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Sample records for 4-mediated c-jun n-terminal

  1. c-Jun N-terminal kinase mediates constitutive human eosinophil apoptosis

    OpenAIRE

    Hasala, Hannele; Zhang, Xianzhi; Saarelainen, Seppo; Moilanen, Eeva; Kankaanranta, Hannu

    2007-01-01

    c-Jun N-terminal kinase mediates constitutive human eosinophil apoptosis correspondence: Corresponding author. Tel.: +358335517318; fax: +358335518082. (Kankaanranta, Hannu) (Kankaanranta, Hannu) The Immunopharmacology Research Group--> , Medical School--> , University of Tampere--> , Tampere--> - FINLAND (Hasala, Hannele) The Immunopharmacology Research Group--> , Medical School--...

  2. HEPATIC APOPTOSIS POST-BURN IS MEDIATED BY C-JUN N-TERMINAL KINASE-2

    OpenAIRE

    Marshall, Alexandra H; Brooks, Natasha C; Hiyama, Yaeko; Qa’aty, Nour; Al-mousawi, Ahmed; Finnerty, Celeste C.; Jeschke, Marc G.

    2013-01-01

    The trauma of a severe burn injury induces a hypermetabolic response that increases morbidity and mortality. Previously, our group showed that insulin resistance post-burn injury is associated with endoplasmic reticulum (ER) stress. Evidence suggests that c-jun N-terminal kinase (JNK) -2 may be involved in ER stress-induced apoptosis. Here, we hypothesized that JNK2 contributes to the apoptotic response after burn injury downstream of ER stress. To test this, we compared JNK2 knockout mice (−...

  3. Activation of c-Jun N-terminal Kinases by Ribotoxic Stresses

    Institute of Scientific and Technical Information of China (English)

    Dong-Yun Ouyang; Yuan-Yuan Wang; Yong-Tang Zheng

    2005-01-01

    The c-Jun N-terminal kinases (JNKs) are classic stress-activated protein kinases. Many cellular stresses have been shown to stimulate JNK activation. In this review, we focus on ribotoxic stresses based on their multiple biological potencies including anti-HIV-1 activity. Some of the functions of ribotoxins and the signaling transduction pathway that mediated are mentioned. Different from other stimulators, ribotoxic stresses act on special motifs of 28S rRNA in translationally active mammal ribosomes. Binding and damaging on the motif leads to JNK activation and subsequently biological response to the signal initiator, which is named ribotoxic stress response.

  4. c-Jun N-Terminal Phosphorylation: Biomarker for Cellular Stress Rather than Cell Death in the Injured Cochlea.

    Science.gov (United States)

    Anttonen, Tommi; Herranen, Anni; Virkkala, Jussi; Kirjavainen, Anna; Elomaa, Pinja; Laos, Maarja; Liang, Xingqun; Ylikoski, Jukka; Behrens, Axel; Pirvola, Ulla

    2016-01-01

    Prevention of auditory hair cell death offers therapeutic potential to rescue hearing. Pharmacological blockade of JNK/c-Jun signaling attenuates injury-induced hair cell loss, but with unsolved mechanisms. We have characterized the c-Jun stress response in the mouse cochlea challenged with acoustic overstimulation and ototoxins, by studying the dynamics of c-Jun N-terminal phosphorylation. It occurred acutely in glial-like supporting cells, inner hair cells, and the cells of the cochlear ion trafficking route, and was rapidly downregulated after exposures. Notably, death-prone outer hair cells lacked c-Jun phosphorylation. As phosphorylation was triggered also by nontraumatic noise levels and none of the cells showing this activation were lost, c-Jun phosphorylation is a biomarker for cochlear stress rather than an indicator of a death-prone fate of hair cells. Preconditioning with a mild noise exposure before a stronger traumatizing noise exposure attenuated the cochlear c-Jun stress response, suggesting that the known protective effect of sound preconditioning on hearing is linked to suppression of c-Jun activation. Finally, mice with mutations in the c-Jun N-terminal phosphoacceptor sites showed partial, but significant, hair cell protection. These data identify the c-Jun stress response as a paracrine mechanism that mediates outer hair cell death. PMID:27257624

  5. Correlation between spina bifida manifesta in fetal rats and c-Jun N-terminal kinase signaling

    Institute of Scientific and Technical Information of China (English)

    Yinghuan Ma; Yongxin Bao; Chenghao Li; Fubin Jiao; Hongjie Xin; Zhengwei Yuan

    2012-01-01

    Fetal rat models with neural tube defects were established by injection with retinoic acid at 10 days after conception. The immunofluorescence assay and western blot analysis showed that the number of caspase-3 positive cells in myeloid tissues for spina bifida manifesta was increased. There was also increased phosphorylation of c-Jun N-terminal kinase, a member of the mitogen activated protein kinase family. The c-Jun N-terminal kinase phosphorylation level was positively correlated with caspase-3 expression in myeloid tissues for spina bifida manifesta. Experimental findings indicate that abnormal apoptosis is involved in retinoic acid-induced dominant spina bifida formation in fetal rats, and may be associated with the c-Jun N-terminal kinase signal transduction pathway.

  6. HEPATIC APOPTOSIS POST-BURN IS MEDIATED BY C-JUN N-TERMINAL KINASE-2

    Science.gov (United States)

    Marshall, Alexandra H.; Brooks, Natasha C.; Hiyama, Yaeko; Qa’aty, Nour; Al-mousawi, Ahmed; Finnerty, Celeste C.; Jeschke, Marc G.

    2013-01-01

    The trauma of a severe burn injury induces a hypermetabolic response that increases morbidity and mortality. Previously, our group showed that insulin resistance post-burn injury is associated with endoplasmic reticulum (ER) stress. Evidence suggests that c-jun N-terminal kinase (JNK) -2 may be involved in ER stress-induced apoptosis. Here, we hypothesized that JNK2 contributes to the apoptotic response after burn injury downstream of ER stress. To test this, we compared JNK2 knockout mice (−/−) to wildtype mice after inducing a 30% total body surface area thermal injury. Animals were sacrificed after 1, 3 and 5 days. Inflammatory cytokines in the blood were measured by multiplex analysis. Hepatic ER stress and insulin signaling were assessed by Western Blotting and insulin resistance was measured by a peritoneal glucose tolerance test. Apoptosis in the liver was quantified by TUNEL staining. Liver function was quantified by AST and ALT activity assays. ER stress increased after burn in both JNK2−/− and wildtype mice, indicating that JNK2 activation is downstream of ER stress. Knockout of JNK2 did not affect serum inflammatory cytokines; however, the increase in IL-6 mRNA expression was prevented in the knockouts. Serum insulin did not significantly increase in the JNK2−/− group. On the other hand, insulin signaling (PI3K/Akt pathway) and glucose tolerance tests did not improve in JNK2−/−. As expected, apoptosis in the liver increased after burn injury in wildtype mice but not in JNK2−/−. AST/ALT activity revealed that liver function recovered more quickly in JNK2−/−. This study indicates that JNK2 is a central mediator of hepatic apoptosis after a severe burn. PMID:23324888

  7. Regulation of Apoptotic c-Jun N-Terminal Kinase Signaling by a Stabilization-Based Feed-Forward Loop†

    OpenAIRE

    Xu, Zhiheng; Kukekov, Nikolay V.; Greene, Lloyd A.

    2005-01-01

    A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect ...

  8. c-Jun N-terminal Kinase (JNK) signaling as a therapeutic target for Alzheimer’s disease

    OpenAIRE

    Yarza, Ramon; Vela, Silvia; Solas, Maite; Ramirez, Maria J

    2016-01-01

    c-Jun N-terminal kinases (JNKs) are a family of protein kinases that play a central role in stress signaling pathways implicated in gene expression, neuronal plasticity, regeneration, cell death, and regulation of cellular senescence. It has been shown that there is a JNK pathway activation after exposure to different stressing factors, including cytokines, growth factors, oxidative stress, unfolded protein response signals or Aβ peptides. Altogether, JNKs have become a focus of screening str...

  9. c-Jun N-terminal kinase phosphorylates DCP1a to control formation of P bodies

    OpenAIRE

    Rzeczkowski, Katharina; Beuerlein, Knut; Müller, Helmut; Dittrich-Breiholz, Oliver; Schneider, Heike; Kettner-Buhrow, Daniela; Holtmann, Helmut; Kracht, Michael

    2011-01-01

    Cytokines and stress-inducing stimuli signal through c-Jun N-terminal kinase (JNK) using a diverse and only partially defined set of downstream effectors. In this paper, the decapping complex subunit DCP1a was identified as a novel JNK target. JNK phosphorylated DCP1a at residue S315 in vivo and in vitro and coimmunoprecipitated and colocalized with DCP1a in processing bodies (P bodies). Sustained JNK activation by several different inducers led to DCP1a dispersion from P bodies, whereas IL-1...

  10. Melanocortin-4 receptor activation inhibits c-Jun N-terminal kinase activity and promotes insulin signaling

    OpenAIRE

    Chai, Biaoxin; Li, Ji-Yao; Zhang, Weizhen; Wang, Hui; Mulholland, Michael W.

    2009-01-01

    The melanocortin system is crucial to regulation of energy homeostasis. The melanocortin receptor type 4 (MC4R) modulates insulin signaling via effects on c-Jun N-terminal kinase (JNK). The melanocortin agonist NDP-MSH dose-dependently inhibited JNK activity in HEK293 cells stably expressing the human MC4R; effects were reversed by melanocortin receptor antagonist. NDP-MSH time- and dose-dependently inhibited IRS-1ser307 phosphorylation, effects also reversed by a specific melanocortin recept...

  11. Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop.

    Science.gov (United States)

    Xu, Zhiheng; Kukekov, Nikolay V; Greene, Lloyd A

    2005-11-01

    A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death. PMID:16260609

  12. Regulation of Apoptotic c-Jun N-Terminal Kinase Signaling by a Stabilization-Based Feed-Forward Loop†

    Science.gov (United States)

    Xu, Zhiheng; Kukekov, Nikolay V.; Greene, Lloyd A.

    2005-01-01

    A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death. PMID:16260609

  13. c-Jun-N-terminal kinase 1 is necessary for nicotine-induced enhancement of contextual fear conditioning.

    Science.gov (United States)

    Leach, Prescott T; Kenney, Justin W; Gould, Thomas J

    2016-08-01

    Acute nicotine enhances hippocampus-dependent learning. Identifying how acute nicotine improves learning will aid in understanding how nicotine facilitates the development of maladaptive memories that contribute to drug-seeking behaviors, help development of medications to treat disorders associated with cognitive decline, and advance understanding of the neurobiology of learning and memory. The effects of nicotine on learning may involve recruitment of signaling through the c-Jun N-terminal kinase family (JNK 1-3). Learning in the presence of acute nicotine increases the transcription of mitogen-activated protein kinase 8 (MAPK8, also known as JNK1), likely through a CREB-dependent mechanism. The functional significance of JNK1 in the effects of acute nicotine on learning, however, is unknown. The current studies undertook a backward genetic approach to determine the functional contribution JNK1 protein makes to nicotine-enhanced contextual fear conditioning. JNK1 wildtype (WT) and knockout (KO) mice were administered acute nicotine prior to contextual and cued fear conditioning. 24h later, mice were evaluated for hippocampus-dependent (contextual fear conditioning) and hippocampus-independent (cued fear conditioning) memory. Nicotine selectively enhanced contextual conditioning in WT mice, but not in KO mice. Nicotine had no effect on hippocampus-independent learning in either genotype. JNK1 KO and WT mice given saline showed similar levels of learning. These data suggest that JNK1 may be recruited by nicotine and is functionally necessary for the acute effects of nicotine on learning and memory. PMID:27235579

  14. C-jun N-terminal Kinase-mediated Signaling Is Essential for Staphylococcus Aureus-induced U937 Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Jia-he Wang; Bo Yu; Hui-yan Niu; Hui Li; Yi Zhang; Xin Wang; Ping He

    2009-01-01

    Objective To investigate the effect of SP600125, a specific c-jun N-terminal protein kinase (JNK) inhibitor, on Staphylococcus aureus (S. aureus)-induced U937 cell death and the underlying mechanism. Methods The human monocytic U937 cells were treated with S. aureus at different time with or without SP600125. Cell apoptosis was analyzed by flow cytometry. JNK, Bax, and caspase-3 activities were detected by Western blotting. Results S. aureus induced apoptosis in cultured U937 cells in a time-dependent manner. Expression of Bax and phospho-JNK significantly increased in S. aureus-treated U937 cells, and the level of activated caspase-3 also increased in a time-dependent manner. Inhibition of JNK with SP600125 significantly inhibited S. aureus-induced apoptosis in U937 cells. Conclusions S. aureus can induce apoptosis in U937 cells by phosphorylation of JNK and activation of Bax and caspase-3. SP600125 protects U937 cells from apoptosis induced by S. aureus via inhibiting the activity of JNK.

  15. The c-Jun N-terminal Kinase 2 Plays a Dominant Role in Human Epidermal Neoplasia

    Science.gov (United States)

    Ke, Hengning; Harris, Rebecca; Coloff, Jonathan; Jin, Jane Y.; Leshin, Benjamin; de Marval, Paula Miliani; Tao, Shiying; Rathmell, Jeffrey C; Hall, Russell P.; Zhang, Jennifer Y.

    2010-01-01

    The c-Jun N-terminal Kinase (JNK) signaling cascade has been implicated in a wide range of diseases, including cancer. It is unclear how different JNK proteins contribute to human cancer. Here, we report that JNK2 is activated in over 70% of human squamous cell carcinoma (SCC) samples and that inhibition of JNK2 pharmacologically or genetically impairs tumorigenesis of human SCC cells. Most importantly, JNK2, but not JNK1, is sufficient to couple with oncogenic Ras to transform primary human epidermal cells into malignancy with features of SCC. JNK2 prevents Ras-induced cell senescence and growth arrest by reducing the expression levels of the cell cycle inhibitor p16 and NF-κB activation. On the other hand, JNK, along with PI3K, is essential for Ras-induced glycolysis, an energy producing process known to benefit cancer growth. These data indicate that JNK2 collaborates with other oncogenes, such as Ras, at multiple molecular levels to promote tumorigenesis and hence represents a promising therapeutic target for cancer. PMID:20354187

  16. Molecular clone and characterization of c-Jun N-terminal kinases 2 from orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Guo, Minglan; Wei, Jingguang; Zhou, Yongcan; Qin, Qiwei

    2016-02-01

    c-Jun N-terminal kinase 2 (JNK2) is a multifunctional mitogen-activated protein kinases involving in cell differentiation and proliferation, apoptosis, immune response and inflammatory conditions. In this study, we reported a new JNK2 (Ec-JNK2) derived from orange-spotted grouper, Epinephelus coioides. The full-length cDNA of Ec-JNK2 was 1920 bp in size, containing a 174 bp 5'-untranslated region (UTR), 483 bp 3'-UTR, and a 1263 bp open reading frame (ORF), which encoded a putative protein of 420 amino acids. The deduced protein sequence of Ec-JNK2 contained a conserved Thr-Pro-Tyr (TPY) motif in the domain of serine/threonine protein kinase (S-TKc). Ec-JNK2 has been found to involve in the immune response to pathogen challenges in vivo, and the infection of Singapore grouper iridovirus (SGIV) in vitro. Immunofluorescence staining showed that Ec-JNK2 was localized in the cytoplasm of grouper spleen (GS) cells, and moved to the nucleus after infecting with SGIV. Ec-JNK2 distributed in all immune-related tissues examined. After challenging with lipopolysaccharide (LPS), SGIV and polyriboinosinic polyribocytidylic acid (poly I:C), the mRNA expression of Ec-JNK2 was significantly (P orange-spotted grouper. Over-expressing Ec-JNK2 in fathead minnow (FHM) cells increased the SGIV infection and replication, while over-expressing the dominant-negative Ec-JNK2Δ181-183 mutant decreased it. These results indicated that Ec-JNK2 could be an important molecule in the successful infection and evasion of SGIV.

  17. C-Jun N-terminal kinase controls TDP-43 accumulation in stress granules induced by oxidative stress

    Directory of Open Access Journals (Sweden)

    Masters Colin L

    2011-08-01

    Full Text Available Abstract Background TDP-43 proteinopathies are characterized by loss of nuclear TDP-43 expression and formation of C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs in response to cell stresses and this could be associated with subsequent formation of TDP-43 ubiquinated protein aggregates. However, the initial mechanisms controlling endogenous TDP-43 accumulation in SGs during chronic disease are not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in SH-SY5Y neuronal-like cells exposed to chronic oxidative stress. Cell cultures were treated overnight with the mitochondrial inhibitor paraquat and examined for TDP-43 and SG processing. Results We found that mild stress induced by paraquat led to formation of TDP-43 and HuR-positive SGs, a proportion of which were ubiquitinated. The co-localization of TDP-43 with SGs could be fully prevented by inhibition of c-Jun N-terminal kinase (JNK. JNK inhibition did not prevent formation of HuR-positive SGs and did not prevent diffuse TDP-43 accumulation in the cytosol. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. JNK inhibition also inhibited TDP-43 SG localization in cells acutely treated with sodium arsenite and reduced the number of aggregates per cell in cultures transfected with C-terminal TDP-43 162-414 and 219-414 constructs. Conclusions Our studies are the first to demonstrate a critical role for kinase control of TDP-43 accumulation in SGs and may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.

  18. Myocardial protective effects of a c-Jun N-terminal kinase inhibitor in rats with brain death.

    Science.gov (United States)

    Guo, Wenzhi; Cao, Shengli; Yan, Bing; Zhang, Gong; Li, Jie; Zhao, Yongfu; Zhang, Shuijun

    2016-07-01

    To investigate whether the mitochondrial apoptotic pathway mediates myocardial cell injuries in rats under brain death (BD), and observe the effects and mechanisms of the c-Jun N-terminal kinase (JNK) inhibitor SP600125 on cell death in the heart. Forty healthy male Sprague-Dawley (SD) rats were randomized into four groups: sham group (dural external catheter with no BD); BD group (maintain the induced BD state for 6 hrs); BD + SP600125 group (intraperitoneal injection of SP600125 10 mg/kg 1 hr before inducing BD, and maintain BD for 6 hrs); and BD + Dimethyl Sulphoxide (DMSO) group (intraperitoneal injection of DMSO 1 hr before inducing BD, and maintain BD for 6 hrs). Real-time quantitative PCR was used to evaluate mRNA levels of Cyt-c and caspase-3. Western blot analysis was performed to examine the levels of mitochondrial apoptosis-related proteins p-JNK, Bcl-2, Bax, Cyt-c and Caspase-3. TUNEL assay was employed to evaluate myocardial apoptosis. Compared with the sham group, the BD group exhibited increased mitochondrial apoptosis-related gene expression, accompanied by the elevation of p-JNK expression and myocardial apoptosis. As the vehicle control, DMSO had no treatment effects. The BD + SP600125 group had decreased p-JNK expression, and reduced mitochondrial apoptosis-related gene expression. Furthermore, the apoptosis rate of myocardial cells was reduced. The JNK inhibitor SP600125 could protect myocardial cells under BD through the inhibition of mitochondrial apoptosis-related pathways. PMID:27072084

  19. c-Jun N-terminal kinase is required for thermotherapy-induced apoptosis in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Feng Xiao; Bin Liu; Qing-Xian Zhu

    2012-01-01

    AIM:To investigate the role of c-Jun N-terminal kinase (JNK) in thermotherapy-induced apoptosis in human gastric cancer SGC-7901 cells.METHODS:Human gastric cancer SGC-7901 cells were cultured in vitro.Following thermotherapy at 43 ℃ for 0,0.5,1,2 or 3 h,the cells were cultured for a further 24 h with or without the JNK specific inhibitor,SP600125 for 2 h.Apoptosis was evaluated by immunohistochemistry [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)] and flow cytometry (Annexin vs propidium iodide).Cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.The production of p-JNK,Bcl-2,Bax and caspase-3 proteins was evaluated by Western blotting.The expression of JNK at mRNA level was determined by reverse transcription polymerase chain reaction.RESULTS:The Proliferation of gastric carcinoma SGC-7901 cells was significantly inhibited following thermotherapy,and was 32.7%,30.6%,43.8% and 52.9% at 0.5,1,2 and 3 h post-thermotherapy,respectively.Flow cytometry analysis revealed an increased population of SGC-7901 cells in G0/G1 phase,but a reduced population in S phase following therrnotherapy for 1 or 2 h,compared to untreated cells (P < 0.05).The increased number of SGC-7901 cells in G0/G1 phase was consistent with induced apoptosis (flow cytometry) following thermotherapy for 0.5,1,2 or 3 h,compared to the untreated group (46.5% ± 0.23%,39.9% ± 0.53%,56.6% ±0.35% and 50.4% ± 0.29% vs 7.3% ± 0.10%,P < 0.01),respectively.This was supported by the TUNEL assay (48.2% ± 0.4%,40.1% ± 0.2%,61.2% ± 0.29% and 52.0% ± 0.42% vs 12.2% ± 0.22%,P < 0.01) respectively.More importantly,the expression of p-JNK protein and JNK mRNA levels were significantly higher at 0.5 h than at 0 h post-treatment (P < 0.01),and peaked at 2 h.A similar pattem was detected for Bax and caspase-3 proteins.Bcl-2 increased at 0.5 h,peaked at 1 h,and then decreased

  20. Identification of a c-Jun N-terminal kinase-2-dependent signal amplification cascade that regulates c-Myc levels in ras transformation

    DEFF Research Database (Denmark)

    Mathiasen, D.P.; Egebjerg, C.; Andersen, S.H.;

    2012-01-01

    Ras is one of the most frequently activated oncogenes in cancer. Two mitogen-activated protein kinases (MAPKs) are important for ras transformation: extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase 2 (JNK2). Here we present a downstream signal amplification cascade that is...... essential for ras transformation. Previous studies show that ERK-mediated serine 62 phosphorylation protects c-Myc from proteasomal degradation. ERK is, however, not alone sufficient to stabilize c-Myc but requires the cooperation of cancerous inhibitor of protein phosphatase 2A (CIP2A), an oncogene that...

  1. c-Jun N-terminal kinase is required for vitamin E succinate-induced apoptosis in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Kun Wu; Yan Zhao; Gui-Chang Li; Wei-Ping Yu

    2004-01-01

    AIM: To investigate the roles of c-Jun N-terminal kinase (JNK)signaling pathway in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells.METHODS: Human gastric cancer cell lines (SGC-7901)were treated with vitamin E succinate (VES) at 5, 10, 20 mg/L.Succinic acid and vitamin E were used as vehicle controls and condition medium only as an untreated (UT) control.Apoptosis was observed by 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI) staining for morphological changes and by DNA fragmentation for biochemical alterations.Western blot analysis was applied to measure the expression ofJNK and phosphorylated JNK. After the cells were transiently transfected with dominant negative mutant of JNK (DNJNK) followed by treatment of VES, the expression of JNK and c-Jun protein was determined.RESULTS: The apoptotic changes were observed after VES treatment by DNA fragmentation. DNA ladder in the 20 mg/L VES group was more clearly seen than that in 10 mg/L VES group and was not detected following treatment of UT control, succinate and vitamin E. VES at 5, 10 and 20 mg/L increased the expression of p-JNK by 2.5-, 2.8- and 4.2-fold, respectively. VES induced the phosphorylation of JNK beginning at 1.5 h and produced a sustained increase for 24 h with the peak level at 12 h. Transient transfection of DN-JNK blocked VES-triggered apoptosis by 52%. DN-JNK significantly increased the level of JNK, while decreasing the expression of VES-induced c-Jun protein.CONCLUSION: VES-induced apoptosis in human gastric cancer SGC-7901 cells involves JNK signaling pathway via c-Jun and its downstream transcription factor.

  2. Monosodium Urate in the Presence of RANKL Promotes Osteoclast Formation through Activation of c-Jun N-Terminal Kinase

    Directory of Open Access Journals (Sweden)

    Jung-Yoon Choe

    2015-01-01

    Full Text Available The aim of this study was to clarify the role of monosodium urate (MSU crystals in receptor activator of nuclear factor kB ligand- (RANKL- RANK-induced osteoclast formation. RAW 264.7 murine macrophage cells were incubated with MSU crystals or RANKL and differentiated into osteoclast-like cells as confirmed by staining for tartrate-resistant acid phosphatase (TRAP and actin ring, pit formation assay, and TRAP activity assay. MSU crystals in the presence of RANKL augmented osteoclast differentiation, with enhanced mRNA expression of NFATc1, cathepsin K, carbonic anhydrase II, and matrix metalloproteinase-9 (MMP-9, in comparison to RAW 264.7 macrophages incubated in the presence of RANKL alone. Treatment with both MSU crystals and RANKL induced osteoclast differentiation by activating downstream molecules in the RANKL-RANK pathway including tumor necrosis factor receptor-associated factor 6 (TRAF-6, JNK, c-Jun, and NFATc1. IL-1b produced in response to treatment with both MSU and RANKL is involved in osteoclast differentiation in part through the induction of TRAF-6 downstream of the IL-1b pathway. This study revealed that MSU crystals contribute to enhanced osteoclast formation through activation of RANKL-mediated pathways and recruitment of IL-1b. These findings suggest that MSU crystals might be a pathologic causative agent of bone destruction in gout.

  3. S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanism

    Institute of Scientific and Technical Information of China (English)

    María del Pilar Cabrales-Romero; Lucrecia Márquez-Rosado; Samia Fattel-Fazenda; Cristina Trejo-Solís; Evelia Arce-Popoca; Leticia Alemén-Lazarini; Saúl Villa-Trevi(n)o

    2006-01-01

    AIM: To determine the role of c-Jun N-terminal kinase (JNK) activity in ethanol-induced apoptosis and the modulation of this signaling cascade by S-Adenosylmethionine (AdoMet).METHODS: Primary hepatocyte cultures were pretreated with 100 μmol/L SP600125, a selective JNK inhibitor, 1 mL/L DMSO or 4 mmol/L AdoMet and then exposed to 100 mmo/L ethanol. Hepatocyte apoptosis was determined by the TUNEL and DNA ladder assays.JNK activity and its inhibition by SP600125 and AdoMet were determined by Western blot analysis of c-jun phosphorylation and Bid fragmentation. SP600125 and AdoMet effects on the apoptotic signaling pathway were determined by Western blot analysis of cytochrome c release and pro-caspase 3 fragmentation. The AdoMet effect on glutathione levels was measured by Ellman's method and reactive oxygen species (ROS) generation by cell cytometry.RESULTS: The exposure of hepatocytes to ethanol induced JNK activation, c-jun phosphorylation, Bid fragmentation, cytochrome c release and pro-caspase 3 cleavage; these effects were diminished by SP600125, and caused a significant decreasein ethanol-induced apoptosis (P< 0.05). AdoMet exerted an antioxidant effect maintaining glutathione levels and decreasing ROS generation, without a significant effect on JNK activity,and prevented cytochrome c release and pro-caspase 3 cleavage.CONCLUSION: The JNK signaling cascade is a key component of the proapoptotic signaling pathway induced by ethanol. JNK activation may be independent from ROS generation, since AdoMet which exerted antioxidant properties did not have a significant effect on JNK activity. JNK pathway modulator agents and AdoMet may be components of promising therapies for alcoholic liver disease (ALD) treatment.

  4. Apoptosis induced by genipin in human leukemia K562 cells:involvement of C-Jun N-terminal kinase in G2/M arrest

    Institute of Scientific and Technical Information of China (English)

    Qian FENG; Hou-li CAO; Wei XU; Xiao-rong LI; Yan-qin REN; Lin-fang DU

    2011-01-01

    Aim:To investigate the effect of genipin on apoptosis in human leukemia K562 cells in vitro and elucidate the underlying mechanisms.Methods:The effect of genipin on K562 cell viability was measured using trypan blue dye exclusion and cell counting.Morphological changes were detected using phase-contrast microscopy.Apoptosis was analyzed using DNA ladder, propidium iodide(PI)-labeled flow cytometry(FCM)and Hoechst 33258 staining.The infiuence of genipin on cell cycle distribution was determined using Plstaining.Caspase 3 activity was analyzed to detect apoptosis at different time points.Protein levels of phospho-c-Jun,phosphor-C-Jun N-terminal kinase(p-JNK).phosphor-p38-Fas-L,p63,and Bax and the release of cytochrome c were detected using Western blot analysis.Results:Genipin reduced the viability of K562 cells with an IC50 value of approximately 250 μmol/L.Genipin 200-400 μmol/L induced formation of typicaI apoptotic bodies and DNA fragmentation.Additionally,genipin 400 μmol/L significantly increased the caspase 3activity from 8-24 h and arrested the cells in the G2/M phase.After stimulation with genipin 500 μmol/L, the levels of p-JNK, p-c-Jun.Fas-L,Bax.and cytochrome c were remarkably upregulated,but there were no obvious changes of p-p38.Genipin 200-500 μmol/Lsignificantly upregulated the Fas-L expression and downregulated p63 expression.Dicoumarol 100 μmol/L.a JNK1/2 inhibitor,markedly suppressed the formation of apoptotic bodies and JNK activation induced by genipin 400 μmol/L.Conclusion:These results suggest that genipin inhibits the proliferation of K562 cells and induces apoptosis through the activation of JNK and induction of the Fas ligand.

  5. Tumor suppressor BLU inhibits proliferation of nasopharyngeal carcinoma cells by regulation of cell cycle, c-Jun N-terminal kinase and the cyclin D1 promoter

    Directory of Open Access Journals (Sweden)

    Zhang Xiangning

    2012-06-01

    Full Text Available Abstract Background Tumor suppressor genes function to regulate and block tumor cell proliferation. To explore the mechanisms underlying the tumor suppression of BLU/ZMYND10 gene on a frequently lost human chromosomal region, an adenoviral vector with BLU cDNA insert was constructed. Methods BLU was re-expressed in nasopharyngeal carcinoma cells by transfection or viral infection. Clonogenic growth was assayed; cell cycle was analyzed by flow cytometry-based DNA content detection; c-Jun N-terminal kinase (JNK and cyclin D1 promoter activities were measured by reporter gene assay, and phosphorylation was measured by immunoblotting. The data for each pair of groups were compared with Student t tests. Results BLU inhibits clonogenic growth of nasopharyngeal carcinoma cells, arrests cell cycle at G1 phase, downregulates JNK and cyclin D1 promoter activities, and inhibits phosphorylation of c-Jun. Conclusions BLU inhibits growth of nasopharyngeal carcinoma cells by regulation of the JNK-cyclin D1 axis to exert tumor suppression.

  6. Momordica charantia polysaccharides could protect against cerebral ischemia/reperfusion injury through inhibiting oxidative stress mediated c-Jun N-terminal kinase 3 signaling pathway.

    Science.gov (United States)

    Gong, Juanjuan; Sun, Fumou; Li, Yihang; Zhou, Xiaoling; Duan, Zhenzhen; Duan, Fugang; Zhao, Lei; Chen, Hansen; Qi, Suhua; Shen, Jiangang

    2015-04-01

    Momordica charantia (MC) is a medicinal plant for stroke treatment in Traditional Chinese Medicine, but its active compounds and molecular targets are unknown yet. M. charantia polysaccharide (MCP) is one of the important bioactive components in MC. In the present study, we tested the hypothesis that MCP has neuroprotective effects against cerebral ischemia/reperfusion injury through scavenging superoxide (O2(-)), nitric oxide (NO) and peroxynitrite (ONOO(-)) and inhibiting c-Jun N-terminal protein kinase (JNK3) signaling cascades. We conducted experiments with in vivo global and focal cerebral ischemia/reperfusion rat models and in vitro oxygen glucose deprivation (OGD) neural cells. The effects of MCP on apoptotic cell death and infarction volume, the bioactivities of scavenging O2(-), NO and ONOO(-), inhibiting lipid peroxidation and modulating JNK3 signaling pathway were investigated. Major results are summarized as below: (1) MCP dose-dependently attenuated apoptotic cell death in neural cells under OGD condition in vitro and reduced infarction volume in ischemic brains in vivo; (2) MCP had directing scavenging effects on NO, O2(-) and ONOO(-) and inhibited lipid peroxidation; (3) MCP inhibited the activations of JNK3/c-Jun/Fas-L and JNK3/cytochrome C/caspases-3 signaling cascades in ischemic brains in vivo. Taken together, we conclude that MCP could be a promising neuroprotective ingredient of M. charantia and its mechanisms could be at least in part attributed to its antioxidant activities and inhibiting JNK3 signaling cascades during cerebral ischemia/reperfusion injury. PMID:25510970

  7. Stress-induced phosphorylation of c-Jun-N-terminal kinases and nuclear translocation of Hsp70 in the Wistar rat hippocampus

    Directory of Open Access Journals (Sweden)

    Adžić M.

    2009-01-01

    Full Text Available Glucocorticoids are key regulators of the neuroendocrine stress response in the hippocampus. Their action is partly mediated through the subfamily of MAPKs termed c-Jun-N-terminal kinases (JNKs,whose activation correlates with neurodegeneration. The stress response also involves activation of cell protective mechanisms through various heat shock proteins (HSPs that mediate neuroprotection. We followed both JNKs and Hsp70 signals in the cytoplasmic and nuclear compartments of the hippocampus of Wistar male rats exposed to acute, chronic, and combined stress. The activity of JNK1 was decreased in both compartments by all three types of stress, while the activity of cytoplasmic JNK2/3 was elevated in acute and unaltered or lowered in chronic and combined stress. Under all stress conditions, Hsp70 translocation to the nucleus was markedly increased. The results suggest that neurodegenerative signaling of JNKs may be counteracted by increase of nuclear Hsp70,especially under chronic stress.

  8. c-Jun N-terminal kinase 3 expression in the retina of ocular hypertension mice: a possible target to reduce ganglion cell apoptosis

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    Yue He

    2015-01-01

    Full Text Available Glaucoma, a type of optic neuropathy, is characterized by the loss of retinal ganglion cells. It remains controversial whether c-Jun N-terminal kinase (JNK participates in the apoptosis of retinal ganglion cells in glaucoma. This study sought to explore a possible mechanism of action of JNK signaling pathway in glaucoma-induced retinal optic nerve damage. We established a mouse model of chronic ocular hypertension by reducing the aqueous humor followed by photocoagulation using the laser ignition method. Results showed significant pathological changes in the ocular tissues after the injury. Apoptosis of retinal ganglion cells increased with increased intraocular pressure, as did JNK3 mRNA expression in the retina. These data indicated that the increased expression of JNK3 mRNA was strongly associated with the increase in intraocular pressure in the retina, and correlated positively with the apoptosis of retinal ganglion cells.

  9. c-Jun N-terminal kinase 3 expression in the retina of ocular hypertension mice:a possible target to reduce ganglion cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Yue He; Jie Chen; Shu-guang Zhang; Yuan-sheng Yuan; Yan Li; Hong-bin Lv; Jin-hua Gan

    2015-01-01

    Glaucoma, a type of optic neuropathy, is characterized by the loss of retinal ganglion cells. It remains controversial whether c-Jun N-terminal kinase (JNK) participates in the apoptosis of retinal ganglion cells in glaucoma. This study sought to explore a possible mechanism of action of JNK signaling pathway in glaucoma-induced retinal optic nerve damage. We established a mouse model of chronic ocular hypertension by reducing the aqueous humor followed by pho-tocoagulation using the laser ignition method. Results showed signiifcant pathological changes in the ocular tissues after the injury. Apoptosis of retinal ganglion cells increased with increased intraocular pressure, as did JNK3 mRNA expression in the retina. These data indicated that the increased expression of JNK3 mRNA was strongly associated with the increase in intraocular pressure in the retina, and correlated positively with the apoptosis of retinal ganglion cells.

  10. Resveratrol alleviates diabetes-induced testicular dysfunction by inhibiting oxidative stress and c-Jun N-terminal kinase signaling in rats.

    Science.gov (United States)

    Faid, Iman; Al-Hussaini, Heba; Kilarkaje, Narayana

    2015-12-15

    Diabetes adversely affects reproductive functions in humans and animals. The present study investigated the effects of Resveratrol on diabetes-induced alterations in oxidative stress, c-Jun N-terminal kinase (JNK) signaling and apoptosis in the testis. Adult male Wistar rats (13-15 weeks; n=6/group) were segregated into 1) normal control, 2) Resveratrol-treated (5mg/kg; ip; given during last 3 weeks), 3) Streptozotocin-induced diabetic and, 4) Resveratrol-treated diabetic groups, and euthanized on day 42 after the confirmation of diabetes. Resveratrol did not normalize blood glucose levels in diabetic rats. Resveratrol supplementation recovered diabetes-induced decreases in reproductive organ weights, sperm count and motility, intra-testicular levels of superoxide dismutase, catalase, and glutathione peroxidase and an increase in 4-hydroxynonenal activities (Prats. These results suggest that Resveratrol supplementation may be a useful strategy to treat diabetes-induced testicular dysfunction. PMID:26499206

  11. Involvement of c-Jun N-terminal kinase in reversal of multidrug resistance of human leukemia cells in hypoxia by 5-bromotetrandrine.

    Science.gov (United States)

    Zhang, Wei; Chen, Bao-an; Jin, Jun-fei; He, You-ji; Niu, Yi-qi

    2013-11-01

    5-Bromotetrandrine (BrTet), a candidate multidrug resistance (MDR) modulator, is a potential compound for use in cancer therapy when combined with anticancer agents such as daunorubicin (DNR) and paclitaxel. The purposeof this study was to investigate the mechanism of reversal of P-glycoprotein (P-gp)-mediated MDR by BrTet and the involvement of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway in both adriamycin-sensitive K562 and adriamycin-resistant K562 (KA) leukemia cells in hypoxia. The combination of BrTet and DNR decreased both phosphorylated JNK1/2 and MDR1/P-gp levels under hypoxic conditions. Furthermore, a pharmacological inhibitor of JNK, SP600125, or small interfering RNA (siRNA) oligonucleotides to both JNK1 and JNK2 reversed BrTet- or DNR-induced JNK phosphorylation and MDR1/P-gp levels. We further demonstrated that the decreased JNK phosphorylation and MDR1/P-gp levels were associated with a significant increase in intracellular accumulation of DNR, which dramatically enhanced the sensitivity of drug-resistant KA cells to DNR, and led to cellular apoptosis through activation of the caspase-3 pathway. It is concluded that using BrTet in combination with other chemotherapeutic agents and pharmacological inhibitors of JNK can abrogate the P-gp-induced MDR in adriamycin-resistant K562 cells, which has potential clinical relevance in cancer therapy for chemotherapeutic-resistant human leukemia. PMID:23418897

  12. The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

    International Nuclear Information System (INIS)

    The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK. These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis

  13. Inhibition of Apoptosis in Prostate Cancer Cells by Androgens Is Mediated through Downregulation of c-Jun N-terminal Kinase Activation

    Directory of Open Access Journals (Sweden)

    Petra Isabel Lorenzo

    2008-05-01

    Full Text Available Androgen deprivation induces the regression of prostate tumors mainly due to an increase in the apoptosis rate; however, the molecular mechanisms underlying the antiapoptotic actions of androgens are not completely understood. We have studied the antiapoptotic effects of androgens in prostate cancer cells exposed to different proapoptotic stimuli. Terminal deoxynucleotidyl transferase-mediated nick-end labeling and nuclear fragmentation analyses demonstrated that androgens protect LNCaP prostate cancer cells from apoptosis induced by thapsigargin, the phorbol ester 12-O-tetradecanoyl-13-phorbol-acetate, or UV irradiation. These three stimuli require the activation of the c-Jun N-terminal kinase (JNK pathway to induce apoptosis and in all three cases, androgen treatment blocks JNK activation. Interestingly, okadaic acid, a phosphatase inhibitor that causes apoptosis in LNCaP cells, induces JNK activation that is also inhibited by androgens. Actinomycin D, the antiandrogen bicalutamide or specific androgen receptor (AR knockdown by small interfering RNA all blocked the inhibition of JNK activation mediated by androgens indicating that this activity requires AR-dependent transcriptional activation. These data suggest that the crosstalk between AR and JNK pathways may have important implications in prostate cancer progression and may provide targets for the development of new therapies.

  14. TAp73-mediated the activation of c-Jun N-terminal kinase enhances cellular chemosensitivity to cisplatin in ovarian cancer cells.

    Directory of Open Access Journals (Sweden)

    Pingde Zhang

    Full Text Available P73, one member of the tumor suppressor p53 family, shares highly structural and functional similarity to p53. Like p53, the transcriptionally active TAp73 can mediate cellular response to chemotherapeutic agents in human cancer cells by up-regulating the expressions of its pro-apoptotic target genes such as PUMA, Bax, NOXA. Here, we demonstrated a novel molecular mechanism for TAp73-mediated apoptosis in response to cisplatin in ovarian cancer cells, and that was irrespective of p53 status. We found that TAp73 acted as an activator of the c-Jun N-terminal kinase (JNK signaling pathway by up-regulating the expression of its target growth arrest and DNA-damage-inducible protein GADD45 alpha (GADD45α and subsequently activating mitogen-activated protein kinase kinase-4 (MKK4. Inhibition of JNK activity by a specific inhibitor or small interfering RNA (siRNA significantly abrogated TAp73-mediated apoptosis induced by cisplatin. Furthermore, inhibition of GADD45α by siRNA inactivated MKK4/JNK activities and also blocked TAp73-mediated apoptosis induction by cisplatin. Our study has demonstrated that TAp73 activated the JNK apoptotic signaling pathway in response to cisplatin in ovarian cancer cells.

  15. EGCG-targeted p57/KIP2 reduces tumorigenicity of oral carcinoma cells: Role of c-Jun N-terminal kinase

    International Nuclear Information System (INIS)

    The green tea polyphenol epigallocatechin-3-gallate (EGCG) regulates gene expression differentially in tumor and normal cells. In normal human primary epidermal keratinocytes (NHEK), one of the key mediators of EGCG action is p57/KIP2, a cyclin-dependent kinase (CDK) inhibitor. EGCG potently induces p57 in NHEK, but not in epithelial cancer cells. In humans, reduced expression of p57 often is associated with advanced tumors, and tumor cells with inactivated p57 undergo apoptosis when exposed to EGCG. The mechanism of p57 induction by EGCG is not well understood. Here, we show that in NHEK, EGCG-induces p57 via the p38 mitogen-activated protein kinase (MAPK) signaling pathway. In p57-negative tumor cells, JNK signaling mediates EGCG-induced apoptosis, and exogenous expression of p57 suppresses EGCG-induced apoptosis via inhibition of c-Jun N-terminal kinase (JNK). We also found that restoration of p57 expression in tumor cells significantly reduced tumorigenicity in athymic mice. These results suggest that p57 expression may be an useful indicator for the clinical course of cancers, and could be potentially useful as a target for cancer therapies

  16. Inhibition of spinal astrocytic c-Jun N-terminal kinase (JNK activation correlates with the analgesic effects of ketamine in neuropathic pain

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    Wang Wen

    2011-01-01

    Full Text Available Abstract Background We have previously reported that inhibition of astrocytic activation contributes to the analgesic effects of intrathecal ketamine on spinal nerve ligation (SNL-induced neuropathic pain. However, the underlying mechanisms are still unclear. c-Jun N-terminal kinase (JNK, a member of mitogen-activated protein kinase (MAPK family, has been reported to be critical for spinal astrocytic activation and neuropathic pain development after SNL. Ketamine can decrease lipopolysaccharide (LPS-induced phosphorylated JNK (pJNK expression and could thus exert its anti-inflammatory effect. We hypothesized that inhibition of astrocytic JNK activation might be involved in the suppressive effect of ketamine on SNL-induced spinal astrocytic activation. Methods Immunofluorescence histochemical staining was used to detect SNL-induced spinal pJNK expression and localization. The effects of ketamine on SNL-induced mechanical allodynia were confirmed by behavioral testing. Immunofluorescence histochemistry and Western blot were used to quantify the SNL-induced spinal pJNK expression after ketamine administration. Results The present study showed that SNL induced ipsilateral pJNK up-regulation in astrocytes but not microglia or neurons within the spinal dorsal horn. Intrathecal ketamine relieved SNL-induced mechanical allodynia without interfering with motor performance. Additionally, intrathecal administration of ketamine attenuated SNL-induced spinal astrocytic JNK activation in a dose-dependent manner, but not JNK protein expression. Conclusions The present results suggest that inhibition of JNK activation may be involved in the suppressive effects of ketamine on SNL-induced spinal astrocyte activation. Therefore, inhibition of spinal JNK activation may be involved in the analgesic effects of ketamine on SNL-induced neuropathic pain.

  17. Glycogen synthase kinase 3β sustains invasion of glioblastoma via the focal adhesion kinase, Rac1, and c-Jun N-terminal kinase-mediated pathway.

    Science.gov (United States)

    Chikano, Yuri; Domoto, Takahiro; Furuta, Takuya; Sabit, Hemragul; Kitano-Tamura, Ayako; Pyko, Ilya V; Takino, Takahisa; Sai, Yoshimichi; Hayashi, Yutaka; Sato, Hiroshi; Miyamoto, Ken-ichi; Nakada, Mitsutoshi; Minamoto, Toshinari

    2015-02-01

    The failure of current treatment options for glioblastoma stems from their inability to control tumor cell proliferation and invasion. Biologically targeted therapies offer great hope and one promising target is glycogen synthase kinase-3β (GSK3β), implicated in various diseases, including cancer. We previously reported that inhibition of GSK3β compromises the survival and proliferation of glioblastoma cells, induces their apoptosis, and sensitizes them to temozolomide and radiation. Here, we explore whether GSK3β also contributes to the highly invasive nature of glioblastoma. The effects of GSK3β inhibition on migration and invasion of glioblastoma cells were examined by wound-healing and Transwell assays, as well as in a mouse model of glioblastoma. We also investigated changes in cellular microarchitectures, cytoskeletal components, and proteins responsible for cell motility and invasion. Inhibition of GSK3β attenuated the migration and invasion of glioblastoma cells in vitro and that of tumor cells in a mouse model of glioblastoma. These effects were associated with suppression of the molecular axis involving focal adhesion kinase, guanine nucleotide exchange factors/Rac1 and c-Jun N-terminal kinase. Changes in cellular phenotypes responsible for cell motility and invasion were also observed, including decreased formation of lamellipodia and invadopodium-like microstructures and alterations in the subcellular localization, and activity of Rac1 and F-actin. These changes coincided with decreased expression of matrix metalloproteinases. Our results confirm the potential of GSK3β as an attractive therapeutic target against glioblastoma invasion, thus highlighting a second role in this tumor type in addition to its involvement in chemo- and radioresistance. PMID:25504636

  18. Hydrogen-Rich Saline Attenuates Lipopolysaccharide-Induced Heart Dysfunction by Restoring Fatty Acid Oxidation in Rats by Mitigating C-Jun N-Terminal Kinase Activation.

    Science.gov (United States)

    Tao, Bingdong; Liu, Lidan; Wang, Ni; Tong, Dongyi; Wang, Wei; Zhang, Jin

    2015-12-01

    Sepsis is common in intensive care units (ICU) and is associated with high mortality. Cardiac dysfunction complicating sepsis is one of the most important causes of this mortality. This dysfunction is due to myocardial inflammation and reduced production of energy by the heart. A number of studies have shown that hydrogen-rich saline (HRS) has a beneficial effect on sepsis. Therefore, we tested whether HRS prevents cardiac dysfunction by increasing cardiac energy. Four groups of rats received intraperitoneal injections of one of the following solutions: normal saline (NS), HRS, lipopolysaccharide (LPS), and LPS plus HRS. Cardiac function was measured by echocardiography 8 h after the injections. Gene and protein expression related to fatty acid oxidation (FAO) were measured by quantitative polymerase chain reaction (PCR) and Western blot analysis. The injection of LPS compromised heart function through decreased fractional shortening (FS) and increased left ventricular diameter (LVD). The addition of HRS increased FS, palmitate triphosphate, and the ratio of phosphocreatinine (PCr) to adenosine triphosphate (ATP) as well as decreasing LVD. The LPS challenge reduced the expression of genes related to FAO, including perioxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), perioxisome proliferator-activated receptor alpha (PPARα), Estrogen-related receptor alpha (ERRα), and their downstream targets, in mRNA and protein level, which were attenuated by HRS. However, HRS had little effect on glucose metabolism. Furthermore, HRS inhibited c-Jun N-terminal kinase (JNK) activation in the rat heart. Inhibition of JNK by HRS showed beneficial effects on LPS-challenged rats, at least in part, by restoring cardiac FAO.

  19. Colchicine induces apoptosis in HT‑29 human colon cancer cells via the AKT and c-Jun N-terminal kinase signaling pathways.

    Science.gov (United States)

    Huang, Zhen; Xu, Ye; Peng, Wei

    2015-10-01

    Colchicine is a natural compound, which belongs to the botanical family Colchicaceae and prevents growth of cancer cells via antimitotic activity by interacting with microtubules. Although numerous studies have demonstrated that the effect of colchicine on cell apoptosis is mediated by the activation of caspase‑3, the signaling pathways involved in the process remain unknown. In the current study, evidence is presented regarding the missing information using HT‑29 human colon cancer cells. The effect of colchicine on apoptosis in HT‑29 cells and the apoptosis‑associated signaling pathways were determined using various methods, including cell viability assay, Annexin V/propidium idodide (PI) binding, PI staining, Hoechst 33342 staining, mitochondrial membrane potential (Δψm) assay, reactive oxygen species (ROS) assay and western blot analysis. Colchicine was observed to induce a dose‑dependent reduction in cell viability in HT‑29 cells and early apoptosis occurred when the cells were treated with 1 µg/ml colchicine. Furthermore, colchicine treatment induced a loss of Δψm, increased ROS production, activated caspase‑3, upregulated BAX expression and downregulated Bcl‑2 expression, which evidenced the colchicine activity on apoptosis, potentially by acting via the intrinsic apoptotic signaling pathway. Colchicine increased phosphorylation of p38, although not phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase, which indicates that colchicine activates the p38 signaling pathway in order to induce cell apoptosis. Therefore, colchicine exhibited significant growth inhibition of the HT‑29 colon cancer cell line and induced apoptosis in the cells via the mitochondrial pathway, which is regulated by p38 signaling pathways. PMID:26299305

  20. A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-terminal kinase signaling pathway.

    Science.gov (United States)

    Yao, Z; Zhou, G; Wang, X S; Brown, A; Diener, K; Gan, H; Tan, T H

    1999-01-22

    The yeast serine/threonine kinase STE20 activates a signaling cascade that includes STE11 (mitogen-activated protein kinase kinase kinase), STE7 (mitogen-activated protein kinase kinase), and FUS3/KSS1 (mitogen-activated protein kinase) in response to signals from both Cdc42 and the heterotrimeric G proteins associated with transmembrane pheromone receptors. Using degenerate polymerase chain reaction, we have isolated a human cDNA encoding a protein kinase homologous to STE20. This protein kinase, designated HPK/GCK-like kinase (HGK), has nucleotide sequences that encode an open reading frame of 1165 amino acids with 11 kinase subdomains. HGK was a serine/threonine protein kinase that specifically activated the c-Jun N-terminal kinase (JNK) signaling pathway when transfected into 293T cells, but it did not stimulate either the extracellular signal-regulated kinase or p38 kinase pathway. HGK also increased AP-1-mediated transcriptional activity in vivo. HGK-induced JNK activation was inhibited by the dominant-negative MKK4 and MKK7 mutants. The dominant-negative mutant of TAK1, but not MEKK1 or MAPK upstream kinase (MUK), strongly inhibited HGK-induced JNK activation. TNF-alpha activated HGK in 293T cells, as well as the dominant-negative HGK mutants, inhibited TNF-alpha-induced JNK activation. These results indicate that HGK, a novel activator of the JNK pathway, may function through TAK1, and that the HGK --> TAK1 --> MKK4, MKK7 --> JNK kinase cascade may mediate the TNF-alpha signaling pathway. PMID:9890973

  1. The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

    Energy Technology Data Exchange (ETDEWEB)

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Xie, Yuchao [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Farhood, Anwar [Department of Pathology, St. David' s North Austin Medical Center, Austin, TX 78756 (United States); Vinken, Mathieu [Department of Toxicology, Center for Pharmaceutical Sciences, Vrije Universiteit Brussels, 1090 Brussels (Belgium); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2013-12-15

    Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2-APB

  2. Lower susceptibility of female mice to acetaminophen hepatotoxicity: Role of mitochondrial glutathione, oxidant stress and c-jun N-terminal kinase

    International Nuclear Information System (INIS)

    Acetaminophen (APAP) overdose causes severe hepatotoxicity in animals and humans. However, the mechanisms underlying the gender differences in susceptibility to APAP overdose in mice have not been clarified. In our study, APAP (300 mg/kg) caused severe liver injury in male mice but 69–77% lower injury in females. No gender difference in metabolic activation of APAP was found. Hepatic glutathione (GSH) was rapidly depleted in both genders, while GSH recovery in female mice was 2.6 fold higher in the mitochondria at 4 h, and 2.5 and 3.3 fold higher in the total liver at 4 h and 6 h, respectively. This faster recovery of GSH, which correlated with greater induction of glutamate-cysteine ligase, attenuated mitochondrial oxidative stress in female mice, as suggested by a lower GSSG/GSH ratio at 6 h (3.8% in males vs. 1.4% in females) and minimal centrilobular nitrotyrosine staining. While c-jun N-terminal kinase (JNK) activation was similar at 2 and 4 h post-APAP, it was 3.1 fold lower at 6 h in female mice. However, female mice were still protected by the JNK inhibitor SP600125. 17β-Estradiol pretreatment moderately decreased liver injury and oxidative stress in male mice without affecting GSH recovery. Conclusion: The lower susceptibility of female mice is achieved by the improved detoxification of reactive oxygen due to accelerated recovery of mitochondrial GSH levels, which attenuates late JNK activation and liver injury. However, even the reduced injury in female mice was still dependent on JNK. While 17β-estradiol partially protects male mice, it does not affect hepatic GSH recovery. - Highlights: • Female mice are less susceptible to acetaminophen overdose than males. • GSH depletion and protein adduct formation are similar in both genders. • Recovery of hepatic GSH levels is faster in females and correlates with Gclc. • Reduced oxidant stress in females leads to reduced JNK activation. • JNK activation and mitochondrial translocation are critical

  3. Lower susceptibility of female mice to acetaminophen hepatotoxicity: Role of mitochondrial glutathione, oxidant stress and c-jun N-terminal kinase

    Energy Technology Data Exchange (ETDEWEB)

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu

    2014-11-15

    Acetaminophen (APAP) overdose causes severe hepatotoxicity in animals and humans. However, the mechanisms underlying the gender differences in susceptibility to APAP overdose in mice have not been clarified. In our study, APAP (300 mg/kg) caused severe liver injury in male mice but 69–77% lower injury in females. No gender difference in metabolic activation of APAP was found. Hepatic glutathione (GSH) was rapidly depleted in both genders, while GSH recovery in female mice was 2.6 fold higher in the mitochondria at 4 h, and 2.5 and 3.3 fold higher in the total liver at 4 h and 6 h, respectively. This faster recovery of GSH, which correlated with greater induction of glutamate-cysteine ligase, attenuated mitochondrial oxidative stress in female mice, as suggested by a lower GSSG/GSH ratio at 6 h (3.8% in males vs. 1.4% in females) and minimal centrilobular nitrotyrosine staining. While c-jun N-terminal kinase (JNK) activation was similar at 2 and 4 h post-APAP, it was 3.1 fold lower at 6 h in female mice. However, female mice were still protected by the JNK inhibitor SP600125. 17β-Estradiol pretreatment moderately decreased liver injury and oxidative stress in male mice without affecting GSH recovery. Conclusion: The lower susceptibility of female mice is achieved by the improved detoxification of reactive oxygen due to accelerated recovery of mitochondrial GSH levels, which attenuates late JNK activation and liver injury. However, even the reduced injury in female mice was still dependent on JNK. While 17β-estradiol partially protects male mice, it does not affect hepatic GSH recovery. - Highlights: • Female mice are less susceptible to acetaminophen overdose than males. • GSH depletion and protein adduct formation are similar in both genders. • Recovery of hepatic GSH levels is faster in females and correlates with Gclc. • Reduced oxidant stress in females leads to reduced JNK activation. • JNK activation and mitochondrial translocation are critical

  4. The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

    International Nuclear Information System (INIS)

    Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2-APB

  5. Tumor Necrosis Factor-α and Apoptosis Signal-Regulating Kinase 1 Control Reactive Oxygen Species Release, Mitochondrial Autophagy and C-Jun N-Terminal Kinase/P38 Phosphorylation During Necrotizing Enterocolitis

    Directory of Open Access Journals (Sweden)

    Naira Baregamian

    2009-01-01

    Full Text Available Background: Oxidative stress and inflammation may contribute to the disruption of the protective gut barrier through various mechanisms; mitochondrial dysfunction resulting from inflammatory and oxidative injury may potentially be a significant source of apoptosis during necrotizing enterocolitis (NEC. Tumor necrosis factor (TNFα is thought to generate reactive oxygen species (ROS and activate the apoptosis signal-regulating kinase 1 (ASK1-c-Jun N-terminal kinase (JNK/p38 pathway. Hence, the focus of our study was to examine the effects of TNFα/ROs on mitochondrial function, ASK1-JNK/p38 cascade activation in intestinal epithelial cells during NEC.

  6. Growth arrest- and DNA-damage-inducible 45beta gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1beta-induced apoptosis in insulin-producing INS-1E cells

    DEFF Research Database (Denmark)

    Larsen, Claus Morten; Døssing, M G; Papa, S;

    2006-01-01

    IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen......-activated protein kinase (MAPK) family. Inhibition of JNK prevents IL-1beta-mediated beta cell destruction. In mouse embryo fibroblasts and 3DO T cells, overexpression of the gene encoding growth arrest and DNA-damage-inducible 45beta (Gadd45b) downregulates pro-apoptotic JNK signalling. The aim of this study...

  7. Streptococcus pneumoniae induced c-Jun-N-terminal kinase- and AP-1 -dependent IL-8 release by lung epithelial BEAS-2B cells

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    Rosseau Simone

    2006-07-01

    Full Text Available Abstract Background Although pneumococcal pneumonia is one of the most common causes of death due to infectious diseases, little is known about pneumococci-lung cell interaction. Herein we tested the hypothesis that pneumococci activated pulmonary epithelial cell cytokine release by c-Jun-NH2-terminal kinase (JNK Methods Human bronchial epithelial cells (BEAS-2B or epithelial HEK293 cells were infected with S. pneumoniae R6x and cytokine induction was measured by RT-PCR, ELISA and Bioplex assay. JNK-phosphorylation was detected by Western blot and nuclear signaling was assessed by electrophoretic mobility shift assay (EMSA and chromatin immunoprecipitation (ChIP. JNK was modulated by the small molecule inhibitor SP600125 and AP1 by transfection of a dominant negative mutant. Results S. pneumoniae induced the release of distinct CC and CXC, as well as Th1 and Th2 cytokines and growth factors by human lung epithelial cell line BEAS-2B. Furthermore, pneumococci infection resulted in JNK phosphorylation in BEAS-2B cells. Inhibition of JNK by small molecule inhibitor SP600125 reduced pneumococci-induced IL-8 mRNA expression and release of IL-8 and IL-6. One regulator of the il8 promoter is JNK-phosphorylated activator protein 1 (AP-1. We showed that S. pneumoniae time-dependently induced DNA binding of AP-1 and its phosphorylated subunit c-Jun with a maximum at 3 to 5 h after infection. Recruitment of Ser63/73-phosphorylated c-Jun and RNA polymerase II to the endogenous il8 promoter was found 2 h after S. pneumoniae infection by chromatin immunoprecipitation. AP-1 repressor A-Fos reduced IL-8 release by TLR2-overexpressing HEK293 cells induced by pneumococci but not by TNFα. Antisense-constructs targeting the AP-1 subunits Fra1 and Fra2 had no inhibitory effect on pneumococci-induced IL-8 release. Conclusion S. pneumoniae-induced IL-8 expression by human epithelial BEAS-2B cells depended on activation of JNK and recruitment of phosphorylated c-Jun

  8. Calcium has a permissive role in interleukin-1beta-induced c-jun N-terminal kinase activation in insulin-secreting cells

    DEFF Research Database (Denmark)

    Størling, Joachim; Zaitsev, Sergei V; Kapelioukh, Iouri L;

    2005-01-01

    -acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca(2+)/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1beta-stimulated c-jun phosphorylation in INS-1 or betaTC3 cells. Our data suggest that Ca(2+) plays a permissive role in IL-1beta activation of the JNK signaling pathway in insulin......-cells are largely unknown. In this study, we investigated whether Ca(2+) plays a role for IL-1beta-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mm glucose, combined pharmacological blockade of L- and T-type Ca(2+) channels suppressed IL-1beta-induced in vitro...

  9. Highly Oxygenated Sesquiterpene Lactones from Cousinia aitchisonii and their Cytotoxic Properties: Rhaserolide Induces Apoptosis in Human T Lymphocyte (Jurkat) Cells via the Activation of c-Jun n-terminal Kinase Phosphorylation.

    Science.gov (United States)

    Iranshahy, Milad; Tayarani-Najaran, Zahra; Kasaian, Jamal; Ghandadi, Morteza; Emami, Seyed Ahmad; Asili, Javad; Chandran, Jima N; Schneider, Bernd; Iranshahi, Mehrdad

    2016-02-01

    Infrared-guided chromatographic fractionation of sesquiterpene lactones from the extracts of Cousinia aitchisonii and Cousinia concolor led to the isolation of five pure compounds. A new sesquiterpene lactone, namely, aitchisonolide, and two known sesquiterpene lactones (desoxyjanerin and rhaserolide) were isolated from C. aitchisonii and two known lignans (arctiin and arctigenin) from C. concolor. The structures of these compounds were elucidated by one-dimensional and two-dimensional nuclear magnetic resonance techniques, as well as high-resolution mass spectrometry. The purified and characterized compounds were subjected to cytotoxicity assay. The sesquiterpene lactones desoxyjanerin and rhaserolide showed significant cytotoxic activities against five different cancer cell lines and the normal human embryonic kidney cell line. Rhaserolide was chosen to evaluate the possible mechanism of action. Western blot analysis revealed that rhaserolide could induce apoptosis in Jurkat cells via the activation of c-Jun n-terminal kinase phosphorylation. PMID:26581585

  10. Highly Oxygenated Sesquiterpene Lactones from Cousinia aitchisonii and their Cytotoxic Properties: Rhaserolide Induces Apoptosis in Human T Lymphocyte (Jurkat) Cells via the Activation of c-Jun n-terminal Kinase Phosphorylation.

    Science.gov (United States)

    Iranshahy, Milad; Tayarani-Najaran, Zahra; Kasaian, Jamal; Ghandadi, Morteza; Emami, Seyed Ahmad; Asili, Javad; Chandran, Jima N; Schneider, Bernd; Iranshahi, Mehrdad

    2016-02-01

    Infrared-guided chromatographic fractionation of sesquiterpene lactones from the extracts of Cousinia aitchisonii and Cousinia concolor led to the isolation of five pure compounds. A new sesquiterpene lactone, namely, aitchisonolide, and two known sesquiterpene lactones (desoxyjanerin and rhaserolide) were isolated from C. aitchisonii and two known lignans (arctiin and arctigenin) from C. concolor. The structures of these compounds were elucidated by one-dimensional and two-dimensional nuclear magnetic resonance techniques, as well as high-resolution mass spectrometry. The purified and characterized compounds were subjected to cytotoxicity assay. The sesquiterpene lactones desoxyjanerin and rhaserolide showed significant cytotoxic activities against five different cancer cell lines and the normal human embryonic kidney cell line. Rhaserolide was chosen to evaluate the possible mechanism of action. Western blot analysis revealed that rhaserolide could induce apoptosis in Jurkat cells via the activation of c-Jun n-terminal kinase phosphorylation.

  11. Hippocampal activation of c-Jun N-terminal kinase,protein kinase B,and p38 mitogen-activated protein kinase in a chronic stress rat model of depression

    Institute of Scientific and Technical Information of China (English)

    Wei Dai; Weidong Li; Jun Lu; Yingge A; Ya Tu

    2010-01-01

    Recent studies have shown that vaned stress stimuli activate c-Jun N-terminal kinase(JNK),protein kinase B(Akt),and p38 mitogen-activated protein kinase(p38)signal transduction pathway,and also regulate various apoptotic cascades.JNK and p38 promote apoptosis,but Akt protects against apoptosis,in hippocampal neurons.However,changes in the transduction pathway in different regions of brain tissues in a chronic stress rat model of depression remain poorly understood.Results from this study showed that JNK phosphorylation levels were significantly greater in the stress group hippocampus compared with the control group(P 0.05).These results suggested that the JNK signal pathway is activated by JNK phosphorylation and participates in pathophysiological changes in rat models of depression.

  12. Hyperoside Downregulates the Receptor for Advanced Glycation End Products (RAGE and Promotes Proliferation in ECV304 Cells via the c-Jun N-Terminal Kinases (JNK Pathway Following Stimulation by Advanced Glycation End-Products In Vitro

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    Zhengyu Zhang

    2013-11-01

    Full Text Available Hyperoside is a major active constituent in many medicinal plants which are traditionally used in Chinese medicines for their neuroprotective, anti-inflammatory and antioxidative effects. The molecular mechanisms underlying these effects are unknown. In this study, quiescent ECV304 cells were treated in vitro with advanced glycation end products (AGEs in the presence or absence of hyperoside. The results demonstrated that AGEs induced c-Jun N-terminal kinases (JNK activation and apoptosis in ECV304 cells. Hyperoside inhibited these effects and promoted ECV304 cell proliferation. Furthermore, hyperoside significantly inhibited RAGE expression in AGE-stimulated ECV304 cells, whereas knockdown of RAGE inhibited AGE-induced JNK activation. These results suggested that AGEs may promote JNK activation, leading to viability inhibition of ECV304 cells via the RAGE signaling pathway. These effects could be inhibited by hyperoside. Our findings suggest a novel role for hyperoside in the treatment and prevention of diabetes.

  13. c-Jun N-terminal kinase phosphorylation of MARCKSL1 determines actin stability and migration in neurons and in cancer cells.

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    Björkblom, Benny; Padzik, Artur; Mohammad, Hasan; Westerlund, Nina; Komulainen, Emilia; Hollos, Patrik; Parviainen, Lotta; Papageorgiou, Anastassios C; Iljin, Kristiina; Kallioniemi, Olli; Kallajoki, Markku; Courtney, Michael J; Mågård, Mats; James, Peter; Coffey, Eleanor T

    2012-09-01

    Cell migration is a fundamental biological function, critical during development and regeneration, whereas deregulated migration underlies neurological birth defects and cancer metastasis. MARCKS-like protein 1 (MARCKSL1) is widely expressed in nervous tissue, where, like Jun N-terminal protein kinase (JNK), it is required for neural tube formation, though the mechanism is unknown. Here we show that MARCKSL1 is directly phosphorylated by JNK on C-terminal residues (S120, T148, and T183). This phosphorylation enables MARCKSL1 to bundle and stabilize F-actin, increase filopodium numbers and dynamics, and retard migration in neurons. Conversely, when MARCKSL1 phosphorylation is inhibited, actin mobility increases and filopodium formation is compromised whereas lamellipodium formation is enhanced, as is cell migration. We find that MARCKSL1 mRNA is upregulated in a broad range of cancer types and that MARCKSL1 protein is strongly induced in primary prostate carcinomas. Gene knockdown in prostate cancer cells or in neurons reveals a critical role for MARCKSL1 in migration that is dependent on the phosphorylation state; phosphomimetic MARCKSL1 (MARCKSL1(S120D,T148D,T183D)) inhibits whereas dephospho-MARCKSL1(S120A,T148A,T183A) induces migration. In summary, these data show that JNK phosphorylation of MARCKSL1 regulates actin homeostasis, filopodium and lamellipodium formation, and neuronal migration under physiological conditions and that, when ectopically expressed in prostate cancer cells, MARCKSL1 again determines cell movement.

  14. C-Jun N-terminal kinase signal pathway and C-Jun N-terminal kinase inhibitor SP600125 in amygdala kindled rats%c-Jun氨基末端激酶信号通路及其抑制剂SP600125在大鼠杏仁核电刺激癫痫模型中的作用

    Institute of Scientific and Technical Information of China (English)

    吴俊; 陈旭; 舒凯; 肖铮铮; 雷霆; 李龄

    2012-01-01

    Objective By injecting SP600125 into ventricle of amygdale kindled rats,to observe the pathological changes of the hippocampus and the change of C-Jun N-terminal kinase (JNK) phosphorylation,and discuss the action mechanism of SP600125.Methods Forty rats were randomly divided into 4 groups (n =10 each):blank group,kindling group,SP600125 group,DMSO group.Whole-cell extracts of tissues were obtained from the right hippocampus,and Western blotting was used to detect the changes of JNK and phosphorylation of JNK.Pathological changes of the hippocampus and amygdla were observed by GFAP stain and Nissl stain.Results The level of JNK phosphorylation in the hippocampus was significantly higher in the kindling group (0.48 ± 0.04 ) than the blank group (0.38 ± 0.04 ) and the SP600125 group (0.37±0.03).Nissl stain positive cells in the hippocampus of the SP600125 group were significantly more than those in the the DMSO group (20.10 ±5.11 ).The expression of GFAP in the hippocampus of kindling group (65.45 ±4.53 ) and DMSO group (67.18 ± 3.52) was significantly stronger than that in the blank group (40.37 ± 3.82) and the SP600125 group (43.51 ± 1.83).Conclusion The role of repeated activation of JNK can be related to the hippocampal sclerosis in these rats.SP600125 had a protective effect on neurons during the kindling procedure.%目的 通过对杏仁核电刺激癫痫模型大鼠脑室内注射c-Jun氨基末端激酶(JNK)特异性抑制剂SP600125,观察海马区的病理变化和JNK水平的变化,探讨SP600125的作用.方法 将40只Wistar大鼠随机分为4组:空白组、点燃组、加药组和加药对照组各10只,10次癫痫发作后灌注取脑,Western blot法检测JNK的表达变化,进行尼氏和胶原纤维酸性蛋白(GFAP)染色,各组间进行比较.结果 Western blot显示点燃组海马区的JNK磷酸化水平(0.48±0.04)较空白组(0.38±0.04)和加药组(0.37±0.03)显著增高(P<0.05),总JNK水平各组之间差异无统计学意义(P>0

  15. Drosophila endocytic neoplastic tumor suppressor genes regulate Sav/Wts/Hpo signaling and the c-Jun N-terminal kinase pathway.

