Two-colored fluorescence correlation spectroscopy screening for LC3-P62 interaction inhibitors.

The fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen for protein-protein interaction inhibitors is a highly sensitive method as compared with the fluorescent polarization assay used conventionally. However, the FCS assay identifies many false-positive compounds, which requires specifically designed orthogonal screenings. A two-colored application of the FCS-based screening was newly developed, and inhibitors of a protein-protein interaction, involving selective autophagy, were selected. We focused on the interaction of LC3 with the adaptor protein p62, because the interaction is crucial to degrade the specific target proteins recruited by p62. First, about 10,000 compounds were subjected to the FCS-based competitive assay using a TAMRA-labeled p62-derived probe, and 29 hit compounds were selected. Next, the obtained hits were evaluated by the second FCS assay, using an Alexa647-labeled p62-derived probe to remove the false-positive compounds, and six hit compounds inhibited the interaction. Finally, we tested all 29 compounds by surface plasmon resonance-based competitive binding assay to evaluate their inhibition of the LC3-p62 interaction and selected two inhibitors with IC50 values less than 2 µM. The two-colored FCS-based screening was shown to be effective to screen for protein-protein interaction inhibitors.

Effects of curcumin and capsaicin irradiated with visible light on murine oral mucosa.

The purpose of this study was to evaluate the histopathological effects of curcumin and capsaicin, with or without visible light (VL) irradiation for 5 min, on the oral mucous membrane in mice. Capsaicin-treated, but not curcumin-treated, buccal epithelium exhibited slight tissue damage; VL irradiation caused excessive tissue damage, particularly when combined with the former treatment. The TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method demonstrated that both capsaicin and curcumin induced apoptosis, with the apoptotic effect of capsaicin appearing at an early stage of application. VL irradiation increased the number of apoptotic cells, particularly those upon in the capsaicin-treated area. Capsaicin and curcumin acted as photosensitizers exposure to VL, in the presence of oxygen. Curcumin and capsaicin with VL irradiation could thus be used for photodynamic therapy in the clinical setting, especially in precancerous oral diseases.

Effects of catechin-rich green tea on gene expression of gluconeogenic enzymes in rat hepatoma H4IIE cells.

Rat hepatoma H4IIE cells were stimulated with dexamethasone and dibutyryl cAMP to increase gene expressions of gluconeogenic enzymes, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Inclusion of catechin-rich green tea beverage (GTB) in the culture medium reduced the up-regulation of these genes as well as that of hepatocyte nuclear factor 4 alpha (HNF4alpha) gene. GTB was fractionated into chloroform-soluble (Fraction I), ethyl acetatesoluble (Fraction II), methanol-soluble (Fraction III) and residual (Fraction IV) fractions. Fractions II and III containing catechins caused an attenuation of the up-regulated expression of these genes as well as the down-regulation of HNF4alpha gene expression. Fraction IV had a synergistic effect on the up-regulation by dexamethasone/dibutyryl cAMP of the PEPCK gene expression and upregulated HNF4alpha gene expression. These results suggest that GTB down-regulated the expression of the HNF4alpha gene to cause the down-regulated gene expression of gluconeogenic enzymes. One reason why GTB did not down-regulate hepatic PEPCK gene expression in previous animal experiments may be that the component(s) acting to up-regulate PEPCK gene expression was more effective in vivo than in cultured cells.

Differentiation-associated alteration in sensitivity to apoptosis induced by (-)-epigallocatechin-3-O-gallate in HL-60 cells.

Green tea and its constituent (-)-epigallocatechin-3-O-gallate (EGCG) are known to have apoptosis-inducing activity on tumor cells including human leukemia HL-60 cells, providing an explanation for their anti-cancer effects. In the present study, we compared the sensitivity of undifferentiated cells and differentiated HL-60 cells with normal-like phenotypic characters. HL-60 cells treated with three differentiating agents were found to be resistant to EGCG-mediated apoptosis as compared with undifferentiated cells. Gene and protein expression of 67 kDa laminin receptor was down-regulated in differentiated HL-60 cells, suggesting its contribution to the difference in sensitivity in view of the fact that the receptor is a target of EGCG's action to induce apoptosis. The finding supports the view that EGCG induces apoptosis preferentially in cancer cells as compared with normal counterparts.

