Transcriptome Profiling of Anhidrotic Eccrine Sweat Glands Reveals that Olfactory Receptors on Eccrine Sweat Glands Regulate Perspiration in a Ligand-Dependent Manner.

Sweat maintains systemic homeostasis in humans. Although sweating disorders may cause multifaceted health problems, therapeutic options for sweat disorders have not yet been established. To gain new insight into the mechanism underlying the regulation of perspiration, we compared eccrine sweat gland transcriptomes from hidrotic and anhidrotic lesions from patients with anhidrosis and found out that olfactory receptors were expressed differentially in anhidrotic and hidrotic eccrine sweat glands. We then confirmed OR51A7 and OR51E2 expression in human eccrine sweat glands by in situ hybridization and immunohistochemistry. An alkaline phosphatase-TGFα shedding assay revealed that ß-ionone activates G-proteins through OR51A7 or OR51E2. The effect of topically applied ß-ionone on sweating was examined with the quantitative sudomotor axon reflex test, which showed that responses to ß-ionone differed between sexes. Topical ß-ionone attenuated female sweating and augmented male sweating. Taken together, this study suggests that olfactory receptors expressed in eccrine sweat glands may regulate sweating in response to odorous ligands on the basis of sex. These unexpected results indicate that olfactory receptors may modulate sweating and that olfactory receptor modulators may contribute to the management of sweat disorders.

The Transfer of the Hepatocyte Growth Factor Gene by Macrophages Ameliorates the Progression of Peritoneal Fibrosis in Mice.

Growing evidence indicates that hepatocyte growth factor (HGF) possesses potent antifibrotic activity. Furthermore, macrophages migrate to inflamed sites and have been linked to the progression of fibrosis. In this study, we utilized macrophages as vehicles to express and deliver the HGF gene and investigated whether macrophages carrying the HGF expression vector (HGF-M) could suppress peritoneal fibrosis development in mice. We obtained macrophages from the peritoneal cavity of mice stimulated with 3% thioglycollate and used cationized gelatin microspheres (CGMs) to produce HGF expression vector-gelatin complexes. Macrophages phagocytosed these CGMs, and gene transfer into macrophages was confirmed in vitro. Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate (CG) for three weeks; seven days after the first CG injection, HGF-M was administered intravenously. Transplantation of HGF-M significantly suppressed submesothelial thickening and reduced type III collagen expression. Moreover, in the HGF-M-treated group, the number of α-smooth muscle actin- and TGF-ß-positive cells were significantly lower in the peritoneum, and ultrafiltration was preserved. Our results indicated that the transplantation of HGF-M prevented the progression of peritoneal fibrosis and indicated that this novel gene therapy using macrophages may have potential for treating peritoneal fibrosis.

Hydroxychloroquine suppresses anti-GBM nephritis via inhibition of JNK/p38 MAPK signaling.

BACKGROUND: Anti-glomerular basement membrane (anti-GBM) nephritis, characterized by glomerular crescent formation, requires early treatment because of poor prognosis. Hydroxychloroquine (HCQ) is an antimalarial drug with known immunomodulatory, anti-inflammatory, and autophagy inhibitory effects; it is recognized in the treatment of autoimmune diseases such as systemic lupus erythematosus. However, its effect on anti-GBM nephritis remains unknown. In this study, we investigated the effect of HCQ on anti-GBM nephritis in rats. METHODS: Seven-weeks-old male WKY rats were administered anti-GBM serum to induce anti-GBM nephritis. Either HCQ or vehicle control was administered from day 0 to day 7 after the induction of nephritis. Renal function was assessed by measuring serum creatinine, proteinuria, and hematuria. Renal histological changes were assessed by PAS staining and Masson trichrome staining, and infiltration of macrophages was assessed by ED-1 staining. Mitogen-activated protein kinase (MAPK) was evaluated by western blotting, while chemokine and inflammatory cytokines were evaluated by enzyme-linked immunosorbent assay using urine sample. RESULTS: HCQ treatment suppressed the decline in renal function. Histologically, extracapillary and intracapillary proliferations were observed from day 1, while fibrinoid necrosis and ED-1 positive cells were observed from day 3. Rats with anti-GBM nephritis showed high levels of monocyte chemotactic protein-1 and tumor necrosis factor-α. These changes were significantly suppressed following HCQ treatment. In addition, HCQ suppressed JNK/p38 MAPK phosphorylation. CONCLUSION: HCQ attenuates anti-GBM nephritis by exerting its anti-inflammatory effects via the inhibition of JNK/p38 MAPK activation, indicating its therapeutic potential against anti-GBM nephritis.