    Science.gov (United States)

    Robinson, Brian S; Moberg, Kenneth H

    2011-12-01

    Genetic screens in the fruit fly Drosophila melanogaster have identified a class of neoplastic tumor suppressor genes (endocytic nTSGs), which encode proteins that localize to endosomes and facilitate the trafficking of membrane-bound receptors and adhesion molecules into the degradative lysosome. Loss of endocytic nTSGs transforms imaginal disc epithelia into highly proliferative, invasive tissues that fail to differentiate and display defects in cellular apicobasal polarity, adhesion and tissue architecture. As vertebrate homologs of some Drosophila nTSGs are linked to tumor formation, identifying molecular changes in signaling associated with nTSG loss could inform understanding of neoplastic transformation in vertebrates. Here we show that mutations in genes that act at multiple steps of the endolysosomal pathway lead to autonomous activation of the Sav/Wts/Hpo (SWH) transcriptional effector Yki (YAP/TAZ in vertebrates) and the Jun N-terminal kinase (JNK), which is known to promote Yki activity in cells with disrupted polarity. Yki and JNK activity are elevated by mutations at multiple steps in the endolysosomal pathway including mutations in the AP-2σ gene, which encodes a component of the AP-2 adaptor complex that recruits cargoes into clathrin-coated pits for subsequent internalization. Moreover, reduction of JNK activity can decrease elevated Yki-signaling caused by altered endocytosis. These studies reveal a broad requirement for components of the endocytic pathway in regulating SWH and JNK outputs, and place Drosophila endocytic nTSGs into a network that involving two major signaling pathways implicated in oncogenesis. PMID:22101275

  16. Cytotoxic Activity of 3,6-Dihydroxyflavone in Human Cervical Cancer Cells and Its Therapeutic Effect on c-Jun N-Terminal Kinase Inhibition

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    Eunjung Lee

    2014-08-01

    Full Text Available Previously we have shown that 3,6-dihydroxyflavone (3,6-DHF is a potent agonist of the human peroxisome proliferator-activated receptor (hPPAR with cytotoxic effects on human cervical cancer cells. To date, the mechanisms by which 3,6-DHF exerts its antitumor effects on cervical cells have not been clearly defined. Here, we demonstrated that 3,6-DHF exhibits a novel antitumor activity against HeLa cells with IC50 values of 25 μM and 9.8 μM after 24 h and 48 h, respectively. We also showed that the anticancer effects of 3,6-DHF are mediated via the toll-like receptor (TLR 4/CD14, p38 mitogen-activated protein kinase (MAPK, Jun-N terminal kinase (JNK, extracellular-signaling regulated kinase (ERK, and cyclooxygenase (COX-2 pathways in lipopolysaccharide (LPS-stimulated RAW264.7 cells. We found that 3,6-DHF showed a similar IC50 (113 nM value to that of the JNK inhibitor, SP600125 (IC50 = 118 nM in a JNK1 kinase assay. Binding studies revealed that 3,6-DHF had a strong binding affinity to JNK1 (1.996 × 105 M−1 and that the 6-OH and the carbonyl oxygen of the C ring of 3,6-DHF participated in hydrogen bonding interactions with the carbonyl oxygen and the amide proton of Met111, respectively. Therefore, 3,6-DHF may be a candidate inhibitor of JNKs, with potent anticancer effects.

  17. Mesenchymal stem cells promote liver regeneration and prolong survival in small-for-size liver grafts: involvement of C-Jun N-terminal kinase, cyclin D1, and NF-κB.

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    Weijie Wang

    Full Text Available BACKGROUND: The therapeutic potential of mesenchymal stem cells (MSCs has been highlighted recently for treatment of acute or chronic liver injury, by possibly differentiating into hepatocyte-like cells, reducing inflammation, and enhancing tissue repair. Despite recent progress, exact mechanisms of action are not clearly elucidated. In this study, we attempted to explore whether and how MSCs protected hepatocytes and stimulated allograft regeneration in small-for-size liver transplantation (SFSLT. METHODS: SFSLT model was established with a 30% partial liver transplantation (30PLT in rats. The differentiation potential and characteristics of bone marrow derived MSCs were explored in vitro. MSCs were infused transvenously immediately after graft implantation in therapy group. Expressions of apoptosis-, inflammatory-, anti-inflammatory-, and growth factor-related genes were measured by RT-PCR, activities of transcription factors AP-1 and NF-κB were analyzed by EMSA, and proliferative responses of the hepatic graft were evaluated by immunohistochemistry and western blot. RESULTS: MSCs were successfully induced into hepatocyte-like cells, osteoblasts and adipocytes in vitro. MSCs therapy could not only alleviate ischemia reperfusion injury and acute inflammation to promote liver regeneration, but also profoundly improve one week survival rate. It markedly up-regulated the mRNA expressions of HGF, Bcl-2, Bcl-XL, IL-6, IL-10, IP-10, and CXCR2, however, down-regulated TNF-α. Increased activities of AP-1 and NF-κB, as well as elevated expressions of p-c-Jun, cyclin D1, and proliferating cell nuclear antigen (PCNA, were also found in MSCs therapy group. CONCLUSION: These data suggest that MSCs therapy promotes hepatocyte proliferation and prolongs survival in SFSLT by reducing ischemia reperfusion injury and acute inflammation, and sustaining early increased expressions of c-Jun N-terminal Kinase, Cyclin D1, and NF-κB.

  18. c-Jun N-terminal kinases 3 (JNK3) from orange-spotted grouper, Epinephelus coioides, inhibiting the replication of Singapore grouper iridovirus (SGIV) and SGIV-induced apoptosis.

    Science.gov (United States)

    Guo, Minglan; Wei, Jingguang; Zhou, Yongcan; Qin, Qiwei

    2016-12-01

    C-Jun N-terminal kinases (JNKs), a subgroup of serine-threonine protein kinases that activated by phosphorylation, are involve in physiological and pathophysiological processes. JNK3 is one of JNK proteins involved in JNK3 signaling transduction. In the present study, two JNK3 isoforms, Ec-JNK3 X1 and Ec-JNK3 X2, were cloned from orange-spotted grouper, Epinephelus coioides. Both Ec-JNK3 X1 and Ec-JNK3 X2 were mainly expressed in liver, gill, skin, brain and muscle of juvenile grouper. The relative expression of Ec-JNK3 X2 mRNA was much higher in muscle and gill than that of Ec-JNK3 X1. Isoform-specific immune response to challenges was revealed by the expression profiles in vivo. Immunofluorescence staining indicated that JNK3 was localized in the cytoplasm of grouper spleen (GS) cells and shown immune response to SGIV infection in vitro. Over-expressing Ec-JNK3 X1 and/or Ec-JNK3 X2 inhibited the SGIV infection and replication and the SGIV-induced apoptosis. To achieve the antiviral and anti-apoptosis activities, JNK3 promoted the activation of genes ISRE and type I IFN in the antiviral IFN signaling pathway, and inhibited the activation of transcription factors NF-κB and p53 relating to apoptosis, respectively. Ec-JNK3 X2 showed stronger activities in antivirus and anti-apoptosis than that of Ec-JNK3 X1. Our results not only define the characterization of JNK3 but also reveal new immune functions and the molecular mechanisms of JNK3 on iridoviruses infection and the virus-induced apoptosis.

  19. Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase.

    Science.gov (United States)

    Wang, Xiao-Hu; Hong, Xin; Zhu, Lei; Wang, Yun-Tao; Bao, Jun-Ping; Liu, Lei; Wang, Feng; Wu, Xiao-Tao

    2015-04-01

    Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.

  20. The Green Tea Component (--Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity.

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    Jee-Youn Kim

    Full Text Available The green tea component (--epigallocatechin-3-gallate (EGCG has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose polymerase (PARP, activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As treatment significantly induced production of reactive oxygen species (ROS, which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK, which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity.

  1. Neuroprotection by inhibiting the c-Jun N-terminal kinase pathway after cerebral ischemia occurs independently of interleukin-6 and keratinocyte-derived chemokine (KC/CXCL1 secretion

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    Benakis Corinne

    2012-04-01

    Full Text Available Abstract Background Cerebral ischemia is associated with the activation of glial cells, infiltration of leukocytes and an increase in inflammatory mediators in the ischemic brain and systemic circulation. How this inflammatory response influences lesion size and neurological outcome remains unclear. D-JNKI1, an inhibitor of the c-Jun N-terminal kinase pathway, is strongly neuroprotective in animal models of stroke. Intriguingly, the protection mediated by D-JNKI1 is high even with intravenous administration at very low doses with undetectable drug levels in the brain, pointing to a systemic mode of action, perhaps on inflammation. Findings We evaluated whether D-JNKI1, administered intravenously 3 h after the onset of middle cerebral artery occlusion (MCAO, modulates secretion of the inflammatory mediators interleukin-6 and keratinocyte-derived chemokine in the plasma and from the spleen and brain at several time points after MCAO. We found an early release of both mediators in the systemic circulation followed by an increase in the brain and went on to show a later systemic increase in vehicle-treated mice. Release of interleukin-6 and keratinocyte-derived chemokine from the spleen of mice with MCAO was not significantly different from sham mice. Interestingly, the secretion of these inflammatory mediators was not altered in the systemic circulation or brain after successful neuroprotection with D-JNKI1. Conclusions We demonstrate that neuroprotection with D-JNKI1 after experimental cerebral ischemia is independent of systemic and brain release of interleukin-6 and keratinocyte-derived chemokine. Furthermore, our findings suggest that the early systemic release of interleukin-6 and keratinocyte-derived chemokine may not necessarily predict an unfavorable outcome in this model.

  2. Diallyl trisulfide-induced apoptosis in human prostate cancer cells involves c-Jun N-terminal kinase and extracellular-signal regulated kinase-mediated phosphorylation of Bcl-2.

    Science.gov (United States)

    Xiao, Dong; Choi, Sunga; Johnson, Daniel E; Vogel, Victor G; Johnson, Candace S; Trump, Donald L; Lee, Yong J; Singh, Shivendra V

    2004-07-22

    Garlic-derived organosulfides (OSCs) including diallyl trisulfide (DATS) are highly effective in affording protection against chemically induced cancer in animals. Evidence is also mounting to indicate that some naturally occurring OSCs can suppress proliferation of cancer cells by causing apoptosis, but the sequence of events leading to proapoptotic effect of OSCs is poorly defined. Using PC-3 and DU145 human prostate cancer cells as a model, we now demonstrate that DATS is a significantly more potent apoptosis inducer than diallyl sulfide (DAS) or diallyl disulfide (DADS). DATS-induced apoptosis in PC-3 cells was associated with phosphorylation of Bcl-2, reduced Bcl-2 : Bax interaction, and cleavage of procaspase-9 and -3. Bcl-2 overexpressing PC-3 cells were significantly more resistant to apoptosis induction by DATS compared with vector-transfected control cells. DATS treatment resulted in activation of extracellular-signal regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase 1 (JNK1) and/or JNK2, but not p38 mitogen-activated protein kinase. Phosphorylation of Bcl-2 in DATS-treated PC-3 cells was fully blocked in the presence of JNK-specific inhibitor SP600125. Moreover, JNK inhibitor afforded significant protection against DATS-induced apoptosis in both cells. DATS-induced Bcl-2 phosphorylation and apoptosis were partially attenuated by pharmacological inhibition of ERK1/2 using PD98059 or U0126. Overexpression of catalase inhibited DATS-mediated activation of JNK1/2, but not ERK1/2, and apoptosis induction in DU145 cells suggesting involvement of hydrogen peroxide as a second messenger in DATS-induced apoptosis. In conclusion, our data point towards important roles for Bcl-2, JNK and ERK in DATS-induced apoptosis in human prostate cancer cells.

  3. c-Jun N-terminal kinase inhibitor favors transforming growth factor-β to antagonize hepatitis B virus X protein-induced cell growth promotion in hepatocellular carcinoma.

    Science.gov (United States)

    Wu, Yan-Hui; Ai, Xi; Liu, Fu-Yao; Liang, Hui-Fang; Zhang, Bi-Xiang; Chen, Xiao-Ping

    2016-02-01

    Transforming growth factor (TGF)-β induces cell growth arrest in well-differentiated hepatocellular carcinoma (HCC) while hepatitis B virus X protein (HBx) minimizes the tumor suppression of TGF-β signaling in early chronic hepatitis B. However, how to reverse the oncogenic effect of HBx and sustain the tumor-suppressive action of TGF-β has yet to be investigated. The present study examined the effect of TGF-β and a c-Jun N-terminal kinase (JNK) inhibitor on cell growth in HCC cells with forced expression of HBx. It was found that HBx promoted cell growth via activation of the JNK/pSMAD3L pathway and inhibition of the transforming growth factor-beta type I receptor (TβRI)/pSMAD3C pathway. pSMAD3L/SMAD4 and pSMAD3C/SMAD4 complexes antagonized each other to regulate c-Myc expression. In the absence of HBx, TGF-β induced cell growth arrest through activation of the TβRI/pSMAD3C pathway in well-differentiated HCC cells. In the presence of HBx, TGF-β had no effect on cell growth. JNK inhibitor SP600125 significantly reversed the oncogenic action of HBx and favored TGF-β to regain the ability to inhibit the cell growth in HBx-expressing well-differentiated HCC cells. In conclusion, targeting JNK signaling favors TGF-β to block HBx-induced cell growth promotion in well-differentiated HCC cells. As an adjunct to anti-viral therapy, the combination of TGF-β and inhibition of JNK signaling is a potential therapy for HBV-infected HCC.

  4. Cadmium induces apoptosis in pancreatic β-cells through a mitochondria-dependent pathway: the role of oxidative stress-mediated c-Jun N-terminal kinase activation.

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    Kai-Chih Chang

    Full Text Available Cadmium (Cd, one of well-known highly toxic environmental and industrial pollutants, causes a number of adverse health effects and diseases in humans. The growing epidemiological studies have suggested a possible link between Cd exposure and diabetes mellitus (DM. However, the toxicological effects and underlying mechanisms of Cd-induced pancreatic β-cell injury are still unknown. In this study, we found that Cd significantly decreased cell viability, and increased sub-G1 hypodiploid cells and annexin V-Cy3 binding in pancreatic β-cell-derived RIN-m5F cells. Cd also increased intracellular reactive oxygen species (ROS generation and malondialdehyde (MDA production and induced mitochondrial dysfunction (the loss of mitochondrial membrane potential (MMP and the increase of cytosolic cytochrome c release, the decreased Bcl-2 expression, increased p53 expression, poly (ADP-ribose polymerase (PARP cleavage, and caspase cascades, which accompanied with intracellular Cd accumulation. Pretreatment with the antioxidant N-acetylcysteine (NAC effectively reversed these Cd-induced events. Furthermore, exposure to Cd induced the phosphorylations of c-jun N-terminal kinases (JNK, extracellular signal-regulated kinases (ERK1/2, and p38-mitogen-activated protein kinase (MAPK, which was prevented by NAC. Additionally, the specific JNK inhibitor SP600125 or JNK-specific small interference RNA (si-RNA transfection suppressed Cd-induced β-cell apoptosis and related signals, but not ERK1/2 and p38-MAPK inhibitors (PD98059 and SB203580 did not. However, the JNK inhibitor or JNK-specific si-RNA did not suppress ROS generation in Cd-treated cells. These results indicate that Cd induces pancreatic β-cell death via an oxidative stress downstream-mediated JNK activation-triggered mitochondria-regulated apoptotic pathway.

  5. Knockout of the c-Jun N-terminal Kinase 2 aggravates the development of mild chronic dextran sulfate sodium colitis independently of expression of intestinal cytokines TNFα, TGFB1, and IL-6

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    Kersting S

    2013-02-01

    Full Text Available Sabine Kersting,1 Kirstin Reinecke,2 Christoph Hilgert,1 Monika S Janot,1 Elisabeth Haarmann,1 Martin Albrecht,1 Annette M Müller,3 Thomas Herdegen,2 Ulrich Mittelkötter,1 Waldemar Uhl,1 Ansgar M Chromik11Department of General and Visceral Surgery, St Josef Hospital, Ruhr-University of Bochum, Bochum, Germany; 2Institute of Experimental and Clinical Pharmacology, University Hospital of Schleswig-Holstein, Campus Kiel, Germany; 3Department of Pediatric Pathology, Rheinische Friedrich-Wilhems-University of Bonn, Bonn, GermanyIntroduction: The c-Jun N-terminal kinases (JNKs are involved in signal transduction of inflammatory bowel diseases. The aim of this study was to examine the function of JNKs by using a low-dose dextran sulfate sodium (DSS model in JNK1 knockout mice (Mapk8–/–, JNK2 knockout mice (Mapk9–/–, and wild-type controls (WT1, WT2.Methods: The animals were evaluated daily using a disease activity index. After 30 days, the intestine was evaluated histologically with a crypt damage score. CD4+ and CD8+ cells were quantified using immunofluorescence. Analysis of tumor necrosis factor-a (TNFα, interleukin-6 (IL-6, and transforming growth factor ß1 (TGFB1 expression was carried out using LightCycler® real-time polymerase chain reaction.Results: Cyclic administration of low-dose DSS (1% was not able to induce features of chronic colitis in Mapk8–/– WT2 mice. By contrast, DSS administration significantly increased the disease activity index in WT1 and Mapk9–/– mice. In Mapk9–/– mice, the crypt damage score and the number of CD4+ and CD8+ cells as features of chronic colitis/inflammation were also significantly elevated. Expression of TNFα, IL-6, and TGFB1 was not altered by the JNK knockout.Conclusion: Administering DSS at a defined low concentration that is unable to induce colitis in WT animals leads to clinically and histologically detectable chronic colitis in Mapk9–/– mice. The reason for this disease

  6. MORINGA TEA BLOCKS ACUTE LUNG INFLAMMATION INDUCED BY SWINE CONFINEMENT DUST THROUGH A MECHANISM INVOLVING TNF-α EXPRESSION, C-JUN N-TERMINAL KINASE ACTIVATION AND NEUTROPHIL REGULATION

    Directory of Open Access Journals (Sweden)

    Mykea Mcknight

    2014-01-01

    Full Text Available Plant based products represent a promising alternative to conventional treatments for inflammation. Moringa oleifera Lam is a tree rich in proteins, vitamins, minerals and a variety of phytochemcals with health benefits. Among the reported health benefits are antioxidant and anti-inflammatory properties. The purpose of this study was to investigate whether tea brewed from dried Moringa leaves would abrogate inflammation in a mouse model of acute lung inflammation induced by LPS or extracts prepared from dust collected from a swine confinement facility (DE. Mice were offered water or Moringa tea for seven days. Tea consumption was significantly greater than that of water consumption on days 1 and 6, but there were no significant differences in weight gain or food consumption. On day seven, mice from both groups were forced to inhale, via intranasal challenge, either Phosphate Buffered Saline (PBS, Lipopolysaccharide (LPS [10 µg mL-1] or DE [10%]. Compared to mice that drank water, mice that drank Moringa tea had significantly less protein (p<0.05 and cellular influx (p<0.0001 into the lung after inhalation of 10% DE. No difference in neutrophil migration into the lungs of water and M. tea groups after LPS or DE challenge was detected. But mice that drank tea had significantly (p<0.05 more neutrophils with apoptotic morphology after DE challenge. TNF-α expression 24 h after inhalation of 10% DE, was significantly higher (p<0.05 in lungs of M. tea mouse group as compared to water group. This increase in TNF-α was accompanied by higher levels of pro and anti-inflammatory cytokines. Finally, activation of c-Jun N-terminal Kinase (JNK in lungs of M. tea+DE group 24 h post inhalation was decreased. Taken together these results suggest that Moringa oleifera leaf tea exerts anti-inflammatory properties on acute lung inflammation induced by swine confinement dust through a mechanism involving neutrophil regulation and JNK

  7. Construction and identification of dominant-negative c-Jun N-terminal kinase(DN-JNK)recombinant adenovirus%DN-JNK基因重组腺病毒的构建和鉴定

    Institute of Scientific and Technical Information of China (English)

    张佳妮; 刘慧霞; 陈金虎; 郭敏; 全养雅; 谭莺

    2009-01-01

    Objective To construct and identify replication deficient recombinant adenovirus expressing human c-Jun N-terminal kinase(JNK)by homologous recombination adenovirus dominant-negative type JNK(Ad-DN-JNK).Methods The linearized recombinant shuttle vector pAdTrack-CMV-DN-JNK Was co-transformed with backbone vector pAdEasy-l into bacteria BJ5183 for recombinant adenoviral vector.The recombinant adenoviral vector was transfected into HEK293 packing cells tO construct replication deficient recombinant adenovirus,and then the recombinant edenovirns WaS detected by PCR and DNA sequencing.Western blot analysis was utilized to detect the Cxpression of Ad-DN-JNK and the level of insulin receptor substrate l Serine307 phosphorylation.Results JNK recombinant adenoviral vectorcould be effectively transfeeted into HEK 293 cell and successfully packed by intracellular enzyme.The expression of green fluorescent protein(GFP)Was observed on the 5th day after transfection.The fragment of JNK gene waS amplified by PCR and identified by sequencing.The titer of the prepared Ad-DN-JNK is 2.5×1010 pfu/ml.The animal experiment confirmed that constructed Ad-DN-JNK could be effectively expressed in liver tissue.Conclusion The research successfully constructed recombinant adenoviral vector and recombinant adenoviral particle.Animal experiment demonstrated the Ad-DN-JNK could effectively mediated the expression of DN-JNK gene and down-regulated the level of IRSlscfine307 phosphorylation.The achievement laid a foundation for further investigation of the function and application of JNK.%目的 制备表达人c-jun氨基末端激酶(JNK)复制缺陷型重组腺病毒(Ad-DN-JNK).并通过动物实验进行功能鉴定.方法 将重组穿梭载体pAdTraek-CMV-DN-JNK线性化后,与pAdEasy-1共转化大肠杆菌BJ5138,进行同源重组得到重组腺病毒载体.将重组腺病毒栽体转染入包装细胞HEK293内制备复制缺陷型重组腺病毒,并经PCR及DNA测序鉴定.Western blot检

  8. Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death

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    Chang Alice YW

    2012-11-01

    Full Text Available Abstract Background Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2/extracellular signal-regulated kinase 1/2 (ERK1/2/mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2 cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM, the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life and decreases (pro-death to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK and p38 mitogen-activated protein kinase (p38MAPK, the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4 or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2 and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. Results An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol bilaterally into RVLM of Sprague–Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and

  9. Antiepileptic Effect of Uncaria rhynchophylla and Rhynchophylline Involved in the Initiation of c-Jun N-Terminal Kinase Phosphorylation of MAPK Signal Pathways in Acute Seizures of Kainic Acid-Treated Rats

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    Hsin-Cheng Hsu

    2013-01-01

    Full Text Available Seizures cause inflammation of the central nervous system. The extent of the inflammation is related to the severity and recurrence of the seizures. Cell surface receptors are stimulated by stimulators such as kainic acid (KA, which causes intracellular mitogen-activated protein kinase (MAPK signal pathway transmission to coordinate a response. It is known that Uncaria rhynchophylla (UR and rhynchophylline (RP have anticonvulsive effects, although the mechanisms remain unclear. Therefore, the purpose of this study is to develop a novel strategy for treating epilepsy by investigating how UR and RP initiate their anticonvulsive mechanisms. Sprague-Dawley rats were administered KA (12 mg/kg, i.p. to induce seizure before being sacrificed. The brain was removed 3 h after KA administration. The results indicate that pretreatment with UR (1.0 g/kg, RP (0.25 mg/kg, and valproic acid (VA, 250 mg/kg for 3 d could reduce epileptic seizures and could also reduce the expression of c-Jun aminoterminal kinase phosphorylation (JNKp of MAPK signal pathways in the cerebral cortex and hippocampus brain tissues. Proinflammatory cytokines interleukin (IL-1β, IL-6, and tumor necrosis factor-α remain unchanged, indicating that the anticonvulsive effect of UR and RP is initially involved in the JNKp MAPK signal pathway during the KA-induced acute seizure period.

  10. JNK通路在新生鼠坏死性小肠结肠炎模型中的表达及其意义%Expression and Significance of c-Jun N-terminal Kinase in Necrotizing Enterocolitis of Neonatal Rats

    Institute of Scientific and Technical Information of China (English)

    习隽丽; 刘先州

    2010-01-01

    目的 观察新生大鼠坏死性小肠结肠炎(necrotizing enterocolitis, NEC)中c-Jun氨基末端激酶(c-Jun N-terminal Kinase, JNK)的激化及胞内分布规律,探讨其在NEC发病机制中的作用.方法 新生鼠随机分为正常对照组和NEC动物模型组,每组10只,于母鼠喂养3d,第4d处死.取回盲部肠组织1~2cm,用10%的甲醛立即固定,常规石蜡包埋,分别作组织形态学检查和免疫组织化学检测JNK的表达.结果 新生大鼠NEC模型中JNK在肠道细胞的胞浆中黄棕色染色颗粒表达并伴有向核转移,其阳性表达明显高于正常对照组.结论 JNK信号通路可能参与了NEC发病过程,并起着信号传导作用.

  11. The involvement of c-Jun N-terminal kinase signaling pathway in the protective effect of estrogen on attenuating ischemia reperfusion injury in a rat flap model%c-Jun氨基末端激酶通路参与雌激素对大鼠皮瓣缺血再灌注损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    李志敏; 巨积辉; 刘跃飞; 金乾衡; 吴建龙; 侯瑞兴

    2016-01-01

    Objeetive To observe the protective effect of estrogen against ischemia reperfusion injury in axial flaps,and investigate the role of the c-Jun N-terminal kinase signaling pathway in estrogen's protective effect.Methods An ischemia reperfusion injury model in the abdominal flap was created in 40 Wistar rats that were randomly divided into 4 groups:control (group A),ischemia reperfusion injury (group B),estrogen (group C) and JUN inhibitor (group D).Seven days postoperatively,gross observation of the flap,measurement of flap survival area and calculation of flap survival rate were carried out.The flap tissues were harvested for hematoxylin-eosin staining to observe histological changes,and for Westem blot to quantify JNK,p-JNK and MKP-5 expression.The relationship between flap survival and JNK expression was analyzed.Results Flaps in groups C and D grew well.Flap survival rates in these two groups were significantly higher than that in group B,while pathological changes were milder.Expressions of JNK and p-JNK were significantly lower in flaps of groups C and D than in flaps of group B,while expression of JNK negative regulator MKP-5 was the opposite.Conclusion Estrogen can significantly improve the ischemia reperfusion injury in flaps and increase flap survival rate.The potential mechanism of estrogen's protective effect can be through regulating Jun N-terminal kinase signaling pathway.%目的 观察雌激素对皮瓣缺血再灌注损伤的保护作用,初步研究c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)通路与雌激素保护作用的相关性.方法 取40只Wistar大鼠建立大鼠腹部皮瓣缺血再灌注损伤模型,随机分为健康对照组(A组)、缺血再灌注损伤组(B组)、雌激素组(C组)、JNK抑制剂组(D组).术后第七天观察各组皮瓣大体情况,测量皮瓣成活面积并计算成活率,HE染色观察各组皮瓣组织学改变,测定皮瓣组织中JNK、p-JNK、丝裂原活化蛋白激酶磷酸酶-5(MKP-5)的表达.

  12. c-Jun氨基末端激酶1反义真核表达载体及其蛋白缺陷细胞株的构建与鉴定%Construction and identification of antisense c-Jun N-terminal kinase 1 eukaryotic fluorescent expressing plasmids and JNK1+ human embryo lung fibroblasts cell line

    Institute of Scientific and Technical Information of China (English)

    徐辉; 何晓庆; 陈瑞; 尹仕伟; 彭雷; 王国强; 李爱萍; 周建伟; 刘起展

    2008-01-01

    目的 构建反义JNK1荧光真核细胞表达载体,建立JNK1蛋白缺陷人胚肺成纤维细胞(HELF)株.方法 用Trizol试剂抽提HELF细胞中总RNA,以反转录PCR扩增JNK1目的 片断,双酶切,纯化PCR产物后,反向插入pEGFP-C1绿色荧光质粒,构建反义pEGFP-C1-asJNK1真核表达载体;大量抽提质粒并转染至HELF细胞中.24 h后使用G418筛选,挑选单克隆细胞扩大培养,经荧光显微成像和蛋白免疫印迹鉴定.结果 pEGFP-C1-asJNK1表达载体DNA测序结果与预期目的 片断序列一致,且JNK1蛋白表达水平明显抑制.结论 反义pEGFP-C1-asJNK1真核表达载体构建成功,JNK1蛋白质缺陷HELF细胞株成功建立.%Objective To construct antisense c-Jun N-terminal kinase 1 (JNK1) eukaryotic fluorescent expressing vector and JNK1+ human embryo lung fibroblasts cell line. Methods Trizol reagent was used to extract total RNA in HELF. The proper primers of JNK1 were chosen and synthesized. RT-PCR and gene recombinant techniques were used to construct the fragment of JNK1. After purification, the PCR products were cut, and JNK1 were inserted reversely into eukaryotic fluorescent expressing vector pEGFP-C1. Enzyme-cutting and DNA auto-sequencing were used to prove the successful construction of JNK1 eukaryotic expressing vector. Then plasmids were extracted and transfected into HELF cells and screen by G418 24 h later. Monoclone was chosen and cultured. Fluorescent imaging and Western blot were used to identify the JNK+HELF cell line. Results Sequence analysis of pEGFP-C1-as JNK1 plasmids was same as expected. The expression level of JNK1 was inhibited markedly. Conclusion Construction of antisensc JNK1 eukaryotic fluorescent expressing vectors and JNK + HELF cell line is successful.

  13. Toll 样受体2介导的 JNK 信号分子在小鼠支气管哮喘发病中的作用机制%Mechanism of c-Jun N-terminal kinase mediated by Toll like receptor 2 in murine asthma

    Institute of Scientific and Technical Information of China (English)

    沈佩婷; 方磊; 吴惠梅; 沈启英; 何芳; 刘荣玉

    2015-01-01

    目的:探讨 Toll 样受体2(TLR2)介导的 c-Jun 氨基末端激酶(JNK)信号分子参与小鼠支气管哮喘发病的作用机制。方法健康 SPF 级 C57(TLR2野生型)鼠和 TLR2基因缺失(TLR2-/-)鼠各14只,按随机数字表法分为4组:C57对照组、C57哮喘组、TLR2-/-对照组、TLR2-/-哮喘组,每组7只,哮喘组以卵清蛋白(OVA)腹腔注射联合雾化吸入致敏和激发建立哮喘模型,对照组以生理盐水代替 OVA致敏和激发。利用免疫组织化学染色技术( ABC 法)检测TLR2蛋白在 C57对照组、C57哮喘组肺内的表达差异,JNK及磷酸化 JNK(P-JNK)蛋白表达在各组肺内的表达差异。结果 HE 染色提示较其余3组,C57哮喘组有较明显的炎症细胞浸润及呼吸道平滑肌增生。以平均吸光度(mA)衡量各组织蛋白相对表达量,免疫组化结果提示 TLR2蛋白在C57哮喘组表达显著高于 C57对照组(P <0.01),JNK 蛋白在各组的表达差异无统计学意义,P-JNK 蛋白在 C57哮喘组肺组织的表达量显著高于 C57对照组、TLR2-/-哮喘组、TLR2-/-对照组(F =43.261,P <0.01)。结论 TLR2介导的 JNK 信号分子通路可能参与了支气管哮喘的发病过程。%Objective To explore the mechanism of c-Jun N-terminal kinase mediated by Toll like receptor 2 in murine asthma. Methods 14 healthy SPF grade C57 wild-type mice and 14 TLR2 knockout (TLR2 - / - ) mice were randomly divided into four groups: C57 control group, C57 asthma group, TLR2 - / - control group, TLR2 - / - asth-ma group (n = 7). We utilized intraperitoneal injection combined with inhalation of ovalbumin (OVA) to sensitize and challenge the mice, thus establishing the experimental models of asthma. Meanwhile, the control group received normal saline instead of OVA. The protein expression of TLR2 was detected by immunohistochemistry(ABC meth-od) in C57 control group and C57 asthma group,as well as JNK and phosphorylation c-Jun(P-JNK) between each group. Results In C57

  14. P75 and phosphorylated c-Jun are differentially regulated in spinal motoneurons following axotomy in rats

    Institute of Scientific and Technical Information of China (English)

    Qiuju Yuan; Huanxing Su; Wutian Wu; Zhi-Xiu Lin

    2012-01-01

    The neurotrophin receptor (p75) activates the c-Jun N-terminal kinase (JNK) pathway. Activation of JNK and its substrate c-Jun can cause apoptosis. Here we evaluate the role of p75 in spinal motoneurons by comparing immunoreactivity for p75 and phosphorylated c-Jun (p-c-Jun), the production of JNK activation in axotomized motoneurons in postnatal day (PN)1, PN7, PN14 and adult rats. Intensive p-c-Jun was induced in axotomized motoneurons in PN1 and PN7. In PN14, p-c-Jun expression was sharply reduced after the same injury. The decreased expression of p-c-Jun at this age coincided with a developmental switch of re-expression of p75 in axotomized cells. In adult animals, no p-c-Jun but intensive p75 was detected in axotomized motoneurons. These results indicate differential expression or turnover of phosphorylation of c-Jun and p75 in immature versus mature spinal motoneurons in response to axonal injury. The non-co-occurrence of p75 and p-c-Jun in injured motoneurons indicated that p75 may not activate JNK pathway, suggesting that the p75 may not be involved in cell death in axotomized motoneurons.

  15. HSF1/HSP70通路抑制c-Jun氨基末端激酶的活化保护UVA诱导的HaCaT细胞凋亡%Protection of HSF1/HSP70 pathway on UVA-induced HaCaT cells apoptosis via inhibiting the activation of c-Jun N-terminal kinase

    Institute of Scientific and Technical Information of China (English)

    王晓雯; 王春波; 李丙华; 韩彦弢

    2012-01-01

    目的 观察热休克转录因子1( HSF1)与热休克蛋白70( HSP70)对紫外线A(UVA)诱导HaCaT细胞凋亡的保护作用及其机制.方法 建立8mJ/cm2 UVA辐射损伤HaCaT细胞的病理模型.将细胞随机分为对照组、8mJ/cm2 UVA照射组、HSP70转录抑制剂组(50 μmol/L槲皮素).Honechst 33258荧光染色观察细胞凋亡;蛋白质印迹法检测UVA辐射HaCaT细胞后p-HSF1和HSP70蛋白的经时变化及UVA辐射后孵育6h JNK(c-Jun氨基末端激酶)、p-JNK的蛋白表达;Real-Time PCR检测HSP70 mRNA的表达.结果 UVA辐射后HaCaT细胞内p-HSF1、HSP70蛋白表达量均出现先增加后减少的时间依赖性趋势,其中p-HSF1于lh开始增加,3h达高峰,HSP70于6h达高峰,24h基本恢复原始水平;UVA辐射前预先加入HSP70转录抑制剂槲皮素能显著抑制HSP70 mRNA的表达,增加p-JNK的表达量,同时Honechst 33258荧光染色观察其与UVA辐射组比较凋亡率明显升高.结论 8mJ/cm2 UVA辐射HaCaT细胞在一定时间内可使HSF1活化致HSP70表达增加.HSFl/HSP70通路对UVA诱导的HaCaT细胞凋亡具有保护作用,其机制与HSP70大量表达后抑制JNK的活化有关.%Objective To investigate the protective effect of heat shock factorl ( HSF1) and heat shock protein70 ( HSP70) on ultraviolet A ( UVA ) -induced HaCaT cells apoptosis and its mechanism. Methods The apoptotic HaCaT cell model was induced by UVA irradiation (8mJ/cm ). The cells were randomly divided into three groups, including a control group, a model group (8mJ/cm UVA) and a HSP70 transcription inhibitor group (50 μmol/L quercetin). The morphologic alteration of apoptotic cells was investigated by using Hoechst 33258 fluorescent staining. Western blotting was used to investigate protein expression levels of phosphorylated HSF1 and HSP70 at different time points, as well as c-Jun N-terminal kinase ( JNK ) andphosphorylated JNK were investigated after incubating for 6 hours following UVA irradiation. HSP70 mRNA was

  16. 神经干细胞对老化小胶质细胞存活及JNK信号通路的调控作用%Regulation of neural stem cells on viability and c-Jun N-terminal kinase signaling of aging microglia cells

    Institute of Scientific and Technical Information of China (English)

    武爱梅; 赵江明; 吴蕾; 方辉; 王宇; 吴惠梅

    2013-01-01

    Objective To investigate the regulation of neural stem cells (NSCs) on the viability and stress-activated kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling of aging microglia cells.Methods Primary microglia cells were isolated from 12-18 months old ICR mouse and NSCs were isolated from 12.5 days pregnancy ICR mouse.Staining test with Isolectin-B4,the specific marker for microglia,was performed and NSCs were verified by expression of nestin with immunofluorescence.Four groups were chosen in our experiment,including microglia cells group,co-cultured group,Sp600125 stimulated group and Sp600125-stimulation co-cultured group; in the Sp600125 stimulated group,microglia cells were pretreated with 20 ng/mL SP600125,a specific inhibitor of JNK,for four h; in the co-cultured group,microglia and NSC cells (1:4) were co-cultured using a Millicell Hanging Cell Culture Insert plates; and Sp600125-stimulation co-cultured group was also pretreated with 20 ng/mL SP600125 for four h firstly,and then,NSCs were added to co-culture with microglia cells for 3 d.MTT assay was performed to analyze the proliferation ability; Western blotting was used to detect the protein expression level of phosphorylated JNK signaling.Results After culturing for 2 weeks,primary microglia cells had a good adherence ability and strong refractivity.Staining test with Isolectin-B4 showed that the purity reached 80%.Neural stem cells grew like suspended spheres and nestin-positive.As compared with microglia cells group and stem cells group,co-cultured group had a significantly higher proliferation ability in MTT assay (P<0.05).The phosphorylated JNK level in the co-cultured group was significantly up-regulated as compared with that in the microglia cells group (P<0.05);Sp600125-stimulation co-cultured group had obviously higher phosphorylated JNK level than that in the Sp600125-stimulated group (P<0.05).Conclusion NSCs might promote the survival of aging microglia cells through activation of JNK

  17. Selection and characterization of a DNA aptamer that can discriminate between cJun/cJun and cJun/cFos.

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    Ryan D Walters

    Full Text Available The AP-1 family of transcriptional activators plays pivotal roles in regulating a wide range of biological processes from the immune response to tumorigenesis. Determining the roles of specific AP-1 dimers in cells, however, has remained challenging because common molecular biology techniques are unable to distinguish between the role of, for example, cJun/cJun homodimers versus cJun/cFos heterodimers. Here we used SELEX (systematic evolution of ligands by exponential enrichment to identify and characterize DNA aptamers that are >100-fold more specific for binding cJun/cJun compared to cJun/cFos, setting the foundation to investigate the biological functions of different AP-1 dimer compositions.

  18. Ginkgo biloba extract individually inhibits JNK activation and induces c-Jun degradation in human chondrocytes: potential therapeutics for osteoarthritis.

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    Ling-Jun Ho

    Full Text Available Osteoarthritis (OA is a common joint disorder with varying degrees of inflammation. The ideal anti-OA drug should have immunomodulatory effects while at the same time having limited or no toxicity. We examined the anti-inflammatory effects of Ginkgo biloba extract (EGb in interleukin-1 (IL-1-stimulated human chondrocytes. Chondrocytes were prepared from cartilage specimens taken from patients with osteoarthritis who had received total hip or total knee replacement. The concentrations of chemokines and the degree of cell migration were determined by ELISA and chemotaxis assays, respectively. The activation of inducible nitric oxide synthase (iNOS, mitogen-activated protein kinases (MAPKs, activator protein-1 (AP-1, and nuclear factor-kappaB (NF-κB was determined by immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay. We found that EGb inhibited IL-1-induced production of chemokines, which in turn resulted in attenuation of THP-1 cell migration toward EGb-treated cell culture medium. EGb also suppressed IL-1-stimulated iNOS expression and release of nitric oxide (NO. The EGb-mediated suppression of the iNOS-NO pathway correlated with the attenuation of activator protein-1 (AP-1 but not nuclear factor-kappaB (NF-κB DNA-binding activity. Of the mitogen-activated protein kinases (MAPKs, EGb inhibited only c-Jun N-terminal kinase (JNK. Unexpectedly, EGb selectively caused degradation of c-Jun protein. Further investigation revealed that EGb-mediated c-Jun degradation was preceded by ubiquitination of c-Jun and could be prevented by the proteosome inhibitor MG-132. The results imply that EGb protects against chondrocyte degeneration by inhibiting JNK activation and inducing ubiquitination-dependent c-Jun degradation. Although additional research is needed, our results suggest that EGb is a potential therapeutic agent for the treatment of OA.

  19. The regulation of p53 up-regulated modulator of apoptosis by JNK/c-Jun pathway in β-amyloid-induced neuron death.

    Science.gov (United States)

    Akhter, Rumana; Sanphui, Priyankar; Das, Hrishita; Saha, Pampa; Biswas, Subhas Chandra

    2015-09-01

    Neuronal loss in selective areas of brain underlies the pathology of Alzheimer's disease (AD). Recent evidences place oligomeric β-amyloid (Aβ) central to the disease. However, mechanism of neuron death in response to Aβ remains elusive. Activation of the c-Jun N-terminal kinase (JNK) pathway and induction of the AP-1 transcription factor c-Jun are reported in AD. However, targets of JNK/c-Jun in Aβ-induced neuron death are mostly unknown. Our study shows that pro-apoptotic proteins, Bim (Bcl-2 interacting mediator of cell death) and Puma (p53 up-regulated modulator of apoptosis) are targets of c-Jun in Aβ-treated neurons. We demonstrate that the JNK/c-Jun pathway is activated, in cultures of cortical neurons following treatment with oligomeric Aβ and in AD transgenic mice, and that inhibition of this pathway by selective inhibitor blocks induction of Puma by Aβ. We also find that both JNK and p53 pathways co-operatively regulate Puma expression in Aβ-treated neurons. Moreover, we identified a novel AP1-binding site on rat puma gene which is necessary for direct binding of c-Jun with Puma promoter. Finally, we find that knocking down of c-Jun by siRNA provides significant protection from Aβ toxicity and that induction of Bim and Puma by Aβ in neurons requires c-Jun. Taken together, our results suggest that both Bim and Puma are target of c-Jun and elucidate the intricate regulation of Puma expression by JNK/c-Jun and p53 pathways in neurons upon Aβ toxicity. JNK/c-Jun pathway is shown to be activated in neurons of the Alzheimer's disease (AD) brain and plays a vital role in neuron death in AD models. However, downstream targets of c-Jun in this disease have not been thoroughly elucidated. Our study shows that two important pro-apoptotic proteins, Bim (Bcl-2 interacting mediator of cell death) and Puma (p53 up-regulated modulator of apoptosis) are targets of c-Jun in Aβ-treated neurons. We demonstrate that the JNK/c-jun pathway is activated, in cultures

  20. Cucurbitacin-I (JSI-124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B Leukemic Cells

    International Nuclear Information System (INIS)

    Cucurbitacin-I (JSI-124) is potent inhibitor of JAK/STAT3 signaling pathway and has anti-tumor activity in a variety of cancer including B cell leukemia. However, other molecular targets of JSI-124 beyond the JAK/STAT3 pathway are not fully understood. BJAB, I-83, NALM-6 and primary CLL cells were treated with JSI-124 as indicated. Apoptosis was measured using flow cytometry for accumulation of sub-G1 phase cells (indicator of apoptosis) and Annexin V/PI staining. Cell cycle was analyzed by FACS for DNA content of G1 and G2 phases. Changes in phosphorylation and protein expression of p38, Erk1/2, JNK, c-Jun, and XIAP were detected by Western blot analysis. STAT3 and c-Jun genes were knocked out using siRNA transfection. VEGF expression was determined by mRNA and protein levels by RT-PCR and western blotting. Streptavidin Pull-Down Assay was used to determine c-Jun binding to the AP-1 DNA binding site. Herein, we show that JSI-124 activates c-Jun N-terminal kinase (JNK) and increases both the expression and serine phosphorylation of c-Jun protein in the B leukemic cell lines BJAB, I-83 and NALM-6. JSI-124 also activated MAPK p38 and MAPK Erk1/2 albeit at lower levels than JNK activation. Inhibition of the JNK signaling pathway failed to effect cell cycle arrest or apoptosis induced by JSI-124 but repressed JSI-124 induced c-Jun expression in these leukemia cells. The JNK pathway activation c-Jun leads to transcriptional activation of many genes. Treatment of BJAB, I-83, and NALM-6 cells with JSI-124 lead to an increase of Vascular Endothelial Growth Factor (VEGF) at both the mRNA and protein level. Knockdown of c-Jun expression and inhibition of JNK activation significantly blocked JSI-124 induced VEGF expression. Pretreatment with recombinant VEGF reduced JSI-124 induced apoptosis. Taken together, our data demonstrates that JSI-124 activates the JNK signaling pathway independent of apoptosis and cell cycle arrest, leading to increased VEGF expression

  1. Characterization of c-Jun from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.

    Science.gov (United States)

    Wei, Shina; Huang, Youhua; Huang, Xiaohong; Qin, Qiwei

    2015-03-01

    The nuclear phosphoprotein c-Jun is a member of the AP1 family of transcription activating complex, can be induced by various extracellular stimuli such as virus infection. In this study, the c-Jun gene (Ec-c-Jun) was cloned from orange-spotted grouper, Epinephelus coioides. The full-length Ec-c-Jun cDNA is composed of 2046 bp and encodes a polypeptide of 328 amino acids with 81% identity of zebrafish. Amino acid alignment analysis indicated that Ec-c-Jun contained three conserved domains including a transactivation domain (TAD), a DNA-binding domain (DBD) and leucine zipper domain (LZD). RT-PCR results showed that Ec-c-Jun transcript was most abundant in spleen, kidney, heart and gill. The expression of Ec-c-Jun was up-regulated after challenged with Singapore grouper iridovirus (SGIV). To investigate the roles of Ec-c-Jun during SGIV infection, we constructed its dominant-negative mutant (DN-Ec-c-Jun) by deleting the major TAD that lacks amino acids 3-122. Fluorescence microscopy observation revealed that Ec-c-Jun and DN-Ec-c-Jun were expressed predominantly in the nucleus in transfected cells. Interestingly, the green fluorescence of Ec-c-Jun was congregated and co-localized with virus assembly sites at the late stage of SGIV infection. However, in DN-Ec-c-Jun transfected cells, no virus assembly sites were observed, and the distribution of fluorescence remained unchanged. Moreover, overexpression of DN-Ec-c-Jun in vitro delayed the occurrence of CPE induced by SGIV infection and inhibited the virus gene transcription. In addition, ectopic expression of DN-Ec-c-Jun was able to inhibit SGIV induced c-Jun/AP1 promoter activity in GS cells. Thus, we proposed that c-Jun transcription factor was essential for SGIV replication in vitro. Our results will contribute to understanding the crucial roles of JNK signaling pathway in fish virus infection.

  2. c-Jun activation is associated with proliferation and angiogenesis in invasive breast cancer

    NARCIS (Netherlands)

    Vleugel, M.M.; Greijer, A.E.; Bos, R.; Wall, E. van der; Diest, P.J. van

    2006-01-01

    c-Jun is a component of the transcription factor activator protein 1 (AP-1), which binds and activates transcription at TRE/AP-1 elements. Extra- or intracellular signals, including growth factors, transforming oncoproteins, and UV irradiation, stimulate phosphorylation of c-Jun at serine 63/73 and

  3. Rice From Mercury Contaminated Areas in Guizhou Province Induces c-jun Expression in Rat Brain

    Institute of Scientific and Technical Information of China (English)

    JIN-PING CHENG; WEN-HUA WANG; LI-YA QU; JIN-PING JIA; MIN ZHENG; XIU-LING JI; TAO YUAN

    2005-01-01

    Objective Mercury (Hg), as one of the priority pollutants and also a hot topic of frontier environmental research in many countries, has been paid higher attention in the world since the middle of the last century. Guizhou Province (at N24°30′-29°13′, E103°1′-109°30′, 1 100 m above the sea level, with subtropical humid climate) in southwest China is an important mercury production center. It has been found that the mercury content in most media of aquatics, soil, atmosphere and in biomass of corns, plants and animals, is higher than the national standard.The present study aims to explore the influence of mercury pollution on the health of local citizens. Methods The effect of rice from two mercury polluted experimental plots of Guizhou Province on the expression of c-jun mRNA in rat brain and c-jun protein in cortex, hippocampus and ependyma was observed using reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical methods. Results The results showed that the mercury polluted rice induced expression of c-jun mRNA and its protein significantly. Selenium can reduce Hg uptake, an antagonism between selenium and mercury on the expression of c-jun mRNA and c-jun protein. Conclusion c-jun participates in the toxicity process of brain injury by mercury polluted rice, the expression of c- jun mRNA in brain, and c-jun protein in rat cortex and hippocampus can predict neurotoxicity of mercury polluted rice. People should be advised to be cautious in eating any kind of Hg-polluted foods. To reveal the relationship between c-jun induction and apoptosis, further examinations are required.

  4. Mammary gland selective excision of c-jun identifies its role in mRNA splicing

    OpenAIRE

    Katiyar, Sanjay; Jiao, Xuanmao; Addya, Sankar; Ertel, Adam; Rose, Vanessa; Casimiro, Mathew C.; Zhou, Jie; Lisanti, Michael P; Nasim, Talat; Fortina, Paolo; Pestell, Richard G.

    2011-01-01

    The c-jun gene regulates cellular proliferation and apoptosis via direct regulation of cellular gene expression. Alternative splicing of pre-mRNA increases the diversity of protein functions and alternate splicing events occur in tumors. Here, by targeting the excision of the endogenous c-jun gene within the mouse mammary epithelium, we have identified its selective role as an inhibitor of RNA splicing. Microarray-based assessment of gene expression, on laser capture micro-dissected c-jun−/− ...

  5. Persistent induction of c-fos and c-jun expression by asbestos

    Energy Technology Data Exchange (ETDEWEB)

    Heintz, N.H.; Mossman, B.T. (Univ. of Vermont College of Medicine, Burlington (United States)); Janssen, Y.M. (Univ. of Vermont College of Medicine, Burlington (United States) Univ. of Limburg, Maastricht (Netherlands))

    1993-04-15

    To investigate the mechanisms of asbestos-induced carcinogenesis, expression of c-fos and c-jun protooncogenes was examined in rat pleural mesothelial cells and hamster tracheal epithelial cells after exposure to crocidolite or chrysotile asbestos. In contrast to phorbol 12-myristate 13-acetate, which induces rapid and transient increases in c-fos and c-jun mRNA, asbestos causes 2- to 5-fold increases in c-fos and c-jun mRNA that persist for at least 24 hr in mesothelial cells. The induction of c-fos and c-jun mRNA by asbestos in mesothelial cells is dose-dependent and is most pronounced with crocidolite, the type of asbestos most pathogenic in the causation of pleural mesothelioma. Induction of c-jun gene expression by asbestos occurs in tracheal epithelial cells but is not accompanied by a corresponding induction of c-fos gene expression. In both cell types, asbestos induces increases in protein factors that bind specifically to the DNA sites that mediate gene expression by the AP-1 family of transcription factors. The persistent induction of AP-1 transcription factors by asbestos suggests a model of asbestos-induced carcinogenesis involving chronic stimulation of cell proliferation through activation of the early response gene pathway that includes c-jun and/or c-fos. 30 refs., 5 figs.

  6. Parathyroid hormone induces c-fos and c-jun messenger RNA in rat osteoblastic cells

    Science.gov (United States)

    Clohisy, J. C.; Scott, D. K.; Brakenhoff, K. D.; Quinn, C. O.; Partridge, N. C.

    1992-01-01

    PTH is a potent regulator of osteoblast gene expression, yet the nuclear events that mediate PTH action are poorly understood. We were interested in identifying immediate early genes which may regulate PTH-altered gene expression in the osteoblast. Therefore, we examined the effects of PTH on c-fos and c-jun gene expression in a rat osteoblastic cell line (UMR 106-01). Under control conditions, c-fos and c-jun mRNAs were present at low basal levels. After PTH treatment, c-fos mRNA abundance dramatically increased, with a maximal and transient response at 30 min. PTH also stimulated an increase in c-jun mRNA, but in a biphasic manner, with maximal levels at 30 min and 2 h. These responses were dose dependent, not altered by cotreatment with the protein synthesis inhibitor cycloheximide, and preceded PTH-induced expression of matrix metallo-proteinase-1 mRNA. Nuclear run-on assays demonstrated an increased rate of c-fos and c-jun transcription after PTH exposure. To determine the signal transduction pathways involved, second messenger analogs were tested for their ability to mimic the effects of PTH. 8-Bromo-cAMP and phorbol 12-myristate 13-acetate (PMA) caused increases in the abundance of c-fos and c-jun transcripts. Ionomycin had no effect on the expression of these genes. Pretreatment of the cells with PMA resulted in a decrease in basal c-jun expression, but did not alter the PTH-mediated increase in c-fos, c-jun, or matrix metalloproteinase-1 mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS).

  7. G12 Signaling through c-Jun NH2-Terminal Kinase Promotes Breast Cancer Cell Invasion

    OpenAIRE

    Juhi Juneja; Ian Cushman; Casey, Patrick J.

    2011-01-01

    Signaling through the heterotrimeric G protein, G12, via Rho induces a striking increase in breast cancer cell invasion. In this study, evidence is provided that the c-Jun NH(2)-terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively-active Gα12 or activation of G12 signaling by thrombin leads to increased JNK and c-Jun phosphorylation. Pharmacologic inhibition of JNK or knockdown of JNK expression by siRNA significantly decreases G12-induced JN...

  8. c-Jun Promotes whereas JunB Inhibits Epidermal Neoplasia

    OpenAIRE

    Jin, Jane Yingai; Ke, Hengning; Hall, Russell P.; Zhang, Jennifer Y.

    2011-01-01

    Deregulation of the AP1 family gene regulators have been implicated in a wide range of diseases, including cancer. Here, we report that c-Jun was activated in human squamous cell carcinoma (SCC) and coexpression of c-Jun with oncogenic Ras was sufficient to transform primary human epidermal cells into malignancy in a regenerated human skin grafting model. In contrast, JunB was not induced in a majority of human SCC cells. Moreover, exogenous expression of JunB inhibited tumorigenesis driven b...

  9. The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy

    Science.gov (United States)

    Lukey, Michael J.; Greene, Kai Su; Erickson, Jon W.; Wilson, Kristin F.; Cerione, Richard A.

    2016-01-01

    Many transformed cells exhibit altered glucose metabolism and increased utilization of glutamine for anabolic and bioenergetic processes. These metabolic adaptations, which accompany tumorigenesis, are driven by oncogenic signals. Here we report that the transcription factor c-Jun, product of the proto-oncogene JUN, is a key regulator of mitochondrial glutaminase (GLS) levels. Activation of c-Jun downstream of oncogenic Rho GTPase signalling leads to elevated GLS gene expression and glutaminase activity. In human breast cancer cells, GLS protein levels and sensitivity to GLS inhibition correlate strongly with c-Jun levels. We show that c-Jun directly binds to the GLS promoter region, and is sufficient to increase gene expression. Furthermore, ectopic overexpression of c-Jun renders breast cancer cells dependent on GLS activity. These findings reveal a role for c-Jun as a driver of cancer cell metabolic reprogramming, and suggest that cancers overexpressing JUN may be especially sensitive to GLS-targeted therapies. PMID:27089238

  10. c-Jun promotes whereas JunB inhibits epidermal neoplasia.

    Science.gov (United States)

    Jin, Jane Y; Ke, Hengning; Hall, Russell P; Zhang, Jennifer Y

    2011-05-01

    Deregulation of the activator protein 1 (AP1) family gene regulators has been implicated in a wide range of diseases, including cancer. In this study we report that c-Jun was activated in human squamous cell carcinoma (SCC) and coexpression of c-Jun with oncogenic Ras was sufficient to transform primary human epidermal cells into malignancy in a regenerated human skin grafting model. In contrast, JunB was not induced in a majority of human SCC cells. Moreover, exogenous expression of JunB inhibited tumorigenesis driven by Ras or spontaneous human SCC cells. Conversely, the dominant-negative JunB mutant (DNJunB) promoted tumorigenesis, which is in contrast to the tumor-suppressor function of the corresponding c-Jun mutant. At the cellular level, JunB induced epidermal cell senescence and slowed cell growth in a cell-autonomous manner. Consistently, coexpression of JunB and Ras induced premature epidermal differentiation concomitant with upregulation of p16 and filaggrin and downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). These findings indicate that JunB and c-Jun differentially regulate cell growth and differentiation and induce opposite effects on epidermal neoplasia.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub. PMID:21289643

  11. Activation of Tax protein by c-Jun-N-terminal kinase is not dependent on the presence or absence of the early growth response-1 gene product.

    Science.gov (United States)

    Parra, Eduardo; Gutierréz, Luís; Ferreira, Jorge

    2016-02-01

    The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) as an inducer of apoptosis, confirmed that siRNA against EGR-1 failed to suppress ATRA-induced apoptosis in Jurkat and Jurkat-Tax cells, as noted by the low levels of both DEVDase activity and DNA fragmentation, indicating that the induction of apoptosis by ATRA was Egr-1-independent. Finally, our data showed that activation of Tax by JNK-1 was not dependent on the EGR-1 cascade of events, suggesting that EGR-1 is important but not a determinant for the activity for Tax-induced proliferation of Jurkat cells.

  12. TAp73-mediated the activation of C-jun N-terminal kinase enhances cellular chemosensitivity to cisplatin in ovarian cancer cells

    OpenAIRE

    Pingde Zhang; Stephanie Si Liu; Hextan Yuen Sheung Ngan

    2012-01-01

    P73, one member of the tumor suppressor p53 family, shares highly structural and functional similarity to p53. Like p53, the transcriptionally active TAp73 can mediate cellular response to chemotherapeutic agents in human cancer cells by up-regulating the expressions of its pro-apoptotic target genes such as PUMA, Bax, NOXA. Here, we demonstrated a novel molecular mechanism for TAp73-mediated apoptosis in response to cisplatin in ovarian cancer cells, and that was irrespective of p53 status. ...

  13. Downregulation of Cellular c-Jun N-Terminal Protein Kinase and NF-κB Activation by Berberine May Result in Inhibition of Herpes Simplex Virus Replication

    OpenAIRE

    Song, Siwei; Qiu, Min; Chu, Ying; Chen, Deyan; Wang, Xiaohui; Su, Airong; Wu, Zhiwei

    2014-01-01

    Berberine is a quaternary ammonium salt from the protoberberine group of isoquinoline alkaloids. Some reports show that berberine exhibits anti-inflammatory, antitumor, and antiviral properties by modulating multiple cellular signaling pathways, including p53, nuclear factor κB (NF-κB), and mitogen-activated protein kinase. In the present study, we investigated the antiviral effect of berberine against herpes simplex virus (HSV) infection. Current antiherpes medicines such as acyclovir can le...

  14. Increase of RhoB in {gamma}-radiation-induced apoptosis is regulated by c-Jun N-terminal kinase in Jurkat T cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chun-Ho [Laboratory of Cytogenetics and Tissue Regeneration, KIRAMS, Seoul 139-706 (Korea, Republic of); Won, Misun; Choi, Chung-Hae; Ahn, Jiwon; Kim, Bo-Kyung [Genome Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Song, Kyung-Bin [Department of Food Science and Technology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Kang, Chang-Mo, E-mail: kangcm@kcch.re.kr [Laboratory of Cytogenetics and Tissue Regeneration, KIRAMS, Seoul 139-706 (Korea, Republic of); Chung, Kyung-Sook, E-mail: kschung@kribb.re.kr [Genome Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of)

    2010-01-08

    The Ras-related small GTP-binding protein RhoB is known to be a pro-apoptotic protein and immediate-early inducible by genotoxic stresses. In addition, JNK activation is known to function in {gamma}-radiation-induced apoptosis. However, it is unclear how JNK activation and {gamma}-radiation-dependent RhoB induction are related. Here we verified the relationship between JNK activation and RhoB induction. RhoB induction by {gamma}-radiation occurred at the transcriptional level and transcriptional activation of RhoB was concomitant with an increase in RhoB protein. {gamma}-Radiation-induced RhoB expression was markedly attenuated by pretreatment with a JNK-specific inhibitor, SP600125, but not by a p38 MAPK inhibitor, SB203580. Inhibition of JNK caused a decrease in early apoptotic cell death that correlated with RhoB expression. However, PI3K inhibition had no significant effects, indicating that the AKT survival pathway was not involved. The siRNA knockdown of JNK resulted in a decrease in RhoB expression and the siRNA knockdown of RhoB restored cell growth even in the {gamma}-irradiated cells. These results suggest that RhoB regulation involves the JNK pathway and contributes to the early apoptotic response of Jurkat T cells to {gamma}-radiation.

  15. Investigating the role of c-Jun N-terminal kinases in the proliferation of Werner syndrome fibroblasts using diaminopyridine inhibitors

    Directory of Open Access Journals (Sweden)

    Davis Terence

    2011-12-01

    Full Text Available Abstract Fibroblasts derived from the progeroid Werner syndrome show reduced replicative lifespan and a "stressed" morphology, both alleviated using the MAP kinase inhibitor SB203580. However, interpretation of these data is problematical because although SB203580 has the stress-activated kinases p38 and JNK1/2 as its preferred targets, it does show relatively low overall kinase selectivity. Several lines of data support a role for both p38 and JNK1/2 activation in the control of cellular proliferation and also the pathology of diseases of ageing, including type II diabetes, diseases to which Werner Syndrome individuals are prone, thus making the use of JNK inhibitors attractive as possible therapeutics. We have thus tested the effects of the widely used JNK inhibitor SP600125 on the proliferation and morphology of WS cells. In addition we synthesised and tested two recently described aminopyridine based inhibitors. SP600125 treatment resulted in the cessation of proliferation of WS cells and resulted in a senescent-like cellular phenotype that does not appear to be related to the inhibition of JNK1/2. In contrast, use of the more selective aminopyridine CMPD 6o at concentrations that fully inhibit JNK1/2 had a positive effect on cellular proliferation of immortalised WS cells, but no effect on the replicative lifespan of primary WS fibroblasts. In addition, CMPD 6o corrected the stressed WS cellular morphology. The aminopyridine CMPD 6r, however, had little effect on WS cells. CMDP 6o was also found to be a weak inhibitor of MK2, which may partially explain its effects on WS cells, since MK2 is known to be involved in regulating cellular morphology via HSP27 phosphorylation, and is thought to play a role in cell cycle arrest. These data suggest that total JNK1/2 activity does not play a substantial role in the proliferation control in WS cells.

  16. Antiestrogenic activity of flavnoid phytochemicals mediated via c-Jun N-terminal protein kinase pathway. Cell-type specific regulation of estrogen receptor alpha

    Science.gov (United States)

    Flavonoid phytochemicals act as both agonists and antagonists of the human estrogen receptors (ERs). While a number of these compounds act by directly binding to the ER, certain phytochemicals, such as the flavonoid compounds chalcone and flavone, elicit antagonistic effects on estrogen signaling in...

  17. SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling in commercial and wild-type turkey leukocytes

    Science.gov (United States)

    Studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases (PTK) and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on days...

  18. Activation of Tax protein by c-Jun-N-terminal kinase is not dependent on the presence or absence of the early growth response-1 gene product.

    Science.gov (United States)

    Parra, Eduardo; Gutierréz, Luís; Ferreira, Jorge

    2016-02-01

    The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) as an inducer of apoptosis, confirmed that siRNA against EGR-1 failed to suppress ATRA-induced apoptosis in Jurkat and Jurkat-Tax cells, as noted by the low levels of both DEVDase activity and DNA fragmentation, indicating that the induction of apoptosis by ATRA was Egr-1-independent. Finally, our data showed that activation of Tax by JNK-1 was not dependent on the EGR-1 cascade of events, suggesting that EGR-1 is important but not a determinant for the activity for Tax-induced proliferation of Jurkat cells. PMID:26573109

  19. c-jun N-terminal kinase is involved in AUUUA-mediated interleukin-3 mRNA turnover in mast cells.

    OpenAIRE

    Ming, X F; Kaiser, M.; Moroni, C

    1998-01-01

    Whereas signalling pathways involved in transcriptional control have been studied extensively, the pathways regulating mRNA turnover remain poorly understood. We are interested in the role of mRNA stability in cell activation and oncogenesis using PB-3c mast cells as a model system. In these cells the short-lived interleukin-3 (IL-3) mRNA is stabilized by ionomycin treatment and following oncogenesis. To identify the signalling pathways involved in these mechanisms, we analysed the effect of ...

  20. Inhibition of c-Jun N-terminal Kinase Signaling Pathway Alleviates Lipopolysaccharide-induced Acute Respiratory Distress Syndrome in Rats

    Directory of Open Access Journals (Sweden)

    Jian-Bo Lai

    2016-01-01

    Conclusions: Inhibiting JNK alleviated LPS-induced acute lung inflammation and had no effects on pulmonary edema and fibrosis. JNK inhibitor might be a potential therapeutic medication in ARDS, in the context of reducing lung inflammatory.

  1. Ketamine inhibits c-Jun protein expression in mouse hippocampus following cerebral ischemia/reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Feng Xiao; Liangzhi Xiong; Qingxiu Wang; Long Zhou; Qingshan Zhou

    2012-01-01

    A model of cerebral ischemia and reperfusion was established in mice. Mice were treated with ketamine via intraperitoneal injection immediately following ischemia or ischemia/reperfusion. Ketamine did not remarkably change infarct volume in mice immediately following ischemia, but injection immediately following ischemia/reperfusion significantly decreased infarct volume. Ketamine injection immediately after ischemia or ischemia/reperfusion inhibited c-Jun protein expression in mouse hippocampus, but nuclear factor kappa B expression was unaltered. In addition, the Longa scale score for neural impairment was not reduced in mice following cerebral ischemia/reperfusion. These results indicate that ketamine can protect mice against cerebral ischemia and reperfusion injury by modulating c-Jun protein expression in mouse hippocampus.

  2. Modelling the mechanism of GR/c-Jun/Erg crosstalk in apoptosis of acute lymphoblastic leukaemia

    Directory of Open Access Journals (Sweden)

    Daphne eChen

    2012-11-01

    Full Text Available Acute lymphoblastic leukaemia (ALL is one of the most common forms of malignancy that occurs in lymphoid progenitor cells, particularly in children. Synthetic steroid hormones glucocorticoids (GCs are widely used as part of the ALL treatment regimens due to their apoptotic function, but their use also brings about various side effects and drug resistance. The identification of the molecular differences between the GCs responsive and resistant cells therefore are essential to decipher such complexity and can be used to improve therapy. However, the emerging picture is complicated as the activities of genes and proteins involved are controlled by multiple factors. By adapting the systems biology framework to address this issue, we here integrated the available knowledge together with experimental data via the building of a series of mathematical models. This rationale enabled us to unravel molecular interactions involving c-Jun in GC induced apoptosis and identify Erg as determinant for GC resistance. The results revealed an alternative potential mechanism where c-Jun may be an indirect GR target that is controlled via an upstream repressor protein. The models also highlight the importance of Erg for GR function, particularly in GC sensitive C7 cells where Erg directly regulates GR in agreement with our previous experimental results. Our models describe potential GR-controlled molecular mechanisms of c-Jun/Bim and Erg regulation. We also demonstrate the importance of using a systematic approach to translate human disease processes into computational models in order to derive information-driven new hypotheses.

  3. A study on the expression of c-jun mRNA after experimental rat brain concussion%实验性大鼠脑震荡后c-jun mRNA表达

    Institute of Scientific and Technical Information of China (English)

    汪枫; 李永宏

    2011-01-01

    Objective To evaluate changes of c-jun mRNA after brain concussion. Methods Fifty-five rats were randomly divided into brain concussion groups ( 0min, 15 min, 30min, 60min, 3 h, 6h, 12h, 24h, 48 h,96h) and control group. The expression of c-jun mRNA in cortex 、thalamus and brain stem was microscopically observed by In site hybridization method. Results There were weak positive expression of c-jun mRNA in some neutrons and neuroglia cells in control group. In brain concussion group, however,positive expression of c-jun mRNA in some neutrons was seen at 15min after brain concussion,and reach to the peak at 30min after brain concussion the level of expression of c-jun mRNA were as well as control group at 96h. Conclusion There findings suggest that detection of c-jun mRNA could be an index of diagnosis of brain concussion and a sensitive marker of timing of injury after brain concussion.%目的 观察实验性大鼠脑震荡后c-jun mRNA的表达变化规律.方法 55只实验大鼠随机分为脑震荡组(0min、15min、30min、60min、3h、6h、12h、24h、48h、96h)和对照组,用原位杂交法观察大鼠脑震荡后各时间点,大脑皮质、脑干和丘脑神经元c-jun mRNA表达的变化规律.结果 对照组大鼠神经元和胶质细胞均可见c-jun mRNA的弱阳性表达.脑震荡组大鼠损伤后15min神经细胞观察到c-jun mRNA阳性表达,随损伤后经过时间的延长阳性表达逐渐增强;30min时c-jun mRNA阳性反应达高峰,随后逐渐降低,至96h时回落至对照组水平.结论 c-jun mRNA表达水平可成为诊断脑震荡和推断伤后经过时间的一项敏感指标.

  4. HP1a/KDM4A is involved in the autoregulatory loop of the oncogene gene c-Jun.

    Science.gov (United States)

    Liu, Yan; Zhang, Daoyong

    2015-01-01

    The proto-oncogene c-Jun plays crucial roles in tumorigenesis, and its aberrant expression has been implicated in many cancers. Previous studies have shown that the c-Jun gene is positively autoregulated by its product. Notably, it has also been reported that c-Jun proteins are enriched in its gene body region. However, the role of c-Jun proteins in its gene body region has yet to be uncovered. HP1a is an evolutionarily conserved heterochromatin-associated protein, which plays an essential role in heterochromatin-mediated gene silencing. Interestingly, accumulating evidence shows that HP1a is also localized to euchromatic regions to positively regulate gene transcription. However, the underlying mechanism has not been defined. In this study, we demonstrate that HP1a is involved in the positive autoregulatory loop of the Jra gene, the c-Jun homolog in Drosophila. Jra recruits the HP1a/KDM4A complex to its gene body region upon osmotic stress to reduce H3K36 methylation levels and disrupt H3K36 methylation-dependent histone deacetylation, resulting in high levels of histone acetylation in the Jra gene body region, thus promoting gene transcription. These results not only expand our knowledge toward the mechanism of c-Jun regulation, but also reveal the mechanism by which HP1a exerts its positive regulatory function in gene expression.