Effects of chronic ingestion of catechin-rich green tea on hepatic gene expression of gluconeogenic enzymes in rats.

Normal rats were given catechin-rich green tea as drinking fluid and the effects on hepatic gene expression were examined. The results of DNA microarray analysis and quantitative real-time reverse transcription-polymerase chain reaction indicated the down-regulated expression of genes for glucose-6-phosphatase (G6Pase) and fatty acid synthase, and the up-regulated expression of peroxisome proliferator activated receptor alpha in the rats given green tea for 4 weeks as compared with the water-given animals. One may expect anti-diabetic activity by catechin-rich green tea through its chronic down-regulatory effect on G6Pase expression.

Importins and exportins in cellular differentiation.

The importin/exportin transport system provides the machinery involved in nucleocytoplasmic transport. Alterations of the levels of importins and exportins may play crucial roles in development, differentiation and transformation. Employing human leukaemia HL-60 cells, we and others have revealed the differentiation-associated changes in the protein and gene expression of these factors. The recent finding that a switch to the importin-alpha subtype triggers neural differentiation of embryonic stem cells underscores the importance of nucleocytoplasmic transport factors in cellular events. This review focuses on current research into the roles of importins and exportins in cell differentiation.

Differentiation-associated alteration in gene expression of importins and exportins in human leukemia HL-60 cells.

Employing the DNA microarray technique, we previously reported the alteration in gene expression of nucleocytoplasmic transport factors, importins and exportins, induced by 1,25-dihydroxyvitamin D3 (DVD) in human leukemia HL-60 cells. Here, we used the quantitative reverse transcription-polymerase chain reaction method to confirm such previous findings, and compared them with those from the cells treated with all-trans-retinoic acid (ATRA). The results indicated that the gene expression of the transport factors examined was mostly down-regulated following differentiation induced by DVD and ATRA, but importin alpha5 gene expression was up-regulated in either case. The differences were found in the gene expression of importin alpha3 and exportin 6 between the cells after treatments with DVD and ATRA. These variations may be related to the difference between HL-60 cell lineages differentiating into monocytes/macrophages and granulocytes. The present findings provide further evidence to support the important roles of importins and exportins in cell differentiation.

Different effects of all-trans-retinoic acid on phorbol ester-stimulated and phytohemagglutinin-stimulated interleukin-2 expression in human T-cell lymphoma HUT-78 cells.

In view of the importance of vitamin A in the human immune system and the central role of interleukin-2 (IL-2) in the proliferation of T-lymphocytes, we examined the effects of all-trans-retinoic acid (ATRA) on the protein and gene expression of IL-2 in the human T-cell line HUT-78 when stimulated with either 12-O-tetradecanoylphorbol-13-acetate (TPA) or phytohemagglutinin (PHA). ATRA enhanced the production of IL-2 stimulated by TPA, but suppressed that stimulated by PHA. These findings were consistent with the results of a reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction examining IL-2 gene expression. ATRA augmented the gene expression of PKC-beta1 up-regulated by TPA and restored that suppressed by PHA but to below the control level. ATRA suppressed the c-fos gene expression up-regulated by PHA to a level of 36% of the control whereas it had no effect on the up-regulation by TPA. Since PKC- beta1 has been suggested to be important for the secretion and gene expression of IL-2 and since the activator protein-l binding site is present in the promoter of the IL-2 gene, these findings may explain the differences in ATRA's effects on TPA- and PHA-stimulated IL-2 expression. These results suggest that ATRA affects the production of IL-2 by T-lymphocytes in a stimulus-dependent manner.

