Redox-dependent Regulation of Gluconeogenesis by a Novel Mechanism Mediated by a Peroxidatic Cysteine of Peroxiredoxin.

Peroxiredoxin is an abundant peroxidase, but its non-peroxidase function is also important. In this study, we discovered that Tsa1, a major peroxiredoxin of budding yeast cells, is required for the efficient flux of gluconeogenesis. We found that the suppression of pyruvate kinase (Pyk1) via the interaction with Tsa1 contributes in part to gluconeogenic enhancement. The physical interactions between Pyk1 and Tsa1 were augmented during the shift from glycolysis to gluconeogenesis. Intriguingly, a peroxidatic cysteine in the catalytic center of Tsa1 played an important role in the physical Tsa1-Pyk1 interactions. These interactions are enhanced by exogenous H2O2 and by endogenous reactive oxygen species, which is increased during gluconeogenesis. Only the peroxidatic cysteine, but no other catalytic cysteine of Tsa1, is required for efficient growth during the metabolic shift to obtain maximum yeast growth (biomass). This Tsa1 function is separable from the peroxidase function as an antioxidant. This is the first report to demonstrate that peroxiredoxin has a novel nonperoxidase function as a redox-dependent target modulator and that pyruvate kinase is modulated via an alternative mechanism.

Requirement of peroxiredoxin on the stationary phase of yeast cell growth.

Toxic chemicals often induce reactive oxygen species (ROS). Although one of the most abundant ROS-sensitive proteins is in the peroxiredoxin (Prx) family, the function of Prx proteins is poorly understood because they are inactivated under high concentrations of hydrogen peroxide. Like mammalian cells, the model eukaryote Saccharomyces cerevisiae possesses multiple Prx proteins. Among the five Prx family proteins, Tsa1 and Ahp1 have the highest and second-highest expression levels, respectively. Here, we focused on a previously uncharacterized phenotype resulting from Tsa1 loss: impaired growth during the late exponential phase. We overexpressed catalase (CTT1) and Ahp1 in cells with disruptions in TSA1 and its homologue, TSA2 (tsa1/2Δ cells), and we found that neither Ctt1 nor Ahp1 overexpression suppressed the impaired cell growth at the stationary phase, although the ROS levels were successfully suppressed. Furthermore, the cell cycle profile was not altered by Tsa1/2 loss, at least in the late exponential phase; however, the glucose consumption rate slowed in the late exponential phase. Our results suggest that ROS levels are not responsible for the growth phenotype. Tsa1 might have a specific function that could not be replaced by Ahp1.

Effect of propranolol on hyphae formation signal in Candida albicans.

Candida albicans (C. albicans) is known as an opportunistic pathogen that changes from a yeast form to a hyphae form in response to various outside environmental signals. The addition of propranolol inhibited hyphae formation of C. albicans. Propranolol inhibited the expression of agglutinin like sequence 3 (ALS3) and ALS8mRNA, which are regulated by the cAMP-EFG1 pathway in C. albicans. Propranolol did not affect the expression of CST20, HST7 or CPH1mRNA, which are components of the mitogen-activated protein (MAP) kinase cascade in C. albicans. The expression of CYR1mRNA, which encodes adenylate cyclase of C. albicans, was not affected by propranolol. These findings indicated that the interruption of hyphae formation by propranolol is caused by inhibition of the cAMP-EFG1 pathway, but not effects on the MAP kinase cascade.

[Inhibitory mechanism of melanin synthesis by glutathione].

Glutathione dose dependently inhibited melanin synthesis in the reaction of tyrosinase and L-DOPA. The inhibition of melanin synthesis was recovered by increasing the concentration of L-DOPA, but not recovered by increasing tyrosinase. Glutathione inhibited the binding between tyrosinase and L-DOPA. Although the synthesized melanin was aggregated within 1 h, the aggregation was inhibited by the addition of glutathione. These results indicate that glutathione inhibits the synthesis and agglutination of melanin by interrupting the function of L-DOPA.

Anti-candida activity of sodium sulfite.