  5. Heterogeneity in c-jun gene expression in normal and malignant cells exposed to either ionizing radiation or hydrogen peroxide

    International Nuclear Information System (INIS)

    We investigated the role of reactive oxygen intermediates and protein kinase C (PKC) in induction of c-jun gene expression in human ML-2 leukemic cells and normal DET-551 fibroblasts by comparing the effects of either ionizing radiation or H2O2 exposure in the presence or absence of appropriate inhibitors. In these cell types, the radiation and H2O2-mediated increase in c-jun mRNA levels could be prevented by pretreatment of the cells with N-acetylcysteine, an antioxidant, or H7, an inhibitor of PKC and cAMP-dependent protein kinase (PKA), but not by HA1004, an inhibitor of PKA. These results suggest a role for PKC and reactive oxygen intermediates in the induction of c-jun gene expression in both normal and tumor cells. We also investigated potential differences in radiation- or H2O2-induced c-jun gene expression in normal and tumor cells by examining steady-state c-jun mRNA levels in a number of human fibroblast, leukemia, melanoma, sarcoma, and carcinoma cell types. We observed heterogeneity in the steady-state level of c-jun mRNA in both the untreated normal and tumor cells and in such cells exposed to ionizing radiation or to H2O2. Exposure to radiation or to hydrogen peroxide produced a varied response which ranged from little or no induction to a more than two orders of magnitude increase in the steady-state level of the c-jun mRNA

  6. c-Jun represses the human insulin promoter activity that depends on multiple cAMP response elements

    Energy Technology Data Exchange (ETDEWEB)

    Inagaki, Nobuya; Seino, Yutaka; Imura, Hiroo (Kyoto Univ. (Japan)); Maekawa, Toshio; Sudo, Tatsuhiko; Ishii, Shunsuke (Inst. of Physical and Chemical Research (RIKEN), Tsukuba (Japan))

    1992-02-01

    Glucose is known to increase the cAMP concentration in pancreatic {beta} cells. To determine the mechanism by which cAMP augments insulin gene expression, the authors first identified the cAMP response elements (CREs) of human insulin gene. In DNase I footprint analysis, the bacterially synthesized CRE-binding protein, CRE-BP1, protected four sites: two sites in the region upstream from the insulin core promoter, one site in the first exon, and one site in the first intron. To examine the roles of those four sites, they constructed a series of DNA plasmids in which the wild-type and mutant insulin promoters were linked to the chloramphenicol acetyltransferase gene. Studies of the transcriptional activity of these plasmids after transfection into hamster insulinoma (HIT) cells showed that these four sites contributed additively to the cAMP inducibility of the insulin promoter. Surprisingly, the c-jun protooncogene product (c-Jun) repressed the cAMP-induced activity of the insulin promoter in a cotransfection assay with the c-Jun expression plasmic. Northern blot analysis demonstrated that the level of c-jun mRNA was dramatically increased by glucose deprivation in HIT cells. These results suggest that glucose deprivation in HIT cells. These results suggest that glucose may regulate expression of the human insulin gene through multiple CREs and c-Jun.

  7. Heterodimer formation between c-Jun and Jun B proteins mediated by Epstein Barr virus encoded latent membrane protein 1

    Institute of Scientific and Technical Information of China (English)

    SONG; Xin; TAO; Yongguang; TAN; Yunnian; Leo; M.; Lee; DENG

    2005-01-01

    Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) may trigger the transcription factor AP-1 including c-Jun and c-fos. In this report, using a Tet-on LMP1 HNE2 cell line which is a dual-stable LMP1 integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 in which could be regulated by the Tet-on system, we show that Jun B can efficiently form a new heterodimeric complex with the c-Jun protein under the regulation of LMP1, phosphorylation of c-Jun (ser 63, ser 73) and Jun B is involved in the process of the new heterodimeric formation. We also find that this heterodimeric form can bind to the AP-1 consensus sequence. Transfection studies suggest that JNK interaction protein (JIP) could inhibit the heterodimer formation of c-Jun and Jun B through blocking the AP-1 signaling pathway triggered by LMP1. The interaction and function between c-Jun protein and Jun B protein increase the repertoire of possible regulatory complexes by LMP1 that could play an important role in the regulation of transcription of specific cellular genes in the process of genesis of nasopharyngeal carcinoma.

  8. Expression of c-fos and c-jun protooncogenes in the uteri of immature mice neonatally exposed to diethylstilbestrol.

    Science.gov (United States)

    Yamashita, S; Takayanagi, A; Shimizu, N

    2003-01-01

    We studied the cell-type-specific and temporal expression of c-fos and c-jun protooncogenes after 17beta-estradiol (E2) stimulation in the uteri of immature 3-week-old mice neonatally exposed to diethylstilbestrol (DES), DES-mice, and the ontogenic expression of these genes in the uteri of DES-mice using immunohistochemistry and in situ hybridization. A single E2 injection induced the transient and rapid expression of c-fos mRNA and c-Fos protein in the endometrial epithelium and endothelial cells of the blood vessels in both 3-week-old vehicle-treated controls and DES-mice; a peak of mRNA expression was 2 hours after E2 injection and that of protein expression was 2 to 3 hours after the injection. The expression of c-fos mRNA and protein after E2 stimulation was lower in the DES-mice than in the control animals. There were no significant differences in the c-jun expression patterns in both experimental groups before and after the E2 injection. The E2 injection transiently down-regulated the c-jun expression in the epithelium and up-regulated it in the stroma and myometrium. The uterine epithelium of DES-mice showed much stronger c-Jun immunostaining on days 4 and 10, compared with those of controls. Neonatal DES treatment reduced c-Jun immunoreactivity in the uterine epithelium on days 4 and 10, and increased the reaction in the stroma on day 4. These results suggested that the neonatal DES treatment induces permanent changes in the c-fos expression pattern independent of the postpuberal secretion of ovarian steroids. The changes in the expression of c-fos and c-jun protooncogenes, particularly during postnatal development, are likely to play important roles in the production of uterine abnormalities in the DES-mice.

  9. The AP-1 Transcription Factor c-Jun Prevents Stress-Imposed Maladaptive Remodeling of the Heart

    Science.gov (United States)

    Windak, Renata; Müller, Julius; Felley, Allison; Akhmedov, Alexander; Wagner, Erwin F.; Pedrazzini, Thierry; Sumara, Grzegorz; Ricci, Romeo

    2013-01-01

    Systemic hypertension increases cardiac workload and subsequently induces signaling networks in heart that underlie myocyte growth (hypertrophic response) through expansion of sarcomeres with the aim to increase contractility. However, conditions of increased workload can induce both adaptive and maladaptive growth of heart muscle. Previous studies implicate two members of the AP-1 transcription factor family, junD and fra-1, in regulation of heart growth during hypertrophic response. In this study, we investigate the function of the AP-1 transcription factors, c-jun and c-fos, in heart growth. Using pressure overload-induced cardiac hypertrophy in mice and targeted deletion of Jun or Fos in cardiomyocytes, we show that c-jun is required for adaptive cardiac hypertrophy, while c-fos is dispensable in this context. c-jun promotes expression of sarcomere proteins and suppresses expression of extracellular matrix proteins. Capacity of cardiac muscle to contract depends on organization of principal thick and thin filaments, myosin and actin, within the sarcomere. In line with decreased expression of sarcomere-associated proteins, Jun-deficient cardiomyocytes present disarrangement of filaments in sarcomeres and actin cytoskeleton disorganization. Moreover, Jun-deficient hearts subjected to pressure overload display pronounced fibrosis and increased myocyte apoptosis finally resulting in dilated cardiomyopathy. In conclusion, c-jun but not c-fos is required to induce a transcriptional program aimed at adapting heart growth upon increased workload. PMID:24039904

  10. Dentin bonding agents induce c-fos and c-jun protooncogenes expression in human gingival fibroblasts.

    Science.gov (United States)

    Huang, Fu-Mei; Chou, Ming-Yung; Chang, Yu-Chao

    2003-01-01

    An important requirement for a dentin bonding agent is biologic compatibility; the bonding agent usually remains in close contact with living dental tissues over a long period of time. Information on the genotoxicity/mutagenicity and cacinogenicity potentials of dentin bonding agents is rare. It has been shown that c-fos and c-jun are induced rapidly by a variety of chemical and physical stimuli. Little is known about the induction of cellular signaling events and specific gene expression after cell exposure to dentin bonding agents. Therefore, we used primary human gingival fibroblasts to examine the effect of six dentin bonding agents on the expression of c-fos and c-jun protooncogenes to evaluate the genotoxicity/mutagenicity and cacinogenicity potential of the dentin bonding agents. The levels of mRNA were measured by the quantitative RT-PCR analysis. c-fos and c-jun mRNA expression in dentin bonding agents-treated cells revealed a rapid accumulation of the transcript, a significant signal first was detectable after 1h of exposure. Persistent induction of c-jun and c-fos protooncogenes by dentine bonding agents may distribute systemically to cause some unexpected adverse effects on human beings. It would be necessary to identify the severely toxic compounds and replace these substances by better biocompatible components. Otherwise, leaching of those genotoxicity/mutagenicity and cacinogenicity components must be minimized or prevented.

  11. Expression of c-jun in brain stem following moderate lateral fluid percussion brain injury in rats

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    AIM: To study the expression of c-jun in brain stem following moderate lateral fluid percussion brain injury in rats, and to observe the temporal patterns of its expressions following percussion.METHODS: Male Sprague-Dawley rats were divided into normal control, sham operation control and injury groups. The rats of injury group subjected to moderate lateral fluid percussion injury (0.2 mPa), and then were subdivided into 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 12 h groups according to the time elapsed after injury. The expression of c-jun was studied by immunohistochemistry and in situ hybridization. RESULTS: After percussion for 15 min, Jun positive neurons increased in brain stem progressively, and peaked at 12h. At 5min after percussion, the induction of c-jun mRNA was increased, and remained elevated up to 1h-2h after brain injury. CONCLUSION: The induction and expression of the c-jun in brain stem after fluid percussion brain injury were increased rapidly and lasted for a long time.

  12. RhoA regulates invasion of glioma cells via the c-Jun NH2-terminal kinase pathway under hypoxia.

    Science.gov (United States)

    Tong, Jiao Jian; Yan, Zhang; Jian, Ren; Tao, Huang; Hui, Ouyang Tao; Jian, Chen

    2012-09-01

    The purpose of this study was to investigate the mechanism of glioma cell invasion in hypoxic conditions. We demonstrated that hypoxia increased cell invasion, matrix metalloproteinase-2 (MMP2) activity and time-dependent expression of hypoxia inducible factor-1α (HIF-1α) in human glioma cells. These data suggest that MMP2 may play a significant role in tumor invasion in hypoxic conditions. We investigated the mechanisms involved in the increased MMP2 activity and cell invasion in hypoxic conditions. Increased expression of phospho-Jun NH2-terminal kinase (p-JNK) and phospho-c-Jun (p-c-Jun) in glioma cells induced by hypoxia was detected. Furthermore, this effect may be reduced by inhibiting the JNK signaling pathway. We found that inhibition of RhoA geranylgeranylation by geranylgeranyltransferase inhibitor-2147 (GGTI-2147) or knockdown of RhoA by siRNA against RhoA reduced the expression of p-JNK and p-c-Jun, and decreased MMP2 activity and glioma cell invasion in hypoxic conditions. These data suggest a link among RhoA, JNK, c-Jun and MMP2 activity that is functionally involved in the increased glioma cell invasion induced by hypoxia. PMID:23741249

  13. Towards the N-terminal acetylome

    DEFF Research Database (Denmark)

    Zhang, Xumin; Højrup, Peter

    2013-01-01

    Protein N-terminal acetylation (N(α)-acetylation) is observed widely from prokaryotes to eukaryotes. It gains increased importance in biological field, due to its multiple roles in many aspects of the protein life, such as assembly, stability, activity, and location. Today, mass spectrometry (MS...

  14. From reptilian phylogenomics to reptilian genomes: analyses of c-Jun and DJ-1 proto-oncogenes.

    Science.gov (United States)

    Katsu, Y; Braun, E L; Guillette, L J; Iguchi, T

    2009-01-01

    Genome projects have revolutionized our understanding of both molecular biology and evolution, but there has been a limited collection of genomic data from reptiles. This is surprising given the pivotal position of reptiles in vertebrate phylogeny and the potential utility of information from reptiles for understanding a number of biological phenomena, such as sex determination. Although there are many potential uses for genomic data, one important and useful approach is phylogenomics. Here we report cDNA sequences for the c-Jun(JUN) and DJ-1(PARK7) proto-oncogenes from 3 reptiles (the American alligator, Nile crocodile, and Florida red-belly turtle), show that both genes are expressed in the alligator, and integrate them into analyses of their homologs from other organisms. With these taxa it was possible to conduct analyses that include all major vertebrate lineages. Analyses of c-Jun revealed an unexpected but well-supported frog-turtle clade while analyses of DJ-1 revealed a topology largely congruent with expectation based upon other data. The conflict between the c-Jun topology and expectation appears to reflect the overlap between c-Jun and a CpG island in most taxa, including crocodilians. This CpG island is absent in the frog and turtle, and convergence in base composition appears to be at least partially responsible for the signal uniting these taxa. Noise reduction approaches can eliminate the unexpected frog-turtle clade, demonstrating that multiple signals are present in the c-Jun alignment. We used phylogenetic methods to visualize these signals; we suggest that examining both historical and non-historical signals will prove important for phylogenomic analyses.

  15. Arsenic trioxide induces apoptosis in human platelets via C-Jun NH2-terminal kinase activation.

    Directory of Open Access Journals (Sweden)

    Yicun Wu

    Full Text Available Arsenic trioxide (ATO, one of the oldest drugs in both Western and traditional Chinese medicine, has become an effective anticancer drug, especially in the treatment of acute promyelocytic leukemia (APL. However, thrombocytopenia occurred in most of ATO-treated patients with APL or other malignant diseases, and the pathogenesis remains unclear. Here we show that ATO dose-dependently induces depolarization of mitochondrial inner transmembrane potential (ΔΨm, up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation, and phosphotidylserine (PS exposure in platelets. ATO did not induce surface expression of P-selectin and PAC-1 binding, whereas, obviously reduced collagen, ADP, and thrombin induced platelet aggregation. ATO dose-dependently induced c-Jun NH2-terminal kinase (JNK activation, and JNK specific inhibitor dicumarol obviously reduced ATO-induced ΔΨm depolarization in platelets. Clinical therapeutic dosage of ATO was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were significantly reduced after five days of continuous injection. The data demonstrate that ATO induces caspase-dependent apoptosis via JNK activation in platelets. ATO does not incur platelet activation, whereas, it not only impairs platelet function but also reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia in patients treated with ATO.

  16. Arsenic trioxide induces apoptosis in human platelets via C-Jun NH2-terminal kinase activation.

    Science.gov (United States)

    Wu, Yicun; Dai, Jin; Zhang, Weilin; Yan, Rong; Zhang, Yiwen; Ruan, Changgeng; Dai, Kesheng

    2014-01-01

    Arsenic trioxide (ATO), one of the oldest drugs in both Western and traditional Chinese medicine, has become an effective anticancer drug, especially in the treatment of acute promyelocytic leukemia (APL). However, thrombocytopenia occurred in most of ATO-treated patients with APL or other malignant diseases, and the pathogenesis remains unclear. Here we show that ATO dose-dependently induces depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation, and phosphotidylserine (PS) exposure in platelets. ATO did not induce surface expression of P-selectin and PAC-1 binding, whereas, obviously reduced collagen, ADP, and thrombin induced platelet aggregation. ATO dose-dependently induced c-Jun NH2-terminal kinase (JNK) activation, and JNK specific inhibitor dicumarol obviously reduced ATO-induced ΔΨm depolarization in platelets. Clinical therapeutic dosage of ATO was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were significantly reduced after five days of continuous injection. The data demonstrate that ATO induces caspase-dependent apoptosis via JNK activation in platelets. ATO does not incur platelet activation, whereas, it not only impairs platelet function but also reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia in patients treated with ATO. PMID:24466103

  17. c-fos/c-jun expression and AP-1 activation in skin fibroblasts from centenarians.

    Science.gov (United States)

    Grassilli, E; Bellesia, E; Salomoni, P; Croce, M A; Sikora, E; Radziszewska, E; Tesco, G; Vergelli, M; Latorraca, S; Barbieri, D; Fagiolo, U; Santacaterina, S; Amaducci, L; Tiozzo, R; Sorbi, S; Franceschi, C

    1996-09-13

    In vitro replicative senescence is characterized by an irreversible growth arrest due to the inability of the cell to induce some key regulators of cell cycle progression, such as c-fos and AP-1, in response to mitogenic stimuli. In vitro replicative senescence and in vivo aging have been assumed to be two related phenomena, likely controlled by overlapping or interacting genes. As a corollary, fibroblasts from centenarians, which have undergone a long process of senescence in vivo should have very limited proliferative capability. On the contrary, in a previous work we found that fibroblasts from centenarians exhibited the same capacity to respond to different mitogenic stimuli as fibroblasts from young donors. Here we provide evidences that the well preserved proliferative response is likely due to the fact that some pivotal regulators- c-fos, c-jun and AP-1-are still fully inducible, despite a long process of in vivo senescence. Our data therefore suggest that in vivo and in vitro aging are separate phenomena whose possible relationships, if any, have to be ascertained very carefully. PMID:8806666

  18. A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun.

    Science.gov (United States)

    Morton, Simon; Davis, Roger J; McLaren, Ann; Cohen, Philip

    2003-08-01

    We have used phospho-specific antibodies to re-examine the multisite phosphorylation of c-Jun in murine RAW macrophages and embryonic fibroblasts. Our results indicate that JNK isoforms are required and sufficient for the phosphorylation of Thr91 and Thr93, as well as the phosphorylation of Ser63 and Ser73, in response to LPS or anisomycin in macrophages and TNFalpha or anisomycin in fibroblasts. However, the phorbol ester (TPA) and EGF-induced phosphorylation of Ser63 and Ser73 is mediated by ERK1/ERK2, as well as JNK1/JNK2, in fibroblasts from wild-type mice and by ERK1/ERK2 alone in fibroblasts from JNK-deficient mice. The phosphorylation of Thr239 is catalysed by GSK3 and the phosphorylation of Ser243 by an as yet unidentified protein kinase. The inhibition of GSK3 is not required for the dephosphorylation of Thr239 in response to LPS, and nor is the phosphorylation of Thr91 and Thr93 required for the TPA- or EGF-induced dephosphorylation of Thr239 in fibroblasts. The agonist-induced dephosphorylation of Thr239 may involve a conformational change that exposes Thr239 to dephosphorylation and/or the activation of a Thr239 phosphatase. PMID:12881422

  19. Effect of growth hormone and serum on the expression of the proto-oncogenes c-jun and c-fos in insulin producing cells

    DEFF Research Database (Denmark)

    Petersen, Elisabeth D.; Billestrup, N; Nielsen, Jens Høiriis

    1990-01-01

    Expression of the proto-oncogenes c-fos and c-jun was analysed in the insulin producing rat tumor cell line, RIN 5AH. Addition of fetal calf serum (FCS) to serum-starved cells in the presence of cycloheximid induced a modest increase in c-fos and c-jun mRNA levels, whereas growth hormone (GH...

  20. Combined Expression of c-jun, c-fos, and p53 Improves Estimation of Prognosis in Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Wang, Shan; Xu, Xin; Xu, Fei; Meng, Yan; Sun, Changsheng; Shi, Lei; Zhao, Eryang

    2016-09-13

    To identify the prognostic value of c-jun, c-fos, and p53 in oral cancer, we examined the impact of immunohistochemical expression of these markers on tumor progression in 157 oral squamous cell carcinoma (OSCC). We found that c-jun or c-fos was significantly associated with lymph node metastasis, and coexpression of c-jun/c-fos, or c-jun/c-fos/p53 were significantly associated with lymph node metastasis, poor differentiation and clinical stage. The coexpression of c-jun/c-fos/p53 was identified as independent prognostic factors for overall survival. Simultaneous coexpression of these markers in OSCCs might prove to be a useful indicator for differentiation of low and high-risk patients.

  1. Fibroin and sericin from Bombyx mori silk stimulate cell migration through upregulation and phosphorylation of c-Jun.

    Directory of Open Access Journals (Sweden)

    Celia Martínez-Mora

    Full Text Available Wound healing is a biological process directed to the restoration of tissue that has suffered an injury. An important phase of wound healing is the generation of a basal epithelium able to wholly replace the epidermis of the wound. A broad range of products derived from fibroin and sericin from Bombyx mori silk are used to stimulate wound healing. However, so far the molecular mechanism underlying this phenomenon has not been elucidated. The aim of this work was to determine the molecular basis underlying wound healing properties of silk proteins using a cell model. For this purpose, we assayed fibroin and sericin in a wound healing scratch assay using MDA-MB-231 and Mv1Lu cells. Both proteins stimulated cell migration. Furthermore, treatment with sericin and fibroin involved key factors of the wound healing process such as upregulation of c-Jun and c-Jun protein phosphorylation. Moreover, fibroin and sericin stimulated the phosphorylation of ERK 1/2 and JNK 1/2 kinases. All these experiments were done in the presence of specific inhibitors for some of the cell signalling pathways referred above. The obtained results revealed that MEK, JNK and PI3K pathways are involved in fibroin and sericin stimulated cells migration. Inhibition of these three kinases prevented c-Jun upregulation and phosphorylation by fibroin or sericin. Fibroin and sericin were tested in the human keratinocyte cell line, HaCaT, with similar results. Altogether, our results showed that fibroin and sericin initiate cell migration by activating the MEK, JNK and PI3K signalling pathways ending in c-Jun activation.

  2. Effect of Jun N-terminal kinase 1 and 2 on the replication of Penicillium marneffei in human macrophages.

    Science.gov (United States)

    Chen, Renqiong; Xi, Liyan; Huang, Xiaowen; Ma, Tuan; Ren, Hong; Ji, Guangquan

    2015-05-01

    Penicillium marneffei (P. marneffei) is a human pathogen which persists in macrophages and threatens the immunocompromised patients. To clarify the mechanisms involved, we evaluated the effect of c-Jun N-terminal kinase 1 and 2 (JNK1/2) on cytokine expression, phagosomal maturation and multiplication of P. marneffei in P. marneffei-stimulated human macrophages. P. marneffei induced the rapid phosphorylation of JNK1/2. Using the specific inhibitor of JNK1/2 (SP600125), we found that the inhibition of JNK1/2 suppressed P. marneffei-induced tumor necrosis factor-α and IL-10 production. In addition, the presence of SP600125 increased phagosomal acidification and maturation and decreased intracellular replication. These data suggest that JNK1/2 may play an important role in promoting the replication of P. marneffei. Our findings further indicate that the pathogen through the JNK1/2 pathway may attenuate the immune response and macrophage antifungal function.

  3. The effects of vitamin E succinate on the expression of c-jun gene and protein in human gastric cancer SGC-7901 cells

    Institute of Scientific and Technical Information of China (English)

    Yan Zhao; Kun Wu; Wei Xia; Yu-Juan Shan; Li-Jie Wu; Wei-Ping Yu

    2002-01-01

    AIM: To investigate the effects of vitamin E succinate (VES) on the expression of c-jun gene and protein in human gastric cancer SGC-7901 cells.METHODS: After SGC-7901 cells were treated with VES at different doses (5,10,20 mg@L-1) at different time, reverse transcription-PCR technique was used to detect the level of c-jun mRNA; Western Blot was applied to measure the expression of c-jun protei n/RESULTS: After the cells were treated with VES at 20 mg@L-1 for 3 h, the expression rapidly reached its maximum that was 3.5 times of UT control (P<0.01). The level of c-jun mRNA was also increased following treatment of VES for 6 h.However, the expression after treatment of VES at 5 mg@L-1for 24 h was 1.6 times compared with UT control (P<0.01).Western blot analysis showed that the level of c-jun protein was obviously elevated in VES-treated SGC-7901 cells at 20 mg@L-1 for 3 h. The expression of c-jun protein was gradually increased after treatment of VES at 20 mg@L-1 for 3, 6, 12 and 24 h, respectively, with an evident time-effect relationship. CONCLUSION: The levels of c-jun mRNA and protein in VES-treated SGC-7901 cells were increased in a dose- and time-dependent manner; the expression of c-jun was prolonged by VES, indicating that c-jun is involved in VESinduced apoptosis in SGC-7901 cells.

  4. The leucine zipper domains of the transcription factors GCN4 and c-Jun have ribonuclease activity.

    Directory of Open Access Journals (Sweden)

    Yaroslav Nikolaev

    Full Text Available Basic-region leucine zipper (bZIP proteins are one of the largest transcription factor families that regulate a wide range of cellular functions. Owing to the stability of their coiled coil structure leucine zipper (LZ domains of bZIP factors are widely employed as dimerization motifs in protein engineering studies. In the course of one such study, the X-ray structure of the retro-version of the LZ moiety of yeast transcriptional activator GCN4 suggested that this retro-LZ may have ribonuclease activity. Here we show that not only the retro-LZ but also the authentic LZ of GCN4 has weak but distinct ribonuclease activity. The observed cleavage of RNA is unspecific, it is not suppressed by the ribonuclease A inhibitor RNasin and involves the breakage of 3',5'-phosphodiester bonds with formation of 2',3'-cyclic phosphates as the final products as demonstrated by HPLC/electrospray ionization mass spectrometry. Several mutants of the GCN4 leucine zipper are catalytically inactive, providing important negative controls and unequivocally associating the enzymatic activity with the peptide under study. The leucine zipper moiety of the human factor c-Jun as well as the entire c-Jun protein are also shown to catalyze degradation of RNA. The presented data, which was obtained in the test-tube experiments, adds GCN4 and c-Jun to the pool of proteins with multiple functions (also known as moonlighting proteins. If expressed in vivo, the endoribonuclease activity of these bZIP-containing factors may represent a direct coupling between transcription activation and controlled RNA turnover. As an additional result of this work, the retro-leucine zipper of GCN4 can be added to the list of functional retro-peptides.

  5. Bile acids-mediated overexpression of MUC4 via FAK-dependent c-Jun activation in pancreatic cancer.

    Science.gov (United States)

    Joshi, Suhasini; Cruz, Eric; Rachagani, Satyanarayana; Guha, Sushovan; Brand, Randall E; Ponnusamy, Moorthy P; Kumar, Sushil; Batra, Surinder K

    2016-08-01

    The majority of pancreatic cancer (PC) patients are clinically presented with obstructive jaundice with elevated levels of circulatory bilirubin and alkaline phosphatases. In the current study, we examined the implications of bile acids (BA), an important component of bile, on the pathophysiology of PC and investigated their mechanistic association in tumor-promoting functions. Integration of results from PC patient samples and autochthonous mouse models showed an elevated levels of BA (p < 0.05) in serum samples compared to healthy controls. Similarly, an elevated BA levels was observed in pancreatic juice derived from PC patients (p < 0.05) than non-pancreatic non-healthy (NPNH) controls, further establishing the clinical association of BA with the pathogenesis of PC. The tumor-promoting functions of BA were established by observed transcriptional upregulation of oncogenic MUC4 expression. Luciferase reporter assay revealed distal MUC4 promoter as the primary responsive site to BA. In silico analysis recognized two c-Jun binding sites at MUC4 distal promoter, which was biochemically established using ChIP assay. Interestingly, BA treatment led to an increased transcription and activation of c-Jun in a FAK-dependent manner. Additionally, BA receptor, namely FXR, which is also upregulated at transcriptional level in PC patient samples, was demonstrated as an upstream molecule in BA-mediated FAK activation, plausibly by regulating Src activation. Altogether, these results demonstrate that elevated levels of BA increase the tumorigenic potential of PC cells by inducing FXR/FAK/c-Jun axis to upregulate MUC4 expression, which is overexpressed in pancreatic tumors and is known to be associated with progression and metastasis of PC. PMID:27185392

  6. Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance▿

    OpenAIRE

    Sabio, Guadalupe; Kennedy, Norman J.; Cavanagh-Kyros, Julie; Jung, Dae Young; Ko, Hwi Jin; Ong, Helena; Barrett, Tamera; Kim, Jason K.; Davis, Roger J

    2009-01-01

    Obesity caused by feeding of a high-fat diet (HFD) is associated with an increased activation of c-Jun NH2-terminal kinase 1 (JNK1). Activated JNK1 is implicated in the mechanism of obesity-induced insulin resistance and the development of metabolic syndrome and type 2 diabetes. Significantly, Jnk1−/− mice are protected against HFD-induced obesity and insulin resistance. Here we show that an ablation of the Jnk1 gene in skeletal muscle does not influence HFD-induced obesity. However, muscle-s...

  7. Function of c-Fos-like and c-Jun-like Proteins on Trichostatin A-induced G2/M Arrest in Physarum polycephalum

    Institute of Scientific and Technical Information of China (English)

    Xiao-Xue LI; Jun LU; Yan-Mei ZHAO; Bai-Qu HUANG

    2005-01-01

    The homologs of transcription factors c-Fos and c-Jun have been detected in slime mold Physarum polycephalum during progression of the synchronous cell cycle. Here we demonstrated that cFos-like and c-Jun-like proteins participated in G2/M transition by the regulation of the level of Cyclin B1 protein in P. polycephalum. The study of antibody neutralization revealed that interruption of the functions of c-Fos-like and c-Jun-like proteins resulted in G2/M transition arrest, implicating their functional roles in cell cycle control. When G2/M transition was blocked by histone deacetylase inhibitor trichostatin A, changes in c-Fos- and c-Jun-like protein levels, and hyperacetylation of c-Jun-like protein, were observed. The data suggest that in P. polycephalum, c-Fos- and c-Jun-like proteins may be the key factors in the regulation of histone acetylation-related G2/M transition, involving the coordinated expression and hyperacetylation of these proteins.

  8. Puerarin reduces increased c-fos, c-jun, and type Ⅳ collagen expression caused by high glucose in glomerular mesangial cells

    Institute of Scientific and Technical Information of China (English)

    Cai-ping MAO; Zhen-lun GU

    2005-01-01

    Aim: Increased expression of c-fos, c-jun and type Ⅳ collagen (CoⅣ) in glomerular mesangial cells (GMC) are important characteristics of diabetic nephropathy.Both c-fos and c-jun regulate the gene expression of extracellular matrix components, and CoⅣ is the main component of the extracellular matrix. It has been reported that puerarin inhibits aggregation of the extracellular matrix in diabetic rats by an as yet unknown mechanism. The aim of this study is to investigate the effect of puerarin on c-fos, c-jun and CoⅣ expression in GMC cultured in medium containing 5.6 or 27.8 mmol/L glucose. Methods: The expressions ofc-fos and c-jun were measured at the protein level using flow cytometry. CoⅣ content was detected using radioimmunoassay. Protein kinase C (PKC) activity was measured using liquid scintillation counting. Results: Puerarin (10-5 mmol/L) significantly ameliorated the high-glucose effect on c-fos, c-jun and CoⅣ expression.This effect is accompanied by a reduced PKC activity in these cells. Conclusion:Our results suggest that reduced PKC activity and expression of c-fos and c-jun in GMC might participate in the mechanisms underlying the therapeutic effect of puerarin on diabetic nephropathy.

  9. Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation

    Science.gov (United States)

    D'Alonzo, Richard C.; Selvamurugan, Nagarajan; Karsenty, Gerard; Partridge, Nicola C.

    2002-01-01

    Previously, we determined that the activator protein-1 (AP-1)-binding site and the runt domain (RD)-binding site and their binding proteins, c-Fos.c-Jun and Cbfa, regulate the collagenase-3 promoter in parathyroid hormone-treated and differentiating osteoblasts. Here we show that Cbfa1 and c-Fos.c-Jun appear to cooperatively bind the RD- and AP-1-binding sites and form ternary structures in vitro. Both in vitro and in vivo co-immunoprecipitation and yeast two-hybrid studies further demonstrate interaction between Cbfa1 with c-Fos and c-Jun in the absence of phosphorylation and without binding to DNA. Additionally, only the runt domain of Cbfa1 was required for interaction with c-Jun and c-Fos. In mammalian cells, overexpression of Cbfa1 enhanced c-Jun activation of AP-1-binding site promoter activity, demonstrating functional interaction. Finally, insertion of base pairs that disrupted the helical phasing between the AP-1- and RD-binding sites also inhibited collagenase-3 promoter activation. Thus, we provide direct evidence that Cbfa1 and c-Fos.c-Jun physically interact and cooperatively bind the AP-1- and RD-binding sites in the collagenase-3 promoter. Moreover, the AP-1- and RD-binding sites appear to be organized in a specific required helical arrangement that facilitates transcription factor interaction and enables promoter activation.

  10. AP-1 Transcription Factors c-FOS and c-JUN Mediate GnRH-Induced Cadherin-11 Expression and Trophoblast Cell Invasion.

    Science.gov (United States)

    Peng, Bo; Zhu, Hua; Ma, Liyang; Wang, Yan-Ling; Klausen, Christian; Leung, Peter C K

    2015-06-01

    GnRH is expressed in first-trimester human placenta and increases cell invasion in extravillous cytotrophoblasts (EVTs). Invasive phenotypes have been reported to be regulated by transcription factor activator protein 1 (AP-1) and mesenchymal cadherin-11. The aim of our study was to investigate the roles of AP-1 components (c-FOS/c-JUN) and cadherin-11 in GnRH-induced cell invasion in human EVT cells. Phosphorylated c-FOS and phosphorylated c-JUN were detected in the cell column regions of human first-trimester placental villi by immunohistochemistry. GnRH treatment increased c-FOS, c-JUN, and cadherin-11 mRNA and protein levels in immortalized EVT (HTR-8/SVneo) cells. Moreover, GnRH treatment induced c-FOS and c-JUN protein phosphorylation and nuclear accumulation. Pretreatment with antide, a GnRH antagonist, attenuated GnRH-induced cadherin-11 expression. Importantly, basal and GnRH-induced cadherin-11 expression and cell invasion were reduced by small interfering RNA-mediated knockdown of c-FOS, c-JUN, and cadherin-11 in HTR-8/SVneo cells. Our results suggest that GnRH induces the expression and phosphorylation of the AP-1 transcription factors c-FOS and c-JUN in trophoblast cells, which contributes to GnRH-induced elevation of cadherin-11 expression and cell invasion. PMID:25794160

  11. Scorpion Venom Heat-Resistant Peptide Attenuates Glial Fibrillary Acidic Protein Expression via c-Jun/AP-1.

    Science.gov (United States)

    Cao, Zhen; Wu, Xue-Fei; Peng, Yan; Zhang, Rui; Li, Na; Yang, Jin-Yi; Zhang, Shu-Qin; Zhang, Wan-Qin; Zhao, Jie; Li, Shao

    2015-11-01

    Scorpion venom has been used in the Orient to treat central nervous system diseases for many years, and the protein/peptide toxins in Buthus martensii Karsch (BmK) venom are believed to be the effective components. Scorpion venom heat-resistant peptide (SVHRP) is an active component of the scorpion venom extracted from BmK. In a previous study, we found that SVHRP could inhibit the formation of a glial scar, which is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, in the epileptic hippocampus. However, the cellular and molecular mechanisms underlying this process remain to be clarified. The results of the present study indicate that endogenous GFAP expression in primary rat astrocytes was attenuated by SVHRP. We further demonstrate that the suppression of GFAP was primarily mediated by inhibiting both c-Jun expression and its binding with AP-1 DNA binding site and other factors at the GFAP promoter. These results support that SVHRP contributes to reducing GFAP at least in part by decreasing the activity of the transcription factor AP-1. In conclusion, the effects of SVHRP on astrocytes with respect to the c-Jun/AP-1 signaling pathway in vitro provide a practical basis for studying astrocyte activation and inhibition and a scientific basis for further studies of traditional medicine.

  12. miR-138 protects cardiomyocytes from hypoxia-induced apoptosis via MLK3/JNK/c-jun pathway

    Energy Technology Data Exchange (ETDEWEB)

    He, Siyi; Liu, Peng; Jian, Zhao; Li, Jingwei; Zhu, Yun; Feng, Zezhou; Xiao, Yingbin, E-mail: xiaoyb@vip.sina.com

    2013-11-29

    Highlights: •First time to find miR-138 is up-regulated in hypoxic cardiomyocytes. •First time to find miR-138 targets MLK3 and regulates JNK/c-jun pathway. •Rare myocardial biopsy of patients with CHD were collected. •Both silence and overexpression of miR-138 were implemented. •Various methods were used to detect cell function. -- Abstract: Cardiomyocytes experience a series of complex endogenous regulatory mechanisms against apoptosis induced by chronic hypoxia. MicroRNAs are a class of endogenous small non-coding RNAs that regulate cellular pathophysiological processes. Recently, microRNA-138 (miR-138) has been found related to hypoxia, and beneficial for cell proliferation. Therefore, we intend to study the role of miR-138 in hypoxic cardiomyocytes and the main mechanism. Myocardial samples of patients with congenital heart disease (CHD) were collected to test miR-138 expression. Agomir or antagomir of miR-138 was transfected into H9C2 cells to investigate its effect on cell apoptosis. Higher miR-138 expression was observed in patients with cyanotic CHD, and its expression gradually increased with prolonged hypoxia time in H9C2 cells. Using MTT and LDH assays, cell growth was significantly greater in the agomir group than in the negative control (NC) group, while antagomir decreased cell survival. Dual luciferase reporter gene and Western-blot results confirmed MLK3 was a direct target of miR-138. It was found that miR-138 attenuated hypoxia-induced apoptosis using TUNEL, Hoechst staining and Annexin V-PE/7-AAD flow cytometry analysis. We further detected expression of apoptosis-related proteins. In the agomir group, the level of pro-apoptotic proteins such as cleaved-caspase-3, cleaved-PARP and Bad significantly reduced, while Bcl-2 and Bcl-2/Bax ratio increased. Opposite changes were observed in the antagomir group. Downstream targets of MLK3, JNK and c-jun, were also suppressed by miR-138. Our study demonstrates that up-regulation of miR-138 plays

  13. Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jin-Sun; Kim, Hee-Sun, E-mail: hskimp@ewha.ac.kr

    2014-05-16

    Highlights: • tBHQ increased HO-1 mRNA and protein levels in rat primary astrocytes. • tBHQ enhanced HO-1 gene transcription in an ARE-dependent manner. • tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. • Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. • Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction. - Abstract: Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigated the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes.

  14. Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes

    International Nuclear Information System (INIS)

    Highlights: • tBHQ increased HO-1 mRNA and protein levels in rat primary astrocytes. • tBHQ enhanced HO-1 gene transcription in an ARE-dependent manner. • tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. • Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. • Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction. - Abstract: Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigated the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes

  15. Voxel-based analysis of the immediate early gene, c-jun, in the honey bee brain after a sucrose stimulus.

    Science.gov (United States)

    McNeill, M S; Robinson, G E

    2015-06-01

    Immediate early genes (IEGs) have served as useful markers of brain neuronal activity in mammals, and more recently in insects. The mammalian canonical IEG, c-jun, is part of regulatory pathways conserved in insects and has been shown to be responsive to alarm pheromone in honey bees. We tested whether c-jun was responsive in honey bees to another behaviourally relevant stimulus, sucrose, in order to further identify the brain regions involved in sucrose processing. To identify responsive regions, we developed a new method of voxel-based analysis of c-jun mRNA expression. We found that c-jun is expressed in somata throughout the brain. It was rapidly induced in response to sucrose stimuli, and it responded in somata near the antennal and mechanosensory motor centre, mushroom body calices and lateral protocerebrum, which are known to be involved in sucrose processing. c-jun also responded to sucrose in somata near the lateral suboesophageal ganglion, dorsal optic lobe, ventral optic lobe and dorsal posterior protocerebrum, which had not been previously identified by other methods. These results demonstrate the utility of voxel-based analysis of mRNA expression in the insect brain.

  16. A feedback inhibition between miRNA-127 and TGFβ/c-Jun cascade in HCC cell migration via MMP13.

    Directory of Open Access Journals (Sweden)

    Zhihong Yang

    Full Text Available Hepatocellular carcinoma (HCC is the fifth most common cancer worldwide and is increasing in frequency in the U.S. The major reason for the low postoperative survival rate of HCC is widespread intrahepatic metastasis or invasion, and activation of TGFβ signaling is associated with the invasive phenotype. This study aims at determining the novel function of miR-127 in modulating HCC migration. Overexpression of miR-127 inhibits HCC cell migration, invasion and tumor growth in nude mice. MiR-127 directly represses matrix metalloproteinase 13 (MMP13 3'UTR activity and protein expression, and diminishes MMP13/TGFβ-induced HCC migration. In turn, TGFβ decreases miR-127 expression by enhancing c-Jun-mediated inhibition of miR-127 promoter activity. In contrast, p53 transactivates miR-127 promoter and induces miR-127 expression, which is antagonized by c-Jun. The inhibition of miR-127 by c-Jun is through TGFβ-mediated ERK and JNK pathways. The lower miR-127 expression shows a negative correlation with the higher MMP13 expression in a subset of human HCC specimens. This is the first report elucidating a feedback regulation between miR-127 and the TGFβ/c-Jun cascade in HCC migration via MMP13 that involves a crosstalk between the oncogene c-Jun and tumor suppressor p53.

  17. Role of TGF-β-induced Claudin-4 expression through c-Jun signaling in non-small cell lung cancer.

    Science.gov (United States)

    Rachakonda, Girish; Vu, Trung; Jin, Lin; Samanta, Debangshu; Datta, Pran K

    2016-10-01

    Claudin-4 has been identified as an integral member of tight junctions and has been found to be upregulated in various types of cancers especially in metastatic cancers. However, the molecular mechanism of the upregulation of Claudin-4 and its role in lung tumorigenesis are unknown. The aim of the present study is to investigate the role of Claudin-4 on migration and tumorigenicity of lung cancer cells and to examine the regulatory effects of TGF-β on Claudin-4 expression. We have observed that TGF-β induces the expression of Claudin-4 dramatically in lung cell lines in a time dependent manner. TGF-β-induced Smad signaling is important for enhancing Claudin-4 mRNA level through inducing its promoter activity. Treatment with curcumin, a c-Jun inhibitor, or stable knockdown of c-Jun abrogates TGF-β-induced Claudin-4 expression suggesting an involvement of the c-Jun pathway. Notably, TGF-β-induced Claudin-4 expression through c-Jun pathway plays a role in TGF-β-mediated motility and tumorigenicity of these cells. In support of these observations, we have uncovered that Claudin-4 is upregulated in 14 of 24 (58%) lung tumors when compared with normal lung tissue. This is the first study to show how TGF-β regulates the expression of Claudin-4 through c-Jun signaling and how this pathway contributes to the migratory and tumorigenic phenotype of lung tumor cells. PMID:27424491

  18. Tristetraprolin induces cell cycle arrest in breast tumor cells through targeting AP-1/c-Jun and NF-κB pathway.

    Science.gov (United States)

    Xu, Li; Ning, Huan; Gu, Ling; Wang, Qinghong; Lu, Wenbao; Peng, Hui; Cui, Weiguang; Ying, Baoling; Ross, Christina R; Wilson, Gerald M; Wei, Lin; Wold, William S M; Liu, Jianguo

    2015-12-01

    The main characteristic of cancers, including breast cancer, is the ability of cancer cells to proliferate uncontrollably. However, the underlying mechanisms of cancer cell proliferation, especially those regulated by the RNA binding protein tristetraprolin (TTP), are not completely understood. In this study, we found that TTP inhibits cell proliferation in vitro and suppresses tumor growth in vivo through inducing cell cycle arrest at the S phase. Our studies demonstrate that TTP inhibits c-Jun expression through the C-terminal Zn finger and therefore increases Wee1 expression, a regulatory molecule which controls cell cycle transition from the S to the G2 phase. In contrast to the well-known function of TTP in regulating mRNA stability, TTP inhibits c-Jun expression at the level of transcription by selectively blocking NF-κB p65 nuclear translocation. Reconstitution of NF-κB p65 completely abolishes the inhibition of c-Jun transcription by TTP. Moreover, reconstitution of c-Jun in TTP-expressing breast tumor cells diminishes Wee1 overexpression and promotes cell proliferation. Our results indicate that TTP suppresses c-Jun expression that results in Wee1 induction which causes cell cycle arrest at the S phase and inhibition of cell proliferation. Our study provides a new pathway for TTP function as a tumor suppressor which could be targeted in tumor treatment. PMID:26497679

  19. Role of TGF-β-induced Claudin-4 expression through c-Jun signaling in non-small cell lung cancer.

    Science.gov (United States)

    Rachakonda, Girish; Vu, Trung; Jin, Lin; Samanta, Debangshu; Datta, Pran K

    2016-10-01

    Claudin-4 has been identified as an integral member of tight junctions and has been found to be upregulated in various types of cancers especially in metastatic cancers. However, the molecular mechanism of the upregulation of Claudin-4 and its role in lung tumorigenesis are unknown. The aim of the present study is to investigate the role of Claudin-4 on migration and tumorigenicity of lung cancer cells and to examine the regulatory effects of TGF-β on Claudin-4 expression. We have observed that TGF-β induces the expression of Claudin-4 dramatically in lung cell lines in a time dependent manner. TGF-β-induced Smad signaling is important for enhancing Claudin-4 mRNA level through inducing its promoter activity. Treatment with curcumin, a c-Jun inhibitor, or stable knockdown of c-Jun abrogates TGF-β-induced Claudin-4 expression suggesting an involvement of the c-Jun pathway. Notably, TGF-β-induced Claudin-4 expression through c-Jun pathway plays a role in TGF-β-mediated motility and tumorigenicity of these cells. In support of these observations, we have uncovered that Claudin-4 is upregulated in 14 of 24 (58%) lung tumors when compared with normal lung tissue. This is the first study to show how TGF-β regulates the expression of Claudin-4 through c-Jun signaling and how this pathway contributes to the migratory and tumorigenic phenotype of lung tumor cells.

  20. Epstein-Barr virus-encoded latent membrane protein 1 modulates cyclin D1 by c-Jun/Jun B heterodimers

    Institute of Scientific and Technical Information of China (English)

    SONG; Xin; TAO; Yongguang; ZENG; Liang; YANG; Jing; TANG; F

    2005-01-01

    In our recent studies, we found that LMP1 encoded by Epstein-Barr virus could accelerate the formation of active c-Jun/Jun B heterodimer. We studied the regulation of cyclinD1 by c-Jun/Jun B heterodimers by laser scanning confocal influorescence microscopy, Western blot, luciferase activity assay, super-EMSA and flow cytometry in the Tet-on-LMP1 HNE2 cell line, in which LMP1 expression was regulated by Tet-on system. c-Jun/Jun B heterodimers induced by LMP1 could up regulate cyclin D1 promoter activity and expression. Overexpression of cyclinD1 accelerated the progression of cell cycle.

  1. NPM-ALK oncogenic kinase promotes cell-cycle progression through activation of JNK/cJun signaling in anaplastic large-cell lymphoma.

    Science.gov (United States)

    Leventaki, Vasiliki; Drakos, Elias; Medeiros, L Jeffrey; Lim, Megan S; Elenitoba-Johnson, Kojo S; Claret, Francois X; Rassidakis, George Z

    2007-09-01

    Anaplastic large-cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35), resulting in aberrant expression of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). We show that in 293T and Jurkat cells, forced expression of active NPM-ALK, but not kinase-dead mutant NPM-ALK (210K>R), induced JNK and cJun phosphorylation, and this was linked to a dramatic increase in AP-1 transcriptional activity. Conversely, inhibition of ALK activity in NPM-ALK(+) ALCL cells resulted in a concentration-dependent dephosphorylation of JNK and cJun and decreased AP-1 DNA-binding. In addition, JNK physically binds NPM-ALK and is highly activated in cultured and primary NPM-ALK(+) ALCL cells. cJun phosphorylation in NPM-ALK(+) ALCL cells is mediated by JNKs, as shown by selective knocking down of JNK1 and JNK2 genes using siRNA. Inhibition of JNK activity using SP600125 decreased cJun phosphorylation and AP-1 transcriptional activity and this was associated with decreased cell proliferation and G2/M cell-cycle arrest in a dose-dependent manner. Silencing of the cJun gene by siRNA led to a decreased S-phase cell-cycle fraction associated with upregulation of p21 and downregulation of cyclin D3 and cyclin A. Taken together, these findings reveal a novel function of NPM-ALK, phosphorylation and activation of JNK and cJun, which may contribute to uncontrolled cell-cycle progression and oncogenesis.

  2. Induction of c-fos and c-jun protooncogenes expression by formaldehyde-releasing and epoxy resin-based root-canal sealers in human osteoblastic cells.

    Science.gov (United States)

    Huang, Fu-Mei; Hsieh, Yih-Shou; Tai, Kuo-Wei; Chou, Ming-Yung; Chang, Yu-Chao

    2002-03-01

    An important requirement for a root-canal sealer is biologic compatibility; most evaluations have focused on general toxicological and local tissue irritating properties. There is only scant information about mutagenicity or carcinogenicity testing for root-canal sealer. It has been shown that c-fos and c-jun are induced rapidly by a variety of chemical and physical stimuli. Numerous works have extensively investigated the induction mechanisms of c-fos and c-jun protooncogenes by these agents; however, little is known about the induction of cellular signaling events and specific gene expression after cell exposure to root-canal sealers. Therefore, we used osteoblastic cell line U2-OS to examine the effect of zinc-oxide eugenol-based (N2 and Endomethasome), epoxy resin-based (AH Plus), and calcium hydroxide-based (Sealapex) root-canal sealers on the expression of c-fos and c-jun protooncogenes to understand in more detail the molecular mechanisms of root-canal sealer-induced genotoxicity. The cytotoxicity decreased in an order of N2 > Endomethasome > AH Plus > Sealapex. In addition, N2, Endomethasome, and AH Plus rapidly induced c-jun and c-fos mRNA levels in cells. However, Sealapex did not induce c-jun and c-fos mRNA expression at detectable levels all time points. Taken together, persistent induction of c-jun and c-fos protooncogenes by formaldehyde-releasing and epoxy resin-based root-canal sealers may be distributed systemically via apex to cause some unexpected adverse effects on human beings. These data should be taken into consideration when choosing a root-canal sealer.

  3. Pregnane and Xenobiotic Receptor gene expression in liver cells is modulated by Ets-1 in synchrony with transcription factors Pax5, LEF-1 and c-jun

    Energy Technology Data Exchange (ETDEWEB)

    Kumari, Sangeeta; Saradhi, Mallampati; Rana, Manjul; Chatterjee, Swagata [Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067 (India); Aumercier, Marc [IRI, CNRS USR 3078, Université de Lille-Nord de France, Parc CNRS de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d’Ascq Cedex (France); Mukhopadhyay, Gauranga [Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067 (India); Tyagi, Rakesh K., E-mail: rktyagi@yahoo.com [Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067 (India)

    2015-01-15

    Nuclear receptor PXR is predominantly expressed in liver and intestine. Expression of PXR is observed to be dysregulated in various metabolic disorders indicating its involvement in disease development. However, information available on mechanisms of PXR self-regulation is fragmentary. The present investigation identifies some of the regulatory elements responsible for its tight regulation and low cellular expression. Here, we report that the PXR-promoter is a target for some key transcription factors like PU.1/Ets-1, Pax5, LEF-1 and c-Jun. Interestingly, we observed that PXR-promoter responsiveness to Pax5, LEF-1 and c-Jun, is considerably enhanced by Ets transcription factors (PU.1 and Ets-1). Co-transfection of cells with Ets-1, LEF-1 and c-Jun increased PXR-promoter activity by 5-fold and also induced expression of endogenous human PXR. Site-directed mutagenesis and transfection studies revealed that two Ets binding sites and two of the three LEF binding sites in the PXR-promoter are functional and have a positive effect on PXR transcription. Results suggest that expression of Ets family members, in conjunction with Pax5, LEF-1 and c-Jun, lead to coordinated up-regulation of PXR gene transcription. Insights obtained on the regulation of PXR gene have relevance in offering important cues towards normal functioning as well as development of several metabolic disorders via PXR signaling. - Highlights: • The study identified cis-regulatory elements in the nuclear receptor PXR promoter. • Several trans-acting factors modulating the PXR-promoter have been identified. • PU.1/Ets-1, Pax5, LEF-1, c-Jun, LyF-VI and NF-1 act as modulators of the PXR-promoter. • Ets-1 in conjunction with LEF-1 and c-Jun exhibit 5-fold activation of the PXR-promoter. • Insights into PXR-regulation have relevance in normal and pathological conditions.

  4. Salmonella induces SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling pathways in commercial and wild-type turkey leukocytes

    Science.gov (United States)

    Previous studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on d...

  5. Induction of apoptosis by casticin in cervical cancer cells: reactive oxygen species-dependent sustained activation of Jun N-terminal kinase

    Institute of Scientific and Technical Information of China (English)

    Fanxiang Zeng; Li Tian; Fei Liu; Jianguo Cao; Meifang Quan; Xifeng Sheng

    2012-01-01

    Casticin,a polymethoxyflavone from Fructus viticis used as an anti-inflammatory agent in Chinese traditional medicine,has been reported to have anti-cancer activity.The purpose of this study was to examine the apoptotic activity of casticin on human cervical cancer cells and its molecular mechanism.We revealed a novel mechanism by which casticin-induced apoptosis occurs and showed for the first time that the apoptosis induced by casticin is mediated through generation of reactive oxygen species (ROS) and sustained activation of c-Jun N-terminal kinase (JNK) in HeLa cells.Casticin markedly increased the levels of intracellular ROS and induced the expression of phosphorylated JNK and cJun protein.Pre-treatment with N-acetylcvsteine and SP600125 effectively attenuated induction of apoptosis by casticin in HeLa cells.Moreover,casticin induced ROS production and apoptotic cell death in other cervical cancer cell lines,such as CasKi and SiHa.Importantly,casticin did not cause generation of ROS or induction of apoptosis in normal human peripheral blood mononuclear cells and embryonic kidney epithelium 293 cells.These results suggest that ROS generation and sustained JNK activation by casticin play a role in casticin-induced apoptosis and raise the possibility that treatment with casticin might be promising as a new therapy against human cervical cancer.

  6. Predicting Virulence of Aeromonas Isolates Based-on Changes in Transcription of c-jun and c-fos in Human Tissue Culture Cells

    Science.gov (United States)

    Aims: To assess virulence of Aeromonas isolates based on the change in regulation of c-jun and c-fos in the human intestinal tissue culture cell line Caco-2. Methods and Results: Aeromonas cells were added to Caco-2 cells at approximately a one to one ratio. After 1, 2 and 3 ...

  7. Pregnane and Xenobiotic Receptor gene expression in liver cells is modulated by Ets-1 in synchrony with transcription factors Pax5, LEF-1 and c-Jun.

    Science.gov (United States)

    Kumari, Sangeeta; Saradhi, Mallampati; Rana, Manjul; Chatterjee, Swagata; Aumercier, Marc; Mukhopadhyay, Gauranga; Tyagi, Rakesh K

    2015-01-15

    Nuclear receptor PXR is predominantly expressed in liver and intestine. Expression of PXR is observed to be dysregulated in various metabolic disorders indicating its involvement in disease development. However, information available on mechanisms of PXR self-regulation is fragmentary. The present investigation identifies some of the regulatory elements responsible for its tight regulation and low cellular expression. Here, we report that the PXR-promoter is a target for some key transcription factors like PU.1/Ets-1, Pax5, LEF-1 and c-Jun. Interestingly, we observed that PXR-promoter responsiveness to Pax5, LEF-1 and c-Jun, is considerably enhanced by Ets transcription factors (PU.1 and Ets-1). Co-transfection of cells with Ets-1, LEF-1 and c-Jun increased PXR-promoter activity by 5-fold and also induced expression of endogenous human PXR. Site-directed mutagenesis and transfection studies revealed that two Ets binding sites and two of the three LEF binding sites in the PXR-promoter are functional and have a positive effect on PXR transcription. Results suggest that expression of Ets family members, in conjunction with Pax5, LEF-1 and c-Jun, lead to coordinated up-regulation of PXR gene transcription. Insights obtained on the regulation of PXR gene have relevance in offering important cues towards normal functioning as well as development of several metabolic disorders via PXR signaling.

  8. Induction of heat shock protein 70 (Hsp70 prevents neuregulin-induced demyelination by enhancing the proteasomal clearance of c-Jun

    Directory of Open Access Journals (Sweden)

    Rick T Dobrowsky

    2012-12-01

    Full Text Available Modulating molecular chaperones is emerging as an attractive approach to treat neurodegenerative diseases associated with protein aggregation, DPN (diabetic peripheral neuropathy and possibly, demyelinating neuropathies. KU-32 [N-(7-((2R,3R,4S,5R-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy-8-methyl-2-oxo-2H-chromen-3-ylacetamide] is a small molecule inhibitor of Hsp90 (heat shock protein 90 and reverses sensory deficits associated with myelinated fibre dysfunction in DPN. Additionally, KU-32 prevented the loss of myelinated internodes induced by treating myelinated SC (Schwann cell-DRG (dorsal root ganglia sensory neuron co-cultures with NRG1 (neuregulin-1 Type 1. Since KU-32 decreased NRG1-induced demyelination in an Hsp70-dependent manner, the goal of the current study was to clarify how Hsp70 may be mechanistically linked to preventing demyelination. The activation of p42/p44 MAPK (mitogen-activated protein kinase and induction of the transcription factor c-Jun serve as negative regulators of myelination. NRG1 activated MAPK, induced c-Jun expression and promoted a loss of myelin segments in DRG explants isolated from both WT (wild-type and Hsp70 KO (knockout mice. Although KU-32 did not block the activation of MAPK, it blocked c-Jun induction and protected against a loss of myelinated segments in WT mice. In contrast, KU-32 did not prevent the NRG1-dependent induction of c-Jun and loss of myelin segments in explants from Hsp70 KO mice. Overexpression of Hsp70 in myelinated DRG explants prepared from WT or Hsp70 KO mice was sufficient to block the induction of c-Jun and the loss of myelin segments induced by NRG1. Lastly, inhibiting the proteasome prevented KU-32 from decreasing c-Jun levels. Collectively, these data support that Hsp70 induction is sufficient to prevent NRG1-induced demyelination by enhancing the proteasomal degradation of c-Jun.

  9. SUMOylation of the inducible (c-Fos:c-Jun)/AP-1 transcription complex occurs on target promoters to limit transcriptional activation.

    Science.gov (United States)

    Tempé, D; Vives, E; Brockly, F; Brooks, H; De Rossi, S; Piechaczyk, M; Bossis, G

    2014-02-13

    The inducible proto-oncogenic (c-Fos:c-Jun)/AP-1 transcription complex binds 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive elements (TRE) in its target genes. It is tightly controlled at multiple levels to avoid the deleterious effects of its inappropriate activation. In particular, SUMOylation represses its transactivation capacity in transient reporter assays using constitutively expressed proteins. This led to the presumption that (c-Fos:c-Jun)/AP-1 SUMOylation would be required to turn-off transcription of its target genes, as proposed for various transcription factors. Instead, thanks to the generation of an antibody specific for SUMO-modified c-Fos, we provide here direct evidence that SUMOylated c-Fos is present on a stably integrated reporter TPA-inducible promoter at the onset of transcriptional activation and colocalizes with RNA polymerase II within chromatin. Interestingly, (c-Fos:c-Jun)/AP-1 SUMOylation limits reporter gene induction, as well as the appearance of active transcription-specific histone marks on its promoter. Moreover, non-SUMOylatable mutant (c-Fos:c-Jun)/AP-1 dimers accumulate to higher levels on their target promoter, suggesting that SUMOylation might facilitate the release of (c-Fos:c-Jun)/AP-1 from promoters. Finally, activation of GADD153, an AP-1 target gene, is also associated with a rapid increase in SUMOylation at the level of its TRE and c-Fos SUMOylation dampens its induction by TPA. Taken together, our data suggest that SUMOylation could serve to buffer transcriptional activation of AP-1 target genes.

  10. A novel function of B-cell translocation gene 1 (BTG1) in the regulation of hepatic insulin sensitivity in mice via c-Jun.

    Science.gov (United States)

    Xiao, Fei; Deng, Jiali; Yu, Junjie; Guo, Yajie; Chen, Shanghai; Guo, Feifan

    2016-01-01

    Insulin resistance is one of the major factors contributing to metabolic diseases, but the underlying mechanisms are still poorly understood. As an important cofactor, B-cell translocation gene 1 (BTG1) is involved in many physiologic processes; however, the direct effect of BTG1 on insulin sensitivity has not been described. In our study, BTG1 overexpression or knockdown improved or impaired insulin signaling in vitro, respectively. In addition, adenovirus-mediated BTG1 overexpression improved insulin sensitivity in wild-type (WT) and insulin-resistant leptin-receptor mutated (db/db) mice. In addition, transgenic BTG1-overexpressing mice were resistant to high-carbohydrate diet-induced insulin resistance. Adenovirus-mediated BTG1 knockdown consistently impaired insulin sensitivity in WT and insulin-sensitive leucine-deprived mice. Moreover, hepatic BTG1 expression was increased by leucine deprivation via the mammalian target of rapamycin/ribosomal protein S6 kinase 1 pathway. Furthermore, c-Jun expression was up-regulated by BTG1, and adenovirus-mediated c-Jun knockdown blocked BTG1-improved insulin signaling and insulin sensitivity in vitro and in vivo. Finally, BTG1 promoted c-Jun expression via stimulating c-Jun and retinoic acid receptor activities. Taken together, these results identify a novel function for BTG1 in the regulation of hepatic insulin sensitivity and provide important insights into the nutritional regulation of BTG1 expression.- Xiao, F., Deng, J., Yu, J., Guo, Y., Chen, S., Guo, F. A novel function of B-cell translocation gene 1 (BTG1) in the regulation of hepatic insulin sensitivity in mice via c-Jun.

  11. Inhibition of spinal c-Jun-NH2-terminal kinase (JNK) improves locomotor activity of spinal cord injured rats.

    Science.gov (United States)

    Martini, Alessandra C; Forner, Stefânia; Koepp, Janice; Rae, Giles Alexander

    2016-05-16

    Mitogen-activated protein kinases (MAPKs) have been implicated in central nervous system injuries, yet the roles within neurodegeneration following spinal cord injury (SCI) still remain partially elucidated. We aimed to investigate the changes in expression of the three MAPKs following SCI and the role of spinal c-jun-NH2-terminal kinase (JNK) in motor impairment following the lesion. SCI induced at the T9 level resulted in enhanced expression of phosphorylated MAPKs shortly after trauma. SCI increased spinal cord myeloperoxidase levels, indicating a local neutrophil infiltration, and elevated the number of spinal apoptotic cells. Intrathecal administration of a specific inhibitor of JNK phosphorylation, SP600125, given at 1 and 4h after SCI, reduced the p-JNK expression, the number of spinal apoptotic cells and many of the histological signs of spinal injury. Notably, restoration of locomotor performance was clearly ameliorated by SP600125 treatment. Altogether, the results demonstrate that SCI induces activation of spinal MAPKs and that JNK plays a major role in mediating the deleterious consequences of spinal injury, not only at the spinal level, but also those regarding locomotor function. Therefore, inhibition of JNK activation in the spinal cord shortly after trauma might constitute a feasible therapeutic strategy for the functional recovery from SCI. PMID:27080425

  12. Speciifc effects of c-Jun NH2-terminal kinase-interacting protein 1 in neuronal axons

    Institute of Scientific and Technical Information of China (English)

    Shu Tang; Qiang Wen; Xiao-jian Zhang; Quan-cheng Kan

    2016-01-01

    c-Jun NH2-terminal kinase (JNK)-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B (TrkB) anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of TrkB anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neuronsin vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed TrkB com-plexesin vitro andin vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of TrkB gradually increased in axon terminals. However, the distribution of TrkB reduced in axon terminals after knocking out JNK-interact-ing protein 1. In addition, there were differences in distribution of TrkB after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of TrkB in dendrites. These ifndings conifrm that JNK-inter-acting protein 1 can interact with TrkB in neuronal cells, and can regulate the transport of TrkB in axons, but not in dendrites.

  13. Metformin prevents endoplasmic reticulum stress-induced apoptosis through AMPK-PI3K-c-Jun NH2 pathway

    Science.gov (United States)

    Jung, T.W.; Lee, M.W.; Lee, Y.-J.; Kim, S.M.

    2012-01-01

    Type 2 diabetes mellitus is thought to be partially associated with endoplasmic reticulum (ER) stress toxicity on pancreatic beta cells and the result of decreased insulin synthesis and secretion. In this study, we showed that a well-known insulin sensitizer, metformin, directly protects against dysfunction and death of ER stress-induced NIT-1 cells (a mouse pancreatic beta cell line) via AMP-activated protein kinase (AMPK) and phosphatidylinositol-3 (PI3) kinase activation. We also showed that exposure of NIT-1 cells to metformin (5mM) increases cellular resistance against ER stress-induced NIT-1 cell dysfunction and death. AMPK and PI3 kinase inhibitors abolished the effect of metformin on cell function and death. Metformin-mediated protective effects on ER stress-induced apoptosis were not a result of an unfolded protein response or the induced inhibitors of apoptotic proteins. In addition, we showed that exposure of ER stressed-induced NIT-1 cells to metformin decreases the phosphorylation of c-Jun NH(2) terminal kinase (JNK). These data suggest that metformin is an important determinant of ER stress-induced apoptosis in NIT-1 cells and may have implications for ER stress-mediated pancreatic beta cell destruction via regulation of the AMPK-PI3 kinase-JNK pathway.

  14. DNA Damage, Apoptosis and C-myc, C-fos, and C-jun Overexpression Induced by Selenium in Rat Hepatocytes

    Institute of Scientific and Technical Information of China (English)

    RI-AN YU; CHENG-FENG YANG; XUE-MIN CHEN

    2006-01-01

    Objective To study the effects of selenium on DNA damage, apoptosis and c-myc, c-fos, and c-jun expression in rat hepatocytes. Methods Sodium selenite at the doses of 5, 10, and 20 μmol/kg was given to rats by i.p. and there were 5 male SD rats in each group. Hepatocellular DNA damage was detected by single cell gel electrophoresis (or comet assay).Hepatocellular apoptosis was determined by TUNEL (TdT-mediated dUTP nick end labelling) and flow cytometry. C-myc,c-fos, and c-jun expression in rat hepatocytes were assayed by Northern dot hybridization. C-myc, c-fos, and c-jun protein were detected by immunohistochemical method. Results At the doses of 5, 10, and 20 μmol/kg, DNA damage was induced by sodium selenite in rat hepatocytes and the rates of comet cells were 34.40%, 74.80%, and 91.40% respectively. Results also showed an obvious dose-response relationship between the rates of comet cells and the doses of sodium selenite (r=0.9501,P<0.01). Sodium selenite at the doses of 5, 10, and 20 μmol/kg caused c-myc, c-fos, and c-jun overexpression obviously. The positive brown-yellow signal for proteins of c-myc, c-fos, and c-jun was mainly located in the cytoplasm of hepatocytes with immunohistochemical method. TUNEL-positive cells were detected in selenium-treated rat livers. Apoptotic rates (%) of selenium-treated liver cells at the doses of 5, 10, and 20 μmol/kg were (3.72±1.76), (5.82±1.42), and (11.76±1.87) respectively, being much higher than those in the control. Besides an obvious dose-response relationship between apoptotic rates and the doses of sodium selenite (r=0.9897, P<0.01), these results displayed a close relationship between DNA damage rates and apoptotic rates, and the relative coefficient was 0.9021, P<0.01. Conclusion Selenium at 5-20 μmol/kg can induce DNA damage, apoptosis, and overexpression of c-myc, c-fos, and c-jun in rat hepatocytes.

  15. Effect of Qi-protecting powder (Huqi San) on expression of c-jun, c-fos and c-myc in diethylnitrosamine-mediated hepatocarcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Xia Li; Zheng-Ming Shi; Ping Feng; Zhao-Yang Wen; Xue-Jiang Wang

    2007-01-01

    AIM: To study the inhibitory effect of Huqi San (Qiprotecting powder) on rat prehepatocarcinoma induced by diethylinitrosamine (DEN) by analyzing the mutational activation of c-fos proto-oncogene and over-expression of c-jun and c-myc oncogenes.METHODS: A Solt-Farber two-step test model of prehepatocarcinoma was induced in rats by DEN and 2-acetylaminofluorene (AAF) to investigate the modifying effects of Huqi San on the expression of c-jun, c-fos and c-myc in DEN-mediated hepatocarcinogenesis. Huqi San was made of eight medicinal herbs containing glycoprival granules, in which each milliliter contains 0.38 g crude drugs. γ-glutamy-transpeptidase-isoenzyme (γ-GTase)was determined with histochemical methods. Level of 8-hydroxydeoxyguanosine (OHdG) formed in liver and c-jun, c-fos and c-myc proto-oncogenes were detected by immunohistochemical methods.RESULTS: The level of 8-OHdG, a mark of oxidative DNA damage, was significantly decreased in the liver of rats with prehepatocarcinoma induced by DEN who received 8 g/kg body weight or 4 g/kg body weight Huqi San before (1 wk) and after DEN exposure (4 wk). Huqi Santreated rats showed a significant decrease in number of γ-GT positive foci (P < 0.001, prevention group: 4.96 ±0.72 vs 29.46 ± 2.17; large dose therapeutic group: 7.53± 0.88 vs 29.46 ± 2.17). On the other hand, significant changes in expression of c-jun, c-fos and c-myc were found in Huqi San-treated rats.CONCLUSION: Activation of c-jun, c-fos and c-myc plays a crucial role in the pathogenesis of liver cancer.Huqi San can inhibit the over-expression of c-jun, c-fos and c-myc oncogenes and liver preneolastic lesionsinduced by DEN.

