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Sample records for neurofilament nf subunits

  1. The C-terminal domains of NF-H and NF-M subunits maintain axonal neurofilament content by blocking turnover of the stationary neurofilament network.

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    Mala V Rao

    Full Text Available Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M(tailΔ] mice that the deletion of both of these domains selectively lowers NF levels 3-6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF. Pulse labeling studies carried out over 90 days revealed a significantly faster rate of disappearance of NF from the stationary NF network of optic axons in NF-(H/M(tailΔ mice. Faster NF disappearance was accompanied by elevated levels of NF-L proteolytic fragments in NF-(H/M(tailΔ axons. We conclude that NF-H and NF-M C-terminal domains do not normally regulate NF transport rates as previously proposed, but instead increase the proteolytic resistance of NF, thereby stabilizing the stationary neurofilament cytoskeleton along axons.

  2. A molecular dissection of the carboxyterminal tails of the major neurofilament subunits NF-M and NF-H.

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    Harris, J; Ayyub, C; Shaw, G

    1991-09-01

    We have initiated a multidisciplinary project that aims to dissect and ultimately define the functions of the long and unusual C-terminal "tail" sequences of the two high molecular weight neurofilament subunits, NF-M and NF-H. A series of recombinant fusion proteins containing selected NF-M and NF-H tail sequences were constructed using appropriate cDNAs. These fusion proteins were used to further define the epitopes for a variety of widely used neurofilament antibodies, including NN18 and N52, which are now available commercially from several companies. We also measured the SDS-PAGE mobility of the fusion proteins and found that, like the native neurofilament tails, the fusion proteins ran considerably slower than predicted from their molecular weight. Since all fusion proteins produced so far exhibit this characteristic we conclude that all segments of the NF-M and NF-H tail share this unusual property. Finally we were able to produce novel and potentially useful polyclonal and monoclonal antibodies to selected segments of NF-M and NF-H sequence. These antibody studies showed that the extreme C-termini of NF-M and NF-H are immunologically absolutely distinct from one another and also indicate that the extreme C-terminus of NF-M is immunologically much more conserved than the analogous region of NF-H. These findings are in complete agreement with our conclusions derived from amino acid sequence analysis, and further underline the possible functional importance of the extreme C-terminus of NF-M. We also show that the unusual immunological properties of the bovine NF-M tail we have previously observed do not extend to the extreme C-terminal region, which appears immunologically no different from the analogous region of other NF-M molecules. The peculiarities of bovine NF-M could be explained by the presence of a KSP motif that resembles the NF-H KSP prototype.

  3. The phosphorylated axonal form of the neurofilament subunit NF-H (pNF-H) as a blood biomarker of traumatic brain injury.

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    Anderson, Kevin J; Scheff, Stephen W; Miller, Kelly M; Roberts, Kelly N; Gilmer, Lesley K; Yang, Cui; Shaw, Gerry

    2008-09-01

    The detection of neuron-specific proteins in blood might allow quantification of the degree of neuropathology in experimental and clinical contexts. We have been studying a novel blood biomarker of axonal injury, the heavily phosphorylated axonal form of the high molecular weight neurofilament subunit NF-H (pNF-H). We hypothesized that this protein would be released from damaged and degenerating neurons following experimental traumatic brain injury (TBI) in amounts large enough to allow its detection in blood and that the levels detected would reflect the degree of injury severity. An enzyme-linked immunosorbent assay (ELISA) capture assay capable of detecting nanogram amounts of pNF-H was used to test blood of rats subjected to experimental TBI using a controlled cortical impact (CCI) device. Animals were subjected to a mild (1.0 mm), moderate (1.5 mm), or severe (2.0 mm) cortical contusion, and blood samples were taken at defined times post-injury. The assay detected the presence of pNF-H as early as 6 h post-injury; levels peaked at 24-48 h, and then slowly decreased to baseline over several days post-injury. No signal above baseline was detectable in control animals. Analysis of variance (ANOVA) showed a significant effect of lesion severity, and post hoc analysis revealed that animals given a moderate and severe contusion showed higher levels of blood pNF-H than controls. In addition, the peak levels of pNF-H detected at both 24 and 48 h post-injury correlated with the degree of injury as determined by volumetric analysis of spared cortical tissue. Relative amounts of pNF-H were also determined in different areas of the central nervous system (CNS) and were found to be highest in regions containing large-diameter axons, including spinal cord and brainstem, and lowest in the cerebral cortex and hippocampus. These findings suggest that the measurement of blood levels of pNF-H is a convenient method for assessing neuropathology following TBI.

  4. Phosphorylated neurofilament heavy subunit (pNF-H) in peripheral blood and CSF as a potential prognostic biomarker in amyotrophic lateral sclerosis.

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    Boylan, Kevin B; Glass, Jonathan D; Crook, Julia E; Yang, Cui; Thomas, Colleen S; Desaro, Pamela; Johnston, Amelia; Overstreet, Karen; Kelly, Crystal; Polak, Meraida; Shaw, Gerry

    2013-04-01

    The phosphorylated neurofilament heavy subunit (pNF-H), a major structural component of motor axons, is a promising putative biomarker in amyotrophic lateral sclerosis (ALS) but has been studied mainly in CSF. We examined pNF-H concentrations in plasma, serum and CSF as a potential biomarker for disease progression and survival in ALS. We measured pNF-H concentration by monoclonal sandwich ELISA in plasma (n=43), serum and CSF (n=20) in ALS patients collected at the Mayo Clinic Florida and Emory University. We included plasma from an ALS cohort (n=20) from an earlier pilot study in order to evaluate baseline pNF-H levels in relation to disease progression using the Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R), survival and anatomical region of ALS onset. Higher pNF-H levels in plasma, serum and CSF showed evidence of association with faster decline in ALSFRS-R. There was evidence for a relationship of higher serum and plasma pNF-H levels with shorter survival, although evidence was weaker for CSF. pNF-H concentration in plasma (n=62) may be higher in patients with bulbar onset than in patients with spinal onset. In ALS, increased pNF-H concentration in plasma, serum and CSF appears to be associated with faster disease progression. Factors affecting pNF-H levels or their detection in serum and plasma in relation to disease course may differ from those in CSF. Data raising the possibility that site of ALS onset (bulbar vs spinal) may influence pNF-H levels in peripheral blood seems noteworthy but requires confirmation. These data support further study of pNF-H in CSF, serum and plasma as a potential ALS biomarker.

  5. Phosphorylated neurofilament subunit NF-H becomes elevated in the cerebrospinal fluid of patients with acutely worsening symptoms of compression myelopathy.

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    Takahashi, Hiroshi; Aoki, Yasuchika; Nakajima, Arata; Sonobe, Masato; Terajima, Fumiaki; Saito, Masahiko; Taniguchi, Shinji; Yamada, Manabu; Watanabe, Fusako; Furuya, Takeo; Koda, Masao; Yamazaki, Masashi; Takahashi, Kazuhisa; Nakagawa, Koichi

    2014-12-01

    It is known that the severity of compression myelopathy sometimes worsens rapidly and results in poor functional recovery because of limited axonal regeneration. Levels of phosphorylated neurofilament subunit NF-H (pNF-H), which indicate axonal degeneration, are elevated in other neurological disorders. To our knowledge, there has been no examination of pNF-H levels in compression myelopathy. Therefore, we conducted a pilot cross-sectional study to evaluate pNF-H levels in the cerebrospinal fluid (CSF) of patients with worsening symptoms of cervical compression myelopathy. From January 2011 to March 2013, 51 samples of CSF were collected from patients at the time of myelography before spinal surgery. The indications for surgery were acutely worsening compression myelopathy (AM) in eight, chronic compression myelopathy (CM) in six, and lumbar canal stenosis (LCS) in 37 patients. The pNF-H levels were measured using a standard enzyme-linked immunosorbent assay. The mean ± standard deviation pNF-H value was 2127.1 ± 556.8 pg/ml in AM patients, 175.8 ± 67.38 pg/ml in CM patients and 518.7 ± 665.7 pg/ml in LCS patients. A significant increase in pNF-H levels was detected in the CSF of patients with AM compared with those with either CM or LCS. The clinical outcome of surgical treatment for patients with cervical myelopathy was satisfactory in both AM and CM patients. Despite the limitations of small sample size and lack of healthy CSF control data due to ethical considerations, our results suggest that pNF-H in CSF can act as a biomarker that reflects the severity of AM. Copyright © 2014. Published by Elsevier Ltd.

  6. Immunoreactivity of the phosphorylated axonal neurofilament H subunit (pNF-H) in blood of ALS model rodents and ALS patients: evaluation of blood pNF-H as a potential ALS biomarker.

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    Boylan, Kevin; Yang, Cui; Crook, Julia; Overstreet, Karen; Heckman, Michael; Wang, Yong; Borchelt, David; Shaw, Gerry

    2009-12-01

    Levels of neurofilament subunits, potential biomarkers of motor axon breakdown, are increased in amyotrophic lateral sclerosis (ALS) patient's CSF but data on blood are not available. We measured blood levels of the phosphorylated axonal form of neurofilament H (pNF-H) by ELISA in transgenic rodent models of superoxide dismutase 1 (SOD1) ALS, and in 20 ALS patients and 20 similar aged controls monthly for 4 months. All symptomatic rodent ALS models showed robust levels of blood pNF-H, while control rodents or mice transgenic for unmutated SOD1 showed no detectable blood pNF-H. Average pNF-H levels in the G93A SOD1 mouse progressively increased from day 74 through death (day approximately 130). Median blood pNF-H level in ALS patients was 2.8-fold higher than controls (p pNF-H level appeared to be associated with faster ALSFRS-R decline over 4 months (p = 0.087). The median rate of decline in ALSFRS-R was 1.9 pt/month in patients with baseline pNF-H levels above the median pNF-H value of 0.53 ng/mL; ALSFRS-R declined at a median of 0.6 pt/month in patients below this level. The pNF-H levels were relatively stable month to month in individual patients, raising questions regarding the molecular pathogenesis of ALS. Baseline control human pNF-H levels were higher in men than women and increased minimally over time. These data suggest that blood pNF-H can be used to monitor axonal degeneration in ALS model rodents and support further study of this protein as a potential biomarker of disease prognosis in ALS patients.

  7. Oxaliplatin-induced loss of phosphorylated heavy neurofilament subunit neuronal immunoreactivity in rat DRG tissue

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    Connor Bronwen

    2009-11-01

    Full Text Available Abstract Background Oxaliplatin and related chemotherapeutic drugs cause painful chronic peripheral neuropathies in cancer patients. We investigated changes in neuronal size profiles and neurofilament immunoreactivity in L5 dorsal root ganglion (DRG tissue of adult female Wistar rats after multiple-dose treatment with oxaliplatin, cisplatin, carboplatin or paclitaxel. Results After treatment with oxaliplatin, phosphorylated neurofilament heavy subunit (pNF-H immunoreactivity was reduced in neuronal cell bodies, but unchanged in nerve fibres, of the L5 DRG. Morphometric analysis confirmed significant changes in the number (-75%; P P P = 0.82, NF-M (-1%, P = 0.96 or NF-H (0%; P = 0.93 after oxaliplatin treatment, although the sizes of parvalbumin (-29%, P = 0.047, NF-M (-11%, P = 0.038 and NF-H (-28%; P = 0.0033 immunoreactive neurons were reduced. In an independent comparison of different chemotherapeutic agents, the number of pNF-H-immunoreactive neurons was significantly altered by oxaliplatin (-77.2%; P P = 0.03 but not by carboplatin or paclitaxel, and their mean cell body area was significantly changed by oxaliplatin (-31.1%; P = 0.008 but not by cisplatin, carboplatin or paclitaxel. Conclusion This study has demonstrated a specific pattern of loss of pNF-H immunoreactivity in rat DRG tissue that corresponds with the relative neurotoxicity of oxaliplatin, cisplatin and carboplatin. Loss of pNF-H may be mechanistically linked to oxaliplatin-induced neuronal atrophy, and serves as a readily measureable endpoint of its neurotoxicity in the rat model.

  8. Phosphorylated neurofilament subunit levels in the serum of cervical compressive myelopathy patients.

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    Kato, So; Chikuda, Hirotaka; Ohya, Junichi; Hayakawa, Kentaro; Takeshita, Katsushi; Tanaka, Sakae; Ogata, Toru

    2015-10-01

    We investigated the serum levels of the phosphorylated form of the high molecular weight neurofilament subunit (pNF-H) in patients with cervical compressive myelopathy. pNF-H is becoming increasingly recognized as a biomarker for axonal injury, however, it remains unclear whether serum pNF-H is elevated in chronic spinal cord compression. We examined 26 patients who underwent surgery for cervical compressive myelopathy. Peripheral blood samples were obtained both preoperatively and 1 week after surgery to evaluate the serum pNF-H levels using an enzyme-linked immunosorbent assay. A history of recent aggravation of myelopathy was also investigated. Of the 26 myelopathy patients, the preoperative serum pNF-H level was negative in 20 patients and moderately elevated in six. Patients who were positive for pNF-H were more likely to have had a recent aggravation of myelopathy compared with the pNF-H negative patients (83 versus 25%; p=0.02). All patients who were positive for pNF-H before surgery remained positive after surgery. Two patients who became positive after surgery demonstrated a neurologic deterioration associated with the surgery. In conclusion, the serum pNF-H level was negative in the majority of patients with cervical compressive myelopathy. Our results suggest that an elevated serum level of pNF-H is associated with an acute worsening of myelopathy and that a positive conversion of pNF-H after surgery is a marker of perioperative neural damage. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Plasma neurofilament pNF-H concentration is not increased in acute equine grass sickness.

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    Stratford, C H; Pemberton, A; Cameron, L; McGorum, B C

    2013-03-01

    Although a presumptive diagnosis of acute grass sickness (AGS) can be made on the basis of clinical signs, a definitive ante mortem diagnosis currently requires histological examination of enteric ganglia. Development of an accurate noninvasive ante mortem diagnostic test is therefore warranted. The objective of this study was to determine whether quantification of the plasma concentrations of the heavily phosphorylated form of major neurofilament subunit NF-H (pNF-H), which mirror the degree of axonal degeneration in some human and animal neurodegenerative disorders, could distinguish AGS-affected and control horses. The pNF-H was quantified in plasma from 20 AGS cases and 20 control horses using a commercial enzyme-linked immunosorbent assay kit. Five AGS and 4 control samples had detectable pNF-H concentrations (>0.0759 ng/ml). There was no significant intergroup difference in pNF-H concentrations. It was concluded that plasma pNF-H is not a useful biomarker for the diagnosis of AGS. © 2012 EVJ Ltd.

  10. Neurofilament subunit (NFL) head domain phosphorylation regulates axonal transport of neurofilaments.

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    Yates, Darran M

    2009-04-01

    Neurofilaments are the intermediate filaments of neurons and are synthesised in neuronal cell bodies and then transported through axons. Neurofilament light chain (NFL) is a principal component of neurofilaments, and phosphorylation of NFL head domain is believed to regulate the assembly of neurofilaments. However, the role that NFL phosphorylation has on transport of neurofilaments is poorly understood. To address this issue, we monitored axonal transport of phosphorylation mutants of NFL. We mutated four known phosphorylation sites in NFL head domain to either preclude phosphorylation, or mimic permanent phosphorylation. Mutation to preclude phosphorylation had no effect on transport but mutation of three sites to mimic permanent phosphorylation inhibited transport. Mutation of all four sites together to mimic permanent phosphorylation proved especially potent at inhibiting transport and also disrupted neurofilament assembly. Our results suggest that NFL head domain phosphorylation is a regulator of neurofilament axonal transport.

  11. Predictive value of phosphorylated axonal neurofilament subunit H for clinical outcome in patients with acute intracerebral hemorrhage.

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    Cai, Jian-Yong; Lu, Chuan; Chen, Mao-Hua; Ba, Hua-Jun; Chen, Xian-Dong; Lin, Jian-Hu; Sun, Jun

    2013-09-23

    Phosphorylated axonal neurofilament subunit H (pNF-H) is a biomarker of axonal injury. We investigated whether plasma pNF-H concentrations were associated with 6-month clinical outcomes and early neurological deterioration (END) of patients with acute intracerebral hemorrhage. Plasma pNF-H concentrations of 112 patients and 112 healthy individuals were quantified by ELISA. Unfavorable outcome was defined as modified Rankin Scale score >2. Associations of plasma pNF-H concentrations with END, 6-month mortality and unfavorable outcome were evaluated. Plasma pNF-H concentrations were increased in patients than in healthy individuals [700.2 (430.8) pg/ml vs. 25.5 (32.4) pg/ml, PpNF-H concentration as an independent predictor for 6-month mortality [OR: 1.287, 95% CI: 1.140-1.524, PpNF-H concentration predicted 6-month clinical outcomes and END with high area under curves (all PpNF-H was similar to that of the National Institutes of Health Stroke Scale score (all P>0.05). In a combined logistic-regression model, pNF-H did not improve the predictive value of National Institutes of Health Stroke Scale score (all P>0.05). Increased plasma pNF-H concentration was highly associated with 6-month clinical outcomes and END of patients with intracerebral hemorrhage. © 2013.

  12. Determination of the phosphorylated neurofilament subunit NF-H (pNF-H in cerebro-spinal fluid as biomarker in acute traumatic spinal cord injuries / Dozarea neurofilamentelor fosforilate (subunitatea pNF-H ȋn LCR ca biomarker ȋn traumatismul vertebro-medular acut

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    Ungureanu Didona

    2014-09-01

    Full Text Available Obiectivul studiului. Obiectivul acestui studiu a fost dozarea neurofilamentelor fosforilate (subunitatea pNF-H în lichidul cefalorahidian al pacienţilor cu leziuni traumatice ale măduvei spinării şi stabilirea unei corelaţii intre valoarea pNF-H şi gravitatea leziunilor medulare. Material şi metode. Studiul a inclus 15 pacienţi cu leziuni traumatice acute medulare: opt pacienţi cu leziuni medulare complete şi şapte pacienţi cu leziuni medulare incomplete. Gravitatea leziunilor medulare a fost apreciată folosind scala ASIA (American Spinal Injury Association scale şi la toti pacientii s-a aplicat tratamentul chirurgical în primele 24 de ore (decompresiune medulară şi stabilizare vertebrală. S-a facut determinarea zilnică a pNF-H din LCR folosind testul ELISA specific şi am corelat aceste valori cu evoluţia clinică. Rezultate. Subunitatea pNF-H a fost evidenţiată în LCR la toţi pacienţii cu traumatisme acute vertebro-medulare şi valorile au fost diferite în cazurile leziunilor medulare complete faţa de leziunile incomplete. Nivelul pNF-H din LCR a fost de zece până la o sută de ori mai mare în leziunile medulare complete fata de cazurile cu leziuni incomplete, unde nivelul acestui biomarker a fost aproape normal. Pacienţii cu o evoluţie neurologică favorabilă după tratament au avut un model specific al valorilor zilnice de pNF-H: o creştere bruscă până la o valoare maximă apoi o scădere progresivă până la normal. Valorile maxime au fost diferite în fiecare caz, de 10 ori până la 170 de ori mai mari faţă de normal. Concluzii. Forma fosforilată a subunităţii neurofilamentelor cu greutate moleculară mare pNF-H din lichidul cefalorahidian poate fi un biomarker specific pentru leziunile acute traumatice ale măduvei spinării corelat cu severitatea leziunilor medulare. pNF-H pare a fi un biomarker predictiv deoarece modelul evolutiv al valorilor sale arată reducerea sau blocarea leziunilor

  13. Combination of serum phosphorylated neurofilament heavy subunit and hyperintensity of intramedullary T2W on magnetic resonance imaging provides better prognostic value of canine thoracolumbar intervertebral disc herniation.

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    Mashita, Tadahisa; Kamishina, Hiroaki; Nakamoto, Yuya; Akagi, Yosuke; Nakanishi, Ataru; Harasaki, Yusuke; Ozawa, Tsuyoshi; Uemura, Takashi; Kobatake, Yui; Shimamura, Shunsuke; Kitamura, Naoki; Maeda, Sadatoshi; Uzuka, Yuji; Shaw, Gerry; Yasuda, Jun

    2015-04-01

    The aim of this study was to evaluate the prognostic value of concurrent measurement of serum phosphorylated neurofilament heavy subunit (pNF-H) concentration and intramedullary T2W hyperintensity in paraplegic to paraplegic dogs. Our hypothesis was that concurrent measurement of these would provide a more accurate prediction of functional outcome in dogs with thoracolumbar intervertebral disc herniation (IVDH). A prospective case-control clinical study was designed using 94 dogs with acute onset of thoracolumbar IVDH. The association of serum pNF-H concentration, T2W hyperintensity on sagittal MRI (T2H/L2), deep pain perception and surgical outcome were evaluated with logistic regression analysis after three months for all 94 surgically treated dogs. Sensitivity to predict non-ambulatory outcome was compared among pNF-H and T2H/L2 and combination of both. Logistic regression analysis indicated that serum pNF-H concentration and T2H/L2 were significantly correlated with surgical outcome (PpNF-H concentration, 1.9 for T2H/L2 and 2.3 for deep pain sensation. The sensitivity and specificity to predict non-ambulatory outcome for using serum parameter pNF-H>2.6 ng/ml, using T2H/L2 value of>0.84 and using both serum pNF-H and T2H/L2, were 95% and 75.7%, 65% and 86.5%, and 90.0% and 97.5%, respectively. Therefore, combined measurements of serum pNF-H and T2H/L2 might be useful for predicting long-term outcome in dogs with thoracolumbar IVDH.

  14. Gel-expanded to gel-condensed transition in neurofilament networks revealed by direct force measurements

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    Beck, Roy; Deek, Joanna; Jones, Jayna B.; Safinya, Cyrus R.

    2010-01-01

    Neurofilaments (NF)-the principal cytoskeletal constituent of myelinated axons in vertebrates-consist of three molecular-weight subunit proteins NF-L (low), NF-M (medium) and NF-H (high), assembled to form mature filaments with protruding unstructured C-terminus side arms. Liquid-crystal gel networks of side-arm-mediated neurofilament assemblies have a key role in the mechanical stability of neuronal processes. Disruptions of the neurofilament network, owing to neurofilament over-accumulation or incorrect side-arm interactions, are a hallmark of motor-neuron diseases including amyotrophic lateral sclerosis. Using synchrotron X-ray scattering, we report on a direct measurement of forces in reconstituted neurofilament gels under osmotic pressure (P). With increasing pressure near physiological salt and average phosphorylation conditions, NF-LMH, comprising the three subunits near in vivo composition, or NF-LH gels, undergo for P>Pc~10kPa, an abrupt non-reversible gel-expanded to gel-condensed transition. The transition indicates side-arm-mediated attractions between neurofilaments consistent with an electrostatic model of interpenetrating chains. In contrast, NF-LM gels remain in a collapsed state for PPc. These findings, which delineate the distinct roles of NF-M and NF-H in regulating neurofilament interactions, shed light on possible mechanisms for disruptions of optimal mechanical network properties.

  15. Phosphorylated neurofilament H (pNF-H) as a potential diagnostic marker for neurological disorders in horses.

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    Intan-Shameha, A R; Divers, Thomas J; Morrow, Jennifer K; Graves, Amy; Olsen, Emil; Johnson, Amy L; Mohammed, Hussni O

    2017-10-01

    The current study aimed at the investigating the potential use of phosphorylated neurofilament H (pNF-H) as a diagnostic biomarker for neurologic disorders in the horse. Paired serum and cerebrospinal fluid (CSF) samples (n=88) and serum only (n=30) were obtained from horses diagnosed with neurologic disorders and clinically healthy horses as control. The neurologic horses consisted of equine protozoal myeloencephalitis (EPM) (38 cases) and cervical vertebral malformation (CVM) (23 cases). Levels of pNF-H were determined using an ELISA. The correlation between CSF and serum concentrations of pNF-H was evaluated using Spearman's Rank test and the significance of the difference among the groups was assessed using a nonparametric test. Horses had higher pNF-H levels in the CSF than serum. Horses afflicted with EPM had significantly higher serum pNF-H levels in comparison to controls or CVM cases. The correlation between CSF and serum pNF-H levels was poor in both the whole study population and among subgroups of horses included in the study. There was significant association between the likelihood of EPM and the concentrations of pNF-H in either the serum or CSF. These data suggest that pNF-H could be detected in serum and CSF samples from neurologic and control horses. This study demonstrated that pNF-H levels in serum and CSF have the potential to provide objective information to help in the early diagnosis of horses afflicted with neurologic disorders. Published by Elsevier Ltd.

  16. Analysis of the neurofilament heavy subunit (NFH) gene in familial amyotrophic lateral sclerosis

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    Rooke, K.; Rouleau, G.A. [McGill Univ., Montreal (Canada); Figlewicz, D.A. [Univ. of Rochester Medical Center, NY (United States)

    1994-09-01

    Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset, degenerative disorder of the motor neurons in the cortex, brainstem and spinal cord. Approximately 10% of ALS cases are familial (FALS) and are inherited as an age-dependent autosomal dominant trait. Mutations in the Cu/Zn superoxide dismutase (SOD-1) gene on chromosome 21 have been found in a subset of cases. However, for the remaining FALS cases, the etiology is unknown. The abnormal accumulation of neurofilaments in the cell body and proximal axon of motor neurons is a characteristic pathological finding in ALS. Furthermore, aberrant neuronal swellings that closely resemble those found in ALS have been reported in transgenic mice overexpressing NFH. The C-terminal region of NFH contains a unique functional domain with multiple repeats of the amino acids (Lys-Ser-Pro) (KSP) and forms the side-arms which appear, at the level of electron microscopy, to cross-link neurofilaments. Recently, deletions in the DSP repeat domain have been identified in five ALS patients diagnosed as sporadic cases of the disease. Based on these findings, we propose to analyze all 4 exons of the NFH gene for variation in FALS. DNA from 110 FALS cases has been amplified by the polymerase chain reaction (PCR) and analyzed by single strand conformation polymorphism (SSCP) analysis. Exon 2, exon 3 and the KSP repeat domain (part of exon 4) appear normal in all our FALS individuals under several different SSCP conditions. The analysis of exon 1 and the remainder of exon 4 has yet to be completed.

  17. Neurofilament Phosphorylation during Development and Disease: Which Came First, the Phosphorylation or the Accumulation?

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    Dale, Jeffrey M; Garcia, Michael L

    2012-01-01

    Posttranslational modification of proteins is a ubiquitous cellular mechanism for regulating protein function. Some of the most heavily modified neuronal proteins are cytoskeletal proteins of long myelinated axons referred to as neurofilaments (NFs). NFs are type IV intermediate filaments (IFs) that can be composed of four subunits, neurofilament heavy (NF-H), neurofilament medium (NF-M), neurofilament light (NF-L), and α-internexin. Within wild type axons, NFs are responsible for mediating radial growth, a process that determines axonal diameter. NFs are phosphorylated on highly conserved lysine-serine-proline (KSP) repeats located along the C-termini of both NF-M and NF-H within myelinated axonal regions. Phosphorylation is thought to regulate aspects of NF transport and function. However, a key pathological hallmark of several neurodegenerative diseases is ectopic accumulation and phosphorylation of NFs. The goal of this review is to provide an overview of the posttranslational modifications that occur in both normal and diseased axons. We review evidence that challenges the role of KSP phosphorylation as essential for radial growth and suggests an alternative role for NF phosphorylation in myelinated axons. Furthermore, we demonstrate that regulation of NF phosphorylation dynamics may be essential to avoiding NF accumulations.

  18. CSF neurofilament proteins in the differential diagnosis of dementia

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    de Jong, D; Jansen, R W M M; Pijnenburg, Y A L; van Geel, W J A; Borm, G F; Kremer, Berry; Verbeek, M.

    BACKGROUND: Neurofilament (NF) proteins are major cytoskeletal constituents of neurons. Increased CSF NF levels may reflect neuronal degeneration. OBJECTIVE: To investigate the diagnostic value of CSF NF analysis to discriminate in relatively young dementia patients between frontotemporal lobe

  19. Structure-function analysis of the glioma targeting NFL-TBS.40-63 peptide corresponding to the tubulin-binding site on the light neurofilament subunit.

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    Raphael Berges

    Full Text Available We previously reported that a 24 amino acid peptide (NFL-TBS.40-63 corresponding to the tubulin-binding site located on the light neurofilament subunit, selectively enters in glioblastoma cells where it disrupts their microtubule network and inhibits their proliferation. Here, we analyzed the structure-function relationships using an alanine-scanning strategy, in order to identify residues essential for these biological activities. We showed that the majority of modified peptides present a decreased or total loss to penetrate in these cells, or to alter microtubules. Correspondingly, circular dichroism measurements showed that this peptide forms either β-sheet or α-helix structures according to the solvent and that alanine substitution modified or destabilized the structure, in relation with changes in the biological activities. Moreover, substitution of serine residues by phosphoserine or aspartic acid concomitantly decreased the cell penetrating activity and the structure stability. These results indicate the importance of structure for the activities, including selectivity to glioblastoma cells of this peptide, and its regulation by phosphorylation.

  20. NG2 cells response to axonal alteration in the spinal cord white matter in mice with genetic disruption of neurofilament light subunit expression

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    Xiao Zhi

    2008-10-01

    Full Text Available Abstract Background Chondroitin sulphate proteoglycan (NG2 expressing cells, morphologically characterized by multi-branched processes and small cell bodies, are the 4th commonest cell population of non-neuronal cell type in the central nervous system (CNS. They can interact with nodes of Ranvier, receive synaptic input, generate action potential and respond to some pathological stimuli, but the function of the cells is still unclear. We assumed the NG2 cells may play an active role in neuropathogenesis and aimed to determine if NG2 cells could sense and response to the alterations in the axonal contents caused by disruption of neurofilament light subunit (NFL expression. Results In the early neuropathological development stage, our study showed that the diameter of axons of upper motor neurons of NFL-/- mice decreased significantly while the thickness of their myelin sheath increased remarkably. Although there was an obvious morphological distortion in axons with occasionally partial demyelination, no obvious changes in expression of myelin proteins was detected. Parallel to these changes in the axons and their myelination, the processes of NG2 cells were disconnected from the nodes of Ranvier and extended further, suggesting that these cells in the spinal cord white matter could sense the alteration in axonal contents caused by disruption of NFL expression before astrocytic and microglial activation. Conclusion The structural configuration determined by the NFL gene may be important for maintenance of normal morphology of myelinated axons. The NG2 cells might serve as an early sensor for the delivery of information from impaired neurons to the local environment.

  1. Serial cerebrospinal fluid neurofilament heavy chain levels in severe Guillain-Barre syndrome

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    Dujmovic, I.; Lunn, M.P.; Reilly, M.M.; Petzold, A.

    2013-01-01

    Introduction: Proximal axonotmesis results in the release of neurofilament (Nf) proteins into the cerebrospinal fluid (CSF) in patients with Guillain-Barré syndrome (GBS). High CSF levels of the phosphorylated form of Nf-heavy chain (NfH

  2. NF-κB p50 subunit knockout impairs late LTP and alters long term memory in the mouse hippocampus

    Directory of Open Access Journals (Sweden)

    Oikawa Kensuke

    2012-07-01

    Full Text Available Abstract Background Nuclear factor kappa B (NF-κB is a transcription factor typically expressed with two specific subunits (p50, p65. Investigators have reported that NF-κB is activated during the induction of in vitro long term potentiation (LTP, a paradigm of synaptic plasticity and correlate of memory, suggesting that NF-κB may be necessary for some aspects of memory encoding. Furthermore, NF-κB has been implicated as a potential requirement in behavioral tests of memory. Unfortunately, very little work has been done to explore the effects of deleting specific NF-κB subunits on memory. Studies have shown that NF-κB p50 subunit deletion (p50−/− leads to memory deficits, however some recent studies suggest the contrary where p50−/− mice show enhanced memory in the Morris water maze (MWM. To more critically explore the role of the NF-κB p50 subunit in synaptic plasticity and memory, we assessed long term spatial memory in vivo using the MWM, and synaptic plasticity in vitro utilizing high frequency stimuli capable of eliciting LTP in slices from the hippocampus of NF-κB p50−/− versus their controls (p50+/+. Results We found that the lack of the NF-κB p50 subunit led to significant decreases in late LTP and in selective but significant alterations in MWM tests (i.e., some improvements during acquisition, but deficits during retention. Conclusions These results support the hypothesis that the NF-κ p50 subunit is required in long term spatial memory in the hippocampus.

  3. RelA NF-κB subunit activation as a therapeutic target in diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Zhang, Mingzhi; Xu-Monette, Zijun Y; Li, Ling

    2016-01-01

    It has been well established that nuclear factor kappa-B (NF-κB) activation is important for tumor cell growth and survival. RelA/p65 and p50 are the most common NF-kB subunits and involved in the classical NF-kB pathway. However, the prognostic and biological significance of RelA/p65 is equivoca...

  4. Diagnostic and prognostic significance of neurofilament light chain NF-L, but not progranulin and S100B, in the course of amyotrophic lateral sclerosis: Data from the German MND-net.

    Science.gov (United States)

    Steinacker, Petra; Huss, André; Mayer, Benjamin; Grehl, Torsten; Grosskreutz, Julian; Borck, Guntram; Kuhle, Jens; Lulé, Dorothée; Meyer, Thomas; Oeckl, Patrick; Petri, Susanne; Weishaupt, Jochen; Ludolph, Albert C; Otto, Markus

    2017-02-01

    There is a need for diagnostic, prognostic, and monitoring blood biomarkers for ALS. We aimed to analyse and compare proposed candidate markers for disease progression in the course of ALS. Blood samples were taken from 125 ALS patients, including nine patients with C9orf72 or SOD1 mutation, at regular intervals of six months. ALS patients were characterized by the ALS functional rating scale (ALSFRS-R) and the Edinburgh Cognitive and Behavioural ALS Screen (ECAS). We quantified neurofilament light chain (NF-L), S100B, and progranulin (PGRN) and analysed it in relation to disease progression. Results showed that, at baseline, serum concentrations of NF-L but not PGRN or S100B discriminated significantly between ALS and controls. Within 24 months follow-up the marker concentrations remained stable. Baseline serum NF-L levels correlated with survival time, which was confirmed in subgroups with fast, intermediate, and slow disease progression and there was a weak association with disease duration. For S100B and PGRN we found an association with ALSFRS-R score changes and a trend for decreased levels in the fast progressor subgroup. In conclusion, serum NF-L in any ALS disease stage is a promising marker to support diagnosis and predict outcome, while serum PGRN and S100B are only of minor prognostic value.

  5. Increased neurofilament light chain blood levels in neurodegenerative neurological diseases

    NARCIS (Netherlands)

    Gaiottino, J.; Norgren, N.; Dobson, R.; Topping, J.; Nissim, A.; Malaspina, A.; Bestwick, J.P.; Monsch, A.U.; Regeniter, A.; Lindberg, R.L.; Kappos, L.; Leppert, D.; Petzold, A.; Giovannoni, G.; Kuhle, J.

    2013-01-01

    Objective:Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf) are cytoskeletal proteins of neurons and their release into cerebrospinal fluid has shown encouraging results as a biomarker for neurodegeneration. This study aimed to validate the

  6. Targeting Activation of Specific NF-κB Subunits Prevents Stress-Dependent Atherothrombotic Gene Expression

    Science.gov (United States)

    Djuric, Zdenka; Kashif, Muhammed; Fleming, Thomas; Muhammad, Sajjad; Piel, David; von Bauer, Rüdiger; Bea, Florian; Herzig, Stephan; Zeier, Martin; Pizzi, Marina; Isermann, Berend; Hecker, Markus; Schwaninger, Markus; Bierhaus, Angelika; Nawroth, Peter P

    2012-01-01

    Psychosocial stress has been shown to be a contributing factor in the development of atherosclerosis. Although the underlying mechanisms have not been elucidated entirely, it has been shown previously that the transcription factor nuclear factor-κB (NF-κB) is an important component of stress-activated signaling pathway. In this study, we aimed to decipher the mechanisms of stress-induced NF-κB-mediated gene expression, using an in vitro and in vivo model of psychosocial stress. Induction of stress led to NF-κB-dependent expression of proinflammatory (tissue factor, intracellular adhesive molecule 1 [ICAM-1]) and protective genes (manganese superoxide dismutase [MnSOD]) via p50, p65 or cRel. Selective inhibition of the different subunits and the respective kinases showed that inhibition of cRel leads to the reduction of atherosclerotic lesions in apolipoprotein−/− (ApoE−/−) mice via suppression of proinflammatory gene expression. This observation may therefore provide a possible explanation for ineffectiveness of antioxidant therapies and suggests that selective targeting of cRel activation may provide a novel approach for the treatment of stress-related inflammatory vascular disease. PMID:23114885

  7. Piperlongumine selectively suppresses ABC-DLBCL through inhibition of NF-κB p65 subunit nuclear import

    Energy Technology Data Exchange (ETDEWEB)

    Niu, Mingshan [Blood Diseases Institute, Xuzhou Medical College, Xuzhou, Jiangsu (China); Jiangsu Key Laboratory of Bone Marrow Stem Cell, Xuzhou Medical College, Xuzhou, Jiangsu (China); Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu (China); Shen, Yangling; Xu, Xiaoyu [Blood Diseases Institute, Xuzhou Medical College, Xuzhou, Jiangsu (China); Jiangsu Key Laboratory of Bone Marrow Stem Cell, Xuzhou Medical College, Xuzhou, Jiangsu (China); Yao, Yao; Fu, Chunling [Blood Diseases Institute, Xuzhou Medical College, Xuzhou, Jiangsu (China); Jiangsu Key Laboratory of Bone Marrow Stem Cell, Xuzhou Medical College, Xuzhou, Jiangsu (China); Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu (China); Yan, Zhiling [Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu (China); Wu, Qingyun [Blood Diseases Institute, Xuzhou Medical College, Xuzhou, Jiangsu (China); Jiangsu Key Laboratory of Bone Marrow Stem Cell, Xuzhou Medical College, Xuzhou, Jiangsu (China); Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu (China); Cao, Jiang; Sang, Wei [Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu (China); Zeng, Lingyu [Blood Diseases Institute, Xuzhou Medical College, Xuzhou, Jiangsu (China); Jiangsu Key Laboratory of Bone Marrow Stem Cell, Xuzhou Medical College, Xuzhou, Jiangsu (China); Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu (China); Li, Zhenyu [Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu (China); Liu, Xuejiao, E-mail: liuxuejiao0923@126.com [Insititute of Nervous System Diseases, Xuzhou Medical College, Xuzhou, Jiangsu (China); and others

    2015-07-10

    Constitutive NF-κB activation is required for survival of activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL). However, current NF-κB targeting strategies lack cancer cell specificity. Here, we identified a novel inhibitor, piperlongumine, features direct binding to NF-κB p65 subunit and suppression of p65 nuclear import. This was accompanied by NF-κB reporter activity suppression and NF-κB target gene downregulation. Moreover, mutation of Cys{sup 38} to Ser in p65 abolished this effect of piperlongumine on inhibition of p65 nuclear import. Furthermore, we show that piperlongumine selectively inhibited proliferation and induced apoptosis of ABC-DLBCL cells. Most notably, it has been reported that piperlongumine did not affect normal cells even at high doses and was nontoxic to animals. Hence, our current study provides new insight into piperlongumine's mechanism of action and novel approach to ABC-DLBCL target therapy. - Highlights: • Current NF-κB targeting strategies lack cancer cell specificity. • Piperlongumine inhibits NF-κB p65 subunit nuclear import via directly binding to p65. • Piperlongumine selectively inhibits proliferation of ABC-DLBCL cells. • This study provides a novel approach to ABC-DLBCL target therapy.

  8. Age-Dependent Changes in the Immunoreactivity for Neurofilaments in Rabbit Hippocampus

    NARCIS (Netherlands)

    Zee, E.A. van der; Naber, P.A.; Disterhoft, J.F.

    1997-01-01

    The distribution of the three subunits of neurofilaments was examined in the hippocampus of young adult rabbits (three months of age), employing a panel of six monoclonal antibodies. Thereafter, age-dependent and subunit-selective changes in neurofilament immunoreactivity in the ageing rabbit

  9. Cloning and developmental expression of the murine neurofilament gene family.

    NARCIS (Netherlands)

    J-P. Julien (Jean-Pierre); D.N. Meijer (Dies); D. Flavell (David); J. Hurst; F.G. Grosveld (Frank)

    1986-01-01

    textabstractDNA clones encoding the 3 mouse neurofilament (NF) genes have been isolated by cross-hybridization with a previously described NF-L cDNA probe from the rat. Screening of a lambda gt10 cDNA library prepared from mouse brain RNA led to the cloning of an NF-L cDNA of 2.0 kb that spans the

  10. Post-Translational Modifications of RelB NF-κB Subunit and Associated Functions

    Directory of Open Access Journals (Sweden)

    Véronique Baud

    2016-05-01

    Full Text Available The family of NF-κB transcription factors plays a key role in diverse biological processes, such as inflammatory and immune responses, cell survival and tumor development. Beyond the classical NF-κB activation pathway, a second NF-κB pathway has more recently been uncovered, the so-called alternative NF-κB activation pathway. It has been shown that this pathway mainly controls the activity of RelB, a member of the NF-κB family. Post-translational modifications, such as phosphorylation, acetylation, methylation, ubiquitination and SUMOylation, have recently emerged as a strategy for the fine-tuned regulation of NF-κB. Our review discusses recent progress in the understanding of RelB regulation by post-translational modifications and the associated functions in normal and pathological conditions.

  11. Mechanisms and Consequences of Dopamine Depletion-Induced Attenuation of the Spinophilin/Neurofilament Medium Interaction

    Directory of Open Access Journals (Sweden)

    Andrew C. Hiday

    2017-01-01

    Full Text Available Signaling changes that occur in the striatum following the loss of dopamine neurons in the Parkinson disease (PD are poorly understood. While increases in the activity of kinases and decreases in the activity of phosphatases have been observed, the specific consequences of these changes are less well understood. Phosphatases, such as protein phosphatase 1 (PP1, are highly promiscuous and obtain substrate selectivity via targeting proteins. Spinophilin is the major PP1-targeting protein enriched in the postsynaptic density of striatal dendritic spines. Spinophilin association with PP1 is increased concurrent with decreases in PP1 activity in an animal model of PD. Using proteomic-based approaches, we observed dopamine depletion-induced decreases in spinophilin binding to multiple protein classes in the striatum. Specifically, there was a decrease in the association of spinophilin with neurofilament medium (NF-M in dopamine-depleted striatum. Using a heterologous cell line, we determined that spinophilin binding to NF-M required overexpression of the catalytic subunit of protein kinase A and was decreased by cyclin-dependent protein kinase 5. Functionally, we demonstrate that spinophilin can decrease NF-M phosphorylation. Our data determine mechanisms that regulate, and putative consequences of, pathological changes in the association of spinophilin with NF-M that are observed in animal models of PD.

  12. Light induces translocation of NF-κB p65 to the mitochondria and suppresses expression of cytochrome c oxidase subunit III (COX III) in the rat retina

    Energy Technology Data Exchange (ETDEWEB)

    Tomita, Hiroshi, E-mail: htomita@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Soft-Path Engineering Research Center (SPERC), Faculty of Science and Engineering, Iwate University, Morioka 020-8551 (Japan); Clinical Research, Innovation and Education Center, Tohoku University Hospital, 1-1 Seiryo, Aoba, Sendai, Miyagi 980-8574 (Japan); Tabata, Kitako, E-mail: ktabata@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Takahashi, Maki, E-mail: mqdelta@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Nishiyama, Fumiaki, E-mail: t2114018@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Sugano, Eriko, E-mail: sseriko@iwate-u.ac.jp [Laboratory of Visual Neuroscience, Graduate Course in Biological Sciences, Iwate University Division of Science and Engineering, 4-3-5 Ueda, Morioka, Iwate 020-8551 (Japan); Soft-Path Engineering Research Center (SPERC), Faculty of Science and Engineering, Iwate University, Morioka 020-8551 (Japan)

    2016-05-13

    The transcription factor nuclear factor kappaB (NF-κB) plays various roles in cell survival, apoptosis, and inflammation. In the rat retina, NF-κB activity increases after exposure to damaging light, resulting in degeneration of photoreceptors. Here, we report that in dark-adapted rats exposed for 6 h to bright white light, the p65 subunit of retinal NF-κB translocates to the mitochondria, an event associated with a decrease in expression of cytochrome c oxidase subunit III (COX III). However, sustained exposure for 12 h depleted p65 from the mitochondria, and enhanced COX III expression. Treatment with the protective antioxidant PBN prior to light exposure prevents p65 depletion in the mitochondria and COX III upregulation during prolonged exposure, and apoptosis in photoreceptor cells. These results indicate that COX III expression is sensitive to the abundance of NF-κB p65 in the mitochondria, which, in turn, is affected by exposure to damaging light. - Highlights: • Damaging light exposure of the retina induces NF-κB p65 mitochondrial translocation. • NF-κB p65 mitochondrial translocation is associated with the decrease of COX III expression. • Prolonged light exposure depletes mitochondrial p65 resulting in the increase in COX III expression. • NF-κB p65 and COX III expression play an important role in the light-induced photoreceptor degeneration.

  13. N-myc suppresses Major Histocompatibility Complex class I gene expression through down-regulation of the p50 subunit of NF-kappaB

    NARCIS (Netherlands)

    Veer, L.J. van 't; Beijersbergen, R.L.; Bernards, R.A.

    1993-01-01

    In neuroblastoma, N-myc suppresses the expression of major histocompatibility complex (MHC) Class I antigens by reducing the binding of a nuclear factor to the enhancer-A element in the MHC Class I gene promoter. We show here that the p50 subunit of NF-kappaB is part of this complex and that

  14. The DEAD-Box RNA Helicase DDX3 Interacts with NF-κB Subunit p65 and Suppresses p65-Mediated Transcription.

    Science.gov (United States)

    Xiang, Nian; He, Miao; Ishaq, Musarat; Gao, Yu; Song, Feifei; Guo, Liang; Ma, Li; Sun, Guihong; Liu, Dan; Guo, Deyin; Chen, Yu

    2016-01-01

    RNA helicase family members exhibit diverse cellular functions, including in transcription, pre-mRNA processing, RNA decay, ribosome biogenesis, RNA export and translation. The RNA helicase DEAD-box family member DDX3 has been characterized as a tumour-associated factor and a transcriptional co-activator/regulator. Here, we demonstrate that DDX3 interacts with the nuclear factor (NF)-κB subunit p65 and suppresses NF-κB (p65/p50)-mediated transcriptional activity. The downregulation of DDX3 by RNA interference induces the upregulation of NF-κB (p65/p50)-mediated transcription. The regulation of NF-κB (p65/p50)-mediated transcriptional activity was further confirmed by the expression levels of its downstream cytokines, such as IL-6 and IL-8. Moreover, the binding of the ATP-dependent RNA helicase domain of DDX3 to the N-terminal Rel homology domain (RHD) of p65 is involved in the inhibition of NF-κB-regulated gene transcription. In summary, the results suggest that DDX3 functions to suppress the transcriptional activity of the NF-κB subunit p65.

  15. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-{kappa}B p65 subunit and cytotoxicity in renal proximal tubule cells

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Yu Jin; Lee, Jung Eun; Lee, Ae Sin [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Lee, Sang Yong [Department of Diagnostic Radiology, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Han, Myung Kwan [Department of Microbiology, Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kim, Duk Hoon [Division of Forensic Medicine, National Forensic Service, Seoul (Korea, Republic of); Kim, Won, E-mail: kwon@jbnu.ac.kr [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Cisplatin increases acetylation of NF-{kappa}B p65 subunit in HK2 cells. Black-Right-Pointing-Pointer SIRT1 overexpression decreases cisplatin-induced p65 acetylation and -cytotoxicity. Black-Right-Pointing-Pointer Resveratrol decreased cisplatin-induced cell viability through deacetylation of p65. -- Abstract: As the increased acetylation of p65 is linked to nuclear factor-{kappa}B (NF-{kappa}B) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD{sup +})-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-{kappa}B p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-{kappa}B during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-{kappa}B p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-{kappa}B through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.

  16. The 11S Proteasome Subunit PSME3 Is a Positive Feedforward Regulator of NF-κB and Important for Host Defense against Bacterial Pathogens

    Directory of Open Access Journals (Sweden)

    Jinxia Sun

    2016-02-01

    Full Text Available The NF-κB pathway plays important roles in immune responses. Although its regulation has been extensively studied, here, we report an unknown feedforward mechanism for the regulation of this pathway by Toll-like receptor (TLR ligands in macrophages. During bacterial infections, TLR ligands upregulate the expression of the 11S proteasome subunit PSME3 via NF-κB-mediated transcription in macrophages. PSME3, in turn, enhances the transcriptional activity of NF-κB by directly binding to and destabilizing KLF2, a negative regulator of NF-κB transcriptional activity. Consistent with this positive role of PSME3 in NF-κB regulation and importance of the NF-κB pathway in host defense against bacterial infections, the lack of PSME3 in hematopoietic cells renders the hosts more susceptible to bacterial infections, accompanied by increased bacterial burdens in host tissues. Thus, this study identifies a substrate for PSME3 and elucidates a proteolysis-dependent, but ubiquitin-independent, mechanism for NF-κB regulation that is important for host defense and innate immunity.

  17. Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins.

    Science.gov (United States)

    Gainer, Harold; House, Shirley; Kim, Dong Sun; Chin, Hemin; Pant, Harish C

    2017-04-01

    When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon's neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia-axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.

  18. Nuclear translocation of the p65 subunit of NF-{kappa}B in L5178Y sublines differing in antioxidant defense

    Energy Technology Data Exchange (ETDEWEB)

    Sochanowicz, B.; Szumiel, I.; Gradzka, I. [Institute of Nuclear Chemistry and Technology, Warsaw (Poland). Dept. of Radiobiology and Health Protection

    1999-07-01

    We examined the induction of nuclear translocation of the p65 subunit of NF-{kappa}B in L5178Y (LY) cells. We used two LY sublines which are inversely cross-sensitive to hydrogen peroxide and X-rays: LY-R cells are radioresistant and oxidant-sensitive, whereas LY-S cells are radiosensitive and oxidant-resistant. Hydrogen peroxide, phorbol ester and X-rays caused a marked translocation of p65-NF-{kappa}B in LY-R cells and a weak translocation in LY-S cells. By manipulating the antioxidant defense status, we obtained an alteration in the p65-NF-{kappa}B translocation induction in LY-R cells. A similar effect was achieved with lovastatin pretreatment (25 {mu}M, 24 h, 37 C). The response of LY-S cells under all these conditions was considerably weaker. We conclude that differential nuclear translocation of p65-NF-{kappa}B in LY sublines is not related to the lethal effect of the activating, damaging agent; rather it is due to the more efficient antioxidant defense in LY-S than in LY-R cells. (orig.)

  19. CSF neurofilament proteins as diagnostic and prognostic biomarkers for amyotrophic lateral sclerosis.

    Science.gov (United States)

    Rossi, Daniela; Volanti, Paolo; Brambilla, Liliana; Colletti, Tiziana; Spataro, Rossella; La Bella, Vincenzo

    2018-01-10

    Elevated cerebrospinal fluid (CSF), Neurofilament Light (NF-L) and phosphorylated Heavy (pNF-H) chain levels have been found in Amyotrophic Lateral Sclerosis (ALS), with studies reporting a correlation of both neurofilaments (NFs) with the disease progression. Here, we measured NF-L and pNF-H concentrations in the CSF of ALS patients from a single tertiary Center and investigated their relationship with disease-related variables. A total of 190 ALS patients (Bulbar, 29.9%; Spinal, 70.1%; M/F = 1.53) and 130 controls with mixed neurological diseases were recruited. Demographic and clinical variables were recorded, and ΔFS was used to rate the disease progression. Controls were divided into two cohorts: (1) patients with non-inflammatory neurological diseases (CTL-1); (2) patients with acute/subacute inflammatory diseases and tumors, expected to lead to significant axonal and tissue damage (CTL-2). For each patient and control, CSF was taken at the time of the diagnostic work-up and stored following the published guidelines. CSF NF-L and pNF-H were assayed with commercially available ELISA-based methods. Standard curves (from independent ELISA kits) were highly reproducible for both NFs, with a coefficient of variation pNF-H levels in ALS were significantly increased when compared to CTL-1 (NF-L: ALS, 4.7 ng/ml vs CTL-1, 0.61 ng/ml, p pNF-H: ALS, 1.7 ng/ml vs CTL-1, 0.03 ng/ml, p pNF-H levels. Our results, from a relatively large ALS cohort, confirm that CSF NF-L and pNF-H represent valuable diagnostic and prognostic biomarkers in ALS.

  20. Cooperative inhibition of RIP1-mediated NF-κB signaling by cytomegalovirus-encoded deubiquitinase and inactive homolog of cellular ribonucleotide reductase large subunit.

    Science.gov (United States)

    Kwon, Ki Mun; Oh, Se Eun; Kim, Young Eui; Han, Tae-Hee; Ahn, Jin-Hyun

    2017-06-01

    Several viruses have been found to encode a deubiquitinating protease (DUB). These viral DUBs are proposed to play a role in regulating innate immune or inflammatory signaling. In human cytomegalovirus (HCMV), the largest tegument protein encoded by UL48 contains DUB activity, but its cellular targets are not known. Here, we show that UL48 and UL45, an HCMV-encoded inactive homolog of cellular ribonucleotide reductase (RNR) large subunit (R1), target receptor-interacting protein kinase 1 (RIP1) to inhibit NF-κB signaling. Transfection assays showed that UL48 and UL45, which binds to UL48, interact with RIP1 and that UL48 DUB activity and UL45 cooperatively suppress RIP1-mediated NF-κB activation. The growth of UL45-null mutant virus was slightly impaired with showing reduced accumulation of viral late proteins. Analysis of a recombinant virus expressing HA-UL45 showed that UL45 interacts with both UL48 and RIP1 during virus infection. Infection with the mutant viruses also revealed that UL48 DUB activity and UL45 inhibit TNFα-induced NF-κB activation at late times of infection. UL48 cleaved both K48- and K63-linked polyubiquitin chains of RIP1. Although UL45 alone did not affect RIP1 ubiquitination, it could enhance the UL48 activity to cleave RIP1 polyubiquitin chains. Consistently, UL45-null virus infection showed higher ubiquitination level of endogenous RIP1 than HA-UL45 virus infection at late times. Moreover, UL45 promoted the UL48-RIP1 interaction and re-localization of RIP1 to the UL48-containing virion assembly complex. The mouse cytomegalovirus (MCMV)-encoded DUB, M48, interacted with mouse RIP1 and M45, an MCMV homolog of UL45. Collectively, our data demonstrate that cytomegalovirus-encoded DUB and inactive R1 homolog target RIP1 and cooperatively inhibit RIP1-mediated NF-κB signaling at the late stages of HCMV infection.

  1. Semi-in situ atomic force microscopy imaging of intracellular neurofilaments under physiological conditions through the 'sandwich' method.

    Science.gov (United States)

    Sato, Fumiya; Asakawa, Hitoshi; Fukuma, Takeshi; Terada, Sumio

    2016-08-01

    Neurofilaments are intermediate filament proteins specific for neurons and characterized by formation of biochemically stable, obligate heteropolymers in vivo While purified or reassembled neurofilaments have been subjected to morphological analyses by electron microscopy and atomic force microscopy, there has been a need for direct imaging of cytoplasmic genuine intermediate filaments with minimal risk of artefactualization. In this study, we applied the modified 'cells on glass sandwich' method to exteriorize intracellular neurofilaments, reducing the risk of causing artefacts through sample preparation. SW13vim(-) cells were double transduced with neurofilament medium polypeptide (NF-M) and alpha-internexin (α-inx). Cultured cells were covered with a cationized coverslip after prestabilization with tannic acid to form a sandwich and then split into two. After confirming that neurofilaments could be deposited on ventral plasma membranes exposed via unroofing, we performed atomic force microscopy imaging semi-in situ in aqueous solution. The observed thin filaments, considered to retain native structures of the neurofilaments, exhibited an approximate periodicity of 50-60 nm along their length. Their structural property appeared to reflect the morphology formed by their constituents, i.e. NF-M and α-inx. The success of semi-in situ atomic force microscopy of exposed bona fide assembled neurofilaments through separating the sandwich suggests that it can be an effective and alternative method for investigating cytoplasmic intermediate filaments under physiological conditions by atomic force microscopy. © The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Increased neurofilament light chain blood levels in neurodegenerative neurological diseases.

