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Ge-Jee-Bok-Ryung-Hwan (GJBRH), a commonly used herb formulation in Korea, Japan and China, caused a decrease of viability in HeLa human cervical carcinoma cells. The treatment of GJBRH resulted in genomic DNA fragmentation as well as the increase of Sub-G1 portion in cell cycle analysis. In this study, GFP-Bax over-expression system showed that Bax, pro-apoptotic Bcl-2 family protein, was translocated to mitochondria by the presence of GJBRH. The treatment of BAPTA-AM, permeable endogenous calcium chelator, inhibited GJBRH-induced caspase-3 and -9 activations, the release of cytochrome c and Smac/DIABLO into cytoplasm and the resultant cell death in HeLa human cervical carcinoma cells. The treatment of BAPTA-AM increased the expression of XIAP, which mediates binding to and inhibiting caspases and showed protective effect, in GJBRH-treated cells. GJBRH induced the expression of Glucose Response Protein 78 (GRP 78), a positive ER stress marker protein. However, BAPTA-AM did not interfere with the ER-stress response pathway that triggers the expression of GRP 78. This study showed that GJBRH induces cell death, which occurs downstream of or parallel to this point in the ER-stress pathway linked to apoptosis. In conclusion, GJBRH induces apoptosis in HeLa cells via ER stress-pathway associated mitochondria-dependent apoptosis mechansim.

Bax inhibitor-1 (BI-1) is an evolutionarily conserved endoplasmic reticulum (ER) protein that suppresses cell death in both animal and plant cells. We characterized mice in which the bi-1 gene was ablated. Cells from BI-1-deficient mice, including fibroblasts, hepatocytes, and neurons, display selective hypersensitivity to apoptosis induced by ER stress agents (thapsigargin, tunicamycin, brefeldin A), but not to stimulators of mitochondrial or TNF/Fas-death receptor apoptosis pathways. Conversely, BI-1 overexpression protects against apoptosis induced by ER stress. BI-1-mediated protection from apoptosis induced by ER stress correlated with inhibition of Bax activation and translocation to mitochondria, preservation of mitochondrial membrane potential, and suppression of caspase activation. BI-1 overexpression also reduces releasable Ca(2+) from the ER. In vivo, bi-1(-/-) mice exhibit increased sensitivity to tissue damage induced by stimuli that trigger ER stress, including stroke and tunicamycin injection. Thus, BI-1 regulates a cell death pathway important for cytopreservation during ER stress.

Substance P (SP) can stimulate secretion of tumour necrosis factor-alpha (TNF-alpha) from astrocytes stimulated with lipopolysaccharide (LPS). In this study, we have examined whether an aqueous extract of Sesim-Tang inhibits the secretion of TNF-alpha from primary cultures of rat astrocytes. Sesim-Tang (10-1000 microg/mL) significantly inhibited the TNF-alpha secretion by astrocytes stimulated with LPS and SP. Interleukin-1 (IL-1) has been shown to elevate TNF-alpha secretion from LPS-stimulated astrocytes while having no effect on astrocytes in the absence of LPS. We therefore examined whether IL-1 mediated inhibition of TNF-alpha secretion from primary astrocytes by Sesim-Tang. Treatment with Sesim-Tang (10-1000 microg/mL) of astrocytes stimulated with both LPS and SP decreased IL-1 secretion significantly. Moreover, the secretion of TNF-alpha by LPS and SP in astrocytes was progressively inhibited with increasing amounts of IL-1 neutralizing antibody. Our results suggest that Sesim-Tang may inhibit TNF-alpha secretion by inhibiting IL-1 secretion and that Sesim-Tang has an antiinflammatory activity in the central nervous system.

