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Although Krüppel-associated box domain-containing zinc-finger proteins (K-ZNFs) may be associated with sophisticated gene regulation in higher organisms, the physiological functions of most K-ZNFs remain unknown. The Zfp212 protein was highly conserved in mammals and abundant in the brain; it was mainly expressed in the cerebellum (Cb). Zfp212 (mouse homolog of human ZNF212) knockout (Zfp212-KO) mice showed a reduction in survival rate compared to wild-type mice after 20 months of age. GABAergic Purkinje cell degeneration in the Cb and aberrant locomotion were observed in adult Zfp212-KO mice. To identify genes related to the ataxia-like phenotype of Zfp212-KO mice, 39 ataxia-associated genes in the Cb were monitored. Substantial alterations in the expression of ataxin 10, protein phosphatase 2 regulatory subunit beta, protein kinase C gamma, and phospholipase D3 (Pld3) were observed. Among them, Pld3 alone was tightly regulated by Flag-tagged ZNF212 overexpression or Zfp212 knockdown in the HT22 cell line. The Cyclic Amplification and Selection of Targets assay identified the TATTTC sequence as a recognition motif of ZNF212, and these motifs occurred in both human and mouse PLD3 gene promoters. Adeno-associated virus-mediated introduction of human ZNF212 into the Cb of 3-week-old Zfp212-KO mice prevented Purkinje cell death and motor behavioral deficits. We confirmed the reduction of Zfp212 and Pld3 in the Cb of an alcohol-induced cerebellar degeneration mouse model, suggesting that the ZNF212–PLD3 relationship is important for Purkinje cell survival.

Aggregates of disease-causing proteins dysregulate cellular functions, thereby causing neuronal cell loss in diverse neurodegenerative diseases. Although many in vitro or in vivo studies of protein aggregate inhibitors have been performed, a therapeutic strategy to control aggregate toxicity has not been earnestly pursued, partly due to the limitations of available aggregate models. In this study, we established a tetracycline (Tet)-inducible nuclear aggregate (β23) expression model to screen potential lead compounds inhibiting β23-induced toxicity. High-throughput screening identified several natural compounds as nuclear β23 inhibitors, including peucedanocoumarin III (PCIII). Interestingly, PCIII accelerates disaggregation and proteasomal clearance of both nuclear and cytosolic β23 aggregates and protects SH-SY5Y cells from toxicity induced by β23 expression. Of translational relevance, PCIII disassembled fibrils and enhanced clearance of cytosolic and nuclear protein aggregates in cellular models of huntingtin and α-synuclein aggregation. Moreover, cellular toxicity was diminished with PCIII treatment for polyglutamine (PolyQ)-huntingtin expression and α-synuclein expression in conjunction with 6-hydroxydopamine (6-OHDA) treatment. Importantly, PCIII not only inhibited α-synuclein aggregation but also disaggregated preformed α-synuclein fibrils in vitro. Taken together, our results suggest that a Tet-Off β23 cell model could serve as a robust platform for screening effective lead compounds inhibiting nuclear or cytosolic protein aggregates. Brain-permeable PCIII or its derivatives could be beneficial for eliminating established protein aggregates.

Transforming growth factor-β (TGF-β) acts as a key cytokine in epithelial−mesenchymal transition (EMT) and myofibroblast differentiation, which are important for normal tissue repair and fibrotic diseases. Ubiquitylation and proteasomal degradation of TGF-β signaling proteins acts as a regulatory mechanism for the precise control of TGF-β signaling. SMAD-specific ubiquitin E3 ligase (SMAD ubiquitination regulatory factor 2, SMURF2) controls TGF-β signaling proteins including the TGF-β receptor (TGFR) and SMAD2/3. Here, we report that tetratricopeptide repeat domain 3 (TTC3), a ubiquitin E3 ligase, positively regulates TGF-β1-induced EMT and myofibroblast differentiation, through inducing ubiquitylation and proteasomal degradation of SMURF2. In human bronchial epithelial cells (BEAS-2B) and normal human lung fibroblasts, TTC3 knockdown suppressed TGF-β1-induced EMT and myofibroblast differentiation, respectively. Similarly, when TTC3 expression was suppressed, the TGF-β1-stimulated elevation of p-SMAD2, SMAD2, p-SMAD3, and SMAD3 were inhibited. In contrast, overexpression of TTC3 caused both EMT and myofibroblast differentiation in the absence of TGF-β1 treatment. TGF-β1 reduced SMURF2 levels and TTC3 overexpression led to a further decrease in SMURF2 levels, while TTC3 knockdown inhibited TGF-β1-induced SMURF2 reduction. In cell and in vitro ubiquitylation assays demonstrated TTC3-mediated SMURF2 ubiquitylation, and coimmunoprecipitation assays established the binding between SMURF2 and TTC3. TGF-β1-induced TTC3 expression was inhibited by the knockdown of SMAD2 and SMAD3. Finally, Ttc3 mRNA levels were significantly increased and Smurf2 protein levels were significantly decreased in the lungs of mice treated with bleomycin as compared with the lungs of control mice. Collectively, these data suggest that TTC3 may contribute to TGF-β1-induced EMT and myofibroblast differentiation, potentially through SMURF2 ubiquitylation/proteasomal degradation and subsequent inhibition of SMURF2-mediated suppression of SMAD2 and SMAD3, which in turn induces TTC3 expression.

Progressive degeneration of dopaminergic neurons characterizes Parkinson’s disease (PD). This neuronal loss occurs through diverse mechanisms, including a form of programmed cell death dependent on poly-(ADP-ribose) polymerase-1 (PARP1) called parthanatos. Deficient activity of the kinase Akt1 and aggregation of the protein α-synuclein are also implicated in disease pathogenesis. Here, we found that Akt1 suppressed parthanatos in dopaminergic neurons through a transcriptional mechanism. Overexpressing constitutively active Akt1 in SH-SY5Y cells or culturing cells with chlorogenic acid (a polyphenol found in coffee that activates Akt1) stimulated the CREB-dependent transcriptional activation of the gene encoding the E3 ubiquitin ligase RNF146. RNF146 inhibited PARP1 not through its E3 ligase function but rather by binding to and sequestering PAR, which enhanced the survival of cultured cells exposed to the dopaminergic neuronal toxin 6-OHDA or α-synuclein aggregation. In mice, intraperitoneal administration of chlorogenic acid activated the Akt1-CREB-RNF146 pathway in the brain and provided neuroprotection against both 6-OHDA and combinatorial α-synucleinopathy in an RNF146-dependent manner. Furthermore, dysregulation of the Akt1-CREB pathway was observed in postmortem brain samples from PD patients. The findings suggest that therapeutic restoration of RNF146 expression, such as by activating the Akt1-CREB pathway, might halt neurodegeneration in PD.

Progressive degeneration of dopaminergic neurons characterizes Parkinson's disease (PD). This neuronal loss occurs through diverse mechanisms, including a form of programmed cell death dependent on poly(ADP-ribose) polymerase-1 (PARP1) called parthanatos. Deficient activity of the kinase Akt1 and aggregation of the protein α-synuclein are also implicated in disease pathogenesis. Here, we found that Akt1 suppressed parthanatos in dopaminergic neurons through a transcriptional mechanism. Overexpressing constitutively active Akt1 in SH-SY5Y cells or culturing cells with chlorogenic acid (a polyphenol found in coffee that activates Akt1) stimulated the CREB-dependent transcriptional activation of the gene encoding the E3 ubiquitin ligase RNF146. RNF146 inhibited PARP1 not through its E3 ligase function but rather by binding to and sequestering PAR, which enhanced the survival of cultured cells exposed to the dopaminergic neuronal toxin 6-OHDA or α-synuclein aggregation. In mice, intraperitoneal administration of chlorogenic acid activated the Akt1-CREB-RNF146 pathway in the brain and provided neuroprotection against both 6-OHDA and combinatorial α-synucleinopathy in an RNF146-dependent manner. Furthermore, dysregulation of the Akt1-CREB pathway was observed in postmortem brain samples from patients with PD. The findings suggest that therapeutic restoration of RNF146 expression, such as by activating the Akt1-CREB pathway, might halt neurodegeneration in PD.