    Directory of Open Access Journals (Sweden)

    Johanna Gaiottino

    Full Text Available Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf are cytoskeletal proteins of neurons and their release into cerebrospinal fluid has shown encouraging results as a biomarker for neurodegeneration. This study aimed to validate the quantification of the Nf light chain (NfL in blood samples, as a biofluid source easily accessible for longitudinal studies.We developed and applied a highly sensitive electrochemiluminescence (ECL based immunoassay for quantification of NfL in blood and CSF.Patients with Alzheimer's disease (AD (30.8 pg/ml, n=20, Guillain-Barré-syndrome (GBS (79.4 pg/ml, n=19 or amyotrophic lateral sclerosis (ALS (95.4 pg/ml, n=46 had higher serum NfL values than a control group of neurological patients without evidence of structural CNS damage (control patients, CP (4.4 pg/ml, n=68, p<0.0001 for each comparison, p=0.002 for AD patients and healthy controls (HC (3.3 pg/ml, n=67, p<0.0001. Similar differences were seen in corresponding CSF samples. CSF and serum levels correlated in AD (r=0.48, p=0.033, GBS (r=0.79, p<0.0001 and ALS (r=0.70, p<0.0001, but not in CP (r=0.11, p=0.3739. The sensitivity and specificity of serum NfL for separating ALS from healthy controls was 91.3% and 91.0%.We developed and validated a novel ECL based sandwich immunoassay for the NfL protein in serum (NfL(Umea47:3; levels in ALS were more than 20-fold higher than in controls. Our data supports further longitudinal studies of serum NfL in neurodegenerative diseases as a potential biomarker of on-going disease progression, and as a potential surrogate to quantify effects of neuroprotective drugs in clinical trials.

  3. Cerebrospinal fluid neurofilament light chain as a biomarker of neurodegeneration in the Tg4510 and MitoPark mouse models

    DEFF Research Database (Denmark)

    Clement, Amalie; Mitchelmore, Cathy; Andersson, Daniel

    2017-01-01

    disorders like Alzheimer's disease (AD), Parkinson's disease (PD) and tauopathies. We hypothesized that CSF neurofilament light (NF-L) can be used to track progression of neurodegeneration and potentially monitor the efficacy of novel therapeutic agents in preclinical development. To substantiate this, we......A challenge in working with preclinical models of neurodegeneration has been how to non-invasively monitor disease progression. Neurofilament proteins are established axonal damage markers and have been found to be elevated in cerebrospinal fluid (CSF) and blood from patients with neurodegenerative...

  4. Recovery of neurofilament following early monocular deprivation

    Directory of Open Access Journals (Sweden)

    Timothy P O'Leary

    2012-04-01

    Full Text Available A brief period of monocular deprivation in early postnatal life can alter the structure of neurons within deprived-eye-receiving layers of the dorsal lateral geniculate nucleus. The modification of structure is accompanied by a marked reduction in labeling for neurofilament, a protein that composes the stable cytoskeleton and that supports neuron structure. This study examined the extent of neurofilament recovery in monocularly deprived cats that either had their deprived eye opened (binocular recovery, or had the deprivation reversed to the fellow eye (reverse occlusion. The degree to which recovery was dependent on visually-driven activity was examined by placing monocularly deprived animals in complete darkness (dark rearing. The loss of neurofilament and the reduction of soma size caused by monocular deprivation were both ameliorated equally following either binocular recovery or reverse occlusion for 8 days. Though monocularly deprived animals placed in complete darkness showed recovery of soma size, there was a generalized loss of neurofilament labeling that extended to originally non-deprived layers. Overall, these results indicate that recovery of soma size is achieved by removal of the competitive disadvantage of the deprived eye, and occurred even in the absence of visually-driven activity. Recovery of neurofilament occurred when the competitive disadvantage of the deprived eye was removed, but unlike the recovery of soma size, was dependent upon visually-driven activity. The role of neurofilament in providing stable neural structure raises the intriguing possibility that dark rearing, which reduced overall neurofilament levels, could be used to reset the deprived visual system so as to make it more ameliorable with treatment by experiential manipulations.

  5. Recovery of neurofilament following early monocular deprivation

    Science.gov (United States)

    O'Leary, Timothy P.; Kutcher, Matthew R.; Mitchell, Donald E.; Duffy, Kevin R.

    2012-01-01

    Postnatal development of the mammalian geniculostriate visual pathway is partly guided by visually driven activity. Disruption of normal visual input during certain critical periods can alter the structure of neurons, as well as their connections and functional properties. Within the layers of the dorsal lateral geniculate nucleus (dLGN), a brief early period of monocular deprivation can alter the structure and soma size of neurons within deprived-eye-receiving layers. This modification of structure is accompanied by a marked reduction in labeling for neurofilament protein, a principle component of the stable cytoskeleton. This study examined the extent of neurofilament recovery in monocularly deprived cats that either had their deprived eye opened (binocular recovery), or had the deprivation reversed to the fellow eye (reverse occlusion). The loss of neurofilament and the reduction of soma size caused by monocular deprivation were ameliorated equally and substantially in both recovery conditions after 8 days. The degree to which this recovery was dependent on visually driven activity was examined by placing monocularly deprived animals in complete darkness. Though monocularly deprived animals placed in darkness showed recovery of soma size in deprived layers, the manipulation catalyzed a loss of neurofilament labeling that extended to non-deprived layers as well. Overall, these results indicate that both recovery of soma size and neurofilament labeling is achieved by removal of the competitive disadvantage of the deprived eye. However, while the former occurred even in the absence of visually driven activity, recovery of neurofilament did not. The finding that a period of darkness produced an overall loss of neurofilament throughout the dLGN suggests that this experiential manipulation may cause the visual pathways to revert to an earlier more plastic developmental stage. It is possible that short periods of darkness could be incorporated as a component of

  6. Neurofilament L gene is not a genetic factor of sporadic and familial Parkinson's disease.

    Science.gov (United States)

    Rahner, Nils; Holzmann, Carsten; Krüger, Rejko; Schöls, Ludger; Berger, Klaus; Riess, Olaf

    2002-09-27

    Mutations in two genes, alpha-synuclein and parkin, have been identified as some rare causes for familial Parkinson's disease (PD). alpha-Synuclein and parkin protein have subsequently been identified in Lewy bodies (LB). To gain further insight into the pathogenesis of PD we investigated the role of neurofilament light (NF-L), another component of LB aggregation. A detailed mutation search of the NF-L gene in 328 sporadic and familial PD patients of German ancestry revealed three silent DNA changes (G163A, C224T, C487T) in three unrelated patients. Analysis of the promoter region of the NF-L gene identified a total of three base pair substitutions defining five haplotypes. Association studies based on these haplotypes revealed no significant differences between PD patients and 344 control individuals. Therefore, NF-L is unlikely to play a major role in the pathogenesis of PD.

  7. Rapid intermittent movement of axonal neurofilaments observed by fluorescence photobleaching

    National Research Council Canada - National Science Library

    Wang, L; Brown, A

    2001-01-01

    Observations on naturally occurring gaps in the axonal neurofilament array of cultured neurons have demonstrated that neurofilament polymers move along axons in a rapid, intermittent, and highly asynchronous manner...

  8. Changes in the distribution of the neuron-specific B-50, neurofilament protein and glial fibrillary acidic proteins following an unilateral mesencephalic lesion in the rat

    NARCIS (Netherlands)

    Gispen, W.H.; Oestreicher, A.B.; Devay, P.; Isaacson, R.L.

    1988-01-01

    Following a unilateral electrolytic lesion in the ventral rat mesencephalon, changes in the immunocytochemical distribution of the neuron-specific B-50, neurofilament (NF) protein and glial fibrillary acidic (GFAP) proteins were studied around the lesion after 0, 3, 10 and 28 days. At all recovery

  9. Quinacrine Inhibits ICAM-1 Transcription by Blocking DNA Binding of the NF-κB Subunit p65 and Sensitizes Human Lung Adenocarcinoma A549 Cells to TNF-α and the Fas Ligand.

    Science.gov (United States)

    Harada, Misuzu; Morimoto, Kyoko; Kondo, Tetsuya; Hiramatsu, Reiko; Okina, Yuji; Muko, Ryo; Matsuda, Iyo; Kataoka, Takao

    2017-12-02

    Quinacrine has been used for therapeutic drugs in some clinical settings. In the present study, we demonstrated that quinacrine decreased the expression of intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor (TNF)-α and interleukin-1 (IL-1) α in human lung adenocarcinoma A549 cells. Quinacrine inhibited ICAM-1 mRNA expression and nuclear factor κB (NF-κB)-responsive luciferase reporter activity following a treatment with TNF-α and IL-1α. In the NF-κB signaling pathway, quinacrine did not markedly affect the TNF-α-induced degradation of the inhibitor of NF-κB or the TNF-α-induced phosphorylation of the NF-κB subunit, p65, at Ser-536 and its subsequent translocation to the nucleus. In contrast, a chromatin immunoprecipitation assay showed that quinacrine prevented the binding of p65 to the ICAM-1 promoter following TNF-α stimulation. Moreover, TNF-α and the Fas ligand effectively reduced the viability of A549 cells in the presence of quinacrine only. Quinacrine down-regulated the constitutive and TNF-α-induced expression of c-FLIP and Mcl-1 in A549 cells. These results revealed that quinacrine inhibits ICAM-1 transcription by blocking the DNA binding of p65 and sensitizes A549 cells to TNF-α and the Fas ligand.

  10. Direct evidence of central nervous system axonal damage in patients with postoperative delirium: A preliminary study of pNF-H as a promising serum biomarker.

    Science.gov (United States)

    Inoue, Reo; Sumitani, Masahiko; Ogata, Toru; Chikuda, Hirotaka; Matsubara, Takehiro; Kato, So; Shimojo, Nobutake; Uchida, Kanji; Yamada, Yoshitsugu

    2017-07-13

    Approximately 50-80% patients experience postoperative delirium, an acute cognitive dysfunction associated with prolonged hospitalization, increased mortality, excess healthcare costs, and persistent cognitive impairment. Elucidation of the mechanism of delirium and associated diagnostic and therapeutic measures are urgently required. Here we investigated the role of phosphorylated neurofilament heavy subunit (pNF-H), a major structural protein in axons, as a predictive maker of postoperative delirium. Twenty-three patients who underwent surgery for abdominal cancer were screened for postoperative delirium, and they were assessed for its severity using the memorial delirium assessment scale (MDAS) at and 48h after delirium onset. Serum pNF-H levels were also measured at both time points. The patients were divided into two groups according to the presence or absence of pNF-H. Clinical variables were compared between groups using the Mann-Whitney U test, and the relationship between pNF-H levels and delirium severity was analyzed using the exponential curve fitting. Fifteen of the 23 (65.2%) patients tested positive for pNF-H, and these patients exhibited significantly higher MDAS scores compared with the pNF-H-negative patients only at the onset of delirium. Although the MDAS score significantly improved over time in the positive group, pNF-H positivity persisted. There was a correlation between the maximum pNF-H level and maximum MDAS score (R2=0.31, p=0.013). More severe postoperative delirium was directly related to higher serum pNF-H levels, suggesting the potential application of pNF-H as a quantitative biomarker of neural damage in postoperative delirium. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Serum phosphorylated neurofilament-heavy chain levels in multiple sclerosis patients.

    Science.gov (United States)

    Gresle, M M; Liu, Y; Dagley, L F; Haartsen, J; Pearson, F; Purcell, A W; Laverick, L; Petzold, A; Lucas, R M; Van der Walt, A; Prime, H; Morris, D R; Taylor, B V; Shaw, G; Butzkueven, H

    2014-11-01

    We evaluated whether the measurement of serum phosphorylated neurofilament heavy chain (pNF-H) titre is likely to be a valid biomarker of axonal injury in multiple sclerosis (MS). Serum pNF-H concentrations were measured by ELISA in cases with relapsing-remitting (RR)-MS (n=81), secondary progressive (SP) MS (n=13) and primary progressive (PP)-MS; n=6) MS; first demyelinating event (FDE; n=82); and unaffected controls (n=135). A subset of MS cases (n=45) were re-sampled on one or multiple occasions. The Multiple Sclerosis Severity Score (MSSS) and MRI measures were used to evaluate associations between serum pNF-H status, disease severity and cerebral lesion load and activity. We confirmed the presence of pNF-H peptides in serum by ELISA. We showed that a high serum pNF-H titre was detectable in 9% of RR-MS and FDE cases, and 38.5% of SP-MS cases. Patients with a high serum pNF-H titre had higher average MSSS scores and T2 lesion volumes than patients with a low serum pNF-H titre. Repeated sampling of a subset of MS cases showed that pNF-H levels can fluctuate over time, likely reflecting temporal dynamics of axonal injury in MS. A subset of FDE/MS cases was found to have a high serum pNF-H titre, and this was associated with changes in clinical outcome measures. We propose that routine measurement of serum pNF-H should be further investigated for monitoring axonal injury in MS. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  12. Serum Phosphorylated Neurofilament-Heavy Chain, a Potential Biomarker, is Associated With Peripheral Neuropathy in Patients With Type 2 Diabetes.

    Science.gov (United States)

    Qiao, Xiaona; Zhang, Shuo; Zhao, Weiwei; Ye, Hongying; Yang, Yehong; Zhang, Zhaoyun; Miao, Qing; Hu, Renming; Li, Yiming; Lu, Bin

    2015-11-01

    Neurofilament (NF), one of the major axonal cytoskeletal proteins, plays a critical role in degenerative diseases in both the central and the peripheral nervous systems. The aim of this study is to explore the relationship between serum phosphorylated neurofilament-heavy chain (pNF-H) and diabetic peripheral neuropathy (DPN) in patients with type 2 diabetes.Serum pNF-H concentrations were measured by ELISA in hospitalized patients with and without DPN (n = 118). DPN was assessed by clinical symptoms, signs, and electromyography.Compared with the non-DPN group (311.98 [189.59-634.12] pg/mL), the confirmed group (605.99 [281.17-1332.78] pg/mL) patients had the higher serum pNF-H levels (P = 0.007). DPN was significantly correlated with C-peptide (r = -0.269), total cholesterol (TC) (r = 0.185), and pNF-H (r = 0.258). Serum pNF-H levels were independently associated with DPN (P = 0.004), even after adjusting for age, sex, duration of diabetes, fasting plasma glucose, glycosylated hemoglobin A1c, TC, C-peptide, urinary albuminto/creatinine ratio, and estimated glomerular filtration rate. Compared with pNF-H quartile 1 (referent), patients in quartile 3 (odds ratio [OR], 3.977; 95% confidence interval [CI], 1.243-12.728; P = 0.021) and quartile 4 (OR, 10.488; 95% CI, 3.020-34.429; P = 0.000) had the higher risk of DPN after adjusting for the confounders.Serum pNF-H levels might be associated with the DPN, and the correlationship between serum pNF-H and DPN should be further studied.

  13. Modification of Cysteine Residue in p65 Subunit of Nuclear Factor-κB by Picroliv Suppresses NF-κB-Regulated Gene Products and Potentiates Apoptosis

    Science.gov (United States)

    Anand, Preetha; Kunnumakkara, Ajaikumar B.; Harikumar, Kuzhuvelil B.; Ahn, Kwang S.; Badmaev, Vladimir

    2009-01-01

    Picroliv, an iridoid glycoside derived from the plant Picrorhiza kurroa, is used traditionally to treat fever, asthma, hepatitis and other inflammatory conditions. However, the exact mechanism of its therapeutic action is still unknown. Because NF-κB activation plays a major role in inflammation and carcinogenesis, we postulated that picroliv must interfere with this pathway by inhibiting the activation of NF-κB mediated signal cascade. Electrophoretic mobility shift assay showed that pretreatment with picroliv abrogated TNF induced activation of NF-κB. The glycoside also inhibited NF-κB activated by carcinogenic and inflammatory agents such as CSC, PMA, OA, H2O2, LPS and EGF. When examined for the mechanism of action, we found that picroliv inhibited activation of IκBα kinase, leading to inhibition of phosphorylation and degradation of IκBα. It also inhibited phosphorylation and nuclear translocation of p65. Further studies revealed that picroliv directly inhibits the binding of p65 to DNA, which was reversed by the treatment with reducing agents, suggesting a role for a cysteine residue in interaction with picroliv. Mutation of cysteine residue 38 in p65 to serine abolished this effect of picroliv. NF-κB inhibition by picroliv lead to suppression of NF-κB-regulated proteins, including those linked with cell survival (IAP1, Bcl-2, Bcl-xL, survivin, and TRAF2), proliferation (cyclin D1 and COX-2), angiogenesis (VEGF), and invasion (ICAM-1 and MMP-9). Suppression of these proteins, enhanced apoptosis induced by TNF. Overall, our results demonstrate that picroliv inhibits the NF-κB activation pathway, which may explain its anti-inflammatory and anticarcinogenic effects. PMID:18974130

  14. Expression of N-methyl D-aspartate receptor subunits in amoeboid microglia mediates production of nitric oxide via NF-κB signaling pathway and oligodendrocyte cell death in hypoxic postnatal rats.

    Science.gov (United States)

    Murugan, Madhuvika; Sivakumar, Viswanathan; Lu, Jia; Ling, Eng-Ang; Kaur, Charanjit

    2011-04-01

    The present study was focused on identifying the expression of N-methyl D-aspartate receptor (NMDAR) subunits on activated microglia and to determine their role in the pathogenesis of periventricular white matter damage (PWMD) in neonatal rats following hypoxia. One day old wistar rats were subjected to hypoxia (5% O(2) ; 95% N(2) ) and the mRNA and protein expression of NMDAR subunits (NR1, NR2A-D, and NR3A) in the periventricular white matter (PWM) was determined at different time points (3,24 h, 3, 7, and 14 days) following hypoxic exposure. Immunoexpression of NR1 and NR2A-D was localized in amoeboid microglial cells (AMC) suggesting the presence of functional NMDARs in them. The expression of NMDAR in primary microglial cultures was ascertained by RT-PCR analysis and double immunofluorescence studies. The functionality of the microglial NMDAR in cultured microglial cells was examined by monitoring calcium movements in cells with fura-2. In primary microglial cultures, hypoxia induced the nuclear translocation of NF-κB which was suppressed by administration of MK801, an NMDAR antagonist. MK801 also down regulated the hypoxia-induced expression of tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase (iNOS), and nitric oxide (NO) production by microglia which may be mediated by the NF-κB signaling pathway. NO produced by microglia is known to cause death of oligodendrocytes in the developing PWM. In this connection, pharmacological agents such as MK801, BAY (NF-κB inhibitor), and 1400w (iNOS inhibitor) proved to be beneficial since they reduced the hypoxia-induced iNOS expression, NO production, and a corresponding reduction in the death of oligodendrocytes following hypoxia. Copyright © 2011 Wiley-Liss, Inc.

  15. Neurofilaments as Biomarkers for Amyotrophic Lateral Sclerosis: A Systematic Review and Meta-Analysis.

    Directory of Open Access Journals (Sweden)

    Zhouwei Xu

    Full Text Available To allow early diagnosis and monitoring of disease progression, there is a need for biomarkers in amyotrophic lateral sclerosis (ALS. Neurofilaments (NF are emerging protein biomarkers in other neurological diseases, and are of possible use in ALS.The aim of this study is to evaluate the utility of NF levels as blood or cerebrospinal fluid (CSF biomarker in patients with ALS.A systematic search of Pubmed, Embase and Scopus was performed. Methodological quality assessment was applied to refine the final search results. Meta-analysis of the data was performed.Level of NF heavy chain and light chains were significantly elevated in the CSF of ALS patients compared to healthy controls/controls without parenchymal central nervous system (CNS involvement and ALS mimic disease patients. NF light chain level in CSF was higher in ALS patients than in neurological patients with CNS involvement (SMD = 1.352, P = 0.01. NF light chain concentration in blood was higher in ALS patients than healthy controls/controls without CNS involvement (SMD = 1.448, P<0.0001. NF heavy chain levels in CSF were negatively correlated disease duration and ALSFRS-R ((r = -0.447, P<0.0001; r = -0.486, P<0.0001. NF light chain levels in CSF were negatively correlated with disease duration (r = -0.273, P = 0.011.NF heavy and light chain levels have potential use as a marker of neural degeneration in ALS, but are not specific for the disease, and are more likely to be used as measures of disease progression.

  16. Cerebrospinal Fluid Levels of Phosphorylated Neurofilament Heavy as a Diagnostic Marker of Canine Degenerative Myelopathy.

    Science.gov (United States)

    Toedebusch, C M; Bachrach, M D; Garcia, V B; Johnson, G C; Katz, M L; Shaw, G; Coates, J R; Garcia, M L

    2017-03-01

    No definitive, antemortem diagnostic test for canine degenerative myelopathy (DM) is available. Phosphorylated neurofilament heavy (pNF-H) is a promising biomarker for nervous system diseases. Cerebrospinal fluid (CSF) and serum pNF-H is a detectable biological marker for diagnosis of canine DM. Fifty-three DM-affected, 27 neurologically normal, 7 asymptomatic at-risk, and 12 DM mimic dogs. Archived CSF and serum pNF-H concentrations were determined by a commercially available ELISA. A receiver-operating characteristic (ROC) curve was generated with CSF values. Compared with old control dogs, median CSF pNF-H concentration was increased in all stages of DM; old dogs 5.1 ng/mL (interquartile range [IQR] 1.4-9.3) versus DM stage 1 23.9 ng/mL (IQR 20.8-29.6; P pNF-H concentrations compared with asymptomatic, at-risk dogs (3.4 ng/mL [IQR 1.5-10.9; P pNF-H concentration >20.25 ng/mL was 80.4% sensitive (confidence interval [CI] 66.09-90.64%) and 93.6% specific (CI 78.58-99.21%) for DM. Area under the ROC curve was 0.9467 (CI 0.92-0.9974). No differences in serum pNF-H concentration were found between control and DM-affected dogs. pNF-H concentration in CSF is a sensitive biomarker for diagnosis of DM. Although there was high specificity for DM in this cohort, further study should focus on a larger cohort of DM mimics, particularly other central and peripheral axonopathies. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  17. A new marker for ischemic cerebrovascular stroke: Phosphorylated Neurofilament H

    Directory of Open Access Journals (Sweden)

    Waheed M. Radwan

    2013-04-01

    Conclusion: Phosphorylated Neurofilament H can be used as a useful tool to assess patients with acute ischemic CVS. Levels of the neurofilament correlated with the degree of conscious level in such patients and with CT findings hence can be used to assess short term prognosis.

  18. Cerebrospinal fluid neurofilament light levels mark grey matter volume in clinically isolated syndrome suggestive of multiple sclerosis.

    Science.gov (United States)

    Tortorella, Carla; Direnzo, Vita; Ruggieri, Maddalena; Zoccolella, Stefano; Mastrapasqua, Mariangela; D'Onghia, Mariangela; Paolicelli, Damiano; Cuonzo, Franca Di; Gasperini, Claudio; Trojano, Maria

    2017-05-01

    Brain atrophy is a known marker of irreversible tissue damage in multiple sclerosis (MS). Cerebrospinal fluid (CSF) osteopontin (OPN) and neurofilament light chain (NF-L) have been proposed as candidate surrogate markers of inflammatory and neurodegenerative processes in MS. To evaluate the relationship between CSF NF-L and OPN levels and brain grey and white matter volumes in patients with clinically isolated syndrome (CIS) suggestive of MS. A total of 41 CIS patients and 30 neurological controls (NCs) were included. CSF NF-L and OPN were measured by commercial ELISA. Measures of brain volume (normalized brain volume (NBV), normalized grey matter volume (NGV), peripheral grey matter volume (PGV), normalized white matter volume (WMV), and ventricular volume) were obtained by SIENAX. Corpus callosum index (CCI) was calculated. Brain volumes were categorized into 'high' and 'low' according to the median value. CSF NF-L and OPN levels were higher in CIS patients in comparison with NCs. CIS patients with 'low' TGV, PGV, and TBV showed higher CSF NF-L levels than CIS patients with 'high' brain volumes. TGV and PGV correlated inversely with NF-L levels, whereas CCI was inversely related to OPN levels. CSF NF-L was the only independent predictor of TGV and PGV. CSF NF-L tracks mainly grey matter damage in patients with CIS suggestive of MS.

  19. Phosphorylated neurofilament heavy chain is a marker of neurodegeneration in Leber hereditary optic neuropathy (LHON).

    Science.gov (United States)

    Guy, John; Shaw, Gerry; Ross-Cisneros, Fred N; Quiros, Peter; Salomao, Solange R; Berezovsky, Adriana; Carelli, Valerio; Feuer, William J; Sadun, Alfredo A

    2008-01-01

    To determine the profile of neurodegeneration in Leber hereditary optic neuropathy (LHON). We quantitated serum levels of phosphorylated neurofilament heavy chain (pNF-H) in a Brazilian pedigree of 16 affected patients and 59 carriers with LHON, both molecularly characterized as harboring the G to A mutation at nucleotide 11,778 of the mitochondrial genome. The association of subject characteristics to pNF-H levels was studied with multiple regression; pNF-H data were square-root transformed to effect normality of distribution of residuals. Relationships between the square-root of pNF-H and age and sex were investigated within groups with Pearson correlation and the two-sample t-test. Linear regression was used to assess the difference between groups and to determine if the relationship of age was different between affected individuals and carriers. Results of plotting pNF-H levels by age suggested a nonlinear, quadratic association so age squared was used in the statistical analysis. ANCOVA was used to assess the influence of age and group on pNF-H levels. In the carrier group, there was a significant correlation of square-root pNF-H (mean=0.24 ng/ml(2)) with age (r=0.30, p=0.022) and a stronger correlation with quadratic age (r=0.37, p=0.003). With a higher mean pNF-H (0.33 ng/ml(2)) for the affected group, correlations were of similar magnitude, although they were not statistically significant: age (r=0.22, p=0.42), quadratic age (r=0.22, p=0.45). There was no correlation between age and pNF-H levels (mean=0.34 ng/ml(2)) in the off-pedigree group: age (r=0.03, p=0.87), quadratic age (r=0.04, p=0.84). There was no difference between sexes and pNF-H levels in any of the groups (affected, p=0.65; carriers, p=0.19; off-pedigree, p=0.93). Elevated pNF-H released into the serum of some affected LHON patients may suggest that axonal degeneration occurs at some point after loss of visual function. Increases in pNF-H levels of carriers with increasing age, not seen in

  20. Advanced Glycation End Products (AGEs Antibody Protects Against AGEs-induced Apoptosis and NF-ĸB p65 Subunit Overexpression in Rat Glomerular Culture

    Directory of Open Access Journals (Sweden)

    Oktavia Rahayu Adianingsih

    2016-11-01

    Full Text Available Advanced glycation end products (AGEs have been thought to be a major cause of diabetic nephropathy (DN. The mechanisms underlying the involvement of AGEs antibody in diabetic nephropathy are not fully understood. The present study was designed to investigate the protective effect of AGEs antibody on AGEs-induced glomerular damage. Isolated glomeruli were pre-incubated either with 10 µg/mL polyclonal anti-AGEs antibody (AGE-pAb or monoclonal anti-Nɜ -carboxymethyl-lysine antibody (CML-mAb as a model of AGEs antibody to block interaction of AGEs with receptor for AGEs (RAGE and incubated afterwards either with 100 µg/mL bovine serum albumin (BSA or AGE-modified bovine serum albumin (AGE-BSA for 48 h. Annexin V/nephrin doublestaining was performed to determine apoptosis. Using immunofluorescence, we found that administration of AGE-BSA not only significantly increased glomerular cells apoptosis and nuclear factor kappa B (NF-ĸB p65 expression, but also reduced expression of nephrin, an important structural and signal molecule of podocytes slit diaphragm. Blocking the interaction of AGE-RAGE with AGEs antibody significantly protected glomerular cells from AGEs-induced apoptosis and NF-ĸB p65 overexpression. We found that AGE-pAb conferred superior protective effect compared with CmL-mAb for the same reduction in apoptosis and NF-ĸB p65 expression. In sharp contrast, CmL-mAb led to preserve expression of podocytes nephrin better than AGE-pAb. These results demonstrate that the antibody against AGEs may be beneficial for preventing the glomerular damage in DN.

  1. Phosphorylated Neurofilament Heavy Chain Correlations to Visual Function, Optical Coherence Tomography, and Treatment

    Science.gov (United States)

    Pasol, Joshua; Feuer, William; Yang, Cui; Shaw, Gerry; Kardon, Randy; Guy, John

    2010-01-01

    Objective. To correlate visual and neurologic clinical scores and treatment of optic neuritis and multiple sclerosis (MS) patients with assays of serum phosphorylated neurofilament heavy chain (pNF-H) and optical coherence tomography (OCT) measurements of axonal loss. Design/Methods. The Optic Neuritis Treatment Trial (ONTT) randomized 457 patients with acute optic neuritis to intravenous methylprednisolone (IVMP) followed by oral prednisone, oral prednisone or placebo treatment arms. We quantified serum pNF-H levels in 175 ONTT patients 5 years after study entry. We performed OCT measurements of macular volume and the retinal nerve fiber layer (RNFL) in a subset of 51 patients at year 15. Results. Elevated pNF-H levels at year 5 correlated to poorer visual function at study entry. Lower 15 year macular volumes and RNFL thickness correlated better with follow-up than with baseline visual function measures. With IVMP treatment, 15 year RNFL differences of the fellow eye (FE) minus the affected eye (SE) RNFLFEmSE correlated with five-year pNF-H levels. PNF-H was reduced by half with IVMP relative to placebo or by 40% relative to prednisone. Conclusions/Relevance. Acute optic neuritis patients who have more severe visual loss during initial presentation have a higher incidence of axonal loss that was slightly suppressed with IVMP treatment. PMID:22096624

  2. Potential role of pNF-H, a biomarker of axonal damage in the central nervous system, as a predictive marker of chemotherapy-induced cognitive impairment.

    Science.gov (United States)

    Natori, Akina; Ogata, Toru; Sumitani, Masahiko; Kogure, Takamichi; Yamauchi, Teruo; Yamauchi, Hideko

    2015-03-15

    Chemotherapy-induced cognitive impairment (CICI) is a clinically significant problem. Previous studies using magnetic resonance imaging indicated structural changes in the cerebral white matter of patients with CICI. Phosphorylated neurofilament heavy subunit (pNF-H), a major structural protein in axons, was recently reported to be elevated in the serum of patients with some central nervous system disorders. We performed a cross-sectional analysis of neuropsychological test results and serum pNF-H levels in patients undergoing adjuvant chemotherapy for breast cancer. Our hypothesis was that CICI is accompanied by axonal damage that can be detected by elevated serum pNF-H levels. Seventy-six patients with early breast cancer in various phases of treatment (naïve to chemotherapy; after one, three, or seven cycles of chemotherapy; or with a history of chemotherapy) were assessed by self-administered neuropsychological tests and a single pNF-H measurement. The χ(2) and Mann-Whitney tests were used for statistical analysis. Increased pNF-H levels were observed in 28.8% of the patients who underwent chemotherapy, but in none of the chemotherapy-naïve patients or patients with a history of chemotherapy. The pNF-H-positive rate increased significantly in proportion to the number of chemotherapy cycles (one cycle, 5.0%; three cycles, 31.6%; seven cycles, 55.0%; P pNF-H level in patients undergoing chemotherapy for breast cancer increased in a cumulative dose-dependent manner, suggesting its potential application as a biomarker of neural damage after chemotherapy. ©2015 American Association for Cancer Research.

  3. Biomarker report from the phase II lamotrigine trial in secondary progressive MS - neurofilament as a surrogate of disease progression.

    Directory of Open Access Journals (Sweden)

    Sharmilee Gnanapavan

    Full Text Available Lamotrigine trial in SPMS was a randomised control trial to assess whether partial blockade of sodium channels has a neuroprotective effect. The current study was an additional study to investigate the value of neurofilament (NfH and other biomarkers in predicting prognosis and/or response to treatment.SPMS patients who attended the NHNN or the Royal Free Hospital, UK, eligible for inclusion were invited to participate in the biomarker study. Primary outcome was whether lamotrigine would significantly reduce detectable serum NfH at 0-12, 12-24 and 0-24 months compared to placebo. Other serum/plasma and CSF biomarkers were also explored.Treatment effect by comparing absolute changes in NfH between the lamotrigine and placebo group showed no difference, however based on serum lamotrigine adherence there was significant decline in NfH (NfH 12-24 months p=0.043, Nfh 0-24 months p=0.023. Serum NfH correlated with disability: walking times, 9-HPT (non-dominant hand, PASAT, z-score, MSIS-29 (psychological and EDSS and MRI cerebral atrophy and MTR. Other biomarkers explored in this study were not found to be significantly associated, aside from that of plasma osteopontin.The relations between NfH and clinical scores of disability and MRI measures of atrophy and disease burden support NfH being a potential surrogate endpoint complementing MRI in neuroprotective trials and sample sizes for such trials are presented here. We did not observe a reduction in NfH levels between the Lamotrigine and placebo arms, however, the reduction in serum NfH levels based on lamotrigine adherence points to a possible neuroprotective effect of lamotrigine on axonal degeneration.

  4. Activation of RAW264.7 mouse macrophage cells in vitro through treatment with recombinant ricin toxin-binding subunit B: involvement of protein tyrosine, NF-κB and JAK-STAT kinase signaling pathways.

    Science.gov (United States)

    Xu, Na; Yuan, Hongyan; Liu, Wensen; Li, Songyan; Liu, Yang; Wan, Jiayu; Li, Xiaoyan; Zhang, Rui; Chang, Yaping

    2013-09-01

    Ricin toxin-binding subunit B (RTB) is a galactose-binding lectin protein. In the present study, we investigated the effects of RTB on inducible nitric oxide (NO) synthase (iNOS), interleukin (IL)-6 and tumor necrosis factor (TNF)-α, as well as the signal transduction mechanisms involved in recombinant RTB-induced macrophage activation. RAW264.7 macrophages were treated with RTB. The results revealed that the mRNA and protein expression of iNOS was increased in the recombinant RTB-treated macrophages. TNF-α production was observed to peak at 20 h, whereas the production of IL-6 peaked at 24 h. In another set of cultures, the cells were co-incubated with RTB and the tyrosine kinase inhibitor, genistein, the phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, the p42/44 inhibitor, PD98059, the p38 inhibitor, SB203580, the JNK inhibitor, SP600125, the protein kinase C (PKC) inhibitor, staurosporine, the JAK2 inhibitor, tyrphostin (AG490), or the NOS inhibitor, L-NMMA. The recombinant RTB-induced production of NO, TNF-α and IL-6 was inhibited in the macrophages treated with the pharmacological inhibitors genistein, LY294002, staurosporine, AG490, SB203580 and BAY 11-7082, indicating the possible involvement of protein tyrosine kinases, PI3K, PKC, JAK2, p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in the above processes. A phosphoprotein analysis identified tyrosine phosphorylation targets that were uniquely induced by recombinant RTB and inhibited following treatment with genistein; some of these proteins are associated with the downstream cascades of activated JAK-STAT and NF-κB receptors. Our data may help to identify the most important target molecules for the development of novel drug therapies.

  5. Diagnostic Value of Serum Levels of GFAP, pNF-H, and NSE Compared With Clinical Findings in Severity Assessment of Human Traumatic Spinal Cord Injury.

    Science.gov (United States)

    Ahadi, Reza; Khodagholi, Fariba; Daneshi, Abdolhadi; Vafaei, Ali; Mafi, Amir Ali; Jorjani, Masoumeh

    2015-07-15

    An analytical cohort study. This study aimed to evaluate severity of traumatic spinal cord injury (SCI) based on the serum levels of phosphorylated form of heavy subunit of neurofilament (pNF-H), neuron-specific enolase (NSE), and glial fibrillary acidic protein (GFAP), which are axonal, neural cell body, and glial cell injury markers, respectively. Prior studies have reported elevated serum levels of pNF-H, NSE, and GFAP as biomarkers for the detection of traumatic SCI in animals. However, in this study, these biomarkers were studied in humans and with an extended level of timing. The study included 35 patients with SCI with a mean age of 36.5 years. All patients were evaluated using the American Spinal Injury Association Impairment Scale, followed by examinations including radiography and spinal computed tomography for determining the injury level. Serum levels of NSE, pNF-H, and GFAP were determined using enzyme-linked immunosorbent assay. The mean serum level of GFAP was significantly higher in patients with SCI than in the control group. Mean serum levels of pNF-H and NSE were significantly higher during 24 and 48 hours after injury in patients with SCI than in the control group. The serum level of GFAP was appropriate for estimating the severity of SCI in the first 24 hours after injury. Our findings suggest that increased serum levels of GFAP, NSE, and pNF-H can be used for the diagnosis and degree of SCI severity in trauma patients. During 48 hours after injury, estimation of serum levels of pNF-H, NSE, and GFAP, combined with neurological testing, could predict the presence of SCI and severity prior to spinal computed tomography and surgical or conservative interventions. 2.

  6. Co-Administration of Molecular Adjuvants Expressing NF-Kappa B Subunit p65/RelA or Type-1 Transactivator T-bet Enhance Antigen Specific DNA Vaccine-Induced Immunity

    Directory of Open Access Journals (Sweden)

    Devon J. Shedlock

    2014-03-01

    Full Text Available DNA vaccine-induced immunity can be enhanced by the co-delivery of synthetic gene-encoding molecular adjuvants. Many of these adjuvants have included cytokines, chemokines or co-stimulatory molecules that have been demonstrated to enhance vaccine-induced immunity by increasing the magnitude or type of immune responses and/or protective efficacy. In this way, through the use of adjuvants, immune responses can be highly customizable and functionally tailored for optimal efficacy against pathogen specific (i.e., infectious agent or non-pathogen (i.e., cancer antigens. In the novel study presented here, we examined the use of cellular transcription factors as molecular adjuvants. Specifically the co-delivery of (a RelA, a subunit of the NF-κB transcription complex or (b T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated. As well, all of the vaccines and adjuvants were administered to mice using in vivo electroporation (EP, a technology demonstrated to dramatically increase plasmid DNA transfection and subsequent transgene expression with concomitant enhancement of vaccine induced immune responses. As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses. This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity.

  7. Serum neurofilament light in familial Alzheimer disease: A marker of early neurodegeneration.

    Science.gov (United States)

    Weston, Philip S J; Poole, Teresa; Ryan, Natalie S; Nair, Akshay; Liang, Yuying; Macpherson, Kirsty; Druyeh, Ronald; Malone, Ian B; Ahsan, R Laila; Pemberton, Hugh; Klimova, Jana; Mead, Simon; Blennow, Kaj; Rossor, Martin N; Schott, Jonathan M; Zetterberg, Henrik; Fox, Nick C

    2017-11-21

    To investigate whether serum neurofilament light (NfL) concentration is increased in familial Alzheimer disease (FAD), both pre and post symptom onset, and whether it is associated with markers of disease stage and severity. We recruited 48 individuals from families with PSEN1 or APP mutations to a cross-sectional study: 18 had symptomatic Alzheimer disease (AD) and 30 were asymptomatic but at 50% risk of carrying a mutation. Serum NfL was measured using an ultrasensitive immunoassay on the single molecule array (Simoa) platform. Cognitive testing and MRI were performed; 33 participants had serial MRI, allowing calculation of atrophy rates. Genetic testing established mutation status. A generalized least squares regression model was used to compare serum NfL among symptomatic mutation carriers, presymptomatic carriers, and noncarriers, adjusting for age and sex. Spearman coefficients assessed associations between serum NfL and (1) estimated years to/from symptom onset (EYO), (2) cognitive measures, and (3) MRI measures of atrophy. Nineteen of the asymptomatic participants were mutation carriers (mean EYO -9.6); 11 were noncarriers. Compared with noncarriers, serum NfL concentration was higher in both symptomatic ( p < 0.0001) and presymptomatic mutation carriers ( p = 0.007). Across all mutation carriers, serum NfL correlated with EYO (ρ = 0.81, p < 0.0001) and multiple cognitive and imaging measures, including Mini-Mental State Examination (ρ = -0.62, p = 0.0001), Clinical Dementia Rating Scale sum of boxes (ρ = 0.79, p < 0.0001), baseline brain volume (ρ = -0.62, p = 0.0002), and whole-brain atrophy rate (ρ = 0.53, p = 0.01). Serum NfL concentration is increased in FAD prior to symptom onset and correlates with measures of disease stage and severity. Serum NfL may thus be a feasible biomarker of early AD-related neurodegeneration. Copyright © 2017 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.

  8. Quantitative study of neurofilament-positive fiber length in rat spinal cord lesions using isotropic virtual planes

    DEFF Research Database (Denmark)

    von Euler, Mia; Larsen, Jytte Overgaard; Janson, A M

    1998-01-01

    analysis after spinal cord injury is needed. Length quantification of the putatively spontaneously regenerating fibers has been difficult until recently, when two length estimators based on sampling with isotropic virtual planes within thick physical sections were introduced. The applicability......Spontaneous reocurrence of neurofilament (NF)-positive fibers has been described after spinal cord lesions in rats. However, previously introduced methods to evaluate the lesion and the regenerative fiber outgrowth suffer from several biases, why a new concept of quantitative, morphological...... of these techniques to estimate the total length of NF-positive fibers was evaluated in photochemically induced ischemic lesions of thoracic spinal cords in young rats 6 weeks postlesion. Fiber length was found to be the most consistent measure with a mean of 3.71 m (coefficient of variation, CV = 0.16) in the 0...

  9. Lipid Head Group Charge and Fatty Acid Configuration Dictate Liposome Mobility in Neurofilament Networks

    NARCIS (Netherlands)

    Arends, F.; Chaudhary, Himanshu; Janmey, P.; Claessens, Mireille Maria Anna Elisabeth; Lieleg, O.

    Intermediate filaments constitute a class of biopolymers whose function is still poorly understood. One example for such intermediate filaments is given by neurofilaments, large macromolecules that fill the axon of neurons. Here, reconstituted networks of purified porcine neurofilaments are studied

  10. An enzyme immunoassay to quantify neurofilament light chain in cerebrospinal fluid.

    NARCIS (Netherlands)

    Geel, W.J.A. van; Rosengren, L.E.; Verbeek, M.M.

    2005-01-01

    Neurofilament light chain is a component of the axonal cytoskeleton. The concentration of the neurofilament light chain in cerebrospinal fluid may reflect axonal damage or the extent of white matter damage. In this study we describe a sensitive immunoassay for the detection of neurofilament light

  11. Functional conservation of rice OsNF-YB/YC and Arabidopsis AtNF-YB/YC proteins in the regulation of flowering time

    KAUST Repository

    Hwang, Yoon-Hyung

    2016-01-11

    Plant NUCLEAR FACTOR Y (NF-Y) transcription factors play important roles in plant development and abiotic stress. In Arabidopsis thaliana, two NF-YB (AtNF-YB2 and AtNF-YB3) and five NF-YC (AtNF-YC1, AtNF-YC2, AtNF-YC3, AtNF-YC4, and AtNF-YC9) genes regulate photoperiodic flowering by interacting with other AtNF-Y subunit proteins. Three rice NF-YB (OsNF-YB8, OsNF-YB10, and OsNF-YB11) and five rice OsNF-YC (OsNF-YC1, OsNF-YC2, OsNF-YC4, OsNF-YC6, and OsNF-YC7) genes are clustered with two AtNF-YB and five AtNF-YC genes, respectively. To investigate the functional conservation of these NF-YB and NF-YC genes in rice and Arabidopsis, we analyzed the flowering phenotypes of transgenic plants overexpressing the respective OsNF-YB and OsNF-YC genes in Arabidopsis mutants. Overexpression of OsNF-YB8/10/11 and OsNF-YC2 complemented the late flowering phenotype of Arabidopsis nf-yb2 nf-yb3 and nf-yc3 nf-yc4 nf-yc9 mutants, respectively. The rescued phenotype of 35S::OsNF-YC2 nf-yc3 nf-yc4 nf-yc9 plants was attributed to the upregulation of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). In vitro and in planta protein–protein analyses revealed that OsNF-YB8/10/11 and OsNF-YC1/2/4/6/7 interact with AtNF-YC3/4/9 and AtNF-YB2/3, respectively. Our data indicate that some OsNF-YB and OsNF-YC genes are functional equivalents of AtNF-YB2/3 and AtNF-YC3/4/9 genes, respectively, and suggest functional conservation of Arabidopsis and rice NF-Y genes in the control of flowering time.

  12. Time-related morphometric studies of neurofilaments in brain contusions

    Directory of Open Access Journals (Sweden)

    Mariusz Kobek

    2016-03-01

    Full Text Available In forensic pathology age determination of injuries is of key importance. The purpose of the study was to analyze morphometrically changes in neurofilaments following the brain contusion and relate them to the length of the time of survival. To do this, the authors analyzed specimens of brains collected during medicolegal autopsies. According to the available literature, no such study involving material from deceased humans was conducted. The researched material was divided into nine subgroups (10 cases each according to the time of death of persons: immediately at the crime site, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days and 7 days after head trauma. Neurofilaments were immunohistochemically stained and evaluated quantitatively using the Met-Ilo computer application. The initial results were then analyzed statistically with the one way analysis of variance (ANOVA and the least significant difference (LSD tests. It was calculated that there are significant differences in numbers and area fractions of neurofilaments within 7 days after head trauma. It must be concluded that morphometric analysis of neurofilaments is a promising method but further studies are required.

  13. Riluzole protects against glutamate-induced slowing of neurofilament axonal transport.

    LENUS (Irish Health Repository)

    Stevenson, Alison

    2009-04-24

    Riluzole is the only drug approved for the treatment of amyotrophic lateral sclerosis (ALS) but its precise mode of action is not properly understood. Damage to axonal transport of neurofilaments is believed to be part of the pathogenic mechanism in ALS and this has been linked to defective glutamate handling and increased phosphorylation of neurofilament side-arm domains. Here, we show that riluzole protects against glutamate-induced slowing of neurofilament transport. Protection is associated with decreased neurofilament side-arm phosphorylation and inhibition of the activities of two neurofilament kinases, ERK and p38 that are activated in ALS. Thus, the anti-glutamatergic properties of riluzole include protection against glutamate-induced changes to neurofilament phosphorylation and transport.

  14. Neurofilament light protein in blood as a potential biomarker of neurodegeneration in Huntington's disease: a retrospective cohort analysis.

    Science.gov (United States)

    Byrne, Lauren M; Rodrigues, Filipe B; Blennow, Kaj; Durr, Alexandra; Leavitt, Blair R; Roos, Raymund A C; Scahill, Rachael I; Tabrizi, Sarah J; Zetterberg, Henrik; Langbehn, Douglas; Wild, Edward J

    2017-08-01

    Blood biomarkers of neuronal damage could facilitate clinical management of and therapeutic development for Huntington's disease. We investigated whether neurofilament light protein NfL (also known as NF-L) in blood is a potential prognostic marker of neurodegeneration in patients with Huntington's disease. We did a retrospective analysis of healthy controls and carriers of CAG expansion mutations in HTT participating in the 3-year international TRACK-HD study. We studied associations between NfL concentrations in plasma and clinical and MRI neuroimaging findings, namely cognitive function, motor function, and brain volume (global and regional). We used random effects models to analyse cross-sectional associations at each study visit and to assess changes from baseline, with and without adjustment for age and CAG repeat count. In an independent London-based cohort of 37 participants (23 HTT mutation carriers and 14 controls), we further assessed whether concentrations of NfL in plasma correlated with those in CSF. Baseline and follow-up plasma samples were available from 97 controls and 201 individuals carrying HTT mutations. Mean concentrations of NfL in plasma at baseline were significantly higher in HTT mutation carriers than in controls (3·63 [SD 0·54] log pg/mL vs 2·68 [0·52] log pg/mL, pdisease stage to the next. At any given timepoint, NfL concentrations in plasma correlated with clinical and MRI findings. In longitudinal analyses, baseline NfL concentration in plasma also correlated significantly with subsequent decline in cognition (symbol-digit modality test r=-0·374, pHuntington's disease, NfL concentration in plasma at baseline was associated with subsequent clinical onset during the 3-year follow-up period (hazard ratio 3·29 per log pg/mL, 95% CI 1·48-7·34, p=0·0036). Concentrations of NfL in CSF and plasma were correlated in mutation carriers (r=0·868, pdisease onset and progression in Huntington's disease. Medical Research Council, Glaxo

  15. p38alpha stress-activated protein kinase phosphorylates neurofilaments and is associated with neurofilament pathology in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Ackerley, Steven; Grierson, Andrew J; Banner, Steven; Perkinton, Michael S; Brownlees, Janet; Byers, Helen L; Ward, Malcolm; Thornhill, Paul; Hussain, Kader; Waby, Jennifer S; Anderton, Brian H; Cooper, Jonathan D; Dingwall, Colin; Leigh, P Nigel; Shaw, Christopher E; Miller, Christopher C J

    2004-06-01

    Neurofilament middle and heavy chains (NFM and NFH) are heavily phosphorylated on their carboxy-terminal side-arm domains in axons. The mechanisms that regulate this phosphorylation are complex. Here, we demonstrate that p38alpha, a member of the stress-activated protein kinase family, will phosphorylate NFM and NFH on their side-arm domains. Aberrant accumulations of neurofilaments containing phosphorylated NFM and NFH side-arms are a pathological feature of amyotrophic lateral sclerosis (ALS) and we also demonstrate that p38alpha and active forms of p38 family kinases are associated with these accumulations. This is the case for sporadic and familial forms of ALS and also in a transgenic mouse model of ALS caused by expression of mutant superoxide dismutase-1 (SOD1). Thus, p38 kinases may contribute to the aberrant phosphorylation of NFM and NFH side-arms in ALS. Copyright 2004 Elsevier Inc.

  16. A New Variant of Charcot-Marie-Tooth Disease Type 2 Is Probably the Result of a Mutation in the Neurofilament-Light Gene

    Science.gov (United States)

    Mersiyanova, Irina V.; Perepelov, Alexander V.; Polyakov, Alexander V.; Sitnikov, Vladimir F.; Dadali, Elena L.; Oparin, Roman B.; Petrin, Alexander N.; Evgrafov, Oleg V.

    2000-01-01

    Charcot-Marie-Tooth (CMT) disease is the most common inherited motor and sensory neuropathy. The axonal form of the disease is designated as “CMT type 2” (CMT2). Although four loci known to be implicated in autosomal dominant CMT2 have been mapped thus far (on 1p35-p36, 3q13.1, 3q13-q22, and 7p14), no one causative gene is yet known. A large Russian family with CMT2 was found in the Mordovian Republic (Russia). Affected members had the typical CMT2 phenotype. Additionally, several patients suffered from hyperkeratosis, although the association, if any, between the two disorders is not clear. Linkage with the CMT loci already known (CMT1A, CMT1B, CMT2A, CMT2B, CMT2D, and a number of other CMT-related loci) was excluded. Genomewide screening pinpointed the disease locus in this family to chromosome 8p21, within a 16-cM interval between markers D8S136 and D8S1769. A maximum two-point LOD score of 5.93 was yielded by a microsatellite from the 5′ region of the neurofilament-light gene (NF-L). Neurofilament proteins play an important role in axonal structure and are implicated in several neuronal disorders. Screening of affected family members for mutations in the NF-L gene and in the tightly linked neurofilament-medium gene (NF-M) revealed the only DNA alteration linked with the disease: a A998C transversion in the first exon of NF-L, which converts a conserved Gln333 amino acid to proline. This alteration was not found in 180 normal chromosomes. Twenty unrelated CMT2 patients, as well as 26 others with an undetermined form of CMT, also were screened for mutations in NF-L, but no additional mutations were found. It is suggested that Gln333Pro represents a rare disease-causing mutation, which results in the CMT2 phenotype. PMID:10841809

  17. Cloning of a cDNA encoding the smallest neurofilament protein from the rat

    NARCIS (Netherlands)

    J-P. Julien (Jean-Pierre); K. Ramachadran; F.G. Grosveld (Frank)

    1985-01-01

    textabstractWe have cloned a cDNA coding for the smallest rat neurofilament protein. The cDNA is 861 nucleotides long coding for 287 amino acids from the internal alpha-helical region and the carboxy-terminal tail domain of the neurofilament protein. Comparison of the porcine, mouse and rat

  18. Identification and characterization of NF-Y transcription factor families in Canola (Brassica napus L.).

    Science.gov (United States)

    Liang, Mingxiang; Yin, Xiangzhen; Lin, Zhongyuan; Zheng, Qingsong; Liu, Guohong; Zhao, Gengmao

    2014-01-01

    NF-Y (NUCLEAR FACTOR-Y), a heterotrimeric transcription factor, is composed of NF-YA, NF-YB, and NF-YC proteins in yeast, animal, and plant systems. In plants, each of the NF-YA/B/C subunit forms a multi-member family. NF-Ys are key regulators with important roles in many physiological processes, such as drought tolerance, flowering time, and seed development. In this study, we identified, annotated, and further characterized 14 NF-YA, 14 NF-YB, and 5 NF-YC proteins in Brassica napus (canola). Phylogenetic analysis revealed that the NF-YA/B/C subunits were more closely clustered with the Arabidopsis thaliana (Arabidopsis) homologs than with rice OsHAP2/3/5 subunits. Analyses of the conserved domain indicated that the BnNF-YA/B/C subfamilies, respectively, shared the same conserved domains with those in other organisms, including Homo sapiens, Saccharomyces cerevisiae, Arabidopsis, and Oryza sativa (rice). An examination of exon/intron structures revealed that most gene structures of BnNF-Y were similar to their homologs in Arabidopsis, a model dicot plant, but different from those in the model monocot plant rice, suggesting that plant NF-Ys diverged before monocot and dicot plants differentiated. Spatial-tempo expression patterns, as determined by qRT-PCR, showed that most BnNF-Ys were widely expressed in different tissues throughout the canola life cycle and that several closely related BnNF-Y subunits had similar expression profiles. Based on these findings, we predict that BnNF-Y proteins have functions that are conserved in the homologous proteins in other plants. This study provides the first extensive evaluation of the BnNF-Y family, and provides a useful foundation for dissecting the functions of BnNF-Y.

  19. Plasma neurofilament heavy chain levels correlate to markers of late stage disease progression and treatment response in SOD1(G93A mice that model ALS.

    Directory of Open Access Journals (Sweden)

    Ching-Hua Lu

    Full Text Available Amyotrophic lateral sclerosis (ALS is an incurable neurodegenerative disorder characterised by progressive degeneration of motor neurons leading to death, typically within 3-5 years of symptom onset. The diagnosis of ALS is largely reliant on clinical assessment and electrophysiological findings. Neither specific investigative tools nor reliable biomarkers are currently available to enable an early diagnosis or monitoring of disease progression, hindering the design of treatment trials.In this study, using the well-established SOD1(G93A mouse model of ALS and a new in-house ELISA method, we have validated that plasma neurofilament heavy chain protein (NfH levels correlate with both functional markers of late stage disease progression and treatment response. We detected a significant increase in plasma levels of phosphorylated NfH during disease progression in SOD1(G93A mice from 105 days onwards. Moreover, increased plasma NfH levels correlated with the decline in muscle force, motor unit survival and, more significantly, with the loss of spinal motor neurons in SOD1 mice during this critical period of decline. Importantly, mice treated with the disease modifying compound arimoclomol had lower plasma NfH levels, suggesting plasma NfH levels could be validated as an outcome measure for treatment trials.These results show that plasma NfH levels closely reflect later stages of disease progression and therapeutic response in the SOD1(G93A mouse model of ALS and may potentially be a valuable biomarker of later disease progression in ALS.