The aim of this study is to elucidate the possible mechanism of apoptosis in response to hypoxia in MC3T3E1 osteoblasts. MC3T3E1 osteoblasts under hypoxic conditions (2% oxygen) resulted in apoptosis in a time-dependent manner estimated by DNA fragmentation assay and nuclear morphologystained with fluorescent dye, Hoechst 33258. Pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, or Z-DEVD-CHO, a specific caspase-3 inhibitor, completely suppressed the DNA ladder in response to hypoxia. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase- activity (YVADase) was detected. To confirm what caspases are involved in apoptosis, western blot analysis was performed using anti-caspase-3 or -6 antibody. The 10-kDa protein, corresponding to the active products of caspase-3 and the 10-kDA protein of the active protein of caspase-6 were generated in hypoxia-challenged cells in which processing of the full length form of caspase-3 and -6 was evident. With a time course similar to this caspase-3 and -6 activation was evident, hypoxic stress caused the cleavage of lamin A, typical of caspase-6 activity. In addition, the stress elicited the release of cytochrome c into the cytosol during apoptosis. Furthermore, we have observed that pre-treatment with SB203580, a selective p38 MAP kinase (p38 MAPK) inhibitor, attenuated the hypoxia-induced apoptosis. The addition of SB203580 suppressed caspase-3 and -6-like protease activity by hypoxia up to 50%. In contrast, PD98059 had no effect on the hypoxia-induced apoptosis. To confirm the involvement of MAP kinase, JNK/SAPK, ERK, or p38 kinase assay was performed. Although p38 MAPK was activated in response to hypoxic treatment, the other MAP kinase -JNK/SAPK or ERK- was not or modestly activated. These results suggest that p38 MAPK positively regulates hypoxia-induced apoptosis in MC3T3E1 osteoblasts.

The objective of the currently study was to determine the effect of Kunbi-Boshin-Hangam-Tang (KBH-Tang) on the production of nitric oxide (NO). Stimulation of RAW 264.7 cells with KBH-Tang after the treatment of recombinant interferon-gamma (rIFN-gamma) resulted in increased NO synthesis. KBH-Tang partially increased NO synthesis by itself. When KBH-Tang was used in combination with rIFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) protein. NO production was inhibited by NG-monomethyl-L-arginine (NGMMA). Furthermore, activation of nuclear factor (NF)-kappaB was increased by KBH-Tang. These results suggest that KBH-Tang may stimulate the NO production through the activation of the NF-kappaB.

Nitric oxide (NO) induces apoptotic cell death and cAMP has a significantly protective effect on NO-induced cytotoxicity in human osteoblasts, MG-63 cells. Treatment with S-nitroso-N-acetylpenicillamine (SNAP) (0.6 mM) resulted in genomic DNA fragmentation, characteristic of apoptosis. However, concomitant incubation of the cells with either DBcAMP or forskolin markedly inhibited SNAP-induced apoptosis in a dose-dependent manner. Furthermore, pretreatment of MG-63 cells with H-89 or KT5720, which is known to inhibit cAMP-dependent protein kinase (PKA), abolished the protective effect of DBcAMP and forskolin on SNAP-induced apoptosis. In this study, we explored the involvement of caspases in the regulatory mechanism of SNAP-induced apoptosis by cAMP. Our data show that DBcAMP or forskolin blocked SNAP-induced caspase-3-like cysteine protease activation and that H-89, a PKA inhibitor, reversed the cAMP-induced regulatory effect of caspase-3 like protease. Consistent with the results, cAMP inhibited the proteolytic cleavage of caspase-3, -6, -9 and cytochrome c release to cytoplasm. The inhibition of caspase-3 activation did not block SNAP-induced cytochrome c release to cytoplasm, suggesting that caspase-3 activation may occur downstream of cytochrome c release. In summary, these findings show that the exposure of MG-63 cells to cAMP analogs renders them more resistant to NO-induced damage and suggests the presence of regulatory mechanisms of the cell death pathway by cAMP in which caspase-3, -6, and -9 and cytochrome c release serves to mediate NO-induced apoptosis.

The induction of interleukin-6 (IL-6), using a proinflammatory cytokine (tumor necrosis factor-alpha), was studied in a human osteoblast cell line (MG-63) in relation to p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB transcription factor. When added to MG-63 cells, tumor necrosis factor-alpha (TNF-alpha) had a stimulatory effect on the production of IL-6, and this elevation was significantly reduced by SB203580, a specific p38 MAPK inhibitor. In addition, the stimulation of IL-6 release was also reduced by pyrrolidine dithiocarbamate (PDTC) or NF-kappaB SN50, which has been reported to be a potent NF-kappaB inhibitor. Both the NF-kappaB inhibitors in the presence of SB203580 had a more inhibitory effect on IL-6 release. In this study, TNF-alpha stimulated NF-kappaB binding affinity as well as p38 MAP kinase activation, leading to the release of IL-6. However, the specific inhibitor of p38 MAPK, SB203580, had no effect on TNF-alpha-induced NF-kappaB activation and both NF-kappaB inhibitors failed to reduce the p38 MAPK activation in the TNF-alpha-stimulated osteoblasts. In addition, inhibition of p38 MAPK partially, but significantly, impaired TNF-alpha-regulated release of osteocalcin, an important differentiation marker in osteoblasts. These results strongly suggest that both p38 MAPK and NF-kappaB are required in TNF-alpha-induced IL-6 synthesis and that these two TNF-alpha-activated pathways can be primarily dissociated. Furthermore, p38 MAPK may play a significant role in differentiation in MG-63 cells.