Parkinson&rsquo, s disease (PD) is the most common neurodegenerative movement disorder, characterized by olfactory dysfunction in the early stages. &alpha, Synuclein pathologies in the olfactory organs are shown to spread to the brain through the nose-brain axis. We first developed a nasal epithelial PD cellular model by treating RPMI-2650 cells with &alpha, synuclein preformed fibrils (PFF). Upon uptake of PFF, RPMI-2650 cells showed mitochondrial proteome alteration and downregulation of parkin, which has previously been identified as a nasal biomarker of PD. Functional cluster analysis of differentially expressed genes in RPMI-2650 cells revealed various pathways affected by &alpha, synuclein pathology, including the detection of chemical stimulus involved in sensory perception, olfactory receptor activity, and sensory perception of smell. Among genes that were most affected, we validated, by real-time quantitative PCR, the downregulation of MAP3K8, OR10A4, GRM2, OR51B6, and OR9A2, as well as upregulation of IFIT1B, EPN1, OR1D5, LCN, and OTOL1 in PFF-treated RPMI-2650 cells. Subsequent analyses of clinical samples showed a downregulation of OR10A4 and OR9A2 transcripts and an upregulation of IFIT1B in cells isolated from the nasal fluid of PD patients, as compared to those from the controls (cutoff value = 0.5689 for OR9A2, with 72.4% sensitivity and 75% specificity, and 1.4658 for IFIT1B, with 81.8% sensitivity and 77.8% specificity). Expression levels of these nasal PD markers were not altered in nasal fluid cells from SWEDD (scans without evidence of dopaminergic deficits) patients with PD-like motor symptoms. These nasal markers were significantly altered in patients of PD with hyposmia compared to the control hyposmic subjects. Our results validated the &alpha, synuclein-treated nasal epithelial cell model to identify novel biomarkers for PD and suggest the utility of olfactory transcripts, along with olfactory dysfunction, in the diagnosis of PD.

// Hojin Kang 1,2,* , Rin Khang 1,2,* , Sangwoo Ham 1,* , Ga Ram Jeong 3 , Hyojung Kim 1 , Minkyung Jo 1 , Byoung Dae Lee 3 , Yun Il Lee 4 , Areum Jo 1,2 , ChiHu Park 5,6 , Hyein Kim 1,5 , Jeongkon Seo 7 , Sun Ha Paek 8 , Yun-Song Lee 1 , Jeong-Yun Choi 1 , Yunjong Lee 1 and Joo-Ho Shin 1,2 1 Department of Molecular Cell Biology, Division of Pharmacology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon, South Korea 2 Single Cell Network Research Center, Sungkyunkwan University School of Medicine, Suwon, South Korea 3 Department of Neuroscience, Department of Physiology, Neurodegeneration Control Research Center, Kyung Hee University School of Medicine, Seoul, South Korea 4 Department of New Biology, Daegu Geongbuk Institute of Science and Technology, Daegu, South Korea 5 Research Core Facility, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon, South Korea 6 HuGeX Co., Ltd. Seongnam, South Korea 7 UNIST Central Research Facility, Ulsan National Institute of Science and Technology, Ulsan, South Korea 8 Department of Neurosurgery, Seoul National University College of Medicine, Seoul, South Korea * These authors have contributed equally to this work Correspondence to: Yunjong Lee, email: // Joo-Ho Shin, email: // Keywords : metformin, dopaminergic, Parkinson’s disease, PGC-1α, mitochondria, Gerotarget Received : October 10, 2016 Accepted : April 24, 2017 Published : May 24, 2017 Abstract Progressive dopaminergic neurodegeneration is responsible for the canonical motor deficits in Parkinson’s disease (PD). The widely prescribed anti-diabetic medicine metformin is effective in preventing neurodegeneration in animal models; however, despite the significant potential of metformin for treating PD, the therapeutic effects and molecular mechanisms underlying dopaminergic neuroprotection by metformin are largely unknown. In this study, we found that metformin induced substantial proteomic changes, especially in metabolic and mitochondrial pathways in the substantia nigra (SN). Consistent with this data, metformin increased mitochondrial marker proteins in SH-SY5Y neuroblastoma cells. Mitochondrial protein expression by metformin was found to be brain region specific, with metformin increasing mitochondrial proteins in the SN and the striatum, but not the cortex. As a potential upstream regulator of mitochondria gene transcription by metformin, PGC-1α promoter activity was stimulated by metformin via CREB and ATF2 pathways. PGC-1α and phosphorylation of ATF2 and CREB by metformin were selectively increased in the SN and the striatum, but not the cortex. Finally, we showed that metformin protected dopaminergic neurons and improved dopamine-sensitive motor performance in an MPTP-induced PD animal model. Together these results suggest that the metformin-ATF2/CREB-PGC-1α pathway might be promising therapeutic target for PD.

Activation of the Nlrp3 inflammasome consisting of three major components, Nlrp3, Asc, and pro-caspase-1, results in the activation of caspase-1 and subsequent proteolytic cleavage of pro-IL-1β and pro-IL-18. To avoid excessive inflammatory response, the Nlrp3 inflammasome has to be precisely controlled. In this study, we show that the mouse mitochondrial E3 ubiquitin protein ligase (Mul1) suppresses Nlrp3 inflammasome activation through ubiquitination and degradation of Asc. In J774A.1 cells, Mul1 overexpression attenuated Nlrp3 activation, whereas Mul1 knockdown augmented Nlrp3 activation in terms of IL-1β secretion and cleavage of pro-caspase-1 and pro-IL-1β. Mul1 interacted with Asc, and ubiquitinated it at K21, K22, K26, and K55 residues, in a K48-linked manner, leading to proteasomal degradation. Convincingly, Mul1-mediated suppression of Nlrp3 activation was inhibited by K21R-, K22R-, K26R-, K52R-Asc mutants in RAW264.7 cells, when compared with the wild-type Asc. Furthermore, Aim2 inflammasome activation was also inhibited by Mul1 in the wild-type Asc-, but not in mutant Asc-expressing RAW264.7 cells. Taken together, these data suggest that Mul1 suppresses Nlrp3 inflammasome activation, through Asc ubiquitination and degradation.

