Shigenari Hashimoto

Exercise Effects on Methylation of ASC Gene

Chronic moderate exercise has been reported to reduce pro-inflammatory cytokines. To analyze the molecular mechanisms by which training exerts these effects, the epigenetic influences of age and exercise on the ASC gene, which is responsible for IL-1beta and IL-18 secretion, were investigated by ASC gene methylation. Further, the relationship between carcinogenesis and exercise, and methylation of the P15 tumor suppressive gene was also analyzed. High-intensity interval walking exercise, consisting of 3 min low-intensity walking at 40% of peak aerobic capacity followed by a 3 min high-intensity walking period above 70% of peak aerobic capacity, was continued for 6 months. Peripheral blood DNA extracts from young control (n=34), older control (n=153), and older exercise (n=230) groups were then analyzed by pyrosequencing for DNA methylation. Methylation of ASC decreased significantly with age (young control vs. older control, p<0.01), which is indicative of an age-dependent increase in ASC expression. Compared to the older control group, the degree of ASC methylation was higher in the older exercise group (older control vs. older exercise: p<0.01), and presumably lower ASC expression. Neither exercise nor age affected the methylation of the P15. In summary, chronic moderate exercise appears to attenuate the age-dependent decrease in ASC methylation, implying suppression of excess pro-inflammatory cytokines through reduction of ASC expression.

La autoantigen is cleaved in the COOH terminus and loses the nuclear localization signal during apoptosis.

La autoantigen is a 47-kDa nuclear protein that binds to nascent polymerase III transcripts and a number of viral RNAs. We show that La protein was cleaved to generate a 43-kDa fragment during apoptosis of human leukemic HL-60 cells treated with camptothecin or etoposide. Immunofluorescence microscopy showed that the La protein level was increased in the cytoplasm during apoptosis of HL-60 cells. In addition, UV irradiation of HeLa cells led to the cleavage and redistribution of La protein upon apoptosis. Several lines of evidence show that La protein is cleaved by caspase-3 or closely related proteases at Asp-374 in the COOH terminus. When the full-length (La) and COOH-terminally truncated (LaΔC374) forms of La protein were expressed as fusion proteins with green fluorescence protein (GFP), GFP-LaΔC374 was predominantly cytoplasmic, whereas GFP-La was localized in the nucleus. These results suggest that La protein loses the nuclear localization signal residing in the COOH terminus upon cleavage and is thus redistributed to the cytoplasm during apoptosis.

Clinical features of patients with GJB2 (connexin 26) mutations: severity of hearing loss is correlated with genotypes and protein expression patterns.

AbstractMutations in the GJB2 (connexin 26, Cx26) gene are the major cause of nonsyndromic hearing impairment in many populations. Genetic testing offers opportunities to determine the cause of deafness and predict the course of hearing, enabling the prognostication of language development. In the current study, we compared severity of hearing impairment in 60 patients associated with biallelic GJB2 mutations and assessed the correlation of genotypes and phenotypes. Within a spectrum of GJB2 mutations found in the Japanese population, the phenotype of the most prevalent mutation, 235delC, was found to show more severe hearing impairment than that of V37I, which is the second most frequent mutation. The results of the present study, taken together with phenotypes caused by other types of mutations, support the general rule that phenotypes caused by the truncating GJB2 mutations are more severe than those caused by missense mutations. The present in vitro study further confirmed that differences in phenotypes could be explained by the protein expression pattern.