An Advanced Detection System for In Situ Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe.

In situ hybridization (ISH) is a powerful method for detecting specific RNAs at the cellular level. Although conventional ISH using hapten-labeled probes are useful for detecting multiple RNAs, the detection procedures are still complex and required longer time. Therefore, we introduced a new application of fluorescence resonance energy transfer (FRET)-based molecular beacon (MB) probes for ISH. MCF-7 cells and C57BL/6J mouse uterus were used for ISH. MB probes for ERα mRNA and 28S rRNA were labeled with Cy3/BHQ-2 and 6-FAM/DABCYL, and conventional probes were labeled with digoxigenin. Fluorescence measurements revealed that of more-rapid hybridization kinetics compared to conventional probes. In MCF-7 cells, 28S rRNA was detected in nucleolus and cytoplasm of all cells, whereas ERα mRNA was detected in some nucleolus. In the uterus, 28S rRNA was clearly detected using complementary MB probe, but there were no signals in control slides. Moreover, 28S rRNA was detected in all cells, whereas ERα mRNA was detected mainly in the epithelium. Fluorescence intensity of 28S rRNA was decreased significantly in 1 or 2 base-mismatched sequences, that indicates highly specific detection of target RNAs. In conclusion, the FRET-based MB probes are very useful for ISH, providing rapid hybridization, high sensitivity and specificity.

Compounds in cigarette smoke induce EGR1 expression via the AHR, resulting in apoptosis and COPD.

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide, and pulmonary epithelial cell apoptosis is regarded as one of the most important factors in its pathogenesis. Here, we examined the molecular mechanisms of apoptosis caused by cigarette smoke (CS). In the normal bronchial epithelium cell line BEAS-2B, a CS extract markedly induced apoptosis together with transient early growth response 1 (EGR1) protein expression, which is activated over time via the aryl hydrocarbon receptor (AHR). The CS extract-induced apoptosis decreased cell count of BEAS-2B cells and was significantly reversed by knockdown of either EGR1 or AHR. In vivo, the CS extract caused alveolar wall destruction, mimicking COPD, 1 week after intrathoracic injection. Bronchoalveolar lavage fluid (BALF) from the CS extract-treated mice contained massive numbers of apoptotic epithelial cells. Furthermore, it was found that aminoanthracene induced EGR1 expression and cell apoptosis. By contrast, the AHR antagonist stemregenin 1 (SR1) restored apoptosis upon CS treatment. These results suggest that aryl hydrocarbons, such as aminoanthracene, induce EGR1 expression via the AHR, resulting in cell apoptosis and that this can be prevented by administration of an antagonist of AHR.

Mitochonic acid-5 ameliorates chlorhexidine gluconate-induced peritoneal fibrosis in mice.

