Compound Heterozygous Mutations in SLC30A2/ZnT2 Results in Low Milk Zinc Concentrations: A Novel Mechanism for Zinc Deficiency in a Breast-Fed Infant.

Zinc concentrations in breast milk are considerably higher than those of the maternal serum, to meet the infant's requirements for normal growth and development. Thus, effective mechanisms ensuring secretion of large amounts of zinc into the milk operate in mammary epithelial cells during lactation. ZnT2 was recently found to play an essential role in the secretion of zinc into milk. Heterozygous mutations of human ZnT2 (hZnT2), including H54R and G87R, in mothers result in low (>75% reduction) secretion of zinc into the breast milk, and infants fed on the milk develop transient neonatal zinc deficiency. We identified two novel missense mutations in the SLC30A2/ZnT2 gene in a Japanese mother with low milk zinc concentrations (>90% reduction) whose infant developed severe zinc deficiency; a T to C transition (c.454T>C) at exon 4, which substitutes a tryptophan residue with an arginine residue (W152R), and a C to T transition (c.887C>T) at exon 7, which substitutes a serine residue with a leucine residue (S296L). Biochemical characterization using zinc-sensitive DT40 cells indicated that the W152R mutation abolished the abilities to transport zinc and to form a dimer complex, indicating a loss-of-function mutation. The S296L mutation retained both abilities but was extremely destabilized. The two mutations were found on different alleles, indicating that the genotype of the mother with low milk zinc was compound heterozygous. These results show novel compound heterozygous mutations in the SLC30A2/ZnT2 gene causing zinc deficiency in a breast-fed infant

Suppression of the Fenton Reaction by Ceriporic Acids Produced by a Selective Lignin-Degrading Fungus, Ceriporiopsis subvermispora

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Arginine-Rich Cell-Penetrating Peptide-Mediated Transduction of Mouse Nasal Cells with FOXP3 Protein Alleviates Allergic Rhinitis

Intranasal corticosteroids are effective medications against allergic rhinitis (AR). However, mucociliary clearance promptly eliminates these drugs from the nasal cavity and delays their onset of action. Therefore, a faster, longer-lasting therapeutic effect on the nasal mucosa is required to enhance the efficacy of AR management. Our previous study showed that polyarginine, a cell-penetrating peptide, can deliver cargo to nasal cells; moreover, polyarginine-mediated cell-nonspecific protein transduction into the nasal epithelium exhibited high transfection efficiency with minimal cytotoxicity. In this study, poly-arginine-fused forkhead box P3 (FOXP3) protein, the “master transcriptional regulator” of regulatory T cells (Tregs), was administered into the bilateral nasal cavities of the ovalbumin (OVA)-immunoglobulin E mouse model of AR. The effects of these proteins on AR following OVA administration were investigated using histopathological, nasal symptom, flow cytometry, and cytokine dot blot analyses. Polyarginine-mediated FOXP3 protein transduction induced Treg-like cell generation in the nasal epithelium and allergen tolerance. Overall, this study proposes FOXP3 activation-mediated Treg induction as a novel and potential therapeutic strategy for AR, providing a potential alternative to conventional intranasal drug application for nasal drug delivery

Identification of two missense mutations in the <i>SLC30A2/ZnT2</i> gene in the mother of a zinc-deficient infant.

<p>(<b>A</b>) Photograph of an affected infant with severe zinc deficiency. The dermatitis was erythematous and erosive, particularly around the infant's mouth. (<b>B</b>) Predicted topology of hZnT2 indicating the positions of the W152R and S296L substitutions found in this study. (<b>C</b>) Electropherograms showing SLC30A2/<i>ZnT2</i> mutations in the affected mother. W152R and S296L mutations were found at exons 4 and 7 on different alleles.</p

S296L mutation causes hZnT2 destabilized.