The motor and nonmotor symptoms of Parkinson&rsquo, s disease (PD) correlate with the formation and propagation of aberrant &alpha, synuclein aggregation. This protein accumulation is a pathological hallmark of the disease. Our group recently showed that peucedanocoumarin III (PCIII) possesses the ability to disaggregate &beta, sheet aggregate structures, including &alpha, synuclein fibrils. This finding suggests that PCIII could be a therapeutic lead compound in PD treatment. However, the translational value of PCIII and its safety information have never been explored in relevant animal models of PD. Therefore, we first designed and validated a sequence of chemical reactions for the large scale organic synthesis of pure PCIII in a racemic mixture. The synthetic PCIII racemate facilitated clearance of repeated &beta, sheet aggregate (&beta, 23), and prevented &beta, 23-induced cell toxicity to a similar extent to that of purified PCIII. Given these properties, the synthetic PCIII&rsquo, s neuroprotective function was assessed in 6-hydroxydopamine (6-OHDA)-induced PD mouse models. The PCIII treatment (1 mg/kg/day) in a 6-OHDA-induced PD mouse model markedly suppressed Lewy-like inclusions and prevented dopaminergic neuron loss. To evaluate the safety profiles of PCIII, high dose PCIII (10 mg/kg/day) was administered intraperitoneally to two-month-old mice. Following 7 days of PCIII treatment, PCIII distributed to various tissues, with substantial penetration into brains. The mice that were treated with high dose PCIII had no structural abnormalities in the major organs or neuroinflammation. In addition, high dose PCIII (10 mg/kg/day) in mice had no adverse impact on motor function. These findings suggest that PCIII has a relatively high therapeutic index. Given the favorable safety features of PCIII and neuroprotective function in the PD mouse model, it may become a promising disease-modifying therapy in PD to regulate pathogenic &alpha, synuclein aggregation.

By virtue of the critical roles of Akt in vascular endothelial cell (EC) survival and function, cigarette smoke-induced Akt reduction may contribute to EC death and dysfunction in smokers' lungs. One of the negative Akt regulatory mechanisms is K48-linked Akt ubiquitination and subsequent proteasomal degradation. Here, we assessed the involvement of mitochondrial E3 ubiquitin protein ligase 1 (MUL1), recently revealed as a novel Akt ubiquitin E3 ligase, in cigarette smoke-induced Akt ubiquitination and its contribution to pulmonary EC death and dysfunction. In human lung microvascular ECs (HLMVECs), cigarette smoke extract (CSE) noticeably elevated MUL1 expression and K48-linked Akt ubiquitination, whereas Akt, p-Akt, eNOS, and p-eNOS levels were decreased. MUL1 knockdown suppressed CSE-induced Akt ubiquitination/degradation and cytoplasmic reductions of Akt and p-Akt. Furthermore, MUL1 knockdown attenuated reductions of eNOS and p-eNOS and alleviated EC survival, migration, and tube formation in the presence of CSE exposure. In addition, overexpression of K284R Akt, a mutant for a MUL1-ubiquitination site, produced similar effects. In HLMVECs exposed to CSE, Akt-MUL1 interaction was increased in coimmunoprecipitation and in situ proximity ligation assays. Similarly, the proximity ligation assay signals were elevated in rat lungs exposed to cigarette smoke for 3 months, during which Mul1 levels were noticeably increased. Finally, we found that CSE-mediated MUL1 induction in HLMVECs is mediated by retinoic acid receptor-related orphan receptor α. Taken together, these data suggest that cigarette smoke-induced MUL1 elevation mediates Akt ubiquitination/degradation, potentially leading to pulmonary EC death and functional impairment.

Transforming growth factor-β (TGF-β) acts as a key cytokine in epithelial−mesenchymal transition (EMT) and myofibroblast differentiation, which are important for normal tissue repair and fibrotic diseases. Ubiquitylation and proteasomal degradation of TGF-β signaling proteins acts as a regulatory mechanism for the precise control of TGF-β signaling. SMAD-specific ubiquitin E3 ligase (SMAD ubiquitination regulatory factor 2, SMURF2) controls TGF-β signaling proteins including the TGF-β receptor (TGFR) and SMAD2/3. Here, we report that tetratricopeptide repeat domain 3 (TTC3), a ubiquitin E3 ligase, positively regulates TGF-β1-induced EMT and myofibroblast differentiation, through inducing ubiquitylation and proteasomal degradation of SMURF2. In human bronchial epithelial cells (BEAS-2B) and normal human lung fibroblasts, TTC3 knockdown suppressed TGF-β1-induced EMT and myofibroblast differentiation, respectively. Similarly, when TTC3 expression was suppressed, the TGF-β1-stimulated elevation of p-SMAD2, SMAD2, p-SMAD3, and SMAD3 were inhibited. In contrast, overexpression of TTC3 caused both EMT and myofibroblast differentiation in the absence of TGF-β1 treatment. TGF-β1 reduced SMURF2 levels and TTC3 overexpression led to a further decrease in SMURF2 levels, while TTC3 knockdown inhibited TGF-β1-induced SMURF2 reduction. In cell and in vitro ubiquitylation assays demonstrated TTC3-mediated SMURF2 ubiquitylation, and coimmunoprecipitation assays established the binding between SMURF2 and TTC3. TGF-β1-induced TTC3 expression was inhibited by the knockdown of SMAD2 and SMAD3. Finally, Ttc3 mRNA levels were significantly increased and Smurf2 protein levels were significantly decreased in the lungs of mice treated with bleomycin as compared with the lungs of control mice. Collectively, these data suggest that TTC3 may contribute to TGF-β1-induced EMT and myofibroblast differentiation, potentially through SMURF2 ubiquitylation/proteasomal degradation and subsequent inhibition of SMURF2-mediated suppression of SMAD2 and SMAD3, which in turn induces TTC3 expression.

Cigarette smoking causes apoptotic death, senescence, and impairment of repair functions in lung fibroblasts, which maintain the integrity of alveolar structure by producing extracellular matrix (ECM) proteins. Therefore, recovery of lung fibroblasts from cigarette smoke-induced damage may be crucial in regeneration of emphysematous lung resulting from degradation of ECM proteins and subsequent loss of alveolar cells. Recently, we reported that bone marrow-derived mesenchymal stem cell-conditioned media (MSC-CM) led to angiogenesis and regeneration of lung damaged by cigarette smoke. In this study, to further investigate reparative mechanisms for MSC-CM-mediated lung repair, we attempted to determine whether MSC-CM can recover lung fibroblasts from cigarette smoke-induced damage. In lung fibroblasts exposed to cigarette smoke extract (CSE), MSC-CM, not only inhibited apoptotic death, but also induced cell proliferation and reversed CSE-induced changes in the levels of caspase-3, p53, p21, p27, Akt, and p-Akt. MSC-CM also restored expression of ECM proteins and collagen gel contraction while suppressing CSE-induced expression of cyclooxygenase-2 and microsomal PGE2synthase-2. The CSE-opposing effects of MSC-CM on cell fate, expression of ECM proteins, and collagen gel contraction were partially inhibited by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. In rats, MSC-CM administration also resulted in elevation of p-Akt and restored proliferation of lung fibroblasts, which was suppressed by exposure to cigarette smoke. Taken together, these data suggest that MSC-CM may recover lung fibroblasts from cigarette smoke-induced damage, possibly through inhibition of apoptosis, induction of proliferation, and restoration of lung fibroblast repair function, which are mediated in part by the PI3K/Akt pathway.