High molecular weight water-soluble chitosan (WSC), having an average molecular weight of 300000 Da and a degree of deacethylation over 90%, can be produced using a simple multi-step membrane separation process. In this study, the effect of WSC on the production of nitric oxide (NO) in RAW 264.7 macrophages was evaluated. Water-insoluble chitosan alone has been previously shown to exhibit in vitro stimulatory effect on macrophages NO production. However, WSC had no effect on NO production by itself. When WSC was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of WSC on NO synthesis was shown 24 h after treatment with rIFN-gamma. The increased production of NO from rIFN-gamma plus WSC-stimulated RAW 264.7 macrophages was decreased by the treatment with N(G)-monomethyl-L-arginine (N(G)MMA). The increase in NO synthesis was reflected, as an increased amounts of inducible NO synthase protein. In addition, synergy between rIFN-gamma and WSC was mainly dependent on WSC-induced tumor necrosis factor-alpha (TNF-alpha) and nuclear factor-kappaB (NF-kappaB) activation. The present results indicate that the capacity of WSC to increase NO production from rIFN-gamma-primed RAW 264.7 macrophages is the result of WSC-induced TNF-alpha secretion via the signal transduction pathway of NF-kappaB activation.

The effects of hydrogen peroxide, which fetal bovine serum (FBS) releases, on proliferation have been studied in ROS 17/2.8 osteoblasts. Cell proliferation, when activated by FBS, was inhibited by catalase in ROS 17/2.8 osteoblasts, but did not in primary osteoblast-like cells. Serum-induced extracellular signal-regulated kinase(ERK) activity was reduced by the pretreated catalase in ROS 17/2.8 osteoblasts. In addition, the present studies demonstrate that addition of FBS led to an increase of fluorescence of dihydrorhodamine 123, indicating formation of free radicals including hydrogen peroxide in ROS 17/2.8 osteoblasts, but not in primary osteoblast-like cells. These phenomena may account for the generation of reactive oxygen species during cellular proliferation in ROS 17/2.8 osteoblasts.

Substance P (SP) can stimulate production of tumor necrosis factor-alpha (TNF-alpha) from astrocytes stimulated with lipopolysaccharide (LPS). The objective of the current study was to determine the effect of Taraxacum officinale (TO) on the production of TNF-alpha from primary cultures of rat astrocytes. TO (100 and 1000 microg/ml) significantly inhibited the TNF-alpha production by astrocytes stimulated with LPS and SP. Interleukin-1 (IL-1) has been shown to elevate TNF-alpha production from LPS-stimulated astrocytes while having no effect on astrocytes in the absence of LPS. We therefore examined whether IL-1 mediated inhibition of TNF-alpha production from primary astrocytes by TO. Treatment of TO (100 and 1000 microg/ml) to astrocytes stimulated with both LPS and SP decreased IL-1 production significantly. Moreover, the production of TNF-alpha by LPS and SP in astrocytes was progressively inhibited with increasing amount of IL-1 neutralizing antibody. Our results suggest that TO may inhibit TNF-alpha production by inhibiting IL-1 production and that TO has an antiinflammatory activity in the central nervous system.

Although the acceleration of bone regeneration by radiation has been reported, the mechanisms of action of radiation on bone are unclear. The present results indicate that ionizing radiation-stimulated differentiation could result from the generation of reactive oxygen species during radiation exposure. The free radical release is considered as the most important mechanism of bone effect by radiation treatment. In addition, we report that radiation induced transient activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation and the transcription factor, AP-1. The JNK and AP-1 activation is mediated with radiation-released free radicals in ROS 17/2.8 osteoblasts. These results indicate that ionizing radiation at a single dose of up to 5 Gray stimulates differentiation of ROS 17/2.8 osteoblasts via free radial release which may affect JNK/SAPK and AP-1 activities.