Effects of visible light-irradiated camphorquinone and 9-fluorenone on murine oral mucosa.

The purpose of this study was to evaluate the histopathological effects of camphorquinone (CQ) and 9-fluorenone (9F) with or without visible light (VL) irradiation on the oral mucous membranes of mice. VL irradiation resulted in a higher degree of tissue damage after CQ or 9F application, particularly the latter. Necrosis and apoptosis were responsible for the tissue damage after application of either agent in the presence of VL irradiation.

Radical-scavenging activity of natural methoxyphenols vs. synthetic ones using the induction period method.

The radical-scavenging activities of the synthetic antioxidants 2-allyl-4-X-phenol (X = NO2, Cl, Br, OCH3, COCH3, CH3, t-(CH3)3, C6H5) and 2,4-dimethoxyphenol, and the natural antioxidants eugenol and isoeugenol, were investigated using differential scanning calorimetry (DSC) by measuring their anti-1,1-diphenyl-2-picrylhydrazyl (DPPH) radical activity and the induction period for polymerization of methyl methacrylate (MMA) initiated by thermal decomposition of 2,2'-azobisisobutyronitrile (AIBN) and benzoyl peroxide (BPO). 2-Allyl-4-methoxyphenol and 2,4-dimethoxy-phenol scavenged not only oxygen-centered radicals (PhCOO*) derived from BPO, but also carbon-centered radicals (R*) derived from the AIBN and DPPH radical much more efficiently, in comparison with eugenol and isoeugenol. 2-Allyl-4-methoxyphenol may be useful for its lower prooxidative activity.

Free radical interaction between vitamin E (alpha-, beta-, gamma- and delta-tocopherol), ascorbate and flavonoids.

Despite a large number of previous studies, the mechanism of free radical interaction between vitamin E (VE) (alpha-, beta-, gamma- and delta-tocopherol) and ascorbate or flavonoids as coantioxidants remains unclear. VE, particularly alpha-tocopherol, shows less antioxidant activity against peroxyl radicals, suggesting that VE possesses functions that are independent of its antioxidant/radical-scavenging activity. The synergistic antioxidant effect of VE or L-ascorbyl 2,6-dibutyrate (ASDB, an ascorbate derivative) with the flavonoids (-)-epicatechin (EC) and (-)-epigallocatechin gallate (EGCG) was investigated using the induction period method in the polymerization of methyl methacrylate initiated by thermal decomposition of benzoyl peroxide (an oxygen-centered radical, PhCOO*) under nearly anaerobic conditions. For delta-tocopherol, a synergistic antioxidant effect was observed in the presence of both EC and EGCG, whereas antioxidant activity for alpha-, beta- and gamma-tocopherol was decreased by addition of EC and EGCG. This suggested that the partial regeneration between VE and flavonoids may depend on the chemical structure of VE, i.e., monomethyl, dimethyl, or trimethyl tocol. The regeneration of delta-tocopherol, a monomethyl tocol, by flavonoids may be due to the lower steric effect of tocol. For ASDB, regeneration of vitamin E, which is well-known for a VE/ascorbate mixture, was not observed, possibly due to the anaerobic experimental conditions. The radical interaction between VE and EC, EGCG or ASDB suggests reactivity of VE with biological systems.

Screening of novel cellulose-degrading bacterium and its application to denitrification of groundwater.

To establish an environmentally friendly groundwater bioremediation process using a cellulose carrier combined with cellulose-utilizing, denitrifying microorganisms, a novel psychrophilic bacterium, designated CL-5, which can degrade a commercial-based cellulose carrier as the sole carbon source, was screened. Since the denitrification capability of CL-5 is low, complex microbial systems were constructed together with other denitrifying bacteria designated NR-1 and NR-2 that were also isolated from soil. The nitrate-reducing activities of mixed cultures were much higher than those of the pure cultures of CL-5, NR-1 and NR-2. The highest N(2)O and N(2) formation activities were observed in the mixed culture of CL-5+NR-2.