The growth of Candida albicans in RPMI1640 medium was inhibited by sodium sulfite between pH 3-6. Under the condition of pH 7, growth of C. albicans was not inhibited by sodium sulfite. Under an acidic pH condition, sodium sulfite had a candidacidal effect and the activity was expressed within 150 min. The concentration of ATP in C. albicans was decreased by sodium sulfite. Ethanol production by C. albicans was inhibited by sodium sulfite at pH 5. These results indicated that the candidacidal effect of sodium sulfite under acidic conditions was caused by interruption of alcohol fermentation and aerobic respiration.

Antifungal mechanism of hinokitiol against Candida albicans.

The growth of Candida albicans was dose-dependently inhibited by addition of hinokitiol. The sensitivity of C. albicans to hinokitiol under aerobic conditions was higher than that under anaerobic conditions. Amount of ATP in C. albicans was not inhibited by hinokitiol under both conditions. The expression of mRNAs related to the growth signal, CYR1 and RAS1, was inhibited by hinokitiol. These findings suggested that the growth inhibition of C. albicans by hinokitiol was due to the interruption of RAS-signal transmission, such as the cAMP pathway.

[Growth-inhibitory activity of Cladosporium cladosporioides by cysteine].

When Cladosporium cladosporioides was cultured with cysteine, its growth was completely inhibited statically. The growth of C. cladosporioides cultured on potato-dextrose agar plates was also inhibited by the addition of cysteine. The production of ATP in C. cladosporioides was inhibited by cysteine. When a silicone block was incubated with C. cladosporioides, the surface of the block was coated with the biofilm of C. cladosporioides. However, the block containing cysteine was not covered with biofilm. These results indicate that cysteine is useful as a material to prevent the growth of C. cladosporioides.

Chitohexose induce the yeast proliferation of Candida albicans.

Candida albicans generally grows in the hyphae form in RPMI1640 medium. Chitohexose (COS-6) induced the yeast form of C. albicans dose-dependently under this condition. When COS-6 was exposed to C. albicans, yeast proliferation was observed 6 h after starting the incubation. When we observed the growth form of C. albicans cultured with COS-6, yeast proliferation was observed at log-phase. These results showed that COS-6 was useful for the yeast form inducer of C. albicans.

Hyphal formation of Candida albicans is inhibited by salivary mucin.

When mucin was added to Candida albicans under hyphal growth conditions, the hyphal formation was inhibited. After the 24 h incubation, the ratio of hyphal cells was 95.7+/-1.13% in the absence of mucin and no hyphal cells were observed in the presence of 1000 microg/ml mucin. The ratio of hyphal cells began to decreases at 6 h in the mucin addition group. Although mucin has antifungal activity, the concentration of mucin used in this assay did not inhibit the growth of C. albicans, indicating that the inhibition of hyphal formation was not due to the inhibition of germination by its antifungal activity. Expression of RAS1mRNA in C. albicans was inhibited by mucin. These results suggest that the inhibition of hyphal formation by mucin was caused by interruption of the hyphal formation signal of C. albicans.

Hyphal formation of Candida albicans is controlled by electron transfer system.

Most Candida albicans cells cultured in RPMI1640 medium at 37 degrees C grow in hyphal form in aerobic conditions, but they grow in yeast form in anaerobic conditions. The hyphal growth of C. albicans was inhibited in glucose-deficient conditions. Malonic acid, an inhibitor of succinate dehydrogenase, enhanced the yeast proliferation of C. albicans, indicating that the hyphal-formation signal was derived from the glycolysis system and the signal was transmitted to the electron transfer system via the citric acid cycle. Thenoyl trifluoro acetone (TTFA), an inhibitor of the signal transmission between complex II and Co Q, significantly inhibited the hyphal growth of C. albicans. Antimycin, KCN, and oligomycin, inhibitors of complex III, IV, and V, respectively, did not inhibit the hyphal growth of C. albicans. The production of mRNAs for the hyphal formation signal was completely inhibited in anaerobic conditions. These results indicate that the electron transfer system functions upstream of the RAS1 signal pathway and activates the expression of the hyphal formation signal. Since the electron transfer system is inactivated in anaerobic conditions, C. albicans grew in yeast form in this condition.

Effects of antifungal drugs on proliferation signals in Candida albicans.