  16. The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Pozo-Yauner, Luis del, E-mail: ldelpozo@inmegen.gob.mx [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); Wall, Jonathan S. [Departments of Radiology and Medicine, The University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN (United States); González Andrade, Martín [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); Sánchez-López, Rosana [Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa Cuernavaca, Morelos C.P. 62210 (Mexico); Rodríguez-Ambriz, Sandra L. [Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, Calle CEPROBI No. 8, Col. San Isidro, Yautepec, Morelos C.P. 62731 (Mexico); Pérez Carreón, Julio I. [Instituto Nacional de Medicina Genómica, Periférico Sur No. 4809, Col. Arenal Tepepan, Delegación Tlalpan, México, D.F. C.P. 14610 (Mexico); and others

    2014-01-10

    Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.

  17. Constitutive hypophosphorylation of extracellular signal-regulated kinases-1/2 and down-regulation of c-Jun in human gastric adenocarcinoma

    International Nuclear Information System (INIS)

    Hyperphosphorylation of extracellular signal-regulated protein kinases-1/2 (ERK1/2) is known to promote cancer cell proliferation. We therefore investigated the constitutive phosphorylation levels of ERK1/2 and the expression of its downstream targets c-Fos, c-Jun, and cyclooxygenase-2 (COX-2) in biopsied human gastric cancer tissues. Results showed that ERK1/2 phosphorylation and c-Jun expression were significantly lowered in gastric cancer compared with the non-cancer adjacent tissues. The expression of c-Fos, however, was not altered while COX-2 was significantly up-regulated. To conclude, we demonstrate that hypophosphorylation of ERK1/2 may occur in gastric cancer. Such discovery may have implication in the application of pathway-directed therapy for this malignant disease

  18. β-glucan reduces exercise-induced stress through downregulation of c-Fos and c-Jun expression in the brains of exhausted rats.

    Science.gov (United States)

    Hong, Heeok; Kim, Chang-Ju; Kim, Jae-Deung; Seo, Jin-Hee

    2014-05-01

    Immediate-early genes are involved in acute stress responses in the central nervous system. β-glucan stimulates innate immune defenses, exerts an anti-tumor response and increases resistance to a wide variety of types of infection. To date, the effect of β-glucan on the expression of immediate-early genes under stressful conditions has not been elucidated. In the present study, the effects of β-glucan on the expression of the oncogenes c-Fos and c-Jun in the hypothalamus, dentate gyrus and dorsal raphe in rats following exhaustive treadmill running were investigated. Male Sprague Dawley rats were randomly divided into five groups (n=10 in each group) as follows: Control, exercise, exercise and 50 mg/kg β-glucan treatment, exercise and 100 mg/kg β-glucan treatment, and exercise and 200 mg/kg β-glucan treatment. Rats in the β-glucan‑treated groups were administered β-glucan at the respective dose once per day for seven days. Rats in the exercise groups performed treadmill running once per day for six days. On the seventh day of the experiment, the time to exhaustion in response to treadmill running was determined for the exercise groups. The expression of c-Fos and c-Jun in the hypothalamus, dorsal raphe and hippocampus was enhanced by exhaustive treadmill running. Administration of β-glucan resulted in an increase in the time to exhaustion and the suppression of the exercise-induced increment in c-Fos and c-Jun expression. In conclusion, β-glucan may exert an alleviating effect on exercise-induced stress through the suppression of c-Fos and c-Jun expression in the brains of exhausted rats. PMID:24604295

  19. Effect of normothermic liver ischemic preconditioning on the expression of apoptosis-regulating genes C-jun and Bcl-XL in rats

    Institute of Scientific and Technical Information of China (English)

    Guo-Huang Hu; Xin-Sheng Lü

    2005-01-01

    AIM: To explore the expression of apoptosis-regulatinggenes C-jun and Bcl-XL after normothermic liver ischemic preconditioning and its protective effect on hepatocytes in the rat.METHODS: Wistar rats are randomly divided into sham operation group (S group, n = 10), ischemic reperfusion group (IR group, n = 10) and ischemic preconditioning group (IP group, n = 10). After dissection of the hepatoduodenal ligament in S group, and after 30-min reperfusion in IR group and in IP group, the samples of liver tissue were taken for studying the hepatocellular apoptosis, theexpressions of C-jun mRNA, Bcl-XL mRNA and their proteins, and morphologic changes at 0, 3, 6, 20 h. Meanwhile the venous blood samples were drawn at 3, 6 and 20 h for testing ALT, AST and LDH.RESULTS: The levels of ALT, AST and LDH in IR group and IP group were significantly higher than those in S group. Hepatocellular apoptosis was significantly increased in both IR group and IP group, especially in IR group.Expressions of C-jun mRNA and protein were significantly increased in IR group compared with those in both IP group and S group, but no significant difference between IP group and S group (P>0.05). Expressions of Bcl-XL mRNA and protein in IR group and S group were not significant (P>0.05), but were significantly increased in IP group compared with those in both S group and IR group. Patch necrosis of hepatocytes because of severe injury could be seen in IR group microscopically, and the ultrastructural changes were irreversible. Meanwhile in IP group, no hepatocellular necrosis occurred, and the ultrastructural changes were reversible because of mild injury. CONCLUSION: (1) IP can protect the rat liver from normothermic IR injury by modulation of the expressionof apoptosis-regulating genes C-jun and Bcl-XL; (2) IR injury may activate the apoptosis of hepatocytes by increasing the expression of apoptosis-inducing gene C-jun; (3) IP may prohibit the apoptosis of hepatocytes by increasing the

  20. Nerve growth factor downregulates c-jun mRNA and Caspase-3 in striate cortex of rats after transient global cerebral ischemia/reperfusion

    Institute of Scientific and Technical Information of China (English)

    Dacheng Jin; Tiemin Wang; Xiubin Fang

    2006-01-01

    BACKGROUND: Immediate early gene (LEG) c-jun is a sensitive marker for functional status of nerve cells.Caspase-3 is a cysteine protease,which is a critical regulator of apoptosis. The effect of exogenous nerve growth factor (NGF) on the expression of c-jun Mrna and Caspase-3 protein in striate cortex of rats with transient global cerebral ischemia/reperfusion (IR) is unclear.OBJECTIVE: To study the protective effect of exogenous NGF on the brain of rats with transient global cerebral IR and its effecting pathway by observing the expression of c-jun Mrna and Caspase-3 protein.DESIGN: Randomized controlled animal trial.SETTING: Department of Neural Anatomy, Institute of Brain,China Medical University.MATERTALS:Eighteen healthy male SD rats of clean grade, aged 1 to 3 months, with body mass of 250 to 300 g, were involved in this study. NGF was provided by Dalian Svate Pharmaceutical Co.,Ltd, c-jun in situ hybridization detection kit, Caspase-3 antibody and SABC kit were purchased from Boster Biotechnology Co. ,Ltd.METHODS: This trial was carried out in the Department of Neural Anatomy, Institute of Brain, China Medical University during September 2003 to April 2005. ①Experimental animals were randomized into three groups with 6 in each: sham-operation group,IR group and NGF group. ②After the rats were anesthetized,the bilateral common carotid arteries and right external carotid arteries of rats were bluntly dissected and bilateral common carotid arteries were clamped for 30 minutes with bulldog clamps. Reperfusion began after buldog clamps were removed. Normal saline of 1mL and NGF (1×106 U/L) of 1 Ml was injected into the common carotid artery of rats via right external carotid arteries in the IR group and NGF group respectively.The injection was conducted within 30 minutes, and then the right external carotid arteries were ligated. In the sham-operation group, occlusion of bilateral common carotid arteries and administration of drugs were phosphate buffer

  1. Relations of transcription expression of IL-2 with nuclear factor of activated T cells as well as changes of C-Fos and C-Jun after trauma

    Institute of Scientific and Technical Information of China (English)

    罗艳; 梁华平; 胡承香; 徐祥; 王正国

    2002-01-01

    Objective: To observe the relations among expression of interleukin-2 (IL-2) in spleen lymphocytes, DNA binding activity of nuclear factor of activated T cells (NFAT) and expression of the partly family members C-Fos, C-Jun after trauma. Methods: A murine closed trauma model was used, animals were sacrificed 6, 12 hours and 1, 4, 7, 10, 14 days, respectively after injury. Spleen lymphocytes were isolated from injured mice and stimulated with concanavalin-A. The culture supernatants were harvested and assayed for IL-2 activity. Total RNA was extracted from spleen lymphocytes and assayed for IL-2 mRNA. Nuclear protein was extracted, and the DNA binding activity of NFAT was measured using an electrophoretic mobility shift assay (EMSA), the expressions of C-Fos, C-Jun protein determined by Western blot analysis. Results: The expressions of IL-2 activity and IL-2 mRNA in spleen lymphocytes were decreased in injured mice compared with those in control mice, and the most obvious decrease appeared on the 4th day after injury. The DNA binding activity of NFAT decreased gradually and reached the minimum that was only 41% of the control on the 4th day after injury, which was closely associated with the decline of IL-2 activity and IL-2 mRNA. An decrease in the expression of C-Fos on the 1st and 4th day after injury, trauma had no significant effect on the C-Jun expression.Conclusions: These results suggest that the inhibition of IL-2 expression is partly due to the impairment in the activation of NFAT in injured mice; and the decline in the DNA binding activity of NFAT is partly due to trauma block in the C-Fos expression.

  2. c-Jun NH2-terminal kinase-dependent upregulation of DR5 mediates cooperative induction of apoptosis by perifosine and TRAIL

    Directory of Open Access Journals (Sweden)

    Chen Georgia Z

    2010-12-01

    Full Text Available Abstract Background Perifosine, an alkylphospholipid tested in phase II clinical trials, modulates the extrinsic apoptotic pathway and cooperates with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL to augment apoptosis. The current study focuses on revealing the mechanisms by which perifosine enhances TRAIL-induced apoptosis. Results The combination of perifosine and TRAIL was more active than each single agent alone in inducing apoptosis of head and neck squamous cell carcinoma cells and inhibiting the growth of xenografts. Interestingly, perifosine primarily increased cell surface levels of DR5 although it elevated the expression of both DR4 and DR5. Blockade of DR5, but not DR4 upregulation, via small interfering RNA (siRNA inhibited perifosine/TRAIL-induced apoptosis. Perifosine increased phosphorylated c-Jun NH2-terminal kinase (JNK and c-Jun levels, which were paralleled with DR4 and DR5 induction. However, only DR5 upregulaiton induced by perifosine could be abrogated by both the JNK inhibitor SP600125 and JNK siRNA. The antioxidants, N-acetylcysteine and glutathione, but not vitamin C or tiron, inhibited perifosine-induced elevation of p-c-Jun, DR4 and DR5. Moreover, no increased production of reactive oxygen species was detected in perifosine-treated cells although reduced levels of intracellular GSH were measured. Conclusions DR5 induction plays a critical role in mediating perifosine/TRAIL-induced apoptosis. Perifosine induces DR5 expression through a JNK-dependent mechanism independent of reactive oxygen species.

  3. Aryl hydrocarbon receptor pathway activation enhances gastric cancer cell invasiveness likely through a c-Jun-dependent induction of matrix metalloproteinase-9

    Directory of Open Access Journals (Sweden)

    Song Xin

    2009-04-01

    Full Text Available Abstract Background Abberant aryl hydrocarbon receptor (AhR expression and AhR pathway activation are involved in gastric carcinogenesis. However, the relationship between AhR pathway activation and gastric cancer progression is still unclear. In present study, we used 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD, a classic and most potent ligand of AhR, to activate AhR pathway and investigated the effect of AhR pathway activation on human gastric cancer AGS cell invasion and explored the corresponding mechanism. Results To determine whether AhR pathway can be activated in AGS cells, we examined the expression of CYP1A1, a classic target gene of AhR pathway, following TCDD exposure. RT-PCR and western blot analysis showed that both CYP1A1 mRNA and protein expression were increased in a dose-dependent manner following TCDD treatment and AhR antagonist resveratrol (RSV could reverse this TCDD-induced CYP1A1 expression. To determine whether TCDD treatment of AGS cells results in an induction of MMP-9 expression, we detected MMP-9 mRNA using RT-PCR and detected MMP-9 enzymatic activity using gelatin zymography. The results showed that both MMP-9 mRNA expression and enzymatic activity were gradually increased with the concentration increase of TCDD in media and these changes could be reversed by RSV treatment in a dose-dependent manner. To examine whether AhR activation-induced MMP-9 expression and activity in AGS cells results in increased migration and invasion, we performed wound healing migration assay and transwell migration and invasion assay. After TCDD treatment, the migration distance and the migration and invasion abilities of AGS cells were increased with a dose-dependent manner. To demonstrate AhR activation-induced MMP-9 expression is mediated by c-Jun, siRNA transfection was performed to silence c-Jun mRNA in AGS cells. The results showed that MMP-9 mRNA expression and activity in untreated control AGS cells were very weak; After TCDD

  4. Effect of ketamine anesthesia in early pregnancy on the c-fos mRNA and c-jun mRNA expression in offsprings of rats%孕早期氯胺酮麻醉对子代大鼠海马c-fos mRNA和c-jun mRNA表达的影响

    Institute of Scientific and Technical Information of China (English)

    李钢; 赵为禄; 罗佛全

    2010-01-01

    目的 探讨孕早期氯胺酮麻醉对子代大鼠海马c-fos mRNA和c-jun mRNA表达的影响.方法 孕5~13 d的SD大鼠30只,体重250~300 g,随机分为2组(n=15):对照组(C组)和氯胺酮组(K组).K组经尾静脉注射氯胺酮20 mg/kg,随后以130 mg·kg-1·h-1的速率静脉输注2 h;C组以等量生理盐水替代氯胺酮.子代大鼠于出生后20和30 d时测定认知功能,取海马组织,测定c-fosmRNA和c-jun mRNA表达水平并观察超微结构.结果 与C组比较,K组子代大鼠出生后30 d时认知功能测定第2天逃避潜伏期延长(P<0.05),海马c-fos mRNA和c-jun mRNA的表达水平差异无统计学意义,出生后20 d上述指标差异无统计学意义(P>0.05).K组海马神经元发生损伤.结论 孕早期氯胺酮麻醉抑制子代大鼠认知功能的机制与海马神经元受损有关,但与海马c-fos mRNA和c-jun mRNA表达无关.%Objective To investigate the effect of ketamine anesthesia in the early pregnancy on the c-fos mRNA and c-jun mRNA expression in the offsprings of rats. Methods Thirty pregnant SD rats at 5-13 days of gestation were randomly divided into control group and ketamine group (n = 15 each). Ketamine 20 mg/kg was injected intravenously through tail vein followed by 2 h infusion at a rate of 130 mg·kg-1 ·h-1 in ketmine group.While the equal volume of normal saline was given instead of ketamine in control group. The learning and memory function of the offsprings were tested by Morris water maze test on postnatal day 20 and 30. The hippocampal tissues were taken to detect the expression of c-fos mRNA and c-jun mRNA and to observe the ultrastructure. Results Compared with group C, the escape latency was significantly prolonged at 2 days during the test which was performed on postnatal day 30, but there was no significant difference in the expression of c-fos mRNA and c-jun mRNA on postnatal day 20 and 30 and in the indices mentioned above on postnatal day 20 in ketamine group (P >0.05). The

  5. Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase pathway

    International Nuclear Information System (INIS)

    Highlights: ► The expression levels of Bex2 markedly increased in glioma tissues. ► Bex2 over-expression promoted cell proliferation, while its down-regulation inhibited cell growth. ► Bex2 down-regulation promoted cell apoptosis via JNK/c-Jun signaling pathway. -- Abstract: The function of Bex2, a member of the Brain Expressed X-linked gene family, in glioma is controversial and its mechanism is largely unknown. We report here that Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase (JNK) pathway. The expression level of Bex2 is markedly increased in glioma tissues. We observed that Bex2 over-expression promotes cell proliferation, while down-regulation of Bex2 inhibits cell growth. Furthermore, Bex2 down-regulation promotes cell apoptosis and activates the JNK pathway; these effects were abolished by administration of the JNK specific inhibitor, (SP600125). Thus, Bex2 may be an important player during the development of glioma.

  6. Dynamic acetylation of all lysine 4-methylated histone H3 in the mouse nucleus: analysis at c-fos and c-jun.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4-methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9-methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9-methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.

  7. Expressão dos protooncogenes c-fos, c-myc e c-jun em miométrio normal e mioma humanos Expression of the protooncogenes c-fos, c-myc and c-jun in human normal miometrium and leiomyoma

    Directory of Open Access Journals (Sweden)

    Ana Luiza Ferrari

    2006-10-01

    Full Text Available OBJETIVO: Comparar a expressão gênica (mRNA e protéica dos protooncogenes c-fos, c-myc e c-jun em miométrio normal e mioma humanos. MÉTODOS: Foi realizado um estudo do tipo caso-controle. O material foi coletado de 12 pacientes submetidas a histerectomia no Hospital de Clínicas de Porto Alegre. A expressão do mRNA específico para c-myc, c-fos, c-jun e beta-microglobulina foi avaliada pela técnica de RT-PCR, utilizando primers específicos para cada gene. A expressão protéica destes protooncogenes foi avaliada através de Western blot com anticorpos específicos. RESULTADOS: Não houve diferença significativa para expressão gênica desses protooncogenes entre miométrio normal e mioma (c-myc: 0,87 ± 0,08 vs 0,87 ± 0,08, p = 0,952; c-fos: 1,10 ± 0,17 vs 1,01 ± 0,11, p = 0,21; c-jun: 1,03 ± 0,12 vs 0,96 ± 0,09, p = 0,168, respectivamente. Não houve diferença significativa para expressão protéica desses protooncogenes entre miométrio normal e mioma (c-myc: 1,36 ± 0,48 vs 1,53 ± 0,29, p = 0,569; c-fos: 8,85 ± 5,5 vs 6,56 ± 4,22, p = 0,434; e c-jun: 6,47 ± 3,04 vs 5,42 ± 2,03, p = 0,266, respectivamente. CONCLUSÃO: A expressão gênica (transcrição e a expressão protéica (tradução dos protooncogenes c-myc, c-fos e c-jun em mioma e miométrio normal são semelhantes.Uterine myomas are common benign tumors of the female genital tract. The expression of growth factor signal transduction cascade components including the protooncogenes c-myc, c-fos, and c-jun seem to be involved in the development of myomas. PURPOSE: To compare the gene (mRNA and protein expression of the protooncogenes c-fos, c-myc, and c-jun in human normal myometrium and leiomyoma. METHOD: A case-control study was performed. Samples were collected from 12 patients submitted to hysterectomy at the Hospital de Clínicas at Porto Alegre. The expression of the specific mRNA for c-myc, c-fos, c-jun, and beta-microglobulin was assessed through the RT

  8. The activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 to the distal CCAAT box of the RhoB promoter

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Jiwon [Genome Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Department of Microbiology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Choi, Jeong-Hae; Won, Misun [Genome Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Kang, Chang-Mo [Laboratory of Cytogenetics and Tissue Regeneration, KIRAMS, Seoul 139-706 (Korea, Republic of); Gyun, Mi-Rang [Genome Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Functional Genomics, Korea University of Science and Technology, Daejeon 305-350 (Korea, Republic of); Park, Hee-Moon [Department of Microbiology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Kim, Chun-Ho, E-mail: chkim@kirams.re.kr [Laboratory of Cytogenetics and Tissue Regeneration, KIRAMS, Seoul 139-706 (Korea, Republic of); Chung, Kyung-Sook, E-mail: kschung@kribb.re.kr [Genome Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of)

    2011-06-03

    Highlights: {yields} Regulation of transcriptional activation of RhoB is still unclear. {yields} We examine the effect of p38 MAPK inhibition, and c-Jun and RhoB depletion on UV-induced RhoB expression and apoptosis. {yields} We identify the regions of RhoB promoter necessary to confer UV responsiveness using pRhoB-luciferase reporter assays. {yields} c-Jun, ATF2 and p300 are dominantly associated with NF-Y on the distal CCAAT box. {yields} The activation of p38 MAPK primarily contribute to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins on distal CCAAT box of RhoB promoter. -- Abstract: The Ras-related small GTP-binding protein RhoB is rapidly induced in response to genotoxic stresses caused by ionizing radiation. It is known that UV-induced RhoB expression results from the binding of activating transcription factor 2 (ATF2) via NF-Y to the inverted CCAAT box (-23) of the RhoB promoter. Here, we show that the association of c-Jun with the distal CCAAT box (-72) is primarily involved in UV-induced RhoB expression and p38 MAPK regulated RhoB induction through the distal CCAAT box. UV-induced RhoB expression and apoptosis were markedly attenuated by pretreatment with the p38 MAPK inhibitor. siRNA knockdown of RhoB, ATF2 and c-Jun resulted in decreased RhoB expression and eventually restored the growth of UV-irradiated Jurkat cells. In the reporter assay using luciferase under the RhoB promoter, inhibition of RhoB promoter activity by the p38 inhibitor and knockdown of c-Jun using siRNA occurred through the distal CCAAT box. Immunoprecipitation and DNA affinity protein binding assays revealed the association of c-Jun and p300 via NF-YA and the dissociation of histone deacetylase 1 (HDAC1) via c-Jun recruitment to the CCAAT boxes of the RhoB promoter. These results suggest that the activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins to the distal CCAAT box of the RhoB promoter in

  9. Effect of different therapies of Chinese medicine on the expressions of c-Fos and c-Jun proteins in hippocampus of rats with post-stroke depression

    Institute of Scientific and Technical Information of China (English)

    Hongyan Wang; Mei Chen; Binhui Zhang

    2006-01-01

    BACKGROUND: c-fos and c-jun, the important immediate early genes (IEG), are regarded as the markers for the location and function of neuronal activity, as well as the third signal messengers, they couple the stress stimulation and the gene expression in neuron, and hippocampus is involved in the process of signal transmission after stress stimulation induced depression.OBJECTIVE: To observe the therapeutic effects of Bushen Yiqi (tonifying kidney to benefit qi), Huoxue Huayu (promoting blood circulation to dissipate blood stasis) and Ditan Kaiqiao (eliminating phlegm for resuscitation) on the expressions of c-Fos and c-Jun proteins in hippocampus and spontaneous behaviors of rats with post-stroke depression (PSD), and compare the results with those of fluoxetine, which is known to have definite effect on depression.DESIGN: A randomized controlled trial.SETrING: Zhejiang College of Traditional Chinese Medicine.MATERIALS: The trial was completed in Zhejiang College of Traditional Chinese Medicine from January to July in 2003. Fifty-six healthy adult Wistar male rats of clean grade, weighing (250±50) g, were randomly divided into 7 groups with 8 rats in each group: control group, model group, forced swimming group,Bushen Yiqi group; Huoxue Huayu, Ditan Kaiqiao group and fluoxetine group. The Bushen Yiqi Tang con tained Renshen, Huangqi, Heshouwu, Gouqi, Shudi, etc., crude drugs 1 800 g/L. The Huoxue Huayu Tang contained Danshen, Chuanxiong, Chishao, Yujin, etc., crude drugs 3 600 g/L. The Dian Kaiqiao Tang contained Banxia, Danxing, Changpu, Yuanzhi, etc., crude drug 1 000 g/L.METHODS: ① Except the control group and forced swimming group, rats in the other groups were made into PSD models by deligating the bilateral common carotid arteries permanently. ② Rats in the control group, model group and forced swimming group were intragastrically perfused by saline (3 mL for each time); those in the Bushen Yiqi group, Huoxue Huayu, Ditan Kaiqiao group and fluoxetine

  10. Influence of thermalization on A549 cells growth, c-Jun N-terminal kinase phosphorylation and expression of heat shock protein 70 in patients with lung cancer%热化联合对肺癌患者A549细胞生长、c-Jun N-末端激酶磷酸化及热休克蛋白70表达的影响

    Institute of Scientific and Technical Information of China (English)

    吴海乔; 田甜; 胡君程; 林蓁

    2015-01-01

    目的:观察热化联合对肺癌患者A549细胞生长的影响及机制探讨。方法对A549细胞分别进行单独热疗、单独化疗,热化联合干预及热化联合并SP600125干预,同时选取未做任何处理的A549细胞作为对照组。观察各组细胞增殖率、细胞侵袭力的变化。同时采用蛋白免疫印记法(Western Bolt)检测JNK磷酸化以及热休克蛋白70(HSP70)的表达。结果热化联合组的A549细胞增值率明显低于单独热疗、单独化疗和热化联合并SP600125组(P<0.05)。热化联合组JNK磷酸化表达明显高于对照组及单独化疗组(P<0.05),热化联合组HSP70表达明显低于单独热疗组(P<0.05)。热化联合干预下,p-JNK表达水平出现上升,与对照组、单独热疗组和单独化疗组相比,差异均具有统计学意义(P<0.05);热化联合并SP600125组的p-JNK的表达水平较热化联合组显著下降(P<0.05)。结论热化联合抑制A549细胞增殖的效果优于单独热疗或单独化疗,作用机制可能与激活JNK信号通路或抑制HSP70表达有关。%Objective To investigate the effect of thermalization on A549 cells growth in patients with lung cancer and its mechanism. Methods A549 cells were given thermotherapy alone (group A), chemotherapy alone (group B), and thermotherapy combined with chemotherapy (group C), thermotherapy combined with chemotherapy and SP600125 intervention (group D). Untreated A549 cells were selected as the control group. The changes of cell in-vasion, proliferation rate of the cells in each group were observed. Phosphorylated JNK and expression of heat shock protein 70 (HSP70) were detected by Western blot. Results A549 cell proliferation rate of group C was significantly lower than that of group A, group B and group D (P<0.05). The expression of group C was significantly higher than that of control group and group B (P<0.05), and the expression of HSP70 in group C was significantly lower than that in group A (P<0.05). In group C, the expression level of p-JNK increased, compared with the control group, group A and group B, with statistically significant difference (P<0.05). In group D, the expression level of p-JNK decreased significantly compared with group C (P<0.05). Conclusion Thermotherapy combined with chemotherapy had better effects in inhibiting proliferation of A549 cells than thermotherapy or chemotherapy alone. The mechanism may be re-lated to the activation of JNK signal pathway or inhibiting expression of HSP70.

  11. Potentiation of the action of phorbol ester by heparin in human CNE2 cells: Association with enhanced expression of c-jun,p53 and p21%肝素增强佛波酯对人鼻咽癌CNE2细胞的作用:调c-jun,p5 3,p21的表达

    Institute of Scientific and Technical Information of China (English)

    李红良; 陈丹丹; 张海伟; 钟玲; 李小红; 罗英儒; 任先达; 司徒锐

    2002-01-01

    目的:观察肝素联合佛波酯对人鼻咽癌细胞的增殖与凋亡的影响及其可能的分子机制.方法: 用细胞记数法、流式细胞术观察肝素联合佛波酯对人鼻咽癌细胞的增殖及细胞周期的影响;而用TUNEL、琼脂糖凝胶电泳、Westernblot等方法观察肝素与佛波酯联合应用对人鼻咽癌细胞凋亡的作用.结果: 肝素与佛波酯联合应用后对鼻咽癌细胞的生长抑制及其凋亡具有显著增强的作用,同佛波酯单独应用于诱导细胞凋亡相比,低剂量的肝素与佛波酯联合应用后发现 :TUNEL阳性细胞明显增多;G1期细胞阻止,S期细胞明显减少,凋亡率增加;DNA"梯形 " 变化更加明显;c-jun,p53,p21基因表达明显升高,而c-fos在整个用药过程中没有改变. 结论: 肝素增强佛波酯对人鼻咽癌细胞的抗增殖及促凋亡,这可能与c- jun,p53,p21的表达上凋有关.%AIM: To measure the effect of addition of hep arin to TPA on cell proli feration and apoptosis in CNE2 cells and investigate the possible molecular mech anisms underlying heparin and TPA interaction on cell proliferation and apoptosi s. METHODS: Cell viability and cell cycle were determined by cel l counting and flow cytometry. Apoptosis was evaluated by terminal deoxynucleotidyl transferase -mediated dUPT nick-end labeling (TUNEL) and agarose gel electrophoresis. The ex pression of c-jun,c-fos,p21 and p53 was examined by Western blot. RESU LTS: TPA alone inhibited CNE2 cell proliferation and evoked apoptosis associated with ty pical morphological changes and DNA fragmentation,which was augmented when hepa rin was added. Compared with TPA or heparin alone,TPA plus heparin obviously en hanced the number of TUNEL-positive cells from 23%±1.2% to 51%±0.9%. After exp os ure to different concentrations of heparin (with or without TPA) for 24 h,CNE2 cells were accumulated G 0/G 1 phase. There was a decrease in the number of ce lls in S phase by the combined heparin and TPA

  12. N-terminal protein processing: A comparative proteogenomic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Bonissone, Stefano; Gupta, Nitin; Romine, Margaret F.; Bradshaw, Ralph A.; Pevzner, Pavel A.

    2013-01-01

    N-Terminal Methionine Excision (NME) is a universally conserved mechanism with the same specificity across all life forms that removes the first Methionine in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val. In spite of its necessity for proper cell functioning, the functional role of NME remains unclear. In 1988, Arfin and Bradshaw connected NME with the N-end protein degradation rule and postulated that the role of NME is to expose the stabilizing residues with the goal to resist protein degradation. While this explanation (that treats 7 stabilizing residues in the same manner) has become the de facto dogma of NME, comparative proteogenomics analysis of NME tells a different story. We suggest that the primary role of NME is to expose only two (rather than seven) amino acids Ala and Ser for post-translational modifications (e.g., acetylation) rather than to regulate protein degradation. We argue that, contrary to the existing view, NME is not crucially important for proteins with 5 other stabilizing residue at the 2nd positions that are merely bystanders (their function is not affected by NME) that become exposed to NME because their sizes are comparable or smaller than the size of Ala and Ser.

  13. c-Jun NH2-terminal kinase activity in subcutaneous adipose tissue but not nuclear factor-kappaB activity in peripheral blood mononuclear cells is an independent determinant of insulin resistance in healthy individuals

    DEFF Research Database (Denmark)

    Sourris, Karly C; Lyons, Jasmine G; de Courten, Maximilian;

    2009-01-01

    Chronic low-grade activation of the immune system (CLAIS) predicts type 2 diabetes via a decrease in insulin sensitivity. Our study investigated potential relationships between nuclear factor-kappaB (NF-kappaB) and c-Jun NH(2)-terminal kinase (JNK) pathways-two pathways proposed as the link between...

  14. Systematic analysis of BRAF(V600E) melanomas reveals a role for JNK/c-Jun pathway in adaptive resistance to drug-induced apoptosis.

    Science.gov (United States)

    Fallahi-Sichani, Mohammad; Moerke, Nathan J; Niepel, Mario; Zhang, Tinghu; Gray, Nathanael S; Sorger, Peter K

    2015-03-26

    Drugs that inhibit RAF/MEK signaling, such as vemurafenib, elicit profound but often temporary anti-tumor responses in patients with BRAF(V) (600E) melanoma. Adaptive responses to RAF/MEK inhibition occur on a timescale of hours to days, involve homeostatic responses that reactivate MAP kinase signaling and compensatory mitogenic pathways, and attenuate the anti-tumor effects of RAF/MEK inhibitors. We profile adaptive responses across a panel of melanoma cell lines using multiplex biochemical measurement, single-cell assays, and statistical modeling and show that adaptation involves at least six signaling cascades that act to reduce drug potency (IC50) and maximal effect (i.e., Emax ≪ 1). Among these cascades, we identify a role for JNK/c-Jun signaling in vemurafenib adaptation and show that RAF and JNK inhibitors synergize in cell killing. This arises because JNK inhibition prevents a subset of cells in a cycling population from becoming quiescent upon vemurafenib treatment, thereby reducing drug Emax. Our findings demonstrate the breadth and diversity of adaptive responses to RAF/MEK inhibition and a means to identify which steps in a signaling cascade are most predictive of phenotypic response.

  15. Enediyne lidamycin induces apoptosis in human multiple myeloma cells through activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase.

    Science.gov (United States)

    Zhen, Yong-Zhan; Lin, Ya-Jun; Shang, Bo-Yang; Zhen, Yong-Su

    2009-07-01

    In the present study, the effects of lidamycin (LDM), a member of the enediyne antibiotic family, on two human multiple myeloma (MM) cell lines, U266 and SKO-007, were evaluated. In MTS assay, LDM showed much more potent cytotoxicity than conventional anti-MM agents to both cell lines. The IC(50) values of LDM for the U266 and SKO-007 cells were 0.0575 +/- 0.0015 and 0.1585 +/- 0.0166 nM, respectively, much lower than those of adriamycin, dexamethasone, and vincristine. Mechanistically, LDM triggered MM cells apoptosis by increasing the levels of cleaved poly ADP-ribose polymerase (PARP) and caspase-3/7. In addition, activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) was a critical mediator in LDM-induced cell death. Inhibition of the expression of p38 MAPK and JNK by pharmacological inhibitors reversed the LDM-induced apoptosis through decreasing the level of cleaved PARP and caspase-3/7. Interestingly, phosphorylation of extracellular signal-related kinase was increased by LDM; conversely, MEK inhibitor synergistically enhanced LDM-induced cytotoxicity and apoptosis in MM cells. The results demonstrated that LDM suppresses MM cell growth through the activation of p38 MAPK and JNK, with the potential to be developed as a chemotherapeutic agent for MM. PMID:19468799

  16. Naringin Mitigates Cardiac Hypertrophy by Reducing Oxidative Stress and Inactivating c-Jun Nuclear Kinase-1 Protein in Type I Diabetes.

    Science.gov (United States)

    Adebiyi, A Olubunmi; Adebiyi, Oluwafeysetan O; Owira, Peter M O

    2016-02-01

    Cardiac hypertrophy (CH) in type 1 diabetes mellitus is attributed to increased oxidative stress-associated activation of c-Jun Nuclear Kinase (JNK). We investigated the effects of naringin on hyperglycemia-associated oxidative stress, activation of JNK-1, and CH. Male Sprague-Dawley rats (225-250 g) (n = 7) were divided into 6 groups. Groups I and II were orally treated with distilled water [3.0 mL/kg body weight/day (BW)] and naringin (50 mg/kg BW), respectively. Groups III-VI were rendered diabetic by a single intraperitoneal injection of 65 mg/kg BW of streptozotocin. Groups III, IV, and V were further treated with insulin (4.0 I.U, s.c, twice daily), naringin (50 mg/kg BW), and ramipril (3.0 mg/kg BW), respectively. After 56 days, the animals were sacrificed and then plasma and cardiac tissues obtained for further analysis. Naringin treatment of diabetic rats significantly reversed oxidative stress, lipid peroxidation, proteins oxidation, CH indices, and JNK protein activation compared with untreated diabetic animals. Our results do suggest that naringin mitigates CH by inhibiting oxidative stress leading to inactivation of JNK-1. Naringin supplements could therefore ameliorate CH in diabetic patients. PMID:26421421

  17. Antral content, secretion and peripheral metabolism of N-terminal progastrin fragments

    DEFF Research Database (Denmark)

    Goetze, Jens Peter; Hansen, Carsten Palnaes; Rehfeld, Jens F

    2006-01-01

    OBJECTIVES: In addition to the acid-stimulatory gastrins, progastrin also release N-terminal fragments. In order to examine the cellular content, secretion and peripheral metabolism of these fragments, we developed an immunoassay specific for the N-terminal sequence of human progastrin. RESULTS: ...

  18. A comparative study of fibrous dysplasia and osteofibrous dysplasia with regard to expressions of c-fos and c-jun products and bone matrix proteins: a clinicopathologic review and immunohistochemical study of c-fos, c-jun, type I collagen, osteonectin, osteopontin, and osteocalcin.

    Science.gov (United States)

    Sakamoto, A; Oda, Y; Iwamoto, Y; Tsuneyoshi, M

    1999-12-01

    Fibrous dysplasia and osteofibrous dysplasia are both benign fibro-osseous lesions of the bone and are generally seen during childhood or adolescence. Histologically, the features of these bone lesions sometimes look quite similar, but their precise nature remains controversial. We retrospectively studied clinicopathologic findings in 62 cases of fibrous dysplasia and 20 cases of osteofibrous dysplasia with regard to their anatomic location and histological appearance. From among these cases, the immunohistochemical expressions of c-fos and c-jun proto-oncogene products and bone matrix proteins of type I collagen, osteonectin, osteopontin, and osteocalcin were evaluated in 20 typical fibrous dysplasias and 17 osteofibrous dysplasias using paraffin sections, and these expressions were then assessed semiquantitatively. Microscopically, fibrous dysplasia showed various secondary changes, such as hyalinization, hemorrhage, xanthomatous reaction, and cystic change in 22 of the 62 cases (35%). This was a higher incidence than in osteofibrous dysplasia, in which only 2 of the 20 cases (10%) showed such changes. In the elderly fibrous dysplasia cases, the cellularity of fibroblast-like cells was rather low, and those cases were hyalinized. Almost all of the cases of fibrous dysplasia and osteofibrous dysplasia showed positive expressions of c-fos and c-jun products. The expressions of type I collagen and osteopontin showed no difference between fibrous dysplasia and osteofibrous dysplasia. Immunoreactivity for osteonectin in bone matrix was detected in only 1 case of fibrous dysplasia (1 of 20), whereas it was recognized in 14 of the 17 cases of osteofibrous dysplasia. Furthermore, the immunoreactivity for osteocalcin in bone matrix and fibroblast-like cells was higher in fibrous dysplasia than it was in osteofibrous dysplasia, semiquantitatively. Our immunohistochemical results regarding osteonectin and osteocalcin suggest that the bone matrix of fibrous dysplasia is

  19. Activation of the KCa3.1 channel contributes to traumatic scratch injury-induced reactive astrogliosis through the JNK/c-Jun signaling pathway.

    Science.gov (United States)

    Yi, Mengni; Dou, Fangfang; Lu, Qin; Yu, Zhihua; Chen, Hongzhuan

    2016-06-15

    Reactive astrogliosis is widely considered to contribute to pathogenic responses to stress and brain injury and to diseases as diverse as ischemia and neurodegeneration. We previously found that expression of the intermediate-conductance calcium-activated potassium channel (KCa3.1) involved in TGF-β-activated astrogliosis. In the present study, we investigated whether migration of cortical astrocytes following mechanical scratch injury involves the KCa3.1 channel, which contributes to Ca(2+)-mediated migration in other cells. We found that scratch injury increased the expression of KCa3.1 protein in reactive astrocytes. Application of the KCa3.1 blocker TRAM-34 decreased glial fibrillary acidic protein (GFAP) expression and slowed migration in a concentration-dependent manner. Application of the Ca(2+) chelators, EGTA and BAPTA-AM, also slowed the migration of astrocytes. Blockade or genetic deletion of KCa3.1 both slowed and dramatically reduced the scratch injuries induced the sharp rise in astrocytes Ca(2+) concentrations. The scratch injury-induced phosphorylation of JNK and c-Jun proteins was also attenuated both by blockade of KCa3.1 with TRAM-34 and in KCa3.1(-/-) astrocytes. Using KCa3.1 knockout mice, we further confirmed that deletion of KCa3.1 reduced expression of GFAP in an in vivo stab wound model. Taken together, our findings highlight a novel role for KCa3.1 in phenotypic modulation of reactive astrocytes and in astrocyte mobilization in response to mechanical stress, providing a potential target for therapeutic intervention in brain injuries.

  20. (-)-Epigallocatechin-3-gallate decreases thrombin/paclitaxel-induced endothelial tissue factor expression via the inhibition of c-Jun terminal NH2 kinase phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Huang-Joe [Institute of Biotechnology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan (China); Division of Cardiology, Department of Medicine, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan (China); Lo, Wan-Yu [Department of Medical Research, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan (China); Graduate Integration of Chinese and Western Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan (China); Lu, Te-Ling [School of Pharmacy, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan (China); Huang, Haimei, E-mail: hmhuang@life.nthu.edu.tw [Institute of Biotechnology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan (China)

    2010-01-01

    Patients with paclitaxel-eluting stents are concerned with stent thrombosis caused by premature discontinuation of dual antiplatelet therapy or clopidogrel resistance. This study investigates the effect of (-)-epigallocatechin-3-gallate (EGCG) on the expression of thrombin/paclitaxel-induced endothelial tissue factor (TF) expressions in human aortic endothelial cells (HAECs). EGCG was nontoxic to HAECs at 6 h up to a concentration of 25 {mu}mol/L. At a concentration of 25 {mu}mol/L, EGCG pretreatment potently inhibited both thrombin-stimulated and thrombin/paclitaxel-stimulated endothelial TF protein expression. Thrombin and thrombin/paclitaxel-induced 2.6-fold and 2.9-fold increases in TF activity compared with the control. EGCG pretreatment caused a 29% and 38% decrease in TF activity on thrombin and thrombin/paclitaxel treatment, respectively. Real-time polymerase chain reaction (PCR) showed that thrombin and thrombin/paclitaxel-induced 3.0-fold and 4.6-fold TF mRNA expressions compared with the control. EGCG pretreatment caused an 82% and 72% decrease in TF mRNA expression on thrombin and thrombin/paclitaxel treatment, respectively. The c-Jun terminal NH2 kinase (JNK) inhibitor SP600125 reduced thrombin/paclitaxel-induced TF expression. Furthermore, EGCG significantly inhibited the phosphorylation of JNK to 49% of thrombin/paclitaxel-stimulated HAECs at 60 min. Immunofluorescence assay did not show an inhibitory effect of EGCG on P65 NF-{kappa}B nuclear translocation in the thrombin/paclitaxel-stimulated endothelial cells. In conclusion, EGCG can inhibit TF expression in thrombin/paclitaxel-stimulated endothelial cells via the inhibition of JNK phosphorylation. The unique property of EGCG may be used to develop a new drug-eluting stent by co-coating EGCG and paclitaxel.

  1. Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress

    Science.gov (United States)

    Go, Y. M.; Boo, Y. C.; Park, H.; Maland, M. C.; Patel, R.; Pritchard, K. A. Jr; Fujio, Y.; Walsh, K.; Darley-Usmar, V.; Jo, H.

    2001-01-01

    Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.

  2. An apoptotic signaling pathway in the interferon antiviral response mediated by RNase L and c-Jun NH2-terminal kinase.

    Science.gov (United States)

    Li, Geqiang; Xiang, Ying; Sabapathy, Kanaga; Silverman, Robert H

    2004-01-01

    Cellular stress responses induced during viral infections are critical to the health and survival of organisms. In higher vertebrates, interferons (IFNs) mediate the innate antiviral response in part through the action of RNase L, a uniquely regulated enzyme. RNase L is activated by 5'-phosphorylated, 2'-5' oligoadenylates (2-5A) produced from IFN-inducible and double stranded RNA-dependent synthetases. We show that viral activation of the c-Jun NH2-terminal kinases (JNK) family of MAP kinases and viral induction of apoptosis are both deficient in mouse cells lacking RNase L. Also, JNK phosphorylation in response to 2-5A was greatly reduced in RNase L-/- mouse cells. In addition, 2-5A treatment of the human ovarian carcinoma cell line, Hey1b, resulted in specific ribosomal RNA cleavage products coinciding with JNK activation. Furthermore, suppression of JNK activity with the chemical inhibitor, SP600125, prevented apoptosis induced by 2-5A. In contrast, inhibition of alternative MAP kinases, p38 and ERK, failed to prevent 2-5A-mediated apoptosis. Short interfering RNA to JNK1/JNK2 mRNAs resulted in JNK ablation while also suppressing 2-5A-mediated apoptosis. Moreover, Jnk1-/- Jnk2-/- cells were highly resistant to the apoptotic effects of IFN and 2-5A. These findings suggest that JNK and RNase L function in an integrated signaling pathway during the IFN response that leads to elimination of virus-infected cells through apoptosis. PMID:14570908

  3. Function of the N-terminal segment of the RecA-dependent nuclease Ref.

    Science.gov (United States)

    Gruber, Angela J; Olsen, Tayla M; Dvorak, Rachel H; Cox, Michael M

    2015-02-18

    The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA-dependent, HNH endonuclease. It can be directed to create targeted double-strand breaks within a displacement loop formed by RecA. The 76 amino acid N-terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N-terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization and is necessary for efficient Ref-mediated DNA cleavage. Specifically, Ref N-terminal truncation variants lacking between 21 and 47 amino acids are more effective RecA-mediated targeting nucleases. We propose a more refined set of options for the Ref-mediated cleavage mechanism, featuring the N-terminal region as an anchor for at least one of the DNA strand cleavage events.

  4. Mitogen-activated protein kinases (p38 and c-Jun NH2-terminal kinase) are differentially regulated during cardiac volume and pressure overload hypertrophy.

    Science.gov (United States)

    Sopontammarak, Somkiat; Aliharoob, Assad; Ocampo, Catherina; Arcilla, Rene A; Gupta, Mahesh P; Gupta, Madhu

    2005-01-01

    Chronic pressure overload (PO) and volume overload (VO) result in morphologically and functionally distinct forms of myocardial hypertrophy. However, the molecular mechanism initiating these two types of hypertrophy is not yet understood. Data obtained from different cell types have indicated that the mitogen-activated protein kinases (MAPKs) comprising c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 play an important role in transmitting signals of stress stimuli to elicit the cellular response. We tested the hypothesis that early induction of MAPKs differs in two types of overload on the heart and associates with distinct expression of hypertrophic marker genes, namely ANF, alpha-myosin heavy chain (alpha-MHC), and beta-MHC. In rats, VO was induced by aortocaval shunt and PO by constriction of the abdominal aorta. The PO animals were further divided into two groups depending on the severity of the constriction, mild (MPO) and severe pressure overload (SPO), having 35 and 85% aortic constriction, respectively. Early changes in MAPK activity (2-120 min and 1 to 2 d) were analyzed by the in vitro kinase assay using kinase-specific antibodies for p38, JNK, and ERK2. The change in expression of hypertrophy marker genes was examined by Northern blot analysis. In VO hypertrophy, the activity of p38 was markedly increased (10-fold), without changing the activity of ERK and JNK. However, during PO hypertrophy, the activity of JNK was significantly increased (two- to sixfold) and depended on the severity of the load. The activity of p38 was not changed in MPO hypertrophy, whereas it was slightly elevated (50%) in hearts with SPO. Similarly, ERK activity was not changed in hearts with MPO, but a transient rise in activity was observed in hearts with SPO. The expression of ANF and beta-MHC genes was elevated in both PO and VO hypertrophy; however, this change was much greater in hearts subjected to PO than VO hypertrophy. Alpha

  5. Identification and functional characterization of N-terminally acetylated proteins in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Sandra Goetze

    2009-11-01

    Full Text Available Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (XPX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (XPX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species.

  6. Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

    Energy Technology Data Exchange (ETDEWEB)

    Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Cheng, Tian-Lu [Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Lin, Shinne-Ren [Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, Long-Sen, E-mail: lschang@mail.nsysu.edu.tw [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China)

    2015-04-01

    Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.

  7. Rs6295 promoter variants of the serotonin type 1A receptor are differentially activated by c-Jun in vitro and correlate to transcript levels in human epileptic brain tissue.

    Science.gov (United States)

    Pernhorst, Katharina; van Loo, Karen M J; von Lehe, Marec; Priebe, Lutz; Cichon, Sven; Herms, Stefan; Hoffmann, Per; Helmstaedter, Christoph; Sander, Thomas; Schoch, Susanne; Becker, Albert J

    2013-03-01

    Many brain disorders, including epilepsy, migraine and depression, manifest with episodic symptoms that may last for various time intervals. Transient alterations of neuronal function such as related to serotonin homeostasis generally underlie this phenomenon. Several nucleotide polymorphisms (SNPs) in gene promoters associated with these diseases have been described. For obvious reasons, their regulatory roles on gene expression particularly in human brain tissue remain largely enigmatic. The rs6295 G-/C-allelic variant is located in the promoter region of the human HTR1a gene, encoding the G-protein-coupled receptor for 5-hydroxytryptamine (5HT1AR). In addition to reported transcriptional repressor binding, our bioinformatic analyses predicted a reduced binding affinity of the transcription factor (TF) c-Jun for the G-allele. In vitro luciferase transfection assays revealed c-Jun to (a) activate the rs6295 C- significantly stronger than the G-allelic variant and (b) antagonize efficiently the repressive effect of Hes5 on the promoter. The G-allele of rs6295 is known to be associated with aspects of major depression and migraine. In order to address a potential role of rs6295 variants in human brain tissue, we have isolated DNA and mRNA from fresh frozen hippocampal tissue of pharmacoresistant temporal lobe epilepsy (TLE) patients (n=140) after epilepsy surgery for seizure control. We carried out SNP genotyping studies and mRNA analyses in order to determine HTR1a mRNA expression in human hippocampal samples stratified according to the rs6295 allelic variant. The mRNA expression of HTR1a was significantly more abundant in hippocampal mRNA of TLE patients homozygous for the rs6295 C-allele as compared to those with the GG-genotype. These data may point to a novel, i.e., rs6295 allelic variant and c-Jun dependent transcriptional 5HT1AR 'receptoropathy'. PMID:23333373

  8. N-terminal acetylation inhibits protein targeting to the endoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Gabriella M A Forte

    2011-05-01

    Full Text Available Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%-80% of proteins reportedly altered in this way. Here we report a systematic analysis of the predicted N-terminal processing of cytosolic proteins versus those destined to be sorted to the secretory pathway. While cytosolic proteins were profoundly biased in favour of processing, we found an equal and opposite bias against such modification for secretory proteins. Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery. Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species.

  9. Secretin N-terminal hexapeptide potentiates insulin release in mouse islets

    DEFF Research Database (Denmark)

    Kofod, Hans

    1986-01-01

    Peptides representing the N-terminal part of secretin were synthesized and their effects tested on column-perifused isolated mouse pancreatic islets. Insulin release induced by D-glucose was potentiated by the two peptides His-Ser-Asp-Gly-Thr-Phe-OMe (S1-6) and Ser-Asp-Gly-Thr-Phe-OMe (S2......-6). The consecutive smaller N-terminal peptides Asp-Gly-Thr-Phe-OMe (S3-6) and Gly-Thr-Phe-OMe (S4-6) had no effects while the dipeptide ester Thr-Phe-OMe (S5-6) also potentiated the release of insulin. The results suggest that the N-terminal part of secretin may be involved in the marked in vitro glucose......-dependent insulin release induced by secretin....

  10. Role of C-Jun N-terminal kinase signal pathway in amygdala kindled rats%c-Jun氨基末端激酶信号通路在大鼠杏仁核电点燃癫痫模型的发病机制中的作用

    Institute of Scientific and Technical Information of China (English)

    刘平; 刘红朝; 李学慧; 王宝峰; 卢俊章; 吴俊; 陈旭; 舒凯

    2014-01-01

    目的 观察大鼠杏仁核电点燃癫痫模型海马区的JNK水平及病理变化.方法 将健康雄性wistar大鼠,随机分为空白对照组、手术对照组和点燃组.10次癫痫发作后灌注取脑,Western blot法检测JNK的表达,进行胶原纤维酸性蛋白(GFAP)和尼氏染色,各组间进行比较.结果 Western blot显示点燃组海马区的JNK磷酸化水平(0.537±0.050)较空白组(0.379±0.050)和手术对照组(0.387±0.043)显著增高(P<0.05),总JNK水平各组之间差异无统计学意义(P>0.05);点燃组GFAP阳性细胞计数(68.12±5.36)较空白组(39.83±3.90)和手术对照组(40.26±4.51)显著增加(P<0.05);尼氏染色阳性细胞计数点燃组(19.14±3.87)较空白对照组(43.15±5.62)手术对照组(42.91±4.25)显著减少(P<0.05).结论 JNK信号通路可能参与颞叶癫痫海马硬化形成,表现为磷酸化JNK水平的升高.

  11. 大鼠脑挫裂伤后c-jun和c-fos基因快速反应的特点%Quick response of genes c-jun and c-fos after brain injury in rats

    Institute of Scientific and Technical Information of China (English)

    薄爱华; 薛贵平; 张辉; 董建峰; 邢立强; 米坤龙

    2004-01-01

    BACKGROUND: The importance of gene expressions of c-jun and c-fos has been widely recognized. However, there are very few reports on the characters of the expressions of these two genes after brain contusion.OBJECTIVE: To explore the characters of gene expressions of c-jun and c-fos.DESIGN: Randomized case control study.SETTING and MATERIALS: Experimental location: Experimental Centre of Zhangjiakou Medical College. Forty-five adult Wistar rats, with a boby mass about 200 g to 310 g.INTERVENTIONS: Forty-five rats were randomly divided into model group, sham operation group and normal control group. No treatment to normal group. The skulls were opened without strike for sham operation group. Free falling body method was used to make the brain injury model for model group. Rats were executed in 0. 5, 1, 2, 4, 8, 24 and 72 hours. Brain tissues were taken out to make paraffin sections and dyed by PV6000 immunohistochemistry.MAIN OUTCOME MEASURES: The similarities and differences of gene expressions of c-jun and c-fos.RESULTS: There was no positive expression of c-fos in the normal and sham group. However, there was weakly positive of c-jun. In the model group, the positive neurons distributed in the 1.0 - 2.0 mm cortex area surrounding the focus and the hippocampus of the same side. c-jun positive neurons were widely found in the cortex of injury side and hippocampus. The color of c-jun positive cells was dark, especially in hippocampus which c-jun positive cells concentrated. There was hardly expression of c-fos in 72 hours, but there were obvious positive neurons of c-jun existed.CONCLUSION: The gene expression of c-jun in the same side of brain injury is much stronger and more extensive than that of c-fos. The expressive time is much longer.%背景:目前关于c-jun和c-fos基因表达的重要性已逐渐被人们认识,而关于脑挫伤后两种基因表达的特点报道较少.目的:探讨脑挫裂伤后c-jun和c-fos基因表达的特点.设计:随机

  12. Effect of Serum from Overfatigue Rats on JNK/c-Jun/HO-1 Pathway in Human Umbilical Vein Endothelial Cells and the Intervening Effect of Tongxinluo(通心络)Superfine Powder

    Institute of Scientific and Technical Information of China (English)

    梁俊清; 徐海波; 吴以岭; 孙士然; 贾振华; 魏聪; 游佳华

    2009-01-01

    Objective:To cultivate human umbilical vein endothelial cells (HUVECs) in the serum of overfatigue rats with the intervention of Tongxinluo (通心络) superfine powder (TXLSP).By examining the variation of the activity of JNK/c-Jun/HO-1 pathway,the possible mechanisms of vascular endothelial dysfunction under overfatigue conditions and the intervening effect of TXLSP were explored.Methods:The HUVECs were randomly divided into the normal control group,the model group,the SP600125 (a specific antagonist of JNK)...

  13. Expression and Biochemical Characterization of the Human Enzyme N-Terminal Asparagine Amidohydrolase (hNTAN1)

    OpenAIRE

    Cantor, Jason R.; Stone, Everett M.; Georgiou, George

    2011-01-01

    The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein we report the bacterial expression, purification, and biochemical characterization of the human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl L-Asn, but fails to de...

  14. Growth hormone induces expression of c-jun and jun B oncogenes and employs a protein kinase C signal transduction pathway for the induction of c-fos oncogene expression.

    Science.gov (United States)

    Slootweg, M C; de Groot, R P; Herrmann-Erlee, M P; Koornneef, I; Kruijer, W; Kramer, Y M

    1991-04-01

    Although the structure of several members of the GH receptor family has been defined, signal transduction following GH binding to its receptor has not been elucidated. Mouse osteoblasts were used to study the effect of GH on immediate early gene expression and, subsequently, the cellular signal(s) mediating this expression were analysed. GH rapidly and transiently induced the expression of c-jun and jun B in concert with the already reported expression of c-fos. The GH-induced expression of c-fos was completely blocked by the protein kinase inhibitors staurosporine and H7, indicating that the action of GH is mediated by one or several protein kinases. We next analysed the identity of the putative protein kinases in more detail by using a more specific protein kinase inhibitor, namely the ether-lipid 1-O-alkyl-2-O-methylglycerol, understood to be an inhibitor of protein kinase C (PKC). Data obtained from these studies revealed that GH-induced expression of c-fos is mediated by PKC. In addition, we observed a profound increase in formation of the PKC activator diacyglycerol upon addition of GH, a natural activator of PKC. In conclusion, upon binding of GH to mouse osteoblasts, the receptor-mediated cellular signal involves diacyglycerol formation and activation of PKC, leading to the induction of oncogene expression. Finally, the expression of c-fos, c-jun and jun B results in an increased binding of protein complexes to AP-1 binding sites.

  15. NF-kB and c-Jun induce the expression of the oncogenic miR-221 and miR-222 in prostate carcinoma and glioblastoma cells

    Science.gov (United States)

    Galardi, Silvia; Mercatelli, Neri; Farace, Maria G.; Ciafrè, Silvia A.

    2011-01-01

    MicroRNAs (miRNAs) are potent negative regulators of gene expression involved in all aspects of cell biology. They finely modulate virtually all physiological pathways in metazoans, and are deeply implicated in all main pathologies, among which cancer. Mir-221 and miR-222, two closely related miRNAs encoded in cluster from a genomic region on chromosome X, are strongly upregulated in several forms of human tumours. In this work, we report that the ectopic modulation of NF-kB modifies miR-221/222 expression in prostate carcinoma and glioblastoma cell lines, where we had previously shown their oncogenic activity. We identify two separate distal regions upstream of miR-221/222 promoter which are bound by the NF-kB subunit p65 and drive efficient transcription in luciferase reporter assays; consistently, the site-directed mutagenesis disrupting p65 binding sites or the ectopical inhibition of NF-kB activity significantly reduce luciferase activity. In the most distal enhancer region, we also define a binding site for c-Jun, and we show that the binding of this factor cooperates with that of p65, fully accounting for the observed upregulation of miR-221/222. Thus our work uncovers an additional mechanism through which NF-kB and c-Jun, two transcription factors deeply involved in cancer onset and progression, contribute to oncogenesis, by inducing miR-221/222 transcription. PMID:21245048

  16. NF-kB and c-Jun induce the expression of the oncogenic miR-221 and miR-222 in prostate carcinoma and glioblastoma cells.

    Science.gov (United States)

    Galardi, Silvia; Mercatelli, Neri; Farace, Maria G; Ciafrè, Silvia A

    2011-05-01

    MicroRNAs (miRNAs) are potent negative regulators of gene expression involved in all aspects of cell biology. They finely modulate virtually all physiological pathways in metazoans, and are deeply implicated in all main pathologies, among which cancer. Mir-221 and miR-222, two closely related miRNAs encoded in cluster from a genomic region on chromosome X, are strongly upregulated in several forms of human tumours. In this work, we report that the ectopic modulation of NF-kB modifies miR-221/222 expression in prostate carcinoma and glioblastoma cell lines, where we had previously shown their oncogenic activity. We identify two separate distal regions upstream of miR-221/222 promoter which are bound by the NF-kB subunit p65 and drive efficient transcription in luciferase reporter assays; consistently, the site-directed mutagenesis disrupting p65 binding sites or the ectopical inhibition of NF-kB activity significantly reduce luciferase activity. In the most distal enhancer region, we also define a binding site for c-Jun, and we show that the binding of this factor cooperates with that of p65, fully accounting for the observed upregulation of miR-221/222. Thus our work uncovers an additional mechanism through which NF-kB and c-Jun, two transcription factors deeply involved in cancer onset and progression, contribute to oncogenesis, by inducing miR-221/222 transcription. PMID:21245048

  17. H-ras transfection of the rat kidney cell line NRK-52E results in increased induction of c-fos, c-jun and hsp70 following sulofenur treatment.

    Science.gov (United States)

    Gu, H; Smith, M W; Phelps, P C; Berezesky, I K; Merriman, R L; Boder, G B; Trump, B F

    1996-09-10

    The effect of the antineoplastic drug sulofenur on the induction of the immediate-early genes (IEG) c-fos and c-jun and the stress gene hsp70 was compared in the rat kidney epithelial-like cell line NRK-52E and a derivative H-ras-transfected (H/1.2NRK-52E) cell line. Fold induction for each gene after sulofenur (500 microM) treatment was greater in H/1.2NRK-52E. The maximum increases for NRK-2E and H/1.2NRK-52E were as follows: c-fos, approximately 10-fold and approximately 18-fold; c-jun, approximately 2.5-fold and approximately 3.6-fold; hsp70, approximately 13-fold and approximately 30-fold. In cells loaded with EGTA/AM or treated in low or no Ca2+ HBSS, c-fos induction was reduced similarly in both cell types. However, inhibition of protein kinases with staurosporin and calphostin C reduced c-fos by 80% in NRK-52E but by only 10-20% in H/1.2NRK.52E. These results indicate that sulofenur-induced IEG elevation is Ca(2+)-dependent and that the requirement for protein kinase C activation is bypassed in H-ras-transfected cells.

  18. Localization of the N-terminal domain of cauliflower mosaic virus coat protein precursor

    International Nuclear Information System (INIS)

    Cauliflower mosaic virus (CaMV) open reading frame (ORF) IV encodes a coat protein precursor (pre-CP) harboring an N-terminal extension that is cleaved off by the CaMV-encoded protease. In transfected cells, pre-CP is present in the cytoplasm, while the processed form (p44) of CP is targeted to the nucleus, suggesting that the N-terminal extension might be involved in keeping the pre-CP in the cytoplasm for viral assembly. This study reports for the first time the intracellular localization of the N-terminal extension during CaMV infection in Brassica rapa. Immunogold-labeling electron microscopy using polyclonal antibodies directed to the N-terminal extension of the pre-CP revealed that this region is closely associated with viral particles present in small aggregates, which we called small bodies, adjacent to the main inclusion bodies typical of CaMV infection. Based on these results, we propose a model for viral assembly of CaMV

  19. Supramolecular hydrogelators of N-terminated dipeptides selectively inhibit cancer cells.

    Science.gov (United States)

    Kuang, Yi; Gao, Yuan; Xu, Bing

    2011-12-21

    Consisting of N-terminated diphenylalanine, a new type of supramolecular hydrogelators forms hydrogels within a narrow pH window (pH 5.0 to 6.0) and selectively inhibits growth of HeLa cells, which provides important and useful insights for designing molecular nanofibers as potential nanomedicines. PMID:22037699

  20. Supramolecular hydrogelators of N-terminated dipeptides selectively inhibit cancer cells

    OpenAIRE

    Kuang, Yi; Gao, Yuan; Xu, Bing

    2011-01-01

    Consisting of N-terminated diphenylalanine, a new type of supramolecular hydrogelators forms hydrogels within a narrow pH window (pH 5.0 to 6.0) and selectively inhibits growth of HeLa cells, which provides important and useful insights for designing molecular nanofibers as potential nanomedicines.

  1. Selecting protein N-terminal peptides by combined fractional diagonal chromatography.

    Science.gov (United States)

    Staes, An; Impens, Francis; Van Damme, Petra; Ruttens, Bart; Goethals, Marc; Demol, Hans; Timmerman, Evy; Vandekerckhove, Joël; Gevaert, Kris

    2011-07-14

    In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.

  2. 高+Gx环境对猴咬肌肌细胞c-jun表达影响的研究%Effect of high +Gx on c-jun expression in masseter muscle cell of Rhesus macaque

    Institute of Scientific and Technical Information of China (English)

    施生根; 张建中; 汤楚华; 牛忠英; 张铭

    2008-01-01

    目的 观察高+Gx环境对猴咬肌组织病理学及c-jun表达的影响. 方法以9只雄性猕猴为对象,按受试猴暴露的+Gx环境及持续时间随机分4组,对照组为+1 Gx/300 s;实验组为3组,分别为+15 Gx/200 s、+18 Gx/165 s、+21 Gx/140 s.采用病理学和免疫组织化学方法,观察模拟高正加速度环境下猴咬肌肌细胞组织及c-jun表达的变化. 结果组织病理学观察:对照组猴咬肌肌细胞无明显变化;实验组肌细胞结构趋紊乱,偶见少量间质出血.免疫组织化学观察:对照组咬肌肌细胞c-jun呈阴性或弱阳性表达,主要位于肌细胞核,胞质着色不明显;实验组咬肌细胞核c-jun着色明显,呈强阳性表达,不同高+Gx暴露组之间无明显差别. 结论高于+15 Gx的模拟环境可引起猴咬肌肌细胞c-jun表达增强.%Objective To investigate the changes of c-jun expression in the monkey masseter muscles induced by stress reaction under +Gx loads. Methods Nine male Rhesus macaques were randomly divided in to four groups according to Gx loads and duration: control group was exposed to+1 Gx/300 s overloads, and experimental groups 1, 2 and 3 were exposed to +15 Gx/200 s,+18 Gx/165 s and + 21 Gx/140 s respectively. Masseter tissue was fixed with 10% buffered formaldehyde and embedded with paraffin, and histopathological changes were observed under microscope. The expression of c-jun was detected by immunohistochemical PicTureTM method on the masseters. Results Gross and histological analyses showed that no significant pathological changes were observed in the masseters in control group; there was structural disturbance of masseter with slight stromal hemorrhage in the different experimental groups. Immunohistochemical staining demonstrated that over-expression of c-jun was detected in the nuclei of the muscles in the experimental groups, and negative staining was also observed in the nuclei of muscles in controlanimals. There was no obvious difference in c-jun

  3. Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

    Energy Technology Data Exchange (ETDEWEB)

    Couto, Sheila G. [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Grupo de Biofisica e Fisica Aplicada a Medicina, Instituto de Fisica, Universidade Federal de Goias, Campus Samambaia, C.P. 131, 74001-970, Goiania, GO (Brazil); Cristina Nonato, M. [Laboratorio de Cristalografia de Proteinas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe S/N, 14040-903, Ribeirao Preto, SP (Brazil); Costa-Filho, Antonio J., E-mail: ajcosta@ffclrp.usp.br [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Departamento de Fisica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Av. Bandeirantes 3900, 14040-901, Ribeirao Preto, SP (Brazil)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

  4. N-terminal pro-C-type natriuretic peptide in serum associated with bone destruction in patients with multiple myeloma

    DEFF Research Database (Denmark)

    Mylin, Anne K; Gøtze, Jens P.; Heickendorff, Lene;

    2015-01-01

    AIM: To examine whether N-terminal proCNP concentrations in serum is associated with bone destruction in patients with multiple myeloma. MATERIALS & METHODS: N-terminal proCNP and biochemical bone markers were measured in 153 patients. Radiographic bone disease and skeletal-related events were ev...

  5. Effect of estrogen and tamoxifen on the expression pattern of AP-1 factors in MCF-7 cells: role of c-Jun, c-Fos, and Fra-1 in cell cycle regulation.

    Science.gov (United States)

    Babu, R L; Naveen Kumar, M; Patil, Rajeshwari H; Devaraju, K S; Ramesh, Govindarajan T; Sharma, S Chidananda

    2013-08-01

    The activated transcription factor ERα plays an important role in the breast development and progression of cancer. In a non-classical pathway ER interacts with other transcription factors AP-1, NFkB, SP1, etc. AP-1 transcription factors control rapid responses of mammalian cells to stimuli that impact proliferation, differentiation, and transformation. AP-1 factors are leucine zipper proteins belonging to members of the Jun family (c-Jun, JunB, and JunD) and Fos family (c-Fos, FosB, Fra-1, and Fra-2) proteins. Although AP-1 factors are well characterized, not much is known about the expression pattern of the AP-1 factors in breast cancer cells. Hence to determine which AP-1 factors are expressed and regulated by estrogen, we used human breast cancer MCF-7 cells as in vitro model system. The MCF-7 cells were treated with or without estradiol-17β (E2) or antiestrogen tamoxifen (TMX) and the cell proliferation and viability was assessed by MTT assay. The expression of different AP-1 factors was analyzed by semi-quantitative RT-PCR. The cells treated with E2 found to increase the cell proliferation by more than 35 % and TMX an antiestrogen decreased by 29 % compared to control. The E2 found to induce the expression of c-Jun, Fra-1, and c-Fos, while TMX decreased the expression. In addition TMX also decreased the mRNA levels of Jun-D and Fra-2. These results suggest that the AP-1 factors c-Jun, c-Fos, and Fra-1 may be involved in the proliferation and transformation of MCF-7 cells. E2 also found to induce cyclin D1 and cyclin E1 mRNA transcripts of cell cycle regulators while TMX significantly decreased compared to control. Further E2 induced the anti-apoptotic Bcl-2 and TMX decreased mRNA transcripts. The data presented here support the E2-ERα-mediated MCF-7 cell proliferation and confirms the role of AP-1 factors in cell cycle regulation. PMID:23625206

  6. Roles of PI3K/Akt and c-Jun signaling pathways in human papillomavirus type 16 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression and in vitro angiogenesis in non-small cell lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Erying Zhang

    Full Text Available Human papillomavirus (HPV-16 infection may be related to non-smoking associated lung cancer. Our previous studies have found that HPV-16 oncoproteins promoted angiogenesis via enhancing hypoxia-inducible factor-1α (HIF-1α, vascular endothelial growth factor (VEGF, and interleukin-8 (IL-8 expression in non-small cell lung cancer (NSCLC cells. In this study, we further investigated the roles of PI3K/Akt and c-Jun signaling pathways in it.Human NSCLC cell lines, A549 and NCI-H460, were stably transfected with pEGFP-16 E6 or E7 plasmids. Western blotting was performed to analyze the expression of HIF-1α, p-Akt, p-P70S6K, p-P85S6K, p-mTOR, p-JNK, and p-c-Jun proteins. VEGF and IL-8 protein secretion and mRNA levels were determined by ELISA and Real-time PCR, respectively. The in vitro angiogenesis was observed by human umbilical vein endothelial cells (HUVECs tube formation assay. Co-immunoprecipitation was performed to analyze the interaction between c-Jun and HIF-1α.HPV-16 E6 and E7 oncoproteins promoted the activation of Akt, P70S6K, P85S6K, mTOR, JNK, and c-Jun. LY294002, a PI3K inhibitor, inhibited HPV-16 oncoprotein-induced activation of Akt, P70S6K, and P85S6K, expression of HIF-1α, VEGF, and IL-8, and in vitro angiogenesis. c-Jun knockdown by specific siRNA abolished HPV-16 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression and in vitro angiogenesis. Additionally, HPV-16 oncoproteins promoted HIF-1α protein stability via blocking proteasome degradation pathway, but c-Jun knockdown abrogated this effect. Furthermore, HPV-16 oncoproteins increased the quantity of c-Jun binding to HIF-1α.PI3K/Akt signaling pathway and c-Jun are involved in HPV-16 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression and in vitro angiogenesis. Moreover, HPV-16 oncoproteins promoted HIF-1α protein stability possibly through enhancing the interaction between c-Jun and HIF-1α, thus making a contribution to angiogenesis in NSCLC cells.

