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.

Enhancement of paraben-fungicidal activity by sulforaphane, a cruciferous vegetable-derived isothiocyanate, via membrane structural damage in Saccharomyces cerevisiae.

Parabens have been widely used as antimicrobial preservatives in cosmetics, pharmaceuticals, foods and beverages. Commonly, methyl-, ethyl-, propyl- and butylparaben are used independently or in combination to maintain the quality of industrial products, and they are considered to have low toxicity. However, recent evidence has suggested that parabens are toxic in mammalian cells, and parabens have been associated with allergic-contact dermatitis, breast cancer and changes in testosterone levels. Sulforaphane, a cruciferous vegetable-derived isothiocyanate, was effective in decreasing the growth inhibitory concentrations of ethyl-, propyl-, butyl- and methylparaben in the yeast Saccharomyces cerevisiae. The sulforaphane-enhanced fungicidal effects of methylparaben were deemed to be caused by drastic cell membrane damage and the leakage of internal substances, such as nucleotides, from S. cerevisiae cells. Moreover sulforaphane markedly decreased the minimum concentration of methyl- and ethylparaben required to inhibit the growth of various microbes, such as the pathogenic yeast that causes severe mycosis, Candida albicans; the filamentous fungi Aspergillus niger; and the Gram-negative bacterium Escherichia coli. Enhanced antimicrobial activity from the beneficial components of edible plants may increase paraben efficacy at low concentrations and minimize preservative-induced side effects in consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: Sulforaphane, a natural and beneficial cruciferous vegetable-derived isothiocyanate, increased the ability of parabens to disrupt fungal cell membranes. Paraben-containing products have been reported to cause allergic contact dermatitis and drug hypersensitivity; therefore, methods to preserve organic products that may reduce the concentrations of parabens are both timely and necessary. In this study, we found that the combined antimicrobial effects of sulforaphane and parabens had the potential to reduce the paraben concentration needed to preserve organic products, thereby indicating that paraben toxicity may be reduced without affecting its activity as a preservative.

Curcumin potentiates the fungicidal effect of dodecanol by inhibiting drug efflux in wild-type budding yeast.

Drug resistance commonly occurs when treating immunocompromised patients who have fungal infections. Curcumin, is a compound isolated from Curcuma longa, has been reported to inhibit drug efflux in several human cell lines and nonpathogenic budding yeast Saccharomyces cerevisiae cells that overexpresses the ATP-binding cassette (ABC) transporters S. cerevisiae Pdr5p and pathogenic Candida albicans Cdr1p and Cdr2p. The aim of this study was to examine the effects of curcumin on multidrug resistance in a wild-type strain of the budding yeast with an intrinsic expression system of multidrug efflux-related genes. The antifungal activity of dodecanol alone was temporary against S. cerevisiae; however, restoration of cell viability was completely inhibited when the cells were co-treated with dodecanol and curcumin. Furthermore, restriction of rhodamine 6G (R6G) efflux from the cells and intracellular accumulation of R6G were observed with curcumin treatment. Reverse transcription-polymerase chain reaction analysis revealed that curcumin reduced the dodecanol-induced overexpression of the ABC transporter-related genes PDR1, PDR3 and PDR5 to their control levels in untreated cells. Curcumin can directly restrict the glucose-induced drug efflux and inhibits the expression of the ABC transporter gene PDR5, and can thereby inhibit the efflux of dodecanol from S. cerevisiae cells. Curcumin is effective in potentiating the efficacy of antifungal drugs via its effects on ABC transporters. SIGNIFICANCE AND IMPACT OF THE STUDY: Drug resistance is common in immunocompromised patients with fungal infections. Curcumin, isolated from Curcuma longa, inhibits drug efflux in nonpathogenic budding yeast Saccharomyces cerevisiae cells overexpressing ABC transporters S. cerevisiae Pdr5p and pathogenic Candida albicans Cdr1p and Cdr2p. We examined the effects of curcumin on multidrug resistance in a wild-type strain of the budding yeast with an intrinsic expression system of multidrug efflux-related genes. Curcumin directly inhibited drug efflux and also suppressed the PDR5 expression, thereby enhancing the antifungal effects. Thus, curcumin potentially promotes the efficacy of antifungals via its effects on ABC transporters in wild-type fungal strains.

Spasmolytic effect of peppermint oil in barium during double-contrast barium enema compared with Buscopan.

AIM: To evaluate the efficacy of peppermint oil in barium as a spasmolytic agent during a double-contrast barium enema (DCBE). MATERIALS AND METHODS: A total of 383 DCBEs with positive results from occult blood tests were assessed. Patients were assigned to one of four groups: peppermint in barium (n=91), peppermint in tube (n=90), Buscopan (n=105), or no treatment (n=97). After a screening sigmoidoscopy, the DCBEs were performed using air as a distending gas. In the Buscopan group, the DCBE was performed with an intramuscular injection of 20mg Buscopan at the start of the examination. Patients in the no-treatment group underwent DCBE without any spasmolytic agent. A peppermint oil preparation (30ml) was mixed in the barium solution for patients in the peppermint-in-barium group, and the same dose of peppermint oil was included in the enema tube in the peppermint-in-tube group. The presence of spasm on a series of spot films was evaluated without information about the type of spasmolytic agent used. RESULTS: The percentage of patients in the four groups (no treatment, Buscopan, peppermint in tube, and peppermint in barium) with absence of spasm in the entire colon on the series of spot films was 13.4, 38.1, 41.8, and 37.8%, respectively. In the group using peppermint oil or Buscopan, the rate of patients with non-spasm examination was higher than that in no-treatment group (p<0.0005). Peppermint oil had the same spasmolytic effect as the systemic administration of Buscopan in the transverse and descending colon. Peppermint oil had a stronger effect in the caecum and the ascending colon than a Buscopan injection (p<0.005). There was no advantage to placing peppermint oil in the enema tube over mixing it in the barium solution. A total of 157 polyps were found during the DCBE procedures, and no differences were observed in the number of lesions among the four groups. Peppermint oil did not impair image quality. CONCLUSION: Barium solution mixed with peppermint oil was safe and effective for the elimination of colonic spasm during the DCBE procedure, and it could be used instead of Buscopan.

