Abstract-Melanocytes and melanoma cells contain melanin, a complex polymer that modulates redox changes in these cells. Relative intracellular hydrogen peroxide levels measured by dichlorodihydrofluorescein are similar in the two cell types, but the levels of superoxide anion measured by dihydroethidium were markedly increased in melanoma cells. Chelator-induced oxidative stress is efficiently suppressed by melanocytes without substantial recruitment of the transcription factors NF-B and AP-1 as measured by electrophoretic mobility shift assay and quantitated by densitometry or by a change in frequency of apoptosis as determined by annexin V binding. In contrast, NF-B in melanoma cells is strongly recruited by changes in redox status and exhibits a correlative relationship to intracellular hydrogen peroxide (but not superoxide anion). However, the response of the NF-B pathway to intracellular hydrogen peroxide is anomalous, including downregulation of p65 and IB␣ RNA expression (Northern blot). Additionally, recruitment of AP-1 binding in melanoma cells was directly correlated with intracellular levels of superoxide anion (but not hydrogen peroxide). Neither the degree of NF-B nor AP-1 binding in melanoma cells was related to the frequency of apoptosis. The responsiveness of NF-B and AP-1 recruitment to intracellular levels of hydrogen peroxide and superoxide anion without concomitant control of apoptosis provides a general mechanism by which these cells can escape noxious injury (e.g., chemotherapy). The marked enhancement of apoptosis in melanoma cells by chelators indicates, however, that this alteration can be circumvented and offers a unique therapeutic window to explore.
Sex reversal can occur in XY humans with only a single functional WT1 or SF1 allele or a duplication of the chromosome region containing WNT4. In contrast, XY mice with only a single functional Wt1, Sf1, or Wnt4 allele, or mice that over-express Wnt4 from a transgene, reportedly are not sex-reversed. Because genetic background plays a critical role in testis differentiation, particularly in C57BL/6J (B6) mice, we tested the hypothesis that Wt1, Sf1, and Wnt4 are dosage sensitive in B6 XY mice. We found that reduced Wt1 or Sf1 dosage in B6 XYB6 mice impaired testis differentiation, but no ovarian tissue developed. If, however, a YAKR chromosome replaced the YB6 chromosome, these otherwise genetically identical B6 XY mice developed ovarian tissue. In contrast, reduced Wnt4 dosage increased the amount of testicular tissue present in Sf1+/− B6 XYAKR, Wt1+/− B6 XYAKR, B6 XYPOS, and B6 XYAKR fetuses. We propose that Wt1B6 and Sf1B6 are hypomorphic alleles of testis-determining pathway genes and that Wnt4B6 is a hypermorphic allele of an ovary-determining pathway gene. The latter hypothesis is supported by the finding that expression of Wnt4 and four other genes in the ovary-determining pathway are elevated in normal B6 XX E12.5 ovaries. We propose that B6 mice are sensitive to XY sex reversal, at least in part, because they carry Wt1B6 and/or Sf1B6 alleles that compromise testis differentiation and a Wnt4B6 allele that promotes ovary differentiation and thereby antagonizes testis differentiation. Addition of a “weak” Sry allele, such as the one on the YPOS chromosome, to the sensitized B6 background results in inappropriate development of ovarian tissue. We conclude that Wt1, Sf1, and Wnt4 are dosage-sensitive in mice, this dosage-sensitivity is genetic background-dependant, and the mouse strains described here are good models for the investigation of human dosage-sensitive XY sex reversal.
Mammalian gonad differentiation involves sexually dimorphic cell-fate decisions within the bipotential gonadal primordia. Testis differentiation is initiated by a center-to-poles wave of Sry expression that induces supporting cell precursors (SCPs) to become Sertoli rather than granulosa cells. The initiation of ovary differentiation is less well understood. We identified two novel SCP markers, 1700106J16Rik and Sprr2d, whose expression is ovary-biased during early gonad development, and altered in Wnt4, Sf1, Wt1 and Fog2 mutant gonads. In XX and XY gonads, both genes were upregulated at ~E11 in a center-to-poles wave, and then rapidly downregulated in XY gonads in a center-to-poles wave, which is reminiscent of Sry expression in XY gonads. Our data suggest that 1700106J16Rik and Sprr2d may have important roles in early gonad development, and are consistent with the hypothesis that ovarian SCP differentiation occurs in a center-to-poles wave with similar timing to that of testicular SCP differentiation.
In women with signs and symptoms of ischemia but no obstructive coronary artery disease, CMD and elevated LVEDP are prevalent. While non-endothelial dependent CMD may be related to diastolic dysfunction, further investigation is needed regarding links between CMD, diastolic dysfunction and the development of heart failure with preserved LVEF.
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