Seong-Beom Lee

Hydrogen peroxide induces autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway

Oxidative stress by exposure to H(2)O(2) induces various types of cell death depending on cell type and conditions. We report herein on a study of the mechanisms underlying H(2)O(2)-induced cell death in C6 glioma cells. The findings show that H(2)O(2) triggers a caspase-independent autophagic cell death in these cells. The findings also show that H(2)O(2) induces the dephosphorylation of the mammalian target of rapamycin (mTOR) at Ser 2481 and the p70 ribosomal protein S6 kinase (p70S6K) at Thr389 in a Bcl-2/E1B 19kDa interacting protein 3 (BNIP3)-dependent manner. BNIP3 has the capacity to inhibit mTOR activity and mTOR inhibition plays a role in autophagic induction. This suggests that BNIP3 may mediate H(2)O(2)-induced autophagic cell death through the suppression of mTOR. The findings show that the down-regulation of BNIP3 by BNIP3 siRNA prevents C6 cells from undergoing H(2)O(2)-induced autophagic cell death. Collectively, these results suggest that H(2)O(2) induces autophagic cell death in C6 cells via the BNIP3-mediated suppression of the mTOR pathway.

Hydrogen peroxide induces Beclin 1-independent autophagic cell death by suppressing the mTOR pathway via promoting the ubiquitination and degradation of Rheb in GSH-depleted RAW 264.7 cells

Abstract A novel mechanism for H2O2-induced autophagic cell death in GSH-depleted RAW 264.7 cells, a murine macrophage cell line, is proposed. Under GSH-depleted conditions, H2O2-induced autophagic cell, characterized by an increased LC3-II/I ratio, a decreased level of p62 and the formation of autophagic vacuoles, was inhibited by bafilomycin A1 and by Atg5 siRNA transfection, whereas the cell death was not inhibited by zVAD-fmk, by PI3K inhibitors or by Beclin 1 siRNA transfection. In addition, H2O2 treatment reduced the activity of mTOR and promoted the ubiquitination and degradation of Rheb, a key upstream activator of mTOR. Furthermore, proteasome inhibition with MG132 restored the expression of Rheb and increased mTOR activity, resulting in an increased viability of H2O2-treated cells. Collectively, these findings demonstrate that H2O2 induces Beclin 1-independent autophagic cell death by suppressing the mTOR pathway via promoting the ubiquitination and degradation of Rheb in GSH-depleted RAW 264.7 cells.

Bis deficiency results in early lethality with metabolic deterioration and involution of spleen and thymus

Bcl-2 interacting cell death suppressor (Bis), also known as Bag3 or CAIR-1, is involved in antistress and antiapoptotic pathways. In addition to Bcl-2, Bis binds to several proteins, suggesting it has diverse functions in normal and pathological conditions. To better define the physiological function of Bis in vivo, we developed bis-deficient mice with a cre-loxP system. Targeted disruption of exon 4 of the bis gene was demonstrated by Southern blotting and PCR, and Western blotting showed that no intact or truncated Bis protein was synthesized in bis(-/-) mice. While heterozygotes were fertile and appeared normal, Bis-deficient mice showed growth retardation and died by 3 wk after birth. The relative weight of the thymus and spleen was reduced and the total numbers of white blood cells, splenocytes, and thymocytes were significantly reduced compared with wild-type littermates. Serum profiles indicated significant hypoglycemia as well as decrease in triglyceride and cholesterol levels. Expression profiles of metabolic genes indicated that gluconeogenesis and beta-oxidation are activated in the liver of bis(-/-) mice. This activation, as well as a decrease in peripheral fat and an induction of fatty liver, appears to be an adaptive response to hypoglycemia. Our study reveals that the absence of Bis has considerable influences on postnatal growth and survival, possibly due to a nutritional impairment.

DNA-PCR and RT-PCR for the 18-kDa gene of Mycobacterium leprae to assess the efficacy of multi-drug therapy for leprosy.

