Elevated amyloid β production in senescent retinal pigment epithelium, a possible mechanism of subretinal deposition of amyloid β in age-related macular degeneration

Age-related macular degeneration (AMD) is the most common cause of legal blindness in the elderly individuals in developed countries. Subretinally-deposited amyloid β (Aβ) is a main contributor of developing AMD. However, the mechanism causing Aβ deposition in AMD eyes is unknown. Aging is the most significant risk of AMD, thus, we examined the effect of aging on subretinal Aβ deposition. mRNAs and cell lysates were isolated from retinal pigment epithelial (RPE) cells derived from 24-month-old (24M RPE) and 2-month-old (2M RPE) C57BL/6 mice. Aβ concentration in culture supernatants was measured by ELISA. Activity and expression of proteins that regulate Aβ level were examined by activity assay and real time PCR. Effect of β-secretase (BACE) on Aβ production was examined by siRNA silencing. Aβ amounts in supernatants of 24M RPE were significantly higher than 2M RPE. Activity and mRNA levels of neprilysin, an Aβ degrading enzyme, were significantly decreased in 24M RPE compared to 2M RPE. PCR analysis found that BACE2 was significantly more abundantly expressed than BACE1 in RPE cells, however, inactivation of BACE2 gene did not affect Aβ production. BACE1 protein amounts did not differ between 24M and 2M RPE, however, BACE1 activity was significantly higher in 24M RPE compared to 2M RPE. There were no significant changes in the activities of α- or γ-secretase between 2M and 24M RPE. In conclusion, RPE cells produce more amounts of Aβ when they are senescent, and this is probably caused by a decrease in Aβ degradation due to a reduction in the expression and activity of neprilysin and an increase in Aβ synthesis due to increased activity of BACE1.     

Discharge of solubilized and Dectin-1-reactive β-glucan from macrophage cells phagocytizing insoluble β-glucan particles: involvement of reactive oxygen species (ROS)-driven degradation

Phagocytes engulf pathogenic microbes, kill them and degrade their cellular macromolecules by hydrolytic enzymes in phagolysosomes. However, such enzymes are unable to degrade some microbial polysaccharides, and fate of such indigestible polysaccharides in phagocytes remains uncertain. Using the extracellular domain of Dectin-1 as β-glucan-specific probes, we succeeded in detection of soluble and Dectin-1-reactive β-glucan discharged from mouse RAW 264.7 and human THP-1 macrophage cell lines as well as mouse peritoneal macrophages, which had phagocytized insoluble β-glucan particles. The RAW 264.7 cell culture-supernatant containing the discharged β-glucan stimulated naïve RAW 264.7 cells, resulting in the induction of cytokine expression. Such discharge of Dectin-1-reactive β-glucan from macrophage cells was inhibited by either NADPH oxidase inhibitors (apocynin and diphenylene iodonium) or radical scavengers (N-acetyl cysteine and MCI-186). Moreover, reactive oxygen species (ROS) produced by a Cu²⁺/ascorbic acid system solubilized insoluble β-glucan particles in vitro, and a part of the solubilized β-glucan was Dectin-1 reactive and biologically active in macrophage activation. The soluble and biologically active β-glucan was degraded further during prolonged exposure to ROS. These results suggest that degraded but Dectin-1-reactive β-glucan is discharged from macrophage cells phagocytizing insoluble β-glucan particles and stimulates not only themselves again but also the other naïve phagocytes, leading to the effective elimination of infecting microbes and the ultimate breakdown and inactivation of metabolically resistant β-glucan.     

Differential requirements of mammalian ESCRTs in multivesicular body formation, virus budding and cell division

The endosomal sorting complexes required for transport (ESCRTs) mediate membrane fission from the cytoplasmic face of the bud neck. ESCRTs were originally identified as factors involved in multivesicular body vesicle biogenesis in yeast but have since been shown to function in other membrane fission events in mammalian cells, including enveloped virus budding and the abscission step of cytokinesis. Several recent studies have revealed that not all ESCRT factors are required for each of these biological processes, and this review summarizes our current understanding of the different requirements for ESCRT factors in these three different ESCRT-mediated mammalian membrane fission processes.     

