Quantitative evaluation of intraspecific genetic diversity in a natural fish population using environmental DNA analysis.

Recent advances in environmental DNA (eDNA) analysis using high-throughput sequencing (HTS) enable evaluation of intraspecific genetic diversity in a population. As the intraspecific genetic diversity provides invaluable information for wildlife conservation and management, there is an increasing demand to apply eDNA analysis to population genetics and the phylogeography by quantitative evaluation of intraspecific diversity. However, quantitative evaluations of intraspecific genetic diversity using eDNA is not straightforward because the number of eDNA sequence reads obtained by HTS may not be an index of the quantity of eDNA. In this study, to quantitatively evaluate genetic diversity using eDNA analysis, we applied a quantitative eDNA metabarcoding method using the internal standard DNAs. We targeted Ayu (Plecoglossus altivelis altivelis) and added internal standard DNAs with known copy numbers to each eDNA sample obtained from three rivers during the library preparation process. The sequence reads of each Ayu haplotype were successfully converted to DNA copy numbers based on the relationship between the copy numbers and sequence reads of the internal standard DNAs. In all rivers, the calculated copy number of each haplotype showed a significant positive correlation with the haplotype frequency estimated by a capture-based survey. Furthermore, estimates of genetic indicators such as nucleotide diversity based on the eDNA copy numbers were comparable with those estimated based on a capture-based study. Our results demonstrate that eDNA analysis with internal standard DNAs enables reasonable quantification of intraspecific genetic diversity, and this method could thus be a promising tool in the field of population genetics and phylogeography.

Isotope analysis reveals proportional change and site-selection variation of river- and lake-produced eggs of a landlocked migratory fish.

Changes in the proportions of river- and lake-produced eggs of a landlocked amphidromous fish, ayu (Plecoglossus altivelis altivelis) in the Lake Biwa water system, Japan, were monitored by stable isotope analysis, based on different δ15 N and δ13 C values of prey organisms between the lake and its tributaries. During the 3 month reproduction season, the δ15 N values of spawned eggs decreased with time. This result implies that there was a shift from lake-produced eggs to river-produced eggs within a reproductive season, based on the observation that adult fish in the lake had previously been shown to have eggs with distinctly higher δ15 N values in their ovaries than those in the tributaries. This explanation was also supported by the change in δ13 C values of the spawned eggs. Furthermore, eggs with lower δ15 N and higher δ13 C values tended to be spawned at less variable depths, suggesting that females spawning river-produced eggs selected the spawning sites from a narrower range. We conclude that stable isotope ratios of spawned eggs can be indicators of the relative contributions of different food chains and can enable comparisons of reproductive characteristics between types of egg.

Hairs in old books isotopically reconstruct the eating habits of early modern Japan.

To complement literature-based historical knowledge of the eating habits of 17th- and 18th-century Japan, we analysed carbon and nitrogen isotope ratios (δ13C and δ15N, respectively) of human hairs embedded in cover paper of Japanese books printed during 1690s-1890s, taking regional and temporal variations into consideration. We purchased 24 book sets from second-hand book markets. Twenty-three sets contained enough human hairs, which were non-destructively extracted from the thick, recycled paper of the book covers and used to measure the δ13C and δ15N values, found to be identical within each book set. Relatively low δ13C values and high δ15N values suggested that people depended on rice, C3 vegetables, and fish, more exclusively than contemporary Japanese people. The relatively high δ13C values found in Edo (Tokyo) might be associated with the preference for C4 millets by Edo people as a measure against beriberi (locally recognised as the Edo affliction). The δ15N values gradually increased over 200 years, indicating an increase in the contribution of marine fish both as food and fertiliser for rice fields as suggested by literature-based studies. Further collection of hairs from books will enable a thorough examination of regional and temporal variations to better understand the pre-globalised food culture.

Magnesium deficiency suppresses cell cycle progression mediated by increase in transcriptional activity of p21(Cip1) and p27(Kip1) in renal epithelial NRK-52E cells.

Lack of magnesium suppresses cell growth, but the molecular mechanism is not examined in detail. We examined the effect of extracellular magnesium deficiency on cell cycle progression and the expression of cell cycle regulators in renal epithelial NRK-52E cells. In synchronized cells caused by serum-starved method, over 80% cells were distributed in G1 phase. Cell proliferation and percentage of the cells in S phase in the presence of MgCl(2) were higher than those in the absence of MgCl(2) , suggesting that magnesium is involved in the cell cycle progression from G1 to S phase. After serum addition, the expression levels of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The exogenous expression of p21(Cip1) or p27(Kip1) increased the percentage in G1 phase, whereas it decreased that in S phase. The mRNA levels and promoter activities of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The phosphorylated p53 (p-p53) level was decreased by MgCl(2) addition. Pifithrin-α, a p53 inhibitor, decreased the p-p53, p21(Cip1) and p27(Kip1) levels, and the percentage in G1 phase in the absence of MgCl(2) . Rotenone, a mitochondrial respiratory inhibitor, decreased ATP content and increased the p-p53 level in the presence of MgCl(2) . Together, lack of magnesium may increase p21(Cip1) and p27(Kip1) levels mediated by the decrease in ATP content and the activation of p53, resulting in the suppression of cell cycle progression from G1 to S phase in NRK-52E cells.