Peritoneal fibrosis is a serious complication of long-term peritoneal dialysis, attributable to inflammation and mitochondrial dysfunction. Mitochonic acid-5 (MA-5), an indole-3-acetic acid derivative, improves mitochondrial dysfunction and has therapeutic potential against various diseases including kidney diseases. However, whether MA-5 is effective against peritoneal fibrosis remains unclear. Therefore, we investigated the effect of MA-5 using a peritoneal fibrosis mouse model. Peritoneal fibrosis was induced in C57BL/6 mice via intraperitoneal injection of chlorhexidine gluconate (CG) every other day for 3 weeks. MA-5 was administered daily by oral gavage. The mice were divided into control, MA-5, CG, and CG + MA-5 groups. Following treatment, immunohistochemical analyses were performed. Fibrotic thickening of the parietal peritoneum induced by CG was substantially attenuated by MA-5. The number of α-smooth muscle actin-positive myofibroblasts, transforming growth factor ß-positive cells, F4/80-positive macrophages, monocyte chemotactic protein 1-positive cells, and 4-hydroxy-2-nonenal-positive cells was considerably decreased. In addition, reduced ATP5a1-positive and uncoupling protein 2-positive cells in the CG group were notably increased by MA-5. MA-5 may ameliorate peritoneal fibrosis by suppressing macrophage infiltration and oxidative stress, thus restoring mitochondrial function. Overall, MA-5 has therapeutic potential against peritoneal fibrosis.

Vitamin K-Dependent γ-Glutamyl Carboxylase in Sertoli Cells Is Essential for Male Fertility in Mice.

γ-Glutamyl carboxylase (GGCX) is a vitamin K (VK)-dependent enzyme that catalyzes the γ-carboxylation of glutamic acid residues in VK-dependent proteins. The anticoagulant warfarin is known to reduce GGCX activity by inhibiting the VK cycle and was recently shown to disrupt spermatogenesis. To explore GGCX function in the testis, here, we generated Sertoli cell-specific Ggcx conditional knockout (Ggcx scKO) mice and investigated their testicular phenotype. Ggcx scKO mice exhibited late-onset male infertility. They possessed morphologically abnormal seminiferous tubules containing multinucleated and apoptotic germ cells, and their sperm concentration and motility were substantially reduced. The localization of connexin 43 (Cx43), a gap junction protein abundantly expressed in Sertoli cells and required for spermatogenesis, was distorted in Ggcx scKO testes, and Cx43 overexpression in Sertoli cells rescued the infertility of Ggcx scKO mice. These results highlight GGCX activity within Sertoli cells, which promotes spermatogenesis by regulating the intercellular connection between Sertoli cells and germ cells.

An inhibitor of Krüppel-like factor 5 suppresses peritoneal fibrosis in mice.

BACK GROUND: Krüppel-like transcription factor 5 (KLF5) is a transcription factor regulating cell proliferation, angiogenesis and differentiation. It has been recently reported that Am80, a synthetic retinoic acid receptor α-specific agonist, inhibits the expression of KLF5. In the present study, we have examined the expression of KLF5 in fibrotic peritoneum induced by chlorhexidine gluconate (CG) in mouse and evaluated that Am80, as an inhibitor of KLF5, can reduce peritoneal fibrosis. METHODS: Peritoneal fibrosis was induced by intraperitoneal injection of CG into peritoneal cavity of ICR mice. Am80 was administered orally for every day from the start of CG injection. Control mice received only a vehicle (0.5% carboxymethylcellulose solution). After 3 weeks of treatment, peritoneal equilibration test (PET) was performed and peritoneal tissues were examined by immunohistochemistry. RESULTS: The expression of KLF5 was less found in the peritoneal tissue of control mice, while KLF5 was expressed in the thickened submesothelial area of CG-injected mice receiving the vehicle. Am80 treatment reduced KLF5 expression and remarkably attenuated peritoneal thickening, accompanied with the reduction of type III collagen expression. The numbers of transforming growth factor ß-positive cells, α-smooth muscle actin-positive cells and infiltrating macrophages were significantly decreased in Am80-treated group. PET revealed the increased peritoneal permeability in CG mice, whereas Am80 administration significantly improved the peritoneal high permeability state. CONCLUSIONS: These results indicate the involvement of KLF5 in the progression of experimental peritoneal fibrosis and suggest that Am80 may be potentially useful for the prevention of peritoneal fibrosis through inhibition of KLF5 expression.

Nuclear Expression of Pygo2 Correlates with Poorly Differentiated State Involving c-Myc, PCNA and Bcl9 in Myanmar Hepatocellular Carcinoma.

