Enhancing the fungicidal activity of amphotericin B via vacuole disruption by benzyl isothiocyanate,a cruciferous plant constituent

Amphotericin B (AmB), a typical polyene macrolide antifungal agent, is widely used to treat systemic mycoses. In the present study, we show that the fungicidal activity of AmB was enhanced by benzyl isothiocyanate (BITC), a cruciferous plant-derived compound, in the budding yeast, Saccharomyces cerevisiae. In addition to forming a molecular complex with ergosterol present in fungal cell membranes to form K⁺-permeable ion channels, AmB has been recognized to mediate vacuolar membrane disruption resulting in lethal effects. BITC showed no effect on AmB-induced plasma membrane permeability; however, it amplified AmB-induced vacuolar membrane disruption in S. cerevisiae. Furthermore, the BITC-enhanced fungicidal effects of AmB significantly decreased cell viability due to the disruption of vacuoles in the pathogenic fungus Candida albicans. The application of the combinatorial antifungal effect of AmB and BITC may aid in dose reduction of AmB in clinical antifungal therapy and consequently decrease side effects in patients. These results also have significant implications for the development of vacuole-targeting chemotherapy against fungal infections.     

A PNA-DNA hybridization chip approach for the detection of beta-secretase activity

Developed was the addressable chip technology based on the PNA-DNA complementary hybridization equipped with short seven-mer PNA-encoded peptides that can be a versatile scaffold to monitor on-chip immunoassays. We also developed and validated a methodology to perform beta-secretase enzyme assay with a highly sensitive fashion, resulting that a peptide substrate tethering dual fluorescent probes allowed us to detect beta-secretase activity 10 times more sensitively than assays in solution.     

Electron transfer complex formation between oxygenase and ferredoxin components in Rieske nonheme iron oxygenase system

Carbazole 1,9a-dioxygenase (CARDO), a member of the Rieske nonheme iron oxygenase system (ROS), consists of a terminal oxygenase (CARDO-O) and electron transfer components (ferredoxin [CARDO-F] and ferredoxin reductase [CARDO-R]). We determined the crystal structures of the nonreduced, reduced, and substrate-bound binary complexes of CARDO-O with its electron donor, CARDO-F, at 1.9, 1.8, and 2.0 A resolutions, respectively. These structures provide the first structure-based interpretation of intercomponent electron transfer between two Rieske [2Fe-2S] clusters of ferredoxin and oxygenase in ROS. Three molecules of CARDO-F bind to the subunit boundary of one CARDO-O trimeric molecule, and specific binding created by electrostatic and hydrophobic interactions with conformational changes suitably aligns the two Rieske clusters for electron transfer. Additionally, conformational changes upon binding carbazole resulted in the closure of a lid over the substrate-binding pocket, thereby seemingly trapping carbazole at the substrate-binding site.     

Two-dimensional gel analysis of zymogen-activating factors in the small intestine of the mouse

Zymogen-activating factors in the mouse were investigated by two-dimensional electrophoresis. Mouse pancreatic zymogens--trypsinogen-I group (Try G-I group), trypsinogen-II (Try G-II), and chymotrypsinogen (Chy G)--were purified using DEAE-cellulose column chromatography. Analysis by two-dimensional electrophoresis, using the purified zymogens as substrates, revealed enterokinase isozymes and chymotrypsinogen-activating factors in both the intestinal extract and luminal fluid. Mouse enterokinase was separated into at least two bands in the first-dimensional gel, each able to activate both trypsinogens Try G-I group and Try G-II. Chymotrypsinogen-activating factors were separated into several bands in the first-dimensional gel. Some activating factors showed mobilities similar to those of mouse enterokinase isozymes. Moreover, other activating factors that can activate chymotrypsinogen were present only in the more anodal area of the first-dimensional gel. These findings indicate that at least two enterokinases and several chymotrypsinogen-activating factors play an important role in the process of activating digestive enzymes.     

Synthesis and structure-activity relationships of 2-amino-8-hydroxyadenines as orally active interferon inducing agents

Recently, we have reported the 8-hydroxyadenine derivatives (2–4) as a novel class of interferon (IFN) inducing agents. In the present study, a series of 8-hydroxyadenines, which possess various amino moieties at the adenine C(2)-position, were synthesized and evaluated for their ability to induce endogenous IFN in comparison to the known active agent, Imiquimod. Among the compounds prepared, compound 9o possessing a 2-methoxyethylamino group at C(2)-position of adenine was found to exhibit potent IFN inducing activity in vivo. Compound 9o induced IFN from the dosage of 0.1 mg/kg, which was 30-fold potent than that of Imiquimod, and showed a good oral bioavailability (F=81%).     

Calpain inhibitors stimulate phagocyte functions via activation of human formyl peptide receptors

Calpain inhibitors induce pertussis toxin (PTx)-sensitive chemotaxis in human neutrophils and monocytes. Here, we show that various calpain inhibitors (PD150606, PD151746, N-acetyl-Leu-Leu-Nle-CHO [ALLN], N-acetyl-Leu-Leu-Met-CHO [ALLM], and calpeptin) and γ-secretase inhibitor I induced PTx-sensitive increase in cytoplasmic free Ca²⁺ ([Ca²⁺]_i) in human neutrophils and neutrophil migration. HEK-293 cells stably expressing human formyl peptide receptor (hFPR) or hFPR-like 1 (hFPRL1) displayed stimulus-specific increase in [Ca²⁺]_i in response to calpain inhibitors (PD150606, PD151746, ALLN, ALLM, MG-132, and calpeptin), γ-secretase inhibitor I, and N-formyl-Met-Leu-Phe. Parent HEK-293 cells also displayed PTx-sensitive increase in [Ca²⁺]_i in response to calpeptin and γ-secretase inhibitor I, whereas they displayed PTx-resistant increase in [Ca²⁺]_i in response to MG-132. MDL-28170 induced neither an increase in [Ca²⁺]_i in neutrophils and HEK-293 cells nor neutrophil migration. Ionomycin-induced cleavage of talin (a substrate of calpain) in neutrophils was inhibited by all inhibitors used here. These findings suggest that potent calpain inhibitors could stimulate phagocyte functions via activation of hFPR, hFPRL1 and/or other G-protein coupled receptors depending on the inhibitors used.     

Cell fingerprint patterns using designed α-helical peptides to screen for cell-specific toxicity

We conducted cell-based cytotoxicity screening of a 101-membered α-helical peptide library using cell fingerprints (CFPs). The CFP data suggested that there is a relationship between cytotoxicity and peptide characteristics, such as hydrophobicity, charge, and amino acid composition. In spite of the small size of the library used in this study, several peptides demonstrated cell-specific toxicity. The strategy of combining a designed peptide library with CFP thus shows real promise for peptide-based screening with cells.     

Nectins and nectin-like molecules: roles in contact inhibition of cell movement and proliferation

Nectins and nectin-like molecules (Necls) are immunoglobulin-like transmembrane cell adhesion molecules that are expressed in various cell types. Homophilic and heterophilic engagements between family members provide cells with molecular tools for intercellular communications. Nectins primarily regulate cell-cell adhesions, whereas Necls are involved in a greater variety of cellular functions. Recent studies have revealed that nectins and NECL-5, in cooperation with integrin alphavbeta3 and platelet-derived growth factor receptor, are crucial for the mechanisms that underlie contact inhibition of cell movement and proliferation; this has important implications for the development and tissue regeneration of multicellular organisms and the phenotypes of cancer cells.