  20. The human neurofilament gene (NEFL) is located on the short arm of chromosome 8.

    NARCIS (Netherlands)

    J. Hurst; D. Flavell (David); J-P. Julien (Jean-Pierre); D.N. Meijer (Dies); W. Mushynski (Walter); F.G. Grosveld (Frank)

    1987-01-01

    textabstractWe have localized the gene coding for the human neurofilament light chain (NEFL) to chromosome band 8p2.1 by Southern blotting of DNA from hybrid cell panels and in situ hybridization to metaphase chromosomes.

  1. Role of Phosphorylated Neurofilament H as a diagnostic and prognostic marker in traumatic brain injury

    Directory of Open Access Journals (Sweden)

    Moh Omar Ghonemi

    2013-09-01

    Conclusion: Phosphorylated Neurofilament H can be used as a diagnostic and prognostic marker in patients with TBI as seen by the presence of significant correlations between the marker levels and different clinical and radiological tools.

  2. The Arabidopsis NF-YA3 and NF-YA8 genes are functionally redundant and are required in early embryogenesis.

    Directory of Open Access Journals (Sweden)

    Monica Fornari

    Full Text Available Nuclear factor Y (NF-Y is a trimeric transcription factor composed of three distinct subunits called NF-YA, NF-YB and NF-YC. In Arabidopsis thaliana, NF-Y subunits are known to play roles in many processes, such as gametogenesis, embryogenesis, seed development, drought resistance, ABA signaling, flowering time, primary root elongation, Endoplasmic Reticulum (ER stress response and blue light responses. Here, we report that the closely related NF-YA3 and NF-YA8 genes control early embryogenesis. Detailed GUS and in situ analyses showed that NF-YA3 and NF-YA8 are expressed in vegetative and reproductive tissues with the highest expression being during embryo development from the globular to the torpedo embryo stage. Plants from the nf-ya3 and nf-ya8 single mutants do not display any obvious phenotypic alteration, whereas nf-ya3 nf-ya8 double mutants are embryo lethal. Morphological analyses showed that the nf-ya3 nf-ya8 embryos fail to undergo to the heart stage and develop into abnormal globular embryos with both proembryo and suspensor characterized by a disordered cell cluster with an irregular shape, suggesting defects in embryo development. The suppression of both NF-YA3 and NF-YA8 gene expression by RNAi experiments resulted in defective embryos that phenocopied the nf-ya3 nf-ya8 double mutants, whereas complementation experiments partially rescued the abnormal globular nf-ya3 nf-ya8 embryos, confirming that NF-YA3 and NF-YA8 are required in early embryogenesis. Finally, the lack of GFP expression of the auxin responsive DR5rev::GFP marker line in double mutant embryos suggested that mutations in both NF-YA3 and NF-YA8 affect auxin response in early developing embryos. Our findings indicate that NF-YA3 and NF-YA8 are functionally redundant genes required in early embryogenesis of Arabidopsis thaliana.

  3. Overexpression of neurofilament H disrupts normal cell structure and function

    Science.gov (United States)

    Szebenyi, Gyorgyi; Smith, George M.; Li, Ping; Brady, Scott T.

    2002-01-01

    Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

  4. A hereditary spastic paraplegia mutation in kinesin-1A/KIF5A disrupts neurofilament transport

    Directory of Open Access Journals (Sweden)

    Brown Anthony

    2010-11-01

    Full Text Available Abstract Background Hereditary spastic paraplegias are a group of neurological disorders characterized by progressive distal degeneration of the longest ascending and descending axons in the spinal cord, leading to lower limb spasticity and weakness. One of the dominantly inherited forms of this disease (spastic gait type 10, or SPG10 is caused by point mutations in kinesin-1A (also known as KIF5A, which is thought to be an anterograde motor for neurofilaments. Results We investigated the effect of an SPG10 mutation in kinesin-1A (N256S-kinesin-1A on neurofilament transport in cultured mouse cortical neurons using live-cell fluorescent imaging. N256S-kinesin-1A decreased both anterograde and retrograde neurofilament transport flux by decreasing the frequency of anterograde and retrograde movements. Anterograde velocity was not affected, whereas retrograde velocity actually increased. Conclusions These data reveal subtle complexities to the functional interdependence of the anterograde and retrograde neurofilament motors and they also raise the possibility that anterograde and retrograde neurofilament transport may be disrupted in patients with SPG10.

  5. Neurofilaments in CSF as diagnostic biomarkers in motor neuron disease: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Dawei Li

    2016-11-01

    Full Text Available AbstractObjective: Neurofilaments in CSF are promising biomarkers which might help in the diagnosis of motor neuron disease (MND. We aim to assess the diagnostic value of neurofilaments in CSF for MND.Methods: Pubmed, Emabase and Web of Science were searched for relevant studies systematically. Articles in English that evaluated the utility of neurofilaments in CSF in the diagnosis of MND were included. Data were extracted by two independent investigators. Diagnostic indexes for neurofilament light chain (NFL and phosphorylated neurofilament heavy chain (pNFH were calculated separately. Stata 12.0 software with a bivariate mixed-effects model was used to summarize the diagnostic indexes from eligible studies.Results: Five studies on NFL and eight studies on pNFH met inclusion criteria. For NFL, the pooled sensitivity and specificity were 81% (95% confidence interval CI, 72%-88% and 85% (95%CI, 76%-91%, respectively; the positive likelihood ratio (PLR and negative likelihood ratio (NLR were 5.5 (95%CI, 3.1-9.8 and 0.22 (95%CI, 0.14-0.35, respectively; the summary diagnostic odds ratio (DOR was 25 (95%CI, 9-70, and the area under summary receiver operator characteristic curve (AUC was 0.90 (95%CI, 0.87-0.92. For pNFH, the pooled sensitivity, specificity, PLR and NLR were 85% (95% CI, 80%-88%, 85% (95%CI, 77%-90%, 5.5 (95%CI, 3.6-8.4 and 0.18 (95%CI, 0.13-0.25 respectively; the DOR was 30 (95%CI, 16-58, and the AUC was 0.91 (95%CI, 0.88-0.93.Conclusion: Neurofilaments in CSF have a high value in the diagnosis of MND, though the optimal cutoff value remains to be further investigated.

  6. Increased expressions of ADAMTS-13, neuronal nitric oxide synthase, and neurofilament correlate with severity of neuropathology in Border disease virus-infected small ruminants.

    Directory of Open Access Journals (Sweden)

    Gungor Cagdas Dincel

    Full Text Available Border Disease (BD, caused by Pestivirus from the family Flaviviridae, leads to serious reproductive losses and brain anomalies such as hydranencephaly and cerebellar hypoplasia in aborted fetuses and neonatal lambs. In this report it is aimed to investigate the expression of neuronal nitric oxide synthase (nNOS, A Disintegrin And Metalloprotease with Thrombospondin type I repeats-13 (ADAMTS-13, and neurofilament (NF in the brain tissue in small ruminants infected with Border Disease Virus (BDV and to identify any correlation between hypomyelinogenesis and BD neuropathology. Results of the study revealed that the levels of ADAMTS-13 (p<0.05, nNOS (p<0.05, and NF (p<0.05 were remarkably higher in BDV-infected brain tissue than in the uninfected control. It was suggested that L-arginine-NO synthase pathway is activated after infection by BDV and that the expression of NF and nNOS is associated with the severity of BD. A few studies have focused on ADAMTS-13 expression in the central nervous system, and its function continues to remain unclear. The most prominent finding from our study was that ADAMTS-13, which contain two CUB domains, has two CUB domains and its high expression levels are probably associated with the development of the central nervous system (CNS. The results also clearly indicate that the interaction of ADAMTS-13 and NO may play an important role in the regulation and protection of the CNS microenvironment in neurodegenerative diseases. In addition, NF expression might indicate the progress of the disease. To the best of the authors'knowledge, this is the first report on ADAMTS-13 expression in the CNS of BDV-infected small ruminants.

  7. Neurofilament light chain and oligoclonal bands are prognostic biomarkers in radiologically isolated syndrome.

    Science.gov (United States)

    Matute-Blanch, Clara; Villar, Luisa M; Álvarez-Cermeño, José C; Rejdak, Konrad; Evdoshenko, Evgeniy; Makshakov, Gleb; Nazarov, Vladimir; Lapin, Sergey; Midaglia, Luciana; Vidal-Jordana, Angela; Drulovic, Jelena; García-Merino, Antonio; Sánchez-López, Antonio J; Havrdova, Eva; Saiz, Albert; Llufriu, Sara; Alvarez-Lafuente, Roberto; Schroeder, Ina; Zettl, Uwe K; Galimberti, Daniela; Ramió-Torrentà, Lluís; Robles, René; Quintana, Ester; Hegen, Harald; Deisenhammer, Florian; Río, Jordi; Tintoré, Mar; Sánchez, Alex; Montalban, Xavier; Comabella, Manuel

    2018-02-14

    The prognostic role of cerebrospinal fluid molecular biomarkers determined in early pathogenic stages of multiple sclerosis has yet to be defined. In the present study, we aimed to investigate the prognostic value of chitinase 3 like 1 (CHI3L1), neurofilament light chain, and oligoclonal bands for conversion to clinically isolated syndrome and to multiple sclerosis in 75 patients with radiologically isolated syndrome. Cerebrospinal fluid levels of CHI3L1 and neurofilament light chain were measured by enzyme-linked immunosorbent assay. Uni- and multivariable Cox regression models including as covariates age at diagnosis of radiologically isolated syndrome, number of brain lesions, sex and treatment were used to investigate associations between cerebrospinal fluid CHI3L1 and neurofilament light chain levels and time to conversion to clinically isolated syndrome and multiple sclerosis. Neurofilament light chain levels and oligoclonal bands were independent risk factors for the development of clinically isolated syndrome (hazard ratio = 1.02, P = 0.019, and hazard ratio = 14.7, P = 0.012, respectively) and multiple sclerosis (hazard ratio = 1.03, P = 0.003, and hazard ratio = 8.9, P = 0.046, respectively). The best cut-off to classify cerebrospinal fluid neurofilament light chain levels into high and low was 619 ng/l, and high neurofilament light chain levels were associated with a trend to shorter time to clinically isolated syndrome (P = 0.079) and significant shorter time to multiple sclerosis (P = 0.017). Similarly, patients with radiologically isolated syndrome presenting positive oligoclonal bands converted faster to clinically isolated syndrome and multiple sclerosis (P = 0.005 and P = 0.008, respectively). The effects of high neurofilament light chain levels shortening time to clinically isolated syndrome and multiple sclerosis were more pronounced in radiologically isolated syndrome patients with ≥37 years compared to younger patients. Cerebrospinal fluid

  8. OsNF-YC2 and OsNF-YC4 proteins inhibit flowering under long-day conditions in rice

    KAUST Repository

    Kim, SoonKap

    2015-11-05

    OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response through the modulation of three flowering-time genes ( Ehd1, Hd3a , and RFT1 ) in rice. Plant NUCLEAR FACTOR Y (NF-Y) transcription factors control numerous developmental processes by forming heterotrimeric complexes, but little is known about their roles in flowering in rice. In this study, it is shown that some subunits of OsNF-YB and OsNF-YC interact with each other, and among them, OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response of rice. Protein interaction studies showed that the physical interactions occurred between the three OsNF-YC proteins (OsNF-YC2, OsNF-YC4 and OsNF-YC6) and three OsNF-YB proteins (OsNF-YB8, OsNF-YB10 and OsNF-YB11). Repression and overexpression of the OsNF-YC2 and OsNF-YC4 genes revealed that they act as inhibitors of flowering only under long-day (LD) conditions. Overexpression of OsNF-YC6, however, promoted flowering only under LD conditions, suggesting it could function as a flowering promoter. These phenotypes correlated with the changes in the expression of three rice flowering-time genes [Early heading date 1 (Ehd1), Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1)]. The diurnal and tissue-specific expression patterns of the subsets of OsNF-YB and OsNF-YC genes were similar to those of CCT domain encoding genes such as OsCO3, Heading date 1 (Hd1) and Ghd7. We propose that OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response by interacting directly with OsNF-YB8, OsNF-YB10 or OsNF-YB11 proteins in rice.

  9. OsNF-YC2 and OsNF-YC4 proteins inhibit flowering under long-day conditions in rice.

    Science.gov (United States)

    Kim, Soon-Kap; Park, Hyo-Young; Jang, Yun Hee; Lee, Keh Chien; Chung, Young Soo; Lee, Jeong Hwan; Kim, Jeong-Kook

    2016-03-01

    OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response through the modulation of three flowering-time genes ( Ehd1, Hd3a , and RFT1 ) in rice. Plant NUCLEAR FACTOR Y (NF-Y) transcription factors control numerous developmental processes by forming heterotrimeric complexes, but little is known about their roles in flowering in rice. In this study, it is shown that some subunits of OsNF-YB and OsNF-YC interact with each other, and among them, OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response of rice. Protein interaction studies showed that the physical interactions occurred between the three OsNF-YC proteins (OsNF-YC2, OsNF-YC4 and OsNF-YC6) and three OsNF-YB proteins (OsNF-YB8, OsNF-YB10 and OsNF-YB11). Repression and overexpression of the OsNF-YC2 and OsNF-YC4 genes revealed that they act as inhibitors of flowering only under long-day (LD) conditions. Overexpression of OsNF-YC6, however, promoted flowering only under LD conditions, suggesting it could function as a flowering promoter. These phenotypes correlated with the changes in the expression of three rice flowering-time genes [Early heading date 1 (Ehd1), Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1)]. The diurnal and tissue-specific expression patterns of the subsets of OsNF-YB and OsNF-YC genes were similar to those of CCT domain encoding genes such as OsCO3, Heading date 1 (Hd1) and Ghd7. We propose that OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response by interacting directly with OsNF-YB8, OsNF-YB10 or OsNF-YB11 proteins in rice.

  10. Arabidopsis transcription factor genes NF-YA1, 5, 6, and 9 play redundant roles in male gametogenesis, embryogenesis, and seed development.

    Science.gov (United States)

    Mu, Jinye; Tan, Helin; Hong, Sulei; Liang, Yan; Zuo, Jianru

    2013-01-01

    Nuclear factor Y (NF-Y) is a highly conserved transcription factor presented in all eukaryotic organisms, and is a heterotrimer consisting of three subunits: NF-YA, NF-YB, and NF-YC. In Arabidopsis, these three subunits are encoded by multigene families. The best-studied member of the NF-Y transcription factors is LEAFY COTYLEDON1 (LEC1), a NF-YB family member, which plays a critical role in embryogenesis and seed maturation. However, the function of most NF-Y genes remains elusive. Here, we report the characterization of four genes in the NF-YA family. We found that a gain-of-function mutant of NF-YA1 showed defects in male gametogenesis and embryogenesis. Consistently, overexpression of NF-YA1, 5, 6, and 9 affects male gametogenesis, embryogenesis, seed morphology, and seed germination, with a stronger phenotype when overexpressing NF-YA1 and NF-YA9. Moreover, overexpression of these NF-YA genes also causes hypersensitivity to abscisic acid (ABA) during seed germination, retarded seedling growth, and late flowering at different degrees. Intriguingly, overexpression of NF-YA1, 5, 6, and 9 is sufficient to induce the formation of somatic embryos from the vegetative tissues. However, single or double mutants of these NF-YA genes do not have detectable phenotype. Collectively, these results provide evidence that NF-YA1, 5, 6, and 9 play redundant roles in male gametophyte development, embryogenesis, seed development, and post-germinative growth.

  11. Neurofilament-tubulin binding site peptide NFL-TBS.40-63 increases the differentiation of oligodendrocytes in vitro and partially prevents them from lysophosphatidyl choline toxiciy.

    Science.gov (United States)

    Fressinaud, Catherine; Eyer, Joël

    2014-02-01

    During multiple sclerosis (MS), the main axon cystoskeleton proteins, neurofilaments (NF), are altered, and their release into the cerebrospinal fluid correlates with disease severity. The role of NF in the extraaxonal location is unknown. Therefore, we tested whether synthetic peptides corresponding to the tubulin-binding site (TBS) sequence identified on light NF chain (NFL-TBS.40-63) and keratin (KER-TBS.1-24), which could be released during MS, modulate remyelination in vitro. Biotinylated NFL-TBS.40-63, NFL-Scramble2, and KER-TBS.1-54 (1-100 μM, 24 hr) were added to rat oligodendrocyte (OL) and astrocyte (AS) cultures, grown in chemically defined medium. Proliferation and differentiation were characterized by using specific antibodies (A2B5, CNP, MBP, GFAP) and compared with untreated cultures. Lysophosphatidyl choline (LPC; 2 × 10(-5) M) was used to induce OL death and to test the effects of TBS peptides under these conditions. NFL-TBS.40-63 significantly increased OL differentiation and maturation, with more CNP(+) and MBP(+) cells characterized by numerous ramified processes, along with myelin balls. When OL were challenged with LPC, concomitant treatment with NFL-TBS.40-63 rescued more than 50% of OL compared with cultures treated with LPC only. Proliferation of OL progenitors was not affected, nor were AS proliferation and differentiation. NFL-TBS.40-63 peptide induces specific effects in vitro, increasing OL differentiation and maturation without altering AS fate. In addition, it partially protects OL from demyelinating injury. Thus release of NFL-TBS.40-63 caused by axonal damage in vivo could improve repair through increased OL differentiation, which is a prerequisite for remyelination. Copyright © 2013 Wiley Periodicals, Inc.

  12. Interactions of the CCAAT-binding trimer NF-Y with nucleosomes.

    Science.gov (United States)

    Motta, M C; Caretti, G; Badaracco, G F; Mantovani, R

    1999-01-15

    NF-Y is a sequence-specific evolutionary conserved activator binding to CCAAT boxes with high affinity and specificity. It is a trimer formed by NF-YA and two putative histone-like subunits, NF-YB and NF-YC, showing similarity to histones H2B and H2A, respectively. We investigated the relationships between NF-Y and chromatin using an Artemia franciscana chromatin assembly system with plasmids containing the Major HistoCompatibility complex class II Ea promoter. The NF-Y trimer, but not single subunits, protects the Y box in the presence of reconstituted chromatin, and it can bind the target sequence during and after assembly. Using reconstitution assays with purified chicken histones, we show that NF-Y associates with preformed nucleosomes. Translational analysis of various Ea fragments of identical length in which the CCAAT box is at different positions indicated that the lateral fragment was slightly more prone to NF-Y binding. In competition experiments, NF-Y is able to prevent formation of nucleosomes significantly. These data support the idea that NF-Y is a gene-specific activator with a built-in capacity to interface with chromatin structures.

  13. Plant NF-Y transcription factors: Key players in plant-microbe interactions, root development and adaptation to stress.

    Science.gov (United States)

    Zanetti, María Eugenia; Rípodas, Carolina; Niebel, Andreas

    2017-05-01

    NF-Ys are heterotrimeric transcription factors composed by the NF-YA, NF-YB and NF-YC subunits. In plants, NF-Y subunits are encoded by multigene families whose members show structural and functional diversifications. An increasing number of NF-Y genes has been shown to play key roles during different stages of root nodule and arbuscular mycorrhizal symbiosis, as well as during the interaction of plants with pathogenic microorganisms. Individual members of the NF-YA and NF-YB families have also been implicated in the development of primary and lateral roots. In addition, different members of the NF-YA and NF-YB gene families from mono- and di-cotyledonous plants have been involved in plant responses to water and nutrient scarcity. This review presents the most relevant and striking results concerning these NF-Y subunits. A phylogenetic analysis of the functionally characterized NF-Y genes revealed that, across plant species, NF-Y proteins functioning in the same biological process tend to belong to common phylogenetic groups. Finally, we discuss the forthcoming challenges of plant NF-Y research, including the detailed dissection of expression patterns, the elucidation of functional specificities as well as the characterization of the potential NF-Y-mediated epigenetic mechanisms by which they control the expression of their target genes. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Genome-wide identification and characterization of the NF-Y gene family in grape (vitis vinifera L.).

    Science.gov (United States)

    Ren, Chong; Zhang, Zhan; Wang, Yi; Li, Shaohua; Liang, Zhenchang

    2016-08-11

    Nuclear factor Y (NF-Y) transcription factor is composed of three distinct subunits: NF-YA, NF-YB and NF-YC. Many members of NF-Y family have been reported to be key regulators in plant development, phytohormone signaling and drought tolerance. However, the function of the NF-Y family is less known in grape (Vitis vinifera L.). A total of 34 grape NF-Y genes that distributed unevenly on grape (V. vinifera) chromosomes were identified in this study. Phylogenetic analysis was performed to predict functional similarities between Arabidopsis thaliana and grape NF-Y genes. Comparison of the structures of grape NF-Y genes (VvNF-Ys) revealed their functional conservation and alteration. Furthermore, we investigated the expression profiles of VvNF-Ys in response to various stresses, phytohormone treatments, and in leaves and grape berries with various sugar contents at different developmental stages. The relationship between VvNF-Y transcript levels and sugar content was examined to select candidates for exogenous sugar treatments. Quantitative real-time PCR (qPCR) indicated that many VvNF-Ys responded to different sugar stimuli with variations in transcript abundance. qPCR and publicly available microarray data suggest that VvNF-Ys exhibit distinct expression patterns in different grape organs and developmental stages, and a number of VvNF-Ys may participate in responses to multiple abiotic and biotic stresses, phytohormone treatments and sugar accumulation or metabolism. In this study, we characterized 34 VvNF-Ys based on their distributions on chromosomes, gene structures, phylogenetic relationship with Arabidopsis NF-Y genes, and their expression patterns. The potential roles of VvNF-Ys in sugar accumulation or metabolism were also investigated. Altogether, the data provide significant insights on VvNF-Ys, and lay foundations for further functional studies of NF-Y genes in grape.

  15. Axonal loss and neurofilament phosphorylation changes accompany lesion development and clinical progression in multiple sclerosis.

    Science.gov (United States)

    Schirmer, Lucas; Antel, Jack P; Brück, Wolfgang; Stadelmann, Christine

    2011-07-01

    Neuroaxonal damage and loss are increasingly recognized as disability determining features in multiple sclerosis (MS) pathology. However, little is known about the long-term sequelae of inflammatory demyelination on neurons and axons. Spinal cord tissue of 31 MS patients was compared to three amyotrophic lateral sclerosis (ALS) and 10 control subjects. MS lesions were staged according to the density of KiM-1P positive macrophages and microglia and the presence of myelin basic protein (MBP) positive phagocytes. T cells were quantified in the parenchyma and meninges. Neuroaxonal changes were studied by immunoreactivity (IR) for amyloid precursor protein (APP) and variably phosphorylated neurofilaments (SMI312, SMI31, SMI32). Little T cell infiltration was still evident in chronic inactive lesions. The loss of SMI32 IR in ventral horn neurons correlated with MS lesion development and disease progression. Similarly, axonal loss in white matter (WM) lesions correlated with disease duration. A selective reduction of axonal phosphorylated neurofilaments (SMI31) was observed in WM lesions. In ALS, the loss of neuronal SMI32 IR was even more pronounced, whereas the relative axonal reduction resembled that found in MS. Progressive neuroaxonal neurofilament alterations in the context of chronic inflammatory demyelination may reflect changes in neuroaxonal metabolism and result in chronic neuroaxonal dysfunction as a putative substrate of clinical progression. © 2011 The Authors; Brain Pathology © 2011 International Society of Neuropathology.

  16. Morphology of mouse sinoatrial node and its expression of NF-160 and HCN4

    Science.gov (United States)

    Wen, Yu; Li, Bin

    2015-01-01

    Objective: To explore the morphology and cell component of the sinoatrial node in adult mice and to observe the expressions of neurofilament protein (NF-160) and hyperpolarization-activated cyclic nucleotide regulation of cation channel 4 (HCN4) in sinus node cells. Methods: The right and left atria were collected from 10 adult mice. After paraffin embedding and serial section, these sections underwent Masson trichrome staining and NF-160 immunohistochemical staining, respectively, followed by observation under a light microscope. The right and left atria from another 5 mice were used for immunofluorescence staining using NF-160 and HCN4 antibodies, respectively, followed by observation under a confocal microscope. Results: The mouse sinoatrial node was close to the superior vena cava and below the epicardium. The shape of the mouse sinoatrial node was ovoid or irregular in the horizontal sections. We clearly saw a sinus node artery passing through the sinoatrial node, so the artery can be used as a location marker of the sinoatrial node. Immunofluorescence displayed the expression of NF-160 and HCN4 in both pacemaker cells and transitional cells of the sinoatrial node. Finally, sinoatrial node migrated along with Purkinje fibers of the right atrium. Conclusion: The position of mouse sinoatrial node is more stable. The distal end of sinoatrial node extends to the right subendocardial layer and migrates with Purkinje fibers. In mouse sinoatrial node, both pacemaker cells and transitional cells are regularly distributed, and all have HCN4 and NF-160 expression. PMID:26550270

  17. The overexpression of an Amaranthus hypochondriacus NF-YC gene modifies growth and confers water deficit stress resistance in Arabidopsis.

    Science.gov (United States)

    Palmeros-Suárez, Paola A; Massange-Sánchez, Julio A; Martínez-Gallardo, Norma A; Montero-Vargas, Josaphat M; Gómez-Leyva, Juan F; Délano-Frier, John P

    2015-11-01

    Nuclear factor-Y (NF-Y), is a plant heterotrimeric transcription factor constituted by NF-YA, NF-YB and NF-YC subunits. The function of many NF-Y subunits, mostly of the A and B type, has been studied in plants, but knowledge regarding the C subunit remains fragmentary. Here, a water stress-induced NF-YC gene from Amaranthus hypochondriacus (AhNF-YC) was further characterized by its overexpression in transgenic Arabidospis thaliana plants. A role in development was inferred from modified growth rates in root, rosettes and inflorescences recorded in AhNF-YC overexpressing Arabidopsis plants, in addition to a delayed onset of flowering. Also, the overexpression of AhNF-YC caused increased seedling sensitivity to abscisic acid (ABA), and influenced the expression of several genes involved in secondary metabolism, development and ABA-related responses. An altered expression of the latter in water stressed and recovered transgenic plants, together with the observed increase in ABA sensitivity, suggested that their increased water stress resistance was partly ABA-dependent. An untargeted metabolomic analysis also revealed an altered metabolite pattern, both in normal and water stress/recovery conditions. These results suggest that AhNF-YC may play an important regulatory role in both development and stress, and represents a candidate gene for the engineering of abiotic stress resistance in commercial crops. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Identification and characterization of NF-YB family genes in tung tree.

    Science.gov (United States)

    Yang, Susu; Wang, Yangdong; Yin, Hengfu; Guo, Haobo; Gao, Ming; Zhu, Huiping; Chen, Yicun

    2015-12-01

    The NF-YB transcription factor gene family encodes a subunit of the CCAAT box-binding factor (CBF), a highly conserved trimeric activator that strongly binds to the CCAAT box promoter element. Studies on model plants have shown that NF-YB proteins participate in important developmental and physiological processes, but little is known about NF-YB proteins in trees. Here, we identified seven NF-YB transcription factor-encoding genes in Vernicia fordii, an important oilseed tree in China. A phylogenetic analysis separated the genes into two groups; non-LEC1 type (VfNF-YB1, 5, 7, 9, 11, 13) and LEC1-type (VfNF-YB 14). A gene structure analysis showed that VfNF-YB 5 has three introns and the other genes have no introns. The seven VfNF-YB sequences contain highly conserved domains, a disordered region at the N terminus, and two long helix structures at the C terminus. Phylogenetic analyses showed that VfNF-YB family genes are highly homologous to GmNF-YB genes, and many of them are closely related to functionally characterized NF-YBs. In expression analyses of various tissues (root, stem, leaf, and kernel) and the root during pathogen infection, VfNF-YB1, 5, and 11 were dominantly expressed in kernels, and VfNF-YB7 and 9 were expressed only in the root. Different VfNF-YB family genes showed different responses to pathogen infection, suggesting that they play different roles in the pathogen response. Together, these findings represent the first extensive evaluation of the NF-YB family in tung tree and provide a foundation for dissecting the functions of VfNF-YB genes in seed development, stress adaption, fatty acid synthesis, and pathogen response.

  19. NF-κB inhibition delays DNA damage–induced senescence and aging in mice

    Science.gov (United States)

    Tilstra, Jeremy S.; Robinson, Andria R.; Wang, Jin; Gregg, Siobhán Q.; Clauson, Cheryl L.; Reay, Daniel P.; Nasto, Luigi A.; St Croix, Claudette M.; Usas, Arvydas; Vo, Nam; Huard, Johnny; Clemens, Paula R.; Stolz, Donna B.; Guttridge, Denis C.; Watkins, Simon C.; Garinis, George A.; Wang, Yinsheng; Niedernhofer, Laura J.; Robbins, Paul D.

    2012-01-01

    The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB–activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging. PMID:22706308

  20. Activation of the niacin receptor HCA2 reduces demyelination and neurofilament loss, and promotes functional recovery after spinal cord injury in mice.

    Science.gov (United States)

    Yang, Ruilin; He, Jiyong; Wang, Yuliang

    2016-11-15

    After spinal cord injury (SCI), there is an acute phase of alternatively activated (M2) macrophage infiltration, followed by a long-lasting phase of classically activated (M1) macrophage accumulation in the wound, which is believed to derail healing and compromize organ functions. Thus, agents which are able to modulate macrophage phenotypes may provide significant benefits to SCI patients. In the present study, we demonstrate that the niacin receptor HCA2 is specifically expressed on the cell surface of M1 but not M2 macrophages. Treatment of M1 macrophages with niacin (300μM) resulted in down-regulation of the p65 NF-κB phosphorylation, associated with a marked decrease in the levels of M1 markers, including CD86, IL-12, and IL-6, and a significant increase in the expressions of M2 markers, such as CD206, IL-10, and IL-13, suggesting that niacin causes a shift of M1 to M2. Moreover, treatment of the M1-oligodendrocyte precursor cell (OPC) co-cultures with niacin markedly promoted the expression of myelin binding protein (MBP). After SCI in C57/BL6 mice for a week, a marked accumulation of M1 macrophages, which expressed HCA2 receptor, was evident in the wound. Treatment of the SCI mice with niacin (100mg/kg) resulted in a dramatic decrease in the number of M1 macrophages and a significant increase in the number of M2 macrophages in the wound. This was associated with a robust inflammation resolution, attenuation of demyelination and neurofilament loss, and significant improvement of locomotor function. Thus, HCA2 receptor may serve as a therapeutic target to promote post-SCI recovery. Copyright © 2016. Published by Elsevier B.V.

  1. Neonatal High Bone Mass With First Mutation Of The NF-κB Complex

    DEFF Research Database (Denmark)

    Frederiksen, Anja Lisbeth; Larsen, Martin Jakob; Brusgaard, Klaus

    2016-01-01

    from in utero increased OB function rather than failed OC action. These findings demonstrate the importance of the Rela/p65 subunit within the NF-κB pathway for human skeletal homeostasis and represent a new genetic cause of HBM. © 2015 American Society for Bone and Mineral Research....

  2. NF-κB p65 recruited SHP regulates PDCD5-mediated apoptosis in cancer cells.

    Science.gov (United States)

    Murshed, Farhan; Farhana, Lulu; Dawson, Marcia I; Fontana, Joseph A

    2014-03-01

    Transcription factor NF-κB promotes cell proliferation in response to cell injury. Increasing evidence, however, suggests that NF-κB can also play an apoptotic role depending on the stimulus and cell type. We have previously demonstrated that novel retinoid 4-[3-Cl-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC)-mediated apoptosis in breast carcinoma cells requires activation of canonical and non-canonical NF-κB pathways. The mechanism NF-κB uses to induce apoptosis remains largely unknown. NF-κB subunit p65 (RelA) was identified as one potent transcriptional activator in 3-Cl-AHPC-mediated apoptosis in cells. Here we used ChIP-on-chip to identify NF-κB p65 genes activated in 3-Cl-AHPC mediated apoptosis. This paper focuses on one hit: pro-apoptotic protein programmed cell death 5 (PDCD5). 3-Cl-AHPC mediated apoptosis in MDA-MB-468 had three related effects on PDCD5: NF-κB p65 binding to the PDCD5 gene, enhanced PDCD5 promoter activity, and increased PDCD5 protein expression. Furthermore, 3-Cl-AHPC increased orphan nuclear receptor small heterodimer partner (SHP) mRNA expression, increased SHP protein bound to NF-κB p65, and found the SHP/NF-κB p65 complex attached to the PDCD5 gene. PDCD5 triggered apoptosis through increased Bax protein and release of cytochrome C from mitochondria to cytosol. Lastly, knockdown of PDCD5 protein expression blocked 3-Cl-AHPC mediated apoptosis, while over-expression of PDCD5 enhanced apoptosis, suggesting PDCD5 is necessary and sufficient for NF-κB p65 mediated apoptosis. Our results demonstrate a novel pathway for NF-κB p65 in regulating apoptosis through SHP and PDCD5.

  3. Increased CSF levels of phosphorylated neurofilament heavy protein following bout in amateur boxers.

    Directory of Open Access Journals (Sweden)

    Sanna Neselius

    Full Text Available INTRODUCTION: Diagnosis of mild TBI is hampered by the lack of imaging or biochemical measurements for identifying or quantifying mild TBI in a clinical setting. We have previously shown increased biomarker levels of protein reflecting axonal (neurofilament light protein and tau and glial (GFAP and S-100B damage in cerebrospinal fluid (CSF after a boxing bout. The aims of this study were to find other biomarkers of mild TBI, which may help clinicians diagnose and monitor mild TBI, and to calculate the role of APOE ε4 allele genotype which has been associated with poor outcome after TBI. MATERIALS AND METHODS: Thirty amateur boxers with a minimum of 45 bouts and 25 non-boxing matched controls were included in a prospective cohort study. CSF and blood were collected at one occasion between 1 and 6 days after a bout, and after a rest period for at least 14 days (follow up. The controls were tested once. CSF levels of neurofilament heavy (pNFH, amyloid precursor proteins (sAPPα and sAPPβ, ApoE and ApoA1 were analyzed. In blood, plasma levels of Aβ42 and ApoE genotype were analyzed. RESULTS: CSF levels of pNFH were significantly increased between 1 and 6 days after boxing as compared with controls (p<0.001. The concentrations decreased at follow up but were still significantly increased compared to controls (p = 0.018. CSF pNFH concentrations correlated with NFL (r =  0.57 after bout and 0.64 at follow up, p<0.001. No significant change was found in the other biomarkers, as compared to controls. Boxers carrying the APOE ε4 allele had similar biomarker concentrations as non-carriers. CONCLUSIONS: Subconcussive repetitive trauma in amateur boxing causes a mild TBI that may be diagnosed by CSF analysis of pNFH, even without unconsciousness or concussion symptoms. Possession of the APOE ε4 allele was not found to influence biomarker levels after acute TBI.

  4. Cerebrospinal fluid neurofilament light chain levels predict visual outcome after optic neuritis

    DEFF Research Database (Denmark)

    Modvig, Signe; Degn, M; Sander, B

    2016-01-01

    -L), myelin basic protein, osteopontin and chitinase-3-like-1) predict visual outcome after optic neuritis. METHODS: We included 47 patients with optic neuritis as a first demyelinating episode. Patients underwent visual tests, optical coherence tomography (OCT), magnetic resonance imaging (MRI) and lumbar......-L, β=-1.1, p=0.0150 for GC-IPL). Complete/incomplete remission was determined based on LCVA from 30 healthy controls. NF-L had a positive predictive value of 91% and an area under the curve (AUC) of 0.79 for incomplete remission. CONCLUSION: CSF NF-L is a promising biomarker of visual outcome after...

  5. Combination of neurofilament heavy chain and complement c3 as CSF biomarkers for ALS

    Science.gov (United States)

    Ganesalingam, Jeban; An, Jiyan; Shaw, Christopher E; Shaw, Gerry; Lacomis, David; Bowser, Robert

    2011-01-01

    Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease with an average survival of 3 years from symptom onset. Rapid and conclusive early diagnosis is essential if interventions with disease-modifying therapies are to be successful. Cytoskeletal modification and inflammation are known to occur during the pathogenesis of ALS. We measured levels of cytoskeletal proteins and inflammatory markers in the cerebrospinal fluid (CSF) of ALS, disease controls and healthy subjects. We determined threshold values for each protein that provided the optimal sensitivity and specificity for ALS within a training set, as determined by receiver operating characteristic (ROC) analysis. Interestingly, the optimal assay was a ratio of the levels for phosphorylated neurofilament heavy chain and complement C3 (pNFH/C3). We next applied this assay to a separate test set of CSF samples to verify our results. Overall, the predictive pNFH/C3 ratio identified ALS with 87.3% sensitivity and 94.6% specificity in a total of 71 ALS subjects, 52 disease control subjects and 40 healthy subjects. In addition, the level of CSF pNFH correlated with survival of ALS patients. We also detected increased pNFH in the plasma of ALS patients and observed a correlation between CSF and plasma pNFH levels within the same subjects. These findings support large-scale prospective biomarker studies to determine the clinical utility of diagnostic and prognostic signatures in ALS. PMID:21418221

  6. Association of Plasma Neurofilament Light With Neurodegeneration in Patients With Alzheimer Disease.

    Science.gov (United States)

    Mattsson, Niklas; Andreasson, Ulf; Zetterberg, Henrik; Blennow, Kaj

    2017-05-01

    Existing cerebrospinal fluid (CSF) or imaging (tau positron emission tomography) biomarkers for Alzheimer disease (AD) are invasive or expensive. Biomarkers based on standard blood test results would be useful in research, drug development, and clinical practice. Plasma neurofilament light (NFL) has recently been proposed as a blood-based biomarker for neurodegeneration in dementias. To test whether plasma NFL concentrations are increased in AD and associated with cognitive decline, other AD biomarkers, and imaging evidence of neurodegeneration. In this prospective case-control study, an ultrasensitive assay was used to measure plasma NFL concentration in 193 cognitively healthy controls, 197 patients with mild cognitive impairment (MCI), and 180 patients with AD dementia from the Alzheimer's Disease Neuroimaging Initiative. The study dates were September 7, 2005, to February 13, 2012. The plasma NFL analysis was performed in September 2016. Associations were tested between plasma NFL and diagnosis, Aβ pathologic features, CSF biomarkers of neuronal injury, cognition, brain structure, and metabolism. Among 193 cognitively healthy controls, 197 patients with mild cognitive impairment, and 180 patients with AD with dementia, plasma NFL correlated with CSF NFL (Spearman ρ = 0.59, P disease. This finding implies a potential usefulness for plasma NFL as a noninvasive biomarker in AD.

  7. Inhibition of NF-κB promotes autophagy via JNK signaling pathway in porcine granulosa cells

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    Gao, Hui; Lin, Lu; Haq, Ihtesham Ul; Zeng, Shen-ming, E-mail: zengshenming@gmail.com

    2016-04-22

    The transcription factor nuclear factor-κB (NF-κB) plays an important role in diverse processes, including cell proliferation and differentiation, apoptosis and inflammation. However, the role of NF-κB in porcine follicle development is not clearly elucidated. In this study, we demonstrated that follicle stimulating hormone (FSH) increased the level of inhibitor of NF-κB (IκB) protein and promoted the cytoplasmic localization of p65, indicating that FSH inhibits the activation of NF-κB in porcine granulosa cells. Moreover, inhibition of NF-κB by FSH or another specific inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), could activate JNK signaling and enhance autophagic activity in porcine granulosa cells. Knockdown of RelA (p65) Subunit of NF-κB by RNA interference abrogated the activation of JNK signaling pathway and the increase of autophagic protein expression by FSH. Meanwhile, the functional significance of FSH or PDTC-mediated autophagy were further investigated. Our results demonstrated that the increased autophagy promoted progesterone secretion in porcine granulosa cells. Blockage of autophagy by chloroquine obviated the FSH or PDTC-induced progesterone production. Taken together, these results indicate that inhibition of NF-κB increased autophagy via JNK signaling, and promote steroidogenesis in porcine granulosa cells. Our results provide new insights into the regulation and function of autophagy in mammalian follicle development. - Highlights: • FSH inhibits the activation of NF-κB in porcine primary granulosa cells. • Inhibition of NF-κB by FSH promotes autophagy via JNK signaling in granulosa cells. • Increased autophagy contributes to progesterone production in granulosa cells. • This is the first report against beclin1 regulation in porcine granulosa cells.

  8. Dimethyl sulfoxide modulates NF-kappa B and cytokine activation in lipopolysaccharide-treated murine macrophages.

    Science.gov (United States)

    Kelly, K A; Hill, M R; Youkhana, K; Wanker, F; Gimble, J M

    1994-01-01

    Antioxidants are protective against septic shock in animal models. Recently, free radical scavengers have been found to inhibit the activation of the NF-kappa B protein in a number of cell lines. This transcriptional regulatory protein binds to the promoters of the proinflammatory cytokines tumor necrosis factor, interleukin-6, and the macrophage inflammatory proteins. The current work examined lipopolysaccharide-induced NF-kappa B activation in the J774 macrophage-like cell line and primary peritoneal macrophages from lipopolysaccharide-responsive (C3HeB/Fej) and -nonresponsive (C3H/HeJ) murine strains. The DNA-binding activity of the NF-kappa B protein directly correlated with mRNA expression for the genes encoding the proinflammatory cytokines and the free radical scavenging enzyme, superoxide dismutase. Both the p50 and p65 NF-kappa B subunits were detected on gel supershift assays. Minimal NF-kappa B activity was observed following exposure of C3H/HeJ macrophages to lipopolysaccharide. The antioxidant dimethyl sulfoxide decreased the level of NF-kappa B activation in the J774 cells. This correlated with decreased expression of cytokine mRNAs and tumor necrosis factor bioactivity. These results suggest that modulation of NF-kappa B activation may provide a mechanism through which antioxidants protect against endotoxemia in murine models. Images PMID:8039880

  9. Direct non transcriptional role of NF-Y in DNA replication.

    Science.gov (United States)

    Benatti, Paolo; Belluti, Silvia; Miotto, Benoit; Neusiedler, Julia; Dolfini, Diletta; Drac, Marjorie; Basile, Valentina; Schwob, Etienne; Mantovani, Roberto; Blow, J Julian; Imbriano, Carol

    2016-04-01

    NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Segmental NF: A Guide for Patients

    Science.gov (United States)

    ... the body can show signs of segmental NF. Segmental NF1 - Individuals with segmental NF1 most commonly have the skin findings associated with ... cases, can include severe complications. Many individuals with segmental NF1 never develop any complications other than café-au- ...

  11. Nonphosphorylated neurofilament protein is expressed by scattered neurons in the vestibular and precerebellar brainstem.

    Science.gov (United States)

    Baizer, Joan S

    2009-11-17

    Vestibular information is essential for the control of posture, balance, and eye movements. The vestibular nerve projects to the four nuclei of the vestibular nuclear complex (VNC), as well as to several additional brainstem nuclei and the cerebellum. We have found that expression of the calcium-binding proteins calretinin (CR) and calbindin (CB), and the synthetic enzyme for nitric oxide synthase (nNOS) define subdivisions of the medial vestibular nucleus (MVe) and the nucleus prepositus (PrH), in cat, monkey, and human. We have asked if the pattern of expression of nonphosphorylated neurofilament protein (NPNFP) might define additional subdivisions of these or other nuclei that participate in vestibular function. We studied the distribution of cells immunoreactive to NPNFP in the brainstems of 5 cats and one squirrel monkey. Labeled cells were scattered throughout the four nuclei of the VNC, as well as in PrH, the reticular formation (RF) and the external cuneate nucleus. We used double-label immunofluorescence to visualize the distribution of these cells relative to other neurochemically defined subdivisions. NPNFP cells were excluded from the CR and CB regions of the MVe. In PrH, NPNFP and nNOS were not colocalized. Cells in the lateral vestibular nucleus and RF colocalized NPNFP and a marker for glutamatergic neurons. We also found that the cholinergic cells and axons of cranial nerve nuclei 3, 4, 6, 7,10 and 12 colocalize NPNFP. The data suggest that NPNFP is expressed by a subset of glutamatergic projection neurons of the vestibular brainstem. NPNFP may be a marker for those cells that are especially vulnerable to the effects of normal aging, neurological disease or disruption of sensory input.

  12. CSF neurofilament light chain but not FLT3 ligand discriminates Parkinsonian disorders

    Directory of Open Access Journals (Sweden)

    Megan Kristy Herbert

    2015-05-01

    Full Text Available The differentiation between multiple system atrophy (MSA and Parkinson’s disease (PD is difficult, particularly in early disease stages. Therefore, we aimed to evaluate the diagnostic value of neurofilament light chain (NFL, fms-like tyrosine kinase ligand (FLT3L and total tau protein (t-tau in cerebrospinal fluid (CSF as biomarkers to discriminate MSA from PD. Using commercially available enzyme-linked immunoassays (ELISAs, we measured CSF levels of NFL, FLT3L and t-tau in a discovery cohort of 36 PD patients, 27 MSA patients and 57 non-neurological controls and in a validation cohort of 32 PD patients, 25 MSA patients, 15 PSP patients, 5 CBS patients, and 56 non-neurological controls. Cut-offs obtained from individual assays and binary logistic regression models developed from combinations of biomarkers were assessed. CSF levels of NFL were substantially increased in MSA and discriminated between MSA and PD with a sensitivity of 74% and specificity of 92% (AUC = 0.85 in the discovery cohort and with 80% sensitivity and 97% specificity (AUC = 0.94 in the validation cohort. FLT3L levels in CSF were significantly lower in both PD and MSA compared to controls in the discovery cohort, but not in the validation cohort. T-tau levels were significantly higher in MSA than PD and controls. Addition of either FLT3L or t-tau to NFL did not improve discrimination of PD from MSA above NFL alone. Our findings show that increased levels of NFL in CSF offer clinically relevant, high accuracy discrimination between PD and MSA.

  13. Serum Neurofilament Light in American Football Athletes over the Course of a Season.

    Science.gov (United States)

    Oliver, Jonathan M; Jones, Margaret T; Kirk, K Michele; Gable, David A; Repshas, Justin T; Johnson, Torie A; Andréasson, Ulf; Norgren, Niklas; Blennow, Kaj; Zetterberg, Henrik

    2016-10-01

    Despite being underreported, American football boasts the highest incidence of concussion among all team sports, likely due to exposure to head impacts that vary in number and magnitude over the season. This study compared a biological marker of head trauma in American football athletes with non-contact sport athletes and examined changes over the course of a season. Baseline serum neurofilament light polypeptide (NFL) was measured after 9 weeks of no contact and compared with a non-contact sport. Serum NFL was then measured over the course of the entire season at eight time-points coincident with expected changes in likelihood of increased head impacts. Data were compared between starters (n = 11) and non-starters (n = 9). Compared with non-starters (mean ± standard deviation) (7.30 ± 3.57 pg•mL(-1)) and controls (6.75 ± 1.68 pg•mL(-1)), serum NFL in starters (8.45 ± 5.90 pg•mL(-1)) was higher at baseline (mean difference; ±90% confidence interval) (1.69;  ± 1.96 pg•mL(-1) and 1.15;  ± 1.4 pg•mL(-1), respectively). Over the course of the season, an increase (effect size [ES] = 1.8; p non-starters resulted in substantial differences between starters and non-starters over the course of the season. These data suggest that a season of collegiate American football is associated with elevations in serum NFL, which is indicative of axonal injury, as a result of head impacts.

  14. Postnatal development of cerebellar zones revealed by neurofilament heavy chain protein expression

    Directory of Open Access Journals (Sweden)

    Joshua J White

    2013-05-01

    Full Text Available The cerebellum is organized into parasagittal zones that control sensory-motor behavior. Although the architecture of adult zones is well understood, very little is known about how zones emerge during development. Understanding the process of zone formation is an essential step towards unraveling how circuits are constructed to support specific behaviors. Therefore, we focused this study on postnatal development to determine the spatial and temporal changes that establish zonal patterns during circuit formation. We used a combination of wholemount and tissue section immunohistochemistry in mice to show that the cytoskeletal protein neurofilament heavy chain (NFH is a robust marker for postnatal cerebellar zonal patterning. The patterned expression of NFH is initiated shortly after birth, and compared to the domains of several known zonal markers such as zebrin II, HSP25, neurogranin, and phospholipase Cβ4 (PLCβ4, NFH does not exhibit transient expression patterns that are typically remodeled between stages, and the adult zones do not emerge after a period of uniform expression in all lobules. Instead, we found that throughout postnatal development NFH gradually reveals distinct zones in each cerebellar lobule. The boundaries of individual NFH zones sharpen over time, as zones are refined during the second and third weeks after birth. Double labeling with neurogranin and PLCβ4 further revealed that although the postnatal expression of NFH is spatially and temporally unique, its pattern of zones respects a fundamental and well-known molecular topography in the cerebellum. The dynamics of NFH expression support the hypothesis that adult circuits are derived from an embryonic map that is refined into zones during the first three-weeks of life.

  15. Serum neurofilament light chain levels are increased in patients with a clinically isolated syndrome

    DEFF Research Database (Denmark)

    Disanto, Giulio; Adiutori, Rocco; Dobson, Ruth

    2016-01-01

    with a short conversion interval to clinically definite multiple sclerosis (MS) (fast converters (FC), median (IQR) conversion time: 110 days (79-139)); 98 patients with non-converting CIS (non-converters (NC), follow-up: 6.5 years (5.3-7.9)); and 92 healthy controls. RESULTS: NfL levels were higher in FC (24...... with increasing numbers of T2 hyperintense MRI lesions (OR=2.36; 95% CI 1.21 to 4.59; p=0.011), gadolinium-enhancing lesions (OR=2.69; 95% CI 1.13 to 6.41; p=0.026) and higher disability scores (OR=2.54; 95% CI 1.21 to 5.31; p=0.013) at CIS diagnosis. CONCLUSIONS: If replicated in future studies, serum NfL may...

  16. Biliverdin inhibits activation of NF-kappaB: reversal of inhibition by human biliverdin reductase.

    Science.gov (United States)

    Gibbs, Peter E M; Maines, Mahin D

    2007-12-01

    hBVR functions in the cell as a reductase and as a kinase. In the first capacity, it reduces biliverdin, the product of HO activity, to the effective intracellular antioxidant, bilirubin; as a dual-specificity kinase (S/T/Y) it activates the MAPK and IGF/IRK receptor signal transduction pathways. NF-kappaB and the MAPK pathway are activated by ROS, which results in the activation of stress-inducible genes, including ho-1. Presently, we report on the negative effect of biliverdin on NF-kappaB activation and the converse effect of hBVR. Biliverdin, in a concentration- and time-dependent manner, inhibited transcriptional activity of NF-kappaB in HEK293A cells. Nuclear extracts from biliverdin-treated cells show reduced DNA binding of NF-kappaB in an electromobility shift assay, whereas extracts from cells treated with TNF-alpha showed enhanced binding. Coimmunoprecipitation data show hBVR binds to the 65 kDa subunit of NF-kappaB, and that this is dependent on activation by TNF-alpha. Overexpression of hBVR enhanced both the basal and TNF-alpha-mediated activation of NF-kappaB and also that of the NF-kappaB-activated iNOS gene. Also, overexpression of hBVR arrested the cell cycle in the G(1)/G(0) phase and reduced the number of cells in S phase. Similar results were observed with MCF-7 cells. Because of the Janus nature of NF-kappaB activity in the cell and the inhibitory action of biliverdin, the present findings provide a foundation for therapeutic intervention in inflammatory diseases and cancer that may be attained by preventing reduction of biliverdin. On the other hand, by increasing BVR levels beneficial functions of NF-kappaB might be augmented. (c) 2007 Wiley-Liss, Inc.

  17. Cerebrospinal fluid levels of chitinase 3-like 1 and neurofilament light chain predict multiple sclerosis development and disability after optic neuritis

    DEFF Research Database (Denmark)

    Modvig, S; Degn, M; Roed, H

    2015-01-01

    predicted CDMS best. Neurofilament light-chain predicted long-term disability by the multiple sclerosis severity scale (p=0.0111) and nine-hole-peg-test (p=0.0202). Chitinase-3-like-1 predicted long-term cognitive impairment by the paced auditory serial addition test (p=0.0150). CONCLUSION: Neurofilament......BACKGROUND: Cerebrospinal fluid (CSF) biomarkers have been suggested to predict multiple sclerosis (MS) after clinically isolated syndromes, but studies investigating long-term prognosis are needed. OBJECTIVE: To assess the predictive ability of CSF biomarkers with regard to MS development and long......-term disability after optic neuritis (ON). METHODS: Eighty-six patients with ON as a first demyelinating event were included retrospectively. Magnetic resonance imaging (MRI), CSF leukocytes, immunoglobulin G index and oligoclonal bands were registered. CSF levels of chitinase-3-like-1, osteopontin, neurofilament...

  18. ANKRD1 modulates inflammatory responses in C2C12 myoblasts through feedback inhibition of NF-κB signaling activity

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xin-Hua [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Bauman, William A. [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Cardozo, Christopher, E-mail: chris.cardozo@va.gov [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States)

    2015-08-14

    Transcription factors of the nuclear factor-kappa B (NF-κB) family play a pivotal role in inflammation, immunity and cell survival responses. Recent studies revealed that NF-κB also regulates the processes of muscle atrophy. NF-κB activity is regulated by various factors, including ankyrin repeat domain 2 (AnkrD2), which belongs to the muscle ankyrin repeat protein family. Another member of this family, AnkrD1 is also a transcriptional effector. The expression levels of AnkrD1 are highly upregulated in denervated skeletal muscle, suggesting an involvement of AnkrD1 in NF-κB mediated cellular responses to paralysis. However, the molecular mechanism underlying the interactive role of AnkrD1 in NF-κB mediated cellular responses is not well understood. In the current study, we examined the effect of AnkrD1 on NF-κB activity and determined the interactions between AnkrD1 expression and NF-κB signaling induced by TNFα in differentiating C2C12 myoblasts. TNFα upregulated AnkrD1 mRNA and protein levels. AnkrD1-siRNA significantly increased TNFα-induced transcriptional activation of NF-κB, whereas overexpression of AnkrD1 inhibited TNFα-induced NF-κB activity. Co-immunoprecipitation studies demonstrated that AnkrD1 was able to bind p50 subunit of NF-κB and vice versa. Finally, CHIP assays revealed that AnkrD1 bound chromatin at a NF-κB binding site in the AnrkD2 promoter and required NF-κB to do so. These results provide evidence of signaling integration between AnkrD1 and NF-κB pathways, and suggest a novel anti-inflammatory role of AnkrD1 through feedback inhibition of NF-κB transcriptional activity by which AnkrD1 modulates the balance between physiological and pathological inflammatory responses in skeletal muscle. - Highlights: • AnkrD1 is upregulated by TNFα and represses NF-κB-induced transcriptional activity. • AnkrD1 binds to p50 subunit of NF-κB and is recruited to NF-κB bound to chromatin. • AnkrD1 mediates a feed-back inhibitory loop

  19. Effect of natural and semisynthetic pseudoguianolides on the stability of NF-κB:DNA complex studied by agarose gel electrophoresis.

    Science.gov (United States)

    Villagomez, Rodrigo; Hatti-Kaul, Rajni; Sterner, Olov; Almanza, Giovanna; Linares-Pastén, Javier A

    2015-01-01

    The nuclear factor κB (NF-κB) is a promising target for drug discovery. NF-κB is a heterodimeric complex of RelA and p50 subunits that interact with the DNA, regulating the expression of several genes; its dysregulation can trigger diverse diseases including inflammation, immunodeficiency, and cancer. There is some experimental evidence, based on whole cells studies, that natural sesquiterpene lactones (Sls) can inhibit the interaction of NF-κB with DNA, by alkylating the RelA subunit via a Michael addition. In the present work, 28 natural and semisynthetic pseudoguianolides were screened as potential inhibitors of NF-κB in a biochemical assay that was designed using pure NF-κB heterodimer, pseudoguianolides and a ~1000 bp palindromic DNA fragment harboring two NF-κB recognition sequences. By comparing the relative amount of free DNA fragment to the NF-κB - DNA complex, in a routine agarose gel electrophoresis, the destabilizing effect of a compound on the complex is estimated. The results of the assay and the following structure-activity relationship study, allowed the identification of several relevant structural features in the pseudoguaianolide skeleton, which are necessary to enhance the dissociating capacity of NF-κB-DNA complex. The most active compounds are substituted at C-3 (α-carbonyl), in addition to having the α-methylene-γ-lactone moiety which is essential for the alkylation of RelA.