  7. Ibrutinib inhibits SDF1/CXCR4 mediated migration in AML

    OpenAIRE

    Zaitseva, Lyubov; Murray, Megan Y; Shafat, Manar S.; Lawes, Matthew J.; MacEwan, David J.; Bowles, Kristian M.; Rushworth, Stuart A.

    2014-01-01

    Pharmacological targeting of BTK using ibrutinib has recently shown encouraging clinical activity in a range of lymphoid malignancies. Recently we reported that ibrutinib inhibits human acute myeloid leukemia (AML) blast proliferation and leukemic cell adhesion to the surrounding bone marrow stroma cells. Here we report that in human AML ibrutinib, in addition, functions to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable in vivo. It has previously been shown that SDF1/C...

  8. Plasma N-terminal pro-B-type natriuretic peptide and mortality in type 2 diabetes

    DEFF Research Database (Denmark)

    Tarnow, L; Gall, M-A; Hansen, B V;

    2006-01-01

    AIMS/HYPOTHESIS: Raised N-terminal pro-B-type natriuretic peptide (NT-proBNP) is associated with a poor cardiac outcome in non-diabetic populations. Elevated NT-proBNP predicts excess morbidity and mortality in diabetic patients with an elevated urinary albumin excretion rate. This study investig......AIMS/HYPOTHESIS: Raised N-terminal pro-B-type natriuretic peptide (NT-proBNP) is associated with a poor cardiac outcome in non-diabetic populations. Elevated NT-proBNP predicts excess morbidity and mortality in diabetic patients with an elevated urinary albumin excretion rate. This study...... investigated the prognostic value of NT-proBNP in a cohort of type 2 diabetic patients. SUBJECTS, MATERIALS AND METHODS: In a prospective observational follow-up study, 315 type 2 diabetic patients with normoalbuminuria (n=188), microalbuminuria (n=80) and macroalbuminuria (n=47) at baseline were followed...

  9. PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

    Science.gov (United States)

    Sur, Surojit; Qiao, Yuan; Fries, Anja; O'Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

    2015-12-01

    Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics.

  10. N-terminal-pro-B-type natriuretic peptide during pharmacological heart rate reduction in hyperthyroidism

    DEFF Research Database (Denmark)

    Schultz, M; Kistorp, C; Corell, P;

    2009-01-01

    ; resting heart rate [from mean 97 to 80 beats per min (17.5%), p...-index decreased from median 319 to 315 arbitrary units (p=0.039) and free triiodothyronine-index increased from 8.6 to 9.9 arbitrary units (p=0.010). No changes in echocardiographic parameters were observed. A decrease in resting heart rate in untreated hyperthyroidism due to verapamil treatment did not result......We hypothesized that elevated N-terminal-pro-B-type natriuretic peptide levels in hyperthyroidism are mainly driven by increased metabolism due to excess thyroid hormones. Therefore, serum levels of N-terminal-pro-B-type natriuretic peptide were studied during reduced cardiac work load by means...

  11. Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.; Moore, Ronald J.; Camp, David G.; Baker, Scott E.; Smith, Richard D.; Qian, Weijun

    2013-06-17

    Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significant improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.

  12. Label-Free Quantitative Proteomics and N-terminal Analysis of Human Metastatic Lung Cancer Cells

    OpenAIRE

    Min, Hophil; Han, Dohyun; Kim, Yikwon; Cho, Jee Yeon; Jin, Jonghwa; Kim, Youngsoo

    2014-01-01

    Proteomic analysis is helpful in identifying cancer-associated proteins that are differentially expressed and fragmented that can be annotated as dysregulated networks and pathways during metastasis. To examine meta-static process in lung cancer, we performed a proteomics study by label-free quantitative analysis and N-terminal analysis in 2 human non-small-cell lung cancer cell lines with disparate metastatic potentials—NCI-H1703 (primary cell, stage I) and NCI-H1755 (metastatic cell, stage ...

  13. The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.

    Science.gov (United States)

    Absmeier, Eva; Wollenhaupt, Jan; Mozaffari-Jovin, Sina; Becke, Christian; Lee, Chung-Tien; Preussner, Marco; Heyd, Florian; Urlaub, Henning; Lührmann, Reinhard; Santos, Karine F; Wahl, Markus C

    2015-12-15

    The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation. PMID:26637280

  14. Receptor binding and adenylate cyclase activities of glucagon analogues modified in the N-terminal region

    Energy Technology Data Exchange (ETDEWEB)

    McKee, R.L.; Pelton, J.T.; Trivedi, D.; Johnson, D.G.; Coy, D.H.; Sueiras-Diaz, J.; Hruby, V.J.

    1986-04-08

    In this study, we determined the ability of four N-terminally modified derivatives of glucagon, (3-Me-His1,Arg12)-, (Phe1,Arg12)-, (D-Ala4,Arg12)-, and (D-Phe4)glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, (3-Me-His1,Arg12)glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. (Phe1,Arg12)glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. (D-Ala4,Arg12)glucagon and (D-Phe4)glucagon, analogues designed to examine the possible importance of a beta-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. (D-Ala4,Arg12)glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the (D-Phe4) derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal beta-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists.

  15. N-terminal Pro-B-type natriuretic peptide: a measure of significant patent cuctus arteriosus

    LENUS (Irish Health Repository)

    OFarombi-Oghuvbu, IO

    2008-01-24

    Background: B type natriuretic peptide (BNP) is a marker for ventricular dysfunction secreted as a pre-prohormone, Pro-B-type natriuretic peptide (ProBNP), and cleaved into BNP and a biologically inactive fragment, N-terminal pro-B-type natriuretic peptide (NT-proBNP). Little is known about the clinical usefulness of NT-proBNP in preterm infants.\\r\

  16. Expanding the Phenotype Associated with NAA10-Related N-Terminal Acetylation Deficiency.

    Science.gov (United States)

    Saunier, Chloé; Støve, Svein Isungset; Popp, Bernt; Gérard, Bénédicte; Blenski, Marina; AhMew, Nicholas; de Bie, Charlotte; Goldenberg, Paula; Isidor, Bertrand; Keren, Boris; Leheup, Bruno; Lampert, Laetitia; Mignot, Cyril; Tezcan, Kamer; Mancini, Grazia M S; Nava, Caroline; Wasserstein, Melissa; Bruel, Ange-Line; Thevenon, Julien; Masurel, Alice; Duffourd, Yannis; Kuentz, Paul; Huet, Frédéric; Rivière, Jean-Baptiste; van Slegtenhorst, Marjon; Faivre, Laurence; Piton, Amélie; Reis, André; Arnesen, Thomas; Thauvin-Robinet, Christel; Zweier, Christiane

    2016-08-01

    N-terminal acetylation is a common protein modification in eukaryotes associated with numerous cellular processes. Inherited mutations in NAA10, encoding the catalytic subunit of the major N-terminal acetylation complex NatA have been associated with diverse, syndromic X-linked recessive disorders, whereas de novo missense mutations have been reported in one male and one female individual with severe intellectual disability but otherwise unspecific phenotypes. Thus, the full genetic and clinical spectrum of NAA10 deficiency is yet to be delineated. We identified three different novel and one known missense mutation in NAA10, de novo in 11 females, and due to maternal germ line mosaicism in another girl and her more severely affected and deceased brother. In vitro enzymatic assays for the novel, recurrent mutations p.(Arg83Cys) and p.(Phe128Leu) revealed reduced catalytic activity. X-inactivation was random in five females. The core phenotype of X-linked NAA10-related N-terminal-acetyltransferase deficiency in both males and females includes developmental delay, severe intellectual disability, postnatal growth failure with severe microcephaly, and skeletal or cardiac anomalies. Genotype-phenotype correlations within and between both genders are complex and may include various factors such as location and nature of mutations, enzymatic stability and activity, and X-inactivation in females. PMID:27094817

  17. N-terminal of L protein of vesicular stomatitis virus contains a new signal sequence

    Institute of Scientific and Technical Information of China (English)

    NIE Yuchun; KE Yeyan; WANG Zai; YU Xiang; DENG Hongkui; DING Mingxiao

    2003-01-01

    The L protein (241 kD) of vesicular stomatitis virus (VSV) is the mostimportant subunit of the replication complex. The existence of specific localization signal in the L protein was investigated by making recombinant constructs expressing truncated mutants of the L protein fused to green fluorescent protein(GFP) in transient transfection assays. The chimeric genes encoding varied N-terminal of L and GFP gene were put under the control of T7 promoter or CMV promoter. The fusion proteins were transiently expressed in BHK-21, COS-7, CHO or Hep G2 cells. When more than 120 residues were deleted or only 96 residues were kepton the N-terminal, the fusion proteins were shown to be distributed throughout the cells, cytoplasm and nucleus under the confocal microscope. However, other chimeric proteins with 120 or more amino acids were dotted and distributed in theperinuclear regions. And the fusion protein with 96-120 aa has the similar distribution. A thirteen-residue peptide QGYSFLHEVDKEA (108-120) was identified as localization signal, whose function would be absolutely distributed with the deficiency of D or V. Our results show that there is an independent localizing signal in N-terminal domain of L protein of VSV and this functional signal is conserved in different cell lines.

  18. Structure of the human histone chaperone FACT Spt16 N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk [Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, Scotland (United Kingdom)

    2016-01-22

    The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

  19. Two N-Terminal Acetyltransferases Antagonistically Regulate the Stability of a Nod-Like Receptor in Arabidopsis

    Science.gov (United States)

    Li, Lin; Gannon, Patrick; Linster, Eric; Huber, Monika; Kapos, Paul; Bienvenut, Willy; Giglione, Carmela; Zhang, Yuelin; Chen, She

    2015-01-01

    Nod-like receptors (NLRs) serve as immune receptors in plants and animals. The stability of NLRs is tightly regulated, though its mechanism is not well understood. Here, we show the crucial impact of N-terminal acetylation on the turnover of one plant NLR, Suppressor of NPR1, Constitutive 1 (SNC1), in Arabidopsis thaliana. Genetic and biochemical analyses of SNC1 uncovered its multilayered regulation by different N-terminal acetyltransferase (Nat) complexes. SNC1 exhibits a few distinct N-terminal isoforms generated through alternative initiation and N-terminal acetylation. Its first Met is acetylated by N-terminal acetyltransferase complex A (NatA), while the second Met is acetylated by N-terminal acetyltransferase complex B (NatB). Unexpectedly, the NatA-mediated acetylation serves as a degradation signal, while NatB-mediated acetylation stabilizes the NLR protein, thus revealing antagonistic N-terminal acetylation of a single protein substrate. Moreover, NatA also contributes to the turnover of another NLR, RESISTANCE TO P. syringae pv maculicola 1. The intricate regulation of protein stability by Nats is speculated to provide flexibility for the target protein in maintaining its homeostasis. PMID:25966763

  20. N-terminal amino acid sequences and some characteristics of fibrinolytic/hemorrhagic metalloproteinases purified from Bothrops jararaca venom.

    Science.gov (United States)

    Maruyama, Masugi; Sugiki, Masahiko; Anai, Keita; Yoshida, Etsuo

    2002-08-01

    We determined the N-terminal amino acid sequences of the fibrinolytic/hemorrhagic metalloproteinases (jararafibrases I, III and IV) purified from Bothrops jararaca venom. The N-terminal amino acid sequences of jararafibrase I and its degradation products were identical to those of jararhagin, another hemorrhagic metalloproteinase purified from the same snake venom. Together with enzymatic and immunological properties, we concluded that those two enzymes are identical. The N-terminal amino acid sequence of jararafibrase III was quite similar to C-type lectin isolated from Crotalus atrox, and the protein had a hemagglutinating activity on intact rat red blood cells. PMID:12165326

  1. Cyclization of the N-Terminal X-Asn-Gly Motif during Sample Preparation for Bottom-Up Proteomics

    DEFF Research Database (Denmark)

    Zhang, Xumin; Højrup, Peter

    2010-01-01

    We, herein, report a novel -17 Da peptide modification corresponding to an N-terminal cyclization of peptides possessing the N-terminal motif of X-Asn-Gly. The cyclization occurs spontaneously during sample preparation for bottom-up proteomics studies. Distinct from the two well-known N......-terminal cyclizations, cyclization of N-terminal glutamine and S-carbamoylmethylcysteine, it is dependent on pH instead of [NH(4)(+)]. The data set from our recent study on large-scale N(α)-modified peptides revealed a sequence requirement for the cyclization event similar to the well-known deamidation of Asn to iso......Asp and Asp. Detailed analysis using synthetic peptides confirmed that the cyclization forms between the N-terminus and its neighboring Asn residue, and the reaction shares the same succinimide intermediate with the Asn deamidation event. As a result, we, here, propose a molecular mechanism for this specific...

  2. Relationship between N-terminal pro-B-type natriuretic peptide levels and metabolic syndrome

    OpenAIRE

    Bao, Yuanyuan; Shang, Xiliang; Zhou, Linuo; Hu, Renming; Li, Yiming; Ding, Wei

    2011-01-01

    Introduction Previous studies have shown that obese individuals have reduced natriuretic peptide levels. But conflicting data exist on the relation of natriuretic peptide levels to other metabolic risk factors. Material and methods We investigated the relationship between plasma N-terminal pro-B-type natriuretic peptide levels (NT-proBNP) and metabolic syndrome (MetS) and metabolic risk factors in 469 patients free of heart failure. Two hundred thirty diagnosed MetS cases and 239 non-MetS cas...

  3. The N-terminal domain of apolipoprotein B-100: structural characterization by homology modeling

    Directory of Open Access Journals (Sweden)

    Khachfe Hassan M

    2007-07-01

    Full Text Available Abstract Background Apolipoprotein B-100 (apo B-100 stands as one of the largest proteins in humans. Its large size of 4536 amino acids hampers the production of X-ray diffraction quality crystals and hinders in-solution NMR analysis, and thus necessitates a domain-based approach for the structural characterization of the multi-domain full-length apo B. Results The structure of apo B-17 (the N-terminal 17% of apolipoprotein B-100 was predicted by homology modeling based on the structure of the N-terminal domain of lipovitellin (LV, a protein that shares not only sequence similarity with B17, but also a functional aspect of lipid binding and transport. The model structure was first induced to accommodate the six disulfide bonds found in that region, and then optimized using simulated annealing. Conclusion The content of secondary structural elements in this model structure correlates well with the reported data from other biophysical probes. The overall topology of the model conforms with the structural outline corresponding to the apo B-17 domain as seen in the EM representation of the complete LDL structure.

  4. Design, synthesis and aphicidal activity of N-terminal modified insect kinin analogs.

    Science.gov (United States)

    Zhang, Chuanliang; Qu, Yanyan; Wu, Xiaoqing; Song, Dunlun; Ling, Yun; Yang, Xinling

    2015-06-01

    The insect kinins are a class of multifunctional insect neuropeptides present in a diverse variety of insects. Insect kinin analogs showed multiple bioactivities, especially, the aphicidal activity. To find a biostable and bioactive insecticide candidate with simplified structure, a series of N-terminal modified insect kinin analogs was designed and synthesized based on the lead compound [Aib]-Phe-Phe-[Aib]-Trp-Gly-NH2. Their aphicidal activity against the soybean aphid Aphis glycines was evaluated. The results showed that all the analogs maintained the aphicidal activity. In particular, the aphicidal activity of the pentapeptide analog X Phe-Phe-[Aib]-Trp-Gly-NH2 (LC50=0.045mmol/L) was similar to the lead compound (LC50=0.048mmol/L). This indicated that the N-terminal protective group may not play an important role in the activity and the analogs structure could be simplified to pentapeptide analogs while retaining good aphicidal activity. The core pentapeptide analog X can be used as the lead compound for further chemical modifications to discover potential insecticides. PMID:25116632

  5. Plasma biomarker screening for liver fibrosis with the N-terminal isotope tagging strategy.

    Science.gov (United States)

    Li, ShuLong; Liu, Xin; Wei, Lai; Wang, HuiFen; Zhang, JiYang; Wei, HanDong; Qian, XiaoHong; Jiang, Ying; He, FuChu

    2011-05-01

    A non-invasive diagnostic approach is crucial for the evaluation of severity of liver disease, treatment decisions, and assessing drug efficacy. This study evaluated plasma proteomic profiling via an N-terminal isotope tagging strategy coupled with liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry measurement to detect liver fibrosis staging. Pooled plasma from different liver fibrosis stages, which were assessed in advance by the current gold-standard of liver biopsy, was quantitatively analyzed. A total of 72 plasma proteins were found to be dysregulated during the fibrogenesis process, and this finding constituted a valuable candidate plasma biomarker bank for follow-up analysis. Validation results of fibronectin by Western blotting reconfirmed the mass-based data. Ingenuity Pathways Analysis showed four types of metabolic networks for the functional effect of liver fibrosis disease in chronic hepatitis B patients. Consequently, quantitative proteomics via the N-terminal acetyl isotope labeling technique provides an effective and useful tool for screening plasma candidate biomarkers for liver fibrosis. We quantitatively monitored the fibrogenesis process in CHB patients. We discovered many new valuable candidate biomarkers for the diagnosis of liver fibrosis and also partly identified the mechanism involved in liver fibrosis disease. These results provide a clearer understanding of liver fibrosis pathophysiology and will also hopefully lead to improvement of clinical diagnosis and treatment.

  6. Recombinant N-Terminal Slit2 Inhibits TGF-β-Induced Fibroblast Activation and Renal Fibrosis.

    Science.gov (United States)

    Yuen, Darren A; Huang, Yi-Wei; Liu, Guang-Ying; Patel, Sajedabanu; Fang, Fei; Zhou, Joyce; Thai, Kerri; Sidiqi, Ahmad; Szeto, Stephen G; Chan, Lauren; Lu, Mingliang; He, Xiaolin; John, Rohan; Gilbert, Richard E; Scholey, James W; Robinson, Lisa A

    2016-09-01

    Fibrosis and inflammation are closely intertwined injury pathways present in nearly all forms of CKD for which few safe and effective therapies exist. Slit glycoproteins signaling through Roundabout (Robo) receptors have been described to have anti-inflammatory effects through regulation of leukocyte cytoskeletal organization. Notably, cytoskeletal reorganization is also required for fibroblast responses to TGF-β Here, we examined whether Slit2 also controls TGF-β-induced renal fibrosis. In cultured renal fibroblasts, which we found to express Slit2 and Robo-1, the bioactive N-terminal fragment of Slit2 inhibited TGF-β-induced collagen synthesis, actin cytoskeletal reorganization, and Smad2/3 transcriptional activity, but the inactive C-terminal fragment of Slit2 did not. In mouse models of postischemic renal fibrosis and obstructive uropathy, treatment with N-terminal Slit2 before or after injury inhibited the development of renal fibrosis and preserved renal function, whereas the C-terminal Slit2 had no effect. Our data suggest that administration of recombinant Slit2 may be a new treatment strategy to arrest chronic injury progression after ischemic and obstructive renal insults by not only attenuating inflammation but also, directly inhibiting renal fibrosis.

  7. Effect of N-Terminal Acylation on the Activity of Myostatin Inhibitory Peptides.

    Science.gov (United States)

    Takayama, Kentaro; Nakamura, Akari; Rentier, Cédric; Mino, Yusaku; Asari, Tomo; Saga, Yusuke; Taguchi, Akihiro; Yakushiji, Fumika; Hayashi, Yoshio

    2016-04-19

    Inhibition of myostatin, which negatively regulates skeletal muscle growth, is a promising strategy for the treatment of muscle atrophic disorders, such as muscular dystrophy, cachexia and sarcopenia. Recently, we identified peptide A (H-WRQNTRYSRIEAIKIQILSKLRL-NH2 ), the 23-amino-acid minimum myostatin inhibitory peptide derived from mouse myostatin prodomain, and highlighted the importance of its N-terminal tryptophan residue for the effective inhibition. In this study, we synthesized a series of acylated peptide derivatives focused on the tryptophan residue to develop potent myostatin inhibitors. As a result of the investigation, a more potent derivative of peptide A was successfully identified in which the N-terminal tryptophan residue is replaced with a 2-naphthyloxyacetyl moiety to give an inhibitory peptide three times (1.19±0.11 μm) more potent than parent peptide A (3.53±0.25 μm). This peptide could prove useful as a new starting point for the development of improved inhibitory peptides. PMID:26954624

  8. N-terminal palmitoylation is required for Toxoplasma gondii HSP20 inner membrane complex localization.

    Science.gov (United States)

    De Napoli, M G; de Miguel, N; Lebrun, M; Moreno, S N J; Angel, S O; Corvi, M M

    2013-06-01

    Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis. Protein palmitoylation is known to play roles in signal transduction and in enhancing the hydrophobicity of proteins thus contributing to their membrane association. Global inhibition of protein palmitoylation has been shown to affect T. gondii physiology and invasion of the host cell. However, the proteins affected by this modification have been understudied. This paper shows that the small heat shock protein 20 from T. gondii (TgHSP20) is synthesized as a mature protein in the cytosol and is palmitoylated in three cysteine residues. However, its localization at the inner membrane complex (IMC) is dependent only on N-terminal palmitoylation. Absence or incomplete N-terminal palmitoylation causes TgHSP20 to partially accumulate in a membranous structure. Interestingly, TgHSP20 palmitoylation is not responsible for its interaction with the daughter cells IMCs. Together, our data describe the importance of palmitoylation in protein targeting to the IMC in T. gondii.

  9. Role of the N-terminal seven residues of surfactant protein B (SP-B.

    Directory of Open Access Journals (Sweden)

    Mahzad Sharifahmadian

    Full Text Available Breathing is enabled by lung surfactant, a mixture of proteins and lipids that forms a surface-active layer and reduces surface tension at the air-water interface in lungs. Surfactant protein B (SP-B is an essential component of lung surfactant. In this study we probe the mechanism underlying the important functional contributions made by the N-terminal 7 residues of SP-B, a region sometimes called the "insertion sequence". These studies employed a construct of SP-B, SP-B (1-25,63-78, also called Super Mini-B, which is a 41-residue peptide with internal disulfide bonds comprising the N-terminal 7-residue insertion sequence and the N- and C-terminal helices of SP-B. Circular dichroism, solution NMR, and solid state (2H NMR were used to study the structure of SP-B (1-25,63-78 and its interactions with phospholipid bilayers. Comparison of results for SP-B (8-25,63-78 and SP-B (1-25,63-78 demonstrates that the presence of the 7-residue insertion sequence induces substantial disorder near the centre of the lipid bilayer, but without a major disruption of the overall mechanical orientation of the bilayers. This observation suggests the insertion sequence is unlikely to penetrate deeply into the bilayer. The 7-residue insertion sequence substantially increases the solution NMR linewidths, most likely due to an increase in global dynamics.

  10. Specificity of N-terminal methionyl peptidase: analysis by site-directed mutagenesis

    International Nuclear Information System (INIS)

    The start site of eukaryotic translation is normally an AUG codon. The corresponding N-terminal methionine is most often removed when the nascent chain reaches about 30 residues. Data from a survey of 1764 eukaryotic protein sequences suggest that the residue adjacent to the initiator Met determines Met cleavage. In order to investigate the mechanism of this reaction, the authors have prepared oligonucleotide-directed mutants of human β-globin from gapped heteroduplexes of a T3/T7 plasmid containing a globin cDNA clone. To date, the authors have produced mutants encoding for 15 of 19 possible amino acid replacements at position 1 in the β-globin chain. These mutants have been confirmed by dideoxy sequencing, transcribed in vitro, and translated in a rabbit reticulocyte lysate in the presence of 35S-methionine. Labeled translation products were then isolated by cation exchange HPLC, and tryptic peptides were analyzed by RP-HPLC. Thus far, this structural analysis has shown that for β-1 Val, Ala, and Ser, the initiator Met is cleaved, whereas for β-1 Lys, Met, Glu, Trp, Asn, Tyr, and Glu, initiator Met is retained. For β-1 Leu initiator Met is cleaved with a frequency of about 50%. These results are consistent with the data obtained from the previous survey. The expression of site-directed mutants in a cell-free system can also be used to investigate other N-terminal processing events, such as acetylation and myristylation

  11. Directed evolution of the TALE N-terminal domain for recognition of all 5' bases.

    Science.gov (United States)

    Lamb, Brian M; Mercer, Andrew C; Barbas, Carlos F

    2013-11-01

    Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5'-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5' T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5' T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes.

  12. STAT6: its role in interleukin 4-mediated biological functions.

    Science.gov (United States)

    Takeda, K; Kishimoto, T; Akira, S

    1997-05-01

    Interleukin (IL) 4 is known to be a cytokine which plays a central role in the regulation of immune response. Studies on cytokine signal transduction have clarified the mechanism by which IL4 exerts its functions. Two cytoplasmic proteins, signal transducer and activator of transcription (STAT) 6 and IL4-induced phosphotyrosine substrate/insulin receptor substrate 2 (4PS/IRS2), are activated in IL4 signal transduction. Recent studies from STAT6-deficient mice have revealed the essential role of STAT6 in IL4-mediated biological actions. In addition, STAT6 has also been demonstrated to be important for the functions mediated by IL13, which is related to IL4. IL4 and IL13 have been shown to induce the production of IgE, which is a major mediator in an allergic response. These findings indicate that STAT6 activation is involved in IL4- and IL13-mediated disorders such as allergy.

  13. N-Terminal methionine processing by the zinc-activated Plasmodium falciparum methionine aminopeptidase 1b.

    Science.gov (United States)

    Calcagno, Sarah; Klein, Christian D

    2016-08-01

    The methionine aminopeptidase 1b from Plasmodium falciparum (PfMetAP 1b) was cloned, expressed in Escherichia coli and characterized. Surprisingly, and in contrast to other methionine aminopeptidases (MetAPs) that require heavy-metal cofactors such as cobalt, the enzyme is reliably activated by zinc ions. Immobilization of the enzyme is possible by His-tag metal chelation to iminodiacetic acid-agarose and by covalent binding to chloroacetamido-hexyl-agarose. The covalently immobilized enzyme shows long-term stability, allowing a continuous, heterogenous processing of N-terminal methionines, for example, in recombinant proteins. Activation by zinc, instead of cobalt as for other MetAPs, avoids the introduction of heavy metals with toxicological liabilities and oxidative potential into biotechnological processes. The PfMetAP 1b therefore represents a useful tool for the enzymatic, posttranslational processing of recombinant proteins. PMID:27023914

  14. Structural polymorphism in the N-terminal oligomerization domain of NPM1.

    Science.gov (United States)

    Mitrea, Diana M; Grace, Christy R; Buljan, Marija; Yun, Mi-Kyung; Pytel, Nicholas J; Satumba, John; Nourse, Amanda; Park, Cheon-Gil; Madan Babu, M; White, Stephen W; Kriwacki, Richard W

    2014-03-25

    Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer-pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.

  15. Partial N-terminal sequence analysis of human class II molecules expressing the DQw3 determinant.

    Science.gov (United States)

    Obata, F; Endo, T; Yoshii, M; Otani, F; Igarashi, M; Takenouchi, T; Ikeda, H; Ogasawara, K; Kasahara, M; Wakisaka, A

    1985-09-01

    HLA-DQ molecules were isolated from DRw9-homozygous and DR4-homozygous cell lines by using a monoclonal antibody HU-18, which recognizes class II molecules carrying the conventional DQw3 determinant. The partial N-terminal sequence analysis of the DQw3 molecules revealed that they have sequences homologous to those of murine I-A molecules. Within the limits of our sequence analysis, the DQw3 molecules from the two cell lines are identical to each other in both the alpha and beta chains. The DQ alpha as well as DQ beta chains were found to have amino acid substitutions when compared to other I-A-like molecules whose sequences have been reported. These differences may contribute to the DQw supertypic specificity. The polymorphic nature of DQ molecules is in marked contrast to that of DR molecules where DR alpha chains are highly conserved while DR beta chains have easily detectable amino acid substitutions. PMID:2411700

  16. Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae.

    Science.gov (United States)

    Edwin, Aaron; Persson, Cecilia; Mayzel, Maxim; Wai, Sun Nyunt; Öhman, Anders; Karlsson, B Göran; Sauer-Eriksson, A Elisabeth

    2015-12-01

    The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N-terminal domain (residues 23-103) that contains two short α-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.

  17. Copper binding triggers compaction in N-terminal tail of human copper pump ATP7B.

    Science.gov (United States)

    Mondol, Tanumoy; Åden, Jörgen; Wittung-Stafshede, Pernilla

    2016-02-12

    Protein conformational changes are fundamental to biological reactions. For copper ion transport, the multi-domain protein ATP7B in the Golgi network receives copper from the cytoplasmic copper chaperone Atox1 and, with energy from ATP hydrolysis, moves the metal to the lumen for loading of copper-dependent enzymes. Although anticipated, conformational changes involved in ATP7B's functional cycle remain elusive. Using spectroscopic methods we here demonstrate that the four most N-terminal metal-binding domains in ATP7B, upon stoichiometric copper addition, adopt a more compact arrangement which has a higher thermal stability than in the absence of copper. In contrast to previous reports, no stable complex was found in solution between the metal-binding domains and the nucleotide-binding domain of ATP7B. Metal-dependent movement of the first four metal-binding domains in ATP7B may be a trigger that initiates the overall catalytic cycle.

  18. N-Terminal Truncation of TACO Inhibits PMA-Induced U937 Cell Adhesion

    Institute of Scientific and Technical Information of China (English)

    LIU Changzhen; SUI Senfang

    2005-01-01

    The effect of TACO1-299, the N-terminal truncation of TACO, on phorbol 12-myristate 13-acetate (PMA)-induced U937 cell adhesion was investigated. Full-length TACO and several truncations were overexpressed in U937 cells. The effects of the expressed proteins on U937 cell adhesion mediated by PMA-induced differentiation were observed by fluorescence microscopy. The results show that the overexpression of TACO1-299 inhibits cell adhesion while overexpressions of the other proteins do not have this effect. The actin-binding capability of TACO1-299 was investigated and the results show that TACO1-299 lacks the ability of TACO to bind F-actin. The inhibitive effect of TACO1-299, the functional domain of TACO, suggests that TACO may play a role in cell differentiation mediating adhesion of monoblastic leukemia cells.

  19. The vasorelaxant effect of adrenomedullin, proadrenomedullin N-terminal 20 peptide and amylin in human skin

    DEFF Research Database (Denmark)

    Hasbak, Philip; Eskesen, Karen; Lind, Peter Henrik;

    2006-01-01

    of the peptides. The mRNA expression was assessed by real-time reverse transcriptase-polymerase chain reaction (real-time PCR). CGRP, adrenomedullin and amylin induced concentration-dependent, long-lasting increases in skin blood flow. The response to PAMP was shorter in duration appearing similar......In this study we aimed to assess in vivo, the vasodilator effects of adrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP) and amylin in human skin vasculature and compare the responses to the effects mediated by the endogenous neuropeptides calcitonin gene-related peptide (CGRP......) and substance P and to examine the mRNA expression of calcitonin receptor-like receptor (CL-R) and receptor-activity modifying proteins, RAMP1, RAMP 2 and RAMP3 in human subcutaneous arteries. Changes in skin blood flow of the forearm were measured using a Laser Doppler Imager after intradermal injection...

  20. Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide.

    Science.gov (United States)

    Nguyen, Giang K T; Cao, Yuan; Wang, Wei; Liu, Chuan Fa; Tam, James P

    2015-12-21

    An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields. PMID:26563575

  1. Analysis of the secondary structure of a protein's N-terminal

    Science.gov (United States)

    Floare, C. G.; Bogdan, M.; Horovitz, O.; Mocanu, A.; Tomoaia-Cotisel, M.

    2009-08-01

    The major protein component from aleurone cells of barley (Hordeum vulgare L.), PACB, is related to 7S globulins present in other cereals and to the vicilin-type 7S globulins of legumes and cotton seed. It contains 4 subunits of about 20, 25, 40 and 50 kDa molecular weights. The N-terminal sequence of 16 amino acids (over 260 atoms) in the protein was previously determined, and our aim is the prediction of its secondary structure. The empirical Chou-Fasman method was applied in an improved version as well as the empirical DSC method (discrimination of protein secondary structure class) with quite similar results. A molecular dynamics simulation was also performed, using the FF99SB forcefield within AMBER version 9.0. Solvation effects were incorporated using the Born model. The results are compared and a 3D model is proposed.

  2. Analysis of the secondary structure of a protein's N-terminal

    Energy Technology Data Exchange (ETDEWEB)

    Floare, C G; Bogdan, M [National Institute for R and D of Isotopic and Molecular Technologies, 65-103 Donath, 400293 Cluj-Napoca (Romania); Horovitz, O; Mocanu, A; Tomoaia-Cotisel, M, E-mail: calin.floare@itim-cj.r [Babes-Bolyai University, Faculty of Chemistry and Chemical Engineering, Department of Physical Chemistry, 11 Arany Janos, 400028 Cluj-Napoca (Romania)

    2009-08-01

    The major protein component from aleurone cells of barley (Hordeum vulgare L.), PACB, is related to 7S globulins present in other cereals and to the vicilin-type 7S globulins of legumes and cotton seed. It contains 4 subunits of about 20, 25, 40 and 50 kDa molecular weights. The N-terminal sequence of 16 amino acids (over 260 atoms) in the protein was previously determined, and our aim is the prediction of its secondary structure. The empirical Chou-Fasman method was applied in an improved version as well as the empirical DSC method (discrimination of protein secondary structure class) with quite similar results. A molecular dynamics simulation was also performed, using the FF99SB forcefield within AMBER version 9.0. Solvation effects were incorporated using the Born model. The results are compared and a 3D model is proposed.

  3. 157 nm Photodissociation of Dipeptide Ions Containing N-Terminal Arginine

    Science.gov (United States)

    Webber, Nathaniel; He, Yi; Reilly, James P.

    2013-12-01

    Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m2 and m2+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.

  4. Reaction of the N-terminal methionine residues in cyanase with diethylpyrocarbonate.

    Science.gov (United States)

    Anderson, P M; Korte, J J; Holcomb, T A

    1994-11-29

    Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The enzyme is a decamer of identical subunits (M(r) = 17,000). Previous studies have shown that modification of either the single cysteine residue or the single histidine residue in each subunit gives an active decameric derivative that dissociates reversibly to inactive dimer derivative, indicating that decameric structure is required for activity and that the SH and imidazole groups are not required for catalytic activity [Anderson, P. M., Korte, J. J., Holcomb, T. A., Cho, Y.-G., Son, C.-M., & Sung, Y.-C. (1994) J. Biol. Chem. 269, 15036-15045]. Here the effects of reaction of the reagent diethylpyrocarbonate (DEPC) with cyanase or mutant cyanases are reported. DEPC reacts stoichiometrically with the histidine residue and at one additional site in each subunit when the enzyme is in the inactive dimer form, preventing reactivation. DEPC reacts stoichiometrically (with the same result on reactivation) at only one site per subunit with the inactive dimer form of cyanase mutants in which the single histidine residue has been replaced by one of several different amino acids by site-directed mutagenesis; the site of the reaction was identified as the amino group of the N-terminal methionine. DEPC does not react with the histidine residue of the active decameric form of wild-type cyanase and does not affect activity of the active decameric form of wild-type or mutant cyanases. Reaction with the N-terminal amino group of methionine apparently prevents reactivation of the mutant enzymes by blocking association to decamer.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Powdery mildew fungal effector candidates share N-terminal Y/F/WxC-motif

    Directory of Open Access Journals (Sweden)

    Emmersen Jeppe

    2010-05-01

    Full Text Available Abstract Background Powdery mildew and rust fungi are widespread, serious pathogens that depend on developing haustoria in the living plant cells. Haustoria are separated from the host cytoplasm by a plant cell-derived extrahaustorial membrane. They secrete effector proteins, some of which are subsequently transferred across this membrane to the plant cell to suppress defense. Results In a cDNA library from barley epidermis containing powdery mildew haustoria, two-thirds of the sequenced ESTs were fungal and represented ~3,000 genes. Many of the most highly expressed genes encoded small proteins with N-terminal signal peptides. While these proteins are novel and poorly related, they do share a three-amino acid motif, which we named "Y/F/WxC", in the N-terminal of the mature proteins. The first amino acid of this motif is aromatic: tyrosine, phenylalanine or tryptophan, and the last is always cysteine. In total, we identified 107 such proteins, for which the ESTs represent 19% of the fungal clones in our library, suggesting fundamental roles in haustoria function. While overall sequence similarity between the powdery mildew Y/F/WxC-proteins is low, they do have a highly similar exon-intron structure, suggesting they have a common origin. Interestingly, searches of public fungal genome and EST databases revealed that haustoria-producing rust fungi also encode large numbers of novel, short proteins with signal peptides and the Y/F/WxC-motif. No significant numbers of such proteins were identified from genome and EST sequences from either fungi which do not produce haustoria or from haustoria-producing Oomycetes. Conclusion In total, we identified 107, 178 and 57 such Y/F/WxC-proteins from the barley powdery mildew, the wheat stem rust and the wheat leaf rust fungi, respectively. All together, our findings suggest the Y/F/WxC-proteins to be a new class of effectors from haustoria-producing pathogenic fungi.

  6. Antimicrobial activity of human prion protein is mediated by its N-terminal region.

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    Mukesh Pasupuleti

    Full Text Available BACKGROUND: Cellular prion-related protein (PrP(c is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP(c, and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypothesize that PrP(c could exert antimicrobial activity. METHODOLOGY AND PRINCIPAL FINDINGS: Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-alpha in vitro. CONCLUSIONS: The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense.

  7. N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis.

    Science.gov (United States)

    Rada, Petr; Makki, Abhijith Radhakrishna; Zimorski, Verena; Garg, Sriram; Hampl, Vladimír; Hrdý, Ivan; Gould, Sven B; Tachezy, Jan

    2015-12-01

    Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PPi-dependent phosphofructokinase (TvPPi-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPPi-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to "short" bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.

  8. Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates.

    Science.gov (United States)

    Howard, R F; Ardeshir, F; Reese, R T

    1986-01-01

    Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium falciparum, were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichia coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the K1 isolate [Hall et al., Nature 311 (1984) 379-382], codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 317 (1985) 270-273]. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of greater than or equal to 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous K1 isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. falciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15% of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

    Energy Technology Data Exchange (ETDEWEB)

    Auperin,T.; Bolduc, G.; Baron, M.; Heroux, A.; Filman, D.; Madoff, L.; Hogle, J.

    2005-01-01

    Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-{angstrom} resolution crystal structure of NtACP comprising residues Ser{sup 52} through Leu{sup 225} of the full-length ACP. NtACP has two domains, an N-terminal {beta}-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the {beta}-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp{sup 146}, Arg{sup 110}, and Asp{sup 118}. A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.

  10. Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.

    Directory of Open Access Journals (Sweden)

    Boris Zybailov

    Full Text Available Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP. The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence

  11. Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins.

    Science.gov (United States)

    Song, Albert S; Poor, Taylor A; Abriata, Luciano A; Jardetzky, Theodore S; Dal Peraro, Matteo; Lamb, Robert A

    2016-07-01

    Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design. PMID:27335462

  12. Acquired cancer stem cell phenotypes through Oct4-mediated dedifferentiation

    Science.gov (United States)

    Kumar, Suresh M.; Liu, Shujing; Lu, Hezhe; Zhang, Hongtao; Zhang, Paul J.; Gimotty, Phyllis A.; Guerra, Matthew; Guo, Wei; Xu, Xiaowei

    2012-01-01

    There is enormous interest to target cancer stem cells (CSCs) for clinical treatment because these cells are highly tumorigenic and resistant to chemotherapy. Oct4 is expressed by CSC-like cells in different types of cancer. However, function of Oct4 in tumor cells is unclear. In this study, we showed that expression of Oct4 gene or transmembrane delivery of Oct4 protein promoted dedifferentiation of melanoma cells to CSC-like cells. The dedifferentiated melanoma cells showed significantly decreased expression of melanocytic markers and acquired the ability to form tumor spheroids. They showed markedly increased resistance to chemotherapeutic agents and hypoxic injury. In the subcutaneous xenograft and tail vein injection assays, these cells had significantly increased tumorigenic capacity. The dedifferentiated melanoma cells acquired features associated with CSCs such as multipotent differentiation capacity and expression of melanoma CSC markers such as ABCB5 and CD271. Mechanistically, Oct4 induced dedifferentiation was associated with increased expression of endogenous Oct4, Nanog and Klf4, and global gene expression changes that enriched for transcription factors. RNAi mediated knockdown of Oct4 in dedifferentiated cells led to diminished CSC phenotypes. Oct4 expression in melanoma was regulated by hypoxia and its expression was detected in a subpopulation of melanoma cells in clinical samples. Our data indicate that Oct4 is a positive regulator of tumor dedifferentiation. The results suggest that CSC phenotype is dynamic and may be acquired through dedifferentiation. Oct4 mediated tumor cell dedifferentiation may play an important role during tumor progression. PMID:22286766

  13. Prokaryotic Expression and Purification of Human TLE1 N-terminal Q Domain Fragment and Production of its Polyclonal Antibody

    Directory of Open Access Journals (Sweden)

    Su WANG

    2010-11-01

    Full Text Available Background and objective TLE1 is an important protein in regulating Wnt, Notch and EGFR signaling pathways. The TLE1 N-terminal Q domain regulates the pathways by mediating its oligomerization and interaction with LEF1. The aim of this study is to construct the human TLE1 N-terminal Q domain fragment in prokaryotic expression system, express and purify protein TLE1 N-terminal Q domain and prepare its polyclonal antibody. Methods The sequence of TLE1 N-terminal Q domain obtained by PCR from human lung adenocarcinoma cDNA, was cloned into the prokaryotic expression vector pGEX-4T-1 containing Glutathione S-transferase (GST. Vector pGEX-4T1-TLE1-Q was transformed into E.coli BL21 condon plus. The GST-TLE1-Q(1-136 fusion protein was induced by IPTG, digested by Thrombin, purified with glutathione-sepharose beads and FPLC, identified by SDS-PAGE. Then rabbits were immunized with the purified protein TLE1-Q(1-136 for obtaining the antiserum. The titers and specificity of antibodies were measured by ELISA and Western blot. Results The PCR identification and the sequencing of recombinant plasmid demonstrated that vector pGEX-4T1-TLE1-Q was successfully constructed. The SDS-PAGE shows target protein (14 000 Da is the interest protein TLE1-Q(1-136. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired, with an antibody titer of 1:20 000. Conclusion Expression vector pGEX-4T1-TLE1-Q is correctly constructed. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired. These work established the foundation for further biological study between TLE1 and lung cancers.

  14. 阿朴吗啡诱导黑质毁损大鼠腹侧被盖区c-jun表达%Apomorphine induce c-jun expression in ventral tagmental area of 6-OHDA-lesioned rats

    Institute of Scientific and Technical Information of China (English)

    陈晓宇; 姚玉芹; 沈韶辉; 韩卉

    2006-01-01

    目的:观察6-羟基多巴胺(6-hydroxydopamine,6-OHDA)毁损黑质DA能神经元后,不同时间点腹腔注射阿朴吗啡(Apomorphine,APO)大鼠行为学及中脑腹侧被盖区(ventraltagmental area,VTA)形态学、c-jun表达情况,探讨其可能机制.方法:6-OHDA单侧一点注射大鼠右黑质致密区(substantianigracompacta,SNc),特异性毁损DA能神经元;术后1、3、7、14、21d腹腔注射APO,观察旋转行为;利用电镜、尼氏染色、免疫组织化学ABC法,观察各时间点VTA DA能神经元形态学变化和酪氨酸羟化酶(TH)、c-jun表达情况.结果:毁损侧VTA DA能神经元逐渐减少,超微结构损伤逐渐加重;DA神经元丢失≥75%时,APO诱导的旋转实验≥7r/min,VTA毁损侧c-jun表达.结论:APO能诱导毁损侧VTA表达c-jun;c-jun表达与DA能神经元毁损程度有一定的关系.

  15. Heritability assessment of cartilage metabolism. A twin study on circulating procollagen IIA N-terminal propeptide (PIIANP)

    DEFF Research Database (Denmark)

    Munk, H L; Svendsen, A J; Hjelmborg, J V B;

    2014-01-01

    OBJECTIVE: The aim of this investigation was to estimate the heritability of circulating collagen IIA N-terminal propeptide (PIIANP) by studying mono- and dizygotic healthy twin pairs at different age and both genders. DESIGN: 598 monozygotic (MZ) and dizygotic (DZ) twin individuals aged 18...... the collagen IIA synthesis as assessed by the collagen IIA N-terminal propeptide in serum is attributable to genetic effectors while individual and shared environment account for 24% and 31% respectively. The heritability does not differ between genders or according to age....

  16. NEDD4-mediated HSF1 degradation underlies α-synucleinopathy.

    Science.gov (United States)

    Kim, Eunhee; Wang, Bin; Sastry, Namratha; Masliah, Eliezer; Nelson, Peter T; Cai, Huaibin; Liao, Francesca-Fang

    2016-01-15

    Cellular protein homeostasis is achieved by a delicate network of molecular chaperones and various proteolytic processes such as ubiquitin-proteasome system (UPS) to avoid a build-up of misfolded protein aggregates. The latter is a common denominator of neurodegeneration. Neurons are found to be particularly vulnerable to toxic stress from aggregation-prone proteins such as α-synuclein. Induction of heat-shock proteins (HSPs), such as through activated heat shock transcription factor 1 (HSF1) via Hsp90 inhibition, is being investigated as a therapeutic option for proteinopathic diseases. HSF1 is a master stress-protective transcription factor which activates genes encoding protein chaperones (e.g. iHsp70) and anti-apoptotic proteins. However, whether and how HSF1 is dysregulated during neurodegeneration has not been studied. Here, we discover aberrant HSF1 degradation by aggregated α-synuclein (or α-synuclein-induced proteotoxic stress) in transfected neuroblastoma cells. HSF1 dysregulation via α-synuclein was confirmed by in vivo assessment of mouse and in situ studies of human specimens with α-synucleinopathy. We demonstrate that elevated NEDD4 is implicated as the responsible ubiquitin E3 ligase for HSF1 degradation through UPS. Furthermore, pharmacologically induced SIRT1-mediated deacetylation can attenuate aberrant NEDD4-mediated HSF1 degradation. Indeed, we define the acetylation status of the Lys 80 residue located in the DNA-binding domain of HSF1 as a critical factor in modulating HSF1 protein stability in addition to its previously identified role in the transcriptional activity. Together with the finding that preserving HSF1 can alleviate α-synuclein toxicity, this study strongly suggests that aberrant HSF1 degradation is a key neurodegenerative mechanism underlying α-synucleinopathy.

  17. BETA-N-TERMINAL GLYCOHEMOGLOBINS IN SUBJECTS WITH COMMON HEMOGLOBINOPATHIES - RELATION WITH FRUCTOSAMINE AND MEAN ERYTHROCYTE AGE

    NARCIS (Netherlands)

    MARTINA, WV; MARTIJN, EG; VANDERMOLEN, M; SCHERMER, JG; MUSKIET, FAJ

    1993-01-01

    Amounts of beta-N-terminal glycohemoglobins (HbX1c), serum fructosamine, and erythrocyte polyamines were determined in nondiabetic adults with HbAA, HbAC, HbAS, HbCC, HbSC, HbSS, and HbS/hereditary persistent HbF (HPFH). The groups did not differ in fructosamine concentrations. Mean (95% confidence

  18. Glutamate dehydrogenase isoforms with N-terminal (His)6- or FLAG-tag retain their kinetic properties and cellular localization

    DEFF Research Database (Denmark)

    Pajęcka, Kamilla; Nielsen, Camilla Wendel; Hauge, Anne;

    2014-01-01

    containing N-terminal (His)6 tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni(2+)-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG...

  19. Dimeric structure of the N-terminal domain of PriB protein from Thermoanaerobacter tengcongensis solved ab initio.

    Science.gov (United States)

    Liebschner, Dorothee; Brzezinski, Krzysztof; Dauter, Miroslawa; Dauter, Zbigniew; Nowak, Marta; Kur, Józef; Olszewski, Marcin

    2012-12-01

    PriB is one of the components of the bacterial primosome, which catalyzes the reactivation of stalled replication forks at sites of DNA damage. The N-terminal domain of the PriB protein from the thermophilic bacterium Thermoanaerobacter tengcongensis (TtePriB) was expressed and its crystal structure was solved at the atomic resolution of 1.09 Å by direct methods. The protein chain, which encompasses the first 104 residues of the full 220-residue protein, adopts the characteristic oligonucleotide/oligosaccharide-binding (OB) structure consisting of a five-stranded β-barrel filled with hydrophobic residues and equipped with four loops extending from the barrel. In the crystal two protomers dimerize, forming a six-stranded antiparallel β-sheet. The structure of the N-terminal OB domain of T. tengcongensis shows significant differences compared with mesophile PriBs. While in all other known structures of PriB a dimer is formed by two identical OB domains in separate chains, TtePriB contains two consecutive OB domains in one chain. However, sequence comparison of both the N-terminal and the C-terminal domains of TtePriB suggests that they have analogous structures and that the natural protein possesses a structure similar to a dimer of two N-terminal domains.

  20. Fine tuning of the catalytic activity of colicin e7 nuclease domain by systematic n-terminal mutations

    DEFF Research Database (Denmark)

    Németh, Eszter; Körtvélyesi, Tamás; Thulstrup, Peter W.;

    2014-01-01

    The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446–449NColE75KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuc...

  1. N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes.

    OpenAIRE

    Kuhn, H; Fietzek, P P; Lampen, J. O.

    1982-01-01

    The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.

  2. N-terminal propeptide of type III procollagen as a biomarker of anabolic response to recombinant human GH and testosterone

    Science.gov (United States)

    Context: Biomarkers that predict musculoskeletal response to anabolic therapies should expedite drug development. During collagen synthesis in soft lean tissue, N-terminal propeptide of type III procollagen (P3NP) is released into circulation. We investigated P3NP as a biomarker of lean body mass (L...

  3. Barley polyamine oxidase: Characterisation and analysis of the cofactor and the N-terminal amino acid sequence

    DEFF Research Database (Denmark)

    Radova, A.; Sebela, M.; Galuszka, P.;

    2001-01-01

    was further confirmed by measuring the fluorescence spectra, Barley PAO is an acidic protein (pI 5.4) containing 3% of neutral sugars: its molecular mass determined by SDS-PAGE was 56 kDa, whilst gel permeation chromatography revealed the higher value of 76 kDa. The N-terminal amino acid sequence of barley...

  4. The N-Terminal of Aquareovirus NS80 Is Required for Interacting with Viral Proteins and Viral Replication.

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    Full Text Available Reovirus replication and assembly occurs within viral inclusion bodies that formed in specific intracellular compartments of cytoplasm in infected cells. Previous study indicated that aquareovirus NS80 is able to form inclusion bodies, and also can retain viral proteins within its inclusions. To better understand how NS80 performed in viral replication and assembly, the functional regions of NS80 associated with other viral proteins in aquareovirus replication were investigated in this study. Deletion mutational analysis and rotavirus NSP5-based protein association platform were used to detect association regions. Immunofluorescence images indicated that different N-terminal regions of NS80 could associate with viral proteins VP1, VP4, VP6 and NS38. Further co-immunoprecipitation analysis confirmed the interaction between VP1, VP4, VP6 or NS38 with different regions covering the N-terminal amino acid (aa, 1-471 of NS80, respectively. Moreover, removal of NS80 N-terminal sequences required for interaction with proteins VP1, VP4, VP6 or NS38 not only prevented the capacity of NS80 to support viral replication in NS80 shRNA-based replication complementation assays, but also inhibited the expression of aquareovirus proteins, suggesting that N-terminal regions of NS80 are necessary for viral replication. These results provided a foundational basis for further understanding the role of NS80 in viral replication and assembly during aquareovirus infection.

  5. Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1

    DEFF Research Database (Denmark)

    Feng, Tao; Nyffenegger, Christian; Højrup, Peter;

    2014-01-01

    Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing...

  6. Interaction of the N-terminal segment of pulmonary surfactant protein SP-C with interfacial phospholipid films

    DEFF Research Database (Denmark)

    Plasencia, Inés; Keough, Kevin M W; Perez-Gil, Jesus

    2005-01-01

    Pulmonary surfactant protein SP-C is a 35-residue polypeptide composed of a hydrophobic transmembrane alpha-helix and a polycationic, palmitoylated-cysteine containing N-terminal segment. This segment is likely the only structural motif the protein projects out of the bilayer in which SP-C is ins......Pulmonary surfactant protein SP-C is a 35-residue polypeptide composed of a hydrophobic transmembrane alpha-helix and a polycationic, palmitoylated-cysteine containing N-terminal segment. This segment is likely the only structural motif the protein projects out of the bilayer in which SP......-C is inserted and is therefore a candidate motif to participate in interactions with other bilayers or monolayers. In the present work, we have detected intrinsic ability of a peptide based on the sequence of the N-terminal segment of SP-C to interact and insert spontaneously into preformed zwitterionic....... These results demonstrate that the sequence of the SP-C N-terminal region has intrinsic ability to interact with, insert into, and perturb the structure of zwitterionic and anionic phospholipid films, even in the absence of the palmitic chains attached to this segment in the native protein. This effect has been...

  7. Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC).

    Science.gov (United States)

    Staes, An; Van Damme, Petra; Helsens, Kenny; Demol, Hans; Vandekerckhove, Joël; Gevaert, Kris

    2008-04-01

    We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.

  8. Functional characterization of a special thermophilic multifunctional amylase OPMA-N and its N-terminal domain

    Institute of Scientific and Technical Information of China (English)

    Fan Li; Xuejun Zhu; Yanfei Li; Hao Cao; Yingjiu Zhang

    2011-01-01

    A gene encoding a special thermophilic muitifunetional amylase OPMA-N was cloned from Bacillus sp. ZW25311. OPMA-N has an additional 124-residue N-terminal domain compared with typical amylases and forms a relatively independent domain with a IS-pleated sheet and random coil structure. Here we reported an unusual substrate and product specificities of OPMA-N and the impact of the additional N-terminal domain (1-124 aa) on the function and properties of OPMA-N. Both OPMAN (12.82 U/mg) and its N-terminal domain-truncated AOPMA-N (12.55 U/mg) only degraded starch to produce oligosaccharides including maltose, maltotriose, isomaitotriose, and isomaitotetraose, but not to produce glucose. Therefore, the N-terminal domain did not determine its substrate and product specificities that were probably regulated by its C-terminal IS-pleated sheet structure. However, the N-terminal domain of OPMA-N seemed to modulate its catalytic feature, leading to the production of more isomaitotriose and less maltose, and it seemed to contribute to OPMA-N's thermostability since OPMA-N showed higher activity than AOPMA-N in a temperature range from 40 to 80~C and the halflife (tl) was 5 h for OPMA-N and 2 h for AOPMA-N at 60~C. Both OPMA-N and AOPMA-N were Ca-independent, but their activities could be influenced by Cu2+, Niz+, Zn2+, EDTA, SDS (1 mM), or Triton-X100 (1%). Kinetic analysis and starch-adsorption assay indicated that the N-terminal domain of OPMA-N could increase the OPMA-N-starch binding and subsequently increase the catalytic efficiency of OPMA-N for starch. In particular, the N-terminal domain of OPMA-N did not determine its oligomerization, because both OPMA-N and AOPMA-N could exist in the forms of monomer, homodimer, and homooligomer at the same time.

  9. PrP N-terminal domain triggers PrPSc-like aggregation of Dpl

    International Nuclear Information System (INIS)

    Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrPC, for 'cellular prion protein') into an abnormal state (PrPSc, for 'scrapie prion protein'). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrPC. In contrast to its homologue PrPC, Dpl is unable to participate in prion disease progression or to achieve an abnormal PrPSc-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrPC (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the α-helical monomer forms soluble β-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy

  10. N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis

    International Nuclear Information System (INIS)

    A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1-24) and a basic domain (KKRPKP, residues 25-30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp-DNA-gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide's ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases

  11. Jun N-Terminal Protein Kinase Enhances Middle Ear Mucosal Proliferation during Bacterial Otitis Media▿

    Science.gov (United States)

    Furukawa, Masayuki; Ebmeyer, Jörg; Pak, Kwang; Austin, Darrell A.; Melhus, Åsa; Webster, Nicholas J. G.; Ryan, Allen F.

    2007-01-01

    Mucosal hyperplasia is a characteristic component of otitis media. The present study investigated the participation of signaling via the Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase in middle ear mucosal hyperplasia in animal models of bacterial otitis media. Otitis media was induced by the inoculation of nontypeable Haemophilus influenzae into the middle ear cavity. Western blotting revealed that phosphorylation of JNK isoforms in the middle ear mucosa preceded but paralleled mucosal hyperplasia in this in vivo rat model. Nuclear JNK phosphorylation was observed in many cells of both the mucosal epithelium and stroma by immunohistochemistry. In an in vitro model of primary rat middle ear mucosal explants, bacterially induced mucosal growth was blocked by the Rac/Cdc42 inhibitor Clostridium difficile toxin B, the mixed-lineage kinase inhibitor CEP11004, and the JNK inhibitor SP600125. Finally, the JNK inhibitor SP600125 significantly inhibited mucosal hyperplasia during in vivo bacterial otitis media in guinea pigs. Inhibition of JNK in vivo resulted in a diminished proliferative response, as shown by a local decrease in proliferating cell nuclear antigen protein expression by immunohistochemistry. We conclude that activation of JNK is a critical pathway for bacterially induced mucosal hyperplasia during otitis media, influencing tissue proliferation. PMID:17325051

  12. The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

    International Nuclear Information System (INIS)

    Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation

  13. Transcription-dependent nuclear localization of DAZAP1 requires an N-terminal signal

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yi-Tzu; Wen, Wan-Ching [Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan (China); Yen, Pauline H., E-mail: pyen@ibms.sinica.edu.tw [Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan (China)

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer DAZAP1 shuttles between the nucleus and the cytoplasm. Black-Right-Pointing-Pointer DAZAP1 accumulates in the cytoplasm when the nuclear transcription is inhibited. Black-Right-Pointing-Pointer DAZAP1's transcription-dependent nuclear localization requires N-terminal N42. Black-Right-Pointing-Pointer SLIRP binds to N42 and may be involved in the process. -- Abstract: Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous hnRNP protein required for normal development and spermatogenesis. It resides predominantly in the nucleus and moves between the nucleus and the cytoplasm via a ZNS shuttling signal at its C-terminus. DAZAP1 accumulates in the cytoplasm when RNA polymerase II activity is inhibited by actinomycin D. Here we report the mapping of a 42-amino acid segment (N42) at the N-terminus of DAZAP1 that is both necessary and sufficient for its transcription-dependent nuclear localization. In addition, using a yeast two-hybrid system, we have identified SLIRP as a N42-binding protein which may regulate DAZAP1 subcellular localization.

  14. Tor forms a dimer through an N-terminal helical solenoid with a complex topology.

    Science.gov (United States)

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M; Williams, Roger L

    2016-04-13

    The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended 'railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit.

  15. Tor forms a dimer through an N-terminal helical solenoid with a complex topology

    Science.gov (United States)

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M.; Williams, Roger L.

    2016-04-01

    The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended `railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit.

  16. Serum type III procollagen N-terminal peptide in coal miners.

    Science.gov (United States)

    Janssen, Y M; Engelen, J J; Giancola, M S; Low, R B; Vacek, P; Borm, P J

    1992-01-01

    Health surveillance of workers exposed to fibrogenic agents ideally should identify individuals at risk or detect pulmonary fibrosis in preclinical stages. We investigated serum procollagen type III N-terminal peptide (PIIIP) in several groups of active miners and in a nondust-exposed control group. The purpose of this study was to determine the applicability of PIIIP as an early noninvasive marker of pulmonary fibrosis in workers exposed to coal mine dust. PIIIP levels were significantly elevated in miners without radiological signs of coal workers pneumoconiosis (CWP) as compared with the nonexposed controls. However, in coal miners with CWP beyond ILO classification 1/0, PIIIP levels were not significantly different from nondust-exposed controls. Trend analysis within the miners group indicated a decrease in PIIIP levels with progression of the fibrosis. Our data suggest that detection of early lung fibrosis by measuring serum PIIIP values may be more sensitive than radiological diagnosis of CWP. However, follow-up of the control miners with respect to serum PIIIP and chest radiography is essential to validate PIIIP as a biological marker for CWP. PMID:1572317

  17. PLC-δ1-Lf, a novel N-terminal extended phospholipase C-δ1.

    Science.gov (United States)

    Kim, Na Young; Ahn, Sang Jung; Kim, Moo-Sang; Seo, Jung Soo; Kim, Bo Seong; Bak, Hye Jin; Lee, Jin Young; Park, Myoung-Ae; Park, Ju Hyeon; Lee, Hyung Ho; Chung, Joon Ki

    2013-10-10

    Phospholipase C-δ (PLC-δ), a key enzyme in phosphoinositide turnover, is involved in a variety of physiological functions. The widely expressed PLC-δ1 isoform is the best characterized and the most well understood phospholipase family member. However, the functional and molecular mechanisms of PLC-δ1 remain obscure. Here, we identified that the N-terminal region of mouse PLC-δ1 gene has two variants, a novel alternative splicing form, named as long form (mPLC-δ1-Lf) and the previously reported short form (mPLC-δ1-Sf), having exon 2 and exon 1, respectively, while both the gene variants share exons 3-16 for RNA transcription. Furthermore, the expression, identification and enzymatic characterization of the two types of PLC-δ1 genes were compared. Expression of mPLC-δ1-Lf was found to be tissue specific, whereas mPLC-δ1-Sf was widely distributed. The recombinant mPLC-δ1-Sf protein exhibited higher activity than recombinant mPLC-δ1-Lf protein. Although, the general catalytic and regulatory properties of mPLC-δ1-Lf are similar to those of PLC-δ1-Sf isozyme, the mPLC-δ1-Lf showed some distinct regulatory properties, such as tissue-specific expression and lipid binding specificity, particularly for phosphatidylserine.

  18. The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, J.Y.; Yang, L.X. [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Huang, Z.F. [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Key Laboratory of Tropical Diseases Control, Sun Yat-sen University, Ministry of Education in China, Guangzhou (China)

    2013-12-02

    Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation.

  19. Tor forms a dimer through an N-terminal helical solenoid with a complex topology

    Science.gov (United States)

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M.; Williams, Roger L.

    2016-01-01

    The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor–Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor–Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor–Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended ‘railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit. PMID:27072897

  20. Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter.

    Science.gov (United States)

    Lee, Youngjin; Min, Choon Kee; Kim, Tae Gyun; Song, Hong Ki; Lim, Yunki; Kim, Dongwook; Shin, Kahee; Kang, Moonkyung; Kang, Jung Youn; Youn, Hyung-Seop; Lee, Jung-Gyu; An, Jun Yop; Park, Kyoung Ryoung; Lim, Jia Jia; Kim, Ji Hun; Kim, Ji Hye; Park, Zee Yong; Kim, Yeon-Soo; Wang, Jimin; Kim, Do Han; Eom, Soo Hyun

    2015-10-01

    The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca(2+) uptake in a stable MCU knockdown HeLa cell line and exerted dominant-negative effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.

  1. NMR assignments of the N-terminal domain of Ogataea polymorpha telomerase reverse transcriptase.

    Science.gov (United States)

    Polshakov, Vladimir I; Petrova, Olga A; Parfenova, Yulia Yu; Efimov, Sergey V; Klochkov, Vladimir V; Zvereva, Maria I; Dontsova, Olga A

    2016-04-01

    Telomerase is a ribonucleoprotein enzyme that adds telomeric DNA fragments to the ends of chromosomes. This enzyme is the focus of substantial attention, both because its structure and mechanism of action are still poorly studied, and because of its pivotal roles in aging and cellular proliferation. The use of telomerase as a potential target for the design of new anticancer drugs is also of great interest. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is essential for activity and processivity. Elucidation of the structure and dynamics of TEN in solution is important for understanding the molecular mechanism of telomerase activity and for the design of new telomerase inhibitors. To approach this problem, in this study we report the (1)H, (13)C, and (15)N chemical shift assignments of TEN from Ogataea polymorpha. Analysis of the assigned chemical shifts allowed us to identify secondary structures and protein regions potentially involved in interaction with other participants of the telomerase catalytic cycle. PMID:26721464

  2. NMR structure of the N-terminal domain of the replication initiator protein DnaA

    Energy Technology Data Exchange (ETDEWEB)

    Wemmer, David E.; Lowery, Thomas J.; Pelton, Jeffrey G.; Chandonia, John-Marc; Kim, Rosalind; Yokota, Hisao; Wemmer, David E.

    2007-08-07

    DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.

  3. Cdc13 N-Terminal Dimerization DNA Binding and Telomere Length Regulation

    Energy Technology Data Exchange (ETDEWEB)

    M Mitchell; J Smith; M Mason; S Harper; D Speicher; F Johnson; E Skordalakes

    2011-12-31

    The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that is directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.

  4. Structure of the N-terminal region of Haemophilus Influenzae HI0017: Implications for function

    Energy Technology Data Exchange (ETDEWEB)

    Yu Liping; Mack, Jamey; Hajduk, Phil; Fesik, Stephen W. [Abbott Laboratories, Pharmaceutical Discovery Division, D46Y, AP10/LL (United States)

    2001-06-15

    Haemophilus influenzae is a gram-negative pathogen that causes infections ranging from asymptomatic colonization of the human upper respiratory tract to serious invasive diseases such as meningitis. Although the genome of Haemophilus influenzae has been completely sequenced, the structure and function of many of these proteins are unknown. HI0017 is one of these uncharacterized proteins. Here we describe the three-dimensional solution structure of the N-terminal portion of HI0017 as determined by NMR spectroscopy. The structure consists of a five-stranded antiparallel {beta}-sheet and two short {alpha}-helices. It is similar to the C-terminal domain of Diphtheria toxin repressor (DtxR). The C-terminal portion of HI0017 has an amino acid sequence that closely resembles pyruvate formate-lyase - an enzyme that converts pyruvate and CoA into acetyl-CoA and formate by a radical mechanism. Based on structural and sequence comparisons, we propose that the C-terminus of HI0017 functions as an enzyme with a glycyl radical mechanism, while the N-terminus participates in protein/protein interactions involving an activase (iron-sulfur protein) and/or the substrate.

  5. Tor forms a dimer through an N-terminal helical solenoid with a complex topology.

    Science.gov (United States)

    Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M; Williams, Roger L

    2016-01-01

    The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended 'railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit. PMID:27072897

  6. Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences.

    LENUS (Irish Health Repository)

    Ivanov, Ivaylo P

    2011-05-01

    In eukaryotes, it is generally assumed that translation initiation occurs at the AUG codon closest to the messenger RNA 5\\' cap. However, in certain cases, initiation can occur at codons differing from AUG by a single nucleotide, especially the codons CUG, UUG, GUG, ACG, AUA and AUU. While non-AUG initiation has been experimentally verified for a handful of human genes, the full extent to which this phenomenon is utilized--both for increased coding capacity and potentially also for novel regulatory mechanisms--remains unclear. To address this issue, and hence to improve the quality of existing coding sequence annotations, we developed a methodology based on phylogenetic analysis of predicted 5\\' untranslated regions from orthologous genes. We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences. Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated extensions in 17 other genes. In several instances, we have been able to obtain independent experimental evidence of the expression of non-AUG-initiated products from the previously published literature and ribosome profiling data.

  7. In vivo study on the effects of microcystin extracts on the expression profiles of proto-oncogenes (c-fos, c-jun and c-myc) in liver, kidney and testis of male Wistar rats injected i.v. with toxins.

    Science.gov (United States)

    Li, Huiying; Xie, Ping; Li, Guangyu; Hao, Le; Xiong, Qian

    2009-01-01

    Microcystins (MCs) are a potent liver tumor promoter, possessing potent tumor-promoting activity and weak initiating activity. Proto-oncogenes are known to be involved in the tumor-promoting mechanisms of microcystin-LR. However, few data are available on the effects of MCs on proto-oncogenes in the whole animal. To investigate the effects of MCs on the expression profile of the proto-oncogenes in different organs, male Wistar rats were injected intravenously with microcystin extracts at a dose of 86.7 mug MC-LR eq/kg bw (MC-LR eq, MC-LR equivalents). mRNA levels of three proto-oncogenes c-fos, c-jun and c-myc in liver, kidney and testis were analyzed using quantitative real-time PCR at several time points post-injection. Significant induction of these genes at transcriptional level was observed in the three organs. In addition, the increase of mRNA expression of all three genes was much higher in liver than in kidney and testis. Meanwhile, the protein levels of c-Fos and c-Jun were investigated by western blotting. Both proteins were induced in the three organs. However, elevations of protein levels were much lower than those of mRNA levels. These findings suggest that the expression of c-fos, c-jun and c-myc might be one possible mechanism for the tumor-promoting activity and initiating activity of microcystins.

  8. Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

    Energy Technology Data Exchange (ETDEWEB)

    Borko, Ľubomír; Bauerová-Hlinková, Vladena, E-mail: vladena.bauerova@savba.sk; Hostinová, Eva; Gašperík, Juraj [Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava (Slovakia); Beck, Konrad [Cardiff University School of Dentistry, Heath Park, Cardiff CF14 4XY Wales (United Kingdom); Lai, F. Anthony [Cardiff University School of Medicine, Cardiff CF14 4XN Wales (United Kingdom); Zahradníková, Alexandra, E-mail: vladena.bauerova@savba.sk [Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava (Slovakia); Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava (Slovakia); Ševčík, Jozef, E-mail: vladena.bauerova@savba.sk [Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava (Slovakia)

    2014-11-01

    X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix. Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C{sup α} atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.