Caffeine is known to reduce evoked histamine secretion, but the effects of caffeine on anaphylactic shock have not been clarified. We have investigated the effects of caffeine on anaphylactic shock in rats. Systemic anaphylactic shock by compound 48/80 injection was monitored for 1 h. An IgE-dependent local anaphylactic shock was generated by sensitizing the skin with anti-dinitrophenyl (DNP) IgE followed 48 h later with an injection of antigen. Caffeine inhibited compound 48/80-induced anaphylatic shock to 40% with a dose of 1 mg/kg. Caffeine (0.1 mg/kg) inhibited to 56.4+/-0.4% passive cutaneous anaphylactic shock activated by anti-DNP IgE. Caffeine (5-20 mM) significantly inhibited histamine release from rat peritoneal mast cells (RPMCs) activated by compound 48/80 or anti-DNP IgE. Especially, caffeine (20 mM) inhibited by 96.7+/-0.5% histamine release activated by compound 48/80. Moreover, caffeine (1-20 mM) had a significant inhibitory effect on anti-DNP IgE-induced tumor necrosis factor-alpha production from RPMCs. The level of cAMP in RPMCs, when caffeine (20 mM) was added, increased significantly after 5-60 min compared with that of a normal control. These results indicate that caffeine inhibits immediate-type allergic reactions by inhibition of mast cell degranulation in vivo and in vitro.

Osteotropic hormones and cytokines are involved in the differentiation of osteoclast progenitors from haematopoietic stem cells to multinucleated osteoclasts which mediate bone resorption. Stem cell factor, interleukin-6, nitric oxide, and transforming growth factor-beta are implicated in the regulation of bone resorption by osteoclast. We test whether stem cell factor, interleukin-6, nitric oxide, and transforming growth factor-beta affect the generation of osteoclast-like multi-nucleated cells induced by 1 alpha,25-(OH)2D3. 1 alpha,25-(OH)2D3 increase the generation of osteoclast-like cells retaining osteoclast characteristics including multinuclearity and positive staining for tartrate-resistant acid phosphatase. Combined treatment of stem cell factor with interleukin-6 synergistically potentiates the ability of 1 alpha,25-(OH)2D3 to generate tartrate-resistant acid phosphatase-positive multinucleated cells. However, either stem cell factor or interleukin-6 alone does not induce the generation of tartrate-resistant acid phosphatase-positive multinucleated cells. Transforming growth factor-beta produces a biphasic effect on osteoclast generation induced by 1 alpha,25-(OH)2D3. Transforming growth factor-beta stimulates osteoclast generation at low concentration (0.1 ng/ml) whereas it suppresses the formation of osteoclast-like cell at higher concentration (1 ng/ml). Sodium nitroprusside, a donor of nitric oxide, almost completely inhibits the generation of 1 alpha,25-(OH)2D3-induced osteoclast at high concentration (100 microM), but it significantly enhances the osteoclast generation at low concentrations (3 microM). These results suggest that stem cell factor, interleukin-6, transforming growth factor-beta, and nitric oxide interact with 1 alpha,25-(OH)2D3 to modulate the differentiation of hematopoietic precursors toward committed osteoclast precursors.

Nitric oxide (NO) is known to be implicated in the metabolism of bone, especially as a mediator of cytokine effects on the remodelling of bone tissue. In this study we examine whether NO affects the osteoblast activation or the osteoclast differentiation of primary mouse osteoblast-like and osteosarcoma ROS 17/2.8 cell lines. Primary osteoblast and ROS 17/2.8 cells released NO upon stimulation of interleukin-1β, tumour necrosis factor-α, and interferon-γ. Sodium nitroprusside, a donor of nitric oxide, increased the activity of alkaline phosphatase in ROS 17/2.8 cells as well as the number of calcified nodule formations in primary mouse osteoblast-like cells. Sodium nitroprusside also completely inhibited 1α,25-(OH)2D3-induced osteoclast generation in a high concentration (100 μm). However, a low concentration of sodium nitroprusside (3–30 μm) significantly increased the generation of osteoclasts. These results indicated that NO appears to be an important regulatory molecule in the processes of bone formation and resorption. Hence, NO may be involved in the pathogenesis of bone loss in diseases associated with cytokine activation, such as periodontal disease and rheumatoid arthritis.

