We investigated the significance of endogenous reactive oxygen species (ROS) produced by fungi treated with miconazole. ROS production in Candida albicans was measured by a real-time fluorogenic assay. The level of ROS production was increased by miconazole at the MIC (0.125 g/ml) and was enhanced further in a dose-dependent manner, with a fourfold increase detected when miconazole was used at 12.5 g/ml. This increase in the level of ROS production was completely inhibited by pyrrolidinedithiocarbamate (PDTC), an antioxidant, at 10 M. In a colony formation assay, the decrease in cell viability associated with miconazole treatment was significantly prevented by addition of PDTC. Moreover, the level of ROS production by 10 clinical isolates of Candida species was inversely correlated with the miconazole MIC (r ‫؍‬ ؊0.8818; P < 0.01). These results indicate that ROS production is important to the antifungal activity of miconazole.Candidiasis is a life-threatening disease in patients with immune suppression. Azoles are used widely for the treatment of candidiasis, but some clinical isolates show resistance (11,28). Further investigation of the mechanisms underlying the antifungal effects of azoles may aid in the development of new treatment strategies.Azoles exert a cytostatic or cytotoxic effect via inhibition of synthesis reactions in the metabolic pathways of essential fungal cell membrane components including ergosterol (29,30). Their primary target is the cytochrome P450-catalyzed 14␣-demethylation of ergosterol precursors. Mutations or overexpression of 14␣-demethylase, encoded by the ERG11 gene, as well as changes in the ergosterol synthesis pathway reportedly participates in the induction of resistance (14,19,20,25).Since it was found that miconazole, ketoconazole, and deacetylated ketoconazole were inserted in a lipid layer (6) and miconazole induced the release of K ϩ and intracellular ATP from Candida species (3, 4, 7), these agents may cause direct membrane damage.Recent studies clarified that a decrease in drug concentration brought about by energy-dependent efflux pumps is usually involved in multidrug resistance including azole resistance (1,2,16,26). This pumping system closely resembles a system in cancer cells based on the P-glycoprotein encoded by the MDR-1 gene. Thus, common biologic mechanisms shared with other eukaryotic cells are involved in the antifungal effects of azoles.In eukaryotic cells, mitochondria are common organelles that represent an important source of reactive oxygen species (ROS). Many cellular stresses such as irradiation and cytotoxic drugs cause growth inhibition and the death of mammalian cells via endogenous ROS production (5, 15, 27). ROS produced by granulocytes or monocytes are known to exert activity against fungi (9, 31). Furthermore, recent studies demonstrated that Candida albicans possesses an ROS scavenger, superoxide dismutase; this suggests that fungi may require a cytoprotective mechanism against not only exogenous ROS but also endogenous, fungus-derived ROS (1...

Fas ligand (FasL) plays a pivotal role in lymphocyte cytotoxicity and the maintenance of immunological homeostasis. Since FasL has been implicated in the existence of immunologically privileged body sites by inducing apoptosis of activated T lymphocytes, we investigated the expression of FasL in human colon cancers. We found that two out of seven primary tumors and all four hepatic metastatic tumors of surgically obtained colonic adenocarcinoma expressed FasL mRNA and protein, detected by reverse transcription-coupled PCR and by immunohistochemical staining, respectively. Expression of FasL was not detected in normal colonic epithelial cells. FasL mRNA was also expressed in some human colonic adenocarcinoma cell lines including SW480, SW1116, and LS180 cells. Cell-surfaceassociated FasL was detected in these human colon cancer cells by f luorescence immunocytochemical staining. In addition, the expressed FasL was demonstrated to be functional, since coculture experiments using FasL-expressing SW480 cells resulted in apoptosis of Jurkat T leukemia cells that are sensitive to Fas-mediated apoptosis, and this process was specifically inhibited by the neutralizing anti-human FasL antibody. Thus, our findings and other data suggest an alternative mechanism that enables tumors to evade immune destruction by inducing apoptosis in activated T lymphocytes. Furthermore, constitutive expression of FasL in hepatic metastatic tumors suggests that FasL may also be important in their colonization in the liver through induction of apoptosis in the surrounding Fas-expressing hepatocytes.

A new antibiotic, trichostatin, was isolated from the metabolites of strains of Streptomyces hygroscopicus.It is active against trichophytons and some fungi. The structure was determined to be a derivative of a primary hydroxamic acid by chemical and spectroscopic evidences.

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