Type IX collagen is crucial for normal hearing

cDNA microarray analysis indicated that COL9A1 and COL9A3 are highly expressed in the human inner ear, suggesting that type IX collagen has a crucial functional role in the inner ear. This study further confirmed, by means of real-time PCR, the presence of collagen type IX genes in the mouse inner ear. Immunocytochemical analysis also revealed that type IX collagen is distributed in the tectorial membrane, where it co-localizes with type II collagen, indicating that type IX collagen may contribute to the three-dimensional integrated structure of type II collagen molecules. Mice with targeted disruption of the col9a1 gene were shown through assessment by auditory brain stem response to have hearing loss, suggesting an important role of type IX collagen in maintaining normal hearing. At the light microscopic level, the tectorial membrane of knock-out mice was found to be abnormal in shape, and electron microscopy confirmed disturbance of organization of the collagen fibrils. An antibody against type II collagen failed to detect type II collagen in the tectorial membrane of type IX collagen knock-out mice, suggesting that a lack of type IX collagen may affect the three-dimensional structure of type II collagen molecules. These findings indicate that genes encoding each chain of type IX collagen may fulfill an important function associated with the tectorial membrane in the auditory system.

TRANSPLANTED HUMAN AMNIOTIC EPITHELIAL CELLS EXPRESS CONNEXIN 26 AND NA-K-ADENOSINE TRIPHOSPHATASE IN THE INNER EAR

Cochlear fibrocytes are the crucial component of the inner ear homeostasis and its defect by various causes; GJB2 (connexin [Cx] 26) mutation, for example, leads to hearing loss. In the present study, we investigated the potential use of human amniotic epithelial cells, proposed to possess pluripotential properties, as a source of transplantation therapy in inner ear disease. The mRNA of the gap junction protein Cx26 and Na-K-adenosine triphosphatase, the immunohistologic expression of these proteins, and the cells’ intercellular communication capacity were detected in vitro. Their transplantation into the guinea pig cochlea revealed the survival and expression of the proteins even 3 weeks after transplantation. Transplanted human amniotic epithelial cells were localized at the site where the proteins function, strongly indicating their cooperation in the regional potassium ion recycling. This technology suggests the therapeutic potential for the treatment of hearing loss.

Ubiquinol-10 Supplementation Activates Mitochondria Functions to Decelerate Senescence in Senescence-Accelerated Mice

AIM The present study was conducted to define the relationship between the anti-aging effect of ubiquinol-10 supplementation and mitochondrial activation in senescence-accelerated mouse prone 1 (SAMP1) mice. RESULTS Here, we report that dietary supplementation with ubiquinol-10 prevents age-related decreases in the expression of sirtuin gene family members, which results in the activation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a major factor that controls mitochondrial biogenesis and respiration, as well as superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2), which are major mitochondrial antioxidant enzymes. Ubiquinol-10 supplementation can also increase mitochondrial complex I activity and decrease levels of oxidative stress markers, including protein carbonyls, apurinic/apyrimidinic sites, malondialdehydes, and increase the reduced glutathione/oxidized glutathione ratio. Furthermore, ubiquinol-10 may activate Sirt1 and PGC-1α by increasing cyclic adenosine monophosphate (cAMP) levels that, in turn, activate cAMP response element-binding protein (CREB) and AMP-activated protein kinase (AMPK). INNOVATION AND CONCLUSION These results show that ubiquinol-10 may enhance mitochondrial activity by increasing levels of SIRT1, PGC-1α, and SIRT3 that slow the rate of age-related hearing loss and protect against the progression of aging and symptoms of age-related diseases.

Calponin h1 induced a flattened morphology and suppressed the growth of human fibrosarcoma HT1080 cells

Calponin h1 (CNh1) is an actin-binding protein that is expressed mainly in smooth muscle cells and is known to regulate smooth muscle contraction. Recently, re-expression of CNh1 in leiomyosarcoma cell lines is reported to suppress cell proliferation and tumorigenicity. However, little is known about the associated cellular structural and functional changes. Since CNh1 is also detected in normal fibroblasts, we hypothesised that CNh1 would also inhibit cell proliferation of the fibrosarcoma cells, HT1080, in which CNh1 is suppressed. An expression vector of human CNh1 complementary DNA was transfected into human HT1080 cells by a calcium-phosphate precipitation method. CNh1-transfected cells exhibited a flattened morphology with organised actin filaments, a significant decrease in cell motility and enhancement in adhesion to fibronectin in association with an increase in integrin alpha5beta1 expression. Anchorage-independent growth and tumorigenicity in nude mice were suppressed in the CNh1-transfected cells. Our results suggest that CNh1 may have a role as a tumour suppressor in human fibrosarcoma by influencing cytoskeletal activities.