Background Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance and elevation of pulmonary arterial pressure , leading to right ventricular failure and eventual death. Currently, no curative therapy for PAH is available, and the overall prognosis is very poor. Recently, direct activators of soluble guanylyl cyclase (sGC) have been tested as a novel therapeutic modality in experimental models of pulmonary arterial hypertension (PAH). Objective In this study, we used in vitro and in vivo models to evaluate the therapeutic potential of 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), a dual functioning chemical, as a direct activator of guanylyl cyclase and an inhibitor of hypoxia-inducible factor-1 . Methods We analyzed the effects of YC-1 on cell proliferation and the levels of p21 and p53 in human pulmonary artery smooth muscle cells (HPASMCs) under hypoxia . We also determined the effects of YC-1 on expression of endothelin-1 (ET-1) and phosphorylation status of endothelial nitric oxide synthase (eNOS) at Ser 1179 in human pulmonary artery endothelial cells (HPAECs) under hypoxia. In mice, hypoxic PAH was induced by exposure to normobaric hypoxic conditions for 28 days. To assess preventive or therapeutic effects, randomized mice were subjected to once daily i.p. injections of YC-1 for the entire hypoxic period (5 mg/kg) or for the last seven days of a 28-day hypoxic period (5 and 10 mg/kg). On day 28, we measured the right ventricular systolic pressure (RVSP) and determined the degrees of right ventricular hypertrophy (RVH) and vascular remodeling . Results In HPASMCs, YC-1 inhibited hypoxia-induced proliferation and induction of p53 and p21 in a concentration-dependent manner. Also, YC-1 suppressed the hypoxia-induced expression of ET-1 mRNA and dephosphorylation of eNOS at Ser 1179 in HPAECs. In the preventive in vivo model, a daily dose of 5 mg/kg YC-1 significantly prevented the elevation of RVSP, development of RVH, and pulmonary vascular remodeling, which were caused by hypoxic exposure. In the therapeutic model, YC-1 at daily doses of 5 and 10 mg/kg alleviated RVH and pulmonary vascular remodeling but did not prevent the elevation of RVSP. Conclusions Our results indicate that YC-1 prevents the development of hypoxia-induced PAH in a preventive model and alleviates RVH and pulmonary vascular remodeling in a therapeutic model. Therefore, these data imply that YC-1 has therapeutic potential for use in a single or combination therapy for PAH.

Emphysema is one of the characteristic features of chronic obstructive pulmonary disease, which is caused mainly by cigarette smoking. Recent data have suggested that apoptosis and cell cycle arrest may contribute to the development of emphysema. In this study, we addressed the question of whether and how cigarette smoke affected Akt, which plays a critical role in cell survival and proliferation. In normal human lung fibroblasts, cigarette smoke extract (CSE) caused cell death, accompanying degradation of total and phosphorylated Akt (p-Akt), which was inhibited by MG132. CSE exposure resulted in preferential ubiquitination of the active Akt (myristoylated), rather than the inactive (T308A/S473A double mutant) Akt. Consistent with cytotoxicity, CSE induced a progressive decrease of phosphorylated human homolog of mouse double minute homolog 2 (p-HDM2) and phosphorylated apoptosis signal regulating kinase 1 (p-ASK1) with concomitant elevation of p53, p21, and phosphorylated p38 MAPK. Forced expression of the active Akt reduced both CSE-induced cytotoxicity and alteration in HDM2/p53/p21 and ASK1/p38 MAPK, compared with the inactive Akt. Of note, CSE induced expression of the tetratrico-peptide repeat domain 3 (TTC3), known as a ubiquitin ligase for active Akt. TTC3 siRNAs suppressed not only CSE-induced Akt degradation but also CSE-induced cytotoxicity. Accordingly, rat lungs exposed to cigarette smoke for 3 months showed elevated TTC3 expression and reduced Akt and p-Akt. Taken together, these data suggest that cigarette smoke induces cytotoxicity, partly through Akt degradation via the ubiquitin-proteasome system, in which TTC3 acts as a ubiquitin ligase for active Akt.

Background and objective Expression of peroxisome proliferator-activated receptor gamma (PPARgamma) is decreased in the lungs of patients with pulmonary hypertension, and PPARgamma ligands have been associated with the release of vasoactive substances from vascular endothelial cells and prevention of vascular remodelling. We hypothesized that PPARgamma may play a critical role in the development of pulmonary hypertension induced by chronic hypoxia. Methods Male adult Sprague-Dawley rats were exposed to normoxia, normoxia and rosiglitazone (8 mg/kg orally, 5 days/week), hypoxia (12% inspired O(2) fraction), or hypoxia and rosiglitazone for 4 weeks. On the last day of the fourth week, pulmonary arterial pressure was measured and morphological changes in pulmonary vessels were assessed. The expression of PPARgamma, endothelin (ET)-1 and vascular endothelial growth factor (VEGF) was also analysed. Results Rosiglitazone inhibited the development of pulmonary hypertension, and pulmonary vascular remodelling induced by chronic hypoxia. PPARgamma expression was decreased and expression of ET-1 and VEGF was increased in lung tissues of the hypoxia group. Rosiglitazone treatment prevented the hypoxia-induced reduction in PPARgamma expression, and restored ET-1 and VEGF expression almost to the levels of the normoxia group. Conclusions Rosiglitazone inhibited the development of pulmonary hypertension induced by chronic hypoxia, perhaps by reversing the changes in PPARgamma, ET-1 and VEGF expression induced by hypoxia. These findings indicate that rosiglitazone may be beneficial in the treatment of chronic hypoxic pulmonary hypertension.

IFNgamma is strongly related to mast cell-associated diseases. There are many reports that IFNgamma inhibits mast cell degranulation. However, inflammatory cytokine production in mast cells stimulated with IFNgamma has not yet been clearly investigated. Therefore, we aimed to investigate the signaling pathways of cytokine production in mast cells stimulated with IFNgamma. Human mast cell line (HMC)-1 or mouse bone marrow-derived mast cells (BMMCs) were stimulated with IFNgamma (100 units) for time periods indicated. Expressions of proteins and mRNAs of cytokines were determined by ELISA and RT-PCR, respectively, activities of MAP kinases, PKC, JAK1/2, and STAT1 on tyrosine 701 and serine 727 by immunoblotting, the DNA-binding activity of the transcription factors by electrophoretic mobility shift assay. IFNgamma-stimulated mast cells showed increase in expressions of proteins and mRNAs of inflammatory cytokines, phosphorylations of MAP kinases, PKCalpha and betaI, JAK1/2, and STAT1 on tyrosine 701 and serine 727. JAK inhibitor or PKC inhibitors inhibited the phosphorylations of p38 kinase, STAT1 on serine 727, and activities of NF-kappaB and AP-1 compared to IFNgamma stimulation alone. These data suggest that IFNgamma-stimulated mast cells induce productions of inflammatory cytokines through PKC/p38/NF-kappaB and AP-1 pathways, not through classical JAK/STAT1 pathway, in both mast cells.

SUMMARYPhospholipid hydroperoxide glutathione peroxidase (PHGPx; GPx4) plays unique roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. We characterized 2 novelGPxgenes from a lung fluke,Paragonimus westermani(designatedPwGPx1andPwGPx2). These single copy genes spanned 6559 and 12 371 bp, respectively, and contained each of 5 intervening introns. ThePwGPx2harboured a codon for Sec and a Sec insertion sequence motif. Proteins encoded by theParagonimusgenes demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic and glutathione-binding domains and absence of the subunit interaction domain. Expression ofPwGPx1increased gradually as the parasite matured, whereas that ofPwGPx2was temporally regulated.PwGPx2was expressed at the basal level from the metacercariae to the 3-week-old juveniles; however, the expression was significantly induced in the 7-week-old immature worms and reached a plateau in the 12-week-old adults and eggs. PwGPx1 and PwGPx2 were largely localized in vitellocytes within vitelline glands and eggs. Oxidative stress-inducible paraquat, juglone and H2O2substantially augmented thePwGPx1andPwGPx2expressions in viable worms by 1·5- to 11-fold. Our results strongly suggested that PwGPxs may actively participate in detoxification of oxidative hazards inP. westermani.

