Eun-Yi Moon

Characterization of neural cell types expressing peroxiredoxins in mouse brain

The differential expression patterns of antioxidant enzymes observed in the brains of patients with neurodegenerative diseases suggest an important role for reactive oxygen species and antioxidant enzymes in neurodegeneration. The six mammalian peroxiredoxins (Prxs) comprise a novel family of anti-oxidative proteins that are widely distributed in most tissues, but few studies of Prx in brain tissue have been reported. The specific histology of the neural cell types in which Prxs are expressed is an important issue related to biological function and defense against oxidative stress in the brain. This study analyzed mouse brain neural cell types expressing Prx isoforms using single- or double-label immunohistochemical techniques. In neurons, immunoreactivity for Prx II-V was observed in the cytoplasm. In particular, Prx II was found in the habenular nuclei, and Prx III and V were found in the stratum lucidum of the hippocampus. Astrocytes and microglia were immunoreactive only for Prx VI and Prx I, respectively. Prx I and IV immunoreactivity was apparent in oligodendrocytes, where it was principally localized in the nuclei. The observed distribution of Prx isoforms in the mammalian brain may be indicative of their specific roles in their preferred neural cell types and subcellular locales. The results of this study will help in unraveling the physiological and pathophysiological roles of the different Prx isoforms in neural function.

Inhibitory role of peroxiredoxin II (Prx II) on cellular senescence.

Reactive oxygen species (ROS) were generated in all oxygen‐utilizing organisms. Peroxiredoxin II (Prx II) as one of antioxidant enzymes may play a protective role against the oxidative damage caused by ROS. In order to define the role of Prx II in organismal aging, we evaluated cellular senescence in Prx II−/− mouse embryonic fibroblast (MEF). As compared to wild type MEF, cellular senescence was accelerated in Prx II−/− MEF. Senescence‐associated (SA)‐β‐galactosidase (Gal)‐positive cell formation was about 30% higher in Prx II−/− MEF. N‐Acetyl‐l‐cysteine (NAC) treatment attenuated SA‐β‐Gal‐positive cell formation. Prx II−/− MEF exhibited the higher G2/M (41%) and lower S (1.6%) phase cells as compared to 24% and 7.4% in wild type MEF, respectively. A high increase in the p16 and a slight increase in the p21 and p53 levels were detected in PrxII−/− MEF cells. The cellular senescence of Prx II−/− MEF was correlated with the organismal aging of Prx II−/− mouse skin. While extracellular signal‐regulated kinase (ERK) and p38 activation was detected in Prx II−/− MEF, ERK and c‐Jun N‐terminal kinase (JNK) activation was detected in Prx II−/− skin. These results suggest that Prx II may function as an enzymatic antioxidant to prevent cellular senescence and skin aging.

Reactive oxygen species induced by the deletion of peroxiredoxin II (PrxII) increases the number of thymocytes resulting in the enlargement of PrxII-null thymus.

In the thymus, CD4+ or CD8+ single‐positive (SP) thymocytes develop and mature by positive and negative selection or undergo ‘death by neglect’. CD4+ or CD8+ SP then circulate to other lymphoid tissues. We have investigated the role of reactive oxygen species (ROS) in thymocyte development using peroxiredoxin II (PrxII)‐null mice. The level ofROS in PrxII‐null thymocytes is higher than that in wild‐type mice. Deletion of the PrxII gene leads to enlargement of the thymus in young (9 weeks) and old (64 weeks) mice. The increased number ofthymocytes in PrxII‐null thymus is related to reduced hypodiploid cell formation. For mice on a normal diet, the ratio of SP to double‐positive (DP) thymocytes in thymus of PrxII‐null mice is lower thanthat in wild‐type mice. After food restriction, which leads to increased ROS production, this ratio becomes much higher in PrxII‐null thymus. The amount of apoptosis, induced by food restriction orby the injection of dexamethasone, is consistently lower in PrxII‐null thymocytes than in wild‐type thymocytes. In the presence of low serum concentrations, PrxII‐deleted T cells proliferate more vigorously after stimulation with concanavalin A. Phytohemagglutinin‐ or OKT3‐stimulated proliferation of human peripheral blood mononuclear cells is also higher in the presence of lower serum concentrations. Collectively, the results suggest for the first time that thymocyte maturations and proliferations are regulated by ROS levels induced by the deletion of PrxII gene in vivo.

