Results: 4

1.
Figure 4

Figure 4. Loss of daf-36 impacts DAF-12 activity. From: The Rieske oxygenase DAF-36 functions as a cholesterol 7-desaturase in steroidogenic pathways governing longevity.

daf-36(k114) mutant animals have significantly lower transcript levels of two known targets of DAF-12, mir-84 and mir-241, relative to N2 wild-type (WT) animals, determined by quantitative RT-PCR of L3 stage worms. This reduction is similar to that seen in daf-12(rh61rh411) null mutants (N=3, mean ± SD, *P<0.05, **P<0.005, determined by unpaired t-test).

Joshua Wollam, et al. Aging Cell. ;10(5):879-884.
2.
Figure 2

Figure 2. daf-36 mutant animals are deficient in 7-dehydrocholesterol. From: The Rieske oxygenase DAF-36 functions as a cholesterol 7-desaturase in steroidogenic pathways governing longevity.

(A) LC/MS/MS analysis of lipid extracts from L3 stage animals reveals that 7-dehydrocholesterol levels are significantly reduced in daf-36(k114) mutant animals relative to N2 wild-type (WT) animals, shown quantitatively in (B) (N=4, mean ± SEM, **P<0.005, determined by paired t-test). (C) Analysis of daf-36 lipid extracts by GC/MS/MS shows that cholesterol levels are significantly elevated, whereas animals are deficient in (D) 7-dehydrocholesterol and (E) Δ7-DA (N=5, mean ± SEM, **P<0.005, determined by unpaired t-test, ***below limit of detection).

Joshua Wollam, et al. Aging Cell. ;10(5):879-884.
3.
Figure 3

Figure 3. DAF-36 produces 7-dehydrocholesterol. From: The Rieske oxygenase DAF-36 functions as a cholesterol 7-desaturase in steroidogenic pathways governing longevity.

(A) LC/MS/MS scans of lipid extracts from microsomes expressing either human CYP450 oxidoreductase (hOR) (Control) or DAF-36+hOR (DAF-36(+)), showing that production of 7-dehydrocholesterol is detected in the presence of DAF-36, whereas it is absent in control microsomes, shown quantitatively in (B) (N=3, mean ± SEM, **P<0.001, determined by unpaired t-test). (C) Supplementation of daf-36(k114) with lipid extracts from microsomes expressing human CYP450 oxidoreductase (hOR) (Control) or DAF-36+hOR (DAF-36 (+)) shows DAF-36 and 7-dehydrocholesterol dependent rescue of dauer formation at 27°C (N=2, mean ± range, **P<0.001, determined by Fisher’s exact test of grouped data). (D) DAF-36 acts as a cholesterol 7-desaturase, introducing a double bond at the 7 position, forming 7-dehydrocholesterol, shown highlighted in orange.

Joshua Wollam, et al. Aging Cell. ;10(5):879-884.
4.
Figure 1

Figure 1. Rescue experiments suggest DAF-36 produces 7-dehydrocholesterol. From: The Rieske oxygenase DAF-36 functions as a cholesterol 7-desaturase in steroidogenic pathways governing longevity.

(A) daf-36(k114) Daf-c phenotypes at 27°C are rescued with 7-dehydrocholesterol, the dafachronic acids (DAs) and all proposed precursors of the DAs, but not cholesterol. Partial rescue is seen with 4-cholesten-3-one. Experiments were carried out with a compound concentration of 33 µM and ethanol as the vehicle (N≥3, mean ± SD). (B) daf-9::gfp hypodermal expression is upregulated in daf-36(k114) mutants, suggesting loss of negative feedback on daf-9 expression. 7-dehydrocholesterol and downstream DA precursors fully rescue this upregulation. The fraction of animals with strong (green), weak (yellow), or no (red) hypodermal GFP expression is shown (N≥3, mean ± SD). (C) N2 wild-type (WT) and daf-36(k114) animals expressing daf-9::gfp. Arrowheads indicate the XXX R/L neuroendocrine cells in which daf-9 expression is relatively unchanged, and arrows indicate hypodermal expression.

Joshua Wollam, et al. Aging Cell. ;10(5):879-884.

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