Cyclooxygenase-2 (COX-2) is a key inflammatory response molecule, and associated with many immune functions of monocytes/macrophages. Particularly, interferon gamma (IFNgamma)-induced COX-2 expression appears in inflammatory conditions such as viral infection and autoimmune diseases. Recently, statins have been reported to show variable effects on COX-2 expression, and on their cell and species type dependences. Based on the above description, we compared the effect of simvastatin on IFNgamma-induced COX-2 expression in human monocytes versus murine macrophages. In a result, we found that simvastatin suppresses IFNgamma-induced COX-2 expression in human THP-1 monocytes, but rather, potentiates IFNgamma-induced COX-2 expression in murine RAW264.7 macrophages. However, signal transducer and activator of transcription 1/3 (STAT1/3), known as a transcription factor on COX-2 expression, is inactivated by simvastatin in both cells. Our findings showed that simvastatin is likely to suppress IFNgamma-induced COX-2 expression by inhibiting STAT1/3 activation in human THP-1 cells, but not in murine RAW264.7 cells. Thus, we concluded that IFNgamma-induced COX-2 expression is differently regulated by simvastatin depending on species specific mechanism.

Statins have anti-inflammatory property and immunomodulatory activity. In this study we aimed to investigate the inhibitory mechanism of simvastatin in allergic asthmatic symptoms in mice. BALB/c mice were sensitized and challenged by ovalbumin to induce asthma. Ovalbumin-specific serum IgE levels were measured by enzyme-linked immunosorbent assay (ELISA), and the recruitment of inflammatory cells into bronchoalveolar lavage fluid or lung tissues was measured by Diff-Quik staining and hematoxylin and eosin (HE) staining, respectively, the expressions of CD40, CD40 ligand (CD40L), and vascular cell adhesion molecule-1 (VCAM-1) by immunohistochemistry, the mRNA and protein expressions of cytokines in lung tissues by reverse transcriptase-polymerase chain reaction (RT-PCR) or ELISA, epithelial hyperplasia by periodic acid-Schiff (PAS) staining, activities of matrix metalloproteinases (MMPs) by zymography, the activities of small G proteins, mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-kappaB) in bronchoalveolar lavage cells and lung tissues by western blot and EMSA, respectively. Simvastatin reduced ovalbumin-specific IgE level, the number of total inflammatory cells, macrophages, neutrophils, and eosinophils into bronchoalveolar lavage fluid, the expressions of CD40, CD40L or VCAM-1, the mRNA and protein levels of interleukin (IL)-4, IL-13 and tumor necrosis factor (TNF)-alpha, the numbers of goblet cells, activities of MMPs, and further small G proteins, MAP kinases and NF-kappaB activities in bronchoalveolar lavage cells and lung tissues increased in ovalbumin-induced allergic asthma in mice. Our data suggest that simvastatin may be used as a therapeutic agent in asthma, based on reductions of various allergic responses via regulating small G proteins/MAP kinases/NF-kappaB in mouse allergic asthma.

Paraquat-induced pulmonary fibrosis involves two factors, direct injury by oxygen free radicals and indirect injury by inflammatory cells and fibroblasts. Endothelin-1 (ET-1) has been shown to act as a mediator of pulmonary fibrosis, and its formation increases during oxidative stress. We investigated whether green tea extract (GTE), which has antioxidant properties, inhibits paraquat-induced pulmonary fibrosis and whether ET-1 is involved in this process. Paraquat (0.3 mg/kg) was instilled into the right lungs of rats, following which the rats were either not further treated (Group P, n = 7), or they were administered 1% GTE mixed with feed (Group PG; n = 7) or the ET(A) receptor antagonist ZD2574 (10 mg/kg through gavage; Group PZ; n = 7) for two weeks. As control, we used rats instilled with saline (Group N; n = 6). Two weeks after paraquat instillation, we assayed the degree of pulmonary fibrosis by light microscopic morphometry and hydroxyproline content; lipid peroxidation as a marker of oxidative stresses by measurement of malondialdehyde (MDA); ET-1 by immunohistochemistry; and prepro-ET-1 mRNA expression by reverse transcription-polymerase chain reaction. Compared with Group N, significant pulmonary fibrosis was observed in Group P, accompanied by increases in MDA, ET-1, and prepro-ET-1 mRNA expression. Compared with Group P, Group PG showed significant decreases in pulmonary fibrosis, along with decreases in MDA, ET-1, and prepro-ET-1 mRNA expression. We also observed significant decreases in pulmonary fibrosis in Group PZ compared with Group P. These findings suggest that GTE inhibits paraquat-induced pulmonary fibrosis by suppression of oxidative stress and ET-1 expression.

c-Jun N-terminal kinase (JNK) is activated during hepatic reperfusion, and JNK inhibitors are known to protect other major organs from ischemia-reperfusion (I/R) injury. We attempted to determine the effect of SP600125, a JNK inhibitor, on hepatic I/R injury using a partial ischemia model in mice. Compared to a vehicle-treated group, the SP600125- treated group showed a greater increase in serum ALT levels 24 h after reperfusion with more severe parenchymal destruction and leukocyte infiltration. Similarly, tissue myeloperoxidase and malondialdehyde levels were higher in the SP600125-treated group, and chemokine expression was also higher in the SP600125-treated group. These data, which are contradictory to previous results, indicate that JNK inhibition by SP600125 may be harmful in hepatic I/R injury. Therefore, care must be taken when investigating the therapeutic use of JNK inhibitors in hepatic I/R injury, especially in the context of the effects of JNK inhibition on inflammatory infiltration.

Chronic lymphocytic leukemia (CLL) is frequent in the West, but rare in Korea. In this study, the frequency of chromosome aberration in Korean CLL patients was examined by applying interphase fluorescence in situ hybridization (FISH). Conventional cytogenetic test and FISH were performed on bone marrow aspirates obtained from 16 CLL patients. By applying DNA probes (Vysis, Downers Grove, IL, USA), the deletion in 11q22–23, 13q14, 13q34, and 17p13, and trisomy 12 were examined. With FISH, molecular cytogenetic aberration was detected in 10 of 16 patients [63%, 95% confidence interval (CI) 39–86], whereas with conventional cytogenetic test, chromosomal aberration was detected only in 2 out of 13 cases (15%, 95% CI 0–35). In total, the cases with one or more chromosomal aberrations were 11 out of 16 cases (69%, 95% CI 46–92). The most frequently detected aberration was the 13q14 deletion (69%, 95% CI 44–94), followed by trisomy 12 (19%, 95% CI 0–38) and 11q22 deletion (14%, 95% CI 0–33). No deletion in 17p13 was observed. In conclusion, CLL in Korean is a heterogeneous genetic disorder, showing similar genetic changes in Europe and North America.

Fluorescence in situ hybridization (FISH) can detect minor genetic changes that cytogenetic analysis may miss; however, there are few reports on the kinds of genetic changes that show large dis-crepancies between results obtained with FISH versus G-banding techniques. To investigate genetic changes that tend to be detected with FISH only, we compared the results of cytogenetic study and FISH analysis in 919 consecutive specimens from 304 patients with hematologic malignancies, covering most of the frequent genetic changes by using 18 types of FISH probes. The genetic changes with especially large discrepancy rates at diagnosis were del(7q) (20.0%), PML / RARA (17.6%), and trisomy 21 (12.5%) and, at follow-up, BCR/ABL (28.2%) and AML1/ETO (24.4%); the latter two showed only small discrepancies at diagnosis (4.7 and 4.8%, respectively). The overall discrepancy rate was 6.0% at diagnosis and 11.9% at follow-up, indicating generally greater discrepancy rates at follow-up. In all but one of the cases with discrepant results, G-banding missed the corresponding chromosomal abnormalities revealed with FISH. Considered by type of leukemia, the discrepancy rate at follow-up was higher in acute biphenoptypic leukemia (38%) and acute lymphoblastic leukemia (24.5%) than in acute myelogenous leukemia (10.6%). Given these results, all patients with known genetic changes should have FISH analysis in follow-up, for an accurate assessment of the likelihood of complete remission or recurrence. If this is not practical, then at a minimum FISH analysis should be done in follow-up for patients with genetic changes of BCR / ABL and AML1 / ETO seen at diagnosis.