  20. Effect of natural and semisynthetic pseudoguianolides on the stability of NF-κB:DNA complex studied by agarose gel electrophoresis.

    Directory of Open Access Journals (Sweden)

    Rodrigo Villagomez

    Full Text Available The nuclear factor κB (NF-κB is a promising target for drug discovery. NF-κB is a heterodimeric complex of RelA and p50 subunits that interact with the DNA, regulating the expression of several genes; its dysregulation can trigger diverse diseases including inflammation, immunodeficiency, and cancer. There is some experimental evidence, based on whole cells studies, that natural sesquiterpene lactones (Sls can inhibit the interaction of NF-κB with DNA, by alkylating the RelA subunit via a Michael addition. In the present work, 28 natural and semisynthetic pseudoguianolides were screened as potential inhibitors of NF-κB in a biochemical assay that was designed using pure NF-κB heterodimer, pseudoguianolides and a ~1000 bp palindromic DNA fragment harboring two NF-κB recognition sequences. By comparing the relative amount of free DNA fragment to the NF-κB - DNA complex, in a routine agarose gel electrophoresis, the destabilizing effect of a compound on the complex is estimated. The results of the assay and the following structure-activity relationship study, allowed the identification of several relevant structural features in the pseudoguaianolide skeleton, which are necessary to enhance the dissociating capacity of NF-κB-DNA complex. The most active compounds are substituted at C-3 (α-carbonyl, in addition to having the α-methylene-γ-lactone moiety which is essential for the alkylation of RelA.

  1. Altered neurofilament protein expression in the lateral vestibular nucleus in Parkinson's disease.

    Science.gov (United States)

    Wellings, Thomas P; Brichta, Alan M; Lim, Rebecca

    2017-12-01

    A major cause of morbidity in Parkinson's disease (PD) is postural instability. The neuropathology underlying postural instability is unknown. Postural control is mediated by Deiters' neurons of the lateral vestibular nucleus (LVN), which are the brainstem origin of descending vestibulospinal reflexes. Deiters' neurons express the cytostructural protein, non-phosphorylated neurofilament protein (NPNFP). In PD, reduced expression of NPNFP in substantia nigra (SN) neurons is believed to contribute to dysfunction. It was the aim of this study to determine if there is altered expression of NPNFP in the LVN in PD. We immunolabeled NPNFP in brainstem sections of six aged controls (mean age 92 yo) and six PD donors (mean age 83 yo). Our results show there was a ~ 50% reduction in NPNFP-positive Deiters' neurons compared to controls (13 ± 2.0/section vs 25.7 ± 3.0/section; p < 0.01, repeated measures ANOVA). In contrast, there was no difference in NPNFP-positive counts in the facial nucleus between control and PD. The normalized intensity of NPNFP labeling in LVN was also reduced in PD (0.87 ± 0.05 vs 1.09 ± 0.03; p < 0.01). There was a 35% concurrent reduction in NPNFP-positive neuropil in PD relative to controls (p < 0.01). We also show there was an 84% increase (p < 0.05) in somatic lipofuscin in PD patients compared to control. Lipofuscin aggregation has been shown to increase not only with age but also with neurodegeneration. Furthermore, decreased NPNFP intensity was strongly correlated with increasing lipofuscin autofluorescence across all cases (R 2 = 0.81, p < 0.01). These results show two alterations in cellular content with PD, reduced expression and intensity of NPNFP and increased lipofuscin aggregation in Deiter's neurons. These changes may contribute to degeneration of postural reflexes observed in PD.

  2. Hierarchical development of the primate visual cortex, as revealed by neurofilament immunoreactivity: early maturation of the middle temporal area (MT).

    Science.gov (United States)

    Bourne, James A; Rosa, Marcello G P

    2006-03-01

    It has been suggested that the development of the cerebral cortex reflects its hierarchical organization, with the primary sensory areas being the first to reach structural and functional maturity, and higher-order association areas being the last. In the present study, we labelled the cortex of New World marmoset monkeys of late fetal and early postnatal ages with an antibody to non-phosphorylated neurofilament, a marker of structural maturation of a subset of pyramidal cells. Supporting the concept of hierarchical maturation, we found that at birth labelled cells were found in the primary visual, auditory and somatosensory areas, but not in most other cortical fields. The exception was visual area MT, which revealed an infragranular pattern of labelling comparable to the one observed in the primary areas, as well as some supragranular staining. In MT, an adult-like pattern of labelled cells, including both supragranular and infragranular layer neurons, emerged within the first postnatal month. In comparison, the development of other extrastriate areas was delayed, with the first signs of neurofilament staining not present until the third week. The present results support the concept of MT as another primary visual area, an idea previously advanced on the basis of functional and anatomical evidence.

  3. Beyond NF-κB activation: nuclear functions of IκB kinase α

    Directory of Open Access Journals (Sweden)

    Huang Wei-Chien

    2013-01-01

    Full Text Available Abstract IκB kinase (IKK complex, the master kinase for NF-κB activation, contains two kinase subunits, IKKα and IKKβ. In addition to mediating NF-κB signaling by phosphorylating IκB proteins during inflammatory and immune responses, the activation of the IKK complex also responds to various stimuli to regulate diverse functions independently of NF-κB. Although these two kinases share structural and biochemical similarities, different sub-cellular localization and phosphorylation targets between IKKα and IKKβ account for their distinct physiological and pathological roles. While IKKβ is predominantly cytoplasmic, IKKα has been found to shuttle between the cytoplasm and the nucleus. The nuclear-specific roles of IKKα have brought increasing complexity to its biological function. This review highlights major advances in the studies of the nuclear functions of IKKα and the mechanisms of IKKα nuclear translocation. Understanding the nuclear activity is essential for targeting IKKα for therapeutics.

  4. Interleukin-6 upregulates paraoxonase 1 gene expression via an AKT/NF-κB-dependent pathway

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Chi-Chih [Department of Research, Taichung Veterans General Hospital, Taichung, Taiwan (China); Hsueh, Chi-Mei [Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chen, Chiu-Yuan [Graduate Institute of Natural Healing Sciences, Nanhua University, Chiayi, Taiwan (China); Chen, Tzu-Hsiu, E-mail: hsiu@mail.chna.edu.tw [Department of Health and Nutrition, Chia Nan University of Pharmacy and Science, Tainan, Taiwan (China); Hsu, Shih-Lan, E-mail: h2326@vghtc.gov.tw [Department of Research, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Applied Chemistry, National Chi Nan University, Puli, Nantou, Taiwan (China)

    2013-07-19

    Highlights: •IL-6 could induce PON1 gene expression. •IL-6 increased NF-κB protein expression and NF-κB-p50 and -p65 subunits nuclear translocation. •IL-6-induced PON1 up-regulation was through an AKT/NF-κB pathway. -- Abstract: The aim of this study is to investigate the relationship between paraoxonase 1 (PON1) and atherosclerosis-related inflammation. In this study, human hepatoma HepG2 cell line was used as a hepatocyte model to examine the effects of the pro-inflammatory cytokines on PON1 expression. The results showed that IL-6, but not TNF-α and IL-1β, significantly increased both the function and protein level of PON1; data from real-time RT-PCR analysis revealed that the IL-6-induced PON1 expression occurred at the transcriptional level. Increase of IκB kinase activity and IκB phosphorylation, and reduction of IκB protein level were also observed in IL-6-treated HepG2 cells compared with untreated culture. This event was accompanied by increase of NF-κB-p50 and -p65 nuclear translocation. Moreover, treatment with IL-6 augmented the DNA binding activity of NF-κB. Furthermore, pharmacological inhibition of NF-κB activation by PDTC and BAY 11-7082, markedly suppressed the IL-6-mediated PON1 expression. In addition, IL-6 increased the levels of phosphorylated protein kinase B (PKB, AKT). An AKT inhibitor LY294002 effectively suppressed IKK/IκB/NF-κB signaling and PON1 gene expression induced by IL-6. Our findings demonstrate that IL-6 upregulates PON1 gene expression through an AKT/NF-κB signaling axis in human hepatocyte-derived HepG2 cell line.

  5. Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-κB-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Fitzpatrick, Susan F.; Fábián, Zsolt; Schaible, Bettina; Lenihan, Colin R.; Schwarzl, Thomas [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Rodriguez, Javier [Systems Biology Ireland, University College Dublin, Dublin 4 (Ireland); Zheng, Xingnan; Li, Zongwei [Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC (United States); Tambuwala, Murtaza M. [School of Pharmacy and Pharmaceutical Sciences, Ulster University, Coleraine, BT52 1SA, Northern Ireland (United Kingdom); Higgins, Desmond G.; O' Meara, Yvonne [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Slattery, Craig [School of Biomolecular and Biomedical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Manresa, Mario C. [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); Fraisl, Peter; Bruning, Ulrike [Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, University of Leuven, Vesalius Research Center, VIB, B-3000 (Belgium); Baes, Myriam [Laboratory for Cell Metabolism, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven (Belgium); Carmeliet, Peter; Doherty, Glen [Laboratory of Angiogenesis and Vascular Metabolism, Department of Oncology, University of Leuven, Vesalius Research Center, VIB, B-3000 (Belgium); Kriegsheim, Alex von [Systems Biology Ireland, University College Dublin, Dublin 4 (Ireland); Cummins, Eoin P. [School of Medicine and Medical Science, The Conway Institute, University College Dublin, Belfield, Dublin 4 Ireland (Ireland); and others

    2016-06-03

    Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1{sup −/−} hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease. -- Highlights: •Genetic ablation of PHD1 upregulates NF-kappaB (NF-κB) in hepatocytes. •Activation of NF-κB leads to differential DNA-binding of p50/p65 and results in differential regulation of apoptotic genes. •We identified proline 191 in the beta subunit of the I-kappaB kinase as a target for PHD1-mediated hydroxylation. •Blockade of prolyl-4-hydroxylases has been found cytoprotective in liver cells.

  6. Desflurane preconditioning induces oscillation of NF-κB in human umbilical vein endothelial cells.

    Directory of Open Access Journals (Sweden)

    Juan Yi

    Full Text Available BACKGROUND: Nuclear factor kappa B (NF-κB has been implicated in anesthetic preconditioning (APC induced protection against anoxia and reoxygenation (A/R injury. The authors hypothesized that desflurane preconditioning would induce NF-κB oscillation and prevent endothelial cells apoptosis. METHODS: A human umbilical vein endothelial cells (HUVECs A/R injury model was used. A 30 minute desflurane treatment was initiated before anoxia. NF-κB inhibitor BAY11-7082 was administered in some experiments before desflurane preconditioning. Cells apoptosis was analyzed by flow cytometry using annexin V-fluorescein isothiocyanate staining and cell viability was evaluated by modified tertrozalium salt (MTT assay. The cellular superoxide dismutases (SOD activitiy were tested by water-soluble tetrazolium salt (WST-1 assay. NF-κB p65 subunit nuclear translocation was detected by immunofluorescence staining. Expression of inhibitor of NF-κB-α (IκBα, NF-κB p65 and cellular inhibitor of apoptosis 1 (c-IAP1, B-cell leukemia/lymphoma 2 (Bcl-2, cysteine containing aspartate specific protease 3 (caspases-3 and second mitochondrial-derived activator of caspase (SMAC/DIABLO were determined by western blot. RESULTS: Desflurane preconditioning caused phosphorylation and nuclear translocation of NF-κB before anoxia, on the contrary, induced the synthesis of IκBα and inhibition of NF-κB after reoxygenation. Desflurane preconditioning up-regulated the expression of c-IAP1 and Bcl-2, blocked the cleavage of caspase-3 and reduced SMAC release, and decreased the cell death of HUVECs after A/R. The protective effect was abolished by BAY11-7082 administered before desflurane. CONCLUSIONS: The results demonstrated that desflurane activated NF-κB during the preconditioning period and inhibited excessive activation of NF-κB in reperfusion. And the oscillation of NF-κB induced by desflurane preconditioning finally up-regulated antiapoptotic proteins expression and

  7. p38 mitogen-activated protein kinase up-regulates NF-{kappa}B transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Guoping [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China); Liu, Dongxu [Department of Orthodontics, College of Stomatology, Shandong University, Jinan, Shandong Province 250012 (China); Liu, Jing [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Gao, Hui [Department of Orthodontics, Tianjin Stomatological Hospital, Tianjin 300041 (China); Yuan, Xiao, E-mail: yuanxiaoqd@163.com [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Shen, Gang, E-mail: ganshen2007@163.com [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China)

    2010-01-01

    p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-{kappa}B in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-{kappa}B activation during myogenesis, not through down-regulation of degradation of I{kappa}B-{alpha}, and consequent translocation of the p65 subunit of NF-{kappa}B to the nucleus. It is likely that stretch-induced NF-{kappa}B activation by phosphorylation of p65 NF-{kappa}B. Moreover, depletion of p38{alpha} using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-{kappa}B activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-{kappa}B signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The {alpha} isoform of p38MAP kinase regulates the transcriptional activation of NF-{kappa}B following stimulation with cyclic stretch.

  8. Transcriptional Factor NF-κB as a Target for Therapy in Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Patrick M. Flood

    2011-01-01

    Full Text Available Parkinson's disease (PD is a neurodegenerative condition characterized by chronic inflammation. Nuclear factor κB (NF-κB is a family of inducible transcription factors that are expressed in a wide variety of cells and tissues, including microglia, astrocytes, and neurons, and the classical NF-κB pathway plays a key role in the activation and regulation of inflammatory mediator production during inflammation. Activation of the classical NF-κB pathway is mediated through the activity of the IKK kinase complex, which consists of a heterotrimer of IKKα, IKKβ, and IKKγ subunits. Targeting NF-κB has been proposed as an approach to the treatment of acute and chronic inflammatory conditions, and the use of inhibitors specific for either IKKβ or IKKγ has now been found to inhibit neurodegeneration of TH+ DA-producing neurons in murine and primate models of Parkinson's disease. These studies suggest that targeting the classical pathway of NF-κB through the inhibition of the IKK complex can serve as a useful therapeutic approach to the treatment of PD.

  9. Evolutionary conserved regulation of HIF-1β by NF-κB.

    Directory of Open Access Journals (Sweden)

    Patrick van Uden

    2011-01-01

    Full Text Available Hypoxia Inducible Factor-1 (HIF-1 is essential for mammalian development and is the principal transcription factor activated by low oxygen tensions. HIF-α subunit quantities and their associated activity are regulated in a post-translational manner, through the concerted action of a class of enzymes called Prolyl Hydroxylases (PHDs and Factor Inhibiting HIF (FIH respectively. However, alternative modes of HIF-α regulation such as translation or transcription are under-investigated, and their importance has not been firmly established. Here, we demonstrate that NF-κB regulates the HIF pathway in a significant and evolutionary conserved manner. We demonstrate that NF-κB directly regulates HIF-1β mRNA and protein. In addition, we found that NF-κB-mediated changes in HIF-1β result in modulation of HIF-2α protein. HIF-1β overexpression can rescue HIF-2α protein levels following NF-κB depletion. Significantly, NF-κB regulates HIF-1β (tango and HIF-α (sima levels and activity (Hph/fatiga, ImpL3/ldha in Drosophila, both in normoxia and hypoxia, indicating an evolutionary conserved mode of regulation. These results reveal a novel mechanism of HIF regulation, with impact in the development of novel therapeutic strategies for HIF-related pathologies including ageing, ischemia, and cancer.

  10. Evolutionary conserved regulation of HIF-1β by NF-κB.

    Science.gov (United States)

    van Uden, Patrick; Kenneth, Niall S; Webster, Ryan; Müller, H Arno; Mudie, Sharon; Rocha, Sonia

    2011-01-27

    Hypoxia Inducible Factor-1 (HIF-1) is essential for mammalian development and is the principal transcription factor activated by low oxygen tensions. HIF-α subunit quantities and their associated activity are regulated in a post-translational manner, through the concerted action of a class of enzymes called Prolyl Hydroxylases (PHDs) and Factor Inhibiting HIF (FIH) respectively. However, alternative modes of HIF-α regulation such as translation or transcription are under-investigated, and their importance has not been firmly established. Here, we demonstrate that NF-κB regulates the HIF pathway in a significant and evolutionary conserved manner. We demonstrate that NF-κB directly regulates HIF-1β mRNA and protein. In addition, we found that NF-κB-mediated changes in HIF-1β result in modulation of HIF-2α protein. HIF-1β overexpression can rescue HIF-2α protein levels following NF-κB depletion. Significantly, NF-κB regulates HIF-1β (tango) and HIF-α (sima) levels and activity (Hph/fatiga, ImpL3/ldha) in Drosophila, both in normoxia and hypoxia, indicating an evolutionary conserved mode of regulation. These results reveal a novel mechanism of HIF regulation, with impact in the development of novel therapeutic strategies for HIF-related pathologies including ageing, ischemia, and cancer.

  11. AGRO100 inhibits activation of nuclear factor-kappaB (NF-kappaB) by forming a complex with NF-kappaB essential modulator (NEMO) and nucleolin.

    Science.gov (United States)

    Girvan, Allicia C; Teng, Yun; Casson, Lavona K; Thomas, Shelia D; Jüliger, Simone; Ball, Mark W; Klein, Jon B; Pierce, William M; Barve, Shirish S; Bates, Paula J

    2006-07-01

    AGRO100, also known as AS1411, is an experimental anticancer drug that recently entered human clinical trials. It is a member of a novel class of antiproliferative agents known as G-rich oligonucleotides (GRO), which are non-antisense, guanosine-rich phosphodiester oligodeoxynucleotides that form stable G-quadruplex structures. The biological activity of GROs results from their binding to specific cellular proteins as aptamers. One important target protein of GROs has been previously identified as nucleolin, a multifunctional protein expressed at high levels by cancer cells. Here, we report that AGRO100 also associates with nuclear factor-kappaB (NF-kappaB) essential modulator (NEMO), which is a regulatory subunit of the inhibitor of kappaB (IkappaB) kinase (IKK) complex, and also called IKKgamma. In the classic NF-kappaB pathway, the IKK complex is required for phosphorylation of IkappaBalpha and subsequent activation of the transcription factor NF-kappaB. We found that treatment of cancer cells with AGRO100 inhibits IKK activity and reduces phosphorylation of IkappaBalpha in response to tumor necrosis factor-alpha stimulation. Using a reporter gene assay, we showed that AGRO100 blocks both tumor necrosis factor-alpha-induced and constitutive NF-kappaB activity in human cancer cell lines derived from cervical, prostate, breast, and lung carcinomas. In addition, we showed that, in AGRO100-treated cancer cells, NEMO is coprecipitated by nucleolin, indicating that both proteins are present in the same complex. Our studies suggest that abrogation of NF-kappaB activity may contribute to the anticancer effects of AGRO100 and that nucleolin may play a previously unknown role in regulating the NF-kappaB pathway.

  12. Detection of a ternary complex of NF-kappaB and IkappaBalpha with DNA provides insights into how IkappaBalpha removes NF-kappaB from transcription sites.

    Science.gov (United States)

    Sue, Shih-Che; Alverdi, Vera; Komives, Elizabeth A; Dyson, H Jane

    2011-01-25

    It has been axiomatic in the field of NF-κB signaling that the formation of a stable complex between NF-κB and the ankyrin repeat protein IκBα precludes the interaction of NF-κB with DNA. Contradicting this assumption, we present stopped-flow fluorescence and NMR experiments that give unequivocal evidence for the presence of a ternary DNA-NF-κB-IκBα complex in solution. Stepwise addition of a DNA fragment containing the κB binding sequence to the IκBα-NF-κB complex results in changes in the IκBα NMR spectrum that are consistent with dissociation of the region rich in proline, glutamate, serine, and threonine (PEST) and C-terminal ankyrin repeat sequences of IκBα from the complex. However, even at high concentrations of DNA, IκBα remains associated with NF-κB, indicated by the absence of resonances of the free N-terminal ankyrin repeats of IκBα. The IκBα-mediated release of NF-κB from its DNA-bound state may be envisioned as the reverse of this process. The initial step would consist of the coupled folding and binding of the intrinsically disordered nuclear localization sequence of the p65 subunit of NF-κB to the well-structured N-terminal ankyrin repeats of IκBα. Subsequently the poorly folded C-terminal ankyrin repeats of IκBα would fold upon binding to the p50 and p65 dimerization domains of NF-κB, permitting the negatively charged C-terminal PEST sequence of IκBα to displace the bound DNA through a process of local mass action.

  13. Detection of a ternary complex of NF-κB and IκBα with DNA provides insights into how IκBα removes NF-κB from transcription sites

    Science.gov (United States)

    Sue, Shih-Che; Alverdi, Vera; Komives, Elizabeth A.; Dyson, H. Jane

    2011-01-01

    It has been axiomatic in the field of NF-κB signaling that the formation of a stable complex between NF-κB and the ankyrin repeat protein IκBα precludes the interaction of NF-κB with DNA. Contradicting this assumption, we present stopped-flow fluorescence and NMR experiments that give unequivocal evidence for the presence of a ternary DNA–NF-κB–IκBα complex in solution. Stepwise addition of a DNA fragment containing the κB binding sequence to the IκBα–NF-κB complex results in changes in the IκBα NMR spectrum that are consistent with dissociation of the region rich in proline, glutamate, serine, and threonine (PEST) and C-terminal ankyrin repeat sequences of IκBα from the complex. However, even at high concentrations of DNA, IκBα remains associated with NF-κB, indicated by the absence of resonances of the free N-terminal ankyrin repeats of IκBα. The IκBα-mediated release of NF-κB from its DNA-bound state may be envisioned as the reverse of this process. The initial step would consist of the coupled folding and binding of the intrinsically disordered nuclear localization sequence of the p65 subunit of NF-κB to the well-structured N-terminal ankyrin repeats of IκBα. Subsequently the poorly folded C-terminal ankyrin repeats of IκBα would fold upon binding to the p50 and p65 dimerization domains of NF-κB, permitting the negatively charged C-terminal PEST sequence of IκBα to displace the bound DNA through a process of local mass action. PMID:21220295

  14. Ras-Independent Function of NF1

    Science.gov (United States)

    2013-05-01

    months 1-12). Perform immunofluorescence staining on pNF B, CD3 (T-cell), CD4 (helper T cells), CD8 (killer T cells), Gr-1 (granulocytes) and MAC-1...including an increased level of pNF B, increased infiltration of immune cells to the dermis as T-cells, granulocytes and macrophages (figure 4A-D). Moreover...Our staining results proved that loss of NF1 results in increased inflammatory responses only in old mice, including an increased level of pNF B

  15. Novel insights into the role of NF-κB p50 in astrocyte-mediated fate specification of adult neural progenitor cells

    Directory of Open Access Journals (Sweden)

    Valeria Bortolotto

    2017-01-01

    Full Text Available Within the CNS nuclear factor-kappa B (NF-κB transcription factors are involved in a wide range of functions both in homeostasis and in pathology. Over the years, our and other groups produced a vast array of information on the complex involvement of NF-κB proteins in different aspects of postnatal neurogenesis. In particular, several extracellular signals and membrane receptors have been identified as being able to affect neural progenitor cells (NPC and their progeny via NF-κB activation. A crucial role in the regulation of neuronal fate specification in adult hippocampal NPC is played by the NF-κB p50 subunit. NF-κB p50KO mice display a remarkable reduction in adult hippocampal neurogenesis which correlates with a selective defect in hippocampal-dependent short-term memory. Moreover absence of NF-κB p50 can profoundly affect the in vitro proneurogenic response of adult hippocampal NPC (ahNPC to several endogenous signals and drugs. Herein we briefly review the current knowledge on the pivotal role of NF-κB p50 in the regulation of adult hippocampal neurogenesis. In addition we discuss more recent data that further extend the relevance of NF-κB p50 to novel astroglia-derived signals which can influence neuronal specification of ahNPC and to astrocyte-NPC cross-talk.

  16. Expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 in cocultures of dissociated DRG neurons and skeletal muscle cells in administration of NGF or NT-3

    Directory of Open Access Journals (Sweden)

    Weiwei Zhang

    2012-07-01

    Full Text Available Both neurotrophins (NTs and target skeletal muscle (SKM cells are essential for the maintenance of the function of neurons and nerve-muscle communication. However, much less is known about the association of target SKM cells with distinct NTs on the expression of mRNAs for preprotachykinin (PPT, calcitonin-gene related peptide (CGRP, neurofilament 200 (NF-200, and microtubule associated protein 2 (MAP-2 in dorsal root ganglion (DRG sensory neurons. In the present study, a neuromuscular coculture model of dissociated dorsal root ganglion (DRG neurons and SKM cells was established. The morphology of DRG neurons and SKM cells in coculture was observed with an inverted phase contrast microscope. The effects of nerve growth factor (NGF or neurotrophin-3 (NT-3 on the expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 was analyzed by real time-PCR assay. The morphology of DRG neuronal cell bodies and SKM cells in neuromuscular coculture at different conditions was similar. The neurons presented evidence of dense neurite outgrowth in the presence of distinct NTs in neuromuscular cocultures. NGF and NT-3 increased mRNA levels of PPT, CGRP, and NF-200, but not MAP-2, in neuromuscular cocultures. These results offer new clues towards a better understanding of the association of target SKM cells with distinct NTs on the expression of mRNAs for PPT, CGRP, NF-200 and MAP-2, and implicate the association of target SKM cells and NTs with DRG sensory neuronal phenotypes.

  17. Characterization of fimbrial subunits from Bordetella species

    NARCIS (Netherlands)

    Mooi, F.R.; Heide, H.G.J. van der; Avest, A.R. ter; Welinder, K.G.; Livey, I.; Zeijst, B.A.M. van der; Gaastra, W.

    Using antisera raised against serotype 2 and 3 fimbrial subunits from Bordetella pertussis, serologically related polypeptides were detected in Bordetella bronchiseptica, Bordetella parapertussis and Bordetella avium strains. The two B. pertussis fimbrial subunits, and three of the serologically

  18. Subunit mass analysis for monitoring antibody oxidation

    National Research Council Canada - National Science Library

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping

    2017-01-01

    ... (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit...

  19. NF-kappa B modulation is involved in celastrol induced human multiple myeloma cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Haiwen Ni

    Full Text Available Celastrol is an active compound extracted from the root bark of the traditional Chinese medicine Tripterygium wilfordii Hook F. To investigate the effect of celastrol on human multiple myeloma cell cycle arrest and apoptosis and explore its molecular mechanism of action. The activity of celastrol on LP-1 cell proliferation was detected by WST-8 assay. The celastrol-induced cell cycle arrest was analyzed by flow cytometry after propidium iodide staining. Nuclear translocation of the nuclear factor kappa B (NF-κB was observed by fluorescence microscope. Celastrol inhibited cell proliferation of LP-1 myeloma cell in a dose-dependent manner with IC50 values of 0.8817 µM, which was mediated through G1 cell cycle arrest and p27 induction. Celastrol induced apoptosis in LP-1 and RPMI 8226 myeloma cells in a time and dose dependent manner, and it involved Caspase-3 activation and NF-κB pathway. Celastrol down-modulated antiapoptotic proteins including Bcl-2 and survivin expression. The expression of NF-κB and IKKa were decreased after celastrol treatment. Celastrol effectively blocked the nuclear translocation of the p65 subunit and induced human multiple myeloma cell cycle arrest and apoptosis by p27 upregulation and NF-kB modulation. It has been demonstrated that the effect of celastrol on NF-kB was HO-1-independent by using zinc protoporphyrin-9 (ZnPPIX, a selective heme oxygenase inhibitor. From the results, it could be inferred that celastrol may be used as a NF-kB inhibitor to inhibit myeloma cell proliferation.

  20. NF45 and NF90 Bind HIV-1 RNA and Modulate HIV Gene Expression

    Directory of Open Access Journals (Sweden)

    Yan Li

    2016-02-01

    Full Text Available A previous proteomic screen in our laboratory identified nuclear factor 45 (NF45 and nuclear factor 90 (NF90 as potential cellular factors involved in human immunodeficiency virus type 1 (HIV-1 replication. Both are RNA binding proteins that regulate gene expression; and NF90 has been shown to regulate the expression of cyclin T1 which is required for Tat-dependent trans-activation of viral gene expression. In this study the roles of NF45 and NF90 in HIV replication were investigated through overexpression studies. Ectopic expression of either factor potentiated HIV infection, gene expression, and virus production. Deletion of the RNA binding domains of NF45 and NF90 diminished the enhancement of HIV infection and gene expression. Both proteins were found to interact with the HIV RNA. RNA decay assays demonstrated that NF90, but not NF45, increased the half-life of the HIV RNA. Overall, these studies indicate that both NF45 and NF90 potentiate HIV infection through their RNA binding domains.

  1. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

    Boldyreff, B; Piontek, K; Schmidt-Spaniol, I

    1991-01-01

    cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies.......cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

  2. Diagnostic accuracy of CSF neurofilament light chain protein in the biomarker-guided classification system for Alzheimer's disease.

    Science.gov (United States)

    Lista, Simone; Toschi, Nicola; Baldacci, Filippo; Zetterberg, Henrik; Blennow, Kaj; Kilimann, Ingo; Teipel, Stefan J; Cavedo, Enrica; Dos Santos, Antonio Melo; Epelbaum, Stéphane; Lamari, Foudil; Dubois, Bruno; Floris, Roberto; Garaci, Francesco; Hampel, Harald

    2017-09-01

    We assessed the diagnostic accuracy of cerebrospinal fluid (CSF) neurofilament light chain (NFL) protein in the classification of patients with Alzheimer's disease (AD) and cognitively healthy control individuals (HCs) and patients with frontotemporal dementia (FTD) as comparisons. Particularly, we tested the performance of CSF NFL concentration in differentiating patient groups stratified by fluid biomarker profiles, independently of the severity of cognitive impairment (mild cognitive impairment (MCI) and AD dementia individuals), using a biomarker-guided descriptive classification system for AD. CSF NFL concentrations were examined in a multicenter cross-sectional study of 108 participants stratified in AD pathophysiology-negative (both CSF tau and the 42-amino acid-long amyloid-beta (Aβ) peptide (Aβ1-42)) (n = 15), tau pathology-positive only (n = 15), Aβ pathology-positive only (n = 13), AD pathophysiology-positive (n = 33), FTD (n = 9) patients, and HCs (n = 23), according to the biomarker-based classification system. The performance of CSF NFL in discriminating AD pathophysiology-positive patients from HCs is fair, whereas the ability in differentiating tau-positive patients from HCs is poor. The classificatory performance in distinguishing AD pathophysiology-positive patients from FTD is unsatisfactory. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Polymorphisms in NF-κB Inhibitors and Risk of Epithelial Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Kelemen Linda E

    2009-06-01

    Full Text Available Abstract Background The nuclear factor-κB (NF-κB family is a set of transcription factors with key roles in the induction of the inflammatory response and may be the link between inflammation and cancer development. This pathway has been shown to influence ovarian epithelial tissue repair. Inhibitors of κB (IκB prevent NF-κB activation by sequestering NF-κB proteins in the cytoplasm until IκB proteins are phosphorylated and degraded. Methods We used a case-control study to evaluate the association between single nucleotide polymorphisms (SNPs in NFKBIA and NFKBIB (the genes encoding IκBα and IκBβ, respectively and risk of epithelial ovarian cancer. We queried 19 tagSNPs and putative-functional SNPs among 930 epithelial ovarian cancer cases and 1,037 controls from two studies. Results The minor allele for one synonymous SNP in NFKBIA, rs1957106, was associated with decreased risk (p = 0.03. Conclusion Considering the number of single-SNP tests performed and null gene-level results, we conclude that NFKBIA and NFKBIB are not likely to harbor ovarian cancer risk alleles. Due to its biological significance in ovarian cancer, additional genes encoding NF-κB subunits, activating and inhibiting molecules, and signaling molecules warrant interrogation.

  4. An NF-kappaB and slug regulatory loop active in early vertebrate mesoderm.

    Directory of Open Access Journals (Sweden)

    Chi Zhang

    2006-12-01

    Full Text Available In both Drosophila and the mouse, the zinc finger transcription factor Snail is required for mesoderm formation; its vertebrate paralog Slug (Snai2 appears to be required for neural crest formation in the chick and the clawed frog Xenopus laevis. Both Slug and Snail act to induce epithelial to mesenchymal transition (EMT and to suppress apoptosis.Morpholino-based loss of function studies indicate that Slug is required for the normal expression of both mesodermal and neural crest markers in X. laevis. Both phenotypes are rescued by injection of RNA encoding the anti-apoptotic protein Bcl-xL; Bcl-xL's effects are dependent upon IkappaB kinase-mediated activation of the bipartite transcription factor NF-kappaB. NF-kappaB, in turn, directly up-regulates levels of Slug and Snail RNAs. Slug indirectly up-regulates levels of RNAs encoding the NF-kappaB subunit proteins RelA, Rel2, and Rel3, and directly down-regulates levels of the pro-apopotic Caspase-9 RNA.These studies reveal a Slug/Snail-NF-kappaB regulatory circuit, analogous to that present in the early Drosophila embryo, active during mesodermal formation in Xenopus. This is a regulatory interaction of significance both in development and in the course of inflammatory and metastatic disease.

  5. Mediator Subunit Med28 Is Essential for Mouse Peri-Implantation Development and Pluripotency.

    Directory of Open Access Journals (Sweden)

    Lin Li

    Full Text Available The multi-subunit mammalian Mediator complex acts as an integrator of transcriptional regulation by RNA Polymerase II, and has emerged as a master coordinator of development and cell fate determination. We previously identified the Mediator subunit, MED28, as a cytosolic binding partner of merlin, the Neurofibromatosis 2 (NF2 tumor suppressor, and thus MED28 is distinct in having a cytosolic role as an NF2 interacting protein as well as a nuclear role as a Mediator complex subunit. Although limited in vitro studies have been performed on MED28, its in vivo function remains unknown. Employing a knockout mouse model, we describe for the first time the requirement for Med28 in the developing mouse embryo. Med28-deficiency causes peri-implantation lethality resulting from the loss of pluripotency of the inner cell mass accompanied by reduced expression of key pluripotency transcription factors Oct4 and Nanog. Further, overexpression of Med28 in mouse embryonic fibroblasts enhances the efficiency of their reprogramming to pluripotency. Cre-mediated inactivation of Med28 in induced pluripotent stem cells shows that Med28 is required for their survival. Intriguingly, heterozygous loss of Med28 results in differentiation of induced pluripotent stem cells into extraembryonic trophectoderm and primitive endoderm lineages. Our findings document the essential role of Med28 in the developing embryo as well as in acquisition and maintenance of pluripotency during reprogramming.

  6. NF-kappaB in Lung Tumorigenesis

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Zhenjian [Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, New York University School of Medicine, 462 First Avenue, NBV 7N24, New York, NY 10016 (United States); Tchou-Wong, Kam-Meng; Rom, William N., E-mail: william.rom@nyumc.org [Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, New York University School of Medicine, 462 First Avenue, NBV 7N24, New York, NY 10016 (United States); Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987 (United States)

    2011-12-14

    The development of lung cancer in humans can be divided into three steps initiation, promotion and progression. This process is driven by alterations in related signal transduction pathways. These pathways signal the aberrant activation of NF-kappaB, a transcription factor that regulates the expression of genes important for lung tumorigenesis. Our current knowledge about the role of the NF-kappaB signaling pathway in the development of lung cancer has been bolstered by animal models demonstrating the connection between K-ras and tobacco induced lung transformation with NF-kappaB. Activation of downstream genes leads to cell proliferation, inhibition of apoptosis, angiogenesis, inflammation, invasion, and metastasis.

  7. Stoichiometry of δ subunit containing GABAA receptors

    Science.gov (United States)

    Patel, B; Mortensen, M; Smart, T G

    2014-01-01

    Background and Purpose Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach. Experimental Approach Using site-directed mutagenesis, we inserted a highly characterized 9′ serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits. Key Results Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose–response curves of cells co-expressing WT subunits with their respective L9′S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits. Conclusions and Implications Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ. PMID:24206220

  8. NF1 Signal Transduction and Vascular Dysfunction

    Science.gov (United States)

    2014-05-01

    the effects of losing a second allele of NF1 in the vascular endothelium of the adult mouse. This will be the first model of NF1 loss in the... adult endothelium and can serve as a model system for investigation of both cardiovascular effects and the tumor microenvironment. Body: Aim 1...would be to try and determine if there were defects in TGF-b signaling (Smad activation/EndMT) prior to doing a wholesale catalog of all the

  9. Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association

    Energy Technology Data Exchange (ETDEWEB)

    Ozaki, Hana [Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521 (Japan); Katoh, Tsuyoshi [Department of Biochemistry, Asahikawa Medical University, Asahikawa, 078-8510 (Japan); Nakagawa, Ryoko; Ishihara, Yasuhiro [Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521 (Japan); Sueyoshi, Noriyuki; Kameshita, Isamu [Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa, 761-0795 (Japan); Taniguchi, Takanobu [Department of Biochemistry, Asahikawa Medical University, Asahikawa, 078-8510 (Japan); Hirano, Tetsuo; Yamazaki, Takeshi [Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521 (Japan); Ishida, Atsuhiko, E-mail: aishida@hiroshima-u.ac.jp [Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521 (Japan)

    2016-09-02

    Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr phosphatase that belongs to the PPM family. Growing evidence suggests that PPM phosphatases including CaMKP act as a complex with other proteins to regulate cellular functions. In this study, using the two-dimensional far-western blotting technique with digoxigenin-labeled CaMKP as a probe, in conjunction with peptide mass fingerprinting analysis, we identified neurofilament L (NFL) as a CaMKP-binding protein in a Triton-insoluble fraction of rat brain. We confirmed binding of fluorescein-labeled CaMKP (F-CaMKP) to NFL in solution by fluorescence polarization. The analysis showed that the dissociation constant of F-CaMKP for NFL is 73 ± 17 nM (n = 3). Co-immunoprecipitation assay using a cytosolic fraction of NGF-differentiated PC12 cells showed that endogenous CaMKP and NFL form a complex in cells. Furthermore, the effect of CaMKP on self-assembly of NFL was examined. Electron microscopy revealed that CaMKP markedly prevented NFL from forming large filamentous aggregates, suggesting that CaMKP-binding to NFL inhibits its filament association. These findings may provide new insights into a novel mechanism for regulating network formation of neurofilaments during neuronal differentiation. - Highlights: • NFL was identified as a CaMKP-binding protein in an insoluble fraction of rat brain. • CaMKP bound to NFL in solution with a K{sub d} value of 73 ± 17 nM. • A CaMKP-NFL complex was found in NGF-differentiated PC12 cells. • CaMKP-binding to NFL inhibited its filament association. • CaMKP may regulate network formation of neurofilaments in neurons.

  10. The influence of aging on the number of neurons and levels of non-phosporylated neurofilament proteins in the central auditory system of rats

    Directory of Open Access Journals (Sweden)

    Jana eBurianová

    2015-03-01

    Full Text Available In the present study, an unbiased stereological method was used to determine the number of all neurons in Nissl stained sections of the inferior colliculus (IC, medial geniculate body (MGB and auditory cortex (AC in rats (strains Long Evans and Fischer 344 and their changes with aging. In addition, using the optical fractionator and western blot technique, we also evaluated the number of SMI-32-immunoreactive(-ir neurons and levels of non-phosphorylated neurofilament proteins in the IC, MGB, AC, and visual cortex (VC of young and old rats of the two strains. The SMI-32 positive neuronal population comprises about 10% of all neurons in the rat IC, MGB and AC and represents a prevalent population of large neurons with highly myelinated and projecting processes. In both Long Evans and Fischer 344 rats, the total number of neurons in the IC was roughly similar to that in the AC. With aging, we found a rather mild and statistically non-significant decline in the total number of neurons in all three analyzed auditory regions in both rat strains. In contrast to this, the absolute number of SMI-32-ir neurons in both Long Evans and Fischer 344 rats significantly decreased with aging in all the examined structures. The western blot technique also revealed a significant age-related decline in the levels of non-phosphorylated neurofilaments in the auditory brain structures, 30-35%. Our results demonstrate that presbycusis in rats is not likely to be primarily associated with changes in the total number of neurons. On the other hand, the pronounced age-related decline in the number of neurons containing non-phosphorylated neurofilaments as well as their protein levels in the central auditory system may contribute to age-related deterioration of hearing function.

  11. Glycine suppresses TNF-α-induced activation of NF-κB in differentiated 3T3-L1 adipocytes.

    Science.gov (United States)

    Blancas-Flores, Gerardo; Alarcón-Aguilar, Francisco J; García-Macedo, Rebeca; Almanza-Pérez, Julio C; Flores-Sáenz, José L; Román-Ramos, Rubén; Ventura-Gallegos, José L; Kumate, Jesús; Zentella-Dehesa, Alejandro; Cruz, Miguel

    2012-08-15

    Glycine strongly reduces the serum levels of pro-inflammatory cytokines and increases the levels of anti-inflammatory cytokines. Recently, glycine has been shown to decrease the expression and secretion of pro-inflammatory adipokines in monosodium glutamate-induced obese (MSG/Ob) mice. It has been postulated that these effects may be explained by a reduction in nuclear factor kappa B (NF-κB) activation. NF-κB is a transcription factor, which is crucial to the inflammatory response. Hasegawa et al. (2011 and 2012) recently reported a glycine-dependent reduction in NF-κB levels. Here, we have investigated the role of glycine in the regulation of NF-κB in differentiated 3T3-L1 adipocytes. The results revealed that pretreatment with glycine interfered with the activation of NF-κB, which has been shown to be stimulated by tumor necrosis factor-alpha (TNF-α). Glycine alone stimulated NF-κB activation in an unusual way such that the inhibitor κB-β (IκB-β) degradation was more significant than that of the inhibitor κB-α (IκB-α) and led to NF-κB complexes comprised of p50 and p65 subunits; IκB-ε degradation did not affect by glycine. These findings suggest that glycine could be used as an alternative treatment for chronic inflammation, which is a hallmark of obesity and other comorbidities, and is characterized by an elevated production of pro-inflammatory cytokines. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Subunit heterogeneity in the lima bean lectin.

    Science.gov (United States)

    Roberts, D D; Etzler, M E; Goldstein, I J

    1982-08-10

    Three forms of lectin (components I, II, and III) from lima beans (Phaseolus lunatus) have been purified on an affinity support containing the synthetic type A blood group trisaccharide alpha-D-GalNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to). Conversion of components I and II to component III has been achieved by reduction in 10(-2) M dithiothreitol. Isoelectric focusing of lima bean lectin in the presence of 8 M urea and beta-mercaptoethanol revealed charge heterogeneity of the lectin subunits. Three major subunit classes of apparent pI 7.05, 6.65, and 6.45, designated alpha, beta, and alpha', respectively, were identified; they occur in a relative abundance of 2:5:3. Green lima beans harvested before maturity lacked the alpha' subunit (pI 6.45) which appears to accumulate during seed maturation. The three subunits are glycoproteins of identical size and immunochemical reactivity. Identical NH2-terminal sequences were found for the three subunits. Amino acid analysis and tryptic peptide mapping indicated that the observed charge heterogeneity is probably due to differences in the primary structure of the subunits. Studies of subunit composition of charge isolectins provided evidence of nonrandom subunit assembly. A model is proposed involving pairing of a pI 6.65 subunit with either a pI 7.06 or 6.45 subunit to form dimeric units. Possible roles for subunit heterogeneity and ordered subunit assembly in determining the metal and sugar binding properties of lima bean lectin are discussed.

  13. Regulation of NF-{kappa}B activity in astrocytes: effects of flavonoids at dietary-relevant concentrations

    Energy Technology Data Exchange (ETDEWEB)

    Spilsbury, Alison [Reading School of Pharmacy, University of Reading, Reading RG6 6UB (United Kingdom); Vauzour, David; Spencer, Jeremy P.E. [Molecular Nutrition Group, Centre for Integrative Neuroscience and Neurodynamics, School of Chemistry, Food and Pharmacy, University of Reading, Reading RG6 6AP (United Kingdom); Rattray, Marcus, E-mail: m.a.n.rattray@reading.ac.uk [Reading School of Pharmacy, University of Reading, Reading RG6 6UB (United Kingdom)

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer We tested the hypothesis that low concentrations of flavonoids inhibit NF-{kappa}B in astrocytes. Black-Right-Pointing-Pointer Primary cultured astrocytes possess a functional {kappa}B-system, measured using luciferase assays. Black-Right-Pointing-Pointer Seven flavonoids (100 nM-1 {mu}M) failed to reduce NF-{kappa}B activity in astrocytes. Black-Right-Pointing-Pointer Four flavonoids (100 nM-1 {mu}M) failed to reduce TNFa-stimulated NF-{kappa}B activity in astrocytes. Black-Right-Pointing-Pointer (-)-Epicatechin did not regulate nuclear translocation of the NF-{kappa}B subunit, p65. -- Abstract: Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Sustained activation of nuclear transcription factor {kappa}B (NF-{kappa}B) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-{kappa}B signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNF{alpha}, 150 ng/ml) increased NF-{kappa}B-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative I{kappa}B{alpha} construct. In addition, TNF{alpha} increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-{kappa}B activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((-)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1-1 {mu}M) for 18 h. None of the flavonoids modulated constitutive or

  14. Role of the Rubisco Small Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert Joseph [Univ. of Nebraska, Lincoln, NE (United States)

    2016-11-05

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. However, it is a slow enzyme, and O2 competes with CO2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO2. If carboxylation could be increased or oxygenation decreased, an increase in net CO2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO2/O2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

  15. Introducing spatial information into predictive NF-kappaB modelling--an agent-based approach.

    Directory of Open Access Journals (Sweden)

    Mark Pogson

    2008-06-01

    Full Text Available Nature is governed by local interactions among lower-level sub-units, whether at the cell, organ, organism, or colony level. Adaptive system behaviour emerges via these interactions, which integrate the activity of the sub-units. To understand the system level it is necessary to understand the underlying local interactions. Successful models of local interactions at different levels of biological organisation, including epithelial tissue and ant colonies, have demonstrated the benefits of such 'agent-based' modelling. Here we present an agent-based approach to modelling a crucial biological system--the intracellular NF-kappaB signalling pathway. The pathway is vital to immune response regulation, and is fundamental to basic survival in a range of species. Alterations in pathway regulation underlie a variety of diseases, including atherosclerosis and arthritis. Our modelling of individual molecules, receptors and genes provides a more comprehensive outline of regulatory network mechanisms than previously possible with equation-based approaches. The method also permits consideration of structural parameters in pathway regulation; here we predict that inhibition of NF-kappaB is directly affected by actin filaments of the cytoskeleton sequestering excess inhibitors, therefore regulating steady-state and feedback behaviour.

  16. 28 CFR 51.6 - Political subunits.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All political...

  17. Cervical neurofibromas in children with NF-1

    Energy Technology Data Exchange (ETDEWEB)

    Chung, C.J.; Mukherji, S.K.; Fordham, L.A. [University of North Carolina, Chapel Hill (United States). Dept. of Radiology; Armfield, K.B.; Krause, W.L. [Scottish Rite Children`s Medical Center, AtlantA. GA (United States). Dept. of Genetics

    1999-05-01

    Background. Children with neurofibromatosis type 1 (NF1) are at increased risk of developing plexiform neurofibroma throughout the body, including the cervical soft tissues. However, the incidence of cervical soft tissue tumors and the value of screening MR for children with NF1 are not known. Purpose. The purposes of this study were to determine the incidence and clinical significance of cervical tumors seen on MR imaging in children with NF1. Materials and methods. A retrospective review of the brain and orbit MR with cervical images obtained on 95 children who meet the NIH consensus criteria for NF1 and who are followed at our neurofibromatosis clinic was carried out. Results. Cervical tumors were found on MR imaging in 21 of 95 (22 %) children. Of 21 children with cervical tumors, 14 children were determined to be surgical candidates. In nine children, MR imaging altered the clinical management by demonstrating tumors for which surgery was indicated, but the tumors were not suspected prior to MR imaging. Conclusion. Cervical tumors are commonly seen in children with NF1. MR imaging may demonstrate a significant number of tumors that require surgery, but were not suspected prior to MR imaging. (orig.) With 3 figs., 1 tab., 17 refs.

  18. Lycopene acts through inhibition of IκB kinase to suppress NF-κB signaling in human prostate and breast cancer cells.

    Science.gov (United States)

    Assar, Emelia A; Vidalle, Magdalena Castellano; Chopra, Mridula; Hafizi, Sassan

    2016-07-01

    We studied the effect of the potent dietary antioxidant lycopene on multiple points along the nuclear factor kappa B (NF-κB) signaling pathway in prostate and breast cancer cells. Lycopene significantly inhibited prostate and breast cancer cell growth at physiologically relevant concentrations of ≥1.25 μM. Similar concentrations also caused a 30-40 % reduction in inhibitor of kappa B (IκB) phosphorylation in the cells, as determined by western blotting. Furthermore, the same degree of inhibition by lycopene was observed for NF-κB transcriptional activity, as determined by reporter gene assay. Concomitant with this, immunofluorescence staining of lycopene-treated cells showed a significant suppression (≥25 %) of TNF-induced NF-κB p65 subunit nuclear translocation. Further probing of lycopene's effects on upstream elements of the NF-κB pathway showed a 25 % inhibition of both activity of recombinant IκB kinase β (IKKβ) kinase in a cell-free in vitro assay, as well as activity of IKKβ immunoprecipitated from MDA-MB-231 cells treated with lycopene. In conclusion, the anticancer properties of lycopene may occur through inhibition of the NF-κB signaling pathway, beginning at the early stage of cytoplasmic IKK kinase activity, which then leads to reduced NF-κB-responsive gene regulation. Furthermore, these effects in cancer cells were observed at concentrations of lycopene that are relevant and achievable in vivo.

  19. Selinexor, a Selective Inhibitor of Nuclear Export (SINE) compound, acts through NF-κB deactivation and combines with proteasome inhibitors to synergistically induce tumor cell death.

    Science.gov (United States)

    Kashyap, Trinayan; Argueta, Christian; Aboukameel, Amro; Unger, Thaddeus John; Klebanov, Boris; Mohammad, Ramzi M; Muqbil, Irfana; Azmi, Asfar S; Drolen, Claire; Senapedis, William; Lee, Margaret; Kauffman, Michael; Shacham, Sharon; Landesman, Yosef

    2016-11-29

    The nuclear export protein, exportin-1 (XPO1/CRM1), is overexpressed in many cancers and correlates with poor prognosis. Selinexor, a first-in-class Selective Inhibitor of Nuclear Export (SINE) compound, binds covalently to XPO1 and blocks its function. Treatment of cancer cells with selinexor results in nuclear retention of major tumor suppressor proteins and cell cycle regulators, leading to growth arrest and apoptosis. Recently, we described the selection of SINE compound resistant cells and reported elevated expression of inflammation-related genes in these cells. Here, we demonstrated that NF-κB transcriptional activity is up-regulated in cells that are naturally resistant or have acquired resistance to SINE compounds. Resistance to SINE compounds was created by knockdown of the cellular NF-κB inhibitor, IκB-α. Combination treatment of selinexor with proteasome inhibitors decreased NF-κB activity, sensitized SINE compound resistant cells and showed synergistic cytotoxicity in vitro and in vivo. Furthermore, we showed that selinexor inhibited NF-κB activity by blocking phosphorylation of the IκB-α and the NF-κB p65 subunits, protecting IκB-α from proteasome degradation and trapping IκB-α in the nucleus to suppress NF-κB activity. Therefore, combination treatment of selinexor with a proteasome inhibitor may be beneficial to patients with resistance to either single-agent.

  20. Subunit mass analysis for monitoring antibody oxidation.

    Science.gov (United States)

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping

    2017-04-01

    Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.

  1. A Functional Genomic Analysis of NF1-Associated Learning Disabilities

    National Research Council Canada - National Science Library

    Tang, Shao-Jun

    2008-01-01

    Learning disabilities severely deteriorate the life of many NF1 patients. However, the pathogenic process for NF1-associated learning disabilities has not been fully understood and an effective therapy is not available...

  2. A Functional Genomic Analysis of NF1-Associated Learning Disabilities

    National Research Council Canada - National Science Library

    Tang, Shao-Jun

    2007-01-01

    Learning disabilities severely deteriorate the life of many NF1 patients. However, the pathogenic process for NF1-associated learning disabilities has not been fully understood and an effective therapy is not available...

  3. Metalloprotease NleC suppresses host NF-κB/inflammatory responses by cleaving p65 and interfering with the p65/RPS3 interaction.

    Directory of Open Access Journals (Sweden)

    Andrea Hodgson

    2015-03-01

    Full Text Available Attaching/Effacing (A/E pathogens including enteropathogenic Escherichia coli (EPEC, enterohemorrhagic E. coli (EHEC and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p65¹⁻³⁸ fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p65¹⁻³⁸ fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3, a 'specifier' subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a

  4. Ciliary neurotrophic factor activates NF-κB to enhance mitochondrial bioenergetics and prevent neuropathy in sensory neurons of streptozotocin-induced diabetic rodents.

    Science.gov (United States)

    Saleh, Ali; Roy Chowdhury, Subir K; Smith, Darrell R; Balakrishnan, Savitha; Tessler, Lori; Martens, Corina; Morrow, Dwane; Schartner, Emily; Frizzi, Katie E; Calcutt, Nigel A; Fernyhough, Paul

    2013-02-01

    Diabetes causes mitochondrial dysfunction in sensory neurons that may contribute to peripheral neuropathy. Ciliary neurotrophic factor (CNTF) promotes sensory neuron survival and axon regeneration and prevents axonal dwindling, nerve conduction deficits and thermal hypoalgesia in diabetic rats. In this study, we tested the hypothesis that CNTF protects sensory neuron function during diabetes through normalization of impaired mitochondrial bioenergetics. In addition, we investigated whether the NF-κB signal transduction pathway was mobilized by CNTF. Neurite outgrowth of sensory neurons derived from streptozotocin (STZ)-induced diabetic rats was reduced compared to neurons from control rats and exposure to CNTF for 24 h enhanced neurite outgrowth. CNTF also activated NF-κB, as assessed by Western blotting for the NF-κB p50 subunit and reporter assays for NF-κB promoter activity. Conversely, blockade of NF-κB signaling using SN50 peptide inhibited CNTF-mediated neurite outgrowth. Studies in mice with STZ-induced diabetes demonstrated that systemic therapy with CNTF prevented functional indices of peripheral neuropathy along with deficiencies in dorsal root ganglion (DRG) NF-κB p50 expression and DNA binding activity. DRG neurons derived from STZ-diabetic mice also exhibited deficiencies in maximal oxygen consumption rate and associated spare respiratory capacity that were corrected by exposure to CNTF for 24 h in an NF-κB-dependent manner. We propose that the ability of CNTF to enhance axon regeneration and protect peripheral nerve from structural and functional indices of diabetic peripheral neuropathy is associated with targeting of mitochondrial function, in part via NF-κB activation, and improvement of cellular bioenergetics. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. DC-SCRIPT Regulates IL-10 Production in Human Dendritic Cells by Modulating NF-κBp65 Activation.

    Science.gov (United States)

    Søndergaard, Jonas Nørskov; Poghosyan, Susanna; Hontelez, Saartje; Louche, Pauline; Looman, Maaike W G; Ansems, Marleen; Adema, Gosse J

    2015-08-15

    The balance between tolerance and immunity is important for the outcome of an infection or cancer, and dendritic cells (DCs) are key regulators of this balance. DC-specific transcript (DC-SCRIPT) is a protein expressed by DCs and has been demonstrated to suppress both TLR-mediated expression of IL-10 and glucocorticoid receptor-mediated transcription of glucocorticoid-induced leucine zipper (GILZ). Because GILZ is known to promote IL-10 production, we investigated whether these two processes are linked. Dual-knockdown and inhibition experiments demonstrated that neither GILZ nor glucocorticoid receptor play a role in TLR-induced IL-10 production after DC-SCRIPT knockdown. The NF-κB pathway is another route involved in IL-10 production after DC activation. Strikingly, inhibition of NF-κB led to a decreased TLR-mediated IL-10 production in DC-SCRIPT knockdown DCs. Moreover, DC-SCRIPT knockdown DCs showed enhanced phosphorylation, acetylation, and IL10 enhancer binding of the NF-κB subunit p65. These data demonstrate that besides nuclear receptor regulation, DC-SCRIPT also modulates activation of NF-κBp65 after TLR activation in human DCs. Copyright © 2015 by The American Association of Immunologists, Inc.

