Detection of a pheomelanin-like pigment by EPR spectroscopy in the mycelium of Plenodomus biglobosus.

Melanin occurrence in Plenodomus biglobosus was investigated using electron paramagnetic (spin) resonance (EPR, ESR) spectroscopy. The fungus was isolated from living and dead leaves of European ash (Fraxinus excelsior L.). Dark pigmentation of P. biglobosus mycelium in vitro, especially on the reverse, was observed. The black coloration intensified with the age of the culture and inspired us to check if the analyzed fungus species synthesizes melanin. Melanin contains unpaired electrons, thus, EPR spectroscopy was applied, as a specific technique, to verify its presence in P. biglobosus. The EPR spectrum of the mycelium showed a very strong melanin signal, revealing pheomelanin-like features. Thus, the black pigment of P. biglobosus was clearly identified as melanin. However, no melanin was detected in the apparently dark culture medium even when zinc (II) acetate was added to increase the sensitivity of detection. Pheomelanin has many unusual biological functions but it is not commonly found in fungi. Detection of this type of melanin in P. biglobosus, which can be both endophytic or pathogenic, suggests a closer examination of the potential role of this melanin in host-parasite interaction.

Peroxisome proliferator-activated receptor α (PPARα) contributes to control of melanogenesis in B16 F10 melanoma cells.

Recent studies revealed the cooperation between peroxisome proliferator-activated receptor gamma (PPARγ) and α-MSH signaling, which results in enhanced melanogenesis in melanocytes and melanoma cells. However, the agonists of PPARα, such as fenofibrate, exert depigmenting effect. Therefore, we aimed to check how the PPARα expression level affects the antimelanogenic activity of fenofibrate and whether PPARα modulates melanogenesis independently of its agonist. To answer these questions, we used three B16 F10-derived cell lines, which varied in the PPARα expression level and were developed by stable transfection with plasmids driving shRNA-based PPARα silencing or overexpression of PPARα-emerald GFP fusion protein. Melanin contents were assessed with electron paramagnetic resonance spectroscopy along with color component image analysis-a novel approach to pigment content characteristics in melanoma cells. B16 F10 wt and Ctrl shRNA lines showed intermediate pigmentation, whereas the pigmentation of the B16 F10-derived cell lines was inversely correlated with the PPARα expression level. We observed that cells overexpressing PPARα were almost amelanotic and cells with reduced PPARα protein level were heavily melanized. Furthermore, fenofibrate down-regulated the melanogenic apparatus (MITF, tyrosinase, and tyrosinase-related proteins) in the cells with the regular PPARα expression level resulting in their visibly lower total melanin content in all the cell lines. From these observations, we conclude that fenofibrate works as a strong depigmenting agent, which acts independently of PPARα, but in an additive fashion. Our results also indicate that alterations in PGC-1a acetylation and expression level might contribute to the regulation of melanogenesis by PPARα and fenofibrate.