The therapeutic potential of stem cells in chronic obstructive pulmonary disease is not well known although stem cell therapy is effective in models of other pulmonary diseases. We tested the capacities of bone marrow cells (BMCs), mesenchymal stem cells (MSCs), and conditioned media of MSCs (MSC-CM) to repair cigarette smoke-induced emphysema. Inbred female Lewis rats were exposed to cigarette smoke for 6 mo and then received BMCs, MSCs, or MSC-CM from male Lewis rats. For 2 mo after injection, the BMC treatment gradually alleviated the cigarette smoke-induced emphysema and restored the increased mean linear intercept. The BMC treatment significantly increased cell proliferation and the number of small pulmonary vessels, reduced apoptotic cell death, attenuated the mean pulmonary arterial pressure, and inhibited muscularization in small pulmonary vessels. However, only a few male donor cells were detected from 1 day to 1 mo after BMC administration. The MSCs and cell-free MSC-CM also induced the repair of emphysema and increased the number of small pulmonary vessels. Our data show that BMC, MSCs, and MSC-CM treatment repaired cigarette smoke-induced emphysema. The repair activity of these treatments is consistent with a paracrine effect rather than stem cell engraftment because most of the donor cells disappeared and because cell-free MSC-CM also induced the repair.

Background: Impulse oscillometry (IOS); one of the forced oscillation techniques that is a good method for measuring airway resistance without any special breathing skill. This method might reflect different aspects of airway obstruction compared to conventional spirometry. Object: To evaluate airway characteristics of young asthmatics after exercise bronchial provocation test (EBT) using IOS technique Methods: 47 young adults who were recruited to the Korean military service were enrolled. All of them underwent methacholine bronchial provocation test (MBPT) and EBT (10 minutes of treadmill exercise) for their asthma status evaluation. The IOS and spirometry parameters were collected at baseline, immediate-post EBT, 5 min, 10 min, 20 min and 30 min after EBT. Subjects were divided into two groups according to MBPT results (PC20 > 25.0 mg/mL for airway hyperresponsiveness (AHR) absent group (n=12) and PC20 < 12.0 mg/mL for AHR present group (n=35)). Results: There were differences of FEV1 percent falls between two groups at 5 min (+0.12% vs -9.96%, p

Matrix metalloproteinase-9 (MMP-9) secreted from macrophages plays an important role in tissue destruction and inflammation through degradation of matrix proteins and proteolytic activation of cytokines/chemokines. Whereas the MEK-ERK and PI3K- Akt pathways up-regulate MMP-9 expression, regulation of MMP-9 by JNK remains controversial. Presently, we aimed to determine the role of JNK in MMP-9 regulation in Raw 264.7 cells. Inhibition of JNK by the JNK inhibitor SP600125 induced MMP-9 in the absence of serum and suppressed the expression of TNF-alpha, IL-6 and cyclooxygenase-2 in LPS-treated Raw 264.7 cells. In a knockdown experiment with small interfering RNA, suppression of JNK1 induced MMP-9 expression. Interestingly, mouse serum suppressed SP600125- mediated MMP-9 induction, similar to IFN-gamma. However, the inhibitory activity of mouse serum was not affected by pyridone 6, which inhibits Janus kinase downstream to IFN-gamma. In addition to mouse serum, conditioned media of Raw 264.7 cells contained the inhibitory factor(s) larger than 10 kDa, which suppressed SP600125- or LPS-induced MMP-9 expression. Taken together, these data suggest that JNK1 suppresses MMP-9 expression in the absence of serum. In addition, the inhibitory factor(s) present in serum or secreted from macrophages may negatively control MMP-9 expression.

Neural stem cells (NSC) undergo apoptotic cell death during development of nervous system and in adult. However, little is known about the biochemical regulation of neuroprotection by neurotrophin in these cells. In this report, we demonstrate that Staurosporine (STS) and Etoposide (ETS) induced apoptotic cell death of NSC by a mechanism requiring Caspase 3 activation, poly (ADP-ribose) polymerase and Lamin A/C cleavage. Although C17.2 cells revealed higher mRNA level of p75 neurotrophin receptor (p75(NTR)) compared with TrkA or TrkB receptor, neuroprotective effect of both nerve growth factor (NGF) and brain-derived growth factor (BDNF) mediated through the activation of tropomyosin receptor kinase (Trk) receptors. Moreover, both NGF and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathway. Inhibition of Trk receptor by K252a reduced PARP cleavage as well as cell viability, whereas inhibition of p75(NTR) did not affect the effect of neurotrophin on neurotoxic insults. Thus our studies indicate that the protective effect of NGF and BDNF in NSC against apoptotic stimuli is mediated by the PI3K/Akt and MAPK signaling pathway via Trk receptors.

We previously reported that DA-9601, ethanol herbal extract of Artemisia asiatica, inhibited histamine and leukotriene releases in guinea pig lung mast cells activated with specific antigen/antibody reaction. This study aimed to evaluate the inhibitory effect of DA-9601 on the OVA-induced airway inflammation in allergic asthma mouse model. BALB/c mice were sensitized and challenged with OVA. DA-9601 was administered orally 1 h before every local OVA-challenge. OVA-specific serum IgE was measured by ELISA, recruitment of inflammatory cells in BAL fluids and lung tissues by Diff-Quik and HE staining, respectively, the expressions of CD40, CD40L and VCAM-1 by immunohistochemistry, goblet cell hyperplasia by PAS staining, activities of MMPs by gelatin zymography, expressions of mRNA and proteins of cytokines by RT-PCR and ELISA, activities of MAP kinases by western blot, and activity of NF-KappaB by EMSA. DA-9601 reduced IgE level, recruitment of inflammatory cells into the BAL fluid and lung tissues, expressions of CD40, CD40L and VCAM-1 molecules, goblet cell hyperplasia, MMPs activity, expressions of mRNA and productions of various cytokines, activities of MAP kinases and NK-KappaB increased from OVA-challenged mice. These data suggest that DA-9601 may be developed as a clinical therapeutic agent in allergic diseases due to suppressing the airway allergic inflammation via regulation of various cellular molecules expressed by MAP kinases/NF-KappaB pathway.