  9. Dimeric structure of the N-terminal domain of PriB protein from Thermoanaerobacter tengcongensis solved ab initio

    Energy Technology Data Exchange (ETDEWEB)

    Liebschner, Dorothee [National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States); Brzezinski, Krzysztof [National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States); University of Bialystok, 15-399 Bialystok (Poland); Dauter, Miroslawa [Argonne National Laboratory, Argonne, IL 60439 (United States); Dauter, Zbigniew, E-mail: dauter@anl.gov [National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States); Nowak, Marta; Kur, Józef; Olszewski, Marcin, E-mail: dauter@anl.gov [Gdansk University of Technology, 80-952 Gdansk (Poland); National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States)

    2012-12-01

    The N-terminal domain of the PriB protein from the thermophilic bacterium T. tengcongensis (TtePriB) was expressed and its crystal structure has been solved at the atomic resolution of 1.09 Å by direct methods. PriB is one of the components of the bacterial primosome, which catalyzes the reactivation of stalled replication forks at sites of DNA damage. The N-terminal domain of the PriB protein from the thermophilic bacterium Thermoanaerobacter tengcongensis (TtePriB) was expressed and its crystal structure was solved at the atomic resolution of 1.09 Å by direct methods. The protein chain, which encompasses the first 104 residues of the full 220-residue protein, adopts the characteristic oligonucleotide/oligosaccharide-binding (OB) structure consisting of a five-stranded β-barrel filled with hydrophobic residues and equipped with four loops extending from the barrel. In the crystal two protomers dimerize, forming a six-stranded antiparallel β-sheet. The structure of the N-terminal OB domain of T. tengcongensis shows significant differences compared with mesophile PriBs. While in all other known structures of PriB a dimer is formed by two identical OB domains in separate chains, TtePriB contains two consecutive OB domains in one chain. However, sequence comparison of both the N-terminal and the C-terminal domains of TtePriB suggests that they have analogous structures and that the natural protein possesses a structure similar to a dimer of two N-terminal domains.

  10. The effect of hedgehog signaling pathway on c - jun, uPA expression of breast cancer cells MDA- MB - 231%Hedgehog通路对乳腺癌细胞MDA—MB-231c—jun、uPA表达的影响和机制

    Institute of Scientific and Technical Information of China (English)

    隆玲; 邓华瑜; 姜容; 陈黎

    2011-01-01

    目的:研究Hedgehog信号转导通路对人乳腺癌细胞MDA—MB-231c—jun、uPA表达的影响和作用机制,认识其在侵袭转移中的意义。方法:应用Hedgehog信号通路抑制剂Cyclopamine作用人乳腺癌细胞MDA—MB-231,Transwell小室进行侵袭迁移实验;Western blotting检测P—c—itin、uPA蛋白的水平:RT—PCR检测Gli1、c—jun和uPA的mRNA水平。结果:应用Cyclopamine后,细胞的侵袭迁移能力明显下降;P—c—Jun、uPA蛋白表达减少;Gli1、c—iun和uPAmRNA水平降低。结论:Hedgehog信号通路可通过活化c—jun上调uPA的表达,促进乳腺癌细胞MDA—MB-231细胞的侵袭转移。%Objective: To explore the effect and the possible mechanisms of Hedgehog singaling pathway on expression of c - jun, uPA in MDA - MB - 231 cells. Methods: Invasion and metastasis of MDA- MB- 231 cells were evaluated with transwell chamber.The expression of P- c- jun and uPA were detected by Western blot. The mRNA levels of Gli 1, c - jun and uPA were measured by reverse transcriplion - poly - merage chain reaction ( RT - PCR ). Results : When MDA - MB - 231 cells were treated with Cyclopamine, invasion and metastasis were depressed. The expression level of P- c- Jun and uPA were decreased. The mRNA levels of Ghl, c-jun and uPA were also reduced. Conclusion: invasion, metastasis and uPA level can be induced by activeled Hedgehog singnaling nathwav with activation of c - iun.

  11. Production and applications of an N-terminally-truncated recombinant beta-haemolysin from Staphylococcus aureus.

    Science.gov (United States)

    Singh, M; Singh, A; Sharma, A

    2014-07-01

    The beta-haemolysin of Staphylococcus aureus (SA-hlb) is a secreted neutral sphingomyelinase (nSMase) implicated in the pathogenesis of infection and responsible for the characteristic in vitro 'hot-cold' haemolytic ability of the bacterium. Here, we describe the production of a biologically active N-terminally-truncated recombinant SA-hlb protein for use in in vitro assays and as a research tool. Using local isolates of S. aureus, we PCR-amplified an SA-hlb DNA sequence of 891 nucleotides, 99 nucleotides shorter than the full-length molecule, before cloning and sequencing (GenBank accession no. JN580071). The pQE.TriSystem vector (Qiagen, Germany) was used to express recombinant SA-hlb (r-SA-hlb) with a C-terminal 8xHis tag in Escherichia coli JM107 cells. Both JM107 lysate and the purified r-SA-hlb possessed hot-cold lytic activity against sheep and buffalo erythrocytes, which was abolished by incubation at ≥90 °C for 30 min or exposure to dithiothreitol, and could be neutralized by bovine immune sera. Purified r-SA-hlb was also cytotoxic to buffalo mononuclear cells and was effective as a coating antigen for indirect ELISA to screen for reactive sera. Importantly, the r-SA-hlb was suitable for use as a β-toxin in the modified CAMP test. We conclude that the r-SA-hlb protein produced was functionally active and has numerous potential applications.

  12. Contributions of the RAD51 N-terminal domain to BRCA2-RAD51 interaction.

    Science.gov (United States)

    Subramanyam, Shyamal; Jones, William T; Spies, Maria; Spies, M Ashley

    2013-10-01

    RAD51 DNA strand exchange protein catalyzes the central step in homologous recombination, a cellular process fundamentally important for accurate repair of damaged chromosomes, preservation of the genetic integrity, restart of collapsed replication forks and telomere maintenance. BRCA2 protein, a product of the breast cancer susceptibility gene, is a key recombination mediator that interacts with RAD51 and facilitates RAD51 nucleoprotein filament formation on single-stranded DNA generated at the sites of DNA damage. An accurate atomistic level description of this interaction, however, is limited to a partial crystal structure of the RAD51 core fused to BRC4 peptide. Here, by integrating homology modeling and molecular dynamics, we generated a structure of the full-length RAD51 in complex with BRC4 peptide. Our model predicted previously unknown hydrogen bonding patterns involving the N-terminal domain (NTD) of RAD51. These interactions guide positioning of the BRC4 peptide within a cavity between the core and the NTDs; the peptide binding separates the two domains and restricts internal dynamics of RAD51 protomers. The model's depiction of the RAD51-BRC4 complex was validated by free energy calculations and in vitro functional analysis of rationally designed mutants. All generated mutants, RAD51(E42A), RAD51(E59A), RAD51(E237A), RAD51(E59A/E237A) and RAD51(E42A/E59A/E237A) maintained basic biochemical activities of the wild-type RAD51, but displayed reduced affinities for the BRC4 peptide. Strong correlation between the calculated and experimental binding energies confirmed the predicted structure of the RAD51-BRC4 complex and highlighted the importance of RAD51 NTD in RAD51-BRCA2 interaction. PMID:23935068

  13. Membrane binding of prion protein N-terminal peptides characterised by neutron reflectometry

    International Nuclear Information System (INIS)

    The prion protein (PrP) is widely recognised to mis-fold into the causative agent of the transmissible spongiform encephalopathies, known as Creutzfeldt–Jakob disease (CJD) in humans, scrappie in sheep or Bovine spongiform encephalopathy in cows (BSE, “mad cow disease”). PrP has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the mis-folding events associated with prion pathogenesis, PrP can undergo various post-translational modifications, including internal cleavage events. Alpha and beta-cleavage of PrP produces two N-terminal fragments, N1 and N2 respectively, which interact specifically with negatively charged phospholipids at low pH. Previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption [1]. This work aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, neutron reflectometry was used to define the structural details of the interactions in combination with quartz crystal microbalance interrogation and calcein release assays. Neutron reflectometry confirmed that peptides equivalent to N1 and N2 insert into the interstitial space between the phospholipid headgroups but do not penetrate into the acyl tail region. In accord with previous studies, interactions were stronger for the N1 fragment than for the N2, with more peptide bound per lipid. Neutron reflectometry analysis also detected lengthening of the lipid acyl tails, with a concurrent decrease in lipid area. Overall, the data shows that the N1 and N2 peptides interact with the anionic phospholipid headgroups of supported lipid bilayers, inducing lipid ordering in the absence of significant penetration into the acyl tails or permeation of the membrane.

  14. Characterization of niphatenones that inhibit androgen receptor N-terminal domain.

    Directory of Open Access Journals (Sweden)

    Carmen A Banuelos

    Full Text Available Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC. The androgen receptor (AR remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD. Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD. Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S-niphatenone had significantly better activity against the AR NTD compared to (R-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR activity and covalently bound to GR activation function-1 (AF-1 region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.

  15. Structural modeling of the N-terminal signal–receiving domain of IκBα

    Directory of Open Access Journals (Sweden)

    Samira eYazdi

    2015-06-01

    Full Text Available The transcription factor nuclear factor-κB (NF-κB exerts essential roles in many biological processes including cell growth, apoptosis and innate and adaptive immunity. The NF-kB inhibitor (IκBα retains NF-κB in the cytoplasm and thus inhibits nuclear localization of NF-κB and its association with DNA. Recent protein crystal structures of the C-terminal part of IκBα in complex with NF-κB provided insights into the protein-protein interactions but could not reveal structural details about the N-terminal signal receiving domain (SRD. The SRD of IκBα contains a degron, formed following phosphorylation by IκB kinases (IKK. In current protein X-ray structures, however, the SRD is not resolved and assumed to be disordered. Here, we combined secondary structure annotation and domain threading followed by long molecular dynamics (MD simulations and showed that the SRD possesses well-defined secondary structure elements. We show that the SRD contains 3 additional stable α-helices supplementing the six ARDs present in crystallized IκBα. The IκBα/NF-κB protein-protein complex remained intact and stable during the entire simulations. Also in solution, free IκBα retains its structural integrity. Differences in structural topology and dynamics were observed by comparing the structures of NF-κB free and NF-κB bound IκBα-complex. This study paves the way for investigating the signaling properties of the SRD in the IκBα degron. A detailed atomic scale understanding of molecular mechanism of NF-κB activation, regulation and the protein-protein interactions may assist to design and develop novel chronic inflammation modulators.

  16. N-terminal Slit2 inhibits HIV-1 replication by regulating the actin cytoskeleton

    Directory of Open Access Journals (Sweden)

    Anand Appakkudal R

    2013-01-01

    Full Text Available Abstract Background Slit2 is a ~ 200 kDa secreted glycoprotein that has been recently shown to regulate immune functions. However, not much is known about its role in HIV (human immunodeficiency virus-1 pathogenesis. Results In the present study, we have shown that the N-terminal fragment of Slit2 (Slit2N (~120 kDa inhibits replication of both CXCR4 and CCR5-tropic HIV-1 viruses in T-cell lines and peripheral blood T-cells. Furthermore, we demonstrated inhibition of HIV-1 infection in resting CD4+ T-cells. In addition, we showed that Slit2N blocks cell-to-cell transmission of HIV-1. We have shown that Slit2N inhibits HIV-1 infection by blocking viral entry into T-cells. We also ruled out Slit2N-mediated inhibition of various other steps in the life cycle including binding, integration and viral transcription. Elucidation of the molecular mechanism revealed that Slit2N mediates its functional effects by binding to Robo1 receptor. Furthermore, we found that Slit2N inhibited Gp120-induced Robo1-actin association suggesting that Slit2N may inhibit cytoskeletal rearrangements facilitating HIV-1 entry. Studies into the mechanism of inhibition of HIV-1 revealed that Slit2N abrogated HIV-1 envelope-induced actin cytoskeletal dynamics in both T-cell lines and primary T-cells. We further showed that Slit2N specifically attenuated the HIV-1 envelope-induced signaling pathway consisting of Rac1, LIMK and cofilin that regulates actin polymerization. Conclusions Taken together, our results show that Slit2N inhibits HIV-1 replication through novel mechanisms involving modulation of cytoskeletal dynamics. Our study, thus, provides insights into the role of Slit2N in HIV-1 infection and underscores its potential in limiting viral replication in T-cells.

  17. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    Energy Technology Data Exchange (ETDEWEB)

    Gottipati, Keerthi; Acholi, Sudheer [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States); Ruggli, Nicolas [Institute of Virology and Immunology, CH-3147 Mittelhäusern (Switzerland); Choi, Kyung H., E-mail: kychoi@utmb.edu [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States)

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  18. Endogenous N-terminal Domain Cleavage Modulates α1D-Adrenergic Receptor Pharmacodynamics.

    Science.gov (United States)

    Kountz, Timothy S; Lee, Kyung-Soon; Aggarwal-Howarth, Stacey; Curran, Elizabeth; Park, Ji-Min; Harris, Dorathy-Ann; Stewart, Aaron; Hendrickson, Joseph; Camp, Nathan D; Wolf-Yadlin, Alejandro; Wang, Edith H; Scott, John D; Hague, Chris

    2016-08-26

    The α1D-adrenergic receptor (ADRA1D) is a key regulator of cardiovascular, prostate, and central nervous system functions. This clinically relevant G protein-coupled receptor has proven difficult to study, as it must form an obligate modular homodimer containing the PDZ proteins scribble and syntrophin or become retained in the endoplasmic reticulum as non-functional protein. We previously determined that targeted removal of the N-terminal (NT) 79 amino acids facilitates ADRA1D plasma membrane expression and agonist-stimulated functional responses. However, whether such an event occurs in physiological contexts was unknown. Herein, we report the ADRA1D is subjected to innate NT processing in cultured human cells. SNAP near-infrared imaging and tandem-affinity purification revealed the ADRA1D is expressed as both full-length and NT truncated forms in multiple human cell lines. Serial truncation mapping identified the cleavage site as Leu(90)/Val(91) in the 95-amino acid ADRA1D NT domain, suggesting human cells express a Δ1-91 ADRA1D species. Tandem-affinity purification MS/MS and co-immunoprecipitation analysis indicate NT processing of ADRA1D is not required to form scribble-syntrophin macromolecular complexes. Yet, label-free dynamic mass redistribution signaling assays demonstrate that Δ1-91 ADRA1D agonist responses were greater than WT ADRA1D. Mutagenesis of the cleavage site nullified the processing event, resulting in ADRA1D agonist responses less than the WT receptor. Thus, we propose that processing of the ADRA1D NT domain is a physiological mechanism employed by cells to generate a functional ADRA1D isoform with optimal pharmacodynamic properties. PMID:27382054

  19. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

    Science.gov (United States)

    Obergfell, Kyle P; Seifert, H Steven

    2016-05-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3' third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants

  20. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation

    Science.gov (United States)

    2016-01-01

    The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic

  1. The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

    Directory of Open Access Journals (Sweden)

    Kyle P Obergfell

    2016-05-01

    Full Text Available The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3' third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated

  2. Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

    Science.gov (United States)

    Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

    2014-01-01

    Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS.

  3. N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

    Institute of Scientific and Technical Information of China (English)

    Yan-Chang Lei; Dong-Liang Yang; Yong-Jun Tian; Hong-Hui Ding; Bao-Ju Wang; Yan Yang; You-Hua Hao; Xi-Ping Zhao; Meng-Ji Lu; Fei-Li Gong

    2006-01-01

    AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G(APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo.METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA.The expression of hepatitis B virus core antigen (HBcAg)in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively.RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells,and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly,the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls.CONCLUSION: Our findings provide probably the first

  4. N-terminal arginines modulate plasma-membrane localization of Kv7.1/KCNE1 channel complexes.

    Directory of Open Access Journals (Sweden)

    Zenawit Girmatsion

    Full Text Available BACKGROUND AND OBJECTIVE: The slow delayed rectifier current (I(Ks is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 β-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced I(Ks and atrial fibrillation (a human arrhythmia. Structure-function relationship of the KCNE1 N-terminus for I(Ks modulation is poorly understood and was subject of this study. METHODS: We studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy. RESULTS: All KCNE1 constructs physically interacted with Kv7.1. I(Ks resulting from co-expression of Kv7.1 with non-atrial fibrillation '38S' was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions ('38G-3xA' were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct ('Δ1-38'. Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct. CONCLUSIONS: The results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of I(Ks. Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex.

  5. Structure of a double hexamer of the Pyrococcus furiosus minichromosome maintenance protein N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Meagher, Martin; Enemark, Eric J.

    2016-06-22

    The crystal structure of the N-terminal domain of thePyrococcus furiosusminichromosome maintenance (MCM) protein as a double hexamer is described. The MCM complex is a ring-shaped helicase that unwinds DNA at the replication fork of eukaryotes and archaea. Prior to replication initiation, the MCM complex assembles as an inactive double hexamer at specific sites of DNA. The presented structure is highly consistent with previous MCM double-hexamer structures and shows two MCM hexamers with a head-to-head interaction mediated by the N-terminal domain. Minor differences include a diminished head-to-head interaction and a slightly reduced inter-hexamer rotation.

  6. ELKS controls the pool of readily releasable vesicles at excitatory synapses through its N-terminal coiled-coil domains.

    Science.gov (United States)

    Held, Richard G; Liu, Changliang; Kaeser, Pascal S

    2016-06-02

    In a presynaptic nerve terminal, synaptic strength is determined by the pool of readily releasable vesicles (RRP) and the probability of release (P) of each RRP vesicle. These parameters are controlled at the active zone and vary across synapses, but how such synapse specific control is achieved is not understood. ELKS proteins are enriched at vertebrate active zones and enhance P at inhibitory hippocampal synapses, but ELKS functions at excitatory synapses are not known. Studying conditional knockout mice for ELKS, we find that ELKS enhances the RRP at excitatory synapses without affecting P. Surprisingly, ELKS C-terminal sequences, which interact with RIM, are dispensable for RRP enhancement. Instead, the N-terminal ELKS coiled-coil domains that bind to Liprin-α and Bassoon are necessary to control RRP. Thus, ELKS removal has differential, synapse-specific effects on RRP and P, and our findings establish important roles for ELKS N-terminal domains in synaptic vesicle priming.

  7. N-terminal pro-B-type natriuretic peptide and long-term mortality in stable coronary heart disease

    DEFF Research Database (Denmark)

    Kragelund, Charlotte; Grønning, Bjørn; Køber, Lars;

    2005-01-01

    BACKGROUND: The level of the inactive N-terminal fragment of pro-brain (B-type) natriuretic peptide (BNP) is a strong predictor of mortality among patients with acute coronary syndromes and may be a strong prognostic marker in patients with chronic coronary heart disease as well. We assessed...... the relationship between N-terminal pro-BNP (NT-pro-BNP) levels and long-term mortality from all causes in a large cohort of patients with stable coronary heart disease. METHODS: NT-pro-BNP was measured in baseline serum samples from 1034 patients referred for angiography because of symptoms or signs of coronary...... of myocardial infarction, angina, hypertension, diabetes, or chronic heart failure; creatinine clearance rate; body-mass index; smoking status; plasma lipid levels; LVEF; and the presence or absence of clinically significant coronary artery disease on angiography. CONCLUSIONS: NT-pro-BNP is a marker of long...

  8. [Chemical synthesis of lactococcin B and functional evaluation of the N-terminal domain using a truncated synthetic analogue].

    Science.gov (United States)

    Lasta, S; Fajloun, Z; Mansuelle, P; Sabatier, J M; Boudabous, A; Sampieri, F

    2008-01-01

    The lactococcin B (LnB) is a hydrophobic, positively charged bacteriocin, produced by Lactococcus lactis ssp. cremoris 9B4. It consists of a peptidic chain made up of 47 amino acid residues, and inhibits Lactococcus exclusively. In order to study its biological activity a synthetic lactococcin B (LnBs) was obtained by solid-phase chemical synthesis using a Fmoc strategy. LnBs was shown to be indistinguishable from the natural peptide. In addition, a synthetic (7-47) LnBst analogue was obtained by withdrawal of peptidyl-resin after the 41 cycle of LnBs peptide chain assembly. The synthetic N-terminal truncated (7-47) LnBst analogue was found to be inactive on indicator strains. Our results strongly suggest that the first six N-terminal amino acid residues are involved in the bactericidal activity of LnB.

  9. The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites.

    Science.gov (United States)

    Saur, Isabel Marie-Luise; Conlan, Brendon Francis; Rathjen, John Paul

    2015-05-01

    Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats).

  10. The metalloid arsenite induces nuclear export of Id3 possibly via binding to the N-terminal cysteine residues

    Energy Technology Data Exchange (ETDEWEB)

    Kurooka, Hisanori, E-mail: hkurooka@u-fukui.ac.jp [Division of Molecular Genetics, Department of Biochemistry and Bioinformative Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Research and Education Program for Life Science, University of Fukui, Fukui (Japan); Sugai, Manabu [Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto (Japan); Mori, Kentaro [Division of Molecular Genetics, Department of Biochemistry and Bioinformative Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Yokota, Yoshifumi, E-mail: yokota@u-fukui.ac.jp [Division of Molecular Genetics, Department of Biochemistry and Bioinformative Sciences, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Research and Education Program for Life Science, University of Fukui, Fukui (Japan)

    2013-04-19

    Highlights: •Sodium arsenite induces cytoplasmic accumulation of Id3. •Arsenite binds to closely spaced N-terminal cysteine residues of Id3. •N-terminal cysteines are essential for arsenite-induced nuclear export of Id3. •Nuclear export of Id3 counteracts its transcriptional repression activity. -- Abstract: Ids are versatile transcriptional repressors that regulate cell proliferation and differentiation, and appropriate subcellular localization of the Id proteins is important for their functions. We previously identified distinct functional nuclear export signals (NESs) in Id1 and Id2, but no active NES has been reported in Id3. In this study, we found that treatment with the stress-inducing metalloid arsenite led to the accumulation of GFP-tagged Id3 in the cytoplasm. Cytoplasmic accumulation was impaired by a mutation in the Id3 NES-like sequence resembling the Id1 NES, located at the end of the HLH domain. It was also blocked by co-treatment with the CRM1-specific nuclear export inhibitor leptomycin B (LMB), but not with the inhibitors for mitogen-activated protein kinases (MAPKs). Importantly, we showed that the closely spaced N-terminal cysteine residues of Id3 interacted with the arsenic derivative phenylarsine oxide (PAO) and were essential for the arsenite-induced cytoplasmic accumulation, suggesting that arsenite induces the CRM1-dependent nuclear export of Id3 via binding to the N-terminal cysteines. Finally, we demonstrated that Id3 significantly repressed arsenite-stimulated transcription of the immediate-early gene Egr-1 and that this repression activity was inversely correlated with the arsenite-induced nuclear export. Our results imply that Id3 may be involved in the biological action of arsenite.

  11. The metalloid arsenite induces nuclear export of Id3 possibly via binding to the N-terminal cysteine residues

    International Nuclear Information System (INIS)

    Highlights: •Sodium arsenite induces cytoplasmic accumulation of Id3. •Arsenite binds to closely spaced N-terminal cysteine residues of Id3. •N-terminal cysteines are essential for arsenite-induced nuclear export of Id3. •Nuclear export of Id3 counteracts its transcriptional repression activity. -- Abstract: Ids are versatile transcriptional repressors that regulate cell proliferation and differentiation, and appropriate subcellular localization of the Id proteins is important for their functions. We previously identified distinct functional nuclear export signals (NESs) in Id1 and Id2, but no active NES has been reported in Id3. In this study, we found that treatment with the stress-inducing metalloid arsenite led to the accumulation of GFP-tagged Id3 in the cytoplasm. Cytoplasmic accumulation was impaired by a mutation in the Id3 NES-like sequence resembling the Id1 NES, located at the end of the HLH domain. It was also blocked by co-treatment with the CRM1-specific nuclear export inhibitor leptomycin B (LMB), but not with the inhibitors for mitogen-activated protein kinases (MAPKs). Importantly, we showed that the closely spaced N-terminal cysteine residues of Id3 interacted with the arsenic derivative phenylarsine oxide (PAO) and were essential for the arsenite-induced cytoplasmic accumulation, suggesting that arsenite induces the CRM1-dependent nuclear export of Id3 via binding to the N-terminal cysteines. Finally, we demonstrated that Id3 significantly repressed arsenite-stimulated transcription of the immediate-early gene Egr-1 and that this repression activity was inversely correlated with the arsenite-induced nuclear export. Our results imply that Id3 may be involved in the biological action of arsenite

  12. Crystallization and preliminary X-ray crystallographic analysis of yeast prion protein Ure2p with shortened N-terminal

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    An orthorhombic crystal form of a recombinant yeast prion protein with shortened N-terminal, 90Ure2p, has been obtained. Crystals were grown by the vapordiffusion technique against a mother liquor containing imidazole. Crystals belong to the primitive orthorhombic lattice with the cell parameters a = 54.5 ?, b = 74.7 ?, c = 131.0 ?. The crystals diffract to beyond 3.0 ? resolution at a synchrotron beamline.

  13. Exercise Dependence of N-Terminal Pro-Brain Natriuretic Peptide in Patients with Precapillary Pulmonary Hypertension

    OpenAIRE

    Grachtrup, Sabine; Brügel, Mathias; Pankau, Hans; Halank, Michael; Wirtz, Hubert; Seyfarth, Hans-Jürgen

    2014-01-01

    Background: N-terminal pro-brain natriuretic peptide (NT-proBNP) is secreted by cardiac ventricular myocytes upon pressure and volume overload and is a prognostic marker to monitor the severity of precapillary pulmonary hypertension and the extent of right heart failure. Objectives: The impact of physical exercise on NT-proBNP levels in patients with left heart disease was demonstrated previously. No data regarding patients with isolated right heart failure and the influence of acute exer...

  14. Detection of left ventricular enlargement and impaired systolic function with plasma N-terminal pro brain natriuretic peptide concentrations

    DEFF Research Database (Denmark)

    Grønning, Bjørn Aaris; Nilsson, Jens C.; Søndergaard, Lars;

    2002-01-01

    BACKGROUND: Brain- and N-terminal pro brain natriuretic peptide (NT-proBNP) have been identified as promising markers for heart failure. However, previous studies have revealed that they may hold insufficient diagnostic power for implementation into clinical practice because of a significant......); sensitivity/specificity, 85%/86%), and LV ejection fraction (powerful marker for LV dimensions and systolic function in patients with heart failure and discriminates well between...

  15. The first N-terminal unprotected (Gly-Aib)n peptide: H-Gly-Aib-Gly-Aib-OtBu.

    Science.gov (United States)

    Gessmann, Renate; Brückner, Hans; Petratos, Kyriacos

    2015-12-01

    Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (nonribosomally biosynthesized antibiotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (α-aminoisobutyric acid) is severely restricted by the second methyl group attached to the Cα atom. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N- or C-terminus of peptides may alter the hydrogen-bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid tert-butyl ester, C16H30N4O5, describes the first N-terminal-unprotected (Gly-Aib)n peptide. The achiral peptide could form an intramolecular hydrogen bond between the C=O group of Gly1 and the N-H group of Aib4. This hydrogen bond is found in all tetrapeptides and N-terminal-protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N...O = 5.88 Å). Instead, every molecule is hydrogen bonded to six other symmetry-related molecules with a total of eight hydrogen bonds per molecule. The backbone conformation starts in the right-handed helical region (and the left-handed helical region for the inverted molecule) and reverses the screw sense in the last two residues. PMID:26632841

  16. N-terminal aromatic residues closely impact the cytolytic activity of cupiennin 1a, a major spider venom peptide.

    Science.gov (United States)

    Kuhn-Nentwig, Lucia; Sheynis, Tania; Kolusheva, Sofiya; Nentwig, Wolfgang; Jelinek, Raz

    2013-12-01

    Cupiennins are small cationic α-helical peptides from the venom of the ctenid spider Cupiennius salei which are characterized by high bactericidal as well as hemolytic activities. To gain insight into the determinants responsible for the broad cytolytic activities, two analogues of cupiennin 1a with different N-terminal hydrophobicities were designed. The insecticidal, bactericidal and hemolytic activities of these analogues were assayed and compared to the native peptide. Specifically, substitution of two N-terminal Phe residues by Ala results in less pronounced insecticidal and cytolytic activity, whereas a substitution by Lys reduces strongly its bactericidal activity and completely diminishes its hemolytic activity up to very high tested concentrations. Biophysical analyses of peptide/bilayer membrane interactions point to distinct interactions of the analogues with lipid bilayers, and dependence upon membrane surface charge. Indeed, we find that lower hemolytic activity was correlated with less surface association of the analogues. In contrast, our data indicate that the reduced bactericidal activity of the two cupiennin 1a analogues likely correspond to greater bilayer-surface localization of the peptides. Overall, ultimate insertion and destruction of the host cell membrane is highly dependent on the presence of Phe-2 and Phe-6 (Cu 1a) or Leu-6 (Cu 2a) in the N-terminal sequences of native cupiennins.

  17. The first N-terminal unprotected (Gly-Aib)n peptide: H-Gly-Aib-Gly-Aib-OtBu.

    Science.gov (United States)

    Gessmann, Renate; Brückner, Hans; Petratos, Kyriacos

    2015-12-01

    Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (nonribosomally biosynthesized antibiotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (α-aminoisobutyric acid) is severely restricted by the second methyl group attached to the Cα atom. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N- or C-terminus of peptides may alter the hydrogen-bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid tert-butyl ester, C16H30N4O5, describes the first N-terminal-unprotected (Gly-Aib)n peptide. The achiral peptide could form an intramolecular hydrogen bond between the C=O group of Gly1 and the N-H group of Aib4. This hydrogen bond is found in all tetrapeptides and N-terminal-protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N...O = 5.88 Å). Instead, every molecule is hydrogen bonded to six other symmetry-related molecules with a total of eight hydrogen bonds per molecule. The backbone conformation starts in the right-handed helical region (and the left-handed helical region for the inverted molecule) and reverses the screw sense in the last two residues.

  18. Protective epitopes of the Plasmodium falciparum SERA5 malaria vaccine reside in intrinsically unstructured N-terminal repetitive sequences.

    Directory of Open Access Journals (Sweden)

    Masanori Yagi

    Full Text Available The malaria vaccine candidate antigen, SE36, is based on the N-terminal 47 kDa domain of Plasmodium falciparum serine repeat antigen 5 (SERA5. In epidemiological studies, we have previously shown the inhibitory effects of SE36 specific antibodies on in vitro parasite growth and the negative correlation between antibody level and malaria symptoms. A phase 1 b trial of the BK-SE36 vaccine in Uganda elicited 72% protective efficacy against symptomatic malaria in children aged 6-20 years during the follow-up period 130-365 days post-second vaccination. Here, we performed epitope mapping with synthetic peptides covering the whole sequence of SE36 to identify and map dominant epitopes in Ugandan adult serum presumed to have clinical immunity to P. falciparum malaria. High titer sera from the Ugandan adults predominantly reacted with peptides corresponding to two successive N-terminal regions of SERA5 containing octamer repeats and serine rich sequences, regions of SERA5 that were previously reported to have limited polymorphism. Affinity purified antibodies specifically recognizing the octamer repeats and serine rich sequences exhibited a high antibody-dependent cellular inhibition (ADCI activity that inhibited parasite growth. Furthermore, protein structure predictions and structural analysis of SE36 using spectroscopic methods indicated that N-terminal regions possessing inhibitory epitopes are intrinsically unstructured. Collectively, these results suggest that strict tertiary structure of SE36 epitopes is not required to elicit protective antibodies in naturally immune Ugandan adults.

  19. Protective epitopes of the Plasmodium falciparum SERA5 malaria vaccine reside in intrinsically unstructured N-terminal repetitive sequences.

    Science.gov (United States)

    Yagi, Masanori; Bang, Gilles; Tougan, Takahiro; Palacpac, Nirianne M Q; Arisue, Nobuko; Aoshi, Taiki; Matsumoto, Yoshitsugu; Ishii, Ken J; Egwang, Thomas G; Druilhe, Pierre; Horii, Toshihiro

    2014-01-01

    The malaria vaccine candidate antigen, SE36, is based on the N-terminal 47 kDa domain of Plasmodium falciparum serine repeat antigen 5 (SERA5). In epidemiological studies, we have previously shown the inhibitory effects of SE36 specific antibodies on in vitro parasite growth and the negative correlation between antibody level and malaria symptoms. A phase 1 b trial of the BK-SE36 vaccine in Uganda elicited 72% protective efficacy against symptomatic malaria in children aged 6-20 years during the follow-up period 130-365 days post-second vaccination. Here, we performed epitope mapping with synthetic peptides covering the whole sequence of SE36 to identify and map dominant epitopes in Ugandan adult serum presumed to have clinical immunity to P. falciparum malaria. High titer sera from the Ugandan adults predominantly reacted with peptides corresponding to two successive N-terminal regions of SERA5 containing octamer repeats and serine rich sequences, regions of SERA5 that were previously reported to have limited polymorphism. Affinity purified antibodies specifically recognizing the octamer repeats and serine rich sequences exhibited a high antibody-dependent cellular inhibition (ADCI) activity that inhibited parasite growth. Furthermore, protein structure predictions and structural analysis of SE36 using spectroscopic methods indicated that N-terminal regions possessing inhibitory epitopes are intrinsically unstructured. Collectively, these results suggest that strict tertiary structure of SE36 epitopes is not required to elicit protective antibodies in naturally immune Ugandan adults. PMID:24886718

  20. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA.

    Directory of Open Access Journals (Sweden)

    Gajendradhar R Dwivedi

    Full Text Available DNA processing protein A (DprA plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA and double stranded DNA (dsDNA. Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed.

  1. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA.

    Science.gov (United States)

    Dwivedi, Gajendradhar R; Srikanth, Kolluru D; Anand, Praveen; Naikoo, Javed; Srilatha, N S; Rao, Desirazu N

    2015-01-01

    DNA processing protein A (DprA) plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA) acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA) is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA) and double stranded DNA (dsDNA). Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed. PMID:26135134

  2. Kadar N-Terminal Pro-Brain Natriuretic Peptide sebagai Prediktor Luaran Klinis Sindrom Koroner Akut

    Directory of Open Access Journals (Sweden)

    Florencia Idajanti Tandhana

    2012-06-01

    Full Text Available Plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP levels may reflect the severity of ischemia, although there is no necrosis. A transient ischemia which can increase the heart wall stretch would induces BNP synthesis and release. Synthesis and release of BNP are comparable with the severity of ischemia. The aim of this study was to analyze whether NT-proBNP levels in patients with acute coronary syndrome (ACS can be used as a predictor for clinical outcome. Studies was held since January to March 2010. Subject were patients with ACS who came to emergency room Dr. Hasan Sadikin Hospital Bandung and were clinically diagnosed according to World Health Organization criteria. Subjects which were suited with the inclusion criteria, stored until assayed. NT-pro BNP concentration was examined by electrochemiluminescence immunoassay method along with creatine kinase muscle brain (enzymatic method and cardiac troponin T (quantitative method. Statistical analysis was performed using the one-sample Kolmogorov-Smirnov test for verifying normality, normally distributed data were analyzed using parametric analysis and abnormal distributed data was assayed using multiple logistic regression analysis to determine the parameters which can be used as predictor for clinical outcome in patients with ACS. Multiple logistic regression analysis on 83 subjects showed predictive value of NT-proBNP levels with OR=1.00, which mean there was no different likelihood in patients with high and low concentration of NT-proBNP to have longer hospitality duration. NT-proBNP β coefficient of 0.001 states that every addition of 1,000 pg/mL of NT-proBNP concentration will increase the length of hospitality duration for one day. On convalesce subjects, the most significant predictive value for predicting clinical outcome cTnT was more better than NT-proBNP concentration in patients with ACS (OR=32.53, 95%CI; 0.58–1,819.26. In conclusions, NT-proBNP is not a major

  3. Substance P primes lipoteichoic acid- and Pam3CysSerLys4-mediated activation of human mast cells by up-regulating Toll-like receptor 2.

    Science.gov (United States)

    Tancowny, Brian P; Karpov, Victor; Schleimer, Robert P; Kulka, Marianna

    2010-10-01

    Substance P (SP) is a neuropeptide with neuroimmunoregulatory activity that may play a role in susceptibility to infection. Human mast cells, which are important in innate immune responses, were analysed for their responses to pathogen-associated molecules via Toll-like receptors (TLRs) in the presence of SP. Human cultured mast cells (LAD2) were activated by SP and TLR ligands including lipopolysaccharide (LPS), Pam3CysSerLys4 (Pam3CSK4) and lipoteichoic acid (LTA), and mast cell leukotriene and chemokine production was assessed by enzyme-linked immunosorbent assay (ELISA) and gene expression by quantitative PCR (qPCR). Mast cell degranulation was determined using a β-hexosaminidase (β-hex) assay. SP treatment of LAD2 up-regulated mRNA for TLR2, TLR4, TLR8 and TLR9 while anti-immunoglobulin E (IgE) stimulation up-regulated expression of TLR4 only. Flow cytometry and western blot confirmed up-regulation of TLR2 and TLR8. Pretreatment of LAD2 with SP followed by stimulation with Pam3CSK4 or LTA increased production of leukotriene C4 (LTC(4) ) and interleukin (IL)-8 compared with treatment with Pam3CSK4 or LTA alone (>2-fold; P<0·01). SP alone activated 5-lipoxygenase (5-LO) nuclear translocation but also augmented Pam3CSK4 and LTA-mediated 5-LO translocation. Pam3CSK4, LPS and LTA did not induce LAD2 degranulation. SP primed LTA and Pam3CSK4-mediated activation of JNK, p38 and extracellular-signal-regulated kinase (ERK) and activated the nuclear translocation of c-Jun, nuclear factor (NF)-κB, activating transcription factor 2 (ATF-2) and cyclic-AMP-responsive element binding protein (CREB) transcription factors. Pretreatment with SP followed by LTA stimulation synergistically induced production of chemokine (C-X-C motif) ligand 8 (CXCL8)/IL-8, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1), tumour necrosis factor (TNF) and IL-6 protein. SP primes TLR2-mediated activation of human mast cells by up-regulating TLR expression and

  4. The relationship between N-terminal prosomatostatin, all-cause and cardiovascular mortality in patients with type 2 diabetes mellitus (ZODIAC-35)

    NARCIS (Netherlands)

    van Dijk, Peter R.; Landman, Gijs W. D.; van Essen, Larissa; Struck, Joachim; Groenier, Klaas H.; Bilo, Henk J. G.; Bakker, Stephan J. L.; Kleefstra, Nanne

    2015-01-01

    Background: The hormone somatostatin inhibits growth hormone release from the pituitary gland and is theoretically linked to diabetes and diabetes related complications. This study aimed to investigate the relationship between levels of the stable somatostatin precursor, N-terminal prosomatostatin (

  5. Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites.

    Science.gov (United States)

    Cater, Michael A; Forbes, John; La Fontaine, Sharon; Cox, Diane; Mercer, Julian F B

    2004-01-01

    The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4-6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1-5 (amino acids 64-540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases. PMID:14998371

  6. Affects of N-terminal variation in the SeM protein of Streptococcus equi on antibody and fibrinogen binding.

    Science.gov (United States)

    Timoney, John F; DeNegri, Rafaela; Sheoran, Abhineet; Forster, Nathalie

    2010-02-10

    The clonal Streptococcus equi causes equine strangles, a highly contagious suppurative lymphadenopathy and rhinopharyngitis. An important virulence factor and vaccine component, the antiphagocytic fibrinogen binding SeM of S. equi is a surface anchored fibrillar protein. Two recent studies of N. American, Japanese and European isolates have revealed a high frequency of N-terminal amino acid variation in SeM of S. equi CF32 that suggests this region of the protein is subject to immunologic selection pressure. The aims of the present study were firstly to map regions of SeM reactive with convalescent equine IgG and IgA and stimulatory for lymph node cells and secondly to determine effects of N-terminal variation on the functionality of SeM. Variation did not significantly affect fibrinogen binding or susceptibility of S. equi to an opsonic equine serum. Linear epitopes reactive with convalescent IgG and mucosal IgA were concentrated toward the conserved center of SeM. However, IgA but not IgG from every horse reacted with at least one peptide that contained variable sequence. Lymph node cells (CD4+) from horses immunized with SeM were strongly responsive to a peptide (alphaalpha36-138) encoding the entire variable region. SeM (CF32) specific mouse Mab 04D11 which reacted strongly with this larger peptide but not with shorter peptides within that sequence reacted strongly with whole cells of S. equi CF32 but only weakly with cells of any of 14 isolates of S. equi expressing different variants of SeM. These results in combination suggest that N-terminal variation alters a conformational epitope of significance in mucosal IgA and systemic T cell responses but does not affect antibody mediated phagocytosis and killing.

  7. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.

    Science.gov (United States)

    Beccia, Maria Rosa; Sauge-Merle, Sandrine; Lemaire, David; Brémond, Nicolas; Pardoux, Romain; Blangy, Stéphanie; Guilbaud, Philippe; Berthomieu, Catherine

    2015-07-01

    Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

  8. N-terminal PDZ-like domain of chromatin organizer SATB1 contributes towards its function as transcription regulator

    Indian Academy of Sciences (India)

    Dimple Notani; Praveena L Ramanujam; P Pavan Kumar; Kamalvishnu P Gottimukkala; Chandan Kumar-Sinha; Sanjeev Galande

    2011-08-01

    The special AT-rich DNA-binding protein 1 (SATB1) is a matrix attachment region (MAR)-binding protein that acts as a global repressor via recruitment of CtBP1:HDAC1-containing co-repressors to its binding targets. The N-terminal PSD95/Dlg-A/ZO-1 (PDZ)-like domain of SATB1 mediates interactions with several chromatin proteins. In the present study, we set out to address whether the PDZ-domain-mediated interactions of SATB1 are critical for its in vivo function as a global repressor. We reasoned that since the N-terminal PDZ-like domain (amino acid residues 1–204) lacks DNA binding activity, it would fail to recruit the interacting partners of SATB1 to its genomic binding sites and hence would not repress the SATB1-regulated genes. Indeed, in vivo MAR-linked luciferase reporter assay revealed that overexpression of the PDZ-like domain resulted in de-repression, indicating that the PDZ-like domain exerts a dominant negative effect on genes regulated by SATB1. Next, we developed a stable dominant negative model in human embryonic kidney (HEK) 293T cells that conditionally expressed the N-terminal 1–204 region harbouring the PDZ-like domain of SATB1. To monitor the effect of sequestration of the interaction partners on the global gene regulation by SATB1, transcripts from the induced and uninduced clones were subjected to gene expression profiling. Clustering of expression data revealed that 600 out of 19000 genes analysed were significantly upregulated upon overexpression of the PDZ-like domain. Induced genes were found to be involved in important signalling cascades and cellular functions. These studies clearly demonstrated the role of PDZ domain of SATB1 in global gene regulation presumably through its interaction with other cellular proteins.

  9. Troponin T and N-terminal pro B-Type natriuretic peptide and presence of coronary artery disease

    DEFF Research Database (Denmark)

    Mouridsen, Mette R; Sajadieh, Ahmad; Carlsen, Christian M;

    2015-01-01

    BACKGROUND: We tested the effects of exercise intensity, sampling intervals, degree of coronary artery stenosis, and demographic factors on circulating N-terminal pro B-Type natriuretic peptide (NT-pro-BNP) and cardiac Troponin T (cTnT) in subjects suspected of coronary artery disease (CAD......). MATERIALS AND METHODS: A total of 242 subjects referred for diagnostic evaluation of possible CAD had blood samples obtained before, 5 min after, and again 20 h after a symptom-limited exercise test. RESULTS: Totally 40 subjects had CAD with ≥ 50% stenosis, 115 subjects had no stenosis and 87 subjects...

  10. Solution NMR characterization of WT CXCL8 monomer and dimer binding to CXCR1 N-terminal domain

    OpenAIRE

    Joseph, Prem Raj B.; Rajarathnam, Krishna

    2014-01-01

    Chemokine CXCL8 and its receptor CXCR1 are key mediators in combating infection and have also been implicated in the pathophysiology of various diseases including chronic obstructive pulmonary disease (COPD) and cancer. CXCL8 exists as monomers and dimers but monomer alone binds CXCR1 with high affinity. CXCL8 function involves binding two distinct CXCR1 sites – the N-terminal domain (Site-I) and the extracellular/transmembrane domain (Site-II). Therefore, higher monomer affinity could be due...

  11. Uncoupling Intramolecular Processing and Substrate Hydrolysis in the N-terminal Nucleophile Hydrolase hASRGL1 by Circular Permutation

    OpenAIRE

    Li, Wenzong; Cantor, Jason R.; Yogesha, S.D.; Yang, Shirley; Chantranupong, Lynne; Liu, June Qingxia; Agnello, Giulia; Georgiou, George; Stone, Everett M.; Yan ZHANG

    2012-01-01

    The human asparaginase-like protein 1 (hASRGL1) catalyzes the hydrolysis of l-asparagine and isoaspartyl-dipeptides. As an N-terminal nucleophile (Ntn) hydrolase superfamily member, the active form of hASRGL1 is generated by an intramolecular cleavage step with Thr168 as the catalytic residue. However, in vitro, autoprocessing is incomplete (~50 %), fettering the biophysical characterization of hASRGL1. We circumvented this obstacle by constructing a circularly permuted hASRGL1 that uncoupled...

  12. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA

    OpenAIRE

    Dwivedi, Gajendradhar R.; Kolluru D Srikanth; Praveen Anand; Javed Naikoo; Srilatha, N. S.; Rao, Desirazu N.

    2015-01-01

    DNA processing protein A (DprA) plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA) acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA) is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been ...

  13. Mutational analysis of Escherichia coli elongation factor Tu in search of a role for the N-terminal region

    DEFF Research Database (Denmark)

    Mansilla, Francisco; Knudsen, Charlotte Rohde; Laurberg, M;

    1998-01-01

    We have mutated lysine 2 and arginine 7 in elongation factor Tu from Escherichia coli separately either to alanine or glutamic acid. The aim of the work was to reveal the possible interactions between the conserved N-terminal part of the molecule, which is rich in basic residues and aminoacyl......, thus binding the N-terminus tightly to domain 2. We propose that this interaction is needed for aminoacyl-tRNA binding, and also for completing the structural rearrangement, which takes place when the factor switches from its GDP to its GTP form. Udgivelsesdato: 1997-Aug...

  14. Prognostic usefulness of anemia and N-terminal pro-brain natriuretic peptide in outpatients with systolic heart failure

    DEFF Research Database (Denmark)

    Schou, Morten; Gustafsson, Finn; Kistorp, Caroline N;

    2007-01-01

    .041) was closely associated with NT-pro-BNP levels above the median (1,381 pg/ml) after adjustment for traditional confounders (left ventricular ejection fraction, age, body mass index, atrial fibrillation, chronic kidney disease). In an adjusted Cox proportional hazard model, the 2 parameters were associated......N-terminal pro-brain natriuretic peptide (NT-pro-BNP) and anemia are predictors of outcome in systolic heart failure. It is currently unclear how these 2 markers interact in particular with regard to the prognostic information carried by each risk marker. We therefore tested the hypothesis...... that anemia (World Health Organization criteria, hemoglobin levels

  15. Intrinsic structural differences in the N-terminal segment of pulmonary surfactant protein SP-C from different species

    DEFF Research Database (Denmark)

    Plasencia, I; Rivas, L; Casals, C;

    2001-01-01

    Predictive studies suggest that the known sequences of the N-terminal segment of surfactant protein SP-C from animal species have an intrinsic tendency to form beta-turns, but there are important differences on the probable location of these motifs in different SP-C species. Our hypothesis...... with spontaneous lipid/peptide interactions. Both canine and porcine peptides were able to form monolayers at air-liquid interfaces, the canine peptides occupying lower area/molecule and being compressible to higher pressures than the porcine sequences. The peptides also shifted the isotherms and perturbed...

  16. Basolateral localisation of KCNQ1 potassium channels in MDCK cells: molecular identification of an N-terminal targeting motif

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Rasmussen, Hanne B; Grunnet, Morten;

    2004-01-01

    KCNQ1 potassium channels are expressed in many epithelial tissues as well as in the heart. In epithelia KCNQ1 channels play an important role in salt and water transport and the channel has been reported to be located apically in some cell types and basolaterally in others. Here we show that KCNQ1...... channels are located basolaterally when expressed in polarised MDCK cells. The basolateral localisation of KCNQ1 is not affected by co-expression of any of the five KCNE beta-subunits. We characterise two independent basolateral sorting signals present in the N-terminal tail of KCNQ1. Mutation...

  17. The Drosophila Microtubule-Associated Protein Mars Stabilizes Mitotic Spindles by Crosslinking Microtubules through Its N-Terminal Region

    DEFF Research Database (Denmark)

    Zhang, Gang; Beati, Hamze; Nilsson, Jakob;

    2013-01-01

    reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function....... Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs....

  18. Enhancement of humoral immune responses to HBsAg by heat shock protein gp96 and its N-terminal fragment in mice

    Institute of Scientific and Technical Information of China (English)

    Hong-Tao Li; Jia-Bin Yan; Jing Li; Ming-Hai Zhou; Xiao-Dong Zhu; Yu-Xia Zhang; Po Tien

    2005-01-01

    AIM: Most studies on the immune effect of gp96 were focused on its enhancement of CTLs. It is interesting to know whether gp96 could influence the humoral immune response, and whether the recombinant N-terminal fragment of gp96 could substitute native gp96 to stimulate the immune system.METHODS: gp96 isolated from livers of normal mice and its N-terminal fragment (amino acid 22-355) expressed in E coli were used for immunization of BALb/c mice. Eight groups of mice received one of the following regiments subcutaneously in 100 μL phosphate buffered saline (PBS)at an interval of 3 wk. Group 1: PBS only; group 2:gp96 only; group 3: N-terminal fragment only; group 4: HBsAg only; group 5: HBsAg+gp96; group 6: HBsAg+N-terminalfragment; group 7: HBsAg+incomplete Freud's adjuvant; group 8: HBsAg+N-terminal fragment (95 ℃ heated for 30 min). Serum anti-HBsAg antibody levels were assayed by ELISA. CTL responses in splenocytes were analyzed by ELISPOT after the last vaccination.RESULTS: The average titer of serum anti-HBsAg antibodyin the mice immunized with HBsAg together with gp96 or its N-terminal fragment were much higher than those immunized with HBsAg alone detected by ELISA. The cellular immune response of the mice immunized with HBsAg together with gp96 or its N-terminal fragment was not different with those immunized with HBsAg alone measured by ELISPOT assay.CONCLUSION: gp96 or its N-terminal fragment greatly improved humoral immune response induced by HBsAg, but failed to enhance the CTL response, which demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment humoral immune response against HBV infection.

  19. Regulatory Light Chain Phosphorylation and N-Terminal Extension Increase Cross-Bridge Binding and Power Output in Drosophila at In Vivo Myofilament Lattice Spacing

    OpenAIRE

    Miller, Mark S.; Farman, Gerrie P.; Braddock, Joan M.; Soto-Adames, Felipe N.; Irving, Thomas C.; Vigoreaux, Jim O.; Maughan, David W.

    2011-01-01

    The N-terminal extension and phosphorylation of the myosin regulatory light chain (RLC) independently improve Drosophila melanogaster flight performance. Here we examine the functional and structural role of the RLC in chemically skinned fibers at various thick and thin filament lattice spacings from four transgenic Drosophila lines: rescued null or control (Dmlc2+), truncated N-terminal extension (Dmlc2Δ2-46), disrupted myosin light chain kinase phosphorylation sites (Dmlc2S66A,S67A), and du...

  20. Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

    Full Text Available Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC". The calpain-mediated ACT processing allows trafficking of the "soluble AC" domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools", which would play different roles in the cell pathophysiology.

  1. Ubiquitin proteasome-dependent degradation of the transcriptional coactivator PGC-1{alpha} via the N-terminal pathway.

    Science.gov (United States)

    Trausch-Azar, Julie; Leone, Teresa C; Kelly, Daniel P; Schwartz, Alan L

    2010-12-17

    PGC-1α is a potent, inducible transcriptional coactivator that exerts control on mitochondrial biogenesis and multiple cellular energy metabolic pathways. PGC-1α levels are controlled in a highly dynamic manner reflecting regulation at both transcriptional and post-transcriptional levels. Here, we demonstrate that PGC-1α is rapidly degraded in the nucleus (t(½ 0.3 h) via the ubiquitin proteasome system. An N-terminal deletion mutant of 182 residues, PGC182, as well as a lysine-less mutant form, are nuclear and rapidly degraded (t(½) 0.5 h), consistent with degradation via the N terminus-dependent ubiquitin subpathway. Both PGC-1α and PGC182 degradation rates are increased in cells under low serum conditions. However, a naturally occurring N-terminal splice variant of 270 residues, NT-PGC-1α is cytoplasmic and stable (t(½>7 h), providing additional evidence that PGC-1α is degraded in the nucleus. These results strongly suggest that the nuclear N terminus-dependent ubiquitin proteasome pathway governs PGC-1α cellular degradation. In contrast, the cellular localization of NT-PCG-1α results in a longer-half-life and possible distinct temporal and potentially biological actions.

  2. Direct interaction of the N-terminal domain of ribosomal protein S1 with protein S2 in Escherichia coli.

    Science.gov (United States)

    Byrgazov, Konstantin; Manoharadas, Salim; Kaberdina, Anna C; Vesper, Oliver; Moll, Isabella

    2012-01-01

    Despite of the high resolution structure available for the E. coli ribosome, hitherto the structure and localization of the essential ribosomal protein S1 on the 30 S subunit still remains to be elucidated. It was previously reported that protein S1 binds to the ribosome via protein-protein interaction at the two N-terminal domains. Moreover, protein S2 was shown to be required for binding of protein S1 to the ribosome. Here, we present evidence that the N-terminal domain of S1 (amino acids 1-106; S1(106)) is necessary and sufficient for the interaction with protein S2 as well as for ribosome binding. We show that over production of protein S1(106) affects E. coli growth by displacing native protein S1 from its binding pocket on the ribosome. In addition, our data reveal that the coiled-coil domain of protein S2 (S2α(2)) is sufficient to allow protein S1 to bind to the ribosome. Taken together, these data uncover the crucial elements required for the S1/S2 interaction, which is pivotal for translation initiation on canonical mRNAs in gram-negative bacteria. The results are discussed in terms of a model wherein the S1/S2 interaction surface could represent a possible target to modulate the selectivity of the translational machinery and thereby alter the translational program under distinct conditions.

  3. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel

    Science.gov (United States)

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-06-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

  4. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization

    International Nuclear Information System (INIS)

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND356–58, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. - Highlights: • BEND3 localizes to the nucleus. • The N-terminal 60 amino acids region of BEND3 contains NLS. • Amino acids located between 56 and 58 of BEND3 (KRK) are part of NLS. • KRK motif is highly conserved among BEND3 homologs

  5. A new general pathway for synthesis of reference compounds of N-terminal valine-isocyanate adducts.

    Science.gov (United States)

    Davies, Ronnie; Rydberg, Per; Westberg, Emelie; Motwani, Hitesh V; Johnstone, Erik; Törnqvist, Margareta

    2010-03-15

    Adducts to Hb could be used as biomarkers to monitor exposure to isocyanates. Particularly useful is the measurement of carbamoylation of N-terminal valines in Hb, after detachment as hydantoins. The synthesis of references from the reactive isocyanates, especially diisocyanates, has been problematic due to side reactions and polymerization of the isocyanate starting material. A simpler, safer, and more general method for the synthesis of valine adducts of isocyanates has been developed using N-[(4-nitrophenyl)carbamate]valine methylamide (NPCVMA) as the key precursor to adducts of various mono- and diisocyanates of interest. By reacting NPCVMA with a range of isocyanate-related amines, carbamoylated valines are formed without the use of the reactive isocyanates. The carbamoylated products synthesized here were cyclized with good yields of the formed hydantoins. The carbamoylated derivative from phenyl isocyanate also showed quantitative yield in a test with cyclization under the conditions used in blood. This new pathway for the preparation of N-carbamoylated model compounds overcomes the above-mentioned problems in the synthesis and is a general and simplified approach, which could make such reference compounds of adducts to N-terminal valine from isocyanates accessible for biomonitoring purposes. The synthesized hydantoins corresponding to adducts from isocyanic acid, methyl isocyanate, phenyl isocyanate, and 2,6-toluene diisocyanate were characterized by LC-MS analysis. The background level of the hydantoin from isocyanic acid in human blood was analyzed with the LC-MS conditions developed.

  6. The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme.

    Science.gov (United States)

    Raynaud, Sandy; Ragel, Paula; Rojas, Tomás; Mérida, Ángel

    2016-05-13

    Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date. We show that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium This finding suggests that the mechanism of action of SS4 is conserved among different plant species. PMID:26969163

  7. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel.

    Science.gov (United States)

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-01-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence. PMID:27345869

  8. The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

    Science.gov (United States)

    Ly, Thu; Moroz, Natalia; Pappas, Christopher T; Novak, Stefanie M; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M; Helms, Gregory; Gregorio, Carol C; Kostyukova, Alla S

    2016-08-15

    Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends.

  9. The Dahlia mosaic virus gene VI product N-terminal region is involved in self-association.

    Science.gov (United States)

    Raikhy, Gaurav; Krause, Charles; Leisner, Scott

    2011-07-01

    The genome of the floriculture pathogen Dahlia mosaic caulimovirus (DMV) encodes six open reading frames. Generally, caulimovirus gene VI products (P6s) are thought to be multifunctional proteins required for viral infection and it is likely that self-association is required for some of these functions. In this study, yeast two-hybrid and maltose binding protein (MBP) pull-down assays indicated that full-length DMV P6 specifically self-associates. Further analyses indicated that only the DMV P6 N-terminal region, consisting of 115 amino acids, interacts with full-length P6 and with itself. This distinguishes the DMV P6 from its Cauliflower mosaic virus counterpart, which contains four regions involved in self-association. Thus, our results suggest that each caulimovirus P6 may possess a unique pattern of protein-protein interactions. Bioinformatic tools identified a putative nuclear exclusion signal located between amino acid residues 10-20, suggesting another possible function for the P6 N-terminal region. PMID:21571015

  10. Human IgG responses against the N-terminal region of Merozoite Surface Protein 1 of Plasmodium vivax

    Directory of Open Access Journals (Sweden)

    Hernando Antonio Del Portillo

    1992-01-01

    Full Text Available The complete primary structure of the gene encoding the Merozoite Surface Protein 1 of Plasmodium vivax (PvMSP-1 revealed the existence of interspecies conserved regions among the analogous proteins of other Plasmodia species. Here, three DNA recombinant clones expressing 50, 200 and 500 amino acids from the N-terminal region of the PvMSP-1 protein were used on ELISA and protein immunoblotting assays to look at the IgG antibody responses of malaria patients from the Brasilian amazon region of Rondônia. The results showed the existance of P. vivax and P. falciparum IgG antibodies directed against PvMSP-1 antigenic determinants expressed in the clones containing the first 200 and the following 500 amino acids of the molecule, but not within the one expressing the most N-terminal 50 amino acids. Interestingly, there was no correlation between the levels of these IgG antibodies and the previous number of malaria infections.

  11. Solution structure and membrane-binding property of the N-terminal tail domain of human annexin I.

    Science.gov (United States)

    Yoon, M K; Park, S H; Won, H S; Na, D S; Lee, B J

    2000-11-10

    The conformational preferences of AnxI(N26), a peptide corresponding to residues 2-26 of human annexin I, were investigated using CD and NMR spectroscopy. CD results showed that AnxI(N26) adopts a mainly alpha-helical conformation in membrane-mimetic environments, TFE/water and SDS micelles, while a predominantly random structure with slight helical propensity in aqueous buffer. The helical region of AnxI(N26) showed a nearly identical conformation between in TFE/water and in SDS micelles, except for the orientation of the Trp-12 side-chain, which was quite different between the two. The N-terminal region of the AnxI(N26) helix showed a typical amphipathic nature, which could be stabilized by the neighboring hydrophobic cluster. The helical stability of the peptide in SDS micelles was increased by addition of calcium ions. These results suggest that the N-terminal tail domain of human annexin I interacts with biological membranes in a partially calcium-dependent manner.

  12. Interaction between GInB and the N-terminal domain of NifA in Azospirillum brasilense

    Institute of Scientific and Technical Information of China (English)

    ZHOU XiaoYu; ZOU XiaoXiao; LI JiLun

    2008-01-01

    Azospirillum brasilense is a diazotroph associated with many important agricultural crops and shows potential as a biofertilizer. NifA, the transcriptional activator of nitrogen fixation (nif) genes, and GInB, one of P,, signal transduction family protein, are key proteins in the regulation of nitrogen fixation in A. brasilense. It was previously reported that the regulation of NifA activity in A. brasilense depends on GInB. We report here that GInB was found to interact directly with the N-terminal domain of NifA in vivo under nitrogen-free conditions and the N-terminal mutant of NifA in which the Tyr residues at position 18 and 53 were replaced by Phe (NifA-N-Y18/53F) strengthened the interaction with GInB. Moreover, we also found that the amino acid residues 66-88 and 165-176 in N-terminus of NifA are responsible for the interaction with GInB.

  13. Identification of a Major Dimorphic Region in the Functionally Critical N-Terminal ID1 Domain of VAR2CSA.

    Directory of Open Access Journals (Sweden)

    Justin Doritchamou

    Full Text Available The VAR2CSA protein of Plasmodium falciparum is transported to and expressed on the infected erythrocyte surface where it plays a key role in placental malaria (PM. It is the current leading candidate for a vaccine to prevent PM. However, the antigenic polymorphism integral to VAR2CSA poses a challenge for vaccine development. Based on detailed analysis of polymorphisms in the sequence of its ligand-binding N-terminal region, currently the main focus for vaccine development, we assessed var2csa from parasite isolates infecting pregnant women. The results reveal for the first time the presence of a major dimorphic region in the functionally critical N-terminal ID1 domain. Parasite isolates expressing VAR2CSA with particular motifs present within this domain are associated with gravidity- and parasite density-related effects. These observations are of particular interest in guiding efforts with respect to optimization of the VAR2CSA-based vaccines currently under development.

  14. Identification of a Major Dimorphic Region in the Functionally Critical N-Terminal ID1 Domain of VAR2CSA.

    Science.gov (United States)

    Doritchamou, Justin; Sabbagh, Audrey; Jespersen, Jakob S; Renard, Emmanuelle; Salanti, Ali; Nielsen, Morten A; Deloron, Philippe; Tuikue Ndam, Nicaise

    2015-01-01

    The VAR2CSA protein of Plasmodium falciparum is transported to and expressed on the infected erythrocyte surface where it plays a key role in placental malaria (PM). It is the current leading candidate for a vaccine to prevent PM. However, the antigenic polymorphism integral to VAR2CSA poses a challenge for vaccine development. Based on detailed analysis of polymorphisms in the sequence of its ligand-binding N-terminal region, currently the main focus for vaccine development, we assessed var2csa from parasite isolates infecting pregnant women. The results reveal for the first time the presence of a major dimorphic region in the functionally critical N-terminal ID1 domain. Parasite isolates expressing VAR2CSA with particular motifs present within this domain are associated with gravidity- and parasite density-related effects. These observations are of particular interest in guiding efforts with respect to optimization of the VAR2CSA-based vaccines currently under development. PMID:26393516

  15. Identification and analysis of the acetylated status of poplar proteins reveals analogous N-terminal protein processing mechanisms with other eukaryotes.

    Directory of Open Access Journals (Sweden)

    Chang-Cai Liu

    Full Text Available BACKGROUND: The N-terminal protein processing mechanism (NPM including N-terminal Met excision (NME and N-terminal acetylation (N(α-acetylation represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: To reveal the NPM in poplar, we investigated the N(α-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO2 enrichment of acetylated peptides. We identified 58 N-terminally acetylated (N(α-acetylated proteins. Most proteins (47, >81% are subjected to N(α-acetylation following the N-terminal removal of Met, indicating that N(α-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and N(α-acetylation (NPM to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs and N-terminal acetyltransferase (Nat enzymes in poplar. The N(α-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins. CONCLUSIONS/SIGNIFICANCE: This study represents the first extensive investigation of N(α-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of N(α-acetylation of proteins in poplar.

  16. Structure and dynamics of the N-terminal domain of the Cu(I) binding protein CusB.

    Science.gov (United States)

    Ucisik, Melek N; Chakravorty, Dhruva K; Merz, Kenneth M

    2013-10-01

    CusCFBA is one of the metal efflux systems in Escherichia coli that is highly specific for its substrates, Cu(I) and Ag(I). It serves to protect the bacteria in environments that have lethal concentrations of these metals. The membrane fusion protein CusB is the periplasmic piece of CusCFBA, which has not been fully characterized by crystallography because of its extremely disordered N-terminal region. This region has both structural and functional importance because it has been experimentally proven to transfer the metal by itself from the metallochaperone CusF and to induce a structural change in the rest of CusB to increase Cu(I)/Ag(I) resistance. Understanding metal uptake from the periplasm is critical to gain insight into the mechanism of the whole CusCFBA pump, which makes resolving a structure for the N-terminal region necessary because it contains the metal binding site. We ran extensive molecular dynamics simulations to reveal the structural and dynamic properties of both the apo and Cu(I)-bound versions of the CusB N-terminal region. In contrast to its functional companion CusF, Cu(I) binding to the N-terminus of CusB causes only a slight, local stabilization around the metal site. The trajectories were analyzed in detail, revealing extensive structural disorder in both the apo and holo forms of the protein. CusB was further analyzed by breaking the protein up into three subdomains according to the extent of the observed disorder: the N- and C-terminal tails, the central beta strand motif, and the M21-M36 loop connecting the two metal-coordinating methionine residues. Most of the observed disorder was traced back to the tail regions, leading us to hypothesize that the latter two subdomains (residues 13-45) may form a functionally competent metal-binding domain because the tail regions appear to play no role in metal binding. PMID:23988152

  17. Free cholesterol-induced macrophage apoptosis is mediated by inositol-requiring enzyme 1 alpha-regulated activation of Jun N-terminal kinase

    Institute of Scientific and Technical Information of China (English)

    Fangming Li; Yi Guo; Shenggang Sun; Xin Jiang; Bingshan Tang; Qizhang Wang; Ling Wang

    2008-01-01

    Macrophage death in advanced atherosclerotic lesions leads to iesional necrosis, possible plaque rupture, and acute vascular occlusion. A likely cause of macrophage death is the accumulation of free cholesterol (FC) leading to activation of endoplasmic reticulum (ER) stress-induced apoptosis.Inositol-requiring enzyme 1 alpha (IRE1α) is an integral membrane protein of the ER that is a key signaling step in cholesterol-induced apoptosis in macrophages, activated by stress in the ER. However, the role of IRE1α in the regulation of ER stress-induced macrophage death and the mechanism for this process are largely unclear.In this study,a cell culture model was used to explore the mechanisms involved in the ER stress pathway of FC-induced macrophage death.The results herein showed that FC loading of macrophages leads to an apoptotic response that is partially dependent on initiation by activation of IRE1α.Taken together,these results showed that the IRE1-apoptosis-signaling kinase 1-c-Jun NH2-terminal kinase cascade pathway was required in this process.Moreover,the data suggested a novel cellular mechanism for cholesterol-induced macrophage death in advanced atherosclerotic lesions.The critical function of this signaling cascade is indicated by prevention of ER stress-induced apoptosis after inhibition of IRE1α,or c-Jun NH2-terminal kinase.

  18. The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease

    Science.gov (United States)

    Kereïche, Sami; Kováčik, Lubomír; Bednár, Jan; Pevala, Vladimír; Kunová, Nina; Ondrovičová, Gabriela; Bauer, Jacob; Ambro, Ľuboš; Bellová, Jana; Kutejová, Eva; Raška, Ivan

    2016-01-01

    Lon is an essential, multitasking AAA+ protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon’s N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning. PMID:27632940

  19. The solution structure of the N-terminal domain of human tubulin binding cofactor C reveals a platform for tubulin interaction.

    Directory of Open Access Journals (Sweden)

    Ma Flor Garcia-Mayoral

    Full Text Available Human Tubulin Binding Cofactor C (TBCC is a post-chaperonin involved in the folding and assembly of α- and β-tubulin monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process it collaborates with other cofactors (TBC's A, B, D, and E and forms a supercomplex with TBCD, β-tubulin, TBCE and α-tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-stranded α-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers.

  20. The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease.

    Science.gov (United States)

    Kereïche, Sami; Kováčik, Lubomír; Bednár, Jan; Pevala, Vladimír; Kunová, Nina; Ondrovičová, Gabriela; Bauer, Jacob; Ambro, Ľuboš; Bellová, Jana; Kutejová, Eva; Raška, Ivan

    2016-01-01

    Lon is an essential, multitasking AAA(+) protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon's N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning. PMID:27632940

  1. The Drosophila microtubule-associated protein mars stabilizes mitotic spindles by crosslinking microtubules through its N-terminal region.

    Directory of Open Access Journals (Sweden)

    Gang Zhang

    Full Text Available Correct segregation of genetic material relies on proper assembly and maintenance of the mitotic spindle. How the highly dynamic microtubules (MTs are maintained in stable mitotic spindles is a key question to be answered. Motor and non-motor microtubule associated proteins (MAPs have been reported to stabilize the dynamic spindle through crosslinking adjacent MTs. Mars, a novel MAP, is essential for the early development of Drosophila embryos. Previous studies showed that Mars is required for maintaining an intact mitotic spindle but did not provide a molecular mechanism for this function. Here we show that Mars is able to stabilize the mitotic spindle in vivo. Both in vivo and in vitro data reveal that the N-terminal region of Mars functions in the stabilization of the mitotic spindle by crosslinking adjacent MTs.

  2. Heart murmur and N-terminal pro-brain natriuretic peptide as predictors of death in 2977 consecutive hospitalized patients

    DEFF Research Database (Denmark)

    Iversen, Kasper; Nielsen, O.W.; Kirk, V.;

    2008-01-01

    -pro-BNP, discovery of valvular heart disease by echocardiography yielded no additional prognostic information. Conclusions: Detection of a cardiac murmur during routine medical examination of hospitalized patients is associated with increased risk of death within a year. A blood test for NT-pro-BNP gives significant...... valvular heart disease. We wanted to test whether murmur predicts mortality in unselected patients admitted to the hospital and whether NT-pro-BNP is capable of distinguishing between innocent and significant murmurs. Methods: Consecutive patients (n = 2977) older than 40 years admitted to a local hospital......Background: Little is known about the prognostic importance of murmur in unselected patients. It is difficult to distinguish between innocent and significant murmurs. N-terminal pro-brain natriuretic peptide (NT-pro-BNP) and BNP have recently been shown to be useful in small series of patients with...

