In vivo recycling of nitrate (NO 3 − ) and nitrite (NO 2 − ) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic nitrate–nitrite–NO balance. More than 25% of the circulating NO 3 − is actively removed and secreted by salivary glands. Oral commensal bacteria convert salivary NO 3 − to NO 2 − , which enters circulation and leads to NO generation. The transporters for NO 3 − in salivary glands have not yet been identified. Here we report that sialin ( SLC17A 5 ), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO 3 − /H + cotransporter in the plasma membrane of salivary gland acinar cells. We have identified an extracellular pH-dependent anion current that is carried by NO 3 − or sialic acid (SA), but not by Br − , and is accompanied by intracellular acidification. Both responses were reduced by knockdown of sialin expression and increased by the plasma membrane-targeted sialin mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed reduced SA- and NO 3 − -induced currents compared with healthy controls. Furthermore, expression of disease-associated sialin mutants in fibroblasts and salivary gland cells suppressed the H + -dependent NO 3 − conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO 3 − secretion in saliva after intake of a NO 3 − -rich diet. Taken together, these data demonstrate that sialin mediates nitrate influx into salivary gland and other cell types. We suggest that the 2NO 3 − /H + transport function of sialin in salivary glands can contribute significantly to clearance of serum nitrate, as well as nitrate recycling and physiological nitrite-NO homeostasis.

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These results indicate that the inactivation of ECRG 4 gene by hypermethylation is a frequent molecular event in ESCC and may be involved in the carcinogenesis of this cancer.

Purpose: Inactivation of p16 gene by CpG methylation is a frequent event in oral epithelial dysplasia. To investigate the predictive value of p16 methylation on malignant potential in oral epithelial dysplasia, we carried out the prospective cohort study. Experimental Design: One hundred one patients with histologically confirmed mild or moderate oral epithelial dysplasia were included in the present cohort study. p16 Methylation status of the oral epithelial dysplasia lesions from 93 cases was obtained by methylation-specific PCR. Progression of the oral epithelial dysplasia lesions was examined in 78 cases histologically during a 45.8 months follow-up period. The association between p16 methylation and progression of oral epithelial dysplasia was analyzed.Results: Of the 93 enrolled cases, 15 cases were lost during the follow-up because of changes of contact information, with a compliance of 83.9%. p16 Methylation was detectable in oral epithelial dysplasia lesions from 32 (41.0%) of 78 enrolled patients. Oral epithelial dysplasia-related squamous cell carcinomas were observed in 22 patients (28.2%) during the follow-up. Rate of progression to oral cancer in patients with the p16-methylated oral epithelial dysplasia was significantly higher than that with the p16-unmethylated oral epithelial dysplasia (43.8% versus 17.4%; adjusted odds ratio, 3.7; P = 0.013), especially for patients at the baseline age of ≥60 years (adjusted odds ratio, 12.0; P = 0.003) and patients with moderate oral epithelial dysplasia (adjusted odds ratio, 15.6; P = 0.022). The overall sensitivity and specificity of prediction of malignant transformation of oral epithelial dysplasia by p16 methylation were 63.6% and 67.9%, respectively. Conclusion: p16 Methylation was correlated with malignant transformation of oral epithelial dysplasia and is a potential biomarker for prediction of prognosis of mild or moderate oral epithelial dysplasia. (Clin Cancer Res 2009;15(16):5178-83)

Purpose Metastasis is the leading cause of death for gastric carcinoma (GC). An epigenetic biomarker panel for predicting GC metastasis could have significant clinical impact on the care of GC patients. The main purpose of this study is to characterize the methylation differences between GCs with and without metastasis. Experimental Design Genome-wide DNA methylation profiles between 4 metastatic and 4 non-metastatic GCs and their surgical margins (SM) were analyzed using methylated-CpG island amplification with microarray. The methylation states of 73 candidate genes were further analyzed in GC patients in a discovery cohort (n=108) using DHPLC, bisulfite-sequencing, and MethyLight. The predictive values of potential metastasis-methylation biomarkers were validated in GC patient cohorts in China (n=330), Japan (n=129), and Korea (n=153). Results The GC genome showed significantly higher proportions of hypomethylation in the promoter and exon-1 regions, as well as increased hypermethylation of intragenic fragments when compared to SMs. Significant differential methylation was validated in the CGIs of 15 genes (Ps<0.05) and confirmed using bisulfite-sequencing. These genes included BMP3, BNIP3, CDKN2A, ECEL1, ELK1, GFRA1, HOXD10, KCNH1, PSMD10, PTPRT, SIGIRR, SRF, TBX5, TFPI2, and ZNF382. Methylation changes of GFRA1, SRF and ZNF382 resulted in up- or down-regulation of their transcription. Most importantly, the prevalence of GFRA1, SRF, and ZNF382 methylation alterations was consistently and coordinately associated with GC metastasis and the patients’ overall survival throughout discovery and validation cohorts in China, Japan and Korea. Conclusion Methylation changes of GFRA1, SRF, and ZNF382 may be a potential biomarker set for prediction of GC metastasis.