In Myanmar, hepatocellular carcinoma (HCC) is commonly seen in young adult and associated with poor prognosis, while the molecular mechanisms that characterize HCC in Myanmar are unknown. As co-activation of Wnt/ß-catenin signaling and c-Myc (Myc) are reported to associate with malignancy of HCC, we immunohistochemically investigated the expression of Pygo2 and Bcl9, the co-activators of the Wnt/ß-catenin signaling, Myc and PCNA in 60 cases of Myanmar HCC. Pygo2 expression was confirmed by in situ hybridization. The signal intensity was measured by image analyzer and then statistically analyzed. As a result, the expression of Pygo2 was significantly higher in HCC compared to normal liver tissue and the nuclear signal was the most intense in poorly differentiated HCC. Cytoplasmic Bcl9 was expressed in the normal liver tissue but decreased in HCC with the progression of histopathological grade. Myc was significantly higher in poorly differentiated HCC, whereas PCNA labeling index increased with the progression of histopathological grade. Nuclear Pygo2 showed strong correlation with nuclear Myc (P < 0.01) and PCNA (P < 0.001), and inversely correlated with cytoplasmic Bcl9 (P < 0.01). Our results suggested Wnt/ß-catenin and Myc signaling is commonly activated in Myanmar HCC and that the correlative upregulation of nuclear Pygo2 and Myc characterizes the malignant features of HCC in Myanmar.

Global changes in epigenomes during mouse spermatogenesis: possible relation to germ cell apoptosis.

Mammalian spermatogenesis is characterized by disproportionate germ cell apoptosis. The high frequency of apoptosis is considered a safety mechanism that serves to avoid unfavorable transmission of paternal aberrant genetic information to the offspring as well as elimination mechanism for removal of overproduced immature or damaged spermatogenic cells. The molecular mechanisms involved in the induction of germ cell apoptosis include both intrinsic mitochondrial Bcl-2/Bax and extrinsic Fas/FasL pathways. However, little is known about the nuclear trigger of those systems. Recent studies indicate that epigenomes are essential in the regulation of gene expression through remodeling of the chromatin structure, and are genome-like transmission materials that reflect the effects of various environmental factors. In spermatogenesis, epigenetic errors can act as the trigger for elimination of germ cells with abnormal chromatin structure, abnormal gene expression and/or morphological defects (disordered differentiation). In this review, we focus on the relationship between global changes in epigenetic parameters and germ cell apoptosis in mice and other mammals.

Correction to: Accelerated proliferation of hepatocytes in rats with iron overload after partial hepatectomy.

The figure shown below is the correct version. We apologize for the mistake.

Hexapeptide derived from prothymosin alpha attenuates cisplatin-induced acute kidney injury.

BACKGROUND: Prothymosin alpha (ProTα) is a nuclear protein expressed in virtually all mammalian tissues. Previous studies have shown that ProTα exhibits protective effects against ischemia-induced cell death in various cell types. Recently, the 6-residue peptide P6Q (NEVDQE), the modified form of the active 6-residue core (51-56) in ProTα, has also been shown to have protective effects against retinal ischemia. However, it remains to be elucidated whether P6Q is effective against acute kidney injury (AKI). Therefore, we investigated the renoprotective effect of P6Q on cisplatin-induced AKI. METHODS: Cultured HK-2 cells were treated with cisplatin for 24 h and pretreatment with ProTα or P6Q was carried out 30 min before cisplatin treatment. Cell viability was evaluated using the MTT assay. In an in vivo study, 8-week-old male Wistar rats were divided into control, cisplatin treated, and cisplatin treated with P6Q injection groups. In the last of these, P6Q was injected intravenously before cisplatin treatment. Then, we evaluated the renoprotective effect of P6Q. RESULTS: In the study on cultured cells, pretreatment with ProTα or P6Q prevented cisplatin-induced cell death. In the in vivo study, pretreatment with P6Q significantly attenuated cisplatin-induced increase in serum creatinine and blood urea nitrogen levels, renal tubular cell injury, and apoptosis. Moreover, P6Q attenuated the mitochondrial apoptotic pathway and accelerated Akt phosphorylation after cisplatin-induced renal damage. CONCLUSION: Taken together, our findings indicate that P6Q can attenuate cisplatin-induced AKI and suppress the mitochondrial apoptotic pathway via Akt phosphorylation. These data suggest that P6Q has potential as a preventative drug for cisplatin-induced AKI.