B Cell-Activating Factor Is a Novel Diagnosis Parameter for Asthma

Background: Asthma is a life-threatening immediate-type allergic disease. B cell-activating factor (BAFF) is a key regulator of B lymphocyte development and is required to generate and maintain the mature B cell pool. Objectives: To investigate the level of BAFF in the serum of asthma patients and the role of BAFF on T cells. Methods: The BAFF level was measured by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cells (PBMC) from asthma patients were analyzed by flow cytometry. T8.1 cells were used to test the role of BAFF on T cell-antigen-presenting cell (APC) conjugate formation. Results: The BAFF level in patient serum was elevated relative to normal serum. Immunoglobulin E (IgE) concentration and the percentage of CD3+ T and CD19+ B cells vary according to the serum BAFF level. Patients with high BAFF and high IgE (group II) and those with high BAFF and low IgE (group III) show a high ratio of CD3+ T to CD19+ B cells, and the opposite is seen for patients with low BAFF and high IgE (group I) and those with low BAFF and low IgE (group IV). The addition of BAFF increased PBMC proliferation and T cell-APC conjugate formation. BAFF concentration in serum decreased after treatment with antiasthmatic drugs including glucocorticoids and immunosuppressants. Conclusion: These findings suggest that the serum BAFF level is high in both IgE-mediated asthma and non-IgE-mediated asthma and extend our knowledge about the fact that BAFF may play a stimulatory role on the proliferation of T cells. Thus, BAFF could be a parameter to monitor the severity of asthma symptoms.

Inhibitory effect of peroxiredoxin II (Prx II) on Ras–ERK–NFκB pathway in mouse embryonic fibroblast (MEF) senescence

Intracellular reactive oxygen species (ROS) were attenuated by the expression of peroxiredoxin II (Prx II). Cellular senescence as judged by senescence-associated (SA)-β-galactosidase (Gal) positive cell formation was increased in Prx II-deficient mouse embryonic fibroblast (MEF). Ras expression was increased following passages. The level of Ras expression was higher in Prx II− / − MEF than wild type MEF. ERK activity was also augmented by the deletion of Prx II. SA-β-Gal-positive cell formation was reduced by PD98059, ERK inhibitor. Activated nuclear transcription factor, nuclear factor-kappaB (NFκB) by the deletion of Prx II was inhibited by the treatment with PD98059. In contrast, no changes in SA-β-Gal-positive cell formation were detected by NFκB inhibitor, N-alpha-tosyl-l-phenylalanyl chloromethyl ketone (TPCK). Collectively, results suggest that Prx II deletion activate Ras–ERK–NFκB pathways and cellular senescence in Prx II− / − MEF cells was mediated by ERK activation but not by NFκB activation.

Cytotoxic constituents from the roots ofAnthriscus sylvestris

Activity-guided fractionation of the roots ofAnthriscus sylvestris resulted in the isolation and characterization of five cytotoxic compounds, deoxypodophyllotoxin (1), falcarindiol (2), and angeloyl podophyllotoxin (5) from the hexane soluble fraction and morelensin (3), bursehernin (4) from the chloroform soluble fraction. It is the first report of the occurrence of compound5 in nature.

Synthesis and antitumor activity of 4-phenyl-1-arylsulfonyl imidazolidinones

Novel 1-(1-benzoylindoline-5-sulfonyl)-4-phenyl-4,5-dihydroimidazolon es 3 synthesized show highly potent and broad cytotoxicities. Among them compound 3b (DW2143) exhibits much more potent cytotoxicities than doxorubicin and highly effective antitumor activities against murine (3LL, Colon 26) and human xenograft (NCI-H23, SW620) tumor models.

Mouse Melanoma Cell Migration is Dependent on Production of Reactive Oxygen Species under Normoxia Condition.

