were not observed. Many VR1-positive nerve fibers surrounded the furrows of the circumvallate and foliate papillae, forming plexuses. Some of these VR1-positive fibers penetrated the apical epithelium and the trench wall epithelium, while a few of them entered taste buds. These VR1 distribution patterns resembled those of substance P (SP) and the calcitonin gene-related peptide (CGRP). Double-labeling experiments revealed that most of the VR1-immunoreactivity coexisted with SP- or CGRP-immunoreactivity in the nerve terminals in the circumvallate and foliate papillae. On the other hand, in the fungiform papillae, most of the VR1-IR fibers were positive for SP, but fewer were also positive for CGRP. Although VR1-immunoreactivity was not observed in taste-bud cells, our findings that a large number of VR1-IR fibers concentrated in the taste papillae suggest that capsaicin easily reaches the VR1 nerve terminals because of its lipophilic nature.

Aim: To reveal cholesterol transport via non-liver excretion pathway of plasma HDL-cholesterol, we focused on small intestinal and kidney in hyper- and hypo-lipoproteinemia model mice. Method: Mouse plasma was collected within EDTA coated tubes. Plasma lipoprotein profiles were measured by size exclusion HPLC and plasma preβHDL level was detected by immno-blotting of 2D PAGE by anti-mouse apoA-I peptide polyclonal antibody. Mouse HDL was co-incubated with 3H-cholesteryl oleyl ether (CEt) presence of CETP containing human plasma proteins for 48 hrs. The (100μL) of 3H-CEt-HDL was injected trough tail vein in anesthetized mice. 30μL of blood was collected each time point to chase the radioactivity, Mice organs were harvested after 3 hrs of the injection. Since lack of cellular catabolic pathway, CEt within the cells remained. For the immunohistochemical study, mouse was anesthetized and perfused thoroughly with PBS/5mM EDTA followed by 4% PFA/0.1M PB for the fixation. Leica CM1850 Cryostat was used for sectioning (thickness 16 μm). The sections were incubated in PBS containing 5% skim milk, 0.5% Triton X-100 and 5% inactivated normal horse serum for 3 hrs at room temperature, then incubated with primary antibodies (anti-SR-BI antibody diluted to 1:10,000) for overnight at 4°C. Secondary antibodies (Alexa fluor594) dissolved in the same blocking solution were applied to the sections for 90 min at room temperature. The fluorescence samples were observed by Nikon A1RSi confocal microscopy system. Results and conclusion: HDL derived CEt in intestinal cells were significantly reduced in ABCG1 null and SR-BI null mice during this experimental period. The other hand, kidney radioactivity showed no difference. Interestingly, preβHDL in SR-BI null mice was reduced by 2D western blotting of anti-mouse apoA-I peptide. Indicates that reduced speed of the plasma apoA-I recycling in SR-BI null mice, it may also cause reduced uptake of HDL-CEt by alternative pathway. Here we show that strong SR-BI-immunoreactivity was also found in the basolateral membrane and even higher intensity in some region. SR-BI located at the basolateral membrane may function as HDL-CE receptor in the small intestine.

We have isolated a cDNA encoding a splice variant of ASIC (acid-sensing ion channel)-beta from the rat trigeminal ganglion. This clone, designated ASIC-beta2, showed a 342 base deletion just after the first transmembrane domain in ASIC-beta. RT-PCR experiments revealed that ASIC-beta2 was expressed exclusively in the trigeminal ganglion and dorsal root ganglion. In situ hybridization showed that ASIC-beta2 mRNA was concentrated in both small diameter and large diameter neurons and co-localized with ASIC-beta mRNA within single sensory neurons in the trigeminal ganglion. When expressed in Xenopus oocytes, ASIC-beta2 was inactive by itself. However, it associated with ASIC-beta to form heteromers, which display lower affinity for protons than ASIC-beta alone.

Previous experiments have shown that an amino acid transport system B0+transporter in cultured colonic epithelial cells mediates amino acid absorption. Here we describe the cloning and functional characterization of a system B0+transporter selectively expressed in the colon. Using the combination of an expressed sequence tag database search and RT-PCR approaches, we cloned a mouse colonic amino acid transporter, designated mCATB0+. Northern blot analysis revealed that mCATB0+was selectively expressed in the large intestine. In situ hybridization showed the mCATB0+mRNA to be localized in absorptive epithelial cells. When expressed in Xenopus oocytes, mCATB0+exhibited a Na+-dependent stereoselective uptake and a broad specificity for neutral and cationic amino acids, which is characteristic of amino acid transport system B0+. In vivo [3H]glycine uptake assay demonstrated that a system B0+-like transporter protein was expressed on the apical surface of the colonic absorptive cells. Our data suggest that a mouse colonic amino acid transporter mCATB0+may absorb amino acids from the intestinal contents in the colon.

To clarify functional roles of MDEG2 (mammalian degenerin-2), a modulatory subunit of proton-activated cation channels, in MDEG1/MDEG2 heteromer, we replaced the Gly481 residue in MDEG2 with cysteine or phenylalanine and characterized them electrophysiologically. Expression of MDEGI in Xenopus oocytes elicited proton-activated cation currents that were rapidly desensitized. Co-expression of MDEG 1 and MDEG2 (or MDEG2-G481C) displayed similar current traces as MDEG 1 alone. In contrast, co-expression of MDEG 1 and MDEG2-G481F dramatically attenuated desensitization of the proton-activated currents. Interestingly, the G481 F mutation in MDEG2 did not alter other channel properties including maximal whole-cell currents, ionic selectivity, pH-sensitivity and affinity for amiloride. Thus, Gly481 in MDEG2 specifically controls inactivation process of the MDEG1/MDEG2 channel.