  6. Heterologous SUMO-2/3-ubiquitin chains optimize IκBα degradation and NF-κB activity.

    Directory of Open Access Journals (Sweden)

    Fabienne Aillet

    Full Text Available The NF-κB pathway is regulated by SUMOylation at least at three levels: the inhibitory molecule IκBα, the IKK subunit γ/NEMO and the p52 precursor p100. Here we investigate the role of SUMO-2/3 in the degradation of IκBα and activation of NF-κB mediated by TNFα. We found that under conditions of deficient SUMOylation, an important delay in both TNFα-mediated proteolysis of IκBα and NF-κB dependent transcription occurs. In vitro and ex vivo approaches, including the use of ubiquitin-traps (TUBEs, revealed the formation of chains on IκBα containing SUMO-2/3 and ubiquitin after TNFα stimulation. The integration of SUMO-2/3 appears to promote the formation of ubiquitin chains on IκBα after activation of the TNFα signalling pathway. Furthermore, heterologous chains of SUMO-2/3 and ubiquitin promote a more efficient degradation of IκBα by the 26S proteasome in vitro compared to chains of either SUMO-2/3 or ubiquitin alone. Consistently, Ubc9 silencing reduced the capture of IκBα modified with SUMO-ubiquitin hybrid chains that display a defective proteasome-mediated degradation. Thus, hybrid SUMO-2/3-ubiquitin chains increase the susceptibility of modified IκBα to the action of 26S proteasome, contributing to the optimal control of NF-κB activity after TNFα-stimulation.

  7. Rapamycin Attenuated Cardiac Hypertrophy Induced by Isoproterenol and Maintained Energy Homeostasis via Inhibiting NF-κB Activation

    Directory of Open Access Journals (Sweden)

    Xi Chen

    2014-01-01

    Full Text Available Rapamycin, also known as sirolimus, is an immunosuppressant drug used to prevent rejection organ (especially kidney transplantation. However, little is known about the role of Rapa in cardiac hypertrophy induced by isoproterenol and its underlying mechanism. In this study, Rapa was administrated intraperitoneally for one week after the rat model of cardiac hypertrophy induced by isoproterenol established. Rapa was demonstrated to attenuate isoproterenol-induced cardiac hypertrophy, maintain the structure integrity and functional performance of mitochondria, and upregulate genes related to fatty acid metabolism in hypertrophied hearts. To further study the implication of NF-κB in the protective role of Rapa, cardiomyocytes were pretreated with TNF-α or transfected with siRNA against NF-κB/p65 subunit. It was revealed that the upregulation of extracellular circulating proinflammatory cytokines induced by isoproterenol was able to be reversed by Rapa, which was dependent on NF-κB pathway. Furthermore, the regression of cardiac hypertrophy and maintaining energy homeostasis by Rapa in cardiomyocytes may be attributed to the inactivation of NF-κB. Our results shed new light on mechanisms underlying the protective role of Rapa against cardiac hypertrophy induced by isoproterenol, suggesting that blocking proinflammatory response by Rapa might contribute to the maintenance of energy homeostasis during the progression of cardiac hypertrophy.

  8. Viral safety of C1-inhibitor NF

    NARCIS (Netherlands)

    Terpstra, F. G.; Kleijn, M.; Koenderman, A. H. L.; Over, J.; van Engelenburg, F. A. C.; Schuitemaker, H.; van 't Wout, A. B.

    2007-01-01

    We studied the efficacy of virus reduction by three process steps (polyethylene glycol 4000 (PEG) precipitation, pasteurization, and 15nm virus filtration) in the manufacturing of C1-inhibitor NF. The potential prion removing capacity in this process was estimated based on data from the literature.

  9. NF1 Signal Transduction and Vascular Dysfunction

    Science.gov (United States)

    2015-05-01

    malformations, aneurysms, and hypertension . Consequently there is a markedly elevated risk of cerbrovascular accidents(Friedman et al., 2002). NF1...the endothelium seems to trigger a rapid leukemic crisis . This observation is quite unexpected but consistent with the notion that the endothelial

  10. Risk capital allocation with autonomous subunits

    DEFF Research Database (Denmark)

    Hougaard, Jens Leth; Smilgins, Aleksandrs

    2016-01-01

    the sum of the risks of the individual subunits. The question is how to allocate the risk capital of the group among the subunits in a fair way. In this paper we propose to use the Lorenz set as an allocation method. We show that the Lorenz set is operational and coherent. Moreover, we propose three......Risk capital allocation problems have been widely discussed in the academic literature. We consider a set of independent subunits collaborating in order to reduce risk: that is, when subunit portfolios are merged a diversification benefit arises and the risk of the group as a whole is smaller than...... fairness tests related directly to the problem of risk capital allocation and show that the Lorenz set satisfies all three tests in contrast to other well-known coherent methods. Finally, we discuss how to deal with non-uniqueness of the Lorenz set....

  11. Mosaic type-1 NF1 microdeletions as a cause of both generalized and segmental neurofibromatosis type-1 (NF1)

    OpenAIRE

    Ludwine M. Messiaen; Vogt, Julia; Bengesser, Kathrin; Fu, Chuanhua; Mikhail, Fady; Serra, Eduard; Garcia-Linares, Carles; Cooper, David N; Lázaro, Conxi; Kehrer-Sawatzki, Hildegard

    2011-01-01

    Abstract Mosaicism is an important feature of type-1 neurofibromatosis (NF1) on account of its impact upon both clinical manifestations and transmission risk. Using FISH and MLPA to screen 3500 NF1 patients, we identified 146 individuals harbouring gross NF1 deletions, 14 of whom (9.6%) displayed somatic mosaicism. The high rate of mosaicism in patients with NF1 deletions supports the postulated idea of a direct relationship between the high new mutation rate in this cancer predisp...

  12. Role of Merlin/NF2 inactivation in tumor biology.

    Science.gov (United States)

    Petrilli, A M; Fernández-Valle, C

    2016-02-04

    Merlin (Moesin-ezrin-radixin-like protein, also known as schwannomin) is a tumor suppressor protein encoded by the neurofibromatosis type 2 gene NF2. Loss of function mutations or deletions in NF2 cause neurofibromatosis type 2 (NF2), a multiple tumor forming disease of the nervous system. NF2 is characterized by the development of bilateral vestibular schwannomas. Patients with NF2 can also develop schwannomas on other cranial and peripheral nerves, as well as meningiomas and ependymomas. The only potential treatment is surgery/radiosurgery, which often results in loss of function of the involved nerve. There is an urgent need for chemotherapies that slow or eliminate tumors and prevent their formation in NF2 patients. Interestingly NF2 mutations and merlin inactivation also occur in spontaneous schwannomas and meningiomas, as well as other types of cancer including mesothelioma, glioma multiforme, breast, colorectal, skin, clear cell renal cell carcinoma, hepatic and prostate cancer. Except for malignant mesotheliomas, the role of NF2 mutation or inactivation has not received much attention in cancer, and NF2 might be relevant for prognosis and future chemotherapeutic approaches. This review discusses the influence of merlin loss of function in NF2-related tumors and common human cancers. We also discuss the NF2 gene status and merlin signaling pathways affected in the different tumor types and the molecular mechanisms that lead to tumorigenesis, progression and pharmacological resistance.

  13. The NF1 somatic mutational landscape in sporadic human cancers.

    Science.gov (United States)

    Philpott, Charlotte; Tovell, Hannah; Frayling, Ian M; Cooper, David N; Upadhyaya, Meena

    2017-06-21

    Neurofibromatosis type 1 (NF1: Online Mendelian Inheritance in Man (OMIM) #162200) is an autosomal dominantly inherited tumour predisposition syndrome. Heritable constitutional mutations in the NF1 gene result in dysregulation of the RAS/MAPK pathway and are causative of NF1. The major known function of the NF1 gene product neurofibromin is to downregulate RAS. NF1 exhibits variable clinical expression and is characterized by benign cutaneous lesions including neurofibromas and café-au-lait macules, as well as a predisposition to various types of malignancy, such as breast cancer and leukaemia. However, acquired somatic mutations in NF1 are also found in a wide variety of malignant neoplasms that are not associated with NF1. Capitalizing upon the availability of next-generation sequencing data from cancer genomes and exomes, we review current knowledge of somatic NF1 mutations in a wide variety of tumours occurring at a number of different sites: breast, colorectum, urothelium, lung, ovary, skin, brain and neuroendocrine tissues, as well as leukaemias, in an attempt to understand their broader role and significance, and with a view ultimately to exploiting this in a diagnostic and therapeutic context. As neurofibromin activity is a key to regulating the RAS/MAPK pathway, NF1 mutations are important in the acquisition of drug resistance, to BRAF, EGFR inhibitors, tamoxifen and retinoic acid in melanoma, lung and breast cancers and neuroblastoma. Other curiosities are observed, such as a high rate of somatic NF1 mutation in cutaneous melanoma, lung cancer, ovarian carcinoma and glioblastoma which are not usually associated with neurofibromatosis type 1. Somatic NF1 mutations may be critical drivers in multiple cancers. The mutational landscape of somatic NF1 mutations should provide novel insights into our understanding of the pathophysiology of cancer. The identification of high frequency of somatic NF1 mutations in sporadic tumours indicates that neurofibromin is

  14. Cutting edge: NF-κB p65 and c-Rel control epidermal development and immune homeostasis in the skin.

    Science.gov (United States)

    Grinberg-Bleyer, Yenkel; Dainichi, Teruki; Oh, Hyunju; Heise, Nicole; Klein, Ulf; Schmid, Roland M; Hayden, Matthew S; Ghosh, Sankar

    2015-03-15

    Psoriasis is an inflammatory skin disease in which activated immune cells and the proinflammatory cytokine TNF are well-known mediators of pathogenesis. The transcription factor NF-κB is a key regulator of TNF production and TNF-induced proinflammatory gene expression, and both the psoriatic transcriptome and genetic susceptibility further implicate NF-κB in psoriasis etiopathology. However, the role of NF-κB in psoriasis remains controversial. We analyzed the function of canonical NF-κB in the epidermis using CRE-mediated deletion of p65 and c-Rel in keratinocytes. In contrast to animals lacking p65 or c-Rel alone, mice lacking both subunits developed severe dermatitis after birth. Consistent with its partial histological similarity to human psoriasis, this condition could be prevented by anti-TNF treatment. Moreover, regulatory T cells in lesional skin played an important role in disease remission. Our results demonstrate that canonical NF-κB in keratinocytes is essential for the maintenance of skin immune homeostasis and is protective against spontaneous dermatitis. Copyright © 2015 by The American Association of Immunologists, Inc.

  15. E2F mediates induction of the Sp1-controlled promoter of the human DNA polymerase ɛ B-subunit gene POLE2

    Science.gov (United States)

    Huang, Deqi; Jokela, Maarit; Tuusa, Jussi; Skog, Sven; Poikonen, Kari; Syväoja, Juhani E.

    2001-01-01

    The B-subunits of replicative DNA polymerases from Archaea to humans belong to the same protein family, suggesting that they share a common fundamental function. We report here the gene structure for the B-subunit of human DNA polymerase ɛ (POLE2), whose expression and transcriptional regulation is typical for replication proteins with some unique features. The 75 bp core promoter region, located within exon 1, contains an Sp1 element that is a critical determinant of promoter activity as shown by the luciferase reporter, electrophoretic mobility shift and DNase I footprinting assays. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Binding sites for E2F1 and NF-1 reside immediately downstream from the core promoter region. Our results suggest that human POLE2 is regulated by two E2F–pocket protein complexes, one associated with Sp1 and the other with NF-1. So far, only one replicative DNA polymerase B-subunit gene promoter, POLA2 encoding the B-subunit of DNA polymerase α, has been characterized. Mitogenic activation of the POLE2 promoter by an E2F-mediated mechanism resembles that of POLA2, but the regulation of basal promoter activity is different between these two genes. PMID:11433027

  16. PSMB4 promotes multiple myeloma cell growth by activating NF-κB-miR-21 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Peihao; Guo, Honggang [Department of Hematology, Navy General Hospital, Beijing 100048 (China); Li, Guangchao [School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006 (China); Han, Siqi [Department of Medical Oncology, Jinling Hospital, Nanjing 210002 (China); Luo, Fei [Department of Stomatology, Jinling Hospital, Nanjing 210002 (China); Liu, Yi, E-mail: liuyi2033@163.com [Department of Hematology, Navy General Hospital, Beijing 100048 (China)

    2015-03-06

    Proteasomal subunit PSMB4, was recently identified as potential cancer driver genes in several tumors. However, the regulatory mechanism of PSMB4 on carcinogenesis process remains unclear. In this study, we investigated the expression and roles of PSMB4 in multiple myeloma (MM). We found a significant up-regulation of PSMB4 in MM plasma and cell lines. Ectopic overexpression of PSMB4 promoted cell growth and colony forming ability of MM cells, whereas inhibition of PSMB4 led to a decrease of such events. Furthermore, our results demonstrated the up-regulation of miR-21 and a positive correlation between the levels of miR-21 and PSMB4 in MM. Re-expression of miR-21 markedly rescued PSMB4 knockdown-mediated suppression of cell proliferation and clone-formation. Additionally, while enforced expression of PSMB4 profoundly increased NF-κB activity and the level of miR-21, PSMB4 knockdown or NF-κB inhibition suppressed miR-21 expression in MM cells. Taken together, our results demonstrated that PSMB4 regulated MM cell growth in part by activating NF-κB-miR-21 signaling, which may represent promising targets for novel specific therapies. - Highlights: • First reported upregulation of PSMB4 in MM plasma and cell lines. • PSMB4 promoted MM cell growth and colony forming ability. • Further found miR-21 was up-regulated by PSMB4 in MM plasma and cell lines. • PSMB4-induced miR-21 expression was modulated by NF-κB. • PSMB4-NF-κB-miR-21 axis may be potential therapeutic targets of MM.

  17. Sphingosine kinase 1 mediates diabetic renal fibrosis via NF-κB signaling pathway: involvement of CK2α.

    Science.gov (United States)

    Huang, Junying; Li, Jingyan; Chen, Zhiquan; Li, Jie; Chen, Qiuhong; Gong, Wenyan; Liu, Peiqing; Huang, Heqing

    2017-10-24

    Sphingosine kinase 1 (SphK1) plays a pivotal role in regulating diabetic renal fibrotic factors such as fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1). Especially, activation of SphK1 is closely linked to the body inflammatory reaction. Casein kinase 2α subunit (CK2α), a protein kinase related to inflammatory reaction, influences diabetic renal fibrosis and expressions of FN and ICAM-1 via NF-κB pathway. However, the mechanism by which SphK1 mediates diabetic renal fibrosis has not yet fully elucidated. The current study is aimed to investigate if SphK1 mediates diabetic renal fibrotic pathological process via inflammatory pathway and activation of CK2α. The following findings were observed: (1) Expressions of SphK1 were upregulated in kidneys of diabetic mice and rats; (2) Knockdown of SphK1 expression suppressed high glucose (HG)-induced NF-κB nuclear translocation and expressions of FN and ICAM-1; (3) Compared with C57 diabetic mice, SphK1 -/- diabetic mice exhibited less renal fibrotic lesions, FN accumulation and NF-κB nuclear accumulation in glomeruli of kidneys; (4) SphK1 mediated phosphorylation of CK2α, while CK2α knockdown depressed SphK1-induced activation of NF-κB pathway. This study indicates the essential role of SphK1 in regulating activation of CK2α and diabetic renal fibrotic pathological process via NF-κB.

  18. 27-Oxygenated cholesterol induces expression of CXCL8 in macrophages via NF-κB and CD88

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun-Mi, E-mail: lala1647@hanmail.net [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Lee, Chung Won, E-mail: vasculardoctorlee@gmail.com [Department of Thoracic and Cardiovascular Surgery, Pusan National University Hospital, Pusan 602-739 (Korea, Republic of); Kim, Bo-Young, E-mail: kimboyoung@pusan.ac.kr [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Jung, Young-Suk, E-mail: youngjung@pusan.ac.kr [College of Pharmacy, Pusan National University, Busan 609-735 (Korea, Republic of); Eo, Seong-Kug, E-mail: vetvirus@chonbuk.ac.kr [College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, Jeonbuk 570-752 (Korea, Republic of); Park, Young Chul, E-mail: ycpark@pusan.ac.kr [Department of Microbiology & Immunology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Kim, Koanhoi, E-mail: koanhoi@pusan.ac.kr [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of)

    2015-08-07

    We attempted to determine the effects of a milieu rich in cholesterol molecules on expression of chemokine CXCL8. A high-cholesterol diet led to an increased transcription of the IL-8 gene in the arteries and elevated levels of CXCL8 in sera of ApoE{sup −/−} mice, compared with those of wild-type C57BL/6 mice. Treatment of THP-1 monocyte/macrophage cells with 27-hydroxycholesterol (27OHChol) resulted in transcription of the IL-8 gene and increased secretion of its corresponding gene product whereas cholesterol did not induce expression of CXCL8 in THP-1 cells. 27OHChol-induced transcription of the IL-8 gene was blocked by cycloheximide, but not by polymyxin B. Treatment of THP-1 cells with 27OHChol caused translocation of p65 NF-κB subunit into the nucleus and up-regulation of CD88. Inhibition of NF-κB and CD88 using SN50 and W-54011, respectively, resulted in reduced transcription of the IL-8 gene and attenuated secretion of CXCL8 induced by 27OHChol. We propose that oxidatively modified cholesterol like 27OHChol, rather than cholesterol, is responsible for sustained expression of CXCL8 in monocytes/macrophages in atherosclerotic arteries. - Highlights: • Consumption of a high-cholesterol diet leads to increased CXCL8 expression in ApoE{sup −/−} mice. • 27OHChol, but not cholesterol, up-regulates expression of CXCL8 in macrophages. • 27OHChol enhances nuclear translocation of NF-κB and expression of CD88 in macrophages. • Inhibition of NF-κB or CD88 results in decreased CXCL8 expression induced by 27OHChol. • 27OHChol up-regulates CXCL8 expression via NF-κB and CD88 in macrophages.

  19. Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory protein K13 activates NF-κB pathway independent of TRAF6, TAK1 and LUBAC.

    Directory of Open Access Journals (Sweden)

    Hittu Matta

    Full Text Available Kaposi's sarcoma associated herpesvirus encoded viral FLICE inhibitory protein (vFLIP K13 activates the NF-κB pathway by binding to the NEMO/IKKγ subunit of the IκB kinase (IKK complex. However, it has remained enigmatic how K13-NEMO interaction results in the activation of the IKK complex. Recent studies have implicated TRAF6, TAK1 and linear ubiquitin chains assembled by a linear ubiquitin chain assembly complex (LUBAC consisting of HOIL-1, HOIP and SHARPIN in IKK activation by proinflammatory cytokines.Here we demonstrate that K13-induced NF-κB DNA binding and transcriptional activities are not impaired in cells derived from mice with targeted disruption of TRAF6, TAK1 and HOIL-1 genes and in cells derived from mice with chronic proliferative dermatitis (cpdm, which have mutation in the Sharpin gene (Sharpin(cpdm/cpdm. Furthermore, reconstitution of NEMO-deficient murine embryonic fibroblast cells with NEMO mutants that are incapable of binding to linear ubiquitin chains supported K13-induced NF-κB activity. K13-induced NF-κB activity was not blocked by CYLD, a deubiquitylating enzyme that can cleave linear and Lys63-linked ubiquitin chains. On the other hand, NEMO was required for interaction of K13 with IKK1/IKKα and IKK2/IKKβ, which resulted in their activation by "T Loop" phosphorylation.Our results demonstrate that K13 activates the NF-κB pathway by binding to NEMO which results in the recruitment of IKK1/IKKα and IKK2/IKKβ and their subsequent activation by phosphorylation. Thus, K13 activates NF-κB via a mechanism distinct from that utilized by inflammatory cytokines. These results have important implications for the development of therapeutic agents targeting K13-induced NF-κB for the treatment of KSHV-associated malignancies.

  20. Optimized subunit vaccine protects against experimental leishmaniasis.

    Science.gov (United States)

    Bertholet, Sylvie; Goto, Yasuyuki; Carter, Lauren; Bhatia, Ajay; Howard, Randall F; Carter, Darrick; Coler, Rhea N; Vedvick, Thomas S; Reed, Steven G

    2009-11-23

    Development of a protective subunit vaccine against Leishmania spp. depends on antigens and adjuvants that induce appropriate immune responses. We evaluated a second generation polyprotein antigen (Leish-110f) in different adjuvant formulations for immunogenicity and protective efficacy against Leishmania spp. challenges. Vaccine-induced protection was associated with antibody and T cell responses to Leish-110f. CD4 T cells were the source of IFN-gamma, TNF, and IL-2 double- and triple-positive populations. This study establishes the immunogenicity and protective efficacy of the improved Leish-110f subunit vaccine antigen adjuvanted with natural (MPL-SE) or synthetic (EM005) Toll-like receptor 4 agonists.

  1. Changes of protein oxidation, calpain and cytoskeletal proteins (alpha tubulin and pNF-H) levels in rat brain after nerve agent poisoning.

    Science.gov (United States)

    RamaRao, Golime; Acharya, J N; Bhattacharya, B K

    2011-06-24

    Highly toxic organophosphorus (OP) nerve agents, sarin and soman act by inhibiting acetylcholinesterase (AChE) function at neuronal synapses and cause many toxic effects including death within minutes. The effect of nerve agents on protein oxidation, calpain, and cytoskeletal protein levels was not well known. In the present study we investigated these parameters after subcutaneous injection of sarin (120 μg/kg) and soman (80 μg/kg) in the rat brain. Results indicate that several rat brain proteins were intensely oxidized after nerve agent poisoning. Immunoreactivity levels of μ-calpain were significantly elevated in cerebral cortex and cerebellum regions of rat brain from 2.5 h to 30 days. Alpha tubulin levels reduced from 1 to 7 days in the supernatant and 1 to 3 days in the pellet fractions of cerebellum and cerebral cortex, where as phosphorylation of high molecular weight neurofilament (pNF-H) was increased significantly in nerve agent intoxicated rat brains as compared to control rats. AChE activity was inhibited up to 3 days after nerve agent exposure in plasma and brain. Results suggest that altered protein oxidation, calpain and cytoskeletal protein levels are due to multiple mechanisms of nerve agents actions and these changes might be involved in nerve agent induced complex neurotoxicity. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  2. Eudesmane-Type Sesquiterpene Lactones Inhibit Nuclear Translocation of the Nuclear Factor κB Subunit RelB in Response to a Lymphotoxin β Stimulation.

    Science.gov (United States)

    Quach, Hue Tu; Kondo, Tetsuya; Watanabe, Megumi; Tamura, Ryuichi; Yajima, Yoshiki; Sayama, Shinsei; Ando, Masayoshi; Kataoka, Takao

    2017-01-01

    The transcription factor nuclear factor κB (NF-κB) regulates various biological processes, including inflammatory responses. We previously reported that eudesmane-type sesquiterpene lactones inhibited multiple steps in the canonical NF-κB signaling pathway induced by tumor necrosis factor-α and interleukin-1α. In contrast, the biological activities of eudesmane-type sesquiterpene lactones on the non-canonical NF-κB signaling pathway remain unclear. In the present study, we found that (11S)-2α-bromo-3-oxoeudesmano-12,6α-lactone, designated santonin-related compound 2 (SRC2), inhibited NF-κB luciferase reporter activity induced by lymphotoxin β (LTβ) in human lung carcinoma A549 cells. Although SRC2 did not prevent the processing of the NF-κB subunit p100 induced by LTβ, it inhibited the nuclear translocation of RelB and p52 in response to the LTβ stimulation. In contrast to (-)-dehydroxymethylepoxyquinomicin, SRC2 inhibited the LTβ-induced nuclear translocation of the RelB (C144S) mutant in a manner similar to wild-type RelB. While eudesmane derivatives possessing an α-bromoketone moiety or α,β-unsaturated carbonyl moieties inhibited LTβ-induced NF-κB luciferase reporter activity, eudesmane derivatives possessing an α-bromoketone moiety exhibited stronger inhibitory activity on the LTβ-induced nuclear translocation of RelB than those possessing a single α-methylene-γ-lactone moiety. The results of the present study revealed that SRC2 inhibits the nuclear translocation of RelB in the non-canonical NF-κB signaling pathway induced by LTβ.

  3. Retrograde and transganglionic transport of horseradish peroxidase-conjugated cholera toxin B subunit, wheatgerm agglutinin and isolectin B4 from Griffonia simplicifolia I in primary afferent neurons innervating the rat urinary bladder.

    Science.gov (United States)

    Wang, H F; Shortland, P; Park, M J; Grant, G

    1998-11-01

    In the present study, we investigated and compared the ability of the cholera toxin B subunit, wheat germ agglutinin and isolectin B4 from Griffonia simplicifolia I conjugated to horseradish peroxidase, to retrogradely and transganglionically label visceral primary afferents after unilateral injections into the rat urinary bladder wall. Horseradish peroxidase histochemical or lectin-immunofluorescence histochemical labelling of bladder afferents was seen in the L6-S1 spinal cord segments and in the T13-L2 and L6-S1 dorsal root ganglia. In the lumbosacral spinal cord, the most intense and extensive labelling of bladder afferents was seen when cholera toxin B subunit-horseradish peroxidase was injected. Cholera toxin B subunit-horseradish peroxidase-labelled fibres were found in Lissauer's tract, its lateral and medial collateral projections, and laminae I and IV-VI of the spinal gray matter. Labelled fibres were numerous in the lateral collateral projection and extended into the spinal parasympathetic nucleus. Labelling from both the lateral and medial projections extended into the dorsal grey commissural region. Wheat germ agglutinin-horseradish peroxidase labelling produced a similar pattern but was not as dense and extensive as that of cholera toxin B subunit-horseradish peroxidase. The isolectin B4 from Griffonia simplicifolia I-horseradish peroxidase-labelled fibres, on the other hand, were fewer and only observed in the lateral collateral projection and occasionally in lamina I. Cell profile counts showed that a larger number of dorsal root ganglion cells were labelled with cholera toxin B subunit-horseradish peroxidase than with wheat germ agglutinin- or isolectin B4-horseradish peroxidase. In the L6-S1 dorsal root ganglia, the majority (81%) of the cholera toxin B subunit-, and almost all of the wheat germ agglutinin- and isolectin B4-immunoreactive cells were RT97-negative (an anti-neurofilament antibody that labels dorsal root ganglion neurons with

  4. Human stem cell modeling in neurofibromatosis type 1 (NF1).

    Science.gov (United States)

    Wegscheid, Michelle L; Anastasaki, Corina; Gutmann, David H

    2017-04-06

    The future of precision medicine is heavily reliant on the use of human tissues to identify the key determinants that account for differences between individuals with the same disorder. This need is exemplified by the neurofibromatosis type 1 (NF1) neurogenetic condition. As such, individuals with NF1 are born with a germline mutation in the NF1 gene, but may develop numerous distinct neurological problems, ranging from autism and attention deficit to brain and peripheral nerve sheath tumors. Coupled with accurate preclinical mouse models, the availability of NF1 patient-derived induced pluripotent stem cells (iPSCs) provides new opportunities to define the critical factors that underlie NF1-associated nervous system disease pathogenesis and progression. In this review, we discuss the generation and potential applications of iPSC technology to the study of NF1. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Targeting Signaling to YAP for the Therapy of NF2

    Science.gov (United States)

    2015-10-01

    VIII cranial nerve, but also meningiomas and ependymomas. The only effective current therapy for NF2 involves repeated and debilitating surgeries...clear mechanism of Merlin’s tumor suppressor function . Our studies have shown that inactivation of Merlin/NF2 de-regulates the E3 ubiquitin ligase...indicated that this reported functions as anticipated in response to overexpressed YAP. We therefore generated several reporter-expressing NF2 mutant cell

  6. Cautionary notes on the use of NF-κB p65 and p50 antibodies for CNS studies

    Directory of Open Access Journals (Sweden)

    Listwak Samuel J

    2011-10-01

    with antibodies that are not selective for the NF-κB subunit proteins p65 and p50. The data urge caution in interpreting studies of neuronal NF-κB activity in the brain.

  7. Recent operational experiences of FILMTEC NF270 membrane in Europe.

    Science.gov (United States)

    Majamaa, Katariina; Warczok, Justyna; Lehtinen, Martti

    2011-01-01

    Nanofiltration (NF) is an attractive technology for potable and industrial water treatment because NF operates between ultrafiltration (UF) and reverse osmosis (RO) membranes. NF is designed to remove a high percentage of organic contaminants (humic acids, pesticides, color bodies) while passing a medium to high percentage of salt. Compared to UF membranes, the NF product water quality is significantly better; compared to comparable RO treatment systems NF systems require lower operating pressures. Due to these features, NF is increasingly used in a broad range of water treatment applications. The general applications include softening, as well as color, organics and micro-organism removal. DOW FILMTEC NF270-400 is one the most frequently used elements in water treatment and this paper presents examples of three recent NF270-400 installations in Europe. The first two plants, Eupen and Stembert, are located in Belgium and produce potable water from surface water. The third one is a Scandinavian plant which purifies groundwater for a brewery and soft drink production. The presented operation results prove NF to be a highly competitive technique for low cost water treatment.

  8. The Activation of IL-1-Induced Enhancers Depends on TAK1 Kinase Activity and NF-κB p65

    Directory of Open Access Journals (Sweden)

    Liane Jurida

    2015-02-01

    Full Text Available The inflammatory gene response requires activation of the protein kinase TAK1, but it is currently unknown how TAK1-derived signals coordinate transcriptional programs in the genome. We determined the genome-wide binding of the TAK1-controlled NF-κB subunit p65 in relation to active enhancers and promoters of transcribed genes by chromatin immunoprecipitation sequencing (ChIP-seq experiments. Out of 35,000 active enhancer regions, 410 H3K4me1-positive enhancers show interleukin 1 (IL-1-induced H3K27ac and p65 binding. Inhibition of TAK1 or IKK2 or depletion of p65 blocked inducible enhancer activation and gene expression. As exemplified by the CXC chemokine cluster located on chromosome 4, the TAK1-p65 pathway also regulates the recruitment kinetics of the histone acetyltransferase CBP, of NF-κB p50, and of AP-1 transcription factors to both promoters and enhancers. This study provides a high-resolution view of epigenetic changes occurring during the IL-1 response and allows the genome-wide identification of a distinct class of inducible p65 NF-κB-dependent enhancers in epithelial cells.

  9. Quercetin attenuates atherosclerotic inflammation and adhesion molecule expression by modulating TLR-NF-κB signaling pathway.

    Science.gov (United States)

    Bhaskar, Shobha; Sudhakaran, P R; Helen, A

    2016-12-01

    Adhesion molecules expressed by activated endothelial cells play key role in regulating leukocyte trafficking to sites of inflammation. The present study attempted to explore whether the polyphenolic flavonoid quercetin influence leukocyte endothelial attraction and the involvement of TLR-NF-κB signaling pathway in the expression of adhesion molecules involved in the early development of atherosclerosis. Quercetin at 25μM concentration significantly reduced the HUVEC expression of VCAM-1 and ICAM-1 evidently enhanced by oxLDL. In addition, quercetin significantly downregulated the mRNA expression of MCP-1 and alleviated the nuclear translocation of NF-κB p65 subunit in oxLDL induced HUVECs. Western blot and PCR analyses revealed that quercetin significantly attenuated the expression of both protein and mRNA expression of TLR2 and TLR4. Quercetin supplementation significantly decreased the inflammatory mediators like COX, 5-LOX, MPO, NOS, CRP and the mRNA expression of the cytokine; IL-6 in hypercholesterolemic diet (HCD) fed atherosclerotic rats. The results demonstrate that quercetin is effective to regulate the atherosclerotic inflammatory process by inhibiting oxLDL induced endothelial leukocyte adhesion by attenuating the TLR-NF-κB signaling pathway in endothelial cells and decrease the inflammatory process induced by HCD in rats. Therefore, quercetin acts as anti-inflammatory and anti-atherogenic agent, which may have implications for strategies attenuating endothelial dysfunction-related atherosclerosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. The acetyltransferase HAT1 moderates the NF-κB response by regulating the transcription factor PLZF.

    Science.gov (United States)

    Sadler, Anthony J; Suliman, Bandar A; Yu, Liang; Yuan, Xiangliang; Wang, Die; Irving, Aaron T; Sarvestani, Soroush T; Banerjee, Ashish; Mansell, Ashley S; Liu, Jun-Ping; Gerondakis, Steve; Williams, Bryan R G; Xu, Dakang

    2015-04-13

    To date, the activities of protein kinases have formed the core of our understanding of cell signal transduction. Comprehension of the extent of protein acetylation has raised expectations that this alternate post-transcriptional modification will be shown to rival phosphorylation in its importance in mediating cellular responses. However, limited instances have been identified. Here we show that signalling from Toll-like or TNF-α receptors triggers the calcium/calmodulin-dependent protein kinase (CaMK2) to activate histone acetyltransferase-1 (HAT1), which then acetylates the transcriptional regulator PLZF. Acetylation of PLZF promotes the assembly of a repressor complex incorporating HDAC3 and the NF-κB p50 subunit that limits the NF-κB response. Accordingly, diminishing the activity of CaMK2, the expression levels of PLZF or HAT1, or mutating key residues that are covalently modified in PLZF and HAT1, curtails control of the production of inflammatory cytokines. These results identify a central role for acetylation in controlling the inflammatory NF-κB transcriptional programme.

  11. Sirtuin 6 prevents matrix degradation through inhibition of the NF-κB pathway in intervertebral disc degeneration

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Liang [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hu, Jia [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Weng, Yuxiong [Department of Hand Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Jia, Jie [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Zhang, Yukun, E-mail: zhangyukuncom@126.com [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China)

    2017-03-15

    Intervertebral disc degeneration (IDD) is marked by imbalanced metabolism of the extracellular matrix (ECM) in the nucleus pulposus (NP) of intervertebral discs. This study aimed to determine whether sirtuin 6 (SIRT6), a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases, protects the NP from ECM degradation in IDD. Our study showed that expression of SIRT6 markedly decreased during IDD progression. Overexpression of wild-type SIRT6, but not a catalytically inactive mutant, prevented IL-1β-induced NP ECM degradation. SIRT6 depletion by RNA interference in NP cells caused ECM degradation. Moreover, SIRT6 physically interacted with nuclear factor-κB (NF-κB) catalytic subunit p65, transcriptional activity of which was significantly suppressed by SIRT6 overexpression. These results suggest that SIRT6 prevented NP ECM degradation in vitro via inhibiting NF-κB-dependent transcriptional activity and that this effect depended on its deacetylase activity. - Highlights: • SIRT6 expression is decreased in degenerative nucleus pulposus (NP) tissues. • SIRT6 overexpression lowers IL-1β-induced matrix degradation of NP. • SIRT6 inhibition induces matrix degradation of NP. • SIRT6 prevents matrix degradation of NP via the NF-κB signaling pathway.

  12. Ethanol Decreases Inflammatory Response in Human Lung Epithelial Cells by Inhibiting the Canonical NF-kB-Pathway

    Directory of Open Access Journals (Sweden)

    Katharina Mörs

    2017-08-01

    Full Text Available Background/Aims: Alcohol (ethanol, EtOH as significant contributor to traumatic injury is linked to suppressed inflammatory response, thereby influencing clinical outcomes. Alcohol-induced immune-suppression during acute inflammation (trauma was linked to nuclear factor-kappaB (NF-ĸB. Here, we analyzed alcohol`s effects and mechanisms underlying its influence on NF-ĸB-signaling during acute inflammation in human lung epithelial cells. Methods: A549-cells were stimulated with interleukin (IL-1β, or sera from trauma patients (TP or healthy volunteers, with positive/negative blood alcohol concentrations (BAC, and subsequently exposed to EtOH (170 Mm, 1h. IL-6-release and neutrophil adhesion to A549 were analyzed. Specific siRNA-NIK mediated downregulation of non-canonical, and IKK-NBD-inhibition of canonical NF-ĸB signaling were performed. Nuclear levels of activated p50 and p52 NF-ĸB-subunits were detected using TransAm ELISA. Results: Both stimuli significantly induced IL-6-release (39.79±4.70 vs. 0.58±0.8 pg/ml and neutrophil adhesion (132.30±8.80 vs. 100% control, p<0.05 to A549-cells. EtOH significantly decreased IL-6-release (22.90±5.40, p<0.05 and neutrophil adherence vs. controls (105.40±14.5%, p<0.05. IL-1β-induced significant activation of canonical/p50 and non-canonical/p52 pathways. EtOH significantly reduced p50 (34.90±23.70 vs. 197.70±36.43, p<0.05 not p52 activation. Inhibition of canonical pathway was further increased by EtOH (less p50-activation, while p52 remained unaltered. Inhibition of non-canonical pathway was unchanged by EtOH. Conclusion: Here, alcohol`s anti-inflammatory effects are mediated via decreasing nuclear levels of activated p50-subunit and canonical NF-ĸB signaling pathway.

  13. Structural and functional analysis of the related transcriptional enhancer factor-1 and NF-κB interaction.

    Science.gov (United States)

    Ma, Jieliang; Zhang, Li; Tipton, Aaron R; Wu, Jiaping; Messmer-Blust, Angela F; Philbrick, Melissa J; Qi, Yajuan; Liu, Song-Tao; Liu, Hongsheng; Li, Jian; Guo, Shaodong

    2014-01-15

    The related transcriptional enhancer factor-1 (RTEF-1) increases gene transcription of hypoxia-inducible factor 1α (HIF-1α) and enhances angiogenesis in endothelium. Both hypoxia and inflammatory factor TNF-α regulate gene expression of HIF-1α, but how RTEF-1 and TNF-α coordinately regulate HIF-1α gene transcription is unclear. Here, we found that RTEF-1 interacts with p65 subunit of NF-κB, a primary mediator of TNF-α. RTEF-1 increased HIF-1α promoter activity, whereas expression of p65 subunit inhibited the stimulatory effect. By contrast, knockdown of p65 markedly enhanced RTEF-1 stimulation on the HIF-1α promoter activity (7-fold). A physical interaction between RTEF-1 and p65 was confirmed by coimmunoprecipitation experiments in cells and glutathione S-transferase (GST)-pull-down assays. A computational analysis of RTEF-1 crystal structures revealed that a conserved surface of RTEF-1 potentially interacts with p65 via four amino acid residues located at T347, Y349, R351, and Y352. We performed site-directed mutagenesis and GST-pull-down assays and demonstrated that Tyr352 (Y352) in RTEF-1 is a key site for the formation of RTEF-1 and p65-NF-κB complex. An alanine mutation at Y352 of RTEF-1 disrupted the interaction of RTEF-1 with p65. Moreover, expression of RTEF-1 decreased TNF-α-induced HIF-1α promoter activity, IL-1β, and IL-6 mRNA levels in cells; however, the effect of RTEF-1 was largely lost when Y352 was mutated to alanine. These results indicate that RTEF-1 interacts with p65-NF-κB through Y352 and that they antagonize each other for HIF-1α transcriptional activation, suggesting a novel mechanism by which RTEF-1 regulates gene expression, linking hypoxia to inflammation.

  14. Improved spatial learning and memory by perilla diet is correlated with immunoreactivities to neurofilament and α-synuclein in hilus of dentate gyrus

    Directory of Open Access Journals (Sweden)

    Lee Jinwoo

    2012-12-01

    Full Text Available Abstract Background Perilla (Perilla frutescens oil is very rich in α-linolenic acid, an omega-3 fatty acid. As it is widely reported that omega-3 fatty acid supplementation improves cognitive function in children and adults, feeding rats with perilla diets followed by analysis of proteomic changes in the hippocampus can provide valuable information on the mechanism of learning and memory at the molecular level. To identify proteins playing roles in learning and memory, differentially expressed proteins in the hippocampus of the 5 week old rats fed perilla diets for 3 weeks or 3 months were identified by proteomic analysis and validated by immunological assays. Results The perilla diet groups showed improved spatial learning and memory performances in a T-maze test. They also displayed elevated level of 22:6n-3 fatty acid, an omega-3 fatty acid (p Conclusion Improved cognitive function upon administration of n-3 fatty acid-rich perilla diet is associated with the differential expression of hippocampal proteins related to cytoskeleton, energy metabolism, transport, neuro-projection, and apoptosis. Particularly, the enhanced immunoreactivities to α-synuclein and neurofilament in the hilus of dentate gyrus suggest that perilla diet supplementation promotes neuronal signaling and alters synaptic plasticity for improved learning and memory.

  15. Selective neurofilament (SMI-32, FNP-7 and N200) expression in subpopulations of layer V pyramidal neurons in vivo and in vitro.

    Science.gov (United States)

    Voelker, Courtney C J; Garin, Nathalie; Taylor, Jeremy S H; Gähwiler, Beat H; Hornung, Jean-Pierre; Molnár, Zoltán

    2004-11-01

    There are two main types of layer V pyramidal neurons in rat cortex. Type I neurons have tufted apical dendrites extending into layer I, produce bursts of action potentials and project to subcortical targets (spinal cord, superior colliculus and pontine nuclei). Type II neurons have apical dendrites, which arborize in layers II-IV, do not produce bursts of action potentials and project to ipsilateral and contralateral cortex. The specific expression of different genes and proteins in these two distinct layer V neurons is unknown. To distinguish between distinct subpopulations, fluorescent microspheres were injected into subcortical targets (labeling type I neurons) or primary somatosensory cortex (labeling type II neurons) of adult rats. After transport, cortical sections were processed for immunohistochemistry using various antibodies. This study demonstrated that antigens recognized by SMI-32, N200 and FNP-7 antibodies were only expressed in subcortical (type I)--but not in contralateral (type II)--projecting neurons. NR1, NR2a/b, PLCbeta1, BDNF, NGF and TrkB antigens were highly expressed in all neuronal subpopulations examined. Organotypic culture experiments demonstrated that the development of neurofilament expression and laminar specificity does not depend on the presence of the subcortical targets. This study suggests specific markers for the subcortical projecting layer V neuron subpopulations.

  16. Delayed nerve stimulation promotes axon-protective neurofilament phosphorylation, accelerates immune cell clearance and enhances remyelination in vivo in focally demyelinated nerves.

    Directory of Open Access Journals (Sweden)

    Nikki A McLean

    Full Text Available Rapid and efficient axon remyelination aids in restoring strong electrochemical communication with end organs and in preventing axonal degeneration often observed in demyelinating neuropathies. The signals from axons that can trigger more effective remyelination in vivo are still being elucidated. Here we report the remarkable effect of delayed brief electrical nerve stimulation (ES; 1 hour @ 20 Hz 5 days post-demyelination on ensuing reparative events in a focally demyelinated adult rat peripheral nerve. ES impacted many parameters underlying successful remyelination. It effected increased neurofilament expression and phosphorylation, both implicated in axon protection. ES increased expression of myelin basic protein (MBP and promoted node of Ranvier re-organization, both of which coincided with the early reappearance of remyelinated axons, effects not observed at the same time points in non-stimulated demyelinated nerves. The improved ES-associated remyelination was accompanied by enhanced clearance of ED-1 positive macrophages and attenuation of glial fibrillary acidic protein expression in accompanying Schwann cells, suggesting a more rapid clearance of myelin debris and return of Schwann cells to a nonreactive myelinating state. These benefits of ES correlated with increased levels of brain derived neurotrophic factor (BDNF in the acute demyelination zone, a key molecule in the initiation of the myelination program. In conclusion, the tremendous impact of delayed brief nerve stimulation on enhancement of the innate capacity of a focally demyelinated nerve to successfully remyelinate identifies manipulation of this axis as a novel therapeutic target for demyelinating pathologies.

  17. The Accomplices of NF-κB Lead to Radioresistance.

    Science.gov (United States)

    Bai, Miaomiao; Ma, Xiaoxing; Li, Xiaolei; Wang, Xiaohui; Mei, Qian; Li, Xiang; Wu, Zhiqiang; Han, Weidong

    2015-01-01

    Ionizing radiation (IR) plays an important role in the treatment of epithelial tumors, such as lung and prostate cancer, by wounding and killing cancer cells. However, IR also activates sophisticated anti-apoptotic transcriptional factors such that cancer cells fail to repair DNA damage and obtain resistance to apoptosis under conditions of radiotherapy. Among these transcription factors, the transcription factor nuclear factor kappa B (NF-κB) is recognized as a key feature for protecting cells from apoptosis in most cell types. Moreover, the induction of radioresistance is mediated by several genes that are regulated by NF- κB. The primary purpose of this review is to introduce the studies of the signaling mechanisms of IR in NF-κB activation, such as ROS/NF-κB, ATM or DNA-PK/MAPKK/ p90rsk, PI3K/AKT/IKK and k-ras/c-raf/ MEKK/ NF-κB pathways. Moreover, we describe how the expression of the target genes (e.g., XIAP, A20, FLIP, Bcl-xL) are induced by NF-κB to regulate the activation of survival signaling pathways and to inhibit apoptotic signaling pathways. In addition, IR activates NF-κB to express cell cycle-specific genes, for example cyclin D1, which is associated with reinforcing radioresistance. We exhibit the signaling pathways that are induced by IR stimulation of NF-κB and illustrate the molecular mechanisms of radioresistance.

  18. [NF-kappaB tumorigenesis and drug developement].

    Science.gov (United States)

    Liu, Wei-Feng; Yu, Shan-Shan; Li, Yue-Zhong

    2005-01-01

    NF-kappaB, a collective name of dimeric transcription factors, is composed of members of the Rel family proteins that recognize and bind a specific DNA sequence. It is normally sequestered in the cytoplasm of non-stimulated cells by associating with a family of inhibitor proteins called IkappaBs. Exposure of cells to a variety of extra-and intra-cellular stimuli leads to the rapid proteolytic degradation of IkappaBs, which frees NF-kappaBs allowing them to translocate to the nucleus where it regulates gene transcription. NF-kappaB is involved in a lot of physiological processes such as immunity, inflammation, cell proliferation, apoptosis and even tumorigenesis by regulating the transcription of a larger number of genes. This review introduces the various mechanisms of NF-kappaB activation including a recently reported alternative activation pathway mediated by lymphotoxin alpha/beta, B cell activating factor and CD40 ligand. The signal transduction pathway leading to NF-kappaB activation via IKK in response to proinflammatory factors like TNF-alpha and IL-1 is addressed in more detail concerning the regulation of IKK activity, mechanism of IkappaB degradation and regulation of transactivation activity of NF-kappaB on different levels. Considering the important role of NF-kappaB in cell proliferation and regulation of various genes participating in apoptosis, the involvement of NF-kappaB in tumorigenesis and drug screening is also discussed.

  19. NF-κB Signaling in Gastric Cancer

    Science.gov (United States)

    Sokolova, Olga; Naumann, Michael

    2017-01-01

    Gastric cancer is a leading cause of cancer death worldwide. Diet, obesity, smoking and chronic infections, especially with Helicobacter pylori, contribute to stomach cancer development. H. pylori possesses a variety of virulence factors including encoded factors from the cytotoxin-associated gene pathogenicity island (cagPAI) or vacuolating cytotoxin A (VacA). Most of the cagPAI-encoded products form a type 4 secretion system (T4SS), a pilus-like macromolecular transporter, which translocates CagA into the cytoplasm of the host cell. Only H. pylori strains carrying the cagPAI induce the transcription factor NF-κB, but CagA and VacA are dispensable for direct NF-κB activation. NF-κB-driven gene products include cytokines/chemokines, growth factors, anti-apoptotic factors, angiogenesis regulators and metalloproteinases. Many of the genes transcribed by NF-κB promote gastric carcinogenesis. Since it has been shown that chemotherapy-caused cellular stress could elicit activation of the survival factor NF-κB, which leads to acquisition of chemoresistance, the NF-κB system is recommended for therapeutic targeting. Research is motivated for further search of predisposing conditions, diagnostic markers and efficient drugs to improve significantly the overall survival of patients. In this review, we provide an overview about mechanisms and consequences of NF-κB activation in gastric mucosa in order to understand the role of NF-κB in gastric carcinogenesis. PMID:28350359

  20. Atorvastatin Alleviates Experimental Diabetic Cardiomyopathy by Regulating the GSK-3β-PP2Ac-NF-κB Signaling Axis.

    Directory of Open Access Journals (Sweden)

    Xiao-Min Ren

    Full Text Available Recent studies reported that atorvastatin (ATOR alleviated progression of experimental diabetic cardiomyopathy (DCM, possibly by protecting against apoptosis. However, the underlying mechanisms of this protective effect remain unclear. Therefore, our study investigated the role of the glycogen synthase kinase (GSK-3β-protein phosphatase 2A(PP2A-NF-κB signaling pathway in the anti-apoptotic and cardioprotective effects of ATOR on cardiomyocytes cultured in high glucose (HG and in DCM. Our results showed that, in HG-cultured cardiomyocytes, phosphorylation of GSK-3β was decreased, while that of the PP2A catalytic subunit C (PP2Ac and IKK/IкBα was increased, followed by NF-кB nuclear translocation and apoptosis. IKK/IкBα phosphorylation and NF-кB nuclear translocation were also increased by treatment of cells with okadaic acid (OA, a selective PP2A inhibitor, or by silencing PP2Ac expression. The opposite results were obtained by silencing GSK-3β expression, which resulted in PP2Ac activation. Furthermore, IKK/IкBα phosphorylation and NF-кB nuclear translocation were markedly inhibited and apoptosis attenuated in cells treated with ATOR. These effects occurred through inactivation of GSK-3β and subsequent activation of PP2Ac. They were abolished by treatment of cells with OA or PP2Ac siRNA. In mice with type 1 diabetes mellitus, treatment with ATOR, at 10 mg-kg-1-d-1, significantly suppressed GSK-3β activation, IKK/IкBα phosphorylation, NF-кB nuclear translocation and caspase-3 activation, while also activating PP2Ac. Finally, improvements in histological abnormalities, fibrosis, apoptosis and cardiac dysfunction were observed in diabetic mice treated with ATOR. These findings demonstrated that ATOR protected against HG-induced apoptosis in cardiomyocytes and alleviated experimental DCM by regulating the GSK-3β-PP2A-NF-κB signaling pathway.

  1. NF-κB directly mediates epigenetic deregulation of common microRNAs in Epstein-Barr virus-mediated transformation of B-cells and in lymphomas.

    Science.gov (United States)

    Vento-Tormo, Roser; Rodríguez-Ubreva, Javier; Lisio, Lorena Di; Islam, Abul B M M K; Urquiza, Jose M; Hernando, Henar; López-Bigas, Nuria; Shannon-Lowe, Claire; Martínez, Nerea; Montes-Moreno, Santiago; Piris, Miguel A; Ballestar, Esteban

    2014-01-01

    MicroRNAs (miRNAs) have negative effects on gene expression and are major players in cell function in normal and pathological conditions. Epstein-Barr virus (EBV) infection of resting B lymphocytes results in their growth transformation and associates with different B cell lymphomas. EBV-mediated B cell transformation involves large changes in gene expression, including cellular miRNAs. We performed miRNA expression analysis in growth transformation of EBV-infected B cells. We observed predominant downregulation of miRNAs and upregulation of a few miRNAs. We observed similar profiles of miRNA expression in B cells stimulated with CD40L/IL-4, and those infected with EBNA-2- and LMP-1-deficient EBV particles, suggesting the implication of the NF-kB pathway, common to all four situations. In fact, the NF-kB subunit p65 associates with the transcription start site (TSS) of both upregulated and downregulated miRNAs following EBV infection This occurs together with changes at histone H3K27me3 and histone H3K4me3. Inhibition of the NF-kB pathway impairs changes in miRNA expression, NF-kB binding and changes at the above histone modifications near the TSS of these miRNA genes. Changes in expression of these miRNAs also occurred in diffuse large B cell lymphomas (DLBCL), which are strongly NF-kB dependent. Our results highlight the relevance of the NF-kB pathway in epigenetically mediated miRNA control in B cell transformation and DLBCL. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Morphine exacerbates HIV-1 Tat-induced cytokine production in astrocytes through convergent effects on [Ca(2+](i, NF-kappaB trafficking and transcription.

    Directory of Open Access Journals (Sweden)

    Nazira El-Hage

    Full Text Available Astroglia are key cellular sites where opiate drug signals converge with the proinflammatory effects of HIV-1 Tat signals to exacerbate HIV encephalitis. Despite this understanding, the molecular sites of convergence driving opiate-accelerated neuropathogenesis have not been deciphered. We therefore explored potential points of interaction between the signaling pathways initiated by HIV-1 Tat and opioids in striatal astrocytes. Profiling studies screening 152 transcription factors indicated that the nuclear factor-kappa B (NF-kappaB subunit, c-Rel, was a likely candidate for Tat or Tat plus opiate-induced increases in cytokine and chemokine production by astrocytes. Pretreatment with the NF-kappaB inhibitor parthenolide provided evidence that Tat+/-morphine-induced release of MCP-1, IL-6 and TNF-alpha by astrocytes is NF-kappaB dependent. The nuclear export inhibitor, leptomycin B, blocked the nucleocytoplasmic shuttling of NF-kappaB; causing p65 (RelA accumulation in the nucleus, and significantly attenuated cytokine production in Tat+/-morphine exposed astrocytes. Similarly, chelating intracellular calcium ([Ca(2+](i blocked Tat+/-morphine-evoked MCP-1 and IL-6 release, while artificially increasing the concentration of extracellular Ca(2+ reversed this effect. Taken together, these results demonstrate that: 1 exposure to Tat+/-morphine is sufficient to activate NF-kappaB and cytokine production, 2 the release of MCP-1 and IL-6 by Tat+/-morphine are highly Ca(2+-dependent, while TNF-alpha appears to be less affected by the changes in [Ca(2+](i, and 3 in the presence of Tat, exposure to opiates augments Tat-induced NF-kappaB activation and cytokine release through a Ca(2+-dependent pathway.

  3. NF1 germline mutation differentially dictates optic glioma formation and growth in neurofibromatosis-1

    OpenAIRE

    Toonen, Joseph A.; Anastasaki, Corina; Smithson, Laura J.; Gianino, Scott M.; Li, Kairong; Kesterson, Robert A.; Gutmann, David H.

    2016-01-01

    Neurofibromatosis type 1 (NF1) is a common neurogenetic condition characterized by significant clinical heterogeneity. A major barrier to developing precision medicine approaches for NF1 is an incomplete understanding of the factors that underlie its inherent variability. To determine the impact of the germline NF1 gene mutation on the optic gliomas frequently encountered in children with NF1, we developed genetically engineered mice harboring two representative NF1-patient-derived Nf1 gene m...

  4. Cleft Lip Repair: The Hybrid Subunit Method

    OpenAIRE

    Tollefson, TT

    2016-01-01

    Copyright © 2016 by Thieme Medical Publishers, Inc. The unilateral cleft lip repair is one of the most rewarding and challenging of plastic surgery procedures. Surgeons have introduced a variety of straight line, geometric, and rotation-advancement designs, while in practice the majority of North American surgeons have been using hybrids of the rotation-advancement techniques. The anatomic subunit approach was introduced in 2005 by Fisher and has gained popularity, with early adopters of the ...

  5. allelic variation of hmw glutenin subunits of ethiopian bread wheat ...

    African Journals Online (AJOL)

    journal

    reduced subunits of glutenin proteins bands are separated: the high molecular weight (HMW) and low molecular weight (LMW) subunits (Payne et al.,1980; Jackson et al., 1983). The HMW glutenin subunits (GS) of wheat protein are quantitatively minor, but functionally an important group of gluten proteins in the process of ...

  6. Cistrome-based Cooperation between Airway Epithelial Glucocorticoid Receptor and NF-κB Orchestrates Anti-inflammatory Effects.