Induction of cytotoxicity and apoptosis and inhibition of cyclooxygenase-2 gene expression by eugenol-related compounds.

Induction of cytotoxicity and internucleosomal DNA fragmentation by 4-allyl-2-methoxyphenol (eugenol, EUG), 2-methoxy-4-methylphenol (MMP), 3,3'-dimethoxy-5,5'-di-2-propenyl-1,1'-biphenyl-2,2'-diol (bis-EUG) and 3,3'-di-methoxy-5,5'-dimethyl-1,1'-biphenyl-2,2'-diol (bis-MMP) were investigated in HL-60 leukemia cells. The 50% cytotoxic concentrations (CC50) for EUG, MMP, bis-EUG and bis-MMP were 0.38 mM, 0.38 mM, 0.18 mM and 0.20 mM, respectively. DNA fragmentation was induced most strongly by bis-EUG, followed by EUG, MMP and bis-MMP. The expression of MnSOD and, less strongly, Cu/ZnSOD activity, as assessed by acrylamide gel electrophoresis, was inhibited by EUG, suggesting mitochondrial dysfunction. The expression of the mRNAs for MnSOD and Cu/ZnSOD in HL-60 cells, as assessed by RT-PCR, was significantly inhibited by treatment with 1 mM EUG for 1 hour. Furthermore, inhibition of SOD mRNAs expression by EUG was strongly potentiated by the addition of 5 mM N-acetyl cysteine (NAC) or glutathione (GSH), whereas NAC or GSH alone did not affect the expression of SOD mRNAs. The cytotoxicity of EUG was significantly enhanced by high concentrations of NAC or GSH, which may be attributed to the inhibition of SOD mRNAs expression by the synergistic action of EUG and GSH or NAC. The regulatory effects of eugenol-related compounds on lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2) gene expression in RAW 264.7 cells were investigated by Northern blot analysis. Bis-EUG, MMP and bis-MMP inhibited COX-2 gene expression at concentrations of 300 microM, 500 microM and 500 microM, respectively. In contrast, no inhibitory effect of EUG was found over the wide concentration range of 10-500 microM, possibly as a result of the extensive mitochondrial dysfunction induced by this compound, which possesses potent pro-oxidative activity. Eugenol-related compounds, particularly bis-EUG, may act as nonsteroidal anti-inflammatory drug (NSAID)-like compounds.

Effects of visible light irradiation on eugenol-treated oral mucosa.

The purpose of this study was to evaluate the histopathological effects of eugenol (EUG) and iso-eugenol (IsoEUG)--with or without visible light (VL) irradiation--on oral mucous membranes. Oral mucous membranes of mice were applied with three agents, EUG, IsoEUG, and aceton (as the control) in the absence or presence of VL irradiation. VL irradiation resulted in more tissue damage for EUG- or IsoEUG-treated mucosa compared to corresponding compounds without VL irradiation, and that damage under IsoEUG treatment was greater than that under EUG treatment. Necrosis, but not apoptosis, was preferentially expressed in EUG- or IsoEUG-treated mucous membranes in the presence of VL irradiation.

A simple screening procedure for heterotrophic nitrifying bacteria with oxygen-tolerant denitrification activity.

Various naturally occurring strains of heterotrophic nitrifying bacteria were isolated by enrichment culture using acetamide as the C and N source, and 21 strains were identified as heterotrophic nitrifiers. Using a new simple procedure, these 21 strains were also investigated for the ability to carry out denitrifcation in the presence of oxygen. Several of the nitrifying strains were found to exhibit a distinct activity that allows for denitrifcation via nitrite (NO2-) in the presence of oxygen, indicating that they have an oxygen-tolerant denitrifcation system. A wide variety of bacteria possessing both nitrification and denitrifcation capabilities in the presence of oxygen were isolated and partially characterized by using the simple screening combinatorial procedure described in this paper.