Decrease in transient receptor potential melastatin 6 mRNA stability caused by rapamycin in renal tubular epithelial cells.

Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), is used in treatments for transplantation and cancer. Rapamycin causes hypomagnesemia, although precisely how has not been examined. Here, we investigated the effect of rapamycin on the expression of transient receptor potential melastatin 6 (TRPM6), a Mg2+ channel. Rapamycin and LY-294002, an inhibitor of phosphatidilinositol-3 kinase (PI3K) located upstream of mTOR, inhibited epidermal growth factor (EGF)-induced expression of the TRPM6 protein without affecting TRPM7 expression in rat renal NRK-52E epithelial cells. Both rapamycin and LY-294002 decreased EGF-induced Mg2+ influx. U0126, a MEK inhibitor, inhibited EGF-induced increases in c-Fos, p-ERK, and TRPM6 levels. In contrast, neither rapamycin nor LY-294002 inhibited EGF-induced increases in p-ERK and c-Fos levels. EGF increased p-Akt level, an effect inhibited by LY-294002 and 1L-6-hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (Akt inhibitor). Akt inhibitor decreased TRPM6 level similar to rapamycin and LY-294002. These results suggest that a PI3K/Akt/mTOR pathway is involved in the regulation of TRPM6 expression. Rapamycin inhibited the EGF-induced increase in TRPM6 mRNA but did not inhibit human TRPM6 promoter activity. In the presence of actinomycin D, a transcriptional inhibitor, rapamycin accelerated the decrease in TRPM6 mRNA. Rapamycin decreased the expression and activity of a luciferase linked with the 3'-untranslated region of human TRPM6 mRNA. These results suggest that TRPM6 expression is up-regulated by a PI3K/Akt/mTOR pathway and rapamycin reduces TRPM6 mRNA stability, resulting in a decrease in the reabsorption of Mg2+.

Up-regulation of TRPM6 transcriptional activity by AP-1 in renal epithelial cells.

Transient receptor potential melastatin 6 (TRPM6) channel is involved in the reabsorption of magnesium in the kidney. We recently found that TRPM6 expression is up-regulated by EGF, but the regulatory mechanism has not been clear. TRPM6 mRNA was endogenously expressed in HEK293 cells. TRPM6 mRNA expression was increased by EGF, which was inhibited by U0126, an MEK inhibitor. Promoter activity of human TRPM6 was observed in the TRPM6 5'-flanking region from -1,214 to -718. This promoter activity was enhanced by EGF and inhibited by U0126. Three putative AP-1 binding sites were identified within the region of -1,214/-718. The mutation of the putative AP-1 binding site (-741/-736) completely inhibited the EGF-induced promoter activity. EGF increased p-ERK1/2, c-Fos, c-Jun, and p-c-Jun levels, which were inhibited by U0126. The introduction of c-Fos or c-Jun siRNA inhibited the EGF-induced promoter activity. A chromatin immunoprecipitation assay revealed that c-Fos and c-Jun bind to the AP-1 binding site within the region of -1,214/-718. These results suggest that EGF up-regulates TRPM6 mRNA expression mediate via the activation of ERK/AP-1-dependent pathway.

TRPM6 expression and cell proliferation are up-regulated by phosphorylation of ERK1/2 in renal epithelial cells.

Transient receptor potential melastatin 6 (TRPM6) is a magnesium channel and expressed in the intestine and renal distal tubules. Little is known about the regulatory mechanism of TRPM6 expression and the role of magnesium influx. EGF increased the phosphorylation of ERK1/2 and TRPM6 expression that were inhibited by U0126 in renal epithelial NRK-52E cells. Furthermore, EGF enhanced the influx of magnesium, whereas U0126 and TRPM6 siRNA inhibited it. EGF increased the proportion of cells in S phase, whereas U0126 and TRPM6 siRNA increased the proportion in G1 phase. The phosphorylation of ERK1/2 may up-regulate TRPM6 expression and magnesium influx, resulting in an increase in cell proliferation with a shift from G1 to S phase.

Claudin-16 is directly phosphorylated by protein kinase A independently of a vasodilator-stimulated phosphoprotein-mediated pathway.