  3. FS23 binds to the N-terminal domain of human Hsp90:A novel small inhibitor for Hsp90

    Institute of Scientific and Technical Information of China (English)

    李健; 石峰; 陈丹琦; 曹慧玲; 熊兵; 沈竞康; 何建华

    2015-01-01

    The N-terminal domain of heat shock protein 90 (Hsp90N) is responsible for the catalytic activity of Hsp90. The reported inhibitors of Hsp90 bind to this domain and would inhibit tumor growth and progression. Here, we synthesized FS23, a small molecule inhibitor of hsp90 and collected X-ray diffraction data of the complex crystal of Hsp90-FS23. High resolution X-ray crystallography shows that FS23 interacted with Hsp90N at the nucleotide binding cleft, and this suggests that FS23 may complete with nucleotides to bind to Hsp90N. The crystal structure and the interaction between Hsp90N and FS23 suggest a rational basis for the design of novel antitumor drugs.

  4. The chondroitin sulfate A-binding site of the VAR2CSA protein involves multiple N-terminal domains

    DEFF Research Database (Denmark)

    Dahlbäck, Madeleine; Jørgensen, Lars M; Nielsen, Morten A;

    2011-01-01

    by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been...... of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite...

  5. The influence of anaemia on stroke prognosis and its relation to N-terminal pro-brain natriuretic peptide

    DEFF Research Database (Denmark)

    Nybo, M; Kristensen, S R; Mickley, H;

    2007-01-01

    Anaemia is a negative prognostic factor for patients with heart failure and impaired renal function, but its role in stroke patients is unknown. Furthermore, anaemia has been shown to influence the level of N-terminal pro-brain natriuretic peptide (NT-proBNP), but this is only investigated...... in patients with heart failure, not in stroke patients. Two-hundred-and-fifty consecutive, well-defined ischemic stroke patients were investigated. Mortality was recorded at 6 months follow-up. Anaemia was diagnosed in 37 patients (15%) in whom stroke severity was worse than in the non-anaemic group, whilst...... the prevalence of renal affection, smoking and heart failure was lower. At 6 months follow-up, 23 patients were dead, and anaemia had an odds ratio of 4.7 when adjusted for age, Scandinavian Stroke Scale and a combined variable of heart and/or renal failure and/or elevation of troponin T using logistic...

  6. The cyanobacterial cell division factor Ftn6 contains an N-terminal DnaD-like domain

    Directory of Open Access Journals (Sweden)

    Saguez Cyril

    2009-08-01

    Full Text Available Abstract Background DNA replication and cell cycle as well as their relationship have been extensively studied in the two model organisms E. coli and B. subtilis. By contrast, little is known about these processes in cyanobacteria, even though they are crucial to the biosphere, in utilizing solar energy to renew the oxygenic atmosphere and in producing the biomass for the food chain. Recent studies have allowed the identification of several cell division factors that are specifics to cyanobacteria. Among them, Ftn6 has been proposed to function in the recruitment of the crucial FtsZ proteins to the septum or the subsequent Z-ring assembly and possibly in chromosome segregation. Results In this study, we identified an as yet undescribed domain located in the conserved N-terminal region of Ftn6. This 77 amino-acids-long domain, designated here as FND (Ftn6 N-Terminal Domain, exhibits striking sequence and structural similarities with the DNA-interacting module, listed in the PFAM database as the DnaD-like domain (pfam04271. We took advantage of the sequence similarities between FND and the DnaD-like domains to construct a homology 3D-model of the Ftn6 FND domain from the model cyanobacterium Synechocystis PCC6803. Mapping of the conserved residues exposed onto the FND surface allowed us to identify a highly conserved area that could be engaged in Ftn6-specific interactions. Conclusion Overall, similarities between FND and DnaD-like domains as well as previously reported observations on Ftn6 suggest that FND may function as a DNA-interacting module thereby providing an as yet missing link between DNA replication and cell division in cyanobacteria. Consistently, we also showed that Ftn6 is involved in tolerance to DNA damages generated by UV rays.

  7. Investigation of the N-terminal coding region of MUC7 alterations in dentistry students with and without caries

    Directory of Open Access Journals (Sweden)

    Koç Öztürk L

    2016-07-01

    Full Text Available Human low-molecular weight salivary mucin (MUC7 is a small, secreted glycoprotein coded by MUC7. In the oral cavity, they inhibit the colonization of oral bacteria, including cariogenic ones, by masking their surface adhesions, thus helping saliva to avoid dental caries. The N-terminal domain is important for low-molecular weight (MG2 mucins to contact with oral microorganisms. In this study, we aimed to identify the N-terminal coding region of the MUC7 gene between individuals with and without caries. Forty-four healthy dental students were enrolled in this study; 24 of them were classified to have caries [decayed, missing, filled-teeth (DMFT = 5.6] according to the World Health Organization (WHO criteria, and 20 of them were caries-free (DMFT = 0. Simplified oral hygiene index (OHI-S and gingival index (GI were used to determine the oral hygiene and gingival conditions. Total protein levels and salivary total protein levels and salivary buffer capacity (SBC were determined by Lowry and Ericsson methods. DNA was extracted from peripheral blood cells of all the participants and genotyping was carried out by a polymerase chain reaction (PCR-sequencing method. No statistical differences were found between two groups in the terms of salivary parameters, oral hygiene and gingival conditions. We detected one common single nucleotide polymorphism (SNP that leads to a change of asparagine to lysine at codon 80. This substitution was found in 29.0 and 40.0%, respectively, of the groups with and without caries. No other sequence variations were detected. The SNP found in this study may be a specific polymorphism affecting the Turkish population. Further studies with extended numbers are necessary in order to clarify this finding.

  8. Copper complex species within a fragment of the N-terminal repeat region in opossum PrP protein.

    Science.gov (United States)

    Vagliasindi, Laura I; Arena, Giuseppe; Bonomo, Raffaele P; Pappalardo, Giuseppe; Tabbì, Giovanni

    2011-03-21

    A spectroscopic (UV-Vis, CD and EPR), thermodynamic and voltammetric study of the copper(ii) complexes with the Ac-PHPGGSNWGQ-NH(2) polypeptide (L), a fragment of the opossum PrP protein N-terminal four-repeat region, was carried out in aqueous solution. It suggests the formation of a highly distorted [Cu(L)H(-2)] complex species in the neutral region, the stereochemistry of which is ascribable to a square base pyramid and a CuN(3)O(2) chromophore, resulting from the coordination of a histidine imidazole and two peptide nitrogen atoms and probably oxygen atoms from water molecules. At basic pH values a [Cu(L)H(-3)](-) species with a pseudo-octahedral geometry was also obtained, with four nitrogen donor atoms in its equatorial plane, coming from the histidine residue and from peptidic nitrogen atoms. Interestingly, at pH values relatively higher than the neutrality, the coordination sphere of the copper complex in the [Cu(L)H(-2)] species changes its stereochemistry towards a pseudo-octahedron, as suggested by the change in the parallel copper hyperfine coupling constant of the EPR spectra at low temperature. A slight difference in the redox potentials between this two-faced [Cu(L)H(-2)] complex species seems to confirm this behaviour. Both potentiometric and spectroscopic data were compared with the analogous species obtained with the Ac-PHGGGWGQ-NH(2) peptide, belonging to the octarepeat domain of the human prion protein (hPrP) N-terminal region. The [Cu(L)H(-2)] species formed by the Ac-PHPGGSNWGQ-NH(2) decapeptide, having a slightly lower stability, turned out to be less abundant and to exist within a narrow pH range. PMID:21283898

  9. Characterization of the N-terminal domain of BteA: a Bordetella type III secreted cytotoxic effector.

    Directory of Open Access Journals (Sweden)

    Chen Guttman

    Full Text Available BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Currently there is limited information regarding the structure of BteA or its subdomains, and no insight as to the identity of its eukaryotic partners(s and their modes of interaction with BteA. The mechanisms that lead to BteA dependent cell death also remain elusive. The N-terminal domain of BteA is multifunctional, acting as a docking platform for its cognate chaperone (BtcA in the bacterium, and targeting the protein to lipid raft microdomains within the eukaryotic host cell. In this study we describe the biochemical and biophysical characteristics of this domain (BteA287 and determine its architecture. We characterize BteA287 as being a soluble and highly stable domain which is rich in alpha helical content. Nuclear magnetic resonance (NMR experiments combined with size exclusion and analytical ultracentrifugation measurements confirm these observations and reveal BteA287 to be monomeric in nature with a tendency to oligomerize at concentrations above 200 µM. Furthermore, diffusion-NMR demonstrated that the first 31 residues of BteA287 are responsible for the apparent aggregation behavior of BteA287. Light scattering analyses and small angle X-ray scattering experiments reveal a prolate ellipsoidal bi-pyramidal dumb-bell shape. Thus, our biophysical characterization is a first step towards structure determination of the BteA N-terminal domain.

  10. Zinc(II) interactions with brain-derived neurotrophic factor N-terminal peptide fragments: inorganic features and biological perspectives.

    Science.gov (United States)

    Travaglia, Alessio; La Mendola, Diego; Magrì, Antonio; Pietropaolo, Adriana; Nicoletti, Vincenzo G; Grasso, Giuseppe; Malgieri, Gaetano; Fattorusso, Roberto; Isernia, Carla; Rizzarelli, Enrico

    2013-10-01

    Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for neuronal differentiation, growth, and survival; it is involved in memory formation and higher cognitive functions. The N-terminal domain of BDNF is crucial for the binding selectivity and activation of its specific TrkB receptor. Zn(2+) ion binding may influence BDNF activity. Zn(2+) complexes with the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of the N-terminal domain of BDNF were studied by means of potentiometry, electrospray mass spectrometry, NMR, and density functional theory (DFT) approaches. The predominant Zn(2+) complex species, at physiological pH, is [ZnL] in which the metal ion is bound to an amino, an imidazole, and two water molecules (NH2, N(Im), and 2O(water)) in a tetrahedral environment. DFT-based geometry optimization of the zinc coordination environment showed a hydrogen bond between the carboxylate and a water molecule bound to zinc in [ZnL]. The coordination features of the acetylated form [AcBDNF(1-12)] and of a single mutated peptide [BDNF(1-12)D3N] were also characterized, highlighting the role of the imidazole side chain as the first anchoring site and ruling out the direct involvement of the aspartate residue in the metal binding. Zn(2+) addition to the cell culture medium induces an increase in the proliferative activity of the BDNF(1-12) peptide and of the whole protein on the SHSY5Y neuroblastoma cell line. The effect of Zn(2+) is opposite to that previously observed for Cu(2+) addition, which determines a decrease in the proliferative activity for both peptide and protein, suggesting that these metals might discriminate and modulate differently the activity of BDNF.

  11. PKC phosphorylates residues in the N-terminal of the DA transporter to regulate amphetamine-induced DA efflux.

    Science.gov (United States)

    Wang, Qiang; Bubula, Nancy; Brown, Jason; Wang, Yunliang; Kondev, Veronika; Vezina, Paul

    2016-05-27

    The DA transporter (DAT), a phosphoprotein, controls extracellular dopamine (DA) levels in the central nervous system through transport or reverse transport (efflux). Multiple lines of evidence support the claim that PKC significantly contributes to amphetamine-induced DA efflux. Other signaling pathways, involving CaMKII and ERK, have also been shown to regulate DAT mediated efflux. Here we assessed the contribution of putative PKC residues (S4, S7, S13) in the N-terminal of the DAT to amphetamine-induced DA efflux by transfecting DATs containing different serine to alanine (S-A) point mutations into DA pre-loaded HEK-293 cells and incubating these cells in amphetamine (2μM). The effects of a S-A mutation at the non-PKC residue S12 and a threonine to alanine (T-A) mutation at the ERK T53 residue were also assessed for comparison. WT-DATs were used as controls. In an initial experiment, we confirmed that inhibiting PKC with Go6976 (130nM) significantly reduced amphetamine-induced DA efflux. In subsequent experiments, cells transfected with the S4A, S12A, S13A, T53A and S4,7,13A mutants showed a reduction in amphetamine-induced DA efflux similar to that observed with Go6976. Interestingly, cells transfected with the S7A mutant, identified by some as a PKC-PKA residue, showed unperturbed WT-DAT levels of amphetamine-induced DA efflux. These results indicate that phosphorylation by PKC of select residues in the DAT N-terminal can regulate amphetamine-induced efflux. PKC can act either independently or in concert with other kinases such as ERK to produce this effect.

  12. Investigation of the N-terminal coding region of MUC7 alterations in dentistry students with and without caries

    Science.gov (United States)

    Koç Öztürk, L; Yarat, A; Akyuz, S; Furuncuoglu, H

    2016-01-01

    ABSTRACT Human low-molecular weight salivary mucin (MUC7) is a small, secreted glycoprotein coded by MUC7. In the oral cavity, they inhibit the colonization of oral bacteria, including cariogenic ones, by masking their surface adhesions, thus helping saliva to avoid dental caries. The N-terminal domain is important for low-molecular weight (MG2) mucins to contact with oral microorganisms. In this study, we aimed to identify the N-terminal coding region of the MUC7 gene between individuals with and without caries. Forty-four healthy dental students were enrolled in this study; 24 of them were classified to have caries [decayed, missing, filled-teeth (DMFT) = 5.6] according to the World Health Organization (WHO) criteria, and 20 of them were caries-free (DMFT = 0). Simplified oral hygiene index (OHI-S) and gingival index (GI) were used to determine the oral hygiene and gingival conditions. Total protein levels and salivary total protein levels and salivary buffer capacity (SBC) were determined by Lowry and Ericsson methods. DNA was extracted from peripheral blood cells of all the participants and genotyping was carried out by a polymerase chain reaction (PCR)-sequencing method. No statistical differences were found between two groups in the terms of salivary parameters, oral hygiene and gingival conditions. We detected one common single nucleotide polymorphism (SNP) that leads to a change of asparagine to lysine at codon 80. This substitution was found in 29.0 and 40.0%, respectively, of the groups with and without caries. No other sequence variations were detected. The SNP found in this study may be a specific polymorphism affecting the Turkish population. Further studies with extended numbers are necessary in order to clarify this finding.

  13. c-fos与c-jun癌蛋白在溃疡与增生性瘢痕创面联合表达的特征与意义%Expression of oncoproteins c-fos and c-jun in hypertrophic scars and chronic dermal ulcers and their regulation of basic fibroblast growth factor

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    目的探讨c-fos与c-jun两种原癌基因产物在溃疡与增生性瘢痕创面表达的特征与规律以及与不同组织修复结局发生的关系。 方法 16例标本均取自于外科手术患者,其中增生性瘢痕8例,溃疡创面8例,另有正常对照皮肤5例取自增生性瘢痕患者作为对照。用免疫组化法(ABC法)检测c-fos与c-jun两种癌蛋白以及bFGF在三种组织切片的分布特征。 结果在正常皮肤c-fos与c-jun的阳性表达主要见于表皮基底细胞和少量皮下成纤维细胞,但在相应部位c-jun的表达较c-fos为弱。在增生性瘢痕,c-fos与c-jun均出现强阳性表达,主要见于成纤维细胞。在溃疡组织,c-fos与c-jun的联合表达多见于毛细血管内皮细胞、部分炎症细胞以及成纤维细胞胞浆。 结论增生性瘢痕与溃疡创面c-fos与c-jun表达量与部位同正常皮肤存在显著差异,提示这两种原癌基因产物在影响和调控组织修复中有重要作用。%Objective To explore the characteristics of oncoprotein expression of c-fos and c-jun in hypertrophic scars and chronic dermal ulcers and their regulation of basic fibroblast growth factor (bFGF). Methods Tissues of hypertrophic scars (n=8), chronic dermal ulcers (n=8) and normal skin (n=5) were taken from 21 patients with burns and chronic dermal ulcers in operation. The ABC immunohistochemical method was used to characterize the gene product expression of c-fos, c-jun and bFGF in the above tissues. Results In normal skin, both c-fos and c-jun protein expression and bFGF protein expression were observed. The signals of both oncoproteins were localized mainly in subcutaneous fibroblasts, but, positive expression of the bFGF protein was mainly in keratinocytes. In hypertrophic scars, positive expression of both oncoproteins could be found mainly in fibroblasts, but bFGF was mainly in fibroblasts and endothelial cells. In chronic dermal ulcers, endothelial cells, some

  14. Role of the N-terminal signal peptide in the membrane insertion of Aquifex aeolicus F1F0 ATP synthase c-subunit.

    Science.gov (United States)

    Zhang, Chunli; Marcia, Marco; Langer, Julian D; Peng, Guohong; Michel, Hartmut

    2013-07-01

    Rotary ATPases are membrane protein complexes that couple ATP hydrolysis to ion translocation across the membrane. Overall, they are evolutionarily well conserved, but the N-terminal segments of their rotary subunits (c-subunits) possess different lengths and levels of hydrophobicity across species. By analyzing the N-terminal variability, we distinguish four phylogenetic groups of c-subunits (groups 1-4). We characterize a member of group 2, the c-subunit from Aquifex aeolicus F1F0 ATP synthase, both in native cells and in a heterologous expression system. We demonstrate that its N-terminal segment forms a signal peptide with signal recognition particle (SRP) recognition features and is obligatorily required for membrane insertion. Based on our study and on previous characterizations of c-subunits from other organisms, we propose that c-subunits follow different membrane insertion pathways. PMID:23663226

  15. Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the beta chain

    DEFF Research Database (Denmark)

    Deufel, T; Grove, A; Kofod, Hans;

    1985-01-01

    detected a 29 kDa component in immunoblots of Raji and AL-34 cell plasma membrane proteins separated by SDS gel electrophoresis. The binding of either N-terminal peptide antiserum was selectively inhibited only by the peptide used as antigen. Indirect immunofluorescence analysis by flow cytofluorometry......Antibodies against synthetic peptides representing the class-II antigen HLA-DR and -DQ beta chain N-terminal sequences were prepared in rabbits. The two octapeptides only share two amino acids and enzyme-linked immuno-assays showed the antisera only to bind to its own antigen. Both peptide antisera...... chains of HLA-DR and -DQ have been prepared by the preparation by the production of antibodies against the N-terminal sequences of each polypeptide....

  16. The effect of removing the N-terminal extension of the Drosophila myosin regulatory light chain upon flight ability and the contractile dynamics of indirect flight muscle.

    OpenAIRE

    Moore, J R; Dickinson, M H; Vigoreaux, J O; Maughan, D W

    2000-01-01

    The Drosophila myosin regulatory light chain (DMLC2) is homologous to MLC2s of vertebrate organisms, except for the presence of a unique 46-amino acid N-terminal extension. To study the role of the DMLC2 N-terminal extension in Drosophila flight muscle, we constructed a truncated form of the Dmlc2 gene lacking amino acids 2-46 (Dmlc2(Delta2-46)). The mutant gene was expressed in vivo, with no wild-type Dmlc2 gene expression, via P-element-mediated germline transformation. Expression of the tr...

  17. N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile

    OpenAIRE

    Weltrowska, Grazyna; Nguyen, Thi M.-D.; Chung, Nga N.; Wilkes, Brian C.; Schiller, Peter W.

    2013-01-01

    Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic = 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood-brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/μ partial agonist ...

  18. The methylated N-terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by Ran in live cells

    Directory of Open Access Journals (Sweden)

    Sanderson Helen S

    2010-06-01

    Full Text Available Abstract Background Regulator of chromosome condensation 1 (RCC1 is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA. Results We have investigated the mechanism of the dynamic interaction of the α isoform of human RCC1 (RCC1α with chromatin in live cells using fluorescence recovery after photobleaching (FRAP of green fluorescent protein (GFP fusions. We show that the N-terminal tail stabilises the interaction of RCC1α with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1α with chromatin from being stabilised by RanT24N, a mutant that binds stably to RCC1α. The interaction of RCC1α with chromatin is destabilised by mutation of lysine 4 (K4Q, which abolishes α-N-terminal methylation, and this interaction is no longer stabilised by RanT24N. However, α-N-terminal methylation of RCC1α is not regulated by the binding of RanT24N. Conversely, the association of Ran with precipitated RCC1α does not require the N-terminal tail of RCC1α or its methylation. The mobility of RCC1α on chromatin is increased by mutation of aspartate 182 (D182A, which inhibits guanine-nucleotide exchange activity, but RCC1αD182A can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by RanT24N. Conclusions These results show that the stabilisation of the dynamic interaction of RCC1α with chromatin by Ran in live cells requires the N-terminal tail of RCC1α. α-N-methylation is not regulated by formation of the binary

  19. Mapping of Chlamydia trachomatis proteins by immobiline-polyacrylamide two-dimensional electrophoresis: spot identification by N-terminal sequencing and immunoblotting

    DEFF Research Database (Denmark)

    Bini, L; Sanchez-Campillo, M; Santucci, A;

    1996-01-01

    reproducible and resolve ca. 600 spots. By using immunoblot analysis with specific antibodies and/or N-terminal amino acid sequencing, we established the map positions of a number of described chlamydial proteins, such as the major outer membrane protein (MOMP) the 60 kDa cystein-rich outer membrane protein...... parameters (Mr, pI and N-terminal sequence). This work provides a preliminary basis for a future and progressive compilation of a genome-linked database of chlamydial proteins....

  20. DMPD: Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited signalingpathways. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12960231 Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited sign...82. Epub 2003 Jul 22. (.png) (.svg) (.html) (.csml) Show Macrophage activation through CCR5- and CXCR4-media...on through CCR5- and CXCR4-mediated gp120-elicited signalingpathways. Authors Lee C, Liu QH, Tomkowicz B, Yi

  1. Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

    Science.gov (United States)

    Hwang, I.; Harper, J. F.; Liang, F.; Sze, H.

    2000-01-01

    To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze inverted question mark1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

  2. Modulating the activity of short arginine-tryptophan containing antibacterial peptides with N-terminal metallocenoyl groups

    Directory of Open Access Journals (Sweden)

    H. Bauke Albada

    2012-10-01

    Full Text Available A series of small synthetic arginine and tryptophan containing peptides was prepared and analyzed for their antibacterial activity. The effect of N-terminal substitution with metallocenoyl groups such as ferrocene (FcCO and ruthenocene (RcCO was investigated. Antibacterial activity in different media, growth inhibition, and killing kinetics of the most active peptides were determined. The toxicity of selected derivatives was determined against erythrocytes and three human cancer cell lines. It was shown that the replacement of an N-terminal arginine residue with a metallocenoyl moiety modulates the activity of WRWRW-peptides against Gram-positive and Gram-negative bacteria. MIC values of 2–6 µM for RcCO-W(RW2 and 1–11 µM for (RW3 were determined. Interestingly, W(RW2-peptides derivatized with ferrocene were significantly less active than those derivatized with ruthenocene which have similar structural but different electronic properties, suggesting a major influence of the latter. The high activities observed for the RcCO-W(RW2- and (RW3-peptides led to an investigation of the origin of activity of these peptides using several important activity-related parameters. Firstly, killing kinetics of the RcCO-W(RW2-peptide versus killing kinetics of the (RW3 derivative showed faster reduction of the colony forming units for the RcCO-W(RW2-peptide, although MIC values indicated higher activity for the (RW3-peptide. This was confirmed by growth inhibition studies. Secondly, hemolysis studies revealed that both peptides did not lead to significant destruction of erythrocytes, even up to 500 µg/mL for (RW3 and 250 µg/mL for RcCO-W(RW2. In addition, toxicity against three human cancer cell lines (HepG2, HT29, MCF7 showed that the (RW3-peptide had an IC50 value of ~140 µM and the RcW(RW2 one of ~90 µM, indicating a potentially interesting therapeutic window. Both the killing kinetics and growth inhibition studies presented in this work point to a

  3. The Scavenger Receptor SSc5D Physically Interacts with Bacteria through the SRCR-Containing N-Terminal Domain

    Science.gov (United States)

    Bessa Pereira, Catarina; Bocková, Markéta; Santos, Rita F.; Santos, Ana Mafalda; Martins de Araújo, Mafalda; Oliveira, Liliana; Homola, Jiří; Carmo, Alexandre M.

    2016-01-01

    The scavenger receptor cysteine-rich (SRCR) family comprises a group of membrane-attached or secreted proteins that contain one or more modules/domains structurally similar to the membrane distal domain of type I macrophage scavenger receptor. Although no all-inclusive biological function has been ascribed to the SRCR family, some of these receptors have been shown to recognize pathogen-associated molecular patterns (PAMP) of bacteria, fungi, or other microbes. SSc5D is a recently described soluble SRCR receptor produced by monocytes/macrophages and T lymphocytes, consisting of an N-terminal portion, which contains five SRCR modules, and a large C-terminal mucin-like domain. Toward establishing a global common role for SRCR domains, we interrogated whether the set of five SRCR domains of SSc5D displayed pattern recognition receptor (PRR) properties. For that purpose, we have expressed in a mammalian expression system the N-terminal SRCR-containing moiety of SSc5D (N-SSc5D), thus excluding the mucin-like domain likely by nature to bind microorganisms, and tested the capacity of the SRCR functional groups to physically interact with bacteria. Using conventional protein–bacteria binding assays, we showed that N-SSc5D had a superior capacity to bind to Escherichia coli strains RS218 and IHE3034 compared with that of the extracellular domains of the SRCR proteins CD5 and CD6 (sCD5 and sCD6, respectively), and similar E. coli-binding properties as Spα, a proven PRR of the SRCR family. We have further designed a more sensitive, real-time, and label-free surface plasmon resonance (SPR)-based assay and examined the capacity of N-SSc5D, Spα, sCD5, and sCD6 to bind to different bacteria. We demonstrated that N-SSc5D compares with Spα in the capacity to bind to E. coli and Listeria monocytogenes, and further that it can distinguish between pathogenic E. coli RS218 and IHE3034 strains and the non-pathogenic laboratory E. coli strain BL21(DE3). Our work thus advocates the

  4. Comparison of Midregional Pro-A-Type Natriuretic Peptide and the N-Terminal Pro-B-Type Natriuretic Peptide for Predicting Mortality and Cardiovascular Events

    NARCIS (Netherlands)

    van Hateren, Kornelis J. J.; Alkhalaf, Alaa; Kleefstra, Nanne; Groenier, Klaas H.; de Jong, Paul E.; de Zeeuw, Dick; Gans, Rijk O. B.; Struck, Joachim; Bilo, Henk J. G.; Gansevoort, Ron T.; Bakker, Stephan J. L.

    2012-01-01

    BACKGROUND: N-terminal pro-B-type natriuretic peptide (NT-proBNP) provides prognostic information on mortality and future cardiovascular events for individuals from the general population. A novel immunoassay was recently developed that measures a midregional fragment of pro-A-type natriuretic pepti

  5. Activation of G Protein-Coupled Receptor Kinase 1 Involves Interactions between Its N-Terminal Region and Its Kinase Domain

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chih-chin; Orban, Tivadar; Jastrzebska, Beata; Palczewski, Krzysztof; Tesmer, John J.G. (Case Western); (Michigan)

    2012-03-16

    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its 20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.

  6. The major peanut allergen Ara h 1 and its cleaved-off N-terminal peptide; possible implications for peanut allergen detection

    NARCIS (Netherlands)

    Wichers, H.J.; Beijer, de T.; Savelkoul, H.F.J.; Amerongen, van A.

    2004-01-01

    Ara h 1 was purified from raw peanuts (Arachis hypogaea L.) in the presence or absence of protease inhibitors. N-Terminal amino acid sequences were determined after western blotting. Both purification procedures proved to be very consistent and resulted in identical chromatographic and electrophoret

  7. Unbiased Selective Isolation of Protein N-Terminal Peptides from Complex Proteome Samples Using Phospho Tagging PTAG) and TiO2-based Depletion

    NARCIS (Netherlands)

    Mommen, G.P.M.; Waterbeemd, van de B.; Meiring, H.D.; Kersten, G.; Heck, A.J.R.; Jong, de A.P.J.M.

    2012-01-01

    A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO2) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with formald

  8. Troponin T, N-terminal pro natriuretic peptide and a patent ductus arteriosus scoring system predict death before discharge or neurodevelopmental outcome at 2 years in preterm infants.

    LENUS (Irish Health Repository)

    El-Khuffash, Afif F

    2011-03-01

    There is little consensus regarding the use of echocardiography in patent ductus arteriosus (PDA) treatment in preterm infants. The use of troponin T (cTnT) and N-terminal Pro-BNP (NTpBNP) in combination with echocardiography assessment may facilitate the development of a superior predictive model.

  9. A novel method to isolate protein N-terminal peptides from proteome samples using sulfydryl tagging and gold-nanoparticle-based depletion.

    Science.gov (United States)

    Li, Lanting; Wu, Runqing; Yan, Guoquan; Gao, Mingxia; Deng, Chunhui; Zhang, Xiangmin

    2016-01-01

    A novel method to isolate global N-termini using sulfydryl tagging and gold-nanoparticle-based depletion (STagAu method) is presented. The N-terminal and lysine amino groups were first completely dimethylated at the protein level, after which the proteins were digested. The newly generated internal peptides were tagged with sulfydryl by Traut's reagent through digested N-terminal amines in yields of 96%. The resulting sulfydryl peptides were depleted through binding onto nano gold composite materials. The Au-S bond is stable and widely used in materials science. Nano gold composite materials showed nearly complete depletion of sulfydryl peptides. A set of the acetylated and dimethylated N-terminal peptides were analyzed by liquid chromatography-tandem mass spectrometry. This method was demonstrated to be an efficient N-terminus enrichment method because of the use of an effective derivatization reaction, in combination with robust and relative easy to implement Au-S coupling. We identified 632 N-terminal peptides from 386 proteins in a mouse liver sample. The STagAu approach presented is therefore a facile and efficient method for mass-spectrometry-based analysis of proteome N-termini or protease-generated cleavage products.

  10. Solution structure of N-terminal SH3 domain of Vav and the recognition site for Grb2 C-terminal SH3 domain

    International Nuclear Information System (INIS)

    The three-dimensional structure of the N-terminal SH3 domain (residues 583-660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies

  11. Prognostic assessment of elderly patients with symptoms of heart failure by combining high-sensitivity troponin T and N-terminal pro-B-type natriuretic peptide measurements

    DEFF Research Database (Denmark)

    Alehagen, Urban; Dahlström, Ulf; Rehfeld, Jens F.;

    2010-01-01

    N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a useful biomarker in heart failure assessment, whereas measurement of cardiac troponin is central in the diagnosis of patients with acute coronary syndromes. This report examined the prognostic use of combining high-sensitivity cardiac...

  12. Ultrafast resonance energy transfer from a site-specifically attached fluorescent chromophore reveals the folding of the N-terminal domain of CP29

    NARCIS (Netherlands)

    Oort, van B.F.; Murali, S.; Wientjes, E.; Koehorst, R.B.M.; Spruijt, R.B.; Hoek, van A.; Croce, R.; Amerongen, van H.

    2009-01-01

    The photosynthetic minor antenna complex CP29 of higher plants was singly mutated, overexpressed in Escherichia coli, selectively labeled with the fluorescent dye TAMRA at three positions in the N-terminal domain, and reconstituted with its natural pigments. Picosecond fluorescence experiments revea

  13. Quantification of the N-terminal propeptide of human procollagen type I (PINP): comparison of ELISA and RIA with respect to different molecular forms

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Hansen, M; Brandt, J;

    1998-01-01

    This paper compares the results of procollagen type I N-terminal propeptide (PINP) quantification by radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA). PINP in serum from a patient with uremic hyperparathyroidism was measured in RIA and ELISA to 20 micrograms l-1 and 116 microg...

  14. An Experimental Investigation of the Evolution of Chirality in a Potential Dynamic Peptide System: N-Terminal Epimerization and Degradation into Diketopiperazine

    Science.gov (United States)

    Danger, Grégoire; Plasson, Raphaël; Pascal, Robert

    2010-08-01

    The APED model (activation-polymerization-epimerization-depolymerization) is a unique example of a chemical system that allows symmetry breaking through a dynamic process involving indirect network autocatalysis. In its simplest version, the autocatalytic behavior of this model partly relies on the reproduction of local chiral centers in dipeptides through an epimerization process, with a thermodynamic preference for homochiral chains. We studied the reactivity of di- and tripeptides, containing a N-terminal phenylglycine (Phg) residue, as model compounds for the experimental determination of the kinetic and thermodynamic parameters related to the N-terminal epimerization process. Although the N-terminal residue is prone to spontaneous epimerization, catalysis was required for the epimerization to reach the equilibrium state in reasonable time. Unexpectedly, the observed equilibrium diastereoisomeric excesses have shown a general tendency for more stable heterochiral peptides, especially strong in the case of dipeptides. In parallel to this process, a stereoselective peptide cleavage through diketopiperazine formation was observed. Contrary to the N-terminal epimerization of peptides, the diketopiperazine formation did not need any catalyst, and heterochiral peptides were shown to be dynamically unstabilized, as they were cleaved faster than homochiral peptides. The validity of the extrapolation of these results to other residues and longer peptide chains is discussed, and some directions for future developments of the theoretical model are given.

  15. Plasma Pro-C3 (N-terminal type III collagen propeptide) predicts fibrosis progression in patients with chronic hepatitis C

    DEFF Research Database (Denmark)

    Nielsen, Mette J.; Veidal, Sanne S.; Karsdal, Morten A.;

    2015-01-01

    BACKGROUND & AIMS: Fibrogenesis results in release of certain extracellular matrix protein fragments into the circulation. We evaluated the diagnostic and prognostic performance of two novel serological markers, the precisely cleaved N-terminal propeptide of type III collagen (Pro-C3) and a peptide...

  16. N-Terminal Pro-B Type Natriuretic Peptide as a Marker of Bronchopulmonary Dysplasia or Death in Very Preterm Neonates

    DEFF Research Database (Denmark)

    Sellmer, Anna; Hjortdal, Vibeke Elisabeth; Bjerre, Jesper Vandborg;

    2015-01-01

    BACKGROUND: Bronchopulmonary dysplasia (BPD) is a serious complication of preterm birth. Plasma N-terminal pro-B type natriuretic peptide (NT-proBNP) has been suggested as a marker that may predict BPD within a few days after birth. OBJECTIVES: To investigate the association between NT-proBNP day...

  17. On the structure of the N-terminal domain of the MscL channel: helical bundle or membrane interface.

    Science.gov (United States)

    Iscla, Irene; Wray, Robin; Blount, Paul

    2008-09-01

    The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock and is to date the best characterized mechanosensitive channel. A well-recognized and supported model for Escherichia coli MscL gating proposes that the N-terminal 11 amino acids of this protein form a bundle of amphipathic helices in the closed state that functionally serves as a cytoplasmic second gate. However, a recently reexamined crystal structure of a closed state of the Mycobacterium tuberculosis MscL shows these helices running along the cytoplasmic surface of the membrane. Thus, it is unclear if one structural model is correct or if they both reflect valid closed states. Here, we have systematically reevaluated this region utilizing cysteine-scanning, in vivo functional characterization, in vivo SCAM, electrophysiological studies, and disulfide-trapping experiments. The disulfide-trapping pattern and functional studies do not support the helical bundle and second-gate hypothesis but correlate well with the proposed structure for M. tuberculosis MscL. We propose a functional model that is consistent with the collective data. PMID:18515388

  18. The N-terminal domain is a transcriptional activation domain required for Nanog to maintain ES cell self-renewal

    Institute of Scientific and Technical Information of China (English)

    GUO YunQian; ZHANG Juan; YE Li; CHEN Mo; YAO Dong; PAN GuangJin; ZHANG JieQiong; PEI DuanQing

    2009-01-01

    Nanog is a transcription factor identified by its ability to maintain the self-renewal of ES cells in the absence of leukemia inhibitory factor (LIF). Nanog protein contains an N-terminal domain (ND),a DNA-binding homeobox domain (HD) and a C-terminal domain (CD). We previously reported that the CD in Nanog is a transcriptional activation domain essential for the in vivo function of Nanog. Here we demonstrated that the ND in Nanog is also functionally important. Deletion of the ND reduces the transcriptional activity of Nanog on either artificial reporters or native Nanog promoters. This truncated Nanog is also less effective in regulating the endogenous Nanog target genes. Furthermore,the ND truncation disrupted the ability of Nanog to maintain ES cell self-renewal as well. We found that the ND Is not required for the nuclear localization of Nanog. These results suggest that the regulation of endogenous pluripotent genes such as oct3/4 and rex-1 is required for the in vivo function of Nanog.

  19. Recombinant Expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing Yield and N-terminal Processing.

    Science.gov (United States)

    Tanghe, Magali; Danneels, Barbara; Camattari, Andrea; Glieder, Anton; Vandenberghe, Isabel; Devreese, Bart; Stals, Ingeborg; Desmet, Tom

    2015-12-01

    The auxiliary activity family 9 (AA9, formerly GH61) harbors a recently discovered group of oxidative enzymes that boost cellulose degradation. Indeed, these lytic polysaccharide monooxygenases (LPMOs) are able to disrupt the crystalline structure of cellulose, thereby facilitating the work of hydrolytic enzymes involved in biomass degradation. Since these enzymes require an N-terminal histidine residue for activity, their recombinant production as secreted protein is not straightforward. We here report the expression optimization of Trichoderma reesei Cel61A (TrCel61A) in the host Pichia pastoris. The use of the native TrCel61A secretion signal instead of the alpha-mating factor from Saccharomyces cerevisiae was found to be crucial, not only to obtain high protein yields (>400 mg/L during fermentation) but also to enable the correct processing of the N-terminus. Furthermore, the LPMO activity of the enzyme is demonstrated here for the first time, based on its degradation profile of a cellulosic substrate.

  20. DNA replication checkpoint signaling depends on a Rad53-Dbf4 N-terminal interaction in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chen, Ying-Chou; Kenworthy, Jessica; Gabrielse, Carrie; Hänni, Christine; Zegerman, Philip; Weinreich, Michael

    2013-06-01

    Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.

  1. PredSL: A Tool for the N-terminal Sequence-based Prediction of Protein Subcellular Localization

    Institute of Scientific and Technical Information of China (English)

    Evangelia I. Petsalaki; Pantelis G. Bagos; Zoi I. Litou; Stavros J. Hamodrakas

    2006-01-01

    The ability to predict the subcellular localization of a protein from its sequence is of great importance, as it provides information about the protein's function.We present a computational tool, PredSL, which utilizes neural networks, Markov chains, profile hidden Markov models, and scoring matrices for the prediction of the subcellular localization of proteins in eukaryotic cells from the N-terminal amino acid sequence. It aims to classify proteins into five groups: chloroplast,thylakoid, mitochondrion, secretory pathway, and "other". When tested in a fivefold cross-validation procedure, PredSL demonstrates 86.7% and 87.1% overall accuracy for the plant and non-plant datasets, respectively. Compared with TargetP, which is the most widely used method to date, and LumenP, the results of PredSL are comparable in most cases. When tested on the experimentally verified proteins of the Saccharomyces cerevisiae genome, PredSL performs comparably if not better than any available algorithm for the same task. Furthermore, PredSL is the only method capable for the prediction of these subcellular localizations that is available as a stand-alone application through the URL:http://bioinformatics.biol.uoa.gr/PredSL/.

  2. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

    Directory of Open Access Journals (Sweden)

    Carolin Lübker

    2015-07-01

    Full Text Available Bacillus anthracis adenylyl cyclase toxin edema factor (EF is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH-oxidase, thus reducing production of reactive oxygen species (ROS used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut with Met to leucine (Leu substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.

  3. Identification of a novel family of laminin N-terminal alternate splice isoforms: structural and functional characterization.

    Science.gov (United States)

    Hamill, Kevin J; Langbein, Lutz; Jones, Jonathan C R; McLean, W H Irwin

    2009-12-18

    The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5'-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) alpha3 and LaNt alpha5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt alpha3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt alpha3 and LaNt alpha3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt alpha3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt alpha3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro. PMID:19773554

  4. Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain.

    Science.gov (United States)

    Stroehlein, Andreas J; Young, Neil D; Korhonen, Pasi K; Chang, Bill C H; Sternberg, Paul W; La Rosa, Giuseppe; Pozio, Edoardo; Gasser, Robin B

    2016-01-01

    Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea) represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes) of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan) kinases, as a foundation for their subsequent structural and functional investigation. PMID:27412987

  5. Analyses of Compact Trichinella Kinomes Reveal a MOS-Like Protein Kinase with a Unique N-Terminal Domain

    Directory of Open Access Journals (Sweden)

    Andreas J. Stroehlein

    2016-09-01

    Full Text Available Parasitic worms of the genus Trichinella (phylum Nematoda; class Enoplea represent a complex of at least twelve taxa that infect a range of different host animals, including humans, around the world. They are foodborne, intracellular nematodes, and their life cycles differ substantially from those of other nematodes. The recent characterization of the genomes and transcriptomes of all twelve recognized taxa of Trichinella now allows, for the first time, detailed studies of their molecular biology. In the present study, we defined, curated, and compared the protein kinase complements (kinomes of Trichinella spiralis and T. pseudospiralis using an integrated bioinformatic workflow employing transcriptomic and genomic data sets. We examined how variation in the kinome might link to unique aspects of Trichinella morphology, biology, and evolution. Furthermore, we utilized in silico structural modeling to discover and characterize a novel, MOS-like kinase with an unusual, previously undescribed N-terminal domain. Taken together, the present findings provide a basis for comparative investigations of nematode kinomes, and might facilitate the identification of Enoplea-specific intervention and diagnostic targets. Importantly, the in silico modeling approach assessed here provides an exciting prospect of being able to identify and classify currently unknown (orphan kinases, as a foundation for their subsequent structural and functional investigation.

  6. Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration.

    Science.gov (United States)

    Hayashi, Moyuru; Tamura, Yosuke; Dohmae, Naoshi; Kojima, Soichi; Shimonaka, Motoyuki

    2008-05-01

    Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides. PMID:18294956

  7. Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration

    International Nuclear Information System (INIS)

    Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides

  8. Crystal Structure of the N-Terminal RNA Recognition Motif of mRNA Decay Regulator AUF1

    Directory of Open Access Journals (Sweden)

    Young Jun Choi

    2016-01-01

    Full Text Available AU-rich element binding/degradation factor 1 (AUF1 plays a role in destabilizing mRNAs by forming complexes with AU-rich elements (ARE in the 3′-untranslated regions. Multiple AUF1-ARE complexes regulate the translation of encoded products related to the cell cycle, apoptosis, and inflammation. AUF1 contains two tandem RNA recognition motifs (RRM and a Gln- (Q- rich domain in their C-terminal region. To observe how the two RRMs are involved in recognizing ARE, we obtained the AUF1-p37 protein covering the two RRMs. However, only N-terminal RRM (RRM1 was crystallized and its structure was determined at 1.7 Å resolution. It appears that the RRM1 and RRM2 separated before crystallization. To demonstrate which factors affect the separate RRM1-2, we performed limited proteolysis using trypsin. The results indicated that the intact proteins were cleaved by unknown proteases that were associated with them prior to crystallization. In comparison with each of the monomers, the conformations of the β2-β3 loops were highly variable. Furthermore, a comparison with the RRM1-2 structures of HuR and hnRNP A1 revealed that a dimer of RRM1 could be one of the possible conformations of RRM1-2. Our data may provide a guidance for further structural investigations of AUF1 tandem RRM repeat and its mode of ARE binding.

  9. The N-terminal part of Als1 protein from Candida albicans specifically binds fucose-containing glycans.

    Science.gov (United States)

    Donohue, Dagmara S; Ielasi, Francesco S; Goossens, Katty V Y; Willaert, Ronnie G

    2011-06-01

    The opportunistic pathogen Candida albicans expresses on its surface Als (Agglutinin like sequence) proteins, which play an important role in the adhesion to host cells and in the development of candidiasis. The binding specificity of these proteins is broad, as they can bind to various mammalian proteins, such as extracellular matrix proteins, and N- and E-cadherins. The N-terminal part of Als proteins constitutes the substrate-specific binding domain and is responsible for attachment to epithelial and endothelial cells. We have used glycan array screening to identify possible glycan receptors for the binding domain of Als1p-N. Under those conditions, Als1p-N binds specifically to fucose-containing glycans, which adds a lectin function to the functional diversity of the Als1 protein. The binding between Als1p-N and BSA-fucose glycoconjugate was quantitatively characterized using surface plasmon resonance, which demonstrated a weak millimolar affinity between Als1p-N and fucose. Furthermore, we have also quantified the affinity of Als1p-N to the extracellular matrix proteins proteins fibronectin and laminin, which is situated in the micromolar range. Surface plasmon resonance characterization of Als1p-N-Als1p-N interaction was in the micromolar affinity range.

  10. NAA10 mutation causing a novel intellectual disability syndrome with Long QT due to N-terminal acetyltransferase impairment

    Science.gov (United States)

    Casey, Jillian P.; Støve, Svein I.; McGorrian, Catherine; Galvin, Joseph; Blenski, Marina; Dunne, Aimee; Ennis, Sean; Brett, Francesca; King, Mary D.; Arnesen, Thomas; Lynch, Sally Ann

    2015-01-01

    We report two brothers from a non-consanguineous Irish family presenting with a novel syndrome characterised by intellectual disability, facial dysmorphism, scoliosis and long QT. Their mother has a milder phenotype including long QT. X-linked inheritance was suspected. Whole exome sequencing identified a novel missense variant (c.128 A > C; p.Tyr43Ser) in NAA10 (X chromosome) as the cause of the family’s disorder. Sanger sequencing confirmed that the mutation arose de novo in the carrier mother. NAA10 encodes the catalytic subunit of the major human N-terminal acetylation complex NatA. In vitro assays for the p.Tyr43Ser mutant enzyme showed a significant decrease in catalytic activity and reduced stability compared to wild-type Naa10 protein. NAA10 has previously been associated with Ogden syndrome, Lenz microphthalmia syndrome and non-syndromic developmental delay. Our findings expand the clinical spectrum of NAA10 and suggest that the proposed correlation between mutant Naa10 enzyme activity and phenotype severity is more complex than anticipated; the p.Tyr43Ser mutant enzyme has less catalytic activity than the p.Ser37Pro mutant associated with lethal Ogden syndrome but results in a milder phenotype. Importantly, we highlight the need for cardiac assessment in males and females with NAA10 variants as both patients and carriers can have long QT. PMID:26522270

  11. Regulation of Telomere Length Requires a Conserved N-Terminal Domain of Rif2 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kaizer, Hannah; Connelly, Carla J; Bettridge, Kelsey; Viggiani, Christopher; Greider, Carol W

    2015-10-01

    The regulation of telomere length equilibrium is essential for cell growth and survival since critically short telomeres signal DNA damage and cell cycle arrest. While the broad principles of length regulation are well established, the molecular mechanism of how these steps occur is not fully understood. We mutagenized the RIF2 gene in Saccharomyces cerevisiae to understand how this protein blocks excess telomere elongation. We identified an N-terminal domain in Rif2 that is essential for length regulation, which we have termed BAT domain for Blocks Addition of Telomeres. Tethering this BAT domain to Rap1 blocked telomere elongation not only in rif2Δ mutants but also in rif1Δ and rap1C-terminal deletion mutants. Mutation of a single amino acid in the BAT domain, phenylalanine at position 8 to alanine, recapitulated the rif2Δ mutant phenotype. Substitution of F8 with tryptophan mimicked the wild-type phenylalanine, suggesting the aromatic amino acid represents a protein interaction site that is essential for telomere length regulation.

  12. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    Energy Technology Data Exchange (ETDEWEB)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Cotmore, Susan F. [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Tattersall, Peter [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Departments of Genetics, Yale University Medical School, New Haven, CT 06510 (United States); Zhao, Haiyan, E-mail: zhaohy@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Tang, Liang, E-mail: tangl@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States)

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  13. Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back.

    Science.gov (United States)

    Alamo, Lorenzo; Li, Xiaochuan Edward; Espinoza-Fonseca, L Michel; Pinto, Antonio; Thomas, David D; Lehman, William; Padrón, Raúl

    2015-08-01

    Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence length analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation. PMID:26038302

  14. Role of N-terminal domain of HMW 1Dx5 in the functional and structural properties of wheat dough.

    Science.gov (United States)

    Wang, Jing Jing; Liu, Guang; Huang, Yan-Bo; Zeng, Qiao-Hui; Song, Guo-Sheng; Hou, Yi; Li, Lin; Hu, Song-Qing

    2016-12-15

    Effects of N-terminal domain of high molecular weight glutenin subunit (HMW-GS) 1Dx5 (1Dx5-N) on functional and structural properties of wheat dough were determined by farinographic and rheological analysis, size exclusion chromatography, non-reducing/reducing SDS-PAGE, total free sulfhydryl determination, scanning electron microscopy and Fourier transform infrared spectroscopy. Results showed that 1Dx5-N improved the quality of dough with the increased water absorption, dough stability time, elastic and viscous modulus, and the decreased degree of softening, loss tangent. These improvements could be attributed to the formation of the macro-molecular weight aggregates and massive protein networks, which were favored by 1Dx5-N through disulfide bonds and hydrophobic interactions. Additionally, 1Dx5-N drove the transition of α-helix and random coil conformations to β-sheet and β-turn conformations, further demonstrating the formation of HMW-GS polymers and the enhancement of dough strength. Moreover, all the positive effects of 1Dx5-N were reinforced by edible salt NaCl. PMID:27451235

  15. The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain

    Science.gov (United States)

    Ampah-Korsah, Henry; Anderberg, Hanna I.; Engfors, Angelica; Kirscht, Andreas; Norden, Kristina; Kjellstrom, Sven; Kjellbom, Per; Johanson, Urban

    2016-01-01

    Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel. PMID:27379142

  16. The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain.

    Science.gov (United States)

    Ampah-Korsah, Henry; Anderberg, Hanna I; Engfors, Angelica; Kirscht, Andreas; Norden, Kristina; Kjellstrom, Sven; Kjellbom, Per; Johanson, Urban

    2016-01-01

    Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel. PMID:27379142

  17. Calcium Occupancy of N-terminal Sites within Calmodulin Induces Inhibition of the Ryanodine Receptor Calcium Release Channel

    Energy Technology Data Exchange (ETDEWEB)

    Boschek, Curt B; Jones, Terry E; Squier, Thomas C; Bigelow, Diana J

    2007-08-01

    Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e., K3614 – N3643) located within the central portion of the primary sequence. However, it is currently unclear whether the identified CaM-binding sequence a) senses calcium over the physiological range of calcium-concentrations associated with RyR1 regulation or b) plays a structural role unrelated to the calcium-dependent modulation of RyR1 function. Therefore, we have measured the calcium-dependent activation of the individual domains of CaM in association with RyRp and their relationship to the CaM-dependent regulation of RyR1. These measurements utilize an engineered CaM, permitting the site-specific incorporation of N-(1-pyrene) maleimide at either T34C (PyN-CaM) or T110C (PyC-CaM) in the N- and C-domains, respectively. Consistent with prior measurements, we observe a high-affinity association between both apo- and calcium-activated CaM and RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-activation of these individual domains. Fluorescence changes upon calcium-activation of PyC-CaM in association with RyRp are indicative of high-affinity calcium-dependent activation of the C-terminal domain of CaM bound to RyRp at resting calcium levels and the activation of the N-terminal domain at levels of calcium associated cellular activation. In comparison, occupancy of calcium-binding sites in the N-domain of CaM mirrors the calcium-dependence of RyR1 inhibition observed at activating calcium levels, where [Ca]1/2 = 4.3 0.4 μM, suggesting a direct regulation of Ry

  18. High circulating N-terminal pro-brain natriuretic peptide and tumor necrosis factor-α in mixed cryoglobulinemia

    Institute of Scientific and Technical Information of China (English)

    Alessandro Antonelli; Clodoveo Ferri; Silvia Martina Ferrari; Fabio Galetta; Ferdinando Franzoni; Gino Santoro; Salvatore De Marco; Emiliano Ghiri; Poupak Fallahi

    2009-01-01

    AIM: To evaluate serum levels of N-terminal pro-brain natriuretic peptide (NTproBNP) and tumor necrosis factor α (TNF-α) in a large series of patients with hepatitis C associated with mixed cryoglobulinemia (MC+HCV).METHODS: Serum NTproBNP and TNF-α levels were assayed in 50 patients with MC+HCV, and in 50 sex-and age-matched controls.RESULTS: Cryoglobulinemic patients showed significantly higher mean NTproBNP and TNF-α levels than controls ( P < 0.001; Mann-Whitney U test). By defining high NTproBNP level as a value higher than 125 pg/mL (the single cut-off point for outpatients under 75 years of age), 30% of MC+HCV and 6% of controls had high NTproBNP (χ~2, P < 0.01). With a cut-off point of 300 pg/mL (used to rule out heart failure (HF) in patients under 75 years of age), 8% of MC+HCV and 0 controls had high NTproBNP (χ~2, P < 0.04). With a cut-off point of 900 pg/mL (used for ruling in HF in patients aged 50-75 years; such as the patients of our study), 6% of MC+HCV and 0 controls had high NTproBNP (χ~2, P = 0.08).CONCLUSION: The study demonstrates high levels of circulating NTproBNP and TNF-α in MC+HCV patients.The increase of NTproBNP may indicate the presence of a subclinical cardiac dysfunction.

  19. N-Terminal Domain of Feline Calicivirus (FCV) Proteinase-Polymerase Contributes to the Inhibition of Host Cell Transcription.

    Science.gov (United States)

    Wu, Hongxia; Zu, Shaopo; Sun, Xue; Liu, Yongxiang; Tian, Jin; Qu, Liandong

    2016-01-01

    Feline Calicivirus (FCV) infection results in the inhibition of host protein synthesis, known as "shut-off". However, the precise mechanism of shut-off remains unknown. Here, we found that the FCV strain 2280 proteinase-polymerase (PP) protein can suppress luciferase reporter gene expression driven by endogenous and exogenous promoters. Furthermore, we found that the N-terminal 263 aa of PP (PPN-263) determined its shut-off activity using the expression of truncated proteins. However, the same domain of the FCV strain F9 PP protein failed to inhibit gene expression. A comparison between strains 2280 and F9 indicated that Val27, Ala96 and Ala98 were key sites for the inhibition of host gene expression by strain 2280 PPN-263, and PPN-263 exhibited the ability to shut off host gene expression as long as it contained any two of the three amino acids. Because the N-terminus of the PP protein is required for its proteinase and shut-off activities, we investigated the ability of norovirus 3C-like proteins (3CLP) from the GII.4-1987 and -2012 isolates to interfere with host gene expression. The results showed that 3CLP from both isolates was able to shut off host gene expression, but 3CLP from GII.4-2012 had a stronger inhibitory activity than that from GII.4-1987. Finally, we found that 2280 PP and 3CLP significantly repressed reporter gene transcription but did not affect mRNA translation. Our results provide new insight into the mechanism of the FCV-mediated inhibition of host gene expression. PMID:27447663

  20. Type I Collagen Synthesis Marker Procollagen I N-Terminal Peptide (PINP) in Prostate Cancer Patients Undergoing Intermittent Androgen Suppression

    International Nuclear Information System (INIS)

    Intermittent androgen suppression (IAS) therapy for prostate cancer patients attempts to maintain the hormone dependence of the tumor cells by cycles alternating between androgen suppression (AS) and treatment cessation till a certain prostate-specific antigen (PSA) threshold is reached. Side effects are expected to be reduced, compared to standard continuous androgen suppression (CAS) therapy. The present study examined the effect of IAS on bone metabolism by determinations of serum procollagen I N-terminal peptide (PINP), a biochemical marker of collagen synthesis. A total of 105 treatment cycles of 58 patients with prostate cancer stages ≥pT2 was studied assessing testosterone, PSA and PINP levels at monthly intervals. During phases of AS lasting for up to nine months PSA levels were reversibly reduced, indicating apoptotic regression of the prostatic tumors. Within the first cycle PINP increased at the end of the AS period and peaked in the treatment cessation phase. During the following two cycles a similar pattern was observed for PINP, except a break in collagen synthesis as indicated by low PINP levels in the first months off treatment. Therefore, measurements of the serum PINP concentration indicated increased bone matrix synthesis in response to >6 months of AS, which uninterruptedly continued into the first treatment cessation phase, with a break into each of the following two pauses. In summary, synthesis of bone matrix collagen increases while degradation decreases during off-treatment phases in patients undergoing IAS. Although a direct relationship between bone matrix turnover and risk of fractures is difficult to establish, IAS for treatment of biochemical progression of prostate tumors is expected to reduce osteoporosis in elderly men often at high risk for bone fractures representing a highly suitable patient population for this kind of therapy

  1. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    Directory of Open Access Journals (Sweden)

    Mehmet Karaca

    Full Text Available Reactivation of androgen receptor (AR may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

  2. Ribonucleocapsid Formation of SARS-COV Through Molecular Action of the N-Terminal Domain of N Protein

    Energy Technology Data Exchange (ETDEWEB)

    Saikatendu, K.S.; Joseph, J.S.; Subramanian, V.; Neuman, B.W.; Buchmeier, M.J.; Stevens, R.C.; Kuhn, P.; /Scripps Res. Inst.

    2007-07-12

    Conserved amongst all coronaviruses are four structural proteins, the matrix (M), small envelope (E) and spike (S) that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in their lumen. The N terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C-terminus of N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17A (monoclinic) and 1.85 A (cubic) respectively, solved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core and is oriented similar to that in the IBV N-NTD and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggest a common mode of RNA recognition, but probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs hints that they employ different modes of both RNA recognition as well as oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

  3. N-terminal additions to the WE14 peptide of chromogranin A create strong autoantigen agonists in type 1 diabetes.

    Science.gov (United States)

    Jin, Niyun; Wang, Yang; Crawford, Frances; White, Janice; Marrack, Philippa; Dai, Shaodong; Kappler, John W

    2015-10-27

    Chromogranin A (ChgA) is an autoantigen for CD4(+) T cells in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). The natural ChgA-processed peptide, WE14, is a weak agonist for the prototypical T cell, BDC-2.5, and other ChgA-specific T-cell clones. Mimotope peptides with much higher activity share a C-terminal motif, WXRM(D/E), that is predicted to lie in the p5 to p9 position in the mouse MHC class II, IA(g7) binding groove. This motif is also present in WE14 (WSRMD), but at its N terminus. Therefore, to place the WE14 motif into the same position as seen in the mimotopes, we added the amino acids RLGL to its N terminus. Like the other mimotopes, RLGL-WE14, is much more potent than WE14 in T-cell stimulation and activates a diverse population of CD4(+) T cells, which also respond to WE14 as well as islets from WT, but not ChgA(-/-) mice. The crystal structure of the IA(g7)-RLGL-WE14 complex confirmed the predicted placement of the peptide within the IA(g7) groove. Fluorescent IA(g7)-RLGL-WE14 tetramers bind to ChgA-specific T-cell clones and easily detect ChgA-specific T cells in the pancreas and pancreatic lymph nodes of NOD mice. The prediction that many different N-terminal amino acid extensions to the WXRM(D/E) motif are sufficient to greatly improve T-cell stimulation leads us to propose that such a posttranslational modification may occur uniquely in the pancreas or pancreatic lymph nodes, perhaps via the mechanism of transpeptidation. This modification could account for the escape of these T cells from thymic negative selection. PMID:26453556

  4. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    Science.gov (United States)

    Karaca, Mehmet; Liu, Yuanbo; Zhang, Zhentao; De Silva, Dinuka; Parker, Joel S; Earp, H Shelton; Whang, Young E

    2015-01-01

    Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

  5. Relationship of the plasma urotensin Ⅱ with proadrenomedullin N-terminal 20 peptide in patients with congestive heart failure

    Institute of Scientific and Technical Information of China (English)

    钟萍; 李志樑; 吴宏超; 唐朝枢; 陆青

    2003-01-01

    Objective: To understand the role of urotensin Ⅱ(UⅡ) and proadrenomedullin N-terminal 20 peptide (PAMP), a fragment of proadrenomedullin (proADM) possessing biological activity, in the pathophysiological process of congestive heart failure (CHF) by observing the variation of their plasma levels and exploring their interrelations. Methods: Plasma UⅡ and PAMP levels were measured by radioimmunoassay in 52 patients with CHF and 14 healthy subjects. Left ventricular ejection fraction (LVEF) and the ratio of E/A were determined by echocardiography. Results: The plasma UⅡ level was significantly lower in patients with CHF than the healthy subjects (1.5±1.0 pg/ml vs 4.3±1.2 pg/ml, P<0.05), while plasma PAMP level was significantly higher in the former group (30.6±5.8 pg/ml vs 21.0±6.6 pg/ml P<0.05). The levels of UⅡ and PAMP were parallel with the severity of CHF, and significant correlation of plasma levels of UⅡ with LVEF (r=0.530, P=0.000) and the ratio of E/A (r=0.618, P=0.000) was noted. LVEF and ratio of E/A were found to be inversely correlated with plasma PAMP levels in the patients (r=-0.568, P=0.000; r=-0.350, P=0.004). Also found was the significant correlation between plasma UⅡ and PAMP levels (r=-0.528, P=0.000). The treatment of the patients resulted in increased plasma UⅡ levels and lowered PAMP levels. Conclusion: The variations of plasma levels of UⅡ and PAMP are parallel with the severity of CHF, suggesting their cooperative actions in the pathophysiology of CHF.

  6. Elevated N-terminal pro-brain natriuretic peptide is associated with mortality in tobacco smokers independent of airflow obstruction.

    Directory of Open Access Journals (Sweden)

    Jason A Stamm

    Full Text Available BACKGROUND: Tobacco use is associated with an increased prevalence of cardiovascular disease. N-terminal pro-brain natiuretic peptide (NT-proBNP, a widely available biomarker that is associated with cardiovascular outcomes in other conditions, has not been investigated as a predictor of mortality in tobacco smokers. We hypothesized that NT-proBNP would be an independent prognostic marker in a cohort of well-characterized tobacco smokers without known cardiovascular disease. METHODS: Clinical data from 796 subjects enrolled in two prospective tobacco exposed cohorts was assessed to determine factors associated with elevated NT-proBNP and the relationship of these factors and NT-proBNP with mortality. RESULTS: Subjects were followed for a median of 562 (IQR 252-826 days. Characteristics associated with a NT-proBNP above the median (≥49 pg/mL were increased age, female gender, and decreased body mass index. By time-to-event analysis, an NT-proBNP above the median (≥49 pg/mL was a significant predictor of mortality (log rank p = 0.02. By proportional hazard analysis controlling for age, gender, cohort, and severity of airflow obstruction, an elevated NT-proBNP level (≥49 pg/mL remained an independent predictor of mortality (HR = 2.19, 95% CI 1.07-4.46, p = 0.031. CONCLUSIONS: Elevated NT-proBNP is an independent predictor of mortality in tobacco smokers without known cardiovascular disease, conferring a 2.2 fold increased risk of death. Future studies should assess the ability of this biomarker to guide further diagnostic testing and to direct specific cardiovascular risk reduction inventions that may positively impact quality of life and survival.

  7. Two Distinctive Binding Modes of Endonuclease Inhibitors to the N-Terminal Region of Influenza Virus Polymerase Acidic Subunit.

    Science.gov (United States)

    Fudo, Satoshi; Yamamoto, Norio; Nukaga, Michiyoshi; Odagiri, Takato; Tashiro, Masato; Hoshino, Tyuji

    2016-05-10

    Influenza viruses are global threat to humans, and the development of new antiviral agents are still demanded to prepare for pandemics and to overcome the emerging resistance to the current drugs. Influenza polymerase acidic protein N-terminal domain (PAN) has endonuclease activity and is one of the appropriate targets for novel antiviral agents. First, we performed X-ray cocrystal analysis on the complex structures of PAN with two endonuclease inhibitors. The protein crystallization and the inhibitor soaking were done at pH 5.8. The binding modes of the two inhibitors were different from a common binding mode previously reported for the other influenza virus endonuclease inhibitors. We additionally clarified the complex structures of PAN with the same two endonuclease inhibitors at pH 7.0. In one of the crystal structures, an additional inhibitor molecule, which chelated to the two metal ions in the active site, was observed. On the basis of the crystal structures at pH 7.0, we carried out 100 ns molecular dynamics (MD) simulations for both of the complexes. The analysis of simulation results suggested that the binding mode of each inhibitor to PAN was stable in spite of the partial deviation of the simulation structure from the crystal one. Furthermore, crystal structure analysis and MD simulation were performed for PAN in complex with an inhibitor, which was already reported to have a high compound potency for comparison. The findings on the presence of multiple binding sites at around the PAN substrate-binding pocket will provide a hint for enhancing the binding affinity of inhibitors. PMID:27088785

  8. Arabidopsis MKS1 is involved in basal immunity and requires an intact N-terminal domain for proper function.

    Directory of Open Access Journals (Sweden)

    Klaus Petersen

    Full Text Available BACKGROUND: Innate immune signaling pathways in animals and plants are regulated by mitogen-activated protein kinase (MAPK cascades. MAP kinase 4 (MPK4 functions downstream of innate immune receptors via a nuclear substrate MKS1 to regulate the activity of the WRKY33 transcription factor, which in turn controls the production of anti-microbial phytoalexins. METHODOLOGY/PRINCIPAL FINDINGS: We investigate the role of MKS1 in basal resistance and the importance of its N- and C-terminal domains for MKS1 function. We used the information that mks1 loss-of-function partially suppresses the mpk4 loss-of-function phenotype, and that transgenic expression of functional MKS1 in mpk4/mks1 double mutants reverted the mpk4 dwarf phenotype. Transformation of mks1/mpk4 with mutant versions of MKS1 constructs showed that a single amino acid substitution in a putative MAP kinase docking domain, MKS1-L32A, or a truncated MKS1 version unable to interact with WRKY33, were deficient in reverting the double mutant to the mpk4 phenotype. These results demonstrate functional requirement in MKS1 for the interaction with MPK4 and WRKY33. In addition, nuclear localization of MKS1 was shown to depend on an intact N-terminal domain. Furthermore, loss-of-function mks1 mutants exhibited increased susceptibility to strains of Pseudomonas syringae and Hyaloperonospora arabidopsidis, indicating that MKS1 plays a role in basal defense responses. CONCLUSIONS: Taken together, our results indicate that MKS1 function and subcellular location requires an intact N-terminus important for both MPK4 and WRKY33 interactions.

  9. Runx2-I isoform contributes to fetal bone formation even in the absence of specific N-terminal amino acids.

    Directory of Open Access Journals (Sweden)

    Hideaki Okura

    Full Text Available The Runt-related transcription factor 2 (Runx2 gene encodes the transcription factor Runx2, which is the master regulator of osteoblast development; insufficiency of this protein causes disorders of bone development such as cleidocranial dysplasia. Runx2 has two isoforms, Runx2-II and Runx2-I, and production of each isoform is controlled by a unique promoter: a distal promoter (P1 and a proximal promoter (P2, respectively. Although several studies have focused on differences and similarities between the two Runx2 isoforms, their individual roles in bone formation have not yet been determined conclusively, partly because a Runx2-I-targeted mouse model is not available. In this study, we established a novel Runx2-manipulated mouse model in which the first ATG of Runx2-I was replaced with TGA (a stop codon, and a neomycin-resistant gene (neo cassette was inserted at the first intron of Runx2-I. Homozygous Runx2-Ineo/neo mice showed severely reduced expression of Runx2-I, whereas Runx2-II expression was largely retained. Runx2-Ineo/neo mice showed neonatal lethality, and in these mice, intramembranous ossification was more severely defective than endochondral ossification, presumably because of the greater involvement of Runx2-I, compared with that of Runx2-II in intramembranous ossification. Interestingly, the depletion of neo rescued the above-described phenotypes, indicating that the isoform-specific N-terminal region of Runx2-I is not functionally essential for bone development. Taken together, our results provide a novel clue leading to a better understanding of the roles of Runx2 isoforms in osteoblast development.

  10. High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes

    Institute of Scientific and Technical Information of China (English)

    Linhui Wang; Fang Lin; Junchao Wu; Zhenghong Qin

    2009-01-01

    Huntington's disease (HD) is caused by an expansion of polyglutamine tract in N-terminus of huntingtin (htt).The mutation of htt leads to dysfunction and premature death of striatal and cortical neurons. However, the effects of htt mutation on glia remain largely unknown.This study aimed to establish a glia HD model using an adenoviral vector to express wild-type and mutant N-terminal huntingtin fragment 1-552 amino acids (htt552) in rat primary cortical astrocytes. We have eval-uated optimal conditions for the infection of astrocytes with adenovirai vectors, and the kinetics of the expression of htt552 in astrocytes. The majority of astroeytes expressed the transgene after infection. At 24 h post-infection, the highest rate of infection was 89 + 3% for the wild-type (htt552-18Q) with a multiplicity of infection (m.o.i.) of 80, and the highest rate of infection was 91 +4% for the mutant type (htt552-100Q) with the same viral dose. The duration of expression of htt552 lasted for about 7 days with a relatively high level from 1 to 4 days post-infection. Mutant huntingtin (htt552-100Q) pro-duced the characteristic HD pathology after 3 days by the appearance of cytoplasmic aggregates and intranue-lear inclusions. The result of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu mbromide)assay showed that the inhibition of viability by virus on astrocytes was also dose-dependent. To obtain high infection rate and low toxicity, the viral dose with an m.o.i, of 40 was optimal to our cell model. The present study demonstrates that adenovirai-mediated expression of mutant htt provides an advantageous system for his-tological and biochemical analysis of HD pathogenesis in primary cortical astrocyte cultures.

  11. The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain

    Science.gov (United States)

    Nagel, Claus-Henning; Binz, Anne; Sodeik, Beate; Bauerfeind, Rudolf; Bailer, Susanne M.