DNA-PCR and reverse transcription (RT)-PCR for the 18-kDa protein of Mycobacterium leprae were used to examine the efficacy of multi-drug therapy (MDT) in leprosy. MDT was administered for 0-24 months. Fourteen (63.6%) of 22 patients showed positive PCR results after treatment for 12 months and the positive results decreased to 30% after 24 months of MDT. These results did not correlate with the bacterial index (BI) or the IgM antibody titre for the phenolic glycolipid (PGL)-1. One-dimensional densitometric analysis of agarose gels from PCR from the longitudinal study showed a gradual reduction of the 360-bp band after 12-24 months of MDT. RT-PCR for mRNA of the 18-kDa protein successfully tracked bacterial RNA changes in the biopsies and confirmed a decrease in the RNA of M. leprae in patients after MDT for 12 months. Thus, DNA- and RT-PCR for the 18-kDa protein of M. leprae are effective in assessing the efficacy of MDT for leprosy.

Sodium nitroprusside induces autophagic cell death in glutathione-depleted osteoblasts.

Previous studies reported that high levels of nitric oxide (NO) induce apoptotic cell death in osteoblasts. We examined molecular mechanisms of cytotoxic injury induced by sodium nitroprusside (SNP), a NO donor, in both glutathione (GSH)‐depleted and control U2‐OS osteoblasts. Cell viability was reduced by much lower effective concentrations of SNP in GSH‐depleted cells compared to normal cells. The data suggest that the level of intracellular GSH is critical in SNP‐induced cell death processes of osteoblasts. The level of oxidative stress due to SNP treatments doubled in GSH‐depleted cells when measured with fluorochrome H2DCFDA. Pretreatment with the NO scavenger PTIO preserved the viability of cells treated with SNP. Viability of cells treated with SNP was recovered by pretreatment with Wortmannin, an autophagy inhibitor, but not by pretreatment with zVAD‐fmk, a pan‐specific caspase inhibitor. Large increases of LC3‐II were shown by immunoblot analysis of the SNP‐treated cells, and the increase was blocked by pretreatment with PTIO or Wortmannin; this implies that under GSH‐depleted conditions SNP induces different molecular signaling that lead to autophagic cell death. The ultrastructural morphology of SNP‐treated cells in transmission electron microscopy showed numerous autophagic vacuoles. These data suggest NO produces oxidative stress and cellular damage that culminate in autophagic cell death of GSH‐depleted osteoblasts.

Detection of gene mutations related with drug resistance in Mycobacterium leprae from leprosy patients using Touch-Down (TD) PCR

The lack of methods to identify Mycobacterium leprae with the resistance against multi-drugs quickly and specifically has hindered effective chemotherapy against M. leprae infection. To screen M. leprae with resistance against multi-drugs, the Touch-Down (TD)-PCR has been used in this study. Sequences of the folP, rpoA, B, and gyrA, B genes were analyzed for isolates of M. leprae from leprosy patients in Korea. We amplified designated region of several genes in M. leprae involved in drug resistance and could obtain the PCR products of each gene. The mutations in the particular region of folP, rpoB, and gyrB gene were certified by TD-PCR single-stranded conformational polymorphism and DNA sequencing, respectively.

Rifampicin Inhibits the LPS-induced Expression of Toll-like Receptor 2 via the Suppression of NF-κB DNA-binding Activity in RAW 264.7 Cells

Rifampicin is a macrocyclic antibiotic which is used extensively for treatment against Mycobacterium tuberculosis and other mycobacterial infections. Recently, a number of studies have focused on the immune-regulatory effects of rifampicin. Therefore, we hypothesized that rifampicin may influence the TLR2 expression in LPS-activated RAW 264.7 cells. In this study, we determined that rifampicin suppresses LPS-induced TLR2 mRNA expression. The down-regulation of TLR2 expression coincided with decreased production of TNF-alpha. Since NF-kappaB is a major transcription factor that regulates genes for TLR2 and TNF-alpha, we examined the effect of rifampicin on the LPS-induced NF-kappaB activation. Rifampicin inhibited NF-kappaB DNA-binding activity in LPS-activated RAW 264.7 cells, while it did not affect IKKalpha/beta activity. However, rifampicin slightly inhibited the nuclear translocation of NF-kappaB p65. In addition, rifampicin increased physical interaction between pregnane X receptor, a receptor for rifampicin, and NF-kappaB p65, suggesting pregnane X receptor interferes with NF-kappaB binding to DNA. Taken together, our results demonstrate that rifampicin inhibits LPS-induced TLR2 expression, at least in part, via the suppression of NF-kappaB DNA-binding activity in RAW 264.7 cells. Thus, the present results suggest that the rifampicin-mediated inhibition of TLR2 via the suppression of NF-kappaB DNA-binding activity may be a novel mechanism of the immune-suppressive effects of rifampicin.