Evaluation of Candida peritonitis with underlying peritoneal fibrosis and efficacy of micafungin in murine models of intra-abdominal candidiasis.

Candida peritonitis is a crucial disease, however the optimal antifungal therapy regimen has not been clearly defined. Peritoneal fibrosis (PF) can be caused by abdominal surgery, intra-abdominal infection, and malignant diseases, and is also widely recognized as a crucial complication of long-term peritoneal dialysis. However, the influence of PF on Candida peritonitis prognosis remains unknown. Here, we evaluated the severity of Candida peritonitis within the context of PF and the efficacy of micafungin using mice. A PF mouse model was generated by intraperitoneally administering chlorhexidine gluconate. Candida peritonitis, induced by intraperitoneal inoculation of Candida albicans, was treated with a 7-day consecutive subcutaneous administration of micafungin. Candida infection caused a higher mortality rate in the PF mice compared with the control mice on day 7. Proliferative Candida invasion into the peritoneum and intra-abdominal organs was confirmed pathologically only in the PF mice. However, all mice in both groups treated with micafungin survived until day 20. Micafungin treatment tends to suppress inflammatory cytokines in the plasma 12 h after infection in both groups. Our results suggest that PF enhances early mortality in Candida peritonitis. Prompt initiation and sufficient doses of micafungin had good efficacy for Candida peritonitis, irrespective of the underlying PF.

Immunohistochemical Mapping of Bcl9 Using Two Antibodies that Recognize Different Epitopes Is Useful to Characterize Juvenile Development of Hepatocellular Carcinoma in Myanmar.

B-cell lymphoma 9 (Bcl9) is the core component of Wnt/ß-catenin signaling and overexpressed in nuclei of various tumors, including hepatocellular carcinoma (HCC). However, the extent of Bcl9 expression relative to HCC differentiation stage and its functional aspects are poorly understood. In this study, we examined the expression pattern of Bcl9 immunohistochemically, using two anti-Bcl9 antibodies; one was a conventional polyclonal-antibody (anti-Bcl9ABC) against amino acid no.800-900 of human-Bcl9, while the other (anti-Bcl9BIO) was against amino acid no.50-200, covering Pygopus-binding sites of Bcl9. Immunohistochemistry using anti-Bcl9BIO demonstrated distinctive staining in the cytoplasm, while the anti-Bcl9ABC signal was detected in both cytoplasm and nuclei of HCC cells, reflecting different states of Bcl9 function because Pygopus-binding to Bcl9 is essential to exert its function together with ß-catenin in nucleus. Quantitative analysis revealed a significantly higher immunohistochemical-score by anti-Bcl9BIO in normal liver comparing various differentiation grades of HCC (P < 0.004), whereas no significant difference was noted with anti-Bcl9ABC. Interestingly, immunohistochemical-score of anti-Bcl9BIO in patients aged < 40 years was significantly lower than that of ≥ 40 years group (P < 0.01). The results indicated that anti-Bcl9BIO detected cytoplasmic Bcl9, which does not bind to Pygopus suggesting it could be a useful indicator for development of HCC in young Myanmar patients.

Histone deacetylase inhibitors suppress transdifferentiation of gonadotrophs to prolactin cells and proliferation of prolactin cells induced by diethylstilbestrol in male mouse pituitary.