    2015-01-01

    Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+)Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary envelopment. PMID:26083367

  12. The N-terminal cytoplasmic region of NCBE displays features of an intrinsic disordered structure and represents a novel target for specific drug screening

    Directory of Open Access Journals (Sweden)

    Kaare eBjerregaard-Andersen

    2013-11-01

    Full Text Available The sodium dependent bicarbonate transporter NCBE/NBCn2 is predominantly expressed in the central nervous system (CNS. The highest protein abundance is found in the choroid plexus. The primary function of this integral plasma membrane transport protein is to regulate intracellular neuronal pH and probably to maintain the pH homeostasis across the blood-cerebrospinal fluid barrier (CSFB. NCBE has a transmembrane region consisting of 10 predicted α-helices. The N- and C- termini are both cytoplasmic, with a large N-terminal domain (Nt-NCBE and a relatively small C-terminal domain (Ct-NCBE. The cytoplasmic N-terminal domain is likely involved in bicarbonate recognition and transport and contains key areas of regulation through pH sensing and protein - protein interactions (PPIs. Intrinsic disordered proteins (IDPs and regions (IDPRs are defined as not having any rigid three-dimensional structure under physiological conditions and are believed to be involved in signaling networks in which specific, though with low affinity, PPIs play an important role in the signaling event. We show that NCBE and other SLC4 family members have a high level of predicted intrinsic disorder prevalent in the cytoplasmic regions. To provide biophysical evidence for the IDPR predicted in Nt-NCBE, we isolated recombinant NCBE from E. coli and purified it to >99 % purity and used it to perform differential scanning fluorescence spectroscopy (DSF, in the search for small molecules that induce secondary or tertiary structure. This will promote the current need to develop selective drugs for individual SLC4 family members. We have also determined a low resolution X-ray crystal structure of the N-terminal core domain at 4.0 Å resolution. The N-terminal cytoplasmic domain of AE1 (cdb3 shares a similar fold with the N-terminal core domain of NCBE. The crystal conditions for the full-length N-terminal domain have been explored, however, only the core domain forms diffracting

  13. The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain.

    Directory of Open Access Journals (Sweden)

    Christina Funk

    2015-06-01

    Full Text Available Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary

  14. PAD4-mediated neutrophil extracellular trap formation is not required for immunity against influenza infection.

    Directory of Open Access Journals (Sweden)

    Saskia Hemmers

    Full Text Available During an inflammatory response, neutrophils migrate to the site of infection where they can kill invading pathogens by phagocytosis, secretion of anti-microbicidal mediators or the release of neutrophil extracellular traps (NETs. NETs are specialized anti-microbial structures comprised of decondensed chromatin decorated with microbicidal agents. Increased amount of NETs have been found in patients suffering from the chronic lung inflammatory disease cystic fibrosis, correlating with increased severity of pulmonary obstruction. Furthermore, acute lung inflammation during influenza A infection is characterized by a massive influx of neutrophils into the lung. The role of NETs during virus-mediated lung inflammation is unknown. Peptidylarginine deiminase 4 (PAD4-mediated deimination of histone H3 and H4 is required for NET formation. Therefore, we generated a PAD4-deficient mouse strain that has a striking inability to form NETs. These mice were infected with influenza A/WSN, and the disease was monitored at the level of leukocytic lung infiltration, lung pathology, viral replication, weight loss and mortality. PAD4 KO fared comparable to WT mice in all the parameters tested, but they displayed slight but statistically different weight loss kinetics during infection that was not reflected in enhanced survival. Overall, we conclude that PAD4-mediated NET formation is dispensable in a mouse model of influenza A infection.

  15. Chronic Restraint Stress Promotes Immune Suppression through Toll-like Receptor 4-Mediated Phosphoinositide 3-kinase Signaling

    OpenAIRE

    Zhang, Yi; Zhang, Ying; Miao, JunYing; Hanley, Gregory; Stuart, Charles; Sun, Xiuli; Chen, Tingting; Yin, Deling

    2008-01-01

    Stress, either psychological or physical, can have a dramatic impact on the immune system. Toll-like receptors (TLRs) play a pivotal role in the induction of innate and adaptive immune response. We have reported that stress modulates the immune response in a TLR4-dependent manner. However, the mechanisms underlying TLR4-mediated signaling in stress modulation of immune system have not been identified. Here, we demonstrate an essential role for the TLR4-mediated phosphoinositide 3-kinase (PI3K...

  16. Crystallization and preliminary X-ray crystallographic analysis of a 40 kDa N-terminal fragment of the yeast prion-remodeling factor Hsp104

    International Nuclear Information System (INIS)

    An N-terminal fragment of S. cerevisiae Hsp104 has been crystallized. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones. A 40 kDa N-terminal fragment of Saccharomyces cerevisiae Hsp104 was crystallized in two different crystal forms. Native 1 diffracted to 2.6 Å resolution and belonged to space group P212121, with unit-cell parameters a = 66.6, b = 75.8, c = 235.7 Å. Native 2 diffracted to 2.9 Å resolution and belonged to space group P6122 or P6522, with unit-cell parameters a = 179.1, b = 179.1, c = 69.7 Å. This is the first report of the crystallization of a eukaryotic member of the Hsp100 family of molecular chaperones

  17. The N-terminal domain of NifA determines the temperature sensitivity of Nif A in Klebsiella pneumoniae and Enterobacter cloacae

    Institute of Scientific and Technical Information of China (English)

    顾剑颖; 俞冠翘; 朱家璧; 沈善炯

    2000-01-01

    The NifA protein is the central regulator of the nitrogen fixation genes. It activates transcription of nif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor. The NifA protein from Klebsiella pneumoniae consists of the N-terminal domain of unknown function, the central catalytic domain with ATPase activity and the C-terminal DNA-binding domain. The Kp NifA protein is sensitive to temperature, while the Enterobacter cloacae NifA protein is less sensitive to temperature than Kp NifA. Our results show that the N-terminal domain of NifA plays the decisive role in the temperature sensitivity of the protein.

  18. A highly conserved N-terminal sequence for teleost vitellogenin with potential value to the biochemistry, molecular biology and pathology of vitellogenesis

    Science.gov (United States)

    Folmar, L.D.; Denslow, N.D.; Wallace, R.A.; LaFleur, G.; Gross, T.S.; Bonomelli, S.; Sullivan, C.V.

    1995-01-01

    N-terminal amino acid sequences for vitellogenin (Vtg) from six species of teleost fish (striped bass, mummichog, pinfish, brown bullhead, medaka, yellow perch and the sturgeon) are compared with published N-terminal Vtg sequences for the lamprey, clawed frog and domestic chicken. Striped bass and mummichog had 100% identical amino acids between positions 7 and 21, while pinfish, brown bullhead, sturgeon, lamprey, Xenopus and chicken had 87%, 93%, 60%, 47%, 47-60%) for four transcripts and had 40% identical, respectively, with striped bass for the same positions. Partial sequences obtained for medaka and yellow perch were 100% identical between positions 5 to 10. The potential utility of this conserved sequence for studies on the biochemistry, molecular biology and pathology of vitellogenesis is discussed.

  19. The N-terminal cellulose-binding domain of EGXA increases thermal stability of xylanase and changes its specific activities on different substrates

    Institute of Scientific and Technical Information of China (English)

    Ming Ding; Yigang Teng; Qiuyu Yin; Jie Zhao; Fukun Zhao

    2008-01-01

    A full-length EGXA enzyme from a mollusk, Ampullaria crossean, was cloned into pFastBac vector and then heterogeneously expressed in insect Tn5 cells. Its natural N-terminal signal peptide worked well in the insect Tn5 cells.The recombinant EGXA was a 63 kDa protein and had active endo-β-1,4-glucanase (EC 3.2.1.4) and endo-β-1,4-xylanase (EC 3.2.1.8). The specific activity of endo-β-1,4-xylanase was higher than in the EGX, which was purified from the stomach tissues of Ampullaria crossen. The N-terminal cellulosebinding domain of EGXA made it bind to cellulose and xylan more efficiently. This cellulose-binding domain also increased the thermal stability of this recombinant enzyme and decreased the recombinant EGXA's specific activities on p-nitrophenyi-β-D-cellobioside and sodium carboxymethyl cellulose.

  20. The N-terminal domain of NifA determines the temperature sensitivity of NifA in Klebsiella pneumoniae and Enterobacter cloacae

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The NifA protein is the central regulator of the nitrogen fixation genes.It activates transcription of nif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor.The NifA protein from Klebsiella pneumoniae consists of the N-terminal domain of unknown function,the central catalytic domain with ATPase activity and the C-terminal DNA-binding domain.The Kp NifA protein is sensitive to temperature,while the Enterobacter cloacae NifA protein is less sensitive to temperature than Kp NifA.Our results show that the N-terminal domain of NifA plays the decisive role in the temperature sensitivity of the protein.

  1. Bromodomain protein 4 mediates the papillomavirus E2 transcriptional activation function.

    Science.gov (United States)

    Schweiger, Michal-Ruth; You, Jianxin; Howley, Peter M

    2006-05-01

    The papillomavirus E2 regulatory protein has essential roles in viral transcription and the initiation of viral DNA replication as well as for viral genome maintenance. Brd4 has recently been identified as a major E2-interacting protein and, in the case of the bovine papillomavirus type 1, serves to tether E2 and the viral genomes to mitotic chromosomes in dividing cells, thus ensuring viral genome maintenance. We have explored the possibility that Brd4 is involved in other E2 functions. By analyzing the binding of Brd4 to a series of alanine-scanning substitution mutants of the human papillomavirus type 16 E2 N-terminal transactivation domain, we found that amino acids required for Brd4 binding were also required for transcriptional activation but not for viral DNA replication. Functional studies of cells expressing either the C-terminal domain of Brd4 that can bind E2 and compete its binding to Brd4 or short interfering RNA to knock down Brd4 protein levels revealed a role for Brd4 in the transcriptional activation function of E2 but not for its viral DNA replication function. Therefore, these studies establish a broader role for Brd4 in the papillomavirus life cycle than as the chromosome tether for E2 during mitosis. PMID:16611886

  2. Role of N-terminal extension of Bacillus stearothermophilus RNase H2 and C-terminal extension of Thermotoga maritima RNase H2.

    Science.gov (United States)

    Permanasari, Etin-Diah; Angkawidjaja, Clement; Koga, Yuichi; Kanaya, Shigenori

    2013-10-01

    Bacillus stearothermophilus RNase H2 (BstRNH2) and Thermotoga maritima RNase H2 (TmaRNH2) have N-terminal and C-terminal extensions, respectively, as compared with Aquifex aeolicus RNase H2 (AaeRNH2). To analyze the role of these extensions, BstRNH2 and TmaRNH2 without these extensions were constructed, and their biochemical properties were compared with those of their intact partners and AaeRNH2. The far-UV CD spectra of all proteins were similar, suggesting that the protein structure is not significantly altered by removal of these extensions. However, both the junction ribonuclease and RNase H activities of BstRNH2 and TmaRNH2, as well as their substrate-binding affinities, were considerably decreased by removal of these extensions. The stability of BstRNH2 and TmaRNH2 was also decreased by removal of these extensions. The activity, substrate binding affinity and stability of TmaRNH2 without the C-terminal 46 residues were partly restored by the attachment of the N-terminal extension of BstRNH2. These results suggest that the N-terminal extension of BstRNH2 functions as a substrate-binding domain and stabilizes the RNase H domain. Because the C-terminal extension of TmaRNH2 assumes a helix hairpin structure and does not make direct contact with the substrate, this extension is probably required to make the conformation of the substrate-binding site functional. AaeRNH2 showed comparable junction ribonuclease activity to those of BstRNH2 and TmaRNH2, and was more stable than these proteins, indicating that bacterial RNases H2 do not always require an N-terminal or C-terminal extension to increase activity, substrate-binding affinity, and/or stability. PMID:23937561

  3. Distribution of distances between the tryptophan and the N-terminal residue of melittin in its complex with calmodulin, troponin C, and phospholipids.

    OpenAIRE

    Lakowicz, J.R.; Gryczynski, I.; Laczko, G; Wiczk, W; Johnson, M.L.

    1994-01-01

    We used frequency-domain measurements of fluorescence resonance energy transfer to measure the distribution of distances between Trp-19 of melittin and a 1-dimethylamino-5-sulfonylnaphthalene (dansyl) residue on the N-terminal-alpha-amino group. Distance distributions were obtained for melittin free in solution and when complexed with calmodulin (CaM), troponin C (TnC), or palmitoyloleoyl-L-alpha-phosphatidylcholine (POPC) vesicles. A wide range of donor (Trp-19)-to-acceptor (dansyl) distance...

  4. Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules

    OpenAIRE

    Bruell, Shoni; Kong, Roy C. K.; Petrie, Emma J.; Hoare, Brad; John D Wade; Scott, Daniel J.; Gooley, Paul R.; Bathgate, Ross A. D.

    2013-01-01

    Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are...

  5. Chimeric RXFP1 and RXFP2 receptors highlight the similar mechanism of activation utilizing their N-terminal low density lipoprotein class A modules

    OpenAIRE

    RossBathgate; EmmaJunePetrie; JohnDWade

    2013-01-01

    Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are...

  6. Evaluation of Serum Levels of N-terminal Pro Brain Natriuretic Peptide and Atrial Natriuretic Peptide in Neonates with Respiratory Distress

    OpenAIRE

    Noor Mohammad Noori; Sima Savadkoohi; Alireza Teimouri; Fatemeh Alizadeh

    2016-01-01

    Background: Acute respiratory distress (ARD) is a critical respiratory failure due to lung injury of neonates leading to the clinical appearance of poor lung compliance. The aimed of the study was to evaluate the diagnostic values in differentiating respiratory from heart diseases with using of N-terminal pro brain natriuretic peptide (NT-pro BNP) and Atrial natriuretic peptide(ANP) in neonates. Material and Methods: Ninety  neonates  randomly collected from those who hospitalized in the neon...

  7. Opposing actions of intact and N-terminal fragments of the human prolactin/growth hormone family members on angiogenesis: An efficient mechanism for the regulation of angiogenesis

    OpenAIRE

    Struman, Ingrid; Bentzien, Frauke; Lee, Hsinyu; Mainfroid, Véronique; D’Angelo, Gisela; Goffin, Vincent; Weiner, Richard I.; Martial, Joseph A.

    1999-01-01

    Angiogenesis, the process of development of a new microvasculature, is regulated by a balance of positive and negative factors. We show both in vivo and in vitro that the members of the human prolactin/growth hormone family, i.e., human prolactin, human growth hormone, human placental lactogen, and human growth hormone variant are angiogenic whereas their respective 16-kDa N-terminal fragments are antiangiogenic. The opposite actions are regulated in part via activ...

  8. Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

    International Nuclear Information System (INIS)

    Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with 15N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein

  9. Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Berbís, M. Álvaro [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain); André, Sabine [Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, 80539 Munich (Germany); Cañada, F. Javier [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain); Pipkorn, Rüdiger [Central Peptide Synthesis Unit, German Cancer Research Center, 69120 Heidelberg (Germany); Ippel, Hans [Department of Biochemistry, CARIM, University of Maastricht, Maastricht (Netherlands); Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Mayo, Kevin H. [Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Kübler, Dieter [Biomolecular Interactions, German Cancer Research Center, 69120 Heidelberg (Germany); Gabius, Hans-Joachim [Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, 80539 Munich (Germany); Jiménez-Barbero, Jesús, E-mail: jjbarbero@cib.csic.es [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain)

    2014-01-03

    Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with {sup 15}N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.

  10. Simple Screening Method for Autoantigen Proteins Using the N-Terminal Biotinylated Protein Library Produced by Wheat Cell-Free Synthesis

    OpenAIRE

    Matsuoka, Kazuhiro; Komori, Hiroaki; Nose, Masato; Endo, Yaeta; Sawasaki, Tatsuya

    2010-01-01

    Autoimmune diseases are a heterogeneous group of diseases characterized by immune reactions against either a major or a limited number of the bodies own autoantigens, causing inflammation and damage to tissues and organs. Thus, identification of autoantigens is an important first step to understanding autoimmune diseases. Here we demonstrate a simple screening method for identification of autoantigens reacting with patient serum antibodies by combination of an N-terminal biotinylated protein ...

  11. New approaches for synthesis and analysis of adducts to N-terminal valine in hemoglobin from isocyanates, aldehydes, methyl vinyl ketone and diepoxybutane

    OpenAIRE

    Davies, Ronnie

    2009-01-01

    Human exposure to harmful compounds in the environment, from intake via food, occupational exposures or other sources, could have health implications. Exposure to reactive compounds/metabolites can be identified and quantified as hemoglobin (Hb) adducts by mass spectrometry. This thesis aimed at improved synthetic pathways for reference standards, and improved analytical methods for adducts to N-terminal valine in Hb from a range of reactive compounds; isocyanates, aldehydes, methyl vinyl ket...

  12. Thrombospondin-1-N-Terminal Domain Induces a Phagocytic State and Thrombospondin-1-C-Terminal Domain Induces a Tolerizing Phenotype in Dendritic Cells

    OpenAIRE

    Tabib, Adi; Krispin, Alon; Trahtemberg, Uriel; Verbovetski, Inna; Lebendiker, Mario; Danieli, Tsafi; Mevorach, Dror

    2009-01-01

    In our previous study, we have found that thrombospondin-1 (TSP-1) is synthesized de novo upon monocyte and neutrophil apoptosis, leading to a phagocytic and tolerizing phenotype of dendritic cells (DC), even prior to DC-apoptotic cell interaction. Interestingly, we were able to show that heparin binding domain (HBD), the N-terminal portion of TSP-1, was cleaved and secreted simultaneously in a caspase- and serine protease- dependent manner. In the current study we were interested to examine ...

  13. Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

    Science.gov (United States)

    Tamura, Tomokazu; Ruggli, Nicolas; Nagashima, Naofumi; Okamatsu, Masatoshi; Igarashi, Manabu; Mine, Junki; Hofmann, Martin A; Liniger, Matthias; Summerfield, Artur; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-09-01

    Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs. PMID:26018962

  14. Structural and functional characterization of the Geobacillus copper nitrite reductase: involvement of the unique N-terminal region in the interprotein electron transfer with its redox partner.

    Science.gov (United States)

    Fukuda, Yohta; Koteishi, Hiroyasu; Yoneda, Ryohei; Tamada, Taro; Takami, Hideto; Inoue, Tsuyoshi; Nojiri, Masaki

    2014-03-01

    The crystal structures of copper-containing nitrite reductase (CuNiR) from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426 and the amino (N)-terminal 68 residue-deleted mutant were determined at resolutions of 1.3Å and 1.8Å, respectively. Both structures show a striking resemblance with the overall structure of the well-known CuNiRs composed of two Greek key β-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one β-strand and one α-helix extended to the northern surface of the type-1 copper site. The superposition of the Geobacillus CuNiR model on the electron-transfer complex structure of CuNiR with the redox partner cytochrome c551 in other denitrifier system led us to infer that this region contributes to the transient binding with the partner protein during the interprotein electron transfer reaction in the Geobacillus system. Furthermore, electron-transfer kinetics experiments using N-terminal residue-deleted mutant and the redox partner, Geobacillus cytochrome c551, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition.

  15. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain

    Science.gov (United States)

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-01-01

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23–230) as detected by [1H, 15N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn2+-binding to the octarepeat motif. PMID:27341298

  16. Structural Basis for Recognition of H3T3ph and Smac/DIABLO N-terminal Peptides by Human Survivin

    Energy Technology Data Exchange (ETDEWEB)

    Du, Jiamu; Kelly, Alexander E.; Funabiki, Hironori; Patel, Dinshaw J. (MSKCC); (Rockefeller)

    2012-03-02

    Survivin is an inhibitor of apoptosis family protein implicated in apoptosis and mitosis. In apoptosis, it has been shown to recognize the Smac/DIABLO protein. It is also a component of the chromosomal passenger complex, a key player during mitosis. Recently, Survivin was identified in vitro and in vivo as the direct binding partner for phosphorylated Thr3 on histone H3 (H3T3ph). We have undertaken structural and binding studies to investigate the molecular basis underlying recognition of H3T3ph and Smac/DIABLO N-terminal peptides by Survivin. Our crystallographic studies establish recognition of N-terminal Ala in both complexes and identify intermolecular hydrogen-bonding interactions in the Survivin phosphate-binding pocket that contribute to H3T3ph mark recognition. In addition, our calorimetric data establish that Survivin binds tighter to the H3T3ph-containing peptide relative to the N-terminal Smac/DIABLO peptide, and this preference can be reversed through structure-guided mutations that increase the hydrophobicity of the phosphate-binding pocket.

  17. Modeling of the N-terminal Section and the Lumenal Loop of Trimeric Light Harvesting Complex II (LHCII) by Using EPR.

    Science.gov (United States)

    Fehr, Niklas; Dietz, Carsten; Polyhach, Yevhen; von Hagens, Tona; Jeschke, Gunnar; Paulsen, Harald

    2015-10-23

    The major light harvesting complex II (LHCII) of green plants plays a key role in the absorption of sunlight, the regulation of photosynthesis, and in preventing photodamage by excess light. The latter two functions are thought to involve the lumenal loop and the N-terminal domain. Their structure and mobility in an aqueous environment are only partially known. Electron paramagnetic resonance (EPR) has been used to measure the structure of these hydrophilic protein domains in detergent-solubilized LHCII. A new technique is introduced to prepare LHCII trimers in which only one monomer is spin-labeled. These heterogeneous trimers allow to measure intra-molecular distances within one LHCII monomer in the context of a trimer by using double electron-electron resonance (DEER). These data together with data from electron spin echo envelope modulation (ESEEM) allowed to model the N-terminal protein section, which has not been resolved in current crystal structures, and the lumenal loop domain. The N-terminal domain covers only a restricted area above the superhelix in LHCII, which is consistent with the "Velcro" hypothesis to explain thylakoid grana stacking (Standfuss, J., van Terwisscha Scheltinga, A. C., Lamborghini, M., and Kühlbrandt, W. (2005) EMBO J. 24, 919-928). The conformation of the lumenal loop domain is surprisingly different between LHCII monomers and trimers but not between complexes with and without neoxanthin bound.

  18. Roles of N-terminal fatty acid acylations in membrane compartment partitioning: Arabidopsis h-type thioredoxins as a case study.

    Science.gov (United States)

    Traverso, José A; Micalella, Chiara; Martinez, Aude; Brown, Spencer C; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

    2013-03-01

    N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX-green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane.

  19. Roles of N-Terminal Fatty Acid Acylations in Membrane Compartment Partitioning: Arabidopsis h-Type Thioredoxins as a Case Study[C][W

    Science.gov (United States)

    Traverso, José A.; Micalella, Chiara; Martinez, Aude; Brown, Spencer C.; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

    2013-01-01

    N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX–green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane. PMID:23543785

  20. The N-terminal domains of Vps3 and Vps8 are critical for localization and function of the CORVET tethering complex on endosomes.

    Directory of Open Access Journals (Sweden)

    Nadine Epp

    Full Text Available Endosomal biogenesis depends on multiple fusion and fission events. For fusion, the heterohexameric CORVET complex as an effector of the endosomal Rab5/Vps21 GTPase has a central function in the initial tethering event. Here, we show that the CORVET-specific Vps3 and Vps8 subunits, which interact with Rab5/Vps21, require their N-terminal domains for localization and function. Surprisingly, CORVET may lack either one of the two N-terminal domains, but not both, to promote protein sorting via the endosome. The dually truncated complex mislocalizes to the cytosol and is impaired in endocytic protein sorting, but not in assembly. Furthermore, the endosomal localization can be rescued by overexpression of Vps21 or one of the truncated CORVET subunits, even though CORVET assembly is not impaired by loss of the N-terminal domains or in strains lacking all endosomal Rab5s and Ypt7. We thus conclude that CORVET requires only its C-terminal domains for assembly and has beyond its putative β-propeller domains additional binding sites for endosomes, which could be important to bind Vps21 and other endosome-specific factors for efficient endosome tethering.

  1. Functional analysis of the extended N-terminal region in PLC-δ1 (MlPLC-δ1) from the mud loach, Misgurnus mizolepis.

    Science.gov (United States)

    Kim, Na Young; Ahn, Sang Jung; Kim, Moo-Sang; Seo, Jung Soo; Jung, Se Hwan; Park, Sung Hwan; Lee, Hyung Ho; Chung, Joon Ki

    2014-01-01

    Mud loach phospholipase C-δ1 (MlPLC-δ1) contains all the characteristic domains found in mammalian PLC-δ isozymes (pleckstrin homology domain, EF-hands, X–Y catalytic region, and C2 domain) as well as an extended 26-amino acid (aa)-long N-terminal region that is an alternative splice form of PLC-δ1 and is novel to vertebrate PLC-δ. In the present structure-function analysis, deletion of the extended N-terminal region caused complete loss of phosphatidylinositol (PI)- and phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity in MlPLC-δ1. Additionally, recombinant full-length MlPLC-δ1 PLC activity was reduced in a dose-dependent manner by coincubation with the 26-aa protein fragment. Using a protein-lipid overlay assay, both full-length MlPLC-δ1 and the 26-aa protein fragment had substantial affinity for PIP2, whereas deletion of the 26-aa region from MlPLC-δ1 (MlPLC-δ1-deletion) resulted in lower affinity for PIP2. These results suggest that the novel N-terminal exon of MlPLC-δ1 could play an important role in the regulation of PLC-δ1.

  2. The N-terminal repeat and the ligand binding domain A of SdrI protein is involved in hydrophobicity of S. saprophyticus.

    Science.gov (United States)

    Kleine, Britta; Ali, Liaqat; Wobser, Dominique; Sakιnç, Türkân

    2015-03-01

    Staphylococcus saprophyticus is an important cause of urinary tract infection, and its cell surface hydrophobicity may contribute to virulence by facilitating adherence of the organism to uroepithelia. S. saprophyticus expresses the surface protein SdrI, a member of the serine-aspartate repeat (SD) protein family, which has multifunctional properties. The SdrI knock out mutant has a reduced hydrophobicity index (HPI) of 25%, and expressed in the non-hydrophobic Staphylococcus carnosus strain TM300 causes hydrophobicity. Using hydrophobic interaction chromatography (HIC), we confined the hydrophobic site of SdrI to the N-terminal repeat region. S. saprophyticus strains carrying different plasmid constructs lacking either the N-terminal repeats, both B or SD-repeats were less hydrophobic than wild type and fully complemented SdrI mutant (HPI: 51%). The surface hydrophobicity and HPI of both wild type and the complemented strain were also influenced by calcium (Ca(2+)) and were reduced from 81.3% and 82.4% to 10.9% and 12.3%, respectively. This study confirms that the SdrI protein of S. saprophyticus is a crucial factor for surface hydrophobicity and also gives a first significant functional description of the N-terminal repeats, which in conjunction with the B-repeats form an optimal hydrophobic conformation.

  3. The N-terminal repeat and the ligand binding domain A of SdrI protein is involved in hydrophobicity of S. saprophyticus.

    Science.gov (United States)

    Kleine, Britta; Ali, Liaqat; Wobser, Dominique; Sakιnç, Türkân

    2015-03-01

    Staphylococcus saprophyticus is an important cause of urinary tract infection, and its cell surface hydrophobicity may contribute to virulence by facilitating adherence of the organism to uroepithelia. S. saprophyticus expresses the surface protein SdrI, a member of the serine-aspartate repeat (SD) protein family, which has multifunctional properties. The SdrI knock out mutant has a reduced hydrophobicity index (HPI) of 25%, and expressed in the non-hydrophobic Staphylococcus carnosus strain TM300 causes hydrophobicity. Using hydrophobic interaction chromatography (HIC), we confined the hydrophobic site of SdrI to the N-terminal repeat region. S. saprophyticus strains carrying different plasmid constructs lacking either the N-terminal repeats, both B or SD-repeats were less hydrophobic than wild type and fully complemented SdrI mutant (HPI: 51%). The surface hydrophobicity and HPI of both wild type and the complemented strain were also influenced by calcium (Ca(2+)) and were reduced from 81.3% and 82.4% to 10.9% and 12.3%, respectively. This study confirms that the SdrI protein of S. saprophyticus is a crucial factor for surface hydrophobicity and also gives a first significant functional description of the N-terminal repeats, which in conjunction with the B-repeats form an optimal hydrophobic conformation. PMID:25497915

  4. Dissecting functions of the N-terminal domain and GAS-site recognition in STAT3 nuclear trafficking.

    Science.gov (United States)

    Martincuks, Antons; Fahrenkamp, Dirk; Haan, Serge; Herrmann, Andreas; Küster, Andrea; Müller-Newen, Gerhard

    2016-08-01

    Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous transcription factor involved in many biological processes, including hematopoiesis, inflammation and cancer progression. Cytokine-induced gene transcription greatly depends on tyrosine phosphorylation of STAT3 on a single tyrosine residue with subsequent nuclear accumulation and specific DNA sequence (GAS) recognition. In this study, we analyzed the roles of the conserved STAT3 N-terminal domain (NTD) and GAS-element binding ability of STAT3 in nucleocytoplasmic trafficking. Our results demonstrate the nonessential role of GAS-element recognition for both cytokine-induced and basal nuclear import of STAT3. Substitution of five key amino acids within the DNA-binding domain rendered STAT3 unable to bind to GAS-elements while still maintaining the ability for nuclear localization. In turn, deletion of the NTD markedly decreased nuclear accumulation upon IL-6 treatment resulting in a prolonged accumulation of phosphorylated dimers in the cytoplasm, at the same time preserving specific DNA recognition ability of the truncation mutant. Observed defect in nuclear localization could not be explained by flawed importin-α binding, since both wild-type and NTD deletion mutant of STAT3 could precipitate both full-length and autoinhibitory domain (∆IBB) deletion mutants of importin-α5, as well as ∆IBB-α3 and ∆IBB-α7 isoforms independently of IL-6 stimulation. Despite its inability to translocate to the nucleus upon IL-6 stimulation, the NTD lacking mutant still showed nuclear accumulation in resting cells similar to wild-type upon inhibition of nuclear export by leptomycin B. At the same time, blocking the nuclear export pathway could not rescue cytoplasmic trapping of phosphorylated STAT3 molecules without NTD. Moreover, STAT3 mutant with dysfunctional SH2 domain (R609Q) also localized in the nucleus of unstimulated cells after nuclear export blocking, while upon cytokine treatment the

  5. N-Terminal Prosomatostatin as a Risk Marker for Cardiovascular Disease and Diabetes in a General Population

    Science.gov (United States)

    Almgren, Peter; Nilsson, Peter M.; Melander, Olle

    2016-01-01

    Context: Somatostatin inhibits a range of hormones, including GH, insulin, and glucagon, but little is known about its role in the development of cardiometabolic disease. Objective: The objective of the study was to investigate whether fasting plasma concentration of N-terminal prosomatostatin (NT-proSST) is associated with the development of diabetes, coronary artery disease (CAD), and mortality. Design, Setting, and Participants: NT-proSST was measured in plasma from 5389 fasting participants of the population-based study Malmö Preventive Project, with a mean baseline age of 69.4 ± 6.2 years. Cox proportional hazards models adjusted for traditional cardiovascular risk factors were used to investigate the relationships between baseline NT-proSST and end points, with a mean follow-up of 5.6 ± 1.4 years. Main Outcome Measures: CAD, diabetes, and mortality were measured. Results: Overall, NT-proSST (hazard ratio [HR] per SD increment of log transformed NT-proSST) was unrelated to the risk of incident diabetes (220 events; HR 1.05; 95% confidence interval [CI] 0.91–1.20; P = .531) but was related to the risk of incident CAD (370 events; HR 1.17; 95% CI 1.06–1.30; P = .003), all-cause mortality (756 events; HR 1.24; 95% CI 1.15–1.33; P < .001), and cardiovascular mortality (283 events; HR 1.33; 95% CI 1.19–1.43; P < .001). The relationships were not linear, with most of the excess risk observed in subjects with high values of NT-proSST. Subjects in the top vs bottom decile had a severely increased risk of incident CAD (HR 2.41; 95% CI 1.45–4.01; P < .001), all-cause mortality (HR 1.84; 95% CI 1.33–2.53; P < .001), and cardiovascular mortality (HR 2.44; 95% CI 1.39–4.27; P < .001). Conclusion: NT-proSST was significantly and independently associated with the development of CAD, all-cause mortality, and cardiovascular mortality. PMID:27399347

  6. Correlation of N-terminal pro-B-type natriuretic peptide with clinical parameters in patients with hypertension

    Directory of Open Access Journals (Sweden)

    Pejović Janko

    2013-01-01

    Full Text Available Background/Aim. Identification of patients with arterial hypertension and a possible onset of heart failure by determining the concentration of N-terminal pro-B-type natriuretic peptide (NT-proBNP enables timely intensification of treatment and allows clinicians to prescribe and implement optimal and appropriate care. The aim of this study was to evaluate NT-proBNP in patients with longstanding hypertension and in patients with signs of hypertensive cardiomyopathy. Methods. The study involved 3 groups, with 50 subjects each: “healthy” persons (control group, patients with hypertension and normal left ventricular systolic function (group 1 and patients with longstanding hypertension and signs of hypertensive cardiomyopathy with impaired left ventricular systolic function (group 2. We measured levels of NT-proBNP, Creactive protein and creatinine according to the manufacturer’s instructions. All the patients were clinically examined including physical examination of the heart with blood pressure, pulse rate, electrocardiogram (ECG and echocardiogram. Results. Our results showed that the determined parameters generally differed significantly (Student’s t-test among the groups. The mean (± SD values of NT-proBNP in the control group, group 1 and group 2 were: 2.794 (± 1.515 pmol/L, 9.575 (± 5.449 pmol/L and 204.60 (84,93 pmol/L, respectively. NTproBNP correlated significantly with the determined parameters both in the group 1 and the group 2. In the group 1, the highest correlation was obtained with Creactive protein (r = 0.8424. In the group 2, the highest correlation was obtained with ejection fraction (r = - 0.9111. NT-proBNP showed progressive increase in proportion to the New York Heart Association (NYHA classification. The patients in thegroup 2 who belonged to the II and III NYHA class had significantly higher levels of NTproBNP than those in the NYHA class I (ANOVA test, p = 0.001. Conclusion. The obtained results suggest that

  7. Annexin A1 N-terminal derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions.

    Directory of Open Access Journals (Sweden)

    Valentina Bizzarro

    Full Text Available Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we have evaluated whether Annexin A1 derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions. Using normal human skin fibroblasts WS1 in low glucose (LG or high glucose (HG we observed the enrichment of Annexin A1 protein at cell movement structures like lamellipodial extrusions and interestingly, a significant decrease in levels of the protein in HG conditions. The analysis of the translocation of Annexin A1 to cell membrane showed lower levels of Annexin A1 in both membrane pool and supernatants of WS1 cells treated with HG. Wound-healing assays using cell line transfected with Annexin A1 siRNAs indicated a slowing down in migration speed of cells suggesting that Annexin A1 has a role in the migration of WS1 cells. In order to analyze the role of extracellular Annexin A1 in cell migration, we have performed wound-healing assays using Ac2-26 showing that peptide was able to increase fibroblast cell migration in HG conditions. Experiments on the mobilization of intracellular calcium and analysis of p-ERK expression confirmed the activity of the FPR1 following stimulation with the peptide Ac2-26. A wound-healing assay on WS1 cells in the presence of the FPR agonist fMLP, of the FPR antagonist CsH and in the presence of Ac2-26 indicated that Annexin A1 influences fibroblast cell migration under HG conditions acting through FPR receptors whose expression was slightly increased in HG. In conclusion, these data demonstrate that (i Annexin A1 is involved in migration of WS1 cells, through interaction with FPRs; (ii N- terminal peptide of Annexin A1 Ac2-26 is able to stimulate direct migration of WS1 cells in high glucose treatment possibly due to the increased receptor expression observed in hyperglycemia conditions.

  8. Structure of the recombinant N-terminal lobe of human lactoferrin at 2.0 A resolution.

    Science.gov (United States)

    Day, C L; Anderson, B F; Tweedie, J W; Baker, E N

    1993-08-20

    The three-dimensional structure of the N-terminal half-molecule of human lactoferrin, LfN, prepared by recombinant DNA methods, has been determined by X-ray crystallography at 2.0 A resolution. The protein is in its iron-bound form and is deglycosylated. X-ray diffraction data were obtained by diffractometry to 3.2 A resolution and synchrotron data collection, using Weissenberg photography with imaging plates, to 1.8 A resolution. The structure was solved by molecular replacement, using the N-lobe of native diferric human lactoferrin (Lf) as search model. Restrained least squares refinement (program TNT) has resulted in a model structure with an R-factor of 0.184 for all data 34,180 (reflections) in the resolution range 8.0 to 2.0 A. The model comprises 2490 protein atoms (residues 4 to 327), 1 Fe3+, 1 CO3(2-) and 180 solvent molecules, all regarded as water. The structure of LfN is essentially the same as that of the N-lobe of intact Lf, being folded into two similar alpha/beta domains, with the Fe3+ and CO3(2-) bound in a specific site in the interdomain cleft. These details are not affected by either deglycosylation or expression in a non-native system. At the C terminus, however, the conformation of residues 321 to 333 is changed. Whereas in Lf residues 321 to 332 form a helix crossing between the domains at the back of the iron site, in LfN residues 321 to 326 have an extended conformation, forming a third interdomain beta-strand, and residues 328 to 333 appear disordered. The conformational change is attributed to the loss of stabilizing interactions from the C-lobe and is mediated by two Gly residues, at positions 321 and 323. It is further proposed that the conformational change is responsible for the more facile iron release properties of LfN, by its effect on the hinge mechanism and increased solvent exposure of residues near the back of the iron site. Other details of the polypeptide chain conformation and the binding site have also been analysed. Two

  9. The role of phosphorylation of c-Jun NH2-terminal kinase in airway remodeling of asthmatic rats and the effect of glucocorticoids%c-Jun氨基末端激酶磷酸化在支气管哮喘大鼠气道重塑中的作用及糖皮质激素的影响

    Institute of Scientific and Technical Information of China (English)

    林立; 管小俊; 李昌崇; 苏苗赏; 张维溪; 叶乐平; 王强; 陈小芳

    2010-01-01

    目的 研究c-Jun氨基末端激酶(JNK)磷酸化在支气管哮喘(简称哮喘)气道重塑中的作用,探讨糖皮质激素对白细胞介素(IL)-1β、JNK及哮喘气道重塑的影响.方法 将48只SD大鼠按随机数字表法分为对照组、哮喘组、布地奈德组和地塞米松组,每组12只,实验组以卵清白蛋白致敏和激发复制哮喘气道重塑模型,干预组分别于每次雾化激发前以布地奈德雾化或地塞米松腹腔注射干预,对照组以生理盐水代替卵清白蛋白致敏和激发.采用图像分析技术测定支气管壁厚度(Wat)和平滑肌厚度(Wam),ELISA法测定血清、BALF中IL-1β浓度,免疫组织化学检测肺内磷酸化JNK(P-JNK)及其下游物磷酸化c-Jun蛋白表达,Western blot检测肺匀浆P-JNK表达,直线相关分析Wat、Warn与P-JNK蛋白的平均吸光度(mA)的相关性以及P-JNK蛋白的mA与血清、BALF IL-1β浓度的相关性.结果 哮喘组气道壁厚度较对照组明显增加,其血清和BALF中IL-1β浓度[分别为(81±4)ng/L、(331±15)ns/L]高于对照组[(53±6)ng/L、(130±9)ns/L](t值分别为-8.62、-24.10,均P<0.01);免疫组织化学显示哮喘组P-JNK和P-c-Jun蛋白表达增高;Western blot检测哮喘组P-JNK蛋白的mA为1.66±0.16高于对照组的1.00±0.00(t=-8.35,P<0.01);布地奈德、地塞米松均可抑制JNK的磷酸化;各组Wat、Waln与P-JNK mA均呈高度正相关(r值分别为0.700、0.769,均P<0.01,rg=48),P-JNK的mA与血清、BALF IL-1β浓度均呈显著正相关(r值分别为0.689、0.805,均P<0.01).结论 JNK磷酸化与哮喘气道重塑密切相关;糖皮质激素能抑制JNK磷酸化,其机制之一可能是下调IL-1β表达.%Objective To study the role of phosphorylation of c-Jun NH2-terminal kinase(JNK)in asthmatic airway remodeling and to explore the effect of glucocorticoids on IL-1βJNK and airway remodeling. Methods Forty-eight 4-6 week old male SD rats were randomly divided into 4 groups with 12 rats in each group: the control

  10. Clicinal significance of expression of c-JUN, E-selectin, VEGF-D protein in colorectal carcinoma%结直肠腺癌组织中c-JUN氨基末端激酶、E-选择素、血管内皮生长因子-D蛋白表达的临床意义

    Institute of Scientific and Technical Information of China (English)

    庆琳琳; 胡继春

    2015-01-01

    目的 探讨c-JUN氨基末端激酶(c-JUN)、E-选择素(E-selectin)、血管内皮生长因子-D蛋白(VEGF-D)蛋白在结直肠腺癌发展和转移中的作用.方法 收集2012年8月~2014年9月于北京市海淀医院手术切除的正常大肠黏膜组织标本(20例)和结直肠腺癌标本(50例),应用免疫组化法检测标本中c-JUN、E-selectin、VEGF-D蛋白的表达,观察c-JUN、E-selectin、VEGF-D与临床病理的联系及c-JUN与E-selectin、VEGF-D的相关性.结果 ①结直肠癌组织中,c-JUN、E-selectin、VEGF-D蛋白表达的阳性率(84%、78%、92%)显著高于正常大肠黏膜组织(5%、10%、10%),差异有高度统计学意义(P< 0.01);②c-JUN、E-selectin、VEGF-D蛋白的表达与Dukes分期及有无淋巴结转移有关(P< 0.05);③c-JUN与E-selectin呈正相关(r=0.6394,P<0.05),c-JUN与VEGF-D呈正相关(r=0.6592,P< 0.05).结论 E-selectin、VEGF-D蛋白与结直肠腺癌发展和转移有关,c-JUN可能调控E-selectin、VEGF-D的表达.

  11. Superficial disposition of the N-terminal region of the surfactant protein SP-C and the absence of specific SP-B-SP-C interactions in phospholipid bilayers

    DEFF Research Database (Denmark)

    Plasencia, I; Cruz, A; Casals, C;

    2001-01-01

    A dansylated form of porcine surfactant-associated protein C (Dns-SP-C), bearing a single dansyl group at its N-terminal end, has been used to characterize the lipid-protein and protein-protein interactions of SP-C reconstituted in phospholipid bilayers, using fluorescence spectroscopy...... of the N-terminal segment of the protein into less polar environments that originate during protein lateral segregation. This suggests that conformation and interactions of the N-terminal segment of SP-C could be important in regulating the lateral distribution of the protein in surfactant bilayers...... phosphatidylglycerol (PG), the fluorescence of Dns-SP-C shows a 3-fold increase with respect to the fluorescence of phosphatidylcholine (PC), suggesting that electrostatic lipid-protein interactions induce important effects on the structure and disposition of the N-terminal segment of the protein in these membranes...

  12. The 5-amino acid N-terminal extension of non-sulfated drosulfakinin II is a unique target to generate novel agonists.

    Science.gov (United States)

    Leander, M; Heimonen, J; Brocke, T; Rasmussen, M; Bass, C; Palmer, G; Egle, J; Mispelon, M; Berry, K; Nichols, R

    2016-09-01

    The ability to design agonists that target peptide signaling is a strategy to delineate underlying mechanisms and influence biology. A sequence that uniquely characterizes a peptide provides a distinct site to generate novel agonists. Drosophila melanogaster sulfakinin encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH2) and nsDSK II (GGDDQFDDYGHMRF-NH2). Drosulfakinin is typical of sulfakinin precursors, which are conserved throughout invertebrates. Non-sulfated DSK II is structurally related to DSK I, however, it contains a unique 5-residue N-terminal extension; drosulfakinins signal through G-protein coupled receptors, DSK-R1 and DSK-R2. Drosulfakinin II distinctly influences adult and larval gut motility and larval locomotion; yet, its structure-activity relationship was unreported. We hypothesized substitution of an N-terminal extension residue may alter nsDSK II activity. By targeting the extension we identified, not unexpectedly, analogs mimicking nsDSK II, yet, surprisingly, we also discovered novel agonists with increased (super) and opposite (protean) effects. We determined [A3] nsDSK II increased larval gut contractility rather than, like nsDSK II, decrease it. [N4] nsDSK II impacted larval locomotion, although nsDSK II was inactive. In adult gut, [A1] nsDSK II, [A2] nsDSKII, and [A3] nsDSK II mimicked nsDSK II, and [A4] nsDSK II and [A5] nsDSK II were more potent; [N3] nsDSK II and [N4] nsDSK II mimicked nsDSK II. This study reports nsDSK II signals through DSK-R2 to influence gut motility and locomotion, identifying a novel role for the N-terminal extension in sulfakinin biology and receptor activation; it also led to the discovery of nsDSK II structural analogs that act as super and protean agonists. PMID:27397853

  13. Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating.

    Directory of Open Access Journals (Sweden)

    Jorge Fernández-Trillo

    Full Text Available A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4-S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4-S5 linker.

  14. The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets.

    Science.gov (United States)

    Guzmán Prieto, Ana M; Urbanus, Rolf T; Zhang, Xinglin; Bierschenk, Damien; Koekman, C Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P; Pape, Marieke; Paganelli, Fernanda L; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P A; Bonten, Marc J M; Willems, Rob J L; van Schaik, Willem

    2015-12-17

    Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

  15. Immunization with the DNA-encoding N-terminal domain of proteophosphoglycan of Leishmania donovani generates Th1-type immunoprotective response against experimental visceral leishmaniasis.

    Science.gov (United States)

    Samant, Mukesh; Gupta, Reema; Kumari, Shraddha; Misra, Pragya; Khare, Prashant; Kushawaha, Pramod Kumar; Sahasrabuddhe, Amogh Anant; Dube, Anuradha

    2009-07-01

    Leishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) which exist as secretory as well as surface-bound forms in both promastigotes and amastigotes. The structure and function of PPGs have been reported to be species and stage specific as in the case of Leishmania major and Leishmania mexicana; there has been no such information available for Leishmania donovani. We have recently demonstrated that PPG is differentially expressed in sodium stibogluconate-sensitive and -resistant clinical isolates of L. donovani. To further elucidate the structure and function of the ppg gene of L. donovani, a partial sequence of its N-terminal domain of 1.6 kb containing the majority of antigenic determinants, was successfully cloned and expressed in prokaryotic as well as mammalian cells. We further evaluated the DNA-encoding N-terminal domain of the ppg gene as a vaccine in golden hamsters (Mesocricetus auratus) against the L. donovani challenge. The prophylactic efficacy to the tune of approximately 80% was observed in vaccinated hamsters and all of them could survive beyond 6 mo after challenge. The efficacy was supported by a surge in inducible NO synthase, IFN-gamma, TNF-alpha, and IL-12 mRNA levels along with extreme down-regulation of TGF-beta, IL-4, and IL-10. A rise in the level of Leishmania-specific IgG2 was also observed which was indicative of enhanced cellular immune response. The results suggest the N-terminal domain of L. donovani ppg as a potential DNA vaccine against visceral leishmaniasis.

  16. The N-terminal cleavage of chondromodulin-I in growth-plate cartilage at the hypertrophic and calcified zones during bone development.

    Directory of Open Access Journals (Sweden)

    Shigenori Miura

    Full Text Available Chondromodulin-I (ChM-I is a 20-25 kDa anti-angiogenic glycoprotein in cartilage matrix. In the present study, we identified a novel 14-kDa species of ChM-I by immunoblotting, and purified it by immunoprecipitation with a newly raised monoclonal antibody against ChM-I. The N-terminal amino acid sequencing indicated that it was an N-terminal truncated form of ChM-I generated by the proteolytic cleavage at Asp37-Asp38. This 14-kDa ChM-I was shown by the modified Boyden chamber assay to have very little inhibitory activity on the VEGF-A-induced migration of vascular endothelial cells in contrast to the intact 20-25 kDa form of ChM-I (ID50 = 8 nM. Immunohistochemistry suggested that 20-25 kDa ChM-I was exclusively localized in the avascular zones, i.e. the resting, proliferating, and prehypertrophic zones, of the cartilaginous molds of developing long bone, whereas the 14-kDa form of ChM-I was found in hypertrophic and calcified zones. Immunoblotting demonstrated that mature growth-plate chondrocytes isolated from rat costal cartilage actively secrete ChM-I almost exclusively as the intact 20-25 kDa form into the medium in primary culture. Taken together, our results suggest that intact 20-25 kDa ChM-I is stored as a component of extracellular matrix in the avascular cartilage zones, but it is inactivated by a single N-terminal proteolytic cleavage in the hypertrophic zone of growth-plate cartilage.

  17. Phosphorylation and the N-terminal extension of the regulatory light chain help orient and align the myosin heads in Drosophila flight muscle

    Energy Technology Data Exchange (ETDEWEB)

    Farman, Gerrie P.; Miller, Mark S.; Reedy, Mary C.; Soto-Adames, Felipe N.; Vigoreaux, Jim O.; Maughan, David W.; Irving, Thomas C.; (IIT); (Vermont); (Duke)

    2010-02-02

    X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2{sup {Delta}2-46}) or disruption of the phosphorylation sites by substituting alanines (Dmlc2{sup S66A, S67A}) decreased the equatorial intensity ratio (I{sub 20}/I{sub 10}), indicating decreased myosin mass associated with the thin filaments. Phosphorylation site disruption (Dmlc2{sup S66A, S67A}), but not N-terminal extension truncation (Dmlc2{sup {Delta}2-46}), decreased the 14.5 nm reflection intensity, indicating a spread of the axial distribution of the myosin heads. The arrangement of thick filaments and myosin heads in electron micrographs of the phosphorylation mutant (Dmlc2{sup S66A, S67A}) appeared normal in the relaxed and rigor states, but when calcium activated, fewer myosin heads formed cross-bridges. In transgenic flies with both alterations to the RLC (Dmlc2{sup {Delta}2-46; S66A, S67A}), the effects of the dual mutation were additive. The results suggest that the RLC N-terminal extension serves as a 'tether' to help pre-position the myosin heads for attachment to actin, while phosphorylation of the RLC promotes head orientations that allow optimal interactions with the thin filament.

  18. Schisandra polysaccharide evokes immunomodulatory activity through TLR 4-mediated activation of macrophages.

    Science.gov (United States)

    Zhao, Ting; Feng, Yun; Li, Jing; Mao, Riwen; Zou, Ye; Feng, Weiwei; Zheng, Daheng; Wang, Wei; Chen, Yao; Yang, Liuqing; Wu, Xiangyang

    2014-04-01

    Schisandra chinensis (Turcz.) Baill has been used in traditional Chinese medicine for centuries. Previous studies have shown that Schisandra polysaccharide (SCPP11) has robust antitumor activity in vivo. In this study, the immunomodulatory activity and mechanisms of action of SCPP11 were investigated further to reveal its mechanism of action against tumors. Results showed that SCPP11 increased the thymus and spleen indices, pinocytic activity of peritoneal macrophages, and hemolysin formation in CTX-induced immunosuppressed mice. Moreover, SCPP11 significantly increased immunoglobulin levels, cytokines levels in vivo and induced RAW264.7 cells to secrete cytokines in vitro. RAW264.7 cells pretreated with SCPP11 significantly inhibited the proliferation of HepG-2 cells. In addition, SCPP11 promoted both the expression of iNOS protein and of iNOS and TNF-α mRNA. TLR-4 is a possible receptor for SCPP11-mediated macrophage activation. Therefore, the data suggest that SCPP11 exerted its antitumor activity by improving immune system functions through TLR-4-mediated up-regulation of NO and TNF-α.

  19. Complement regulates TLR4-mediated inflammatory responses during intestinal ischemia reperfusion.

    Science.gov (United States)

    Pope, Michael R; Hoffman, Sara M; Tomlinson, Stephen; Fleming, Sherry D

    2010-01-01

    Innate immune responses including TLR4 and complement activation are required for mesenteric ischemia/reperfusion (IR)-induced tissue damage. We examined the regulation of TLR4 and complement activation in a mouse model of intestinal IR. Intestinal IR-induced C3 deposition in a TLR4 dependent manner. In addition, in wild-type but not TLR4 deficient mice, IR significantly increased C3 and Factor B (FB) mRNA expression within the intestine. To further examine the role of TLR4 and complement, we administered the complement inhibitor, CR2-Crry, to target local complement activation in wild-type C57Bl/10, and TLR4 deficient B10/ScN mice. TLR4 deficient mice sustained less damage and inflammation after IR than wild-type mice, but administration of CR2-Crry did not further reduce tissue damage. In contrast, CR2-Crry treatment of wild-type mice was accompanied by a reduction in complement activation and in C3 and FB transcription in response to IR. CR2-Crry also significantly decreased intestinal IL-6 and IL-12p40 production in both the wild-type and TLR4 deficient mice. These data indicate that TLR4 regulates extrahepatic complement production while complement regulates TLR4-mediated cytokine production during intestinal IR. PMID:20800895

  20. Phosphorylation and the N-terminal Extension of the Regulatory Light Chain Help Orient and Align the Myosin Heads in Drosophila Flight Muscle

    OpenAIRE

    Farman, Gerrie P.; Miller, Mark S.; Reedy, Mary C.; Soto-Adames, Felipe N.; Vigoreaux, Jim O.; Maughan, David W.; Irving, Thomas C.

    2009-01-01

    X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2Δ2-46) or disruption of the phosphorylation sites by substituting alanines (Dmlc2S66A, S67A) decreased the equatorial intensity ratio (I20/I10), indicating decreased myosin mass associated with the thin f...

  1. Evidence for an Interaction between the SH3 Domain and the N-terminal Extension of the Essential Light Chain in Class II Myosins

    OpenAIRE

    Lowey, Susan; Saraswat, Lakshmi D.; Liu, HongJun; Volkmann, Niels; Hanein, Dorit

    2007-01-01

    The function of the src-homology 3 (SH3) domain in class II myosins, a distinct β-barrel structure, remains unknown. Here we provide evidence, using electron cryomicroscopy, in conjunction with light scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41-residue extension contains four conserved lysines followed by a repeating sequence of seven Pro/Ala residues. It is ...

  2. Sepp1UF forms are N-terminal selenoprotein P truncations that have peroxidase activity when coupled with thioredoxin reductase-1

    OpenAIRE

    Kurokawa, Suguru; Eriksson, Sofi; Rose, Kristie L.; Wu, Sen; Motley, Amy K.; Hill, Salisha; Winfrey, Virginia P.; McDonald, W. Hayes; Capecchi, Mario R.; Atkins, John F; Arnér, Elias S. J.; Hill, Kristina E.; Raymond F Burk

    2014-01-01

    Mouse selenoprotein P (Sepp1) consists of an N-terminal domain (residues 1-239) that contains 1 selenocysteine (U) as residue 40 in a proposed redox-active motif (-UYLC-) and a C-terminal domain (residues 240-361) that contains 9 selenocysteines. Sepp1 transports selenium from the liver to other tissues by receptor-mediated endocytosis. It also reduces oxidative stress in vivo by an unknown mechanism. A previously uncharacterized plasma form of Sepp1 is filtered in the glomerulus and taken up...

  3. Enhancing the Secretion Efficiency and Thermostability of a Bacillus deramificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

    OpenAIRE

    Duan, Xuguo; WU, Jing

    2015-01-01

    Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of α-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072–4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated...

  4. The crystal structure of Z-Aib-Gly-Aib-Leu-Aib-OtBu, the synthetic, protected N-terminal pentapeptide of trichotoxin.

    Science.gov (United States)

    Gessmann, R; Brueckner, H; Kokkinidis, M

    1991-01-01

    Z-Aib-Gly-Aib-Leu-Aib-OtBu, the alpha-aminoisobutyric acid (Aib)-containing N-terminal pentapeptide of the antibiotic trichotoxin, has been studied by x-ray crystallography. The molecule forms a right-handed helix with a reversal of the sense of the helix at the C-terminus. Torsion angles and hydrogen bonding pattern are consistent with a mixed 3(10)-/alpha-helical conformation. In the crystal, continuous columns are formed by head-to-tail arrangement of hydrogen-bonded molecules along the helix axis. The helical columns associate via hydrogen bonds forming closely packed parallel pairs.

  5. Structure of the starch-debranching enzyme barley limit dextrinase reveals homology of the N-terminal domain to CBM21

    DEFF Research Database (Denmark)

    Møller, Marie Sofie; Abou Hachem, Maher; Svensson, Birte;

    2012-01-01

    molecule in the active site and is virtually identical to the structures of HvLD in complex with the competitive inhibitors α-cyclodextrin and β-cyclodextrin solved to 2.5 and 2.1 Å resolution, respectively. However, three loops in the N-terminal domain that are shown here to resemble carbohydrate......-binding module family 21 were traceable and were included in the present HvLD structure but were too flexible to be traced and included in the structures of the two HvLD-inhibitor complexes....

  6. Cationic liposomes enhance targeted delivery and expression of exogenous DNA mediated by N-terminal modified poly(L-lysine)-antibody conjugate in mouse lung endothelial cells.

    Science.gov (United States)

    Trubetskoy, V S; Torchilin, V P; Kennel, S; Huang, L

    1992-07-15

    A new and improved system for targeted gene delivery and expression is described. Transfection efficiency of N-terminal modified poly(L-lysine) (NPLL) conjugated with anti-thrombomodulin antibody 34A can be improved by adding to the system a lipophilic component, cationic liposomes. DNA, antibody conjugate and cationic liposomes form a ternary electrostatic complex which preserves the ability to bind specifically to the target cells. At the same time the addition of liposomes enhance the specific transfection efficiency of antibody-polylysine/DNA binary complex by 10 to 20-fold in mouse lung endothelial cells in culture.

  7. Use of N-terminal modified poly(L-lysine)-antibody conjugate as a carrier for targeted gene delivery in mouse lung endothelial cells.

    Science.gov (United States)

    Trubetskoy, V S; Torchilin, V P; Kennel, S J; Huang, L

    1992-01-01

    A DNA targeted delivery and expression system has been designed based on an N-terminal modified poly(L-lysine) (NPLL)-antibody conjugate, which readily forms a complex with plasmid DNA. Monoclonal antibodies against the cell-surface thrombomodulin conjugated with NPLL were used for targeted delivery of foreign plasmid DNA to an antigen-expressing mouse lung endothelial cell line in vitro and to mouse lungs in vivo. In both cases significant amounts of DNA can be specifically bound to the target cells or tissues. Specific gene expression was observed in the treated mouse lung endothelial cells.

  8. Left-handed helical preference in an achiral peptide chain is induced by an L-amino acid in an N-terminal type II β-turn.

    Science.gov (United States)

    De Poli, Matteo; De Zotti, Marta; Raftery, James; Aguilar, Juan A; Morris, Gareth A; Clayden, Jonathan

    2013-03-15

    Oligomers of the achiral amino acid Aib adopt helical conformations in which the screw-sense may be controlled by a single N-terminal residue. Using crystallographic and NMR techniques, we show that the left- or right-handed sense of helical induction arises from the nature of the β-turn at the N terminus: the tertiary amino acid L-Val induces a left-handed type II β-turn in both the solid state and in solution, while the corresponding quaternary amino acid L-α-methylvaline induces a right-handed type III β-turn.

  9. Prevention of KLF4-mediated tumor initiation and malignant transformation by UAB30 rexinoid.

    Science.gov (United States)

    Jiang, Wen; Deng, Wentao; Bailey, Sarah K; Nail, Clint D; Frost, Andra R; Brouillette, Wayne J; Muccio, Donald D; Grubbs, Clinton J; Ruppert, J Michael; Lobo-Ruppert, Susan M

    2009-02-01

    The transcription factor KLF4 acts in post-mitotic epithelial cells to promote differentiation and functions in a context-dependent fashion as an oncogene. In the skin KLF4 is co-expressed with the nuclear receptors RARgamma and RXRalpha, and formation of the skin permeability barrier is a shared function of these three proteins. We utilized a KLF4-transgenic mouse model of skin cancer in combination with cultured epithelial cells to examine functional interactions between KLF4 and retinoic acid receptors. In cultured cells, activation of a conditional, KLF4-estrogen receptor fusion protein by 4-hydroxytamoxifen resulted in rapid upregulation of transcripts for nuclear receptors including RARgamma and RXRalpha. We tested retinoids in epithelial cell transformation assays, including an RAR-selective agonist (all-trans RA), an RXR-selective agonist (9-cis UAB30, rexinoid), and a pan agonist (9-cis RA). Unlike for several other genes, transformation by KLF4 was inhibited by each retinoid, implicating distinct nuclear receptor heterodimers as modulators of KLF4 transforming activity. When RXRalpha expression was suppressed by RNAi in cultured cells, transformation was promoted and the inhibitory effect of 9-cis UAB30 was attenuated. Similarly as shown for other mouse models of skin cancer, rexinoid prevented skin tumor initiation resulting from induction of KLF4 in basal keratinocytes. Rexinoid permitted KLF4 expression and KLF4-induced cell cycling, but attenuated the KLF4-induced misexpression of cytokeratin 1 in basal cells. Neoplastic lesions including hyperplasia, dysplasia and squamous cell carcinoma-like lesions were prevented for up to 30 days. Taken together, the results identify retinoid receptors including RXRalpha as ligand-dependent inhibitors of KLF4-mediated transformation or tumorigenesis. PMID:19197145

  10. Glial cell-expressed mechanosensitive channel TRPV4 mediates infrasound-induced neuronal impairment.

    Science.gov (United States)

    Shi, Ming; Du, Fang; Liu, Yang; Li, Li; Cai, Jing; Zhang, Guo-Feng; Xu, Xiao-Fei; Lin, Tian; Cheng, Hao-Ran; Liu, Xue-Dong; Xiong, Li-Ze; Zhao, Gang

    2013-11-01

    Vibroacoustic disease, a progressive and systemic disease, mainly involving the central nervous system, is caused by excessive exposure to low-frequency but high-intensity noise generated by various heavy transportations and machineries. Infrasound is a type of low-frequency noise. Our previous studies demonstrated that infrasound at a certain intensity caused neuronal injury in rats but the underlying mechanism(s) is still largely unknown. Here, we showed that glial cell-expressed TRPV4, a Ca(2+)-permeable mechanosensitive channel, mediated infrasound-induced neuronal injury. Among different frequencies and intensities, infrasound at 16 Hz and 130 dB impaired rat learning and memory abilities most severely after 7-14 days exposure, a time during which a prominent loss of hippocampal CA1 neurons was evident. Infrasound also induced significant astrocytic and microglial activation in hippocampal regions following 1- to 7-day exposure, prior to neuronal apoptosis. Moreover, pharmacological inhibition of glial activation in vivo protected against neuronal apoptosis. In vitro, activated glial cell-released proinflammatory cytokines IL-1β and TNF-α were found to be key factors for this neuronal apoptosis. Importantly, infrasound induced an increase in the expression level of TRPV4 both in vivo and in vitro. Knockdown of TRPV4 expression by siRNA or pharmacological inhibition of TRPV4 in cultured glial cells decreased the levels of IL-1β and TNF-α, attenuated neuronal apoptosis, and reduced TRPV4-mediated Ca(2+) influx and NF-κB nuclear translocation. Finally, using various antagonists we revealed that calmodulin and protein kinase C signaling pathways were involved in TRPV4-triggered NF-κB activation. Thus, our results provide the first evidence that glial cell-expressed TRPV4 is a potential key factor responsible for infrasound-induced neuronal impairment. PMID:24002225

  11. The role of TG2 in regulating S100A4-mediated mammary tumour cell migration.

    Directory of Open Access Journals (Sweden)

    Zhuo Wang

    Full Text Available The importance of S100A4, a Ca(2+-binding protein, in mediating tumour cell migration, both intracellularly and extracellularly, is well documented. Tissue transglutaminase (TG2 a Ca(2+-dependent protein crosslinking enzyme, has also been shown to enhance cell migration. Here by using the well characterised non-metastatic rat mammary R37 cells (transfected with empty vector and highly metastatic KP1 cells (R37 cells transfected with S100A4, we demonstrate that inhibition of TG2 either by TG2 inhibitors or transfection of cells with TG2 shRNA block S100A4-accelerated cell migration in the KP1cells and in R37 cells treated with exogenous S100A4. Cell migration was also blocked by the treatment with the non-cell permeabilizing TG2 inhibitor R294, in the human breast cancer cell line MDA-MB-231 (Clone 16, which has a high level of TG2 expression. Inhibition was paralleled by a decrease in S100A4 polymer formation. In vitro co-immunoprecipitation and Far Western blotting assays and cross-linking assays showed not only the direct interaction between TG2 and S100A4, but also confirmed S100A4 as a substrate for TG2. Using specific functional blocking antibodies, a targeting peptide and a recombinant protein as a competitive treatment, we revealed the involvement of syndecan-4 and α5β1 integrin co-signalling pathways linked by activation of PKCα in this TG2 and S100A4-mediated cell migration. We propose a mechanism for TG2-regulated S100A4-related mediated cell migration, which is dependent on TG2 crosslinking.

  12. BtcA, A class IA type III chaperone, interacts with the BteA N-terminal domain through a globular/non-globular mechanism.

    Directory of Open Access Journals (Sweden)

    Chen Guttman

    Full Text Available Bordetella pertussis, the etiological agent of "whooping cough" disease, utilizes the type III secretion system (T3SS to deliver a 69 kDa cytotoxic effector protein, BteA, directly into the host cells. As with other T3SS effectors, prior to its secretion BteA binds BtcA, a 13.9 kDa protein predicted to act as a T3SS class IA chaperone. While this interaction had been characterized for such effector-chaperone pairs in other pathogens, it has yet to be fully investigated in Bordetella. Here we provide the first biochemical proof that BtcA is indeed a class IA chaperone, responsible for the binding of BteA's N-terminal domain. We bring forth extensive evidence that BtcA binds its substrate effector through a dual-interface binding mechanism comprising of non-globular and bi-globular interactions at a moderate micromolar level binding affinity. We demonstrate that the non-globular interactions involve the first 31 N-terminal residues of BteA287 and their removal leads to destabilization of the effector-chaperone complex and lower binding affinities to BtcA. These findings represent an important first step towards a molecular understanding of BteA secretion and cell entry.

  13. Specific amplification of gene encoding N-terminal region of catalase-peroxidase protein (KatG-N) for diagnosis of disseminated MAC disease in HIV patients.

    Science.gov (United States)

    Latawa, Romica; Singh, Krishna Kumar; Wanchu, Ajay; Sethi, Sunil; Sharma, Kusum; Sharma, Aman; Laal, Suman; Verma, Indu

    2014-10-01

    Disseminated Mycobacterium avium-intracellulare complex (MAC) infection is considered as severe complication of advanced HIV/AIDS disease. Currently available various laboratory investigations have not only limited ability to discriminate between MAC infection and tuberculosis but are also laborious and time consuming. The aim of this study was, therefore, to design a molecular-based strategy for specific detection of MAC and its differentiation from Mycobacterium tuberculosis (M. tb) isolated from the blood specimens of HIV patients. A simple PCR was developed based on the amplification of 120-bp katG-N gene corresponding to the first 40 amino acids of N-terminal catalase-peroxidase (KatG) protein of Mycobacterium avium that shows only ~13% sequence homology by clustal W alignment to N-terminal region of M. tb KatG protein. This assay allowed the accurate and rapid detection of MAC bacteremia, distinguishing it from M. tb in a single PCR reaction without any need for sequencing or hybridization protocol to be performed thereafter. This study produced enough evidence that a significant proportion of Indian HIV patients have disseminated MAC bacteremia, suggesting the utility of M. avium katG-N gene PCR for early detection of MAC disease in HIV patients.

  14. Structural analysis of the starfish SALMFamide neuropeptides S1 and S2: the N-terminal region of S2 facilitates self-association.

    Science.gov (United States)

    Otara, Claire B; Jones, Christopher E; Younan, Nadine D; Viles, John H; Elphick, Maurice R

    2014-02-01

    The neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide), which share sequence similarity, were discovered in the starfish Asterias rubens and are prototypical members of the SALMFamide family of neuropeptides in echinoderms. SALMFamide neuropeptides act as muscle relaxants and both S1 and S2 cause relaxation of cardiac stomach and tube foot preparations in vitro but S2 is an order of magnitude more potent than S1. Here we investigated a structural basis for this difference in potency using spectroscopic techniques. Circular dichroism spectroscopy showed that S1 does not have a defined structure in aqueous solution and this was supported by 2D nuclear magnetic resonance experiments. In contrast, we found that S2 has a well-defined conformation in aqueous solution. However, the conformation of S2 was concentration dependent, with increasing concentration inducing a transition from an unstructured to a structured conformation. Interestingly, this property of S2 was not observed in an N-terminally truncated analogue of S2 (short S2 or SS2; SFNSGLTFamide). Collectively, the data obtained indicate that the N-terminal region of S2 facilitates peptide self-association at high concentrations, which may have relevance to the biosynthesis and/or bioactivity of S2 in vivo.

  15. Recombinant expression of rat glycine N-methyltransferase and evidence for contribution of N-terminal acetylation to co-operative binding of S-adenosylmethionine.

    Science.gov (United States)

    Ogawa, H; Gomi, T; Takata, Y; Date, T; Fujioka, M

    1997-10-15

    An expression vector was constructed that produced rat glycine N-methyltransferase in Escherichia coli. Recombinant glycine N-methyltransferase was purified to homogeneity by DEAE-cellulose and gel-filtration chromatography, with a yield of more than 80 mg of pure enzyme from a 1 litre culture. HPLC of tryptic peptides and analysis of isolated peptides showed that the recombinant enzyme was structurally identical with the liver enzyme except for the absence of N-terminal blocking. The alpha-amino group of rat glycine N-methyltransferase is blocked by acetylation [Ogawa, Konishi, Takata, Nakashima and Fujioka (1987) Eur. J. Biochem. 168, 141-151]. In contrast with the liver enzyme, which shows sigmoidal kinetics toward S-adenosylmethionine at all pH values tested [Ogawa and Fujioka (1982) J. Biol. Chem. 257, 3447-3452], the recombinant enzyme exhibited hyperbolic kinetics at low pH and sigmoidal rate behaviour at high pH. The Hill coefficient increased with increasing pH and a pKa of 8.11 was obtained in this transition. The values of Vmax and Km for glycine were not different between the two enzymes. These results suggest that elimination of the positive charge at the N-terminal end either by acetylation or deprotonation is required for co-operative behaviour. PMID:9359408

  16. A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.

    Science.gov (United States)

    Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H J; Snel, Berend; Perrakis, Anastassis; Kops, Geert J P L

    2013-04-15

    The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

  17. N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile.

    Science.gov (United States)

    Weltrowska, Grazyna; Nguyen, Thi M-D; Chung, Nga N; Wilkes, Brian C; Schiller, Peter W

    2013-09-15

    Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood-brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/μ partial agonist activity. Guanidinylation of the mixed μ agonist/δ antagonists H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) and H-Dmt-TicΨ[CH2NH]Phe-Phe-NH2 (DIPP-NH2[Ψ]) converted them to mixed μ agonist/δ agonists. A docking study revealed distinct positioning of DIPP-NH2 and Guan-DIPP-NH2 in the δ receptor binding site. Lys(3)-analogues of DIPP-NH2 and DIPP-NH2[Ψ] (guanidinylated or non-guanidinylated) turned out to be mixed μ/κ agonists with δ antagonist-, δ partial agonist- or δ full agonist activity. Compounds with some of the observed mixed opioid activity profiles have therapeutic potential as analgesics with reduced side effects o