Claudin-16 (CLDN-16) is involved in the paracellular reabsorption of Mg(2+) in the thick ascending limb of Henle. The tight junctional localization and Mg(2+) transport of CLDN-16 are regulated by cAMP/PKA-dependent phosphorylation. Here, we examined whether PKA phosphorylates CLDN-16 in a direct or indirect manner. CLDN-16 was stably expressed in Madin-Darby canine kidney (MDCK) cells using a Tet-OFF system. The phosphorylation of CLDN-16 is upregulated by fetal calf serum (FCS). This phosphorylation was completely inhibited by a PKA inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride. Without FCS, dibutyryl cAMP (DBcAMP) increased the phosphoserine level of CLDN-16 in a concentration-dependent manner. The phosphorylated CLDN-16 elicited increases of transepithelial electrical resistance (TER) and transepithelial transport of Mg(2+). Vasodilator-stimulated phosphoprotein (VASP) was also phosphorylated in the presence of FCS or DBcAMP. In the glutathione-S-transferase (GST) pull down assay, a cytosolic carboxyl domain of CLDN-16 was associated with PKA, but not with VASP. Furthermore, PKA was immunoprecipitated with CLDN-16 in MDCK cells, but VASP was not. In cells expressing a dephosphorylated mutant (Ser160Ala) of VASP, CLDN-16 was phosphorylated by DBcAMP and was associated with ZO-1, a tight junctional-scaffolding protein, without integral cell-cell junctions. We suggest that PKA directly phosphorylates CLDN-16, resulting in the localization to tight junctions (TJs) and the maintenance of Mg(2+) reabsorption.

Activation of a polyvalent cation-sensing receptor decreases magnesium transport via claudin-16.

Renal magnesium is mainly reabsorbed by a paracellular pathway in the thick ascending limb of Henle. The expression of claudin-16 increased magnesium transport in Madin-Darby canine kidney (MDCK) cells. Little is known about the regulatory mechanism of magnesium transport via claudin-16. Here we examined the effect of a polyvalent cation-sensing receptor (CaSR) on the intracellular distribution of and transport of magnesium by claudin-16. FLAG-tagged claudin-16 was stably expressed in MDCK cells using a Tet-OFF system. The activation of CaSR by magnesium, calcium, neomycin, and gadolinium did not affect the expression of FLAG-tagged claudin-16, CaSR, or ZO-1, a tight junctional scaffolding protein. These activators decreased the phosphoserine level of FLAG-tagged claudin-16 and the association of FLAG-tagged claudin-16 with ZO-1. The activation of CaSR induced a decrease in PKA activity. Immunofluorescence microscopy revealed that FLAG-tagged claudin-16 is distributed at the cell-cell border under unstimulated conditions, whereas it translocates to the intracellular compartment, mainly lysosome, with the activation of CaSR. In contrast, the distribution of ZO-1 was unaffected by the activation. The expression of FLAG-tagged claudin-16 increased transepithelial electrical resistance (TER) and transepithelial magnesium transport without affecting FITC-dextran (MW 4000) flux. The activation of CaSR decreased TER and magnesium transport, which were recovered by co-treatment with dibutyryl cAMP, a membrane-permeable cAMP analogue. Taken together, CaSR activation may decrease PKA activity, resulting in a decrease in phosphorylated claudin-16, the translocation of claudin-16 to lysosome and a decrease in magnesium reabsorption.

Down-regulation of TRPM6-mediated magnesium influx by cyclosporin A.

Transient receptor potential melastatin 6 (TRPM6) is distributed along the apical membrane of the renal tubular cells and is involved in the reabsorption of magnesium. In this study, we show that TRPM6 expression is suppressed by cyclosporin A (CsA) via a down-regulation of c-Fos expression. TRPM6 was expressed in NRK-52E, but not in Madin-Darby canine kidney cells. In contrast, its homolog, TRPM7, was equally expressed in both cells. In NRK-52E cells, CsA dose-dependently decreased TRPM6 expression without affecting TRPM7 expression. Magnesium load measurements revealed the rise in the intracellular free magnesium concentration ([Mg2+]i) to be inhibited by CsA. The transfection of TRPM6 siRNA decreased TRPM6 expression without affecting TRPM7 expression and inhibited the elevation of [Mg2+]i. CsA did not affect the intracellular distribution of nuclear factor of activated T cells (NFATc). Furthermore, TRPM6 expression was not changed by a NFATc inhibitor. Next, we examined the effect of CsA on the transcription factors c-Fos and c-Jun. CsA decreased c-Fos expression without affecting c-Jun expression. The transfection of c-Fos siRNA suppressed TRPM6 expression without affecting TRPM7 expression. We suggest that CsA decreases TRPM6 expression mediated by inhibition of c-Fos transcription, resulting in a decrease of renal Mg2+ reabsorption.