<p>(<b>A</b>) The expression level of the hZnT2 protein at each time point. The <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells expressing WT hZnT2 or S296L mutant were treated with CHX and collected periodically over 4 h. Immunoblot analysis was performed to monitor hZnT2 levels (<i>left</i> panel). The band intensities of hZnT2 protein (○, WT; •, S296L mutant) are shown as the percentage of the intensity at 0 h (T₀) after normalized by that of tubulin at each time (<i>right</i> panel). * and ** denote a significant difference between expression levels of the WT and S296L mutant hZnT2 at each time point (* P<0.05, ** P<0.01) (<b>B</b>) Lysosome inhibitor bafilomycin A1 and proteasome inhibitor MG132 block the degradation of S296L hZnT2 mutant. Immunoblot analysis (<i>left</i> panel) and the band intensities of hZnT2 protein (○, MG132; Δ, bafilomycin A1; • no inhibitor, <i>right</i> panel) are shown. In the right panels of both (<b>A</b>) and (<b>B</b>), each value is the mean ± SD of triplicate experiments. The same membrane was used for detection of both hZnT2 and tubulin. Tubulin is shown as a loading control. * and ** denote significant differences between expression levels in the absence and presence of bafilomycin A1 or MG132 at each time point (* P<0.05, ** P<0.01).</p

No mutations were found in and around the promoter region of the <i>SLC30A2/ZnT2</i> gene.

<p>Alignment of the sequences of the mother with low milk zinc (“mother”) and the human genomic sequence deposited in the GenBank database (“human”). To show high homology in this region among mammals, where the MRE is completely conserved, the sequences are also aligned with those of rats and mice deposited in the GenBank database (corresponding regions from −110 to +22 of mouse <i>Znt2</i> are shown. The transcription start site is indicated by gray shading). Identical nucleotides are indicated by * and the MRE sequence is indicated with bold letters.</p

W152R hZnT2 mutant is not dominant negative because it fails to form functional dimers.

<p>Tagged-hZnT2 WT or mutants were immunoprecipitated <i>(IP</i>) with antibodies against either the FLAG or HA epitopes. The immunoprecipitates were analyzed by immunoblotting using antibodies against the FLAG or HA tags. To estimate the amount of tagged hZnT2 WT and mutant proteins, 10% of each aliquot was subjected to immunoblot analysis (<i>input</i> panels). The IP experiments were performed four times, which gave the same results. The panels show the representative results.</p

Evaluation of zinc transport activities of hZnT2 proteins using <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells.

<p>Viability of the cells exposed to the indicated concentrations of ZnSO₄ for 72 h was determined by counting the number of viable cells. Relative values presented are evaluations of the averages of three independent experiments. All of hZnT2 mutants were HA-tagged. +++: growing to confluence; ++, +: less growth (20–50% or up to 20% relative to +++); −: not growing.</p

W152R hZnT2 loses the ability to transport zinc, but the S296L hZnT2 does not.

<p><b>(A)</b> The carboxyl-terminal epitope tags do not interfere with hZnT2 expression. Untagged and HA- or FLAG-tagged hZnT2 were stably expressed in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells. Immunoblotting was performed using anti-hZnT2 antibody. (<b>B</b>) Confirmation of stable expression of the WT hZnT2-HA, W152R, S296L and other mutants of hZnT2-HA in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells. Immunoblotting was performed using an anti-HA antibody. In both (<b>A</b>) and (<b>B</b>), 20 µg of total cellular protein was loaded onto each lane, and the same membrane was used for detection of both hZnT2 and tubulin. Tubulin is shown as a loading control. (<b>C</b>) Effects of zinc on MT-luciferase reporter gene expression in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells stably expressing WT hZnT2-HA, W152R or S296L mutant hZnT2-HA. Relative activity of Luc is shown (the luciferase activity of <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells cultured without ZnSO₄ is defined as 1). Each value is the mean ± SD of triplicate experiments. * denotes a significant difference of relative activity of Luc between the cells expressing WT and W152R mutant hZnT2 (P<0.05).</p