    Science.gov (United States)

    Kadiyala, Vineela; Sasse, Sarah K; Altonsy, Mohammed O; Berman, Reena; Chu, Hong W; Phang, Tzu L; Gerber, Anthony N

    2016-06-10

    Antagonism of pro-inflammatory transcription factors by monomeric glucocorticoid receptor (GR) has long been viewed as central to glucocorticoid (GC) efficacy. However, the mechanisms and targets through which GCs exert therapeutic effects in diseases such as asthma remain incompletely understood. We previously defined a surprising cooperative interaction between GR and NF-κB that enhanced expression of A20 (TNFAIP3), a potent inhibitor of NF-κB. Here we extend this observation to establish that A20 is required for maximal cytokine repression by GCs. To ascertain the global extent of GR and NF-κB cooperation, we determined genome-wide occupancy of GR, the p65 subunit of NF-κB, and RNA polymerase II in airway epithelial cells treated with dexamethasone, TNF, or both using chromatin immunoprecipitation followed by deep sequencing. We found that GR recruits p65 to dimeric GR binding sites across the genome and discovered additional regulatory elements in which GR-p65 cooperation augments gene expression. GR targets regulated by this mechanism include key anti-inflammatory and injury response genes such as SERPINA1, which encodes α1 antitrypsin, and FOXP4, an inhibitor of mucus production. Although dexamethasone treatment reduced RNA polymerase II occupancy of TNF targets such as IL8 and TNFAIP2, we were unable to correlate specific binding sequences for GR or occupancy patterns with repressive effects on transcription. Our results suggest that cooperative anti-inflammatory gene regulation by GR and p65 contributes to GC efficacy, whereas tethering interactions between GR and p65 are not universally required for GC-based gene repression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. miR-486 sustains NF-κB activity by disrupting multiple NF-κB-negative feedback loops

    National Research Council Canada - National Science Library

    Song, Libing; Lin, Chuyong; Gong, Hui; Wang, Chanjuan; Liu, Liping; Wu, Jueheng; Tao, Sha; Hu, Bo; Cheng, Shi-Yuan; Li, Mengfeng; Li, Jun

    2013-01-01

    .... Herein, we report that miR-486 directly suppresses NF-κB-negative regulators, CYLD and Cezanne, as well as multiple A20 activity regulators, including ITCH, TNIP-1, TNIP-2 and TNIP-3, resulting in promotion of ubiquitin conjugations in NF-κ...

  8. Mosaic type-1 NF1 microdeletions as a cause of both generalized and segmental neurofibromatosis type-1 (NF1).

    Science.gov (United States)

    Messiaen, Ludwine; Vogt, Julia; Bengesser, Kathrin; Fu, Chuanhua; Mikhail, Fady; Serra, Eduard; Garcia-Linares, Carles; Cooper, David N; Lazaro, Conxi; Kehrer-Sawatzki, Hildegard

    2011-02-01

    Mosaicism is an important feature of type-1 neurofibromatosis (NF1) on account of its impact upon both clinical manifestations and transmission risk. Using FISH and MLPA to screen 3500 NF1 patients, we identified 146 individuals harboring gross NF1 deletions, 14 of whom (9.6%) displayed somatic mosaicism. The high rate of mosaicism in patients with NF1 deletions supports the postulated idea of a direct relationship between the high new mutation rate in this cancer predisposition syndrome and the frequency of mosaicism. Seven of the 14 mosaic NF1 deletions were type-2, whereas four were putatively type-1, and three were atypical. Two of the four probable type-1 deletions were confirmed as such by breakpoint-spanning PCR or SNP analysis. Both deletions were associated with a generalized manifestation of NF1. Independently, we identified a third patient with a mosaic type-1 NF1 deletion who exhibited segmental NF1. Together, these three cases constitute the first proven mosaic type-1 deletions so far reported. In two of these three mosaic type-1 deletions, the breakpoints were located within PRS1 and PRS2, previously identified as hotspots for nonallelic homologous recombination (NAHR) during meiosis. Hence, NAHR within PRS1 and PRS2 is not confined to meiosis but may also occur during postzygotic mitotic cell cycles. © 2011 Wiley-Liss, Inc.

  9. Coix seed emulsion synergistically enhances the antitumor activity of gemcitabine in pancreatic cancer through abrogation of NF-κB signaling.

    Science.gov (United States)

    Qian, Yafang; Yang, Bo; Xiong, Yang; Gu, Mancang

    2016-09-01

    Clinical outcomes in patients with pancreatic cancer (PC) continue to be dismal, in part due to de novo and acquired chemoresistance. In the present study, we provide preclinical evidence that pre-treatment with coix seed emulsion, an injectable agent extracted from coix seeds, synergistically sensitized PC cell lines (BxPC-3, PANC-1 and AsPC-1) to gemcitabine, both in vitro and in vivo. Such pretreatment led to significant induction of pro-apoptosis proteins, including caspase-3, cleaved-PARP and Bax (Pcoix seed emulsion suppressed the constitutive and gemcitabine-induced activation of nuclear factor-κB (NF-κB), as shown with the use of electrophoretic mobility shift, reporter and immunoblotting analyses. Coix seed emulsion pretreatment also downregulated the NF-κB-dependent anti‑apoptotic molecules Bcl-2, survivin and cyclooxygenase-2. In vivo, coix seed emulsion combined with gemcitabine had a much greater antitumor effect than the effect of either agent alone, consistent with the downregulation of the proliferation index, and the results of immunostaining for Ki-67, or for the NF-κB subunit p65. Overall, our data demonstrated that coix seed emulsion abrogated gemcitabine-induced activation of NF-κB, and synergistically sensitized PC cells to gemcitabine therapy.

  10. Cardiac-specific overexpression of catalase prevents diabetes-induced pathological changes by inhibiting NF-κB signaling activation in the heart.

    Science.gov (United States)

    Cong, Weitao; Ruan, Dandan; Xuan, Yuanhu; Niu, Chao; Tao, Youli; Wang, Yang; Zhan, Kungao; Cai, Lu; Jin, Litai; Tan, Yi

    2015-12-01

    Catalase is an antioxidant enzyme that specifically catabolizes hydrogen peroxide (H2O2). Overexpression of catalase via a heart-specific promoter (CAT-TG) was reported to reduce diabetes-induced accumulation of reactive oxygen species (ROS) and further prevent diabetes-induced pathological abnormalities, including cardiac structural derangement and left ventricular abnormity in mice. However, the mechanism by which catalase overexpression protects heart function remains unclear. This study found that activation of a ROS-dependent NF-κB signaling pathway was downregulated in hearts of diabetic mice overexpressing catalase. In addition, catalase overexpression inhibited the significant increase in nitration levels of key enzymes involved in energy metabolism, including α-oxoglutarate dehydrogenase E1 component (α-KGD) and ATP synthase α and β subunits (ATP-α and ATP-β). To assess the effects of the NF-κB pathway activation on heart function, Bay11-7082, an inhibitor of the NF-κB signaling pathway, was injected into diabetic mice, protecting mice against the development of cardiac damage and increased nitrative modifications of key enzymes involved in energy metabolism. In conclusion, these findings demonstrated that catalase protects mouse hearts against diabetic cardiomyopathy, partially by suppressing NF-κB-dependent inflammatory responses and associated protein nitration. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. TNF-α-induced NF-κB activation promotes myofibroblast differentiation of LR-MSCs and exacerbates bleomycin-induced pulmonary fibrosis.

    Science.gov (United States)

    Hou, Jiwei; Ma, Tan; Cao, Honghui; Chen, Yabing; Wang, Cong; Chen, Xiang; Xiang, Zou; Han, Xiaodong

    2018-03-01

    Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible lung disease of unknown cause. It has been reported that both lung resident mesenchymal stem cells (LR-MSCs) and tumor necrosis factor-α (TNF-α) play important roles in the development of pulmonary fibrosis. However, the underlying connections between LR-MSCs and TNF-α in the pathogenesis of pulmonary fibrosis are still elusive. In this study, we found that the pro-inflammatory cytokine TNF-α and the transcription factor nuclear factor kappa B (NF-κB) p65 subunit were both upregulated in bleomycin-induced fibrotic lung tissue. In addition, we discovered that TNF-α promotes myofibroblast differentiation of LR-MSCs through activating NF-κB signaling. Interestingly, we also found that TNF-α promotes the expression of β-catenin. Moreover, we demonstrated that suppression of the NF-κB signaling could attenuate myofibroblast differentiation of LR-MSCs and bleomycin-induced pulmonary fibrosis which were accompanied with decreased expression of β-catenin. Our data implicates that inhibition of the NF-κB signaling pathway may provide a therapeutic strategy for pulmonary fibrosis, a disease that warrants more effective treatment approaches. © 2017 Wiley Periodicals, Inc.

  12. GeNF - Experimental report 2006

    Energy Technology Data Exchange (ETDEWEB)

    Pranzas, P.K.; Schreyer, A.; Willumeit, R. (eds.) [GKSS-Forschungszentrum Geesthacht GmbH (Germany). Inst. of Materials Research

    2007-07-01

    At the Geesthacht Neutron Facility GeNF about 212 experiments were performed in 2006 by GKSS and by or for external users, partners or contractors. In most cases the measurements were performed and analysed in cooperation by the guests and by the GKSS staff or by the permanent external user group staff. The activities, which are based on a proposal procedure and on the in house R and D program, are reported in 71 contributions in the present annual experimental report for the year 2006. The contributions may contain one or also several combined experiments. During 2006 the GKSS research reactor FRG-1 achieved an operation time of 197 days at the full 5 MW reactor power providing a neutron flux of ca. 1.4 x 10{sup 14} thermal neutrons/cm{sup 2}s. The cold neutron source was available during the complete operation time. The focus of the in house R and D work at GeNF instruments was the characterisation of nanostructures in engineering materials, the analysis of stresses and textures in welds and technical structures at ARES-2, TEX-2, DCD and SANS-2, the structural investigation of hydrogen containing substances such as polymers, colloids and biological macromolecules at SANS-1 as well as the characterisation of magnetic thin films at PNR, NeRo, POLDI and ROeDI. The thoroughly upgraded residual stress diffractomer ARES-2 went in full operation in spring 2006 as well as the new neutron tomography device at GENRA-3. The installation of modern experiment control hardware and software based on LabView was completed on all designated instruments. In the appendices I and II the experimental reports of REFSANS at FRM II are attached as well as of the GKSS outstation HARWI-II at DESY. Both instruments started full operation in 2006. (orig.)

  13. GeNF - experimental report 2003

    Energy Technology Data Exchange (ETDEWEB)

    Schreyer, A.; Vollbrandt, J.; Willumeit, R. (eds.) [GKSS-Forschungszentrum Geesthacht GmbH (Germany). Inst. for Materials Research

    2004-07-01

    At the Geesthacht Neutron Facility GeNF about 210 experiments were performed in 2003 by GKSS and by or for external users, partners or contractors. In most cases the measurements were performed and analysed in cooperation by the guest and by the GKSS staff or by the permanent external user group staff. The activities, which are based on a proposal procedure and on the in house R and D program, are reported in 76 contributions in the present annual experimental report for the year 2003. The contributions may contain one or also several combined experiments. During 2003 the GKSS research reactor FRG-1 achieved an operation time of 252 days at the full 5 MW reactor power providing a neutron flux of ca. 1,4 x 10{sup 14} thermal neutrons / cm{sup 2} s. The cold neutron source was available during the complete operation time. The focus of the in house R and D work at GeNF instruments was the characterisation of metal alloys, the analysis of stresses in welds and technical structures at ARES, FSS, DCD and SANS-2, the structural investigation of hydrogen containing substances such as polymers, colloids and biological macromolecules at SANS-1 as well as the characterisation of magnetic thin films at PNR and ROeDI. The reflectomer TOREMA was thoroughly upgraded to the instrument NeRo and now offers new measurement possibilities. In the appendices the progress of the project REFSANS at FRM-II is reported as well as the experimental activities of the newly installed GKSS outstation HARWI-II at DESY. (orig.)

  14. A water-soluble polysaccharide from Grifola frondosa induced macrophages activation via TLR4-MyD88-IKKβ-NF-κB p65 pathways

    Science.gov (United States)

    Hou, Lihua; Meng, Meng; Chen, Yuanyuan; Wang, Chunling

    2017-01-01

    Here, the immunomodulatory effects of water-soluble polysaccharide from Grifola frondosa on RAW264.7 macrophages and its molecular mechanisms were investigated. G. frondosa polysaccharide could obviously enhance immunostimulatory activity such as the release of nitric oxide and cytokine production. Western blotting results showed that G. frondosa polysaccharide elevated the TLR4, which might act as an upstream regulator of MyD88 induced G. frondosa polysaccharide. MyD88 promoted IKKβ in endochylema and translocate NF-κB p65 subunit into the nucleus which increased the NO production and cytokine/chemokines level. The results suggested that G. frondosa polysaccharide activated macrophages through TLR4-MyD88-IKKβ-NF-κBp65 signaling pathways. PMID:29156820

  15. Nicotine Induces the Production of IL-1ß and IL-8 via the a7 nAChR/NF-κB Pathway in Human Periodontal Ligament Cells: an in Vitro Study

    Directory of Open Access Journals (Sweden)

    Lizheng Wu

    2014-07-01

    Full Text Available Background/Aims: Tobacco smoking is a major risk factor for the occurrence and progression of periodontitis. We previously demonstrated that nicotine could induce the expression of a7 nicotinic acetylcholine receptors (a7 nAChR in human and rat periodontal tissues. To further examine the signal pathways mediated by a7 nAChR in periodontal ligament (PDL cells, we investigated whether nicotine affects interleukin-1ß (IL-1ß and interleukin-8 (IL-8 via the a7 nAChR/NF-κB pathway in human PDL cells. Methods: Human PDL cells were pre-incubated with alpha-bungarotoxin (a-BTX or pyrrolidine dithiocarbamate (PDTC, then cultured with nicotine. Then, we used western blotting, a dual-luciferase reporter, real-time quantitative PCR and an enzyme-linked immunoassay to assess expression of the NF-κB p65 subunit, NF-κB activity and production of IL-1ß and IL-8 in human PDL cells. Results: Compared with the control group, nicotine could significantly induce production of IL-1ß and IL-8 in human PDL cells and cause the similar effects on the expression of the NF-κB p65 subunit and NF-κB activity. Conclusion: This study demonstrates that nicotine could induce production of IL-1ß and IL-8 via the a7 nAChR/NF-κB pathway in human PDL cells, providing data for a better understanding of the relationships among smoking, nicotine, and periodontitis.

  16. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    DEFF Research Database (Denmark)

    Schmidt, Signe Tandrup; Foged, Camilla; Korsholm, Karen Smith

    2016-01-01

    The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens...... for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce...... been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly...

  17. IKK- and NF-κB-mediated functions in carcinogenesis.

    Science.gov (United States)

    Arkan, Melek C; Greten, Florian R

    2011-01-01

    Since the initial cloning of RelA and its close relationship to c-Rel, the cellular homolog of the viral oncoprotein v-Rel, the nuclear factor κB (NF-κB) signaling pathway and its upstream activating kinase complex (IκB-kinase) have been suspected to play a major role in tumorigenesis. This was further corroborated by the discovery of oncogenic mutations in NF-κB proteins in certain lymphoid malignancies and the notion that NF-κB is persistently activated in a large variety of solid tumors. With the advent of conditional knockout mice allowing tissue-specific targeting of the various components of the NF-κB signaling pathway, it was possible to genetically test the cell autonomous and non-autonomous functions of NF-κB in inflammation-associated cancer as well as sporadic cancers. Here, we review molecular evidence that demonstrates the various functions of NF-κB during different tumor stages and that supports the rationale to target NF-κB in cancer prevention and therapy.

  18. G protein-gated inwardly rectifying potassium channel subunits 1 and 2 are down-regulated in rat dorsal root ganglion neurons and spinal cord after peripheral axotomy.

    Science.gov (United States)

    Lyu, Chuang; Mulder, Jan; Barde, Swapnali; Sahlholm, Kristoffer; Zeberg, Hugo; Nilsson, Johanna; Århem, Peter; Hökfelt, Tomas; Fried, Kaj; Shi, Tie-Jun Sten

    2015-07-22

    Increased nociceptive neuronal excitability underlies chronic pain conditions. Various ion channels, including sodium, calcium and potassium channels have pivotal roles in the control of neuronal excitability. The members of the family of G protein-gated inwardly rectifying potassium (GIRK) channels, GIRK1-4, have been implicated in modulating excitability. Here, we investigated the expression and distribution of GIRK1 and GIRK2 in normal and injured dorsal root ganglia (DRGs) and spinal cord of rats. We found that ~70% of the DRG neurons expressed GIRK1, while only <10% expressed GIRK2. The neurochemical profiles of GIRK1- and GIRK2-immunoreactive neurons were characterized using the neuronal markers calcitonin gene-related peptide, isolectin-B4 and neurofilament-200, and the calcium-binding proteins calbindin D28k, calretinin, parvalbumin and secretagogin. Both GIRK subunits were expressed in DRG neurons with nociceptive characteristics. However, while GIRK1 was widely expressed in several sensory neuronal subtypes, GIRK2 was detected mainly in a group of small C-fiber neurons. In the spinal dorsal horn, GIRK1- and -2-positive cell bodies and processes were mainly observed in lamina II, but also in superficial and deeper layers. Abundant GIRK1-, but not GIRK2-like immunoreactivity, was found in the ventral horn (laminae VI-X). Fourteen days after axotomy, GIRK1 and GIRK2 were down-regulated in DRG neurons at the mRNA and protein levels. Both after axotomy and rhizotomy there was a reduction of GIRK1- and -2-positive processes in the dorsal horn, suggesting a presynaptic localization of these potassium channels. Furthermore, nerve ligation caused accumulation of both subunits on both sides of the lesion, providing evidence for anterograde and retrograde fast axonal transport. Our data support the hypothesis that reduced GIRK function is associated with increased neuronal excitability and causes sensory disturbances in post-injury conditions, including neuropathic

  19. Levels and Age Dependency of Neurofilament Light and Glial Fibrillary Acidic Protein in Healthy Individuals and Their Relation to the Brain Parenchymal Fraction.

    Directory of Open Access Journals (Sweden)

    Mattias Vågberg

    Full Text Available Neurofilament light (NFL and Glial Fibrillary Acidic Protein (GFAP are integral parts of the axonal and astrocytal cytoskeletons respectively and are released into the cerebrospinal fluid (CSF in cases of cellular damage. In order to interpret the levels of these biomarkers in disease states, knowledge on normal levels in the healthy is required. Another biomarker for neurodegeneration is brain atrophy, commonly measured as brain parenchymal fraction (BPF using magnetic resonance imaging (MRI. Potential correlations between levels of NFL, GFAP and BPF in healthy individuals have not been investigated.To present levels of NFL and GFAP in healthy individuals stratified for age, and investigate the correlation between them as well as their correlation with BPF.The CSF was analysed in 53 healthy volunteers aged 21 to 70 (1 sample missing for GFAP analysis and 48 of the volunteers underwent determination of BPF using MRI.Mean (±SD NFL was 355 ng/L (±214, mean GFAP was 421 ng/L (±129 and mean BPF was 0.867 (±0.035. All three biomarkers correlated with age. NFL also correlated with both GFAP and BPF. When controlled for age, only the correlation between NFL and GFAP retained statistical significance.This study presents data on age-stratified levels of NFL and GFAP in the CSF of healthy individuals. There is a correlation between levels of NFL and GFAP and both increase with age. A correlation between NFL and BPF was also found, but did not retain statistical significance if controlled for age.

  20. Angiogenesis and Therapeutic Approaches to NF1 Tumors

    Science.gov (United States)

    2007-04-01

    Medium Wild-type ECGS Nf1+/- ECGS Wild-type bFGF Nf1+/- bFGF Wild-type VEGF NF1+/- VEGF Mitogen treatment Fig. 1. BrdU incorporation assay of brain...anesthetized, and the sciatic nerves of both legs were exposed at midthigh. The cell suspension (5 ll, 5 3 105 cells) was incrementally injected within the...3. Fascicle 24. Washington, DC: Armed Forces Insti- tute of Pathology. p 385–405. Scott JE, Mowry RW. 1970. Alcian blue—a consumer’s guide. J Histo

  1. Stoichiometry of δ subunit containing GABA(A) receptors.

    Science.gov (United States)

    Patel, B; Mortensen, M; Smart, T G

    2014-02-01

    Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach. Using site-directed mutagenesis, we inserted a highly characterized 9' serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits. Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose-response curves of cells co-expressing WT subunits with their respective L9'S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits. Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ. © 2013 The British Pharmacological Society.

  2. Data of evolutionary structure change: 1AOGA-2J3NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available EVID>EVID> 7 2J3N F 2J3NF...EVID>EVID> 8 2J3N F 2J3NF...1AOGA-2J3NF 1AOG 2J3N A F -SKIFDLVVIGAGSGGLEAAWNAATLYKKRVAVIDVQMV... HHHHEEEEE HHHH EEEEEEEEE EEEEEEEEE HHHHHHHHHHHH HHHHH GGG ---- EVID>...hain> 2J3N F 2J3NF

  3. GeNF - Experimental report 2007

    Energy Technology Data Exchange (ETDEWEB)

    Pranzas, P.K.; Schreyer, A.; Willumeit, R. (eds.) [GKSS-Forschungszentrum Geesthacht GmbH (Germany). Inst. fuer Materialforschung

    2008-11-05

    At the Geesthacht Neutron Facility GeNF about 203 experiments were performed in 2007 by GKSS and by or for external users, partners or contractors. In most cases the measurements were performed and analysed in cooperation by the guests and by the GKSS staff or by the permanent external user group staff. The activities, which are based on a proposal procedure and on the in house R and D program, are reported in 70 contributions in the present annual experimental report for the year 2007. The contributions may contain one or also several combined experiments. During 2007 the GKSS research reactor FRG-1 achieved an operation time of 204 days at the full 5 MW reactor power providing a neutron flux of ca. 1.4 x 10{sup 14} thermal neutrons/cm{sup 2}s. In May/June 2007 the FRG-1 was upgraded with a new cold neutron source yielding a flux increase at the five instruments using cold neutrons of up to 40 %. The focus of the in house R and D work at GeNF instruments in 2007 was the characterisation of nano-structures in engineering materials, the analysis of stresses and textures in welds and technical structures at SANS-2, DCD, ARES-2 and TEX-2, the structural investigation of hydrogen containing substances such as polymers, colloids and biological macromolecules at SANS-1 as well as the characterisation of magnetic thin films at NeRo, PNR, POLDI and ROeDI. The modern experiment control hardware and software based on LabView was continuously improved on all instruments. In the appendices I and II the experimental reports of the GKSS outstation at the FRM II are attached as well as of the GKSS outstation at DESY. At the neutron reflectometer REFSANS at FRM II measurements are possible using a broad range of the scattering vector with reflectivities up to 10{sup -7}. Three reports show the activities of GKSS in the field of texture measurement at the instrument STRESS-SPEC. The instrument HARWI II at DESY is accepted very well by the community and is overbooked in all fields

  4. GeNF - Experimental report 2008

    Energy Technology Data Exchange (ETDEWEB)

    Pranzas, Philipp Klaus; Mueller, Martin; Willumeit, Regine; Schreyer, Andreas (eds.) [GKSS-Forschungszentrum Geesthacht GmbH (Germany). Inst. fuer Materialforschung

    2009-12-11

    At the Geesthacht Neutron Facility GeNF about 182 experiments were performed in 2008 by GKSS and by or for external users, partners or contractors. In most cases the measurements were performed and analysed in cooperation by the guests, by GKSS staff or by the permanent external user group staff. The activities, which are based on a proposal procedure and on the in house R and D program, are reported in 76 contributions in the present annual experimental report for the year 2008. The contributions may contain several combined experiments. During 2008 the GKSS research reactor FRG-1 achieved an operation time of 175 days at the full 5 MW reactor power providing a neutron flux of ca. 1.4.10{sup 14} thermal neutrons/cm{sup 2} s. The focus of the in house R and D work at GeNF instruments in 2008 was the characterisation of nanostructures in engineering materials, the analysis of stresses and textures in welds and technical structures at SANS-2, DCD, ARES-2 and TEX-2, the structural investigation of hydrogen containing substances such as polymers, colloids and biological macromolecules at SANS-1 as well as the characterisation of magnetic thin films at NeRo, PNR, POLDI and ROeDI. The modern experiment control hardware (e.g. sample environments, like magnets, cryostats or furnaces) and software based on LabView was continuously improved on all instruments. In the appendices I and II the experimental reports of the GKSS outstation at the FRM II are attached as well as of the GKSS outstation at DESY. The massive activity at the FRM II outstation is documented by the increasing number of REFSANS reports, accumulated to nine. Three reports show the activities of GKSS in the field of texture measurement at the instrument STRESS-SPEC. The instrument HARWI II at the synchrotron storage ring DORIS III at DESY is accepted very well by the community and is heavily overbooked in all fields (tomography, diffraction, etc.). After an 8-month shutdown period for an upgrade in the frame

  5. Hypersecretion of the alpha-subunit in clinically non-functioning pituitary adenomas: Diagnostic accuracy is improved by adding alpha-subunit/gonadotropin ratio to levels of alpha-subunit

    DEFF Research Database (Denmark)

    Andersen, Marianne; Ganc-Petersen, Joanna; Jørgensen, Jens O L

    2010-01-01

    In vitro, the majority of clinically non-functioning pituitary adenomas (NFPAs) produce gonadotropins or their alpha-subunit; however, in vivo, measurements of alpha-subunit levels may not accurately detect the hypersecretion of the alpha-subunit.......In vitro, the majority of clinically non-functioning pituitary adenomas (NFPAs) produce gonadotropins or their alpha-subunit; however, in vivo, measurements of alpha-subunit levels may not accurately detect the hypersecretion of the alpha-subunit....

  6. Bacterial effector binding to ribosomal protein s3 subverts NF-kappaB function.

    Directory of Open Access Journals (Sweden)

    Xiaofei Gao

    2009-12-01

    Full Text Available Enteric bacterial pathogens cause food borne disease, which constitutes an enormous economic and health burden. Enterohemorrhagic Escherichia coli (EHEC causes a severe bloody diarrhea following transmission to humans through various means, including contaminated beef and vegetable products, water, or through contact with animals. EHEC also causes a potentially fatal kidney disease (hemolytic uremic syndrome for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g., enteropathogenic E. coli (EPEC, Salmonella, Shigella, Yersinia utilize a type III secretion system (T3SS to inject virulence proteins (effectors into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, in many cases, the mechanisms by which these effectors bind to host proteins and disrupt the normal function of intestinal epithelial cells have not been completely characterized. In this study, we present evidence that the E. coli O157:H7 nleH1 and nleH2 genes encode T3SS effectors that bind to the human ribosomal protein S3 (RPS3, a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB transcriptional complexes. NleH1 and NleH2 co-localized with RPS3 in the cytoplasm, but not in cell nuclei. The N-terminal region of both NleH1 and NleH2 was required for binding to the N-terminus of RPS3. NleH1 and NleH2 are autophosphorylated Ser/Thr protein kinases, but their binding to RPS3 is independent of kinase activity. NleH1, but not NleH2, reduced the nuclear abundance of RPS3 without altering the p50 or p65 NF-kappaB subunits or affecting the phosphorylation state or abundance of the inhibitory NF-kappaB chaperone IkappaBalpha NleH1 repressed the transcription of a RPS3/NF-kappaB-dependent reporter plasmid, but did not inhibit the transcription of RPS3-independent reporters. In contrast, NleH2 stimulated RPS3-dependent transcription, as well

  7. Aberrant Gene Expression in NF1-Mediated Oncogenesis. Addendum

    National Research Council Canada - National Science Library

    Shields, Janiel M

    2004-01-01

    Neurofibromatosis type I (NF1) is a familial multisystem disease that occurs in approximately one in 3,500 people designating it as one of the most common autosomal-dominant human diseases. Approximately 5...

  8. Mangiferin suppressed advanced glycation end products (AGEs) through NF-κB deactivation and displayed anti-inflammatory effects in streptozotocin and high fat diet-diabetic cardiomyopathy rats.

    Science.gov (United States)

    Hou, Jun; Zheng, Dezhi; Fung, Gabriel; Deng, Haoyu; Chen, Lin; Liang, Jiali; Jiang, Yan; Hu, Yonghe

    2016-03-01

    Given the importance of the aggregation of advanced glycation end products (AGEs) and cardiac inflammation in the onset and progression of diabetic cardiomyopathy (DCM), our objective in this study was to demonstrate the cardioprotective effect of mangiferin, an antidiabetic and anti-inflammatory agent, on diabetic rat model. The DCM model was established by a high-fat diet and a low dose of streptozotocin. DCM rats were treated orally with mangiferin (20 mg/kg) for 16 weeks. Serum and left ventricular myocardium were collected for determination of inflammatory cytokines. AGEs mRNA and protein expression of nuclear factor kappa B (NF-κB) and receptor for AGEs (RAGE) in myocardium were assayed by real-time PCR and Western blot. ROS levels were measured by dihydroethidium fluorescence staining. NF-κB binding activity was assayed by TransAM NF-κB p65 ELISA kit. Chronic treatment with mangiferin decreased the levels of myocardial enzymes (CK-MB, LDH) and inflammatory mediators (TNF-α, IL-1β). Meanwhile, NF-κB is inhibited by the reduction of nuclear translocation of p65 subunit, and mangiferin reduced AGE production and decreased the mRNA and protein expression of RAGE in DCM rats. Our data indicated that mangiferin could significantly ameliorate DCM by preventing the release of inflammatory cytokines, and inhibiting ROS accumulation, AGE/RAGE production, and NF-κB nuclear translocation, suggesting that mangiferin treatment might be beneficial in DCM.

  9. Nuclear Factor-κB (NF-κB Regulates the Expression of Human Testis-Enriched Leucine-Rich Repeats and WD Repeat Domain Containing 1 (LRWD1 Gene

    Directory of Open Access Journals (Sweden)

    Yo-Wen Liu

    2012-12-01

    Full Text Available The human Leucine-rich Repeats and WD repeat Domain containing 1 (LRWD1 gene was originally identified by cDNA microarray as one of the genes down-regulated in the testicular tissues of patients with severe spermatogenic defects. Human LRWD1 is a testicular-enriched protein that is present predominantly in the cytoplasm of spermatocytes and spermatids and colocalizes with the centrosome at the base of sperm tail. Reporter assay, Chromatin immunoprecipitation (ChIP analysis, and gel electrophoretic mobility shift assay (EMSA were used to identify the core promoter region of LRWD1. A 198 bp segment upstream of the LRWD1 transcription initiation site exhibited promoter activity. The LRWD1 core promoter lacked a TATA box but contained a NF-κB binding site. Chromatin immunoprecipitation (ChIP analysis and gel electrophoretic mobility shift assay (EMSA showed basal binding of the NF-κB subunit to the LRWD1 promoter. LRWD1 promoter activity was positively regulated by NF-κB, and this regulation was dependent on the presence of the conserved κB site in the LRWD1 promoter region. Our data suggest that NF-κB is an important regulator for the expression of LRWD1. This is the first study showing that the expression of the testis-enriched LRWD1 gene is regulated by NF-κB.

  10. Genistein inhibits phorbol ester-induced NF-κB transcriptional activity and COX-2 expression by blocking the phosphorylation of p65/RelA in human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Myung-Hoon; Kim, Do-Hee [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Na, Hye-Kyung [Department of Food and Nutrition, Sungshin Women' s University, Seoul (Korea, Republic of); Kim, Jung-Hwan; Kim, Ha-Na [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Haegeman, Guy [LEGEST, University of Gent (Belgium); Surh, Young-Joon, E-mail: surh@snu.ac.kr [Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul (Korea, Republic of); Cancer Research Institute, Seoul National University, Seoul (Korea, Republic of)

    2014-10-15

    Genistein, an isoflavone present in soy products, has chemopreventive effects on mammary carcinogenesis. In the present study, we have investigated the effects of genistein on phorbol ester-induced expression of cyclooxygenase-2 (COX-2) that plays an important role in the pathophysiology of inflammation-associated carcinogenesis. Pretreatment of cultured human breast epithelial (MCF10A) cells with genistein reduced COX-2 expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). There are multiple lines of evidence supporting that the induction of COX-2 is regulated by the eukaryotic transcription factor NF-κB. Genistein failed to inhibit TPA-induced nuclear translocation and DNA binding of NF-κB as well as degradation of IκB. However, genistein abrogated the TPA-induced transcriptional activity of NF-κB as determined by the luciferase reporter gene assay. Genistein inhibited phosphorylation of the p65 subunit of NF-κB and its interaction with cAMP regulatory element-binding protein-binding protein (CBP)/p300 and TATA-binding protein (TBP). TPA-induced NF-κB phosphorylation was abolished by pharmacological inhibition of extracellular signal-regulated kinase (ERK). Likewise, pharmacologic inhibition or dominant negative mutation of ERK suppressed phosphorylation of p65. The above findings, taken together, suggest that genistein inhibits TPA-induced COX-2 expression in MCF10A cells by blocking ERK-mediated phosphorylation of p65 and its subsequent interaction with CBP and TBP.

  11. The Role of NF1 in Memory Retrieval

    Science.gov (United States)

    2014-09-01

    critical for olfactory memory , didn’t affect LTM, whereas overexpression within octopamine neurons showed a phenotype of enhanced appetitive LTM (Fig... memory .
Our
investigation
over
the
entire
funding
 period
leads
to
following
discoveries.
First,
NF1
is
critical
for
supporting
multiple
types
of
long...term
 memories ,
including
aversive
and
appetitive
classic
conditioning.
Second,
it
regulates
 memory 
 bi‐directionally.
A
loss
of
NF1
functions

  12. Targeting Signaling to YAP for the Therapy of NF2

    Science.gov (United States)

    2016-12-01

    any step of our newly identified pathway, and to test the preclinical efficacy of lead compounds in xenograft models of NF2. During this grant, we have...generated NF2 mutant cell lines expressing two different types of YAP reporters and tested their suitability for high throughput screening. 15...interacts through its coiled-coil segment with the central coiled-coil segment of AMOT proteins, which localize to Adherens Junctions (AJs) and

  13. Exploring Neurofibromin Function in a Yeast Model of NF1

    Science.gov (United States)

    2011-11-01

    to define NF1 disease mechanisms. Budding yeast, Saccharomyces cerevisiae , have two NF1-like genes, called IRA1 and IRA2. In year one of the project...mammalian cells and in Drosophila. References Hohfeld, J., Veenhuis, M., and Kunau, W.H. (1991). PAS3, a Saccharomyces cerevisiae gene...anaerobiosis yes yes PEX 11 Peroxisomal membrane protein required for peroxisome proliferation and medium -chain fatty acid oxidation, most abundant

  14. NF-kappaB signaling in the aging process.

    Science.gov (United States)

    Salminen, Antero; Kaarniranta, Kai

    2009-07-01

    The aging process represents a progressive decline in cellular and organism function. Explaining the aging process has given rise to a cornucopia for different theories in which the basic difference has been the question whether aging is genetically regulated or an entropic degeneration process. Different screening techniques have revealed that mammalian aging is associated with the activation of NF-kappaB transcription factor system. The NF-kappaB system is an ancient host defense system concerned with immune responses and different external and internal dangers, such as oxidative and genotoxic stress. NF-kappaB signaling is not only the master regulator of inflammatory responses but can also regulate several homeostatic responses such as apoptosis, autophagy, and tissue atrophy. We will describe how chronic activation of NF-kappaB signaling has the capacity to induce the senescent phenotype associated with aging. Interestingly, several longevity genes such as SIRT1, SIRT6, and FoxOs can clearly suppress NF-kappaB signaling and in this way delay the aging process and extend lifespan. It seems that the aging process is an entropic degeneration process driven by NF-kappaB signaling. This process can be regulated by a variety of longevity genes along with a plethora of other factors such as genetic polymorphism, immune and dietary aspects, and environmental insults.

  15. Mechanism of hypoxia-induced NF-kappaB.

    Science.gov (United States)

    Culver, Carolyn; Sundqvist, Anders; Mudie, Sharon; Melvin, Andrew; Xirodimas, Dimitris; Rocha, Sonia

    2010-10-01

    NF-κB activation is a critical component in the transcriptional response to hypoxia. However, the underlying mechanisms that control its activity under these conditions are unknown. Here we report that under hypoxic conditions, IκB kinase (IKK) activity is induced through a calcium/calmodulin-dependent kinase 2 (CaMK2)-dependent pathway distinct from that for other common inducers of NF-κB. This process still requires IKK and the IKK kinase TAK1, like that for inflammatory inducers of NF-κB, but the TAK1-associated proteins TAB1 and TAB2 are not essential. IKK complex activation following hypoxia requires Ubc13 but not the recently identified LUBAC (linear ubiquitin chain assembly complex) ubiquitin conjugation system. In contrast to the action of other NF-κB inducers, IKK-mediated phosphorylation of IκBα does not result in its degradation. We show that this results from IκBα sumoylation by Sumo-2/3 on critical lysine residues, normally required for K-48-linked polyubiquitination. Furthermore, inhibition of specific Sumo proteases is sufficient to release RelA from IκBα and activate NF-κB target genes. These results define a novel pathway regulating NF-κB activation, important to its physiological role in human health and disease.

  16. Mechanism of Hypoxia-Induced NF-κB▿ †

    Science.gov (United States)

    Culver, Carolyn; Sundqvist, Anders; Mudie, Sharon; Melvin, Andrew; Xirodimas, Dimitris; Rocha, Sonia

    2010-01-01

    NF-κB activation is a critical component in the transcriptional response to hypoxia. However, the underlying mechanisms that control its activity under these conditions are unknown. Here we report that under hypoxic conditions, IκB kinase (IKK) activity is induced through a calcium/calmodulin-dependent kinase 2 (CaMK2)-dependent pathway distinct from that for other common inducers of NF-κB. This process still requires IKK and the IKK kinase TAK1, like that for inflammatory inducers of NF-κB, but the TAK1-associated proteins TAB1 and TAB2 are not essential. IKK complex activation following hypoxia requires Ubc13 but not the recently identified LUBAC (linear ubiquitin chain assembly complex) ubiquitin conjugation system. In contrast to the action of other NF-κB inducers, IKK-mediated phosphorylation of IκBα does not result in its degradation. We show that this results from IκBα sumoylation by Sumo-2/3 on critical lysine residues, normally required for K-48-linked polyubiquitination. Furthermore, inhibition of specific Sumo proteases is sufficient to release RelA from IκBα and activate NF-κB target genes. These results define a novel pathway regulating NF-κB activation, important to its physiological role in human health and disease. PMID:20696840

  17. Comprehensive phenotypic characterization of PTLD reveals potential reliance on EBV or NF-κB signalling instead of B-cell receptor signalling.

    Science.gov (United States)

    Menter, Thomas; Dickenmann, Michael; Juskevicius, Darius; Steiger, Juerg; Dirnhofer, Stephan; Tzankov, Alexandar

    2017-06-01

    Post-transplant lymphoproliferative disorders (PTLD) are a major problem in transplant medicine. So far, the insights into pathogenesis and potentially druggable pathways in PTLD remain scarce. We investigated a cohort of PTLD patients, consisting of both polymorphic (n = 3) and monomorphic (n = 19) B-cell lymphoproliferations. Several signalling pathways, cell of origin of PTLD and their relation to viruses were analysed by immunohistochemistry and in situ hybridization. Most PTLD were of activated B-cell origin. Two-thirds of cases showed an Epstein-Barr virus (EBV) infection of the neoplastic cells. NF-κB signalling components were present in the majority of cases, except for EBV-infected cases with latency type III lacking CD19 and upstream B-cell signalling constituents. Proteins involved in B-cell receptor signalling like Bruton tyrosine kinase were only present in a minority of cases. Phosphoinositide 3-kinase (PI3K) was expressed in 94% of cases and the druggable PI3K class 1 catalytic subunit p110 in 76%, while proteins of other signalling transduction pathways were expressed only in single cases. Unsupervised cluster analysis revealed three distinct subgroups: (i) related to EBV infection, mainly latency type III and mostly lacking CD19, upstream B-cell signalling and NF-κB constituents; (ii) mostly related to EBV infection with expression of the alternative NF-κB pathway compound RelB, CD10, and FOXP1 or MUM1; and finally, (iii) mostly unrelated to virus infection with expression of the classic NF-κB pathway compound p65. EBV and NF-κB are important drivers in PTLD in contrast to B-cell receptor signalling. The main signal transduction pathway is related to PI3K. This links PTLD to other subgroups of EBV-related lymphomas, highlighting also new potential treatment approaches. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Effects of Pharmacologic and Genetic Inhibition of Alk on Cognitive Impairments in NF1 Mutant Mice

    Science.gov (United States)

    2016-08-01

    genetic interaction between Alk and Nf1 in mice is similar to the behavioral phenotypes of Alk and NF1 mutations in flies and that pharmacologic or...the predicted improvement in hippocampal-dependent cognitive function using both genetic and pharmacologic inhibition of Alk in heterozygous NF1...phenotypes of Alk and NF1 mutations in flies and that pharmacologic or genetic inhibition of Alk in NF1 mutant mice will attenuate or even rescue

  19. Children with 5′-end NF1 gene mutations are more likely to have glioma

    OpenAIRE

    Anastasaki, Corina; Morris, Stephanie M.; Gao, Feng; Gutmann, David H.

    2017-01-01

    Objective: To ascertain the relationship between the germline NF1 gene mutation and glioma development in patients with neurofibromatosis type 1 (NF1). Methods: The relationship between the type and location of the germline NF1 mutation and the presence of a glioma was analyzed in 37 participants with NF1 from one institution (Washington University School of Medicine [WUSM]) with a clinical diagnosis of NF1. Odds ratios (ORs) were calculated using both unadjusted and weighted analyses of this...

  20. Diversity of heterotrimeric G-protein γ subunits in plants

    Directory of Open Access Journals (Sweden)

    Trusov Yuri

    2012-10-01

    Full Text Available Abstract Background Heterotrimeric G-proteins, consisting of three subunits Gα, Gβ and Gγ are present in most eukaryotes and mediate signaling in numerous biological processes. In plants, Gγ subunits were shown to provide functional selectivity to G-proteins. Three unconventional Gγ subunits were recently reported in Arabidopsis, rice and soybean but no structural analysis has been reported so far. Their relationship with conventional Gγ subunits and taxonomical distribution has not been yet demonstrated. Results After an extensive similarity search through plant genomes, transcriptomes and proteomes we assembled over 200 non-redundant proteins related to the known Gγ subunits. Structural analysis of these sequences revealed that most of them lack the obligatory C-terminal prenylation motif (CaaX. According to their C-terminal structures we classified the plant Gγ subunits into three distinct types. Type A consists of Gγ subunits with a putative prenylation motif. Type B subunits lack a prenylation motif and do not have any cysteine residues in the C-terminal region, while type C subunits contain an extended C-terminal domain highly enriched with cysteines. Comparative analysis of C-terminal domains of the proteins, intron-exon arrangement of the corresponding genes and phylogenetic studies suggested a common origin of all plant Gγ subunits. Conclusion Phylogenetic analyses suggest that types C and B most probably originated independently from type A ancestors. We speculate on a potential mechanism used by those Gγ subunits lacking isoprenylation motifs to anchor the Gβγ dimer to the plasma membrane and propose a new flexible nomenclature for plant Gγ subunits. Finally, in the light of our new classification, we give a word of caution about the interpretation of Gγ research in Arabidopsis and its generalization to other plant species.

  1. Hypoxia-inducible factor 1alpha determines gastric cancer chemosensitivity via modulation of p53 and NF-kappaB.

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    Nadine Rohwer

    Full Text Available BACKGROUND: Reduced chemosensitivity of solid cancer cells represents a pivotal obstacle in clinical oncology. Hence, the molecular characterization of pathways regulating chemosensitivity is a central prerequisite to improve cancer therapy. The hypoxia-inducible factor HIF-1alpha has been linked to chemosensitivity while the underlying molecular mechanisms remain largely elusive. Therefore, we comprehensively analysed HIF-1alpha's role in determining chemosensitivity focussing on responsible molecular pathways. METHODOLOGY AND PRINCIPAL FINDINGS: RNA interference was applied to inactivate HIF-1alpha or p53 in the human gastric cancer cell lines AGS and MKN28. The chemotherapeutic agents 5-fluorouracil and cisplatin were used and chemosensitivity was assessed by cell proliferation assays as well as determination of cell cycle distribution and apoptosis. Expression of p53 and p53 target proteins was analyzed by western blot. NF-kappaB activity was characterized by means of electrophoretic mobility shift assay. Inactivation of HIF-1alpha in gastric cancer cells resulted in robust elevation of chemosensitivity. Accordingly, HIF-1alpha-competent cells displayed a significant reduction of chemotherapy-induced senescence and apoptosis. Remarkably, this phenotype was completely absent in p53 mutant cells while inactivation of p53 per se did not affect chemosensitivity. HIF-1alpha markedly suppressed chemotherapy-induced activation of p53 and p21 as well as the retinoblastoma protein, eventually resulting in cell cycle arrest. Reduced formation of reactive oxygen species in HIF-1alpha-competent cells was identified as the molecular mechanism of HIF-1alpha-mediated inhibition of p53. Furthermore, loss of HIF-1alpha abrogated, in a p53-dependent manner, chemotherapy-induced DNA-binding of NF-kappaB and expression of anti-apoptotic NF-kappaB target genes. Accordingly, reconstitution of the NF-kappaB subunit p65 reversed the increased chemosensitivity of

  2. Neurofibromatosis type 2 French cohort analysis using a comprehensive NF2 molecular diagnostic strategy.

    Science.gov (United States)

    Pasmant, E; Louvrier, C; Luscan, A; Cohen, J; Laurendeau, I; Vidaud, M; Vidaud, D; Goutagny, S; Kalamarides, M; Parfait, B

    2015-06-11

    Neurofibromatosis type 2 (NF2) affects about one in 25,000 to 40,000 people. Most NF2 patients have private loss-of-function mutations scattered along the NF2 gene. Here, we present our NF2 investigation strategy. We report a comprehensive NF2 mutation analysis of 221 NF2 French patients: 134 unrelated typical NF2 patients fulfilling the Manchester criteria and 87 unrelated patients presenting symptoms that partially fulfilled the Manchester criteria. A NF2 mutation was identified in 56 of the 221 patients, giving a global mutation detection rate of 25%. This rate reached 37% (49/134) for typical NF2 patients fulfilling the Manchester criteria and only 8% (7/87) for patients presenting symptoms suggestive of NF2. Six of these seven patients were under 25 of age. Our approach showed that 77% of NF2 identified variants were detected by coding exons sequencing. Multiplex ligation-dependent probe amplification allowed the identification of restricted rearrangements (23% of NF2 identified variants corresponding to complete deletion or partial deletion/duplication of NF2). High mutation detection rate can be achieved if well phenotyped NF2 patients are studied with multiple complementary and optimized techniques. NF2 somatic mosaicism detection was improved by frozen tumor samples molecular analysis. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  3. Assembly of catalytic subunits of aspartate transcarbamoylase from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Burns, D.L.; Schachman, H.K.

    1980-10-01

    Although extensive studies have been conducted on the assembly of the allosteric enzyme, aspartate transcarbamoylase (ATCase) from isolate, intact catalytic (C) and regulatory (R) subunits, there has been little research on the formation of these subunits from individual catalytic (c) and regulatory (r) polypeptide chains. Such studies would be useful for evaluating the strengths of the interchain bonding domains within the subunits just as earlier experiments provided valuable data regarding interactions between the subunits in ATCase. The intact enzyme comprising two C trimers and three R dimers is designated as C/sub 2/R/sub 3/ or c/sub 6/r/sub 6/.

  4. Reconstruction of the nasal soft triangle subunit.

    Science.gov (United States)

    Constantine, Fadi C; Lee, Michael R; Sinno, Sammy; Thornton, James F

    2013-05-01

    Of all nine subunits, the soft triangle is perhaps the most challenging to recreate. The complexity of soft triangle reconstruction resides in its proximity to such important structures as the nasal tip, nasal ala, and distal columella. If the soft triangle is not properly reconstructed, problems with nasal function and aesthetics often arise. Anatomical asymmetries in the lower third and abnormal shadowing can occur following insufficient restoration. A retrospective review was completed of all patients undergoing reconstruction of the nasal soft triangle subunit at the University of Texas Southwestern Medical Center in Dallas, Texas, from 1995 to 2010. Defects with only external skin intact were classified as type I. Defects involving both skin and underlying soft tissue with intact mucosa were classified as type II. Finally, transmural defects with violated mucosa were classified as type III. Surgical outcomes were graded on a scale of I to IV. Grades given were based on the complexity of the existing defect and restoration of the soft triangle, with higher grades given when adjacent structures were not distorted. Of the 14 cases reviewed, two (14 percent) were type I defects, nine (64 percent) were type II defects, and three (21 percent) were type III defects. Three patients (21 percent) required revision with subsequent resurfacing and two (14 percent) required resurfacing alone. All but one patient (93 percent) had a grade of 2.0 or better, with the one patient opting not to undergo revision. The authors believe their method of soft triangle reconstruction using the proposed algorithm is an easy approach to soft triangle reconstruction that will yield consistent surgical and clinical success from aesthetic and functional perspectives. Furthermore, the authors were able to achieve excellent aesthetic outcomes without compromise or facing any structural complications. Therapeutic, IV.

  5. Interactions between subunits in heterodimeric Ncd molecules.

    Science.gov (United States)

    Kocik, Elzbieta; Skowronek, Krzysztof J; Kasprzak, Andrzej A

    2009-12-18

    The nonprocessive minus-end-directed kinesin-14 Ncd is involved in the organization of the microtubule (MT) network during mitosis. Only one of the two motor domains is involved in the interaction with the MT. The other head is tethered to the bound one. Here we prepared, purified, and characterized mutated Ncd molecules carrying point mutations in one of the heads, thus producing heterodimeric motors. The mutations tested included substitutions in Switch I and II: R552A, E585A, and E585D; the decoupling mutant N600K; and a deletion in the motor domain in one of the subunits resulting in a single-headed molecule (NcN). These proteins were isolated by two sequential affinity chromatography steps, followed by measurements of their affinities to MT, enzymatic properties, and the velocity of the microtubule gliding test in vitro. A striking observation is a low affinity of the single-headed NcN for MT both without nucleotides and in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate, implying that the tethered head has a profound effect on the structure of the Ncd-MT complex. Mutated homodimers had no MT-activated ATPase and no motility, whereas NcN had motility comparable with that of the wild type Ncd. Although the heterodimers had one fully active and one inactive head, the ATPase and motility of Ncd heterodimers varied dramatically, clearly demonstrating that interactions between motor domains exist in Ncd. We also show that the bulk property of dimeric proteins that interact with the filament with only one of its heads depends also on the distribution of the filament-interacting subunits.

  6. miR-155-5p inhibition promotes the transition of bone marrow mesenchymal stem cells to gastric cancer tissue derived MSC-like cells via NF-κB p65 activation.

    Science.gov (United States)

    Zhu, Mengchu; Wang, Mei; Yang, Fang; Tian, Yiqing; Cai, Jie; Yang, Huan; Fu, Hailong; Mao, Fei; Zhu, Wei; Qian, Hui; Xu, Wenrong

    2016-03-29

    Gastric cancer tissue-derived MSC-like cells (GC-MSC) share similar characteristics to bone marrow MSC (BM-MSC); however, the phenotypical and functional differences and the molecular mechanism of transition between the two cell types remain unclear. Compared to BM-MSC, GC-MSC exhibited the classic phenotype of reactive stroma cells, a stronger gastric cancer promoting capacity and lower expression of miR-155-5p. Inhibition of miR-155-5p by transfecting miRNA inhibitor induced a phenotypical and functional transition of BM-MSC into GC-MSC-like cells, and the reverse experiment deprived GC-MSC of tumor-promoting phenotype and function. NF-kappa B p65 (NF-κB p65) and inhibitor of NF-kappa B kinase subunit epsilon (IKBKE/IKKε) were identified as targets of miR-155-5p and important for miRNA inhibitor activating NF-κB p65 in the transition. Inactivation of NF-κB by pyrrolidine dithiocarbamic acid (PDTC) significantly blocked the effect of miR-155-5p inhibitor on BM-MSC. IKBKE, NF-κB p65 and phospho-NF-κB p65 proteins were highly enriched in MSC-like cells of gastric cancer tissues, and the latter two were correlated with the pathological progression of gastric cancer. In GC-MSC, the expression of miR-155-5p was downregulated and NF-κB p65 protein was increased and activated. NF-κB inactivation by PDTC or knockdown of its downstream cytokines reversed the phenotype and function of GC-MSC. Taken together, our findings revealed that miR-155-5p downregulation induces BM-MSC to acquire a GC-MSC-like phenotype and function depending on NF-κB p65 activation, which suggests a novel mechanism underlying the cancer associated MSC remodeling in the tumor microenvironment and offers an effective target and approach for gastric cancer therapy.

  7. Familial Lymphoproliferative Malignancies and Tandem Duplication of NF1 Gene

    Directory of Open Access Journals (Sweden)

    Gustavo Fernandes

    2014-01-01

    Full Text Available Background. Neurofibromatosis type 1 is a genetic disorder caused by loss-of-function mutations in a tumor suppressor gene (NF1 which codifies the protein neurofibromin. The frequent genetic alterations that modify neurofibromin function are deletions and insertions. Duplications are rare and phenotype in patients bearing duplication of NF1 gene is thought to be restricted to developmental abnormalities, with no reference to cancer susceptibility in these patients. We evaluated a patient who presented with few clinical signs of neurofibromatosis type 1 and a conspicuous personal and familiar history of different types of cancer, especially lymphoproliferative malignancies. The coding region of the NF-1 gene was analyzed by real-time polymerase chain reaction and direct sequencing. Multiplex ligation-dependent probe amplification was performed to detect the number of mutant copies. The NF1 gene analysis showed the following alterations: mosaic duplication of NF1, TRAF4, and MYO1D. Fluorescence in situ hybridization using probes (RP5-1002G3 and RP5-92689 flanking NF1 gene in 17q11.2 and CEP17 for 17q11.11.1 was performed. There were three signals (RP5-1002G3conRP5-92689 in the interphases analyzed and two signals (RP5-1002G3conRP5-92689 in 93% of cells. These findings show a tandem duplication of 17q11.2. Conclusion. The case suggests the possibility that NF1 gene duplication may be associated with a phenotype characterized by lymphoproliferative disorders.

  8. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    Directory of Open Access Journals (Sweden)

    Signe Tandrup Schmidt

    2016-03-01

    Full Text Available The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI. Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs, which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the

  9. TRIM44 Is a Poor Prognostic Factor for Breast Cancer Patients as a Modulator of NF-κB Signaling.

    Science.gov (United States)

    Kawabata, Hidetaka; Azuma, Kotaro; Ikeda, Kazuhiro; Sugitani, Ikuko; Kinowaki, Keiichi; Fujii, Takeshi; Osaki, Akihiko; Saeki, Toshiaki; Horie-Inoue, Kuniko; Inoue, Satoshi

    2017-09-08

    Many of the tripartite motif (TRIM) proteins function as E3 ubiquitin ligases and are assumed to be involved in various events, including oncogenesis. In regard to tripartite motif-containing 44 (TRIM44), which is an atypical TRIM family protein lacking the RING finger domain, its pathophysiological significance in breast cancer remains unknown. We performed an immunohistochemical study of TRIM44 protein in clinical breast cancer tissues from 129 patients. The pathophysiological role of TRIM44 in breast cancer was assessed by modulating TRIM44 expression in MCF-7 and MDA-MB-231 breast cancer cells. TRIM44 strong immunoreactivity was significantly associated with nuclear grade (p = 0.033), distant disease-free survival (p = 0.031) and overall survival (p = 0.027). Multivariate analysis revealed that the TRIM44 status was an independent prognostic factor for distant disease-free survival (p = 0.005) and overall survival (p = 0.002) of patients. siRNA-mediated TRIM44 knockdown significantly decreased the proliferation of MCF-7 and MDA-MB-231 cells and inhibited the migration of MDA-MB-231 cells. Microarray analysis and qRT-PCR showed that TRIM44 knockdown upregulated CDK19 and downregulated MMP1 in MDA-MB-231 cells. Notably, TRIM44 knockdown impaired nuclear factor-kappa B (NF-κB)-mediated transcriptional activity stimulated by tumor necrosis factor α (TNFα). Moreover, TRIM44 knockdown substantially attenuated the TNFα-dependent phosphorylation of the p65 subunit of NF-κB and IκBα in both MCF-7 and MDA-MB-231 cells. TRIM44 would play a role in the progression of breast cancer by promoting cell proliferation and migration, as well as by enhancing NF-κB signaling.