The survival of mast cells are dependent on two kinds of growth factors, one derived from T cells (IL-3) and another derived from fibroblasts (stem cell factor [SCF]). The 3T3 fibroblast cell line derived from WCB6F(1-)+/+ mouse embryos (+/+ 3T3 fibroblasts) supported the proliferation of bone marrow-derived cultured mast cells (BMCMC) in the PWM-stimulated spleen cell conditioned medium (PWM-SCM), whereas the 3T3 fibroblast cell line from WCB6F1-Sl/Sld mouse embryos (Sl/Sld 3T3 fibroblasts) did not. To study the role of nitric oxide (NO) on the growth of mast cells in BMCMC-fibroblast coculture, we used a NO synthase inhibitor, NG-monomethyl-L-arginine (NGMMA). NGMMA recovered survival and maintained proliferation of mast cells in BMCMC-Sl/Sld 3T3 fibroblasts coculture. Sl/Sld 3T3 fibroblasts as well as 3T3 fibroblasts from NIH(-)+/+, BALB(-)+/+ or Swiss(-)+/+ mouse embryos secreted NO in PWM-SCM, but not in alpha-MEM. SCF protected BMCMC from cytotoxicity of exogenous NO in IL-3-supplemented alpha-MEM. We concluded that SCF might protect BMCMC from cytocidal effect of NO in BMCMC-fibroblasts coculture.

The effect of glycyrrhetinic acid (18-O-beta-glycyrrhetinic acid, GA) on histamine metabolism was investigated in cultured mast cells (CMCs) cocultured with Swiss 3T3 fibroblasts. GA strongly inhibited histamine synthesis in the cocultured CMCs. Since 50 microM GA inhibited about 80% of histidine decarboxylase (HDC) activity, the inhibitory activity of GA for histamine synthesis was considered to be derived from the inhibition of HDC activity. The number of berberine-sulfate-positive cells also decreased in the presence of GA, which indicated that maturation of CMCs was inhibited by GA. Furthermore, we examined the effect of GA on the mRNA expression of novel protein kinase C delta (nPKC delta), a major isoform of CMCs, by northern blot analysis. The expression of nPKC delta mRNA in the presence of GA was significantly lower than in the absence of GA. These results suggest the possibility that the inhibition of histamine synthesis by GA is regulated by nPKC delta.

Rubus coreanus fruits have been employed as a traditional medicine for centuries in the Asia-Pacific region. Its pharmacological action differs according to the different extraction methods utilized and the degree of fruit ripening. In this study, we determined the cellular effect of different ethanol extracts of mature and immature Rubus coreanus fruits in human hepatic cell line, HepG2 cells. The antioxidant activity, effect on superoxide dismutase activity and cholesterol biosynthesis efficiency was also evaluated. Immature Rubus coreanus extract showed higher antioxidant capability, compared with that of its mature fractions. Cellular antioxidant proteins including HO-1, Cu/Zn-superoxide dismutase and catalase were highly expressed in the presence of Rubus coreanus. Cholesterol levels in HepG2 cells treated with the water fraction of immature Rubus coreanus were significantly reduced. This antihyperlipidaemic action of Rubus coreanus is a consequence of cholesterol biosynthesis and extracellular secretion in HepG2 cells. These results indicate that among different ethanol fraction of mature and immature Rubus coreanus fruit extracts, water extract of immature fruit extract shows higher antioxidant as well as higher antihyperlipidaemic action.

OBJECTIVES Manganese chloride (MnCl(2)) is one of heavy metals for causing neurogenerative dysfunction like Manganism. The purpose of this study was to determine the acute toxicity of MnCl(2) using different times and various concentrations including whether manganese toxicity may involve in two intrinsic pathways, endoplasmic reticulum (ER) stress and mitochondria dysfunction and lead to neuronal apoptosis mediated by organelle disorders in neuroblastoma cell line SK-N-MC. METHODS In the acute toxicity test, five concentrations (200, 400, 600, 800, 1,000 uM) of MnCl(2) with 3, 6, 12, 24, 48 hours exposure were selected to analyze cell viability. In addition, to better understand their toxicity, acute toxicity was examined with 1,000 uM MnCl(2) for 24 hours exposure via reactive oxygen species (ROS), mitochondria membrane potential, western blotting and mitochondrial complex activities. RESULTS Our results showed that both increments of dose and time prompt the increments in the number of dead cells. Cells treated by 1,000 µM MnCl(2) activated 265% (±8.1) caspase-3 compared to control cell. MnCl(2) induced intracellular ROS produced 168% (±2.3%) compared to that of the control cells and MnCl(2) induced neurotoxicity significantly dissipated 48.9% of mitochondria membrane potential compared to the control cells. CONCLUSIONS This study indicated that MnCl(2) induced apoptosis via ER stress and mitochondria dysfunction. In addition, MnCl(2) affected only complex I except complex II, III or IV activities.

