In the present investigation, we have shown for the first time that the onychomycosis-inducing dermatophyte Trichophyton rubrum was able to metabolize 5-aminolevulinic acid (ALA) to protoporphyrin IX (PpIX) in liquid culture medium. We have established and optimized the culture conditions and could show the typical PpIX-induced red fluorescence which was evaluated qualitatively by Wood's light examination and fluorescent microscopic analysis. The optimum concentration of ALA was in the range of 1-10 mmol l(-1). If used in higher concentrations, ALA leads to a significantly reduced growth rate and absence of PpIX formation due to highly acidic conditions. The first observation of red fluorescence was detected between 10 and 14 days poststimulation with ALA, increasing thereafter. Fluorescent microscopic examinations demonstrated that formation of PpIX was restricted to selected parts of the fungal mycelium. Repeated application of ALA in order to achieve the highest formation of PpIX in T. rubrum failed, probably due to the sustained low pH values. ALA treatment and irradiation of T. rubrum clearly demonstrated the growth-inhibiting effect of ALA PDT, either leading to reduced numbers of colonies or reduced diameters of single fungal colonies. Summarizing our results, ALA PDT might be a promising approach in the reduction of T. rubrum colonization in onychomycosis.
The phosphatidylcholine exchange protein from bovine liver stimulates the specific transfer of phosphatidylcholine (PC) from rat liver microsomes to mitochondria or phospholipid vesicles (Wirtz, K.W.A., Kamp, H.H., and van Deenen, L.L.M. (1972), Biochim. Biophys. Acta 274, 606). In the present study, it has been established which components of the PC molecule are essential to the specific interaction with the protein. Radiochemically labeled analogues of PC have been synthesized with modifications in the polar and apolar moiety, and their transfer was measured between donor and acceptor vesicles. Relative to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (egg yolk PC), transfer is inhibited or abolished when (a) the distance between phosphorus and nitrogen is decreased or increased and (b) a methyl group on the quaternary nitrogen is removed or substituted by an ethyl or propyl group. Transfer is much less affected when (a) the ester bonds are replaced by ether or carbon-carbon bonds, (b) the PC molecule contains two saturated fatty acids, and (c) the D stereoisomer is used. It is concluded that the protein has a binding site which interacts specifically with the phosphorylcholine head group and which cannot accommodate substantial configurational changes. Interaction with the apolar moiety of PC is less specific. However, lyso-PC is not transferred, suggesting that two hydrocarbon chains are required to stabilize the exchange protein-phospholipid complex. Interaction of [14C]PC-labeled exchange protein with vesicles of different phospholipid compositon has been analyzed by measuring the release of [14C]PC into these vesicles. Vesicles of egg PC or dimethylphosphatidylethanolamine function as acceptors, in contrast to vesicles of sphingomyelin or phosphatidylethanolamine.
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