Rationale: In cigarette smoking–induced chronic obstructive pulmonary disease, structural and functional derangements are characterized by parenchymal destruction and pulmonary hypertension. Statins are 3-hydroxy-3-methyl-glutaryl–coenzyme-A reductase inhibitors that have been used as lipid-lowering agents. These drugs also have additional pharmacologic properties, including antiinflammation, scavenging reactive oxygen species, restoring endothelial function, and antithrombogenesis, all of which can counteract the harmful effects of cigarette smoking. Objective: We performed assays to determine whether simvastatin could attenuate lung damage induced by chronic cigarette smoking in rats. Methods: In Sprague-Dawley rats exposed to cigarette smoke for 16 weeks, morphologic changes in the lungs and pulmonary arterial pressure were examined. Main Results: Simvastatin inhibited lung parenchymal destruction and development of pulmonary hypertension, and also inhibited peribronchial and perivascular infiltration of inflammatory cells and induction of matrix metalloproteinase-9 activity in lung tissue. Simvastatin additionally prevented pulmonary vascular remodeling and the changes in endothelial nitric oxide synthase expression induced by smoking. In human lung microvascular endothelial cells, simvastatin increased expression of endothelial nitric oxide synthase mRNA. Conclusions: Simvastatin ameliorated the structural and functional derangements of the lungs caused by cigarette smoking, partly by suppressing inflammation and matrix metalloproteinase-9 induction and preventing pulmonary vascular abnormality. These findings indicate that statins may play a role in the treatment of cigarette smoking–induced chronic obstructive pulmonary disease.

To evaluate the prognostic significance of submicroscopic deletions of the ABL or BCR gene associated with t(9;22) in chronic myelogenous leukemia (CML), we investigated the incidence of an ABL or BCR deletion on derivative chromosome 9 using fluorescence in situ hybridization (FISH). FISH was performed using the LSI BCR/ABL dual-fusion translocation probe on bone marrow cells of 86 patients with CML. Of 86 patients, ABL deletion was detected in 13 (15.1%) patients and BCR deletion in 8 patients (9.3%). Patients with ABL deletion showed shorter event-free survival time (EFS) than those without ABL deletion (P = 0.020). Patients with BCR deletion showed significantly short overall survival time (OS; P = 0.039). Patients with ABL and/or BCR deletion (14/86 patients, 16.3%) showed significantly short OS and EFS (median OS, 43.0 months; median EFS, 40.0 months), compared to the patients without any BCR or ABL gene deletions (median OS, 94.0 months; median EFS, 90.0 months; P = 0.041 for OS, P = 0.008 for EFS). All the patients with BCR deletion, except for one, had a concomitant ABL deletion, suggesting that BCR deletion occurs in conjunction with ABL deletion. In patients with ABL deletion only, BCR/ABL rearrangement with b2a2 mRNA type tended to be more frequent than in patients without any deletion of the two genes (P = 0.073). Deletion of any of the BCR or ABL genes on derivative chromosome 9 was associated with both short OS and EFS. We conclude that deletion of not only the ABL gene, but also of the BCR gene, is a poor prognostic marker that indicates rapid disease progression in CML.

8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.

Human 8-oxo-G-DNA glycosylase 1 (hOGG1) is a DNA glycosylase to cleave 8-oxo-7,8-dihydroguanine (8-oxo-G), a mutagenic DNA adduct formed by oxidant stresses. Here, we examined hOGG1 protein expression and repair activity to nick a DNA strand at the site of 8-oxo-G during differentiation of hematopoietic cells using HL-60 cells. Overall expression of hOGG1 protein was increased during granulocytic differentiation of HL-60 cells induced by DMSO and monocytic differentiation by vitamine D(3). Greater level of hOGG1 protein was expressed in DMSO-treated cells. However, change in the DNA nicking activity was not in parallel with the change in hOGG1 protein expression, especially in PMA-treated cells. In PMA- treated cells, the level of hOGG1 protein was lowered, even though the DNA nicking activity was elevated, in a manner similar to the changes in serum- deprived HL-60 cells. These results indicate that hOGG1 expression change during differentiation of hematopoietic stem cells for adaptation to new environments. And the DNA cleaving activity may require additional factor(s) other than expressed hOGG1 protein, especially in apoptotic cell death.

Determination of the remission of acute promyelocytic leukemia (APL) after chemotherapy can be difficult because many cases of APL show reverse transcription polymerase chain reaction positivity after consolidation treatment. Moreover, the discrimination of leukemic promyelocytes and regenerating promyelocytes by morphology is sometimes difficult. Although PML / RARA fluorescence in situ hybridization (FISH) can help, the major drawback of FISH is its high false positive rate, which reaches up to 5–10%. We used RARA FISH at the initial diagnosis (16 cases) and follow-up of APL patients (21 cases) with t(15;17), though RARA FISH was originally designed to detect translocations involving the RARA gene rather than t(15;17), and compared the results with those of PML / RARA FISH. A reference range for PML / RARA and RARA FISH was set using 50 normal control specimens. Using a RARA split probe, we were able to lower the reference range for RARA rearrangement down to 1.5%, which is significantly lower than that of PML / RARA (8%). Actually 74.2% (46/62 cases) of cases with positive signals of the PML / RARA rearrangement by the PML / RARA probe, showed absolutely negative results with the RARA split probe. By conducting RARA FISH, we were able to significantly resolve the difficulty in interpreting results around cut-off value in PML / RARA FISH. In conclusion, we believe that once the PML / RARA rearrangement is confirmed either by G-banding or FISH, RARA FISH is more effective than PML / RARA during the follow-up of APL after treatment.

7,8-Dihydro-8-oxoguanine (oh8Gua) endonuclease is a DNA repair enzyme in Escherichia coli to remove oh8Gua, a promutagenic DNA adduct. Due to the unique mode of enzyme action and substrate specificity, this DNA repair enzyme has been suggested to be identical to 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy)-DNA glycosylase (Fpg). However, oh8Gua endonuclease had not been definitely identified because it had not been homogeneously purified. In this study, we attempted to purify and identify the enzyme. Through several purification procedures, we obtained two proteins (32 kD and 29 kD). The larger protein co-migrated with Fpg in 12% SDS-PAGE gel. Sequences of N-terminal amino acids of these two proteins were identical to that of Fpg; the smaller one is a degraded product of oh8Gua endonuclease during purification steps. These results indicate that oh8Gua endonuclease is identical to Fpg, implying that oh8Gua in oxidatively damaged DNA rather than Fapy is more physiologically relevant substrate for Fpg.

We found previously that 8-hydroxyguanine (oh 8 Gua) endonuclease in E. coli is induced in response to oxidative stress in a fashion similar to the oxidative response of the Mn-superoxide dismutase (MnSOD). In this study, attempts were made to identify the genes involved in the co-regulation of E. coli endonuclease and MnSOD ( sodA ). oh 8 Gua nuclease is induced by molecular oxygen and a superoxide radical generator (paraquat) but not by H 2 O 2 , suggesting that the regulation of this endonuclease is dependent on SoxRS but independent of OxyR. This enzyme was induced by paraquat in all of the soxRS mutant strains used ( soxR − , soxS − and soxR c ), whereas glucose-6-phosphate dehydrogenase (a member of the soxRS regulon) showed the expected responses; therefore, this possibility was excluded. The presence of metal chelators in the growth medium caused the induction of this enzyme, and this induction was suppressed by the addition of Fe ++ . Consistent with this finding, this enzyme was expressed under anaerobiosis in all of the mutant strains of fnr in particular, as well as fur , arcA , and combinations thereof. These findings suggest that the oxidative regulation of oh 8 Gua endonuclease is under control of fnr , fur , and arcA , where fnr plays a predominant role. The multiple involvement of regulatory genes as well as co-regulation with antioxidant enzyme will enhance the efficiency of cellular growth and survival in the aerobic environment.

