Transcellular Mg2+ transport across epithelia, involving both apical entry and basolateral extrusion, is essential for magnesium homeostasis, but molecules involved in basolateral extrusion have not yet been identified. Here, we show that CNNM4 is the basolaterally located Mg2+ extrusion molecule. CNNM4 is strongly expressed in intestinal epithelia and localizes to their basolateral membrane. CNNM4-knockout mice showed hypomagnesemia due to the intestinal malabsorption of magnesium, suggesting its role in Mg2+ extrusion to the inner parts of body. Imaging analyses revealed that CNNM4 can extrude Mg2+ by exchanging intracellular Mg2+ with extracellular Na+. Furthermore, CNNM4 mutations cause Jalili syndrome, characterized by recessive amelogenesis imperfecta with cone-rod dystrophy. CNNM4-knockout mice showed defective amelogenesis, and CNNM4 again localizes to the basolateral membrane of ameloblasts, the enamel-forming epithelial cells. Missense point mutations associated with the disease abolish the Mg2+ extrusion activity. These results demonstrate the crucial importance of Mg2+ extrusion by CNNM4 in organismal and topical regulation of magnesium.

The extent of lymph node metastasis is a major determinant in the prognosis of oral squamous cell carcinoma (OSCC). Abnormalities of cell adhesion molecules are known to play an important role in invasion and metastasis of cancer cells through the loss of cell-to-cell adhesion. In this study, we isolated highly invasive clones from an OSCC cell line established from a lymph node metastasis by using an in vitro invasion assay method and compared the abnormalities of cell adhesion molecule E-cadherin and ␤-catenin in these cells. The isolated, highly invasive clones showed significant invasive capacity and reduction of E-cadherin and membranous ␤-catenin protein in comparison with parent cells. We found that reduced expression of E-cadherin was due to methylation of its promoter region. In fact, most invasive and metastatic area of OSCCs showed reduced expression and methylation of E-cadherin. Moreover, we found that reduced expression of membranous ␤-catenin was due to its protein degradation. Reduced expression of membranous ␤-catenin was also found frequently in invasive and metastatic areas of OSCCs. In summary, invasion and metastasis of OSCC cells require methylation of Ecadherin and/or degradation of membranous ␤-catenin. In addition, we suggest that the method of isolation of highly invasive clones may be useful for studies aimed at discovering novel genes involved in invasion and metastasis.

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Arthrospira (Spirulina) platensis (Nordstedt) Gomont was cultivated under light-limited conditions in 5-L open tanks by daily supplying NH 4 Cl as nitrogen source. Exponentially increasing feeding rates were adopted to prevent ammonia toxicity. The total feeding time (T) was varied between 12 and 20 days, and the starting (m 0 ) and total (m T ) quantities of the nitrogen source per unit reactor volume were varied in the ranges 0.19-1.7 mM and 2.3-23.1 mM, respectively. This intermittent addition of the nitrogen source prevented ammonia from reaching inhibitory levels and ensured final cell concentrations (X m ) and cell productivities (P x ) comparable with those of batch runs with KNO 3 . Moreover, the lower nitrogen addition due to the use of NH 4 Cl rather than KNO 3 allowed for higher nitrogen-to-cell conversions (Y x/n ). These results were evaluated using three-factor, five-level, central composite experimental planning, combined with the response surface methodology, selecting T, m 0 , and m T as the independent variables and X m , P x , and Y x/n as the response variables. This approach allowed us to identify, through the simultaneous optimization of the variables, T 5 16 days, m 0 5 1.7 mM, and m T 5 21.5 mM as the best conditions for A. platensis cultivation at 72 lmol photons . m À 2 . s À 1 . Under these conditions, a maximum cell concentration of 1239 mg . L À 1 was obtained, which is a value comparable with that obtained using KNO 3 as nitrogen source and nearly coincident with the theoretical one estimated by the response surface methodology.