  10. Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity: a comparative study with the HTLV-1 Tax-1 protein

    Science.gov (United States)

    2012-01-01

    Background Retroviruses HTLV-1 and HTLV-2 have homologous genomic structures but differ significantly in pathogenicity. HTLV-1 is associated with Adult T cell Leukemia (ATL), whereas infection by HTLV-2 has no association with neoplasia. Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax-1 to alter cell survival and cell cycle control mechanisms. Among these functions, Tax-1-mediated activation of cellular gene expression via the NF-κB pathway depends on Tax-1 post-translational modifications by ubiquitination and sumoylation. The Tax-2 protein of HTLV-2B (Tax-2B) is also modified by ubiquitination and sumoylation and activates the NF-κB pathway to a level similar to that of Tax-1. The present study aims to understand whether ubiquitination and sumoylation modifications are involved in Tax-2B-mediated activation of the NF-κB pathway. Results The comparison of Tax-1 and Tax-2B lysine to arginine substitution mutants revealed conserved patterns and levels of ubiquitination with notable difference in the lysine usage for sumoylation. Neither Tax-1 nor Tax-2B ubiquitination and sumoylation deficient mutants could activate the NF-κB pathway and fusion of ubiquitin or SUMO-1 to the C-terminus of the ubiquitination and sumoylation deficient Tax-2B mutant strikingly restored transcriptional activity. In addition, ubiquitinated forms of Tax-2B colocalized with RelA and IKKγ in prominent cytoplasmic structures associated with the Golgi apparatus, whereas colocalization of Tax-2B with the RelA subunit of NF-κB and the transcriptional coactivator p300 in punctate nuclear structures was dependent on Tax-2B sumoylation, as previously observed for Tax-1. Conclusions Both Tax-1 and Tax-2 activate the NF-κB pathway via similar mechanisms involving ubiquitination and sumoylation. Therefore, the different transforming potential of HTLV-1 and HTLV-2 is unlikely to be related to different modes of activation of the canonical NF-κB pathway

  11. Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity: a comparative study with the HTLV-1 Tax-1 protein

    Directory of Open Access Journals (Sweden)

    Turci Marco

    2012-12-01

    Full Text Available Abstract Background Retroviruses HTLV-1 and HTLV-2 have homologous genomic structures but differ significantly in pathogenicity. HTLV-1 is associated with Adult T cell Leukemia (ATL, whereas infection by HTLV-2 has no association with neoplasia. Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax-1 to alter cell survival and cell cycle control mechanisms. Among these functions, Tax-1-mediated activation of cellular gene expression via the NF-κB pathway depends on Tax-1 post-translational modifications by ubiquitination and sumoylation. The Tax-2 protein of HTLV-2B (Tax-2B is also modified by ubiquitination and sumoylation and activates the NF-κB pathway to a level similar to that of Tax-1. The present study aims to understand whether ubiquitination and sumoylation modifications are involved in Tax-2B-mediated activation of the NF-κB pathway. Results The comparison of Tax-1 and Tax-2B lysine to arginine substitution mutants revealed conserved patterns and levels of ubiquitination with notable difference in the lysine usage for sumoylation. Neither Tax-1 nor Tax-2B ubiquitination and sumoylation deficient mutants could activate the NF-κB pathway and fusion of ubiquitin or SUMO-1 to the C-terminus of the ubiquitination and sumoylation deficient Tax-2B mutant strikingly restored transcriptional activity. In addition, ubiquitinated forms of Tax-2B colocalized with RelA and IKKγ in prominent cytoplasmic structures associated with the Golgi apparatus, whereas colocalization of Tax-2B with the RelA subunit of NF-κB and the transcriptional coactivator p300 in punctate nuclear structures was dependent on Tax-2B sumoylation, as previously observed for Tax-1. Conclusions Both Tax-1 and Tax-2 activate the NF-κB pathway via similar mechanisms involving ubiquitination and sumoylation. Therefore, the different transforming potential of HTLV-1 and HTLV-2 is unlikely to be related to different modes of activation of

  12. NleC, a type III secretion protease, compromises NF-κB activation by targeting p65/RelA.

    Directory of Open Access Journals (Sweden)

    Hilo Yen

    Full Text Available The NF-κB signaling pathway is central to the innate and adaptive immune responses. Upon their detection of pathogen-associated molecular patterns, Toll-like receptors on the cell surface initiate signal transduction and activate the NF-κB pathway, leading to the production of a wide array of inflammatory cytokines, in attempt to eradicate the invaders. As a countermeasure, pathogens have evolved ways to subvert and manipulate this system to their advantage. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC are closely related bacteria responsible for major food-borne diseases worldwide. Via a needle-like protein complex called the type three secretion system (T3SS, these pathogens deliver virulence factors directly to host cells and modify cellular functions, including by suppressing the inflammatory response. Using gain- and loss-of-function screenings, we identified two bacterial effectors, NleC and NleE, that down-regulate the NF-κB signal upon being injected into a host cell via the T3SS. A recent report showed that NleE inhibits NF-κB activation, although an NleE-deficient pathogen was still immune-suppressive, indicating that other anti-inflammatory effectors are involved. In agreement, our present results showed that NleC was also required to inhibit inflammation. We found that NleC is a zinc protease that disrupts NF-κB activation by the direct cleavage of NF-κB's p65 subunit in the cytoplasm, thereby decreasing the available p65 and reducing the total nuclear entry of active p65. More importantly, we showed that a mutant EPEC/EHEC lacking both NleC and NleE (ΔnleC ΔnleE caused greater inflammatory response than bacteria carrying ΔnleC or ΔnleE alone. This effect was similar to that of a T3SS-defective mutant. In conclusion, we found that NleC is an anti-inflammatory bacterial zinc protease, and that the cooperative function of NleE and NleC disrupts the NF-κB pathway and accounts for most of the immune

  13. Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development.

    Science.gov (United States)

    Kaeser, Gwendolyn E; Rabe, Brian A; Saha, Margaret S

    2011-04-01

    Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABA(A) and metabotropic GABA(B) receptors. During the development of the nervous system, GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation, and activity-dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABA(A) and GABA(B) receptors during early development of Xenopus laevis. Using in situ hybridization and qRT-PCR, GABA(A) α2 was detected as a maternal mRNA. All other α-subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern, suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner. Copyright © 2011 Wiley-Liss, Inc.

  14. Expression and purification of the central stalk subunits of Na + ...

    African Journals Online (AJOL)

    , NtpD and NtpG subunits. The aim of the present study was cloning and expression of these central stalk subunits of E. hirae V-type Na+-ATPase. Here we cloned the synthesized DNA fragments, corresponding to ntpC, ntpD and ntpG genes, ...

  15. Association study of the NF1 gene and autistic disorder.

    Science.gov (United States)

    Mbarek, O; Marouillat, S; Martineau, J; Barthélémy, C; Müh, J P; Andres, C

    1999-12-15

    Neurofibromatosis type 1 (NF1) is increased about 150-fold in autistic patients. The aim of this study was to test for an association between the NF1 locus and autistic disorder. The allele distributions of three markers of the NF1 gene were studied in 85 autistic patients and 90 controls. No differences in allele distributions were observed. However, we found a new allele (allele 5) of the GXAlu marker in four autistic patients. Allele 5 was absent in a larger control population (213 individuals). The patients with allele 5 had a more severe clinical picture, mainly in the fields of motility and tonus. Our preliminary results suggest that the NF1 region is not a major susceptibility locus for autism. However, the GXAlu marker of the NF1 gene appears as a possible candidate for a susceptibility locus in a small subgroup of severely affected autistic patients. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:729-732, 1999. Copyright 1999 Wiley-Liss, Inc.

  16. INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

    Science.gov (United States)

    Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

    2012-01-01

    SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

  17. [Penicillin acylase from Escherichia coli: catalytically active subunits].

    Science.gov (United States)

    Kabakov, V E; Kliachko, N L; Levashov, A V

    1995-05-01

    Gel filtration under denaturing conditions was used to isolate the alpha- and beta-subunits of penicillin acylase (PA). Refolded subunits were obtained through removing urea by dialysis. Both renatured subunits were catalytically active during hydrolysis of phenylacetic acid p-nitroanilide; this activity decreased after addition of a serine-specific inhibitor--phenylmethanesulfonyl fluoride. The subunits were also active in reversed micelles of Aerosol OT (AOT) in octane, the optimum hydration degree being 11.9 and 17.5 for the light (alpha) and heavy (beta) subunits, respectively. The positions of the maxima were consistent with both theoretically calculated optimum hydration degrees and the earlier reported profile of enzymatic activity for native PA in reversed micelles.

  18. Cepharanthine attenuates lipopolysaccharide-induced mice mastitis by suppressing the NF-κB signaling pathway.

    Science.gov (United States)

    Ershun, Zhou; Yunhe, Fu; Zhengkai, Wei; Yongguo, Cao; Naisheng, Zhang; Zhengtao, Yang

    2014-04-01

    Cepharanthine (CEP), a biscoclaurine alkaloid isolated from Stephania cepharantha Hayata, has been reported to have potent anti-inflammatory properties. However, the anti-inflammatory effects of CEP on a mouse model of lipopolysaccharide (LPS)-induced mastitis and its underlying molecular mechanisms remain to be elucidated. The purpose of the present study was to investigate the effects of CEP on LPS-induced mouse mastitis. The mouse model of mastitis was induced by inoculation of LPS through the canals of the mammary gland. CEP was administered intraperitoneally at 1 h before and 12 h after induction of LPS. The results show that CEP significantly attenuates the infiltration of neutrophils, suppresses myeloperoxidase activity, and reduces the levels of TNF-α, IL-1β, and IL-6 in LPS-induced mouse mastitis. Furthermore, CEP inhibited the phosphorylation of NF-κB p65 subunit and the degradation of its inhibitor IκBα. All the results suggest that CEP exerts potent anti-inflammatory effects on LPS-induced mouse mastitis. Accordingly, CEP might be a potential therapeutic agent for mastitis.

  19. Structures and functions of calcium channel beta subunits.

    Science.gov (United States)

    Birnbaumer, L; Qin, N; Olcese, R; Tareilus, E; Platano, D; Costantin, J; Stefani, E

    1998-08-01

    Calcium channel beta subunits have profound effects on how alpha1 subunits perform. In this article we summarize our present knowledge of the primary structures of beta subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with alpha1 subunits, the effects of beta subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by beta subunits, such as long-lasting prepulse facilitation of alpha1C (absent in alpha1E) and inhibition by G protein betagamma dimer of alpha1E, absent in alpha1C. One beta subunit, a brain beta2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of alpha1 in oocytes requires a beta subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca2+ channels to account for "alpha1 alone" channels seen in cells with limited beta subunit expression. In one model, beta dissociates from the mature alpha1 after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of beta would then have subunit composition alpha1beta. In the other model, the "chaperoning" beta remains associated with the mature channel and "alpha1 alone" channels would in fact be alpha1beta channels. Upon co-expression of high levels of beta the regulated channels would have composition [alpha1beta]beta.

  20. ATM regulates NF-κB-dependent immediate-early genes via RelA Ser 276 phosphorylation coupled to CDK9 promoter recruitment

    Science.gov (United States)

    Fang, Ling; Choudhary, Sanjeev; Zhao, Yingxin; Edeh, Chukwudi B; Yang, Chunying; Boldogh, Istvan; Brasier, Allan R.

    2014-01-01

    Ataxia-telangiectasia mutated (ATM), a member of the phosphatidylinositol 3 kinase-like kinase family, is a master regulator of the double strand DNA break-repair pathway after genotoxic stress. Here, we found ATM serves as an essential regulator of TNF-induced NF-kB pathway. We observed that TNF exposure of cells rapidly induced DNA double strand breaks and activates ATM. TNF-induced ROS promote nuclear IKKγ association with ubiquitin and its complex formation with ATM for nuclear export. Activated cytoplasmic ATM is involved in the selective recruitment of the E3-ubiquitin ligase β-TrCP to phospho-IκBα proteosomal degradation. Importantly, ATM binds and activates the catalytic subunit of protein kinase A (PKAc), ribosmal S6 kinase that controls RelA Ser 276 phosphorylation. In ATM knockdown cells, TNF-induced RelA Ser 276 phosphorylation is significantly decreased. We further observed decreased binding and recruitment of the transcriptional elongation complex containing cyclin dependent kinase-9 (CDK9; a kinase necessary for triggering transcriptional elongation) to promoters of NF-κB-dependent immediate-early cytokine genes, in ATM knockdown cells. We conclude that ATM is a nuclear damage-response signal modulator of TNF-induced NF-κB activation that plays a key scaffolding role in IκBα degradation and RelA Ser 276 phosphorylation. Our study provides a mechanistic explanation of decreased innate immune response associated with A-T mutation. PMID:24957606

  1. Cinnamoyloxy-mammeisin Isolated from Geopropolis Attenuates Inflammatory Process by Inhibiting Cytokine Production: Involvement of MAPK, AP-1, and NF-κB.

    Science.gov (United States)

    Franchin, Marcelo; Rosalen, Pedro Luiz; da Cunha, Marcos Guilherme; Silva, Rangel Leal; Colón, David F; Bassi, Gabriel Shimizu; de Alencar, Severino Matias; Ikegaki, Masaharu; Alves-Filho, José C; Cunha, Fernando Q; Beutler, John A; Cunha, Thiago Mattar

    2016-07-22

    Chemical compounds belonging to the class of coumarins have promising anti-inflammatory potential. Cinnamoyloxy-mammeisin (CNM) is a 4-phenylcoumarin that can be isolated from Brazilian geopropolis. To our knowledge, its anti-inflammatory activity has never been studied. Therefore, the present study investigated the anti-inflammatory activity of CNM and elucidated its mechanism of action on isolated macrophages. Pretreatment with CNM reduced neutrophil migration into the peritoneal and joint cavity of mice. Likewise, CNM reduced the in vitro and in vivo release of TNF-α and CXCL2/MIP-2. Regarding the possible molecular mechanism of action, CNM reduced the phosphorylation of proteins ERK 1/2, JNK, p38 MAPK, and AP-1 (subunit c-jun) in PG-stimulated macrophages. Pretreatment with CNM also reduced NF-κB activation in RAW 264.7 macrophages stably expressing the NF-κB-luciferase reporter gene. On the other hand, it did not alter IκBα degradation or nuclear translocation of p65. Thus, the results of this study demonstrate promising anti-inflammatory activity of CNM and provide an explanation of its mechanism of action in macrophages via inhibition of MAPK signaling, AP-1, and NF-κB.

  2. A novel role of the L-type calcium channel α1D subunit as a gatekeeper for intracellular zinc signaling: zinc wave.

    Directory of Open Access Journals (Sweden)

    Satoru Yamasaki

    Full Text Available Recent studies have shown that zinc ion (Zn can behave as an intracellular signaling molecule. We previously demonstrated that mast cells stimulated through the high-affinity IgE receptor (FcεRI rapidly release intracellular Zn from the endoplasmic reticulum (ER, and we named this phenomenon the "Zn wave". However, the molecules responsible for releasing Zn and the roles of the Zn wave were elusive. Here we identified the pore-forming α(1 subunit of the Cav1.3 (α(1D L-type calcium channel (LTCC as the gatekeeper for the Zn wave. LTCC antagonists inhibited the Zn wave, and an agonist was sufficient to induce it. Notably, α(1D was mainly localized to the ER rather than the plasma membrane in mast cells, and the Zn wave was impaired by α(1D knockdown. We further found that the LTCC-mediated Zn wave positively controlled cytokine gene induction by enhancing the DNA-binding activity of NF-κB. Consistent with this finding, LTCC antagonists inhibited the cytokine-mediated delayed-type allergic reaction in mice without affecting the immediate-type allergic reaction. These findings indicated that the LTCC α(1D subunit located on the ER membrane has a novel function as a gatekeeper for the Zn wave, which is involved in regulating NF-κB signaling and the delayed-type allergic reaction.

  3. NF-kappaB p65-dependent transactivation of miRNA genes following Cryptosporidium parvum infection stimulates epithelial cell immune responses.

    Directory of Open Access Journals (Sweden)

    Rui Zhou

    2009-12-01

    Full Text Available Cryptosporidium parvum is a protozoan parasite that infects the gastrointestinal epithelium and causes diarrheal disease worldwide. Innate epithelial immune responses are key mediators of the host's defense to C. parvum. MicroRNAs (miRNAs regulate gene expression at the posttranscriptional level and are involved in regulation of both innate and adaptive immune responses. Using an in vitro model of human cryptosporidiosis, we analyzed C. parvum-induced miRNA expression in biliary epithelial cells (i.e., cholangiocytes. Our results demonstrated differential alterations in the mature miRNA expression profile in cholangiocytes following C. parvum infection or lipopolysaccharide stimulation. Database analysis of C. parvum-upregulated miRNAs revealed potential NF-kappaB binding sites in the promoter elements of a subset of miRNA genes. We demonstrated that mir-125b-1, mir-21, mir-30b, and mir-23b-27b-24-1 cluster genes were transactivated through promoter binding of the NF-kappaB p65 subunit following C. parvum infection. In contrast, C. parvum transactivated mir-30c and mir-16 genes in cholangiocytes in a p65-independent manner. Importantly, functional inhibition of selected p65-dependent miRNAs in cholangiocytes increased C. parvum burden. Thus, we have identified a panel of miRNAs regulated through promoter binding of the NF-kappaB p65 subunit in human cholangiocytes in response to C. parvum infection, a process that may be relevant to the regulation of epithelial anti-microbial defense in general.

  4. Endotoxin/lipopolysaccharide activates NF-kappa B and enhances tumor cell adhesion and invasion through a beta 1 integrin-dependent mechanism.

    LENUS (Irish Health Repository)

    Wang, Jiang Huai

    2012-02-03

    Beta(1) integrins play a crucial role in supporting tumor cell attachment to and invasion into the extracellular matrix. Endotoxin\\/LPS introduced by surgery has been shown to enhance tumor metastasis in a murine model. Here we show the direct effect of LPS on tumor cell adhesion and invasion in extracellular matrix proteins through a beta(1) integrin-dependent pathway. The human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of the beta(1) subunit, whereas various low levels of alpha(1), alpha(2), alpha(4), and alpha(6) expression were detected. SW480 and SW620 did not express membrane-bound CD14; however, LPS in the presence of soluble CD14 (sCD14) significantly up-regulated beta(1) integrin expression; enhanced tumor cell attachment to fibronectin, collagen I, and laminin; and strongly promoted tumor cell invasion through the Matrigel. Anti-beta(1) blocking mAbs (4B4 and 6S6) abrogated LPS- plus sCD14-induced tumor cell adhesion and invasion. Furthermore, LPS, when combined with sCD14, resulted in NF-kappaB activation in both SW480 and SW620 cells. Inhibition of the NF-kappaB pathway significantly attenuated LPS-induced up-regulation of beta(1) integrin expression and prevented tumor cell adhesion and invasion. These results provide direct evidence that although SW480 and SW620 cells do not express membrane-bound CD14, LPS in the presence of sCD14 can activate NF-kappaB, up-regulate beta(1) integrin expression, and subsequently promote tumor cell adhesion and invasion. Moreover, LPS-induced tumor cell attachment to and invasion through extracellular matrix proteins is beta(1) subunit-dependent.

  5. Effect of natural and semi-synthetic cadinanes from Heterotheca inuloides on NF-κB, Nrf2 and STAT3 signaling pathways and evaluation of their in vitro cytotoxicity in human cancer cell lines.

    Science.gov (United States)

    Egas, Verónica; Millán, Estrella; Collado, Juan A; Ramírez-Apan, Teresa; Méndez-Cuesta, Carlos A; Muñoz, Eduardo; Delgado, Guillermo

    2017-06-15

    The effects of ten natural cadinane sesquiterpenoids isolated from Heterotheca inuloides on the pathways of the NF-κB, Nrf2 and STAT3 transcription factors were studied for the first time. The main constituent in this species, 7-hydroxy-3,4-dihydrocadalene (1), showed anti-NF-κB activity and activated the antioxidant Nrf2 pathway, which may explain the properties reported for the traditional use of the plant. In addition to the main metabolite, a structurally similar compound, 7-hydroxy-cadalene (2), also displayed anti-NF-κB activity. Thus, both natural compounds were used as templates for the preparation of a novel semi-synthetic derivative set, including esters and carbamates, which were evaluated for their potential in vitro antiproliferative activities against six human cancer cell lines. Carbamate derivatives 32 and 33 were found to exhibit potent activity against human colorectal adenocarcinoma and showed important selectivity in cancer cells. Among ester derivatives, compound 13 was determined to be a more potent NF-κB inhibitor and Nrf2 activator than its parent, 7-hydroxy-3,4-dihydrocadalene (1). Furthermore, this compound decreases levels of phospho-IκBα, a protein complex involved in the NF-κB activation pathway. Molecular simulations suggest that all active compounds interact with the activation loop of the IKKβ subunit in the IKK complex, which is the responsible of IκBα phosphorylation. Thus, we identified two natural, and one semi-synthetic, NF-κB and Nrf2 modulators and two new promising cytotoxic compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Social and Emotional Functioning of Children With NF-1 and Their Families; A Case Controlled Study

    National Research Council Canada - National Science Library

    Noll, Robert

    2001-01-01

    ...) of children with NF and explore potential linkages between NF and QOL in these 3 domains. The second objective of this work is to evaluate parental distress, family functioning, and child rearing practices...

  7. Social and Emotional Functioning of Children with NF-1 and Their Families: A Case Controlled Study

    National Research Council Canada - National Science Library

    Noll, Robert

    2000-01-01

    ...) of children with NF and explore potential linkages between NF and QOL in these 3 domains. The second objective of this work is to evaluate parental distress, family functioning, and child rearing practices...

  8. Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

    Science.gov (United States)

    Monteiro, Karina M.; Cardoso, Mateus B.; Follmer, Cristian; da Silveira, Nádya P.; Vargas, Daiani M.; Kitajima, Elliot W.; Zaha, Arnaldo; Ferreira, Henrique B.

    2012-01-01

    Background Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. Methodology/Principal Findings The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. Conclusions/Significance For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the

  9. The NF-κB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells.

    Directory of Open Access Journals (Sweden)

    Michael Poppe

    2017-03-01

    Full Text Available Coronavirus replication takes place in the host cell cytoplasm and triggers inflammatory gene expression by poorly characterized mechanisms. To obtain more insight into the signals and molecular events that coordinate global host responses in the nucleus of coronavirus-infected cells, first, transcriptome dynamics was studied in human coronavirus 229E (HCoV-229E-infected A549 and HuH7 cells, respectively, revealing a core signature of upregulated genes in these cells. Compared to treatment with the prototypical inflammatory cytokine interleukin(IL-1, HCoV-229E replication was found to attenuate the inducible activity of the transcription factor (TF NF-κB and to restrict the nuclear concentration of NF-κB subunits by (i an unusual mechanism involving partial degradation of IKKβ, NEMO and IκBα and (ii upregulation of TNFAIP3 (A20, although constitutive IKK activity and basal TNFAIP3 expression levels were shown to be required for efficient virus replication. Second, we characterized actively transcribed genomic regions and enhancers in HCoV-229E-infected cells and systematically correlated the genome-wide gene expression changes with the recruitment of Ser5-phosphorylated RNA polymerase II and prototypical histone modifications (H3K9ac, H3K36ac, H4K5ac, H3K27ac, H3K4me1. The data revealed that, in HCoV-infected (but not IL-1-treated cells, an extensive set of genes was activated without inducible p65 NF-κB being recruited. Furthermore, both HCoV-229E replication and IL-1 were shown to upregulate a small set of genes encoding immunomodulatory factors that bind p65 at promoters and require IKKβ activity and p65 for expression. Also, HCoV-229E and IL-1 activated a common set of 440 p65-bound enhancers that differed from another 992 HCoV-229E-specific enhancer regions by distinct TF-binding motif combinations. Taken together, the study shows that cytoplasmic RNA viruses fine-tune NF-κB signaling at multiple levels and profoundly reprogram the

  10. Gastrodin stimulates anticancer immune response and represses transplanted H22 hepatic ascitic tumor cell growth: Involvement of NF-κB signaling activation in CD4 + T cells

    Energy Technology Data Exchange (ETDEWEB)

    Shu, Guangwen; Yang, Tianming [College of Pharmacy, South-Central University for Nationalities, Wuhan (China); Wang, Chaoyuan [College of Life Science, South-Central University for Nationalities, Wuhan (China); Su, Hanwen, E-mail: suhanwen-1@163.com [Renmin Hospital of Wuhan University, Wuhan (China); Xiang, Meixian, E-mail: xiangmeixian99@163.com [College of Pharmacy, South-Central University for Nationalities, Wuhan (China)

    2013-06-15

    Gastrodia elata Blume (G. elata) is a famous restorative food in East Asia. It can be used as an auxiliary reagent in hepatocellular carcinoma (HCC) treatment. Previous studies unveiled that G. elata exhibited immunomodulatory activities. To explore the active ingredients contributing to its immunomodulatory activities, gastrodin, vanillin, and parishin B were purified from G. elata and their anti-HCC effects were assessed in vivo. Among these compounds, only gastrodin was capable of repressing transplanted H22 ascitic hepatic tumor cell growth in vivo with low toxicity. Further investigations were designed to explore the effects of gastrodin on the immune system of tumor-bearing mice and potential molecular mechanisms underlying these effects. Our data showed that gastrodin ameliorated tumor cell transplantation-induced activation of endogenous pro-apoptotic pathway in CD4 + T cells and abnormalities in serum cytokine profiles in host animals. These events enhanced cytotoxic activities of natural killer and CD8 + T cells against H22 hepatic cancer cells. Gastrodin administration specifically upregulated mRNA levels of several nuclear factor κB (NF-κB) responsive genes in CD4 + T cells but not in CD8 + T cells. Chromatin immunoprecipitation assay showed that gastrodin increased the association of NF-κB p65 subunit to the promoter regions of IL-2 and Bcl-2 encoding genes in CD4 + T cells. Our investigations demonstrated that gastrodin is the main active ingredient contributing to the anticancer immunomodulatory properties of G. elata. Promoting NF-κB-mediated gene transcription in CD4 + T cells is implicated in its immunomodulatory activity. - Highlights: • Gastrodin stimulates anticancer immune response. • Gastrodin represses tumor transplantation-induced CD4 + T cell apoptosis. • Gastrodin activates NF-κB activity in CD4 + T cells.

  11. Caffeine suppresses lipopolysaccharide-stimulated BV2 microglial cells by suppressing Akt-mediated NF-κB activation and ERK phosphorylation.

    Science.gov (United States)

    Kang, Chang-Hee; Jayasooriya, Rajapaksha Gendara Prasad Tharanga; Dilshara, Matharage Gayani; Choi, Yung Hyun; Jeong, Yong-Kee; Kim, Nam Deuk; Kim, Gi-Young

    2012-12-01

    Since the anti-inflammatory effect of caffeine is unclear in microglial cells, we performed whether caffeine attenuates the expression of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Caffeine substantially suppressed the LPS-induced pro-inflammatory mediators nitric oxide (NO), prostaglandin E(2) (PGE(2)) and tumor necrosis factor-α (TNF-α) in BV2 microglial cells. These effects resulted from the inhibition of their regulatory genes inducible NO synthase (iNOS), cycloxygenase-2 (COX-2) and TNF-α. In addition, caffeine significantly decreased LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) by suppressing the nuclear translocation of p50 and p65 subunits. A specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), attenuated the LPS-induced expression of iNOS, COX-2 and TNF-α genes. In addition, we elucidated that inhibition of Akt phosphorylation plays a crucial role in caffeine-mediated NF-κB regulation in LPS-stimulated BV2 microglial cells. Caffeine also attenuated the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK) and a specific inhibitor of ERK, PD98059, subsequently downregulated the expression of the pro-inflammatory genes iNOS, COX-2 and TNF-α. Taken together, our data indicate that caffeine suppresses the generation of pro-inflammatory mediators, such as NO, PGE(2) and TNF-α as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting Akt-dependent NF-κB activation and the ERK signaling pathway. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. The ROS/NF-κB/NR4A2 pathway is involved in H2O2 induced apoptosis of resident cardiac stem cells via autophagy.

    Science.gov (United States)

    Shi, Xingxing; Li, Wenjing; Liu, Honghong; Yin, Deling; Zhao, Jing

    2017-09-29

    Cardiac stem cells (CSCs)-based therapy provides a promising avenue for the management of ischemic heart diseases. However, engrafted CSCs are subjected to acute cell apoptosis in the ischemic microenvironment. Here, stem cell antigen 1 positive (Sca-1+) CSCs proved to own therapy potential were cultured and treated with H2O2 to mimic the ischemia situation. As autophagy inhibitor, 3-methyladenine (3MA), inhibited H2O2-induced CSCs apoptosis, thus we demonstrated that H2O2 induced autophagy-dependent apoptosis in CSCs, and continued to find key proteins responsible for the crosstalk between autophagy and apoptosis. Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), increased upon cardiomyocyte injury with unknown functions in CSCs, was increased by H2O2. NR4A2 siRNA attenuated H2O2 induced autophagy and apoptosis in CSCs, which suggested an important role of NR4A2 in CSCs survival in ischemia conditions. Reactive oxygen species (ROS) and NF-κB (P65) subunit were both increased by H2O2. Either the ROS scavenger, N-acetyl-l-cysteine (NAC) or NF-κB signaling inhibitor, bay11-7082 could attenuate H2O2-induced autophagy and apoptosis in CSCs, which suggested they were involved in this process. Furthermore, NAC inhibited NF-κB activities, while bay11-7082 inhibited NR4A2 expression, which revealed a ROS/NF-κB/NR4A2 pathway responsible for H2O2-induced autophagy and apoptosis in CSCs. Our study supports a new clue enhancing the survival rate of CSCs in the infarcted myocardium for cell therapy in ischemic cardiomyopathy.

  13. Treponema pallidum flagellins stimulate MMP-9 and MMP-13 expression via TLR5 and MAPK/NF-κB signaling pathways in human epidermal keratinocytes.

    Science.gov (United States)

    Jiang, Chuanhao; Xu, Man; Kuang, Xingxing; Xiao, Jinhong; Tan, Manyi; Xie, Yafeng; Xiao, Yongjian; Zhao, Feijun; Wu, Yimou

    2017-12-01

    Syphilis is a chronic disease caused by Treponema pallidum and the pathogenesis is still unclear. T. pallidum infection induced inflammatory responses are involved in the immunopathological damage in skin and other tissues. Flagellin, the monomeric subunit of bacterial flagella, is a classic pathogen associated molecular patterns (PAMPs) that interacts to TLR5 and induces inflammatory responses. Keratinocytes, as immune sentinels recognize the PAMPs via TLRs, play an important role in skin innate immune response. Matrix metalloproteinases (MMPs) expressed by keratinocytes are involved in skin inflammatory responses and promoting pathogens invasion. In this study, we demonstrate that FlaB1, FlaB2 and FlaB3, the flagellins of T. pallidum, induced MMP-9 and MMP-13 production in human immortalized keratinocytes cell line HaCaT. Silencing of TLR5, but not TLR2 and TLR4 attenuated MMP-9 and MMP-13 expressions induced by T. pallidum flagellins. MMP-9 and MMP-13 expressions were also be abrogated by transfection with a dominant negative (DN) plasmid of MyD88. We also found that treatment of HaCaT cells with FlaB1, FlaB2 and FlaB3 activate the MAPK and NF-κB signaling pathways. Inhibited of ERK, JNK, p38 and NF-κB suppressed MMP-9 expression induced by the FlaB1. MMP-13 expression was found to be suppressed by pretreatment with inhibitors of ERK, JNK and NF-κB, but not p38. These findings demonstrate that T. pallidum flagellins (FlaB1, FlaB2 or FlaB3) can stimulate MMP-9 and MMP-13 expression through TLR5 and MAPK/NF-κB signaling pathways in human epidermal keratinocytes, which could contribute to the pathogenesis of T. pallidum infection. Copyright © 2017. Published by Elsevier Inc.

  14. Okanin, effective constituent of the flower tea Coreopsis tinctoria, attenuates LPS-induced microglial activation through inhibition of the TLR4/NF-κB signaling pathways

    Science.gov (United States)

    Hou, Yue; Li, Guoxun; Wang, Jian; Pan, Yingni; Jiao, Kun; Du, Juan; Chen, Ru; Wang, Bing; Li, Ning

    2017-04-01

    The EtOAc extract of Coreopsis tinctoria Nutt. significantly inhibited LPS-induced nitric oxide (NO) production, as judged by the Griess reaction, and attenuated the LPS-induced elevation in iNOS, COX-2, IL-1β, IL-6 and TNF-α mRNA levels, as determined by quantitative real-time PCR, when incubated with BV-2 microglial cells. Immunohistochemical results showed that the EtOAc extract significantly decreased the number of Iba-1-positive cells in the hippocampal region of LPS-treated mouse brains. The major effective constituent of the EtOAc extract, okanin, was further investigated. Okanin significantly suppressed LPS-induced iNOS expression and also inhibited IL-6 and TNF-α production and mRNA expression in LPS-stimulated BV-2 cells. Western blot analysis indicated that okanin suppressed LPS-induced activation of the NF-κB signaling pathway by inhibiting the phosphorylation of IκBα and decreasing the level of nuclear NF-κB p65 after LPS treatment. Immunofluorescence staining results showed that okanin inhibited the translocation of the NF-κB p65 subunit from the cytosol to the nucleus. Moreover, okanin significantly inhibited LPS-induced TLR4 expression in BV-2 cells. In summary, okanin attenuates LPS-induced activation of microglia. This effect may be associated with its capacity to inhibit the TLR4/NF-κB signaling pathways. These results suggest that okanin may have potential as a nutritional preventive strategy for neurodegenerative disorders.

  15. Coxiella burnetii Employs the Dot/Icm Type IV Secretion System to Modulate Host NF-κB/RelA Activation.

    Science.gov (United States)

    Mahapatra, Saugata; Gallaher, Brandi; Smith, Sydni Caet; Graham, Joseph G; Voth, Daniel E; Shaw, Edward I

    2016-01-01

    Coxiella burnetii is the causative agent of Q fever and an obligate intracellular pathogen in nature that survives and grows in a parasitophorous vacuole (PV) within eukaryotic host cells. C. burnetii promotes intracellular survival by subverting apoptotic and pro-inflammatory signaling pathways that are typically regulated by nuclear transcription factor-κB (NF-κB). We and others have demonstrated that C. burnetii NMII proteins inhibit expression of pro-inflammatory cytokines and induce expression of anti-apoptotic genes during infection. Here, we demonstrate that C. burnetii promotes intracellular survival by modulating NF-κB subunit p65 (RelA) phosphorylation, and thus activation, in a Type Four B Secretion System (T4BSS)-dependent manner. Immunoblot analysis of RelA phosphorylated at serine-536 demonstrated that C. burnetii increases NF-κB activation via the canonical pathway. However, RelA phosphorylation levels were even higher in infected cells where bacterial protein or mRNA synthesis was inhibited. Importantly, we demonstrate that inhibition of RelA phosphorylation impairs PV formation and C. burnetii growth. We found that a T4BSS-defective mutant (CbΔdotA) elicited phosphorylated RelA levels similar to those of wild type C. burnetii infection treated with Chloramphenicol. Moreover, cells infected with CbΔdotA or wild type C. burnetii treated with Chloramphenicol showed similar levels of GFP-RelA nuclear localization, and significantly increased localization compared to wild type C. burnetii infection. These data indicate that without de novo protein synthesis and a functional T4BSS, C. burnetii is unable to modulate NF-κB activation, which is crucial for optimal intracellular growth.

  16. A Polyphenol-rich Pomegranate Fruit Extract Suppresses NF-κB and IL-6 Expression by Blocking the Activation of IKKβ and NIK in Primary Human Chondrocytes.

    Science.gov (United States)

    Haseeb, Abdul; Khan, Nazir M; Ashruf, Omer S; Haqqi, Tariq M

    2017-05-01

    Pomegranate fruit extract (PE) rich in polyphenols has been shown to exert chondroprotective effects, but the mechanism is not established. Here, we used an in vitro model of inflammation in osteoarthritis (OA) to investigate the potential of PE to suppress interleukin 1 beta (IL-1β)-stimulated expression of inflammatory cytokine IL-6, generation of reactive oxygen species (ROS) levels, and investigated the mechanism of NF-κB inhibition by analyzing the activation of the kinases upstream of IκBα in primary human chondrocytes. Total and phosphorylated forms of kinases and expression of IL-6 were determined at protein and mRNA levels by western immunoblotting and Taqman assay, respectively. Dihydrorhodamine 123 staining estimated ROS generation. Pomegranate fruit extract inhibited the mRNA and protein expression of IL-6, generation of ROS, and inhibited the IL-1β-mediated phosphorylation of inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), expression of IKKβ mRNA, degradation of IκBα, and activation and nuclear translocation of NF-κB/p65 in human chondrocytes. Importantly, phosphorylation of NF-κB-inducing kinase was blocked by PE in IL-1β-treated human OA chondrocytes. Taken together, these data suggest that PE exerts the chondroprotective effect(s) by suppressing the production of IL-6 and ROS levels. Inhibition of NF-κB activation by PE was blocked via modulation of activation of upstream kinases in human OA chondrocytes. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  17. DHX15 is associated with poor prognosis in acute myeloid leukemia (AML) and regulates cell apoptosis via the NF-kB signaling pathway.

    Science.gov (United States)

    Pan, Lili; Li, Yang; Zhang, Hai-Ying; Zheng, Yi; Liu, Xiao-Li; Hu, Zheng; Wang, Yi; Wang, Jing; Cai, Yuan-Hua; Liu, Qiao; Chen, Wan-Ling; Guo, Ying; Huang, Yuan-Mao; Qian, Feng; Jin, Li; Wang, Jiucun; Wang, Shao-Yuan

    2017-10-27

    The role of DHX15, a newly identified DEAH-box RNA helicase, in leukemogenesis remains elusive. Here, we identified a recurrent mutation in DHX15 (NM_001358:c.664C>G: p.(R222G)) in one familial AML patient and 4/240 sporadic AML patients. Additionally, DHX15 was commonly overexpressed in AML patients and associated with poor overall survival (OS) (P=0.019) and relapse-free survival (RFS) (P=0.032). In addition, we found a distinct expression pattern of DHX15. DHX15 was highly expressed in hematopoietic stem cells and leukemia cells but was lowly expressed in mature blood cells. DHX15 was down-regulated when AML patients achieved disease remission or when leukemia cell lines were induced to differentiate. DHX15 silencing greatly inhibited leukemia cell proliferation and induced cell apoptosis and G1-phase arrest. In contrast, the restoration of DHX15 expression rescued cell viability and reduced cell apoptosis. In addition, we found that DHX15 was down-regulated when cell apoptosis was induced by ATO (arsenic trioxide); overexpression of DHX15 caused dramatic resistance to ATO-induced cell apoptosis, suggesting an important role for DHX15 in cell apoptosis. We further explored the mechanism of DHX15 in apoptosis and found that overexpression of DHX15 activated NF-kB transcription. Knockdown of DHX15 inhibited the nuclear translocation and activation of the NF-kB subunit P65 in leukemia cells. Several downstream targets of the NF-kB pathway were also down-regulated, and apoptosis-associated genes CASP3 and PARP were activated. In conclusion, this study represents the first demonstration that DHX15 plays an important role in leukemogenesis via the NF-kB signaling pathway and may serve as an independent prognostic marker for AML.

  18. Oleifolioside A, a New Active Compound, Attenuates LPS-Stimulated iNOS and COX-2 Expression through the Downregulation of NF-κB and MAPK Activities in RAW 264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Hai Yang Yu

    2012-01-01

    Full Text Available Oleifolioside A, a new triterpenoid compound isolated from Dendropanax morbifera Leveille (D. morbifera, was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS-stimulated nitric oxide (NO and prostaglandin E2 (PGE2 production in RAW 264.7 macrophages. Consistent with these findings, oleifolioside A was further shown to suppress the expression of LPS-stimulated inducible nitric oxide synthase (iNOS and cyclooxigenase-2 (COX-2 in a dose-dependent manner at both the protein and mRNA levels and to significantly inhibit the DNA-binding activity and transcriptional activity of NF-κB in response to LPS. These results were found to be associated with the inhibition of the degradation and phosphorylation of IκB-α and subsequent translocation of the NF-κB p65 subunit to the nucleus. Inhibition of NF-κB activation by oleifolioside A was also shown to be mediated through the prevention of p38 MAPK and ERK1/2 phosphorylation. Taken together, our results suggest that oleifolioside A has the potential to be a novel anti-inflammatory agent capable of targeting both the NF-κB and MAPK signaling pathways.

  19. Ginsenoside Rd blocks AIF mitochondrio-nuclear translocation and NF-κB nuclear accumulation by inhibiting poly(ADP-ribose) polymerase-1 after focal cerebral ischemia in rats.

    Science.gov (United States)

    Hu, Gengyao; Wu, Zhongliang; Yang, Feng; Zhao, Haibo; Liu, Xuedong; Deng, Yanchun; Shi, Ming; Zhao, Gang

    2013-12-01

    Our previous clinical and basic studies have demonstrated that ginsenoside Rd (GS-Rd) has remarkable neuroprotective effects after cerebral ischemia but the underlying mechanisms are still unknown. In our latest studies, we revealed that GS-Rd could prevent mitochondrial release of apoptosis-inducing factor (AIF) and reduce inflammatory response following transient focal ischemia in rats. Poly(ADP-ribose) polymerase-1 (PARP-1) is required for both AIF release from mitochondria and NF-κB-mediated inflammation. Here, we investigated whether GS-Rd could act on PARP-1 and subsequently affect AIF translocation and NF-κB activation. Sprague-Dawley rats were treated with GS-Rd (10 mg/kg) 30 min before surgery with the right middle cerebral artery occlusion, and at different time points following cerebral ischemia, brain tissues were collected for western blotting analysis. Our results showed that GS-Rd significantly attenuated ischemia-triggered increased levels of Poly(ADP-ribose), an enzymatic product catalyzed by PARP-1, but not altered the expression of PARP-1 per se. Meanwhile, GS-Rd pretreatment reduced AIF mitochondrio-nuclear translocation and inhibited NF-κB p65 subunit nuclear accumulation after cerebral ischemia. Therefore, our findings provide the first evidence that GS-Rd can inhibit PARP-1 activity and sequential AIF translocation and NF-κB nuclear accumulation, which may be responsible for GS-Rd's neuroprotection against both neuronal cell death and inflammation after ischemic stroke.

  20. Photobiomodulation reduces oral mucositis by modulating NF-kB

    Science.gov (United States)

    Curra, Marina; Pellicioli, Ana Carolina Amorim; Filho, Nélson Alexandre Kretzmann; Ochs, Gustavo; Matte, Úrsula; Filho, Manoel Sant'Ana; Martins, Marco Antonio Trevizani; Martins, Manoela Domingues

    2015-12-01

    The aim of this study was to evaluate NF-kB during 5-fluorouracil (FU)-induced oral mucositis and ascertain whether photobiomodulation (PBM), as a preventive and/or therapeutic modality, influences this transcription factor. Ninety-six male golden Syrian hamsters were allocated into four groups: control (no treatment); PBM therapeutic, PBM preventive, and PBM combined. Animals received an injection of 5-FU on days 0 and 2. On days 3 and 4, the buccal mucosa was scratched. Irradiation was carried out using a 660-nm, 40-mW diode laser at 6 J/cm2 during 6 s/point, 0.24 J/point, for a total dose of 1.44 J/day of application. Animals were euthanized on days 0, 5, 10, and 15 (n=6). Buccal mucosa was removed for protein quantification by Western blot. Clinical analysis revealed that PBM groups exhibited less mucositis than controls on day 10. Control animals exhibited lower levels of NF-kB during mucositis development and healing. The preventive and combined protocols were associated with higher NF-kB levels at day 5; however, the therapeutic group had higher levels at days 10 and 15. These findings suggest that the preventive and/or therapeutic PBM protocols reduced the severity of oral mucositis by activating the NF-kB pathway.

  1. NF-κB Inhibitors from Eurycoma longifolia

    Science.gov (United States)

    2014-01-01

    The roots of Eurycoma longifolia have been used in many countries of Southeast Asia to alleviate various diseases including malaria, dysentery, sexual insufficiency, and rheumatism. Although numerous studies have reported the pharmacological properties of E. longifolia, the mode of action of the anti-inflammatory activity has not been elucidated. Bioguided isolation of NF-κB inhibitors using an NF-κB-driven luciferase reporter gene assay led to the identification of a new quassinoid, eurycomalide C (1), together with 27 known compounds including 11 quassinoids (2–12), six alkaloids (13–18), two coumarins (19, 20), a squalene derivative (21), a triterpenoid (22), and six phenolic compounds (23–28) from the extract of E. longifolia. Evaluation of the biological activity revealed that C19-type and C20-type quassinoids, β-carboline, and canthin-6-one alkaloids are potent NF-κB inhibitors, with IC50 values in the low micromolar range, while C18-type quassinoids, phenolic compounds, coumarins, the squalene derivative, and the triterpenoid turned out to be inactive when tested at a concentration of 30 μM. Eurycomalactone (2), 14,15β-dihydroklaieanone (7), and 13,21-dehydroeurycomanone (10) were identified as potent NF-κB inhibitors with IC50 values of less than 1 μM. PMID:24467387

  2. The ORF61 Protein Encoded by Simian Varicella Virus and Varicella-Zoster Virus Inhibits NF-κB Signaling by Interfering with IκBα Degradation.

    Science.gov (United States)

    Whitmer, Travis; Malouli, Daniel; Uebelhoer, Luke S; DeFilippis, Victor R; Früh, Klaus; Verweij, Marieke C

    2015-09-01

    Varicella-zoster virus (VZV) causes chickenpox upon primary infection and establishes latency in ganglia. Reactivation from latency causes herpes zoster, which may be complicated by postherpetic neuralgia. Innate immunity mediated by interferon and proinflammatory cytokines represents the first line of immune defense upon infection and reactivation. VZV is known to interfere with multiple innate immune signaling pathways, including the central transcription factor NF-κB. However, the role of these inhibitory mechanisms in vivo is unknown. Simian varicella virus (SVV) infection of rhesus macaques recapitulates key aspects of VZV pathogenesis, and this model thus permits examination of the role of immune evasion mechanisms in vivo. Here, we compare SVV and VZV with respect to interference with NF-κB activation. We demonstrate that both viruses prevent ubiquitination of the NF-κB inhibitor IκBα, whereas SVV additionally prevents IκBα phosphorylation. We show that the ORF61 proteins of VZV and SVV are sufficient to prevent IκBα ubiquitination upon ectopic expression. We further demonstrate that SVV ORF61 interacts with β-TrCP, a subunit of the SCF ubiquitin ligase complex that mediates the degradation of IκBα. This interaction seems to inactivate SCF-mediated protein degradation in general, since the unrelated β-TrCP target Snail is also stabilized by ORF61. In addition to ORF61, SVV seems to encode additional inhibitors of the NF-κB pathway, since SVV with ORF61 deleted still prevented IκBα phosphorylation and degradation. Taken together, our data demonstrate that SVV interferes with tumor necrosis factor alpha (TNF-α)-induced NF-κB activation at multiple levels, which is consistent with the importance of these countermechanisms for varicella virus infection. The role of innate immunity during the establishment of primary infection, latency, and reactivation by varicella-zoster virus (VZV) is incompletely understood. Since infection of rhesus

  3. Motor dysfunction in NF1: Mediated by attention deficit or inherent to the disorder?

    Science.gov (United States)

    Haas-Lude, Karin; Heimgärtner, Magdalena; Winter, Sarah; Mautner, Victor-Felix; Krägeloh-Mann, Ingeborg; Lidzba, Karen

    2018-01-01

    Attention deficit and compromised motor skills are both prevalent in Neurofibromatosis type 1 (NF1), but the relationship is unclear. We investigated motor function in children with NF1 and in children with Attention Deficit/Hyperactivity Disorder (ADHD), and explored if, in patients with NF1, attention deficit influences motor performance. Motor performance was measured using the Movement Assessment Battery for Children (M-ABC) in 71 children (26 with NF1 plus ADHD, 14 with NF1 without ADHD, and 31 with ADHD without NF1) aged 6-12 years. There was a significant effect of group on motor performance. Both NF1 groups scored below children with ADHD without NF1. Attention performance mediated motor performance in children with ADHD without NF1, but not in children with NF1. Motor function is not mediated by attention performance in children with NF1. While in ADHD, attention deficit influences motor performance, motor problems in NF1 seem to be independent from attention deficit. This argues for different pathomechanisms in these two groups of developmental disorders. Copyright © 2017 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

  4. Data of evolutionary structure change: 1GAZA-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

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  5. Data of evolutionary structure change: 1BFOB-2V7NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1BFOB-2V7NF 1BFO 2V7N B F EVKLLESGGGLVQPGGSMRLSCAGSGFTFTDFYMNWIRQ...E EEE EEEEEEEEEEEE EEEEEEE EEEEEE EVID> 0 F 2V7NF PAVLQSSGLYS EVID>EVID> 1 ...dbChain>F 2V7NF VVTVPSSSLGTQ

  6. Data of evolutionary structure change: 1B2KA-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1B2KA-2G4NF 1B2K 2G4N A F KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFN...G HHHH -GGGG -- EVID>EVID> 0 2G4N F 2G4NF IVQNN...>1.955286979675293 EVID>EVID> 1 2G4N F 2G4NF KKILD-KVGIN

  7. Data of evolutionary structure change: 1CKGB-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available EVID>EVID> 1 2G4N F 2G4NF...1CKGB-2G4NF 1CKG 2G4N B F KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYN... HHHH -GGGG -- EVID>EVID> 0 2G4N F 2G4NF ELKDL-...-0.49799999594688416 2.0951859951019287 4.119585990905762

  8. Data of evolutionary structure change: 1B7RA-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

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  9. Data of evolutionary structure change: 1CJ7A-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

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  10. Data of evolutionary structure change: 1GB0A-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1GB0A-2G4NF 1GB0 2G4N A F KLFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYN...G HHHH - GGGG -- EVID>EVID> 0 2G4N... F 2G4NF IVQN.../rotation> 0.7072579860687256 1.318071961402893 EVID> 1 2G4N F 2G4NF

  11. Data of evolutionary structure change: 1B2KB-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1B2KB-2G4NF 1B2K 2G4N B F KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFN... HHHH - GGGG -- EVID>EVID> 0 2G4N F 2G4NF IVQNN...387972831726 EVID> 1 2G4N... F 2G4NF KKILD-KVGIN

  12. Data of evolutionary structure change: 1B0DA-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available EVID>EVID> 1 2G4N F 2G4NF...1B0DA-2G4NF 1B0D 2G4N A F KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFN...HHHH -GGGG -- EVID>EVID> 0 2G4N F 2G4NF IVQNN-D...z> 1.0584540367126465 1.9556859731674194

  13. Data of evolutionary structure change: 1GBOA-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1GBOA-2G4NF 1GBO 2G4N A F KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYN...HH - GGGG -- EVID>EVID> 0 2G4N F 2G4NF IVQNN--D...0.7297589778900146 1.3745650053024292 EVID> 1 2G4N F 2G4NF

  14. Data of evolutionary structure change: 1CKFA-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1CKFA-2G4NF 1CKF 2G4N A F KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYN... HHHH - GGGG -- EVID>EVID> 0 2G4N F 2G4NF IVQNN...tion> 0.7245799899101257 1.3730000257492065 EVID>... 1 2G4N F 2G4NF

  15. Data of evolutionary structure change: 1B7QA-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1B7QA-2G4NF 1B7Q 2G4N A F KVFERCELARTLKRLGMDGYRGISLALWMCLAKWESGYN... HHHH - GGGG -- EVID>EVID> 0 2G4N F 2G4NF VQNND...tation> 1.5742559432983398 3.2563140392303467 EVI...D> 1 2G4N F 2G4NF

  16. Data of evolutionary structure change: 1GB3A-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1GB3A-2G4NF 1GB3 2G4N A F KFFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYN... HHHH - GGGG -- EVID>EVID> 0 2G4N F 2G4NF IVQNN... 0.6893870234489441 1.2930560111999512 EVID> 1 2G4N F 2G4NF

  17. Data of evolutionary structure change: 1B7NA-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1B7NA-2G4NF 1B7N 2G4N A F KVFERCELARTLKRLGMDGYRGISLANWMCLAKWLSGYN... HHHH - GGGG -- EVID>EVID> 0 2G4N F 2G4NF IVQNN...n> 0.6914700269699097 1.3510249853134155 EVID> 1 2G4N F 2G4NF

  18. Data of evolutionary structure change: 1C46A-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1C46A-2G4NF 1C46 2G4N A F GKVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGY...HHH -GGGG -- EVID>EVID> 0 2G4N F 2G4NF ELKDL--K...tation> 2.0758020877838135 3.9801440238952637 EVI...D> 1 2G4N F 2G4NF

  19. Data of evolutionary structure change: 1GB8A-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1GB8A-2G4NF 1GB8 2G4N A F KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYN...G HHHH - GGGG -- EVID>EVID> 0 2G4N... F 2G4NF IVQN...ation> 0.7009519934654236 1.3157780170440674 EVID...> 1 2G4N F 2G4NF

  20. Data of evolutionary structure change: 1B5UA-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1B5UA-2G4NF 1B5U 2G4N A F KVFERCELARTLKRLGMDGYRGIALANWMCLAKWESGYN...G HHHH - GGGG -- EVID>EVID> 0 2G4N... F 2G4NF ELKD...604645 2.0628468990325928 4.0417399406433105 ...EVID> 1 2G4N F 2G4NF

  1. Data of evolutionary structure change: 1B5ZA-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1B5ZA-2G4NF 1B5Z 2G4N A F KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYN... HHHH -GGGG -- EVID>EVID> 0 2G4N F 2G4NF IVQNN-...tion> 0.6593620181083679 1.2106510400772095 EVID>... 1 2G4N F 2G4NF

  2. Data of evolutionary structure change: 1BFOF-2V7NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1BFOF-2V7NF 1BFO 2V7N F F EVKLLESGGGLVQPGGSMRLSCAGSGFTFTDFYMNWIRQ... EEE EEEEEEEEEEEE EEEEEEE EEEEEE EVID> 0 2V7N F 2V7NF VVTVPSSSLGTQ 2.0099360942840576 EVID>EVID> 1 ... 2V7N F 2V7NF

  3. Data of evolutionary structure change: 1BVXA-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1BVXA-2G4NF 1BVX 2G4N A F KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFN...GG HHHH - GGGG -- EVID>EVID> 0 2G4...N F 2G4NF IVQ...84260559 EVID> 1 2G4N F 2G4NF KILDK-VGINY

  4. Data of evolutionary structure change: 1GB7A-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1GB7A-2G4NF 1GB7 2G4N A F KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYN...HHH - GGGG -- EVID>EVID> 0 2G4N F 2G4NF IVQNN--...n> 0.7096549868583679 1.3399250507354736 EVID> 1 2G4N F 2G4NF

  5. Data of evolutionary structure change: 1BB4B-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1BB4B-2G4NF 1BB4 2G4N B F KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYN...HHHH -GGGG -- EVID>EVID> 0 2G4N F 2G4NF IVQNN--...0688 0.7713919878005981 1.3394720554351807 ...EVID> 1 2G4N F 2G4NF

  6. Data of evolutionary structure change: 1BFOH-2V7NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1BFOH-2V7NF 1BFO 2V7N H F EVKLLESGGGLVQPGGSMRLSCAGSGFTFTDFYMNWIRQ...E EEE EEEEEEEEEEEE EEEEEEE EEEEEE EVID> 0 2V7N F 2V7NF PAVLQSSGLYS1.4589489698410034 EVID> 12V7N F 2V7NF VVTVPSSSLGTQ <

  7. Data of evolutionary structure change: 1AKIA-2G4NF [Confc[Archive

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    Full Text Available 1AKIA-2G4NF 1AKI 2G4N A F KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFN... HHHH -GGGG -- EVID>EVID> 0 2G4N F 2G4NF IVQNN-...807559490203857 EVID>EVID> 1 2G4N F 2G4NF KILDK-VGINY

  8. Data of evolutionary structure change: 1HHLA-2G4NF [Confc[Archive

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    Full Text Available 1HHLA-2G4NF 1HHL 2G4N A F KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFN...G HHHH -GGGG -- EVID>EVID> 0 2G4N F 2G4NF IVQNN...9935073853 EVID> 1 2G4N F 2G4NF KILDK-VGINY

  9. Data of evolutionary structure change: 1JKAA-2G4NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1JKAA-2G4NF 1JKA 2G4N A F KVFERCELARTLKRLGMDGYRGISLANWMCLAKWDSGYN...G HHHH - GGGG -- EVID>EVID> 0 2G4N... F 2G4NF IVQN...rotation> 0.7001109719276428 1.2982909679412842 EVID> 1 2G4N F 2G4NF

  10. Neurofibromatosis type 1 (NF1) gene: Beyond café au lait spots and dermal neurofibromas.

    Science.gov (United States)

    Peltonen, Sirkku; Kallionpää, Roope A; Peltonen, Juha

    2017-07-01

    Neurofibromatosis 1 (NF1) occurs in 1:2000 births. The main diagnostic signs are visible on the skin, and this opens several interesting aspects for dermatological point of view. The NF1 syndrome is caused by mutations in the NF1 gene which encodes the tumor suppressor protein neurofibromin. Neurofibromin functions as a Ras-GTPase-activating protein (RasGAP), and NF1 mutations lead to overactivation of the Ras signalling pathway. The NF1 gene and neurofibromin have intriguing functions in keratinocytes and melanocytes. Neurofibromin regulates melanin synthesis and keratinocyte differentiation in a currently unknown manner. The NF1 gene has also an important but poorly understood role in tumorigenesis and cancer. Compared to the general population, NF1 patients have a fivefold risk for cancer and a more than 2000-fold risk for neurogenic malignancies. Mutations of the NF1 gene are common in numerous cancer types in patients without NF1, and this suggests a more general role for the NF1 gene in oncogenesis. In melanoma, NF1 mutations seem to drive tumorigenesis and contribute to drug resistance. In this article, we review the literature on neurofibromin with special attention to keratinocytes, melanocytes, NF1-related tumors and melanoma. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. A process yields large quantities of pure ribosome subunits

    Science.gov (United States)

    Friedman, M.; Lu, P.; Rich, A.