It has been suggested that 8-hydroxyguanine (oh 8 Gua), a DNA adduct formed by active oxygens, impairs the maintenance of genetic integrity. oh 8 Gua glycosylase removes oh 8 Gua residues as a free base from DNA strands. In E. coli , it has been demonstrated that oh 8 Gua glycosylase is induced in response to oxidative stress, but the oxidative inducibility in mammalian tissues has not yet been studied. In the present study, the inducibility of oh 8 Gua glycosylase was tested by comparing activity changes of this enzyme in the kidney and the liver of rats treated with potassium bromate (KBrO 3 ). KBrO 3 is known to cause oxidative damage to the kidney but not to other organs. With a single dose of KBrO 3 (80 mg/kg, i.p.), activity in the kidney was found to increase significantly at 3 h compared to that at zero time. At 6 h, activity peaked, showing a 6-fold increase over that at zero time. Thereafter, it decreased and returned to its zero time level at 12 h. With increasing doses of KBrO 3 (up to 160 mg/kg, i.p.), activity increased linearly with increased dosage, and over 40 mg/kg, i.p., activity increased to a level significantly higher than that in the control. In contrast to the time- and dose-dependent changes in activity in the kidney, no significant change was observed in the liver under the same conditions as above. These results show that oh 8 Gua glycosylase is also induced oxidatively in mammalian tissues. The induction in this tissue as well as in E. coli indicates that the adaptive response of this enzyme to oxidative stress is a general phenomenon in aerobic organisms and implies that the repair of oh 8 Gua residues in DNA is a process important for the survival of organisms in an aerobic environment.

TGase 2 is over-expressed in a variety of inflammatory diseases including allergic asthma. This study aimed to investigate the role of TGase 2 in signaling pathways in mast cells related to OVA-induced allergic asthma in vitro and in vivo. BMMCs isolated from C57BL/6 WT or TGase 2-/- mice were activated with Ag/Ab. Mice were sensitized and challenged with OVA to induce asthma. [Ca2+]i level were determined by confocal, mast cells by May-Giemsa, LT, IgE and cytokine levels by ELISA, expression of TGase 2, PLA2, EMBP, Muc5ac, collagen, cytokines, MMPs and TIMPs by Western blotting or RT-PCR, NF-kB activity by EMSA, the recruitment of inflammatory cells into BAL fluid or lung tissues by Diff-Quik and H&E, AHR by Fexivent system. Act-WT-BMMCs expressed TGase 2, and increased the [Ca2+]i level, activities of I-kB, NF-kB and PLA2, LTs level, expression of CD40/CD40L, cytokines and MMPs, collagen deposition, and IgE production in act-B cells, whereas act-KO-BMMCs reversed all of them. TGase 2-/- mice protected against OVA-specific IgE production as well as AHR, expression of signaling molecules and mediator release in BAL cells and lung tissues versus those in WT mice. Our data suggest that TGase 2 expression and Ca2+ influx required by bidirectional events in mast cell activation facilitate IgE production in B cells via regulating mast cell CD40L expression, and induce the expression of numerous molecules associated with airway inflammation and remodeling in allergic asthma.

Abstract 2202 Poster Board II-179 Background: Statins (HMG-CoA reductase inhibitors) are known to show anti-proliferative effects and are anticipated as a potential drug in the treatment of malignancies. To investigate the effect simvastatin on chronic myelogenous leukemia cells, we treated simvastatin on 3 kinds of CML cell lines and CD34+ primary CML cells from patients: erythrocytic lineage (K-562), granulocytic lineage (KCL-22), erythroid-megakaryocytic (LAMA-84) cell line. Also, antiproliferative effect on imatinib-resistant CML cell lines was assessed. Mehtods: Anti-proliferative effect was assessed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). Apoptosis was assessed by Annexin V and Western blot study. Killing effect was calculated by CalcuSyn does effect analysis software (Biosoft, Ferguson, MO). CD34+cells for patients with CML were purified using immunomagnetic bead column. Cell cycle analysis was done by flow cytometrc method. Si-RNA transfection study for p27 gene was performed for verification of killing mechanism. Change of intracellular location of BCR/ABL protein was observed by confocal microscopy. Cellular changes of proteins and tyrosin phosphorylation after treatment of simvastain was evaluated by 2 dimensional electrophoresis and MALDI-TOF/TOF mass spectrophotometer. In vivo effect of simvastain was evaluated in BALB/c-nude. Results: Simvastatin inhibited the proliferation of imatinib-sensitive and imatinib-resistant K562 cell line in a dose dependent manner. The IC50 values of simvastatin and imatinib in imatinib-sensitive K562 cells were 14.5 and 0.4 μM. Treatment of simvastain induced apoptosis both in capase-dependent and caspase-independent pathways in all 3 cell lines; apoptosis by Annexin V analysis and increased apoptotic proteins (cytochrome C, AIF, Smac/Diablo, caspase-3, and caspase-9) by western blot. Cell cycle analysis revealed the G1/S arrest on treatment of simvastatin and si-RNA transfection specific for p27 reversed the G1/S arrest, suggesting cell cycle arrest as one of anti-proliferative mechanism. Co-treatment of imatinib and simvastain showed synergistic killing interactions between simvastatin and imatinib in imatinib-resistant K562 cells (mean combination index values were 0.56, P< 0.001). Enhanced killing effect was observed in all 3 imatinib-resistant CML cell lines (K-562: 0.6, KCL-22: 0.42 , LAMA-84; 0.99). Co-treatment with imatinib and simvastatin decreased the amount of Bcr-Abl protein and stimulated the import of Abl protein in the nuclei in K562 cells. In CML cells, simvastatin inhibited tyrosine phosphorylation included protease, cytocrome-c reductase, DNA/RNA processing proteins, oxidoructase protein, chaperones, glycolysis protein, cytoskeleton proteins, microtubule protein. Treatment of simvastain reduced subcutaneous tumor mass in nude mice. Conclusion: We showed that simvastatin killed CML cells in vitro and in vivo animal model and killing effect occurred via the induction of apoptosis, cell cyle arrest via p27 and inhibited BCR/ABL tyrosine kinase (TK) activity. Simvastatin may be a potential candidate for the treatment of imatinib-resistant CML patients and the effective dose of imatinib could be reduced in a combined treatment with simvastatin. Disclosures: No relevant conflicts of interest to declare.

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IkappaB, and nuclear AP-1 or NF-kappaB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IkappaB-NF-kappaB are involved.

To evaluate the prognostic significance of the submicroscopic deletions of ABL or BCR gene associated with t(9;22) in CML, we investigated the incidence of ABL or BCR deletion on derivative chromosome 9 using fluorescent in situ hybridization (FISH), and analyzed the survival. FISH was performed using LSI BCR/ABL, dual fusion translocation probe on bone marrow cells of 86 patients with CML. Of 86 patients, ABL deletion was detected in 13 (15.1%) patients and BCR deletion in 8 patients (9.3%). Patients with ABL deletion showed shorter event-free survival time (EFS) than those without ABL deletion (P=0.020). Patients with BCR deletion showed significantly short overall survival time (OS) (P=0.039). Patients with ABL and/or BCR deletion (14/86 patients, 16.3%) showed significantly short OS and EFS (median OS: 43.0 months, median EFS: 40.0 months), compared to the patients without any deletions of BCR or ABL gene (median OS: 94.0 months, median EFS: 90.0 months)(P=0.041 for OS, P=0.008 for EFS). Patients with BCR deletion, all except one had a concomitant ABL deletion, suggesting that BCR deletion occurs in conjunction with ABL deletion. In patients with ABL deletion only, BCR/ABL rearrangement with b2a2 mRNA type tended to be more frequent than in patients without any deletion of the two genes (P=0.073). Deletion of any of BCR or ABL gene on derivative chromosome 9 was associated with both short OS and EFS. We conclude that deletion of not only ABL gene, but also BCR gene is a poor prognostic marker that indicates rapid disease progression in CML.