Differences in the expression of genes between normal tissue and squamous cell carcinomas of head and neck using cancer-related gene cDNA microarray

Conclusion. This study clearly showed the molecular characteristics of head and neck squamous cell carcinoma (HNSCC) on the basis of gene expression patterns. Objective. cDNA microarray has recently been shown to have the ability to represent the expression patterns of large numbers of genes from a small amount of tissue, potentially enabling definition of groups of patients with similar biological behavior of cancer. Although gene expression profiling using this technique has proven helpful for predicting the prognosis in various cancers, little is known regarding HNSCC. The aim of this study was to investigate the differences in the expression of various genes between normal tissue and cancers of patients with HNSCC by cDNA microarray. Patients and methods. We extracted mRNA from 17 HNSCC patients and used cDNA microarray analysis to investigate the gene expression patterns. The present study was not designed to perform an inclusive search for genes but rather to focus on cancer-related genes. Results. Seven independent genes were found to be up-regulated in cancer tissues: matrix metalloproteinase-1, -3, and -10, interleukin-8, cadherin 3, hexabrachion, and interferon gamma-inducible protein 10. Hyaluronic acid-binding protein 2, keratin 4, and keratin 13 were categorized as down-regulated. The hierarchical clustering and dendrogram for 17 cancer samples and 425 genes could be grouped into three clusters.

Gene Expression Pattern after Insertion of Dexamethasone-Eluting Electrode into the Guinea Pig Cochlea

A cochlear implant is an indispensable apparatus for a profound hearing loss patient. But insertion of the electrode entails a great deal of stress to the cochlea, and may cause irreversible damage to hair cells and related nerve structure. Although damage prevention effects of dexamethasone have been reported, long-term administration is difficult. In this study, we used a dexamethasone-eluting electrode in the guinea pig cochlea, and compared the gene expression after 7 days insertion with that of a normal electrode and non-surgically treated control by microarray. 40 genes were up-regulated 2-fold or more in the normal electrode group compared to the non-surgically treated group. Most of the up-regulated genes were associated with immune response and inflammation. In the dexamethasone-eluting group, compared to the normal electrode group, 7 of the 40 genes were further up-regulated, while 12 of them were down-regulated and there was a tendency to return to the non-surgical condition. 9 genes were down-regulated 2-fold or less with normal electrode insertion, and 4 of the 9 tended to return to the non-surgical condition in the dexamethasone-eluting group. These genes are certainly involved in the maintenance of the physiological functions of the cochlea. Our results indicate that the dexamethasone-eluting electrode will have an effect on the normalization of homeostasis in the cochlea.

TECTA mutations in Japanese with mid-frequency hearing loss affected by zona pellucida domain protein secretion

TECTA gene encodes α-tectorin, the major component of noncollagenous glycoprotein of the tectorial membrane, and has a role in intracochlear sound transmission. The TECTA mutations are one of the most frequent causes of autosomal dominant (AD) hearing loss and genotype–phenotype correlations are associated with mutations of TECTA in exons according to α-tectorin domains. In this study, we investigated the prevalence of hearing loss caused by TECTA mutations in Japanese AD hearing loss families, and confirmed genotype–phenotype correlation, as well as the intracellular localization of missense mutations in the α-tectorin domain. TECTA mutations were detected in 2.9% (4/139) of our Japanese AD hearing loss families, with the prevalence in moderate hearing loss being 7.7% (4/52), and all patients showed typical genotype–phenotype correlations as previously described. The present in vitro study showed differences of localization patterns between wild type and mutants, and suggested that each missense mutation may lead to a lack of assembly of secretion, and may reduce the incorporation of α-tectorin into the tectorial membrane.