Cell migration plays a role in many physiological and pathological processes. Reactive oxygen species (ROS) produced in mammalian cells influence intracellular signaling processes which in turn regulate various biological activities. Here, we investigated whether melanoma cell migration could be controlled by ROS production under normoxia condition. Cell migration was measured by wound healing assay after scratching confluent monolayer of B16F10 mouse melanoma cells. Cell migration was enhanced over 12 h after scratching cells. In addition, we found that ROS production was increased by scratching cells. ERK phosphorylation was also increased by scratching cells but it was decreased by the treatment with ROS scavengers, N-acetylcysteine (NAC). Tumor cell migration was inhibited by the treatment with PD98059, ERK inhibitor, NAC or DPI, well-known ROS scavengers. Tumor cell growth as judged by succinate dehydrogenase activity was inhibited by NAC treatment. When mice were intraperitoneally administered with NAC, the intracellular ROS production was reduced in peripheral blood mononuclear cells. In addition, B16F10 tumor growth was significantly inhibited by in vivo treatment with NAC. Collectively, these findings suggest that tumor cell migration and growth could be controlled by ROS production and its downstream signaling pathways, in vitro and in vivo.

Epac1‐mediated Rap1 activation is not required for the production of nitric oxide in BV2, murine microglial cells

This study demonstrates that cyclic AMP (cAMP) production is induced by lipopolysaccharide (LPS) stimulation and activates two different pathways in murine BV2 microglial cells. Two principal effector proteins for cAMP are protein kinase A (PKA) and cAMP‐responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor. When cells were treated with various cAMP level modulators, nitric oxide (NO) production increased as the result of posttreatment with Type IV phosphodiesterase (PDE4) inhibitor, rolipram or dibutyryl‐cAMP (dbcAMP), at 2 hr after LPS stimulation. Intracellular cAMP increased due to LPS stimulation and the cAMP modulators phosphorylate transcription factor CREB, which is enhanced in turn by posttreatment with dbcAMP. In contrast, the Epac‐specific cAMP analog 8‐(4‐chloro‐phenylthio)‐2′‐O‐methyladenosine‐3′,5′‐cyclic monophosphate (8CPT‐2Me‐cAMP) activates Rap1 in the BV2 cells, but does not induce PKA activation, as judged by CREB phosphorylation. NO production was enhanced by posttreatment with dbcAMP but not by treatment with 8CPT‐2Me‐cAMP. This suggests that LPS‐stimulated NO production is mainly PKA‐dependent and also that Epac1‐mediated Rap1 activation is not required for the induction of NO production.

Dynamic rearrangement of F-actin is required to maintain the antitumor effect of trichostatin A.

Actin plays a role in various processes in eukaryotic cells, including cell growth and death. We investigated whether the antitumor effect of trichostatin A (TSA) is associated with the dynamic rearrangement of F-actin. TSA is an antitumor drug that induces hyper-acetylation of histones by inhibiting histone deacetylase. HeLa human cervical cancer cells were used to measure the antitumor effect of TSA. The percent cell survival was determined by an MTT assay. Hypodiploid cell formation was assessed by flow cytometry. Collapse of the mitochondrial membrane potential (MMP) was identified by a decrease in the percentage of cells with red MitoProbe J-aggregate (JC-1) fluorescence. Cell survival was reduced by treatment with TSA, as judged by an MTT assay and staining with propidium iodide, FITC-labeled annexin V, or 4′,6-diamidino-2-phenylindole (DAPI). TSA also induced an MMP collapse, as judged by the measurement of intracellular red JC-1 fluorescence. In addition, the F-actin depolymerizers cytochalasin D (CytoD) and latrunculin B (LatB) induced an MMP collapse and increased apoptotic cell death in HeLa cells. However, our data show that apoptotic cell death and the MMP collapse induced by TSA were decreased by the co-treatment of cells with CytoD and LatB. These findings demonstrate that the dynamic rearrangement of F-actin might be necessary for TSA-induced HeLa cell apoptosis involving a TSA-induced MMP collapse. They also suggest that actin cytoskeleton dynamics play an important role in maintaining the therapeutic effects of antitumor agents in tumor cells. They further suggest that maintaining the MMP could be a novel strategy for increasing drug sensitivity in TSA-treated tumors.