The aim of this study was to evaluate the preventive role of epigallocatechin-3 gallate (EGCG, a derivative of green tea) in ischemia/reperfusion (I/R) injury of isolated rat hearts. It has been suggested that EGCG has beneficial health effects, including prevention of cancer and heart disease, and it is also a potent antioxidant. Rat hearts were subjected to 20 min of normoxia, 20 min of zero-flow ischemia and then 50 min of reperfusion. EGCG was perfused 10 min before ischemia and during the whole reperfusion period. EGCG significantly increased left ventricular developed pressure (LVDP) and increased maximum positive and negative dP/dt (+/-dP/dtmax). EGCG also significantly increased the coronary flow (CF) at baseline before ischemia and at the onset of the reperfusion period. Moreover, EGCG decreased left ventricular end diastolic pressure (LVEDP). This study showed that lipid peroxydation was inhibited and Mn-SOD and catalase expressions were increased in the presence of EGCG. In addition, EGCG increased levels of Bcl-2, Mn-superoxide dismutase (SOD), and catalase expression and decreased levels of Bax and increased the ratio of Bcl-2/Bax in isolated rat hearts. Cleaved caspase-3 was decreased after EGCG treatment. EGCG markedly decreased the infarct size while attenuating the increase in lactate dehydrogenase (LDH) levels in the effluent. In summary, we suggest that EGCG has a protective effect on I/R-associated hemodynamic alteration and injury by acting as an antioxidant and anti-apoptotic agent in one.

High glucose leads to physio/pathological alterations in diabetes patients. We investigated collagen production in human gingival cells that were cultured in high concentrations of glucose. Collagen synthesis and secretion were increased when the cells were exposed to high concentrations of glucose. We examined endoplasmic reticulum (ER) stress response because glucose metabolism is related to ER functional status. An ER stress response including the expression of glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), inositol requiring enzyme alpha (IRE-1α) and phosphoreukaryotic initiation factor alpha (p-eIF-2α) was activated in the presence of high glucose. Activating transcription factor 4 (ATF-4), a downstream protein of p-eIF-2α as well as a transcription factor for collagen, was also phosphorylated and translocalized into the nucleus. The chemical chaperone 4-PBA inhibited the ER stress response and ATF-4 phosphorylation as well as nuclear translocation. Our results suggest that high concentrations of glucose-induced collagen are linked to ER stress and the associated phosphorylation and nuclear translocation of ATF-4.

Introduction Synovial fibroblasts from rheumatoid arthritis show resistance to apoptotic stimuli, indicating they may be difficult to treat. To clearly understand these mechanisms of resistance, rheumatoid and osteoarthritis synovial fibroblasts (RASF and OASF) were exposed to endoplasmic reticulum (ER) stress such as thapsigargin, Ca2+-ATPase inhibitor. Methods Fibroblasts were assessed microscopically for cell viability by trypan blue exclusion and for autophagic cells by LC-3II formation. Caspase-3 activity was measured as aminomethyl-coumarin (AMC) liberated from AC-DEVD-AMC. Immunoblotting was performed to compare protein expression in OASF and RASF. Results ER stress caused cell death in OASF but not in RASF. Thapsigargin, a Ca2+-ATPase inhibitor, did not change the expression of GRP78, an ER chaperone in OASF and RASF, but induced another ER stress protein, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) differently, showing high levels in OASF and low levels in RASF. Thapsigargin increased the autophagy response in RASF, with autophagosome formation, beclin expression, and LC3-II conversion. Transfection with beclin siRNA inhibited autophagy and increased the susceptibility to ER stress-induced cell death. On the other hand, CHOP siRNA increased autophagy and improved cell survival, especially in RASF, indicating that CHOP is involved in regulation of autophagy and cell death, but that low expression of CHOP protects RASF from apoptosis. Conclusions Autophagy induction and CHOP under-expression increases cell resistance against ER stress-induced cell death in fibroblasts from rheumatoid arthritis patients.

Hypothesis / aims of study Urethral stricture after transurethral resection of the prostate (TURP) is the most common late troublesome complication (1). However, the understanding for pathogenesis or predictive factors of urethral stricture formation is still insufficient. The aim of this study is to evaluate possible factors affecting urethral stricture development after TURP in patients with benign prostatic hyperplasia (BPH).