Studies on the interactions between drugs and estrogen: analytical method for prediction system of gynecomastia induced by drugs on the inhibitory metabolism of estradiol using Escherichia coli coexpressing human CYP3A4 with human NADPH-cytochrome P450 reductase.

To establish a prediction system for drug-induced gynecomastia in clinical fields, a model reaction system was developed to explain numerically this side effect. The principle is based on the assumption that 50% inhibition concentration (IC(50)) of drugs on the in vitro metabolism of estradiol (E2) to its major product 2-hydroxyestradiol (2-OH-E2) can be regarded as the index for achieving this purpose. By using human cytochrome P450s coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli as the enzyme, the reaction was examined. Among the nine enzymes (CYP1A1, 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) tested, CYP3A4 having a V(max)/K(m) (ml/min/nmol P450) value of 0.32 for production of 2-OH-E2 was shown to be the most suitable enzyme as the reagent. The inhibitory effects of ketoconazole, cyclosporin A, and cimetidine toward the 2-hydroxylation of E2 catalyzed by CYP3A4 were obtained, and their IC(50) values were 7 nM, 64 nM, and 290 microM, respectively. The present results suggest that IC(50) values thus obtained can be substituted as the prediction index for gynecomastia induced by drugs, considering the patients' individual information.

The mode of action of AKD-2C, an antifungal antibiotic from Streptomyces sp. OCU-42815.

The mechanism of the antifungal action of AKD-2C was studied by using Torulaspora delbrueckii IFO 1621 as a model. AKD-2C slightly inhibited the incorporation of radioactive precursors into protein, RNA and lipid, but not into DNA. On the other hand, AKD-2C greatly enhanced the leakage of K+ ions from treated cells and showed a potent effect on liposomal glucose leakage. Using electron microscopic studies, though drastic morphological changes were not observed, an increase in cell membrane irregularities and swelling of the mitochondria caused by AKD-2C were demonstrated. These results suggest that the antifungal action of AKD-2C is due to effects on the yeast cell membrane.

Absence of mutations in the analysis of coding sequences of the entire transforming growth factor-beta type II receptor gene in sporadic human breast cancers.

The transforming growth factor-beta (TGFbeta) binds the type II TGFbeta growth factor receptor (TGFbetaRII) to inhibit the growth of most epithelial tissues. Most human colon and gastric cancers with microsatellite instability (MI) have frameshift mutations in polynucleotide repeats within the TGFbetaRII coding region; these mutations truncate the receptor protein and disable the serine/threonine kinase to produce TGF-beta resistance. To further investigate the type, frequency and tissue distribution of TGFbetaRII gene mutations, in this study, we examined 36 sporadic breast cancers. We previously produced eight intron based primer pairs for mutational analysis of the entire coding region of the TGFbetaRII gene. Using these primers, we developed protocols for polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of PCR products from genomic DNA samples of 36 breast cancer patients and we tested them for microsatellite instability (MI) at eight microsatellite loci. One case demonstrated MI (2.8%) and we found no mutations. These and other recent data indicate that TGFbetaRII mutations are essentially confined to colon and gastric cancers with MI. The narrow spectrum of tissues containing RII mutations illustrates the complexity of genetic checkpoints in human carcinogenesis.

Plasma membrane fluidity during regeneration and atrophy of the rat liver following portal branch ligation.

Dynamic changes in liver plasma membrane fluidity caused by regeneration and atrophy were assessed in rats following portal branch ligation (PBL). The portal branch, which perfuses 70% of the liver, was ligated with 5-0 prolene, and liver plasma membranes were isolated by ultracentrifugation. The membrane fluorescence polarization was measured as an index of membrane fluidity using 1,6-diphenyl-1,3,5-hexatriene (DPH) as the probe dye. In nonligated lobes, a significant decrease in fluorescence polarization was observed 12 and 24 h after PBL (0.171 +/- 0.004, p < 0.01 and 0.165 +/- 0.005, p < 0.001, respectively) as compared to the controls (0.181 +/- 0.002). The fluorescence polarization values then gradually returned to near control levels. In contrast, in the ligated lobes, the fluorescence polarization had increased by 12 hours after PBL (0.196 +/- 0.002, p < 0.01), and remained significantly elevated (p < 0.01) for up to 1 week after PBL, gradually returning to control levels within 3 weeks. The membrane composition was also evaluated by analyzing the cholesterol/phospholipid (C/P) ratio. A significant increase in the C/P ratio was detected in the ligated lobes 12 h and 3 days after PBL, but there was no significant difference in fluorescence polarization values between nonligated lobes and controls. These results suggest that alterations in membrane fluidity play an important role in the regenerative and atrophic processes of the liver following portal branch ligation.