Mycobacterial infections in coal workers’ pneumoconiosis patients in South Korea

Coal workers’ pneumoconiosis (CWP) is the most common occupational disease in South Korea and is an important factor in the development of infections with Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM). In the current study, we identified mycobacterial species that cause pulmonary infections in CWP patients, using rpoB DNA-PCR-restriction analysis. Among the 129 CWP patients studied, 35 (27.1%) were diagnosed as having mycobacterial infections. Among these, the proportion of NTM infections (21/35, 60.0%) was higher than that for MTB infections (14/35, 40.0%). Of the 21 NTM strains, the most common was M. intracellulare (6/21, 28.6%), followed by M. avium (5/21, 23.8%). We also compared the characteristics of CWP patients between the MTB and NTM infection groups. A higher proportion of CWP patients with NTM infections compared with those with MTB infections had a history of having been involved in rock work (38.1% vs 21.4%), and had complicated CWP (85.7% vs 35.7%) and a past history of TB treatment (61.9% vs 50.0%). We also discovered 3 MTB mutants that are resistant to first-line anti-TB drugs, in CWP patients. These results demonstrate the features of pulmonary mycobacterial infections with a predominance of NTM in CWP patients in South Korea.

Effect of Rifampicin to Inhibit Rapamycin-Induced Autophagy via the Suppression of Protein Phosphatase 2A Activity

Recently, a number of studies have focused on the secondary effects of rifampicin. In the present study, we assessed whether rifampicin influences the rapamycin-induced autophagy of RAW 264.7 cells. Here, we demonstrate that the rapamycin-induced autophagy is dependent on protein phosphatase (PP) 2A activity and rifampicin inhibits the activity of PP2A by reducing expressions of PP2A subunits A and C. In addition, rifampicin slightly, but significantly, inhibited the rapamycin-induced dephosphorylation of p70 ribosomal protein S6 kinase (p70S6K) at Thr421/Ser424, which are regulated dually by both rapamycin and PP2A, but not at the rapamycin dephosphorylation site located at Thr389. These results show that rifampicin inhibits rapamycin-induced autophagy, at least in part, via the suppression of PP2A activity.

Foreign Body Reaction in Glaucoma Drainage Implant Surgery

PURPOSE To investigate the histopathology of the foreign body reaction (FBR) and the effect of aqueous humor on it in glaucoma drainage implant surgery. METHODS A glaucoma drainage device was implanted into 20 New Zealand white rabbits. We monitored the histopathology of blebs at microscopic levels from 3 days to 8 weeks postoperatively. Hematoxylin and eosin staining, Massons trichrome staining, anti-actin and α-smooth muscle immunofluorescence staining, and antiproliferating cell nuclear antigen immunohistochemistry were performed. To observe effects of aqueous humor on FBR, we designed two implant models. One group received a plate with a tube placed in the anterior chamber (experimental group), whereas the other received the plate cut from the tube (control group). RESULTS Foreign body giant cells were found along the inner border of blebs, and the innermost layer of blebs demonstrated a densely packed collagenous stratum in both groups. The number of foreign body giant cells was suppressed in the experimental group compared with the control group (P < 0.001). Fibroblast division was more active in the experimental group than that in the control group. Massons trichrome staining demonstrated that the innermost avascular collagenous layer was much thicker in the experimental group than that in the control group (P = 0.021). The extent of α-SMA staining was greater in the experimental group than that in the control group. CONCLUSIONS In the aqueous humor environment, wound healing around a glaucoma drainage implant revealed a unique FBR with the relatively small number of foreign body giant cells and reinforced fibrotic encapsulation.