Diethylstilbestrol (DES), an estrogen agonist, increases prolactin (PRL) cells through transdifferentiation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells to PRL cells as well as proliferation of PRL cells in adult male mouse pituitary. Since hyperacetylation of histone H3 is implicated in the regulation of activation of various genes, we examined the effect of DES on the state of histone H3 acetylation. DES significantly reduced the immunohistochemical signal for acetylated histone H3 at lysine 9 (H3K9ac) in PRL, LH and FSH cells, but not for H3K18ac or H3K23ac. DES-treated mice were injected intraperitoneally with HDAC inhibitors (HDACi), sodium phenylbutyrate (NaPB) or valproic acid (VPA), to mimic the acetylation level of histone H3. As expected, HDACi treatment restored the level of H3K9ac expression in these cells, and also inhibited DES-induced increase in PRL cells. Furthermore, NaPB and VPA also abrogated the effects of DES on the population density of both LH and FSH cells. Similarly, the numbers of proliferating and apoptotic cells in the pituitary in NaPB- or VPA-treated mice were comparable to those of the control mice. Considered together, these results indicated that the acetylation level of histone H3 plays an important role in DES-induced transdifferentiation of LH to PRL cells as well as proliferation of PRL cells.

Changes in DNA Methylation of Oocytes and Granulosa Cells Assessed by HELMET during Folliculogenesis in Mouse Ovary.

For a better understanding of epigenetic regulation of cell differentiation, it is important to analyze DNA methylation at a specific site. In this study, we examined changes in the methylation level of CCGG and GATCG sites during mouse folliculogenesis in paraffin-embedded sections of mouse ovaries. For the purpose, we used a new method, histo endonuclease-linked detection of methylation sites of DNA (HELMET), designed to detect methylation sites of DNA with a specific sequence in a tissue section. Unlike the global level of DNA methylation, which was no change in immunohistochemical staining of 5-methylcytosine throughout folliculogenesis, we found that there were hypermethylation of CCGG and GATCG sites in most of the granulosa cells of tertiary follicles compared to that of primary and secondary follicles. Interestingly, TUNEL-positive granulosa cells, which were frequent in mammalian folliculogenesis, became markedly Hpa II-reactive and Sau3A I-reactive, indicating that the CCGG and GATCG sites may be preferentially demethylated during apoptosis.

Analysis of H3K27me3 expression and DNA methylation at CCGG sites in smoking and non-smoking patients with non-small cell lung cancer and their clinical significance.

Smoking frequently leads to epigenetic alterations, including DNA methylation and histone modifications. The effect that smoking has on the DNA methylation levels at CCGG sites, the expression of trimethylation of histone H3 at lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (EZH2), and their interactions in patients with non-small cell lung cancer (NSCLC) were analyzed. There were a total of 42 patients with NSCLC, 22 with adenocarcinomas and 20 with squamous cell carcinomas enrolled in the present study. Expression of H3K27me3, EZH2 and proliferating cellular nuclear antigen (PCNA) were immunohistochemically detected. DNA methylation at CCGG sites was evaluated via histoendonuclease-linked detection of DNA methylation sites. The apoptotic index of cancerous tissues obtained from patients of different smoking statuses was evaluated via the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling method. The association with clinicopathological data was calculated relative to different smoking statuses. Compared with the non-smokers, smokers with NSCLC exhibited a significantly lower apoptotic index (P<0.05), and frequently had a lower level of DNA methylation at CCGG sites, lower H3K27me3 expression and a higher EZH2 expression (P<0.05). DNA methylation levels at CCGG sites were negatively correlated to the Brinkman index (P=0.017). Furthermore, there was a parallel association between the H3K27me3 and EZH2 expression levels in the majority of smokers, whereas in the majority of non-smokers, there was a diverging association (P=0.015). There was a diverging association between the PCNA and EZH2 expression levels in the majority of smokers; however, in the majority of non-smokers, there was a parallel association (P=0.048). In addition, the association between the CCGG methylation ratio and immunohistochemical expression of H3K27me3 was a parallel association in the majority of smokers, while in the majority of non-smokers there was a diverging association (P=0.049). Conclusively, patients with NSCLC and different smoking statuses exhibit different epigenetic characteristics. Additionally, DNA methylation levels at the CCGG sites may have the ability to determine associations between the expression levels of H3K27me3, EZH2 and PCNA.