Nuclear factor-kappa b activation in silica-induced interleukin 8 production by human bronchial epithelial cells

Recent studies indicate that interleukin 8 (IL-8) plays an important role in interstitial lung diseases including silica-induced lung inflammation. To investigate the regulation of IL-8 expression and production in human bronchial epithelial cells, we examined the effects of silica on NF-kappaB activation. Human bronchial epithelial cell line BET-1A was cultured with hormonally defined Ham's F12 medium. The administration of silica induced IL-8 production in BET-1A dose-dependently and time-dependently. Northern blot analysis demonstrated that silica upregulated IL-8 expression in BET-1A. Moreover, electrophoretic mobility shift assays revealed that NF-kappaB activation occurred in the presence of silica, which was inhibited by antioxidants such as N-acetylcysteine (NAC). These data suggest that reactive oxygen species may be involved in the activation of NF-kappaB induced by silica.     

Synthesis and evaluation of 4-deacetoxyagosterol A as an MDR-modulator

4-Deacetoxyagosterol A was synthesized from ergosterol by utilizing reductive regioselective epoxy cleavage as a key reaction. This synthesized congener of agosterol A, a spongean MDR-modulator. showed similar MDR-modulating activity against KB CV-60 cells overexpressing MRP.     

A fluorescence cytophotometer operated under computer control for multi-parameter cell analysis

Summary A new type of fluorescence cytophotometer has been developed for multi-parameter cell analysis (Olympus BH2-QRFL). For multi-color fluorescence cytophotometry, this instrument is equipped with four sets of interchangeable filters, each consisting of an excitation filter, a dichroic mirror with a barrier filter, and a measuring filter. For permitting automatic operation of the filter sets, the cytophotometer is connected on line with a personal computer (HP 85F). A desired sequence of filter sets can be memorized in the software and multiple cellular constituents can be rapidly and consecutively determined on a single cell basis. All data are stored in the same computer and can be retrieved for further statistical analysis and display either in tabular form, or as histogram, correlation histograms, two-dimensional scatter plot, or two-dimensional frequency distribution histogram, on the CRT (cathode ray tube) with simultaneous hard copy. As an example of multiparameter cell analysis, combined protein and DNA measurements were performed on normal, border-line, and cancerous gynecological cytology specimens by using the ninhydrin-Schiff and Feulgen techniques.     

Strategic roles of axial histidines in structure formation and redox regulation of tetraheme cytochrome c3

Tetraheme cytochrome c 3 (cyt c 3) exhibits extremely low reduction potentials and unique properties. Since axial ligands should be the most important factors for this protein, every axial histidine of Desulfovibrio vulgaris Miyazaki F cyt c 3 was replaced with methionine, one by one. On mutation at the fifth ligand, the relevant heme could not be linked to the polypeptide, revealing the essential role of the fifth histidine in heme linking. The fifth histidine is the key residue in the structure formation and redox regulation of a c-type cytochrome. A crystal structure has been obtained for only H25M cyt c 3. The overall structure was not affected by the mutation except for the sixth methionine coordination at heme 3. NMR spectra revealed that each mutated methionine is coordinated to the sixth site of the relevant heme in the reduced state, while ligand conversion takes place at hemes 1 and 4 during oxidation at pH 7. The replacement of the sixth ligand with methionine caused an increase in the reduction potential of the mutated heme of 222-244 mV. The midpoint potential of a triheme H52M cyt c 3 is higher than that of the wild type by approximately 50 mV, suggesting a contribution of the tetraheme architecture to the lowering of the reduction potentials. The hydrogen bonding of Thr24 with an axial ligand induces a decrease in reduction potential of approximately 50 mV. In conclusion, the bis-histidine coordination is strategically essential for the structure formation and the extremely low reduction potential of cyt c 3.     

Adipose expression of catalase is regulated via a novel remote PPARgamma-responsive region

In adipose tissue of obese mice, the expression of catalase, an anti-oxidant enzyme, significantly decreases, which may cause insufficient elimination of hydrogen peroxide, but it does not in liver or skeletal muscle. However, the precise regulatory mechanism of catalase expression in adipocytes has not been fully defined. Here, we demonstrated that adipose tissues highly expressed catalase on the level comparable to liver and kidney, and treatment of mice with PPARγ agonist significantly enhanced catalase expression in adipose tissue but not in liver. In 3T3-L1 cells, mRNA expression of catalase was up-regulated by the induction for adipose differentiation, and down-regulated by TNFα, in parallel with alterations in PPARγ expression. PPARγ agonist significantly enhanced catalase mRNA and activity. Furthermore, we newly identified a remote enhancer region containing two functional PPARγ binding sites in mouse catalase gene. Collectively, our findings suggest that PPARγ plays a crucial role in the expression of catalase in adipocytes.