The sensitivity of Candida albicans to antifungal drugs when cultured under aerobic and anaerobic conditions was measured. Ciclopirox olamine and siccanin were more effective under aerobic than under anaerobic conditions. Terbinafine, neticonazole and amphotericin B showed the same antifungal activity under both aerobic and anaerobic conditions. None of these antifungal activities were affected by the pH conditions. Terbinafine inhibited the elongation of hyphae, while neticonazole and amphotericin B induced proliferation of the yeast form. The expression of RAS1, EFG1 and CPH1 mRNAs was inhibited by these drugs. These results suggested that the inhibition of hyphal formation might be caused by disruption of the RAS1-signal pathway.

Mechanism of Candida albicans transformation in response to changes of pH.

Hyphal growth of Candida albicans was observed at neutral condition, whereas the yeast growth was increased below pH 5. Eight out of 12 strains of C. albicans grow in hyphal form at pH 7, and hyphal formation was inhibited in all strains at pH 4. The addition of GMP, an alternative oxidase (AOX) activator, induced the hyphal growth at pH 4. Although C. albicans grew in hyphal form in pH 7, SHAM, an AOX inhibitor, enhanced the yeast proliferation at this pH. The relative expression level of RAS1 mRNA was higher at pH 7 than at pH 4, indicating that the hyphal formation signal was defective under acidic conditions. Based on these findings, we concluded that AOX-RAS1 signal transduction is the main pathway of hyphal formation of C. albicans, and that the signal was controlled by pH condition.

Change in the respiration system of Candida albicans in the lag and log growth phase.

The growth rate of Candida albicans at the lag growth phase was not influenced by sodium sulfite, but that at the log growth phase was remarkably inhibited. C. albicans was able to grow in aerobic conditions both with and without glucose. Under anaerobic conditions, the growth of C. albicans was completely inhibited in glucose-free RPMI1640 medium. Ethanol production was increased at the lag growth phase, but decreased at the log growth phase. When oligomycin was added to the aerobic growth condition, the growth at log growth phase was remarkably inhibited. The expression of SDH2 mRNA was low in the lag growth phase and high in the log growth phase. We concluded that C. albicans obtained ATP by fermentation at the lag growth phase, and needed oxygen to generate ATP by oxidative phosphorylation at the log growth phase.

[Adherence of Candida albicans is inhibited by yeast mannan].

The adherence of Candida albicans strain NIH A207 to plastic plates was inhibited by the addition of mannan, and plates coated with mannan also inhibited the adherence of C. albicans strains TIMM1768, TIMM2640, and JCM2076. Mannan coated the plastic plates under neutral and acidic conditions, but not under alkaline conditions. These results indicate that C. albicans cannot attach to mannan. Thus mannan-coated medical equipment might be useful to prevent C. albicans adherence.

Hyphae formation of Candida albicans is regulated by polyamines.

Candida albicans generally grows in hyphae form in RPMI1640 medium. However, addition of 1,4-diamino-2-butanone (DAB), a competitive inhibitor of ornithine decarboxylase, decreased the amount of polyamines in C. albicans, and induced the proliferation of the C. albicans yeast form. The expression of CYR1 mRNA was significantly inhibited by the addition of DAB compared with that of the control. The amount of intracellular cAMP was also decreased by the addition of DAB. A specific adenylate cyclase inhibitor, cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2-amine (MDL-12,330A) promoted the growth of the yeast form. These results indicated that polyamines exist upstream of the adenylate cyclase-cAMP signal pathway and regulate the transformation of C. albicans.

Direct determination of benzamides in serum by column-switching high-performance liquid chromatography.

A column-switching high-performance liquid chromatographic method with fluorescence detection was developed for the simultaneous determination of four benzamide-type anti-psychotic drugs: sulpiride, tiapride, sultopride and metoclopramide in human serum. In this method, a TSKgel Super-ODS column was used as an analytical column, and a TSKgel G 2000SW was prepared as a pretreatment column. Under the optimized analytical conditions, four benzamide-type anti-psychotic drugs were eluted within 18 min. The detection limits (S/N = 3) for sulpiride, tiapride, sultopride and metoclopramide are 1 ng/ml, 4 ng/ml, 2 ng/ml and 0.5 ng/ml, respectively. Finally, the method was applied to the determination of sulpiride in human serum samples obtained after a single oral dose of sulpiride.