    1972-01-01

    Development of process for in-vitro protein synthesis from living cells followed by dissociation of ribosomes into subunits is discussed. Process depends on dialysis or use of chelating agents. Operation of process and advantages over previous methods are outlined.

  12. Interactions among oscillatory pathways in NF-kappa B signaling

    Directory of Open Access Journals (Sweden)

    White Michael RH

    2011-02-01

    Full Text Available Abstract Background Sustained stimulation with tumour necrosis factor alpha (TNF-alpha induces substantial oscillations—observed at both the single cell and population levels—in the nuclear factor kappa B (NF-kappa B system. Although the mechanism has not yet been elucidated fully, a core system has been identified consisting of a negative feedback loop involving NF-kappa B (RelA:p50 hetero-dimer and its inhibitor I-kappa B-alpha. Many authors have suggested that this core oscillator should couple to other oscillatory pathways. Results First we analyse single-cell data from experiments in which the NF-kappa B system is forced by short trains of strong pulses of TNF-alpha. Power spectra of the ratio of nuclear-to-cytoplasmic concentration of NF-kappa B suggest that the cells' responses are entrained by the pulsing frequency. Using a recent model of the NF-kappa B system due to Caroline Horton, we carried out extensive numerical simulations to analyze the response frequencies induced by trains of pulses of TNF-alpha stimulation having a wide range of frequencies and amplitudes. These studies suggest that for sufficiently weak stimulation, various nonlinear resonances should be observable. To explore further the possibility of probing alternative feedback mechanisms, we also coupled the model to sinusoidal signals with a wide range of strengths and frequencies. Our results show that, at least in simulation, frequencies other than those of the forcing and the main NF-kappa B oscillator can be excited via sub- and superharmonic resonance, producing quasiperiodic and even chaotic dynamics. Conclusions Our numerical results suggest that the entrainment phenomena observed in pulse-stimulated experiments is a consequence of the high intensity of the stimulation. Computational studies based on current models suggest that resonant interactions between periodic pulsatile forcing and the system's natural frequencies may become evident for sufficiently

  13. Interactions among oscillatory pathways in NF-kappa B signaling.

    Science.gov (United States)

    Wang, Yunjiao; Paszek, Pawel; Horton, Caroline A; Kell, Douglas B; White, Michael Rh; Broomhead, David S; Muldoon, Mark R

    2011-02-03

    Sustained stimulation with tumour necrosis factor alpha (TNF-alpha) induces substantial oscillations--observed at both the single cell and population levels--in the nuclear factor kappa B (NF-kappa B) system. Although the mechanism has not yet been elucidated fully, a core system has been identified consisting of a negative feedback loop involving NF-kappa B (RelA:p50 hetero-dimer) and its inhibitor I-kappa B-alpha. Many authors have suggested that this core oscillator should couple to other oscillatory pathways. First we analyse single-cell data from experiments in which the NF-kappa B system is forced by short trains of strong pulses of TNF-alpha. Power spectra of the ratio of nuclear-to-cytoplasmic concentration of NF-kappa B suggest that the cells' responses are entrained by the pulsing frequency. Using a recent model of the NF-kappa B system due to Caroline Horton, we carried out extensive numerical simulations to analyze the response frequencies induced by trains of pulses of TNF-alpha stimulation having a wide range of frequencies and amplitudes. These studies suggest that for sufficiently weak stimulation, various nonlinear resonances should be observable. To explore further the possibility of probing alternative feedback mechanisms, we also coupled the model to sinusoidal signals with a wide range of strengths and frequencies. Our results show that, at least in simulation, frequencies other than those of the forcing and the main NF-kappa B oscillator can be excited via sub- and superharmonic resonance, producing quasiperiodic and even chaotic dynamics. Our numerical results suggest that the entrainment phenomena observed in pulse-stimulated experiments is a consequence of the high intensity of the stimulation. Computational studies based on current models suggest that resonant interactions between periodic pulsatile forcing and the system's natural frequencies may become evident for sufficiently weak stimulation. Further simulations suggest that the

  14. Succinate Dehydrogenase Subunit B (SDHB Is Expressed in Neurofibromatosis 1-Associated Gastrointestinal Stromal Tumors (Gists: Implications for the SDHB Expression Based Classification of Gists

    Directory of Open Access Journals (Sweden)

    Jeanny H. Wang, Jerzy Lasota, Markku Miettinen

    2011-01-01

    Full Text Available Gastrointestinal Stromal Tumor (GIST is the most common mesenchymal tumor of the digestive tract. GISTs develop with relatively high incidence in patients with Neurofibromatosis-1 syndrome (NF1. Mutational activation of KIT or PDGFRA is believed to be a driving force in the pathogenesis of familial and sporadic GISTs. Unlike those tumors, NF1-associated GISTs do not have KIT or PGDFRA mutations. Similarly, no mutational activation of KIT or PDGFRA has been identified in pediatric GISTs and in GISTs associated with Carney Triad and Carney-Stratakis Syndrome. KIT and PDGFRA-wild type tumors are expected to have lesser response to imatinib treatment. Recently, Carney Triad and Carney-Stratakis Syndrome -associated GISTs and pediatric GISTs have been shown to have a loss of expression of succinate dehydrogenase subunit B (SDHB, a Krebs cycle/electron transport chain interface protein. It was proposed that GISTs can be divided into SDHB- positive (type 1, and SDHB-negative (type 2 tumors because of similarities in clinical features and response to imatinib treatment. In this study, SDHB expression was examined immunohistochemically in 22 well-characterized NF1-associated GISTs. All analyzed tumors expressed SDHB. Based on SDHB-expression status, NF1-associated GISTs belong to type 1 category; however, similarly to SDHB type 2 tumors, they do not respond well to imatinib treatment. Therefore, a simple categorization of GISTs into SDHB-positive and-negative seems to be incomplete. A classification based on both SDHB expression status and KIT and PDGFRA mutation status characterize GISTs more accurately and allow subdivision of SDHB-positive tumors into different clinico-genetic categories.

  15. Regions of IkappaBalpha that are critical for its inhibition of NF-kappaB.DNA interaction fold upon binding to NF-kappaB.

    Science.gov (United States)

    Truhlar, Stephanie M E; Torpey, Justin W; Komives, Elizabeth A

    2006-12-12

    Nuclear factor kappaB (NF-kappaB) transcription factors regulate genes responsible for critical cellular processes. IkappaBalpha, -beta, and -epsilon bind to NF-kappaBs and inhibit their transcriptional activity. The NF-kappaB-binding domains of IkappaBs contain six ankyrin repeats (ARs), which adopt a beta-hairpin/alpha-helix/loop/alpha-helix/loop architecture. IkappaBalpha appears compactly folded in the IkappaBalpha.NF-kappaB crystal structure, but biophysical studies suggested that IkappaBalpha might be flexible even when bound to NF-kappaB. Amide H/(2)H exchange in free IkappaBalpha suggests that ARs 2-4 are compact, but ARs 1, 5, and 6 are conformationally flexible. Amide H/(2)H exchange is one of few techniques able to experimentally identify regions that fold upon binding. Comparison of amide H/(2)H exchange in free and NF-kappaB-bound IkappaBalpha reveals that the beta-hairpins in ARs 5 and 6 fold upon binding to NF-kappaB, but AR 1 remains highly solvent accessible. These regions are implicated in various aspects of NF-kappaB regulation, such as controlling degradation of IkappaBalpha, enabling high-affinity interaction with different NF-kappaB dimers, and preventing NF-kappaB from binding to its target DNA. Thus, IkappaBalpha conformational flexibility and regions of IkappaBalpha folding upon binding to NF-kappaB are important attributes for its regulation of NF-kappaB transcriptional activity.

  16. C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7.

    Science.gov (United States)

    Sadhale, P P; Woychik, N A

    1994-09-01

    We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25. Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.

  17. Constitutional and mosaic large NF1 gene deletions in neurofibromatosis type 1.

    OpenAIRE

    Rasmussen, S. A.; Colman, S. D.; Ho, V.T.; Abernathy, C R; Arn, P H; Weiss, L; Schwartz, C.; Saul, R A; Wallace, M. R.

    1998-01-01

    A set of neurofibromatosis type 1 (NF1) patients was screened for large NF1 gene deletions by comparing patient and parent genotypes at 10 intragenic polymorphic loci. Of 67 patient/parent sets (47 new mutation patients and 20 familial cases), five (7.5%) showed loss of heterozygosity (LOH), indicative of NF1 gene deletion. These five patients did not have severe NF1 manifestations, mental retardation, or dysmorphic features, in contrast to previous reports of large NF1 deletions. All five de...

  18. Merlin isoform 2 in neurofibromatosis type 2-associated polyneuropathy.

    Science.gov (United States)

    Schulz, Alexander; Baader, Stephan L; Niwa-Kawakita, Michiko; Jung, Marie Juliane; Bauer, Reinhard; Garcia, Cynthia; Zoch, Ansgar; Schacke, Stephan; Hagel, Christian; Mautner, Victor-Felix; Hanemann, C Oliver; Dun, Xin-Peng; Parkinson, David B; Weis, Joachim; Schröder, J Michael; Gutmann, David H; Giovannini, Marco; Morrison, Helen

    2013-04-01

    The autosomal dominant disorder neurofibromatosis type 2 (NF2) is a hereditary tumor syndrome caused by inactivation of the NF2 tumor suppressor gene, encoding merlin. Apart from tumors affecting the peripheral and central nervous systems, most NF2 patients develop peripheral neuropathies. This peripheral nerve disease can occur in the absence of nerve-damaging tumors, suggesting an etiology that is independent of gross tumor burden. We discovered that merlin isoform 2 (merlin-iso2) has a specific function in maintaining axonal integrity and propose that reduced axonal NF2 gene dosage leads to NF2-associated polyneuropathy. We identified a merlin-iso2-dependent complex that promotes activation of the GTPase RhoA, enabling downstream Rho-associated kinase to promote neurofilament heavy chain phosphorylation. Merlin-iso2-deficient mice exhibited impaired locomotor capacities, delayed sensory reactions and electrophysiological signs of axonal neuropathy. Sciatic nerves from these mice and sural nerve biopsies from NF2 patients revealed reduced phosphorylation of the neurofilament H subunit, decreased interfilament spacings and irregularly shaped axons.

  19. Comprehensive RNA Analysis of the NF1 Gene in Classically Affected NF1 Affected Individuals Meeting NIH Criteria has High Sensitivity and Mutation Negative Testing is Reassuring in Isolated Cases With Pigmentary Features Only

    Directory of Open Access Journals (Sweden)

    D.G. Evans

    2016-05-01

    Interpretation: RNA analysis in individuals with presumed NF1 has high sensitivity and includes a small subset with DNET without an NF1 variant. Furthermore negative analysis for NF1/SPRED1 provides strong reassurance to children with ≥6 CAL that they are unlikely to have NF1.

  20. HSF1 and NF-κB p65 participate in the process of exercise preconditioning attenuating pressure overload-induced pathological cardiac hypertrophy

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Tongyi [Department of Cardiothoracic Surgery, No. 401 Hospital of PLA, Qingdao (China); Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai (China); Zhang, Ben [Centre of Cardiovascular Surgery, Guangzhou General Hospital of Guangzhou Military Region, Guangzhou (China); Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai (China); Yang, Fan; Cai, Chengliang; Wang, Guokun [Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai (China); Han, Qingqi, E-mail: handoctor@gmail.com [Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai (China); Zou, Liangjian, E-mail: zouliangjiansh@gmail.com [Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai (China)

    2015-05-08

    Pathological cardiac hypertrophy, often accompanied by hypertension, aortic stenosis and valvular defects, is typically associated with myocyte remodeling and cardiac dysfunction. Exercise preconditioning (EP) has been proven to enhance the tolerance of the myocardium to cardiac ischemia-reperfusion injury. However, the effects of EP in pathological cardiac hypertrophy are rarely reported. 10-wk-old male Sprague–Dawley rats (n = 80) were randomly divided into four groups: sham, TAC, EP + sham and EP + TAC. Two EP groups were subjected to 4 weeks of treadmill training, and the EP + TAC and TAC groups were followed by TAC operations. The sham and EP + sham groups underwent the same operation without aortic constriction. Eight weeks after the surgery, we evaluated the effects of EP by echocardiography, morphology, and histology and observed the expressions of the associated proteins. Compared with the respective control groups, hypertrophy-related indicators were significantly increased in the TAC and EP + TAC groups (p < 0.05). However, between the TAC and EP + TAC groups, all of these changes were effectively inhibited by EP treatment (p < 0.05). Furthermore, EP treatment upregulated the expression of HSF1 and HSP70, increased the HSF1 levels in the nuclear fraction, inhibited the expression of the NF-κB p65 subunit, decreased the NF-κB p65 subunit levels in the nuclear fraction, and reduced the IL2 levels in the myocardia of rats. EP could effectively reduce the cardiac hypertrophic responses induced by TAC and may play a protective role by upregulating the expressions of HSF1 and HSP70, activating HSF1 and then inhibiting the expression of NF-κB p65 and nuclear translocation. - Highlights: • EP could effectively reduce the cardiac hypertrophic responses induced by TAC. • EP may play a protective role by upregulating the expressions of HSF1 and HSP70 and then activating HSF1. • EP may play a protective role by inhibiting the expression

  1. Neurofibromatosis type 2 (NF2: A clinical and molecular review

    Directory of Open Access Journals (Sweden)

    Evans D Gareth R

    2009-06-01

    Full Text Available Abstract Neurofibromatosis type 2 (NF2 is a tumour-prone disorder characterised by the development of multiple schwannomas and meningiomas. Prevalence (initially estimated at 1: 200,000 is around 1 in 60,000. Affected individuals inevitably develop schwannomas, typically affecting both vestibular nerves and leading to hearing loss and deafness. The majority of patients present with hearing loss, which is usually unilateral at onset and may be accompanied or preceded by tinnitus. Vestibular schwannomas may also cause dizziness or imbalance as a first symptom. Nausea, vomiting or true vertigo are rare symptoms, except in late-stage disease. The other main tumours are schwannomas of the other cranial, spinal and peripheral nerves; meningiomas both intracranial (including optic nerve meningiomas and intraspinal, and some low-grade central nervous system malignancies (ependymomas. Ophthalmic features are also prominent and include reduced visual acuity and cataract. About 70% of NF2 patients have skin tumours (intracutaneous plaque-like lesions or more deep-seated subcutaneous nodular tumours. Neurofibromatosis type 2 is a dominantly inherited tumour predisposition syndrome caused by mutations in the NF2 gene on chromosome 22. More than 50% of patients represent new mutations and as many as one-third are mosaic for the underlying disease-causing mutation. Although truncating mutations (nonsense and frameshifts are the most frequent germline event and cause the most severe disease, single and multiple exon deletions are common. A strategy for detection of the latter is vital for a sensitive analysis. Diagnosis is based on clinical and neuroimaging studies. Presymptomatic genetic testing is an integral part of the management of NF2 families. Prenatal diagnosis and pre-implantation genetic diagnosis is possible. The main differential diagnosis of NF2 is schwannomatosis. NF2 represents a difficult management problem with most patients facing substantial

  2. Neurofibromatosis type 2 (NF2): a clinical and molecular review.

    Science.gov (United States)

    Evans, D Gareth R

    2009-06-19

    Neurofibromatosis type 2 (NF2) is a tumour-prone disorder characterised by the development of multiple schwannomas and meningiomas. Prevalence (initially estimated at 1: 200,000) is around 1 in 60,000. Affected individuals inevitably develop schwannomas, typically affecting both vestibular nerves and leading to hearing loss and deafness. The majority of patients present with hearing loss, which is usually unilateral at onset and may be accompanied or preceded by tinnitus. Vestibular schwannomas may also cause dizziness or imbalance as a first symptom. Nausea, vomiting or true vertigo are rare symptoms, except in late-stage disease. The other main tumours are schwannomas of the other cranial, spinal and peripheral nerves; meningiomas both intracranial (including optic nerve meningiomas) and intraspinal, and some low-grade central nervous system malignancies (ependymomas). Ophthalmic features are also prominent and include reduced visual acuity and cataract. About 70% of NF2 patients have skin tumours (intracutaneous plaque-like lesions or more deep-seated subcutaneous nodular tumours). Neurofibromatosis type 2 is a dominantly inherited tumour predisposition syndrome caused by mutations in the NF2 gene on chromosome 22. More than 50% of patients represent new mutations and as many as one-third are mosaic for the underlying disease-causing mutation. Although truncating mutations (nonsense and frameshifts) are the most frequent germline event and cause the most severe disease, single and multiple exon deletions are common. A strategy for detection of the latter is vital for a sensitive analysis. Diagnosis is based on clinical and neuroimaging studies. Presymptomatic genetic testing is an integral part of the management of NF2 families. Prenatal diagnosis and pre-implantation genetic diagnosis is possible. The main differential diagnosis of NF2 is schwannomatosis. NF2 represents a difficult management problem with most patients facing substantial morbidity and reduced

  3. Ergolide, sesquiterpene lactone from Inula britannica, inhibits inducible nitric oxide synthase and cyclo-oxygenase-2 expression in RAW 264.7 macrophages through the inactivation of NF-kappaB.

    Science.gov (United States)

    Whan Han, J; Gon Lee, B; Kee Kim, Y; Woo Yoon, J; Kyoung Jin, H; Hong, S; Young Lee, H; Ro Lee, K; Woo Lee, H

    2001-06-01

    We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) by ergolide, sesquiterpene lactone from Inula britannica. iNOS activity in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS. Ergolide markedly decreased the production of prostaglandin E(2) (PGE(2)) in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner, without alteration of the catalytic activity of COX-2 itself. Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA caused by stimulation of LPS/IFN-gamma in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-kappaB as well as IkappaB-alpha degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF-kappaB itself or an upstream molecule of NF-kappaB. Ergolide also directly inhibited the DNA-binding activity of active NF-kappaB in LPS/IFN-gamma-pretreated RAW 264.7 macrophages. These results demonstrate that the suppression of NF-kappaB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF-kappaB resulted from blockade of the degradation of IkappaB and the direct modification of active NF-kappaB, leading to the

  4. Ergolide, sesquiterpene lactone from Inula britannica, inhibits inducible nitric oxide synthase and cyclo-oxygenase-2 expression in RAW 264.7 macrophages through the inactivation of NF-κB

    Science.gov (United States)

    Whan Han, Jeung; Gon Lee, Byeong; Kee Kim, Yong; Woo Yoon, Jong; Kyoung Jin, Hye; Hong, Sungyoul; Young Lee, Hoi; Ro Lee, Kang; Woo Lee, Hyang

    2001-01-01

    We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) by ergolide, sesquiterpene lactone from Inula britannica.iNOS activity in cell-free extract of LPS/IFN-γ-stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN-γ-stimulated RAW 264.7 macrophages in a concentration-dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS.Ergolide markedly decreased the production of prostaglandin E2 (PGE2) in cell-free extract of LPS/IFN-γ-stimulated RAW 264.7 macrophages in a concentration-dependent manner, without alteration of the catalytic activity of COX-2 itself.Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA caused by stimulation of LPS/IFN-γ in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively.Ergolide inhibited nuclear factor-κB (NF-κB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-γ. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-κB as well as IκB-α degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF-κB itself or an upstream molecule of NF-κB.Ergolide also directly inhibited the DNA-binding activity of active NF-κB in LPS/IFN-γ-pretreated RAW 264.7 macrophages.These results demonstrate that the suppression of NF-κB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF-κB resulted from blockade of the degradation of IκB and the direct modification of active NF-κB, leading to the suppression of the expression of iNOS and COX-2, which play important

  5. A bioinformatic and computational study of myosin phosphatase subunit diversity.

    Science.gov (United States)

    Dippold, Rachael P; Fisher, Steven A

    2014-08-01

    Variability in myosin phosphatase (MP) subunits may provide specificity in signaling pathways that regulate muscle tone. We utilized public databases and computational algorithms to investigate the phylogenetic diversity of MP regulatory (PPP1R12A-C) and inhibitory (PPP1R14A-D) subunits. The comparison of exonic coding sequences and expression data confirmed or refuted the existence of isoforms and their tissue-specific expression in different model organisms. The comparison of intronic and exonic sequences identified potential expressional regulatory elements. As examples, smooth muscle MP regulatory subunit (PPP1R12A) is highly conserved through evolution. Its alternative exon E24 is present in fish through mammals with two invariant features: 1) a reading frame shift generating a premature termination codon and 2) a hexanucleotide sequence adjacent to the 3' splice site hypothesized to be a novel suppressor of exon splicing. A characteristic of the striated muscle MP regulatory subunit (PPP1R12B) locus is numerous and phylogenetically variable transcriptional start sites. In fish this locus only codes for the small (M21) subunit, suggesting the primordial function of this gene. Inhibitory subunits show little intragenic variability; their diversity is thought to have arisen by expansion and tissue-specific expression of different gene family members. We demonstrate differences in the regulatory landscape between smooth muscle enriched (PPP1R14A) and more ubiquitously expressed (PPP1R14B) family members and identify deeply conserved intronic sequence and predicted transcriptional cis-regulatory elements. This bioinformatic and computational study has uncovered a number of attributes of MP subunits that supports selection of ideal model organisms and testing of hypotheses regarding their physiological significance and regulated expression. Copyright © 2014 the American Physiological Society.

  6. Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

    Directory of Open Access Journals (Sweden)

    Oliveira M.C.

    1999-01-01

    Full Text Available Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6% presented hypogonadism (which was central in 45 cases and primary in 2, 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01 and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02. Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01 as well as free testosterone levels (r = -0.53, P<0.01. In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

  7. Hypersecretion of the alpha-subunit in clinically non-functioning pituitary adenomas: Diagnostic accuracy is improved by adding alpha-subunit/gonadotropin ratio to levels of alpha-subunit

    DEFF Research Database (Denmark)

    Andersen, Marianne; Ganc-Petersen, Joanna; Jørgensen, Jens Otto Lunde

    2010-01-01

    BACKGROUND: In vitro, the majority of clinically non-functioning pituitary adenomas (NFPAs) produce gonadotropins or their alpha-subunit; however, in vivo, measurements of alpha-subunit levels may not accurately detect the hypersecretion of the alpha-subunit. AIM: We wanted to estimate...

  8. Crystal chemistry of ordered rocksalt-type Ca2NF

    Science.gov (United States)

    Al-Azzawi, Mohanad; Zeller, Matthias; Li, Dingqiang; Wagner, Timothy R.

    2017-10-01

    Ordered rocksalt-type Ca2NF has a cubic unit cell that is doubled along [100] relative to the rocksalt-type polymorph due to N/F ordering. Crystals for the present study were prepared from a pure, dry KCuF3 precursor by reaction with Ca metal under nitrogen, and analyzed via high resolution single crystal X-ray diffraction. From this analysis, as well as qualitative compositional analysis using energy dispersive spectroscopy, it was determined that interstitial fluoride ions previously interpreted as Frenkel defects are actually due to a non-stoichiometric defect. The refined composition is Ca2N0.925F1.23, and the ordered cubic phase has space group Fd 3 bar m (No. 227) with a = 10.0301(5) Å and Z = 16.

  9. Bernstein Algorithm for Vertical Normalization to 3NF Using Synthesis

    Directory of Open Access Journals (Sweden)

    Matija Varga

    2013-07-01

    Full Text Available This paper demonstrates the use of Bernstein algorithm for vertical normalization to 3NF using synthesis. The aim of the paper is to provide an algorithm for database normalization and present a set of steps which minimize redundancy in order to increase the database management efficiency, and specify tests and algorithms for testing and proving the reversibility (i.e., proving that the normalization did not cause loss of information. Using Bernstein algorithm steps, the paper gives examples of vertical normalization to 3NF through synthesis and proposes a test and an algorithm to demonstrate decomposition reversibility. This paper also sets out to explain that the reasons for generating normal forms are to facilitate data search, eliminate data redundancy as well as delete, insert and update anomalies and explain how anomalies develop using examples-

  10. B meson spectrum and decay constant from Nf=2 simulations

    CERN Document Server

    Blossier, Benoit; Della Morte, Michele; Donnellan, Michael; Fritzsch, Patrick; Garron, Nicolas; Heitger, Jochen; von Hippel, Georg; Leder, Bjorn; Simma, Hubert; Sommer, Rainer

    2010-01-01

    We report on the status of an ALPHA Collaboration project to extract quantities for B physics phenomenology from Nf=2 lattice simulations. The framework is Heavy Quark Effective Theory (HQET) expanded up to the first order of the inverse b-quark mass. The couplings of the effective theory are determined by imposing matching conditions of observables computed in HQET with their counterpart computed in QCD. That program, based on Nf=2 simulations in a small physical volume with SF boundary conditions, is now almost finished. On the other side the analysis of configurations selected from the CLS ensembles, in order to measure HQET hadronic matrix elements, has just started recently so that only results obtained at a single lattice spacing, a=0.07 fm, will be discussed. We give our first results for the b-quark mass and for the B meson decay constant.

  11. Alpha2 subunit specificity of cyclothiazide inhibition on glycine receptors.

    Science.gov (United States)

    Zhang, Xiao-Bing; Sun, Guang-Chun; Liu, Lu-Ying; Yu, Fang; Xu, Tian-Le

    2008-04-01

    In the mammalian cortex, alpha2 subunit-containing glycine receptors (GlyRs) mediate tonic inhibition, but the precise functional role of this type of GlyRs is difficult to establish because of the lack of subtype-selective antagonist. In this study, we found that cyclothiazide (CTZ), an epileptogenic agent, potently inhibited GlyR-mediated current (I(Gly)) in cultured rat hippocampal neurons. The inhibition was glycine concentration-dependent, suggesting a competitive mechanism. Note that GlyRs containing the alpha2 but not alpha1 or alpha3 subunits, when being heterologously expressed in human embryonic kidney 293T cells, were inhibited by CTZ, indicating subunit specificity of CTZ action. In addition, the degree of CTZ inhibition on I(Gly) in rat spinal neurons declined with time in culture, in parallel with a decline of alpha2 subunit expression, which is known to occur during spinal cord development. Furthermore, site-directed mutagenesis indicates that a single-amino acid threonine at position 59 near the N terminus of the alpha2 subunit confers the specificity of CTZ action. Thus, CTZ is a potent and selective inhibitor of alpha2-GlyRs, and threonine at position 59 plays a critical role in the susceptibility of GlyR to CTZ inhibition.

  12. Bernstein Algorithm for Vertical Normalization to 3NF Using Synthesis

    OpenAIRE

    Matija Varga

    2013-01-01

    This paper demonstrates the use of Bernstein algorithm for vertical normalization to 3NF using synthesis. The aim of the paper is to provide an algorithm for database normalization and present a set of steps which minimize redundancy in order to increase the database management efficiency, and specify tests and algorithms for testing and proving the reversibility (i.e., proving that the normalization did not cause loss of information). Using Bernstein algorithm steps, the paper gives examples...

  13. Neurofibromatosis type 2 (NF2): A clinical and molecular review

    OpenAIRE

    Evans D Gareth R

    2009-01-01

    Abstract Neurofibromatosis type 2 (NF2) is a tumour-prone disorder characterised by the development of multiple schwannomas and meningiomas. Prevalence (initially estimated at 1: 200,000) is around 1 in 60,000. Affected individuals inevitably develop schwannomas, typically affecting both vestibular nerves and leading to hearing loss and deafness. The majority of patients present with hearing loss, which is usually unilateral at onset and may be accompanied or preceded by tinnitus. Vestibular ...

  14. Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR

    Energy Technology Data Exchange (ETDEWEB)

    Sahu, Geetaram; Farley, Kalamo [Division of Infectious Diseases, Department of Medicine, George Washington University, Washington, DC (United States); El-Hage, Nazira [Virginia Commonwealth University, Richmond, VA (United States); Aiamkitsumrit, Benjamas; Fassnacht, Ryan [Division of Infectious Diseases, Department of Medicine, George Washington University, Washington, DC (United States); Kashanchi, Fatah [George Mason University, Manassas, VA (United States); Ochem, Alex [ICGEB, Wernher and Beit Building, Anzio Road, Observatory, 7925 Cape Town (South Africa); Simon, Gary L. [Division of Infectious Diseases, Department of Medicine, George Washington University, Washington, DC (United States); Karn, Jonathan [Case Western Reserve University, Cleveland, OH (United States); Hauser, Kurt F. [Virginia Commonwealth University, Richmond, VA (United States); Tyagi, Mudit, E-mail: tmudit@email.gwu.edu [Division of Infectious Diseases, Department of Medicine, George Washington University, Washington, DC (United States); Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington, DC 20037 (United States)

    2015-09-15

    Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-ĸB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-ĸB at 276th serine residue. These modifications enhance the interaction of NF-ĸB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication. - Highlights: • Cocaine induces the initiation phase of HIV transcription by activating NF-ĸB. • Cocaine induced NF-ĸB phosphorylation promotes its interaction with P300. • Cocaine enhances the elongation phase of HIV transcription by stimulating MSK1. • Cocaine activated MSK1 catalyzes the phosphorylation of histone H3 at its Ser10. • Cocaine induced H3S10 phosphorylation facilitates the recruitment of P-TEFb at LTR.

  15. Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

    Directory of Open Access Journals (Sweden)

    Keegan J. Baldauf

    2015-03-01

    Full Text Available Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT, which consists of two subunits: the A subunit (CTA and the B subunit (CTB. CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.

  16. INTER-SUBUNIT INTERACTION AND ARRANGEMENT OF THE CENTRAL STALK SUBUNITS OF ENTEROCOCCUS HIRAE V-ATPASE

    Directory of Open Access Journals (Sweden)

    K. M. Mozaffor Hossain1,2

    2017-04-01

    Full Text Available V-ATPases function as ATP-dependent ion pumps in various membrane systems of living organisms. ATP hydrolysis causes rotation of the central rotor complex, which is composed of NtpC, NtpD,and NtpG subunits. We successfully cloned, expressed, and purified the central stalk subunits of Na+- translocating V-type ATPase from E. hirae. Full length DNAs for genes of NtpC, NtpD, and NtpG subunits were amplified by conventional PCR method and cloned into the plasmid vector pET23d. Recombinant DNA transformed E. coli (JM109 cells were grown in LB agar plate containing ampicillin. NtpC and NtpG subunit proteins were expressed as separate His-tagged soluble proteins in E. coli BL21(DE3 cells at 30 °C and NtpD subunit protein was stably expressed in E. coli BL21(DE3 cells at 16 °C. Expressed central stalk subunit proteins were purified by affinity chromatography followed by gel-filtration method. The amount of purified NtpC, NtpD, and NtpG subunit proteins were 13.8 mg/1 liter culture, 16.6 mg/1 liter culture, and 15.2 mg/1 liter culture, respectively. Tobacco etch virus (TEV-protease was also expressed in E. coli BL21(DE3 cells and purified by affinity chromatography. The amount of purified TEV-protease was 8.50 mg/1 liter culture. Here, we report the stable interaction and the complex formation mechanisms of the central stalk subunits of E. hirae V-ATPase. The purified NtpD could interact directly with NtpG and NtpC could bind directly to NtpD-G complex and these three subunits could form NtpD-G-C complex. NtpD could bind to NtpG and formed NtpD-G complex. NtpD or NtpD-G complex could bind to NtpA3-B3 and formed NtpA3-B3-D or NtpA3-B3-D-G complex, respectively. NtpC could bind to NtpA3-B3-D-G and formed NtpA3-B3-D-G-C complex but could not bind to NtpA3-B3, NtpA3-B3-D, NtpD, or NtpG.

  17. Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

    Science.gov (United States)

    Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

    1999-03-05

    Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

  18. Separation and characterization of alpha-chain subunits from tilapia (Tilapia zillii) skin gelatin using ultrafiltration.

    Science.gov (United States)

    Chen, Shulin; Tang, Lanlan; Su, Wenjin; Weng, Wuyin; Osako, Kazufumi; Tanaka, Munehiko

    2015-12-01

    Alpha-chain subunits were separated from tilapia skin gelatin using ultrafiltration, and the physicochemical properties of obtained subunits were investigated. As a result, α1-subunit and α2-subunit could be successfully separated by 100 kDa MWCO regenerated cellulose membranes and 150 kDa MWCO polyethersulfone membranes, respectively. Glycine was the most dominant amino acid in both α1-subunit and α2-subunit. However, the tyrosine content was higher in α2-subunit than in α1-subunit, resulting in strong absorption near 280 nm observed in the UV absorption spectrum. Based on the DSC analysis, it was found that the glass transition temperatures of gelatin, α1-subunit and α2-subunit were 136.48 °C, 126.77 °C and 119.43 °C, respectively. Moreover, the reduced viscosity and denaturation temperature of α1-subunit were higher than those of α2-subunit, and the reduced viscosity reached the highest when α-subunits were mixed with α1/α2 ratio of approximately 2, suggesting that α1-subunit plays a more important role in the thermostability of gelatin than α2-subunit. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Knowledge and Self-Esteem of Individuals with Neurofibromatosis Type 1 (NF1).

    Science.gov (United States)

    Rosnau, Kayla; Hashmi, S Shahrukh; Northrup, Hope; Slopis, John; Noblin, Sarah; Ashfaq, Myla

    2017-06-01

    Neurofibromatosis Type 1 (NF1) is a progressive genetic disorder characterized by physical findings such as café-au-lait macules, Lisch nodules, and neurofibromas in addition to other medical complications. Learning and social problems are more prevalent among individuals affected with NF1. It has been reported that people with NF1 have lower self-esteem (SE) when compared to the general population. Additionally, a study published over 20 years ago found that overall knowledge of NF1 was lacking in individuals affected with the condition. The goals of our study were to evaluate NF1 knowledge in adolescents and adults with the condition, as well as to determine if there is a link between patient knowledge and SE. Furthermore, we explored the impact of other factors, such as attendance at a NF1 support group and having a family history of NF1, on knowledge and SE. A survey comprised of knowledge-based questions and the Rosenberg Self-Esteem Scale was distributed to individuals with NF1 through the Texas NF Foundation. Overall, the 49 respondents (13 to 73 years of age) had a mean knowledge score of 77.9 % correct answers. Consistent with previous studies, the SE of our study population was lower when compared to general population norms. Although no correlation between knowledge and SE was observed, SE scores were on average higher if a person reported the following: having friends with NF1 (p = 0.009); attending a NF1 support group (p = 0.006); receiving care at a NF clinic (p = 0.049); or having received genetic counseling (p = 0.008). Further research is needed to better understand the relationship between these factors and SE in the NF1 population.

  20. Loss of NF2/Merlin expression in advanced sporadic colorectal cancer.

    Science.gov (United States)

    Cačev, Tamara; Aralica, Gorana; Lončar, Božo; Kapitanović, Sanja

    2014-02-01

    NF2/Merlin was first identified through its association with neurofibromatosis type 2 (NF2). However, accumulating evidence suggests a more general involvement in tumorigenesis and, in particular, a broader role in tumor suppression. The aim of this study was to examine NF2/Merlin involvement in sporadic colorectal cancer. This study is the first to examine the role of NF2/Merlin in sporadic colorectal cancer through LOH analysis at the NF2 locus and mRNA expression analysis via quantitative RT-PCR of total NF2, NF2 isoform I and II. In addition, Merlin protein expression was assessed by immunohistochemistry and Western blotting. NF2 LOH was detected in 20.0 % of heterozygous cases and was found to be more frequent in tumors larger than 5 cm in diameter (p = 0.041) and in tumors with a less differentiated phenotype (p = 0.027). No differences were observed in total NF2 and NF2 isoform I/isoform II mRNA expression between the tumors and their corresponding normal mucous tissues. NF2 isoform II was the most predominant isoform in all samples analyzed. mRNA expression levels of total NF2 and isoforms I and II were significantly lower in poorly differentiated tumors (p = 0.033, p = 0.036 and p = 0.044, respectively). Weak Merlin immunostaining was more frequent in poorly differentiated tumors (p = 0.034) and tumors classified as Dukes' C (p = 0.023). A distinct pattern of Merin phosphorylation was observed in tumors compared to normal mucous tissues. Our data indicate that NF2/Merlin may serve as a potential target in the management of colorectal cancer.

  1. Lipoamide Inhibits NF1 Deficiency-induced Epithelial-Mesenchymal Transition in Murine Schwann Cells.

    Science.gov (United States)

    Zhang, Yuchen; Zhou, Rongsheng; Qu, Yiping; Shu, Maoguo; Guo, Shuzhong; Bai, Zhuanli

    2017-11-29

    Neurofibromatosis type I (NF1) is one of the most common neurocutaneous syndromes characterized by development of adult neurofibromas which is mainly made up of Schwann cells. The disease is generally accepted to be caused by inactivation mutation of Nf1 gene. And Nf1 deficiency had been reported to lead to ROS overproduction and epithelial-mesenchymal transition (EMT) phenotype. This study was designed to investigate whether excessive ROS conferred to Nf1 deficiency-induced EMT in Schwann cells. Colony formation, wound healing assay and transwell assay was used to evaluate the effects of stable Nf1 knockdown in SW10 Schwann cells. Western blot and ROS assay was conducted to explore the molecular mechanisms of Nf1 inactivation in tumorigenesis. Animal experiments were performed to assess the inhibitory effects of lipoamide, which is the neutral amide of α-lipoic acid and functions as a potent antioxidant to scavenge ROS, on Nf1-deficiency tumor growth in vivo. Nf1 knockdown enhanced the cellular capacities of proliferation, migration and invasion, promoted ROS generation, decreased the expression of epithelial surface marker E-cadherin, and up-regulated several EMT-associated molecules in Schwann cells. Moreover, lipoamide dose-dependently inhibited not only Nf1 deficiency-induced EMT but also spontaneous EMT. Furthermore, lipoamide markedly suppresses tumor growth in a mouse model of NF1-associated neurofibroma. Our results clearly reveal that ROS overproduction is responsible for Nf1 deficiency-induced EMT and plays a crucial role in NF1 tumor growth. The findings presented herein shed light on the potential of antioxidant therapy to prevent the progression of NF1-associated neurofibroma. Copyright © 2017 IMSS. Published by Elsevier Inc. All rights reserved.

  2. NF-κB in the regulation of epithelial homeostasis and inflammation

    Science.gov (United States)

    Wullaert, Andy; Bonnet, Marion C; Pasparakis, Manolis

    2011-01-01

    The IκB kinase/NF-κB signaling pathway has been implicated in the pathogenesis of several inflammatory diseases. Increased activation of NF-κB is often detected in both immune and non-immune cells in tissues affected by chronic inflammation, where it is believed to exert detrimental functions by inducing the expression of proinflammatory mediators that orchestrate and sustain the inflammatory response and cause tissue damage. Thus, increased NF-κB activation is considered an important pathogenic factor in many acute and chronic inflammatory disorders, raising hopes that NF-κB inhibitors could be effective for the treatment of inflammatory diseases. However, ample evidence has accumulated that NF-κB inhibition can also be harmful for the organism, and in some cases trigger the development of inflammation and disease. These findings suggested that NF-κB signaling has important functions for the maintenance of physiological immune homeostasis and for the prevention of inflammatory diseases in many tissues. This beneficial function of NF-κB has been predominantly observed in epithelial cells, indicating that NF-κB signaling has a particularly important role for the maintenance of immune homeostasis in epithelial tissues. It seems therefore that NF-κB displays two faces in chronic inflammation: on the one hand increased and sustained NF-κB activation induces inflammation and tissue damage, but on the other hand inhibition of NF-κB signaling can also disturb immune homeostasis, triggering inflammation and disease. Here, we discuss the mechanisms that control these apparently opposing functions of NF-κB signaling, focusing particularly on the role of NF-κB in the regulation of immune homeostasis and inflammation in the intestine and the skin. PMID:21151201

  3. Cyclothiazide: a subunit-specific inhibitor of GABAC receptors.

    Science.gov (United States)

    Xie, An; Song, Xiangqian; Ripps, Harris; Qian, Haohua

    2008-06-01

    We tested the effects of cyclothiazide (CTZ), an agent used to block desensitization of AMPA-type glutamate receptors, on heterologously expressed GABA(C) receptors formed by homomeric rho subunits. CTZ inhibition of GABA(C) receptors was subunit specific; it produced a dose-dependent reduction of the GABA-elicited current on homomeric rho2 receptors with an IC(50) of about 12 microm, but had no significant effect on homomeric rho1 receptors. This differential sensitivity was attributable to a single amino acid located on the second transmembrane domain of the rho subunits. Mutating the residue at this position from serine to proline on the rho2 subunit eliminated CTZ sensitivity, whereas switching proline to serine on the rho1 subunit made the receptor CTZ sensitive. The inhibitory properties of CTZ were consistent with its action as a channel blocker on the receptors formed by rho2 subunits. The effect showed a small degree of voltage dependence, and was due mainly to a non-competitive mechanism that reduced the maximum response elicited by GABA. In addition, the prominent membrane current rebound when co-application of GABA and CTZ was terminated suggests that the binding site for CTZ on the GABA(C) receptor is distinct from that for GABA, and that CTZ acts as a non-competitive antagonist on the GABA(C) receptor. CTZ inhibited the open channel of the GABA(C) receptor with a time constant of about 0.4 s, but the kinetics were approximately 10-fold slower when GABA is absent. The ability of CTZ to interact with various types of neurotransmitter receptors indicates that the drug has multiple actions in the CNS.

  4. Localisation of AMPK γ subunits in cardiac and skeletal muscles.

    Science.gov (United States)

    Pinter, Katalin; Grignani, Robert T; Watkins, Hugh; Redwood, Charles

    2013-12-01

    The trimeric protein AMP-activated protein kinase (AMPK) is an important sensor of energetic status and cellular stress, and mutations in genes encoding two of the regulatory γ subunits cause inherited disorders of either cardiac or skeletal muscle. AMPKγ2 mutations cause hypertrophic cardiomyopathy with glycogen deposition and conduction abnormalities; mutations in AMPKγ3 result in increased skeletal muscle glycogen. In order to gain further insight into the roles of the different γ subunits in muscle and into possible disease mechanisms, we localised the γ2 and γ3 subunits, along with the more abundant γ1 subunit, by immunofluorescence in cardiomyocytes and skeletal muscle fibres. The predominant cardiac γ2 variant, γ2-3B, gave a striated pattern in cardiomyocytes, aligning with the Z-disk but with punctate staining similar to T-tubule (L-type Ca(2+) channel) and sarcoplasmic reticulum (SERCA2) markers. In skeletal muscle fibres AMPKγ3 localises to the I band, presenting a uniform staining that flanks the Z-disk, also coinciding with the position of Ca(2+) influx in these muscles. The localisation of γ2-3B- and γ3-containing AMPK suggests that these trimers may have similar functions in the different muscles. AMPK containing γ2-3B was detected in oxidative skeletal muscles which had low expression of γ3, confirming that these two regulatory subunits may be co-ordinately regulated in response to metabolic requirements. Compartmentalisation of AMPK complexes is most likely dependent on the regulatory γ subunit and this differential localisation may direct substrate selection and specify particular functional roles.

  5. Advancements in the development of subunit influenza vaccines

    Science.gov (United States)

    Zhang, Naru; Zheng, Bo-Jian; Lu, Lu; Zhou, Yusen; Jiang, Shibo; Du, Lanying

    2014-01-01

    The ongoing threat of influenza epidemics and pandemics has emphasized the importance of developing safe and effective vaccines against infections from divergent influenza viruses. In this review, we first introduce the structure and life cycle of influenza A viruses, describing major influenza A virus-caused pandemics. We then compare different types of influenza vaccines and discuss current advancements in the development of subunit influenza vaccines, particularly those based on nucleoprotein (NP), extracellular domain of matrix protein 2 (M2e) and hemagglutinin (HA) proteins. We also illustrate potential strategies for improving the efficacy of subunit influenza vaccines. PMID:25529753

  6. A second sequence element located 3' to the NF-kappa B-binding site regulates IL-2 receptor-alpha gene induction.

    Science.gov (United States)

    Pomerantz, J L; Mauxion, F; Yoshida, M; Greene, W C; Sen, R

    1989-12-15

    Transcriptional induction of the gene encoding the alpha-subunit of IL-2R has been shown to be mediated by a sequence element (GGGGAATCTCCC) that is homologous to the NF-kappa B-binding site of the kappa Ig gene enhancer. In this report we demonstrate that the induced transcription of this gene by mitogen and by the tax gene product of the type-I human T cell leukemia virus is dependent upon an additional sequence motif (GGGCGTAGC) located approximately 10 bp downstream of the previously identified site. This newly identified motif binds a factor that is present in extracts derived from different cell types and does not appear to be required for basal promoter activity. We conclude that proteins binding at both sites act coordinately, leading to maximal induction of the receptor gene.

  7. Apolipoprotein E inhibits osteoclast differentiation via regulation of c-Fos, NFATc1 and NF-κB

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Woo-Shin; Kim, Hyung Joon; Lee, Zang Hee [Department of Cell and Developmental Biology, BK21 Program and Dental Research Institute, Seoul National University, 28 Yeongon-Dong, Chongno-Gu, Seoul 110-749 (Korea, Republic of); Lee, Youngkyun [Department of Biochemistry, School of Dentistry, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kim, Hong-Hee, E-mail: hhbkim@snu.ac.kr [Department of Cell and Developmental Biology, BK21 Program and Dental Research Institute, Seoul National University, 28 Yeongon-Dong, Chongno-Gu, Seoul 110-749 (Korea, Republic of)

    2013-02-15

    Apolipoprotein E (ApoE) plays a major role in the transport and metabolism of lipid. Other functions of ApoE include modulation of innate and adaptive immune responses. The expression of ApoE in osteoblasts and its relevance with bone formation have also been reported. However, the effect of ApoE on osteoclasts has not yet been examined. Here, we investigated the role of ApoE in osteoclast differentiation using bone marrow-derived macrophages (BMMs) and RAW264.7 cells. We found a down-regulation of ApoE gene expression during osteoclastic differentiation of those cells. Overexpression of ApoE in BMMs and RAW264.7 cells significantly blocked the induction of c-Fos and nuclear factor of activated T cell c1 (NFATc1), transcription factors critical for expression of osteoclast marker genes, by receptor activator of nuclear factor κB ligand (RANKL), the osteoclast differentiation factor. ApoE inhibited osteoclast differentiation, as measured by decreased number of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs). In addition, ApoE reduced the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and ATPase, H{sup +} transporting, lysosomal 38 kDa, V0 subunit d2 (ATP6v0d2), genes involved in cell–cell fusion during osteoclastogenesis. Knock-down of ApoE using a specific siRNA promoted the RANKL-mediated induction of osteoclast differentiation. While ApoE did not affect the activation of ERK, JNK, and p38 MAPK signaling pathways by RANKL, the phosphorylation of p65 trans-activation domain on serine 536 and transcription activity of NF-κB were reduced by ApoE overexpression. These findings suggest that ApoE plays an inhibitory role in osteoclast differentiation via the suppression of RANKL-dependent activation of NF-κB and induction of c-Fos and NFATc1. - Highlights: ► Apolipoprotein E (ApoE) significantly inhibited osteoclast differentiation and activation of NF-κB. ► ApoE decreased the induction of osteoclast marker

  8. Probing the proton channels in subunit N of Complex I from Escherichia coli through intra-subunit cross-linking.

    Science.gov (United States)

    Tursun, Ablat; Zhu, Shaotong; Vik, Steven B

    2016-12-01

    Respiratory Complex I appears to have 4 sites for proton translocation, which are coupled to the oxidation of NADH and reduction of coenzyme Q. The proton pathways are thought to be made of offset half-channels that connect to the membrane surfaces, and are connected by a horizontal path through the center of the membrane. In this study of the enzyme from Escherichia coli, subunit N, containing one of the sites, was targeted. Pairs of cysteine residues were introduced into neighboring α-helices along the proposed proton pathways. In an effort to constrain conformational changes that might occur during proton translocation, we attempted to form disulfide bonds or methanethiosulfonate bridges between two engineered cysteine residues. Cysteine modification was inferred by the inability of PEG-maleimide to shift the electrophoretic mobility of subunit N, which will occur upon reaction with free sulfhydryl groups. After the cross-linking treatment, NADH oxidase and NADH-driven proton translocation were measured. Ten different pairs of cysteine residues showed evidence of cross-linking. The most significant loss of enzyme activity was seen for residues near the essential Lys 395. This residue is positioned between the proposed proton half-channel to the periplasm and the horizontal connection through subunit N, and is also near the essential Glu 144 of subunit M. The results suggest important conformational changes in this region for the delivery of protons to the periplasm, or for coupling the actions of subunit N to subunit M. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Subunit compensation and plasticity of synaptic GABAA receptors induced by ethanol in α4 subunit knockout mice

    Directory of Open Access Journals (Sweden)

    Asha eSuryanarayanan

    2011-09-01

    Full Text Available There is considerable evidence that ethanol (EtOH potentiates γ-aminobutyric acid type A receptor (GABAAR action, but only GABAARs containing δ subunits appear sensitive to low mM EtOH. The α4 and δ subunits co-assemble into GABAARs which are relatively highly expressed at extrasynaptic locations in the dentate gyrus where they mediate tonic inhibition. We previously demonstrated reversible- and time-dependent changes in GABAAR function and subunit composition in rats after single-dose EtOH intoxication. We concluded that early tolerance to EtOH occurs by over-activation and subsequent internalization of EtOH-sensitive extrasynaptic α4βδ-GABAARs. Based on this hypothesis, any highly EtOH-sensitive GABAARs should be subject to internalization following exposure to suitably high EtOH doses. To test this, we studied the GABAARs in mice with a global deletion of the α4 subunit (KO. The dentate granule cells (DGCs of these mice exhibited greatly reduced tonic currents and greatly reduced potentiation by acutely applied EtOH, whereas synaptic currents showed heightened sensitivity to low EtOH concentrations. The hippocampus of naive KO mice showed reduced δ subunit protein levels, but increased α2, and γ2 levels compared to wild-type (WT controls, suggesting at least partial compensation by these subunits in synaptic, highly EtOH-sensitive GABAARs of KO mice. In WT mice, cross-linking and Western blot analysis at 1 h after an EtOH challenge (3.5 g/kg, i.p. revealed increased intracellular fraction of the α1, α4 and δ, but not α2, α5 or γ2 subunits. By contrast, we observed significant internalization of α1, α2, δ, and γ2 subunits after a similar EtOH challenge in KO mice. Synaptic currents from naïve KO mice were more sensitive to potentiation by zolpidem (0.3 μM, requiring α1/α2, inactive at α4/5 GABAARs than those from naïve WT mice. At 1 h after EtOH, synaptic currents of WT mice were unchanged, whereas those of KO mice

  10. Segmental neurofibromatosis is caused by somatic mutation of the neurofibromatosis type 1 (NF1) gene.

    Science.gov (United States)

    Tinschert, S; Naumann, I; Stegmann, E; Buske, A; Kaufmann, D; Thiel, G; Jenne, D E

    2000-06-01

    Segmental neurofibromatosis (NF) is generally thought to result from a postzygotic NF1 (neurofibromatosis type 1) gene mutation. However, this has not yet been demonstrated at the molecular level. Using fluorescence in situ hybridisation (FISH) we identified an NF1 microdeletion in a patient with segmental NF in whom café-au-lait spots and freckles are limited to a single body region. The mutant allele was present in a mosaic pattern in cultured fibroblasts from a café-au-lait spot lesion, but was absent in fibroblasts from normal skin as well as in peripheral blood leukocytes. These findings prove the hypothesis that the molecular basis of segmental cutaneous NF is a mutation in the NF1 gene and that the regional distribution of manifestations reflects different cell clones, commensurate with the concept of somatic mosaicism.

  11. Polysaccharide from Ganoderma atrum evokes antitumor activity via Toll-like receptor 4-mediated NF-κB and mitogen-activated protein kinase signaling pathways.

    Science.gov (United States)

    Zhang, Shenshen; Nie, Shaoping; Huang, Danfei; Huang, Jianqin; Wang, Yawei; Xie, Mingyong

    2013-04-17

    Ganoderma atrum has been used as a traditional Chinese medicine for centuries. In this study, the antitumor activity of a novel G. atrum polysaccharide (PSG-1) was investigated in vitro and in vivo using S180 tumor-bearing mice. The results showed that PSG-1 significantly inhibited the proliferation of S180 via the activation of macrophages in a dose-dependent manner. PSG-1-primed macrophages exhibited a higher tumoricidal activity than untreated macrophages. Administration of PSG-1 significantly inhibited the growth of transplantable sarcoma S180-bearing mice and increased macrophage phagocytosis and the levels of cytokines and nitride oxide. Expression of Toll-like receptor (TLR) 4 in the membrane was markedly increased in PSG-1-treated groups, suggesting that it may be a possible receptor for PSG-1. PSG-1 also promoted the translocation of the p65 subunit of NF-κB from cytosol to nucleus and the degradation of IκBα. Moreover, the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1/2, and c-Jun N-terminal kinase in macrophages was improved by PSG-1 in a dose-dependent manner. Therefore, it is suggested that PSG-1 may elicit its antitumor effect by improving immune system functions through TLR4-mediated NF-κB and MAPK signaling pathways.

  12. Pomegranate protects liver against cecal ligation and puncture-induced oxidative stress and inflammation in rats through TLR4/NF-κB pathway inhibition.

    Science.gov (United