Dent

Dent. cross the fungal cell membrane and to accumulate inside the cells. The internalization of MUC7 20-mer was inhibited by divalent cations. Confocal microscopy of cells doubly labeled with MUC7 20-mer and a mitochondrion-specific dye indicated that mitochondria are not the target of MUC7 20-mer for either or (23, 36). Further studies showed that Hsn-5 is usually Diflorasone targeted to mitochondria and that its cytotoxic activity depends on the metabolic activity of (12). The killing of by Hsn-5 is usually accomplished by an increase in membrane potential and permeability and the subsequent release of intracellular ATP (15, 16). It was also shown that Hsn-5 and human neutrophil defensin 1 kill via a shared pathway (4). Our laboratory reported that MUC7 domain name 1 (D1), a 51-amino-acid-residue peptide (Table ?(Table1)1) derived from the N terminus of the low-molecular-weight human salivary mucin, MUC7 (comprised of 357 residues), possesses antifungal activity that is comparable to or exceeds the antifungal activity of Hsn-5 (26). It was shown that this peptide is effective against wild-type, azole-resistant, and amphotericin B-resistant and and against (29). It was implicated that this MUC7 D1 (MUC7 51-mer) net Diflorasone positive charge played a key role in its antifungal activity (26, 29). Another, much shorter peptide, MUC7 15-mer (amino acids 3 to 17 of MUC7) (Table ?(Table1),1), which was also derived from the MUC7 N terminus and Diflorasone which showed 53.3% sequence similarity to Hsn-5, was found to be at least sixfold less active against than MUC7 51-mer (8). Because seven out of eight positively charged amino acid residues present in the rest of the MUC7 D1 sequence are located within its C-terminal 20 residues, we investigated this MUC7 20-mer peptide (amino acids 32 to 51 of MUC7) (Table ?(Table1).1). Indeed, our initial studies showed that MUC7 20-mer displayed fungicidal activities comparable to or better than those of MUC7 51-mer against and (cariogenic bacteria) and (30). TABLE 1. Amino acid sequences and charges of peptides under study and since candidiasis and cryptococcosis, caused by these organisms, are the most common opportunistic infections in immunocompromised patients, especially patients with human immunodeficiency computer virus or AIDS. MUC7 20-mer, like Hsn-5, is usually a basic salivary antimicrobial peptide (pI, 10.58). Thus, it was of interest to determine whether MUC7 20-mer and Hsn-5 kill fungi by comparable mechanisms. We also examined and compared the dependence of 20-mer and Hsn-5 fungicidal activities around the metabolic state of the cells; the ability of the 20-mer to cross the plasma membrane and accumulate intracellularly; and the effects of heat, metabolic inhibitors, and divalent cations on 20-mer internalization. Lastly, we studied the possible intracellular target(s) of the 20-mer. MATERIALS AND METHODS Materials. (i) Peptides. Unlabeled MUC7 20-mer and fluorescein isothiocyanate (FITC)-labeled MUC7 20-mer were purchased from Bio-Synthesis Inc., (Lewisville, Tex.). High-pressure liquid Diflorasone chromatography and mass spectrometry were Kdr performed by Diflorasone the company to analyze the purity of the peptides. Recombinant Hsn-5 was produced in by using vector pET-30b(+). The cloning, expression, and purification of this peptide were done as previously described (34). Bovine insulin chain A (Ins-A) (21 amino acid residues) was purchased from Sigma Chemical Co. (St. Louis, Mo.). (ii) Other materials. Carbonyl cyanide strain DIS, a clinical isolate from a patient with denture-induced stomatitis, was provided by M. Edgerton (Department of Oral Biology, University at Buffalo), and an azole-resistant clinical isolate (no. 12-99) of was a gift from Theodore C. White (University of Washington and Seattle Biomedical Research Institute, Seattle, Wash.). Azole-sensitive was purchased from ATCC (ATCC 90030), and its azole-resistant counterpart, clinical isolate 65C, was obtained from John E. Bennett (National Institute of Allergy and Infectious Diseases, Bethesda, Md.). A clinical isolate of was obtained from the Erie County Medical Center, Buffalo, N.Y. Amphotericin B-sensitive (CN2) and amphotericin B-resistant (CN2843) strains were obtained from AIDS patients with cryptococcal meningitis and were generously provided by John H. Rex (University of Texas Medical School, Houston). Additionally, strain S288C was provided by D. Kosman, Department of Biochemistry, University at Buffalo. All were streaked and produced on SAB plates at 37C, except for (DIS)5.85 (4.17-8.67)6.68 (6.05-7.37)????(CN2)4.05 (3.16-5.81)3.71 (1.92-5.60)????(azole resistant)2.40 (1.73-3.08)6.40 (5.59-7.32)????(fluconazole resistant)12.3 (9.06-17.1)85.3 (78.4-94.0)????(amphotericin B resistant)4.29 (3.59-4.26)3.72 (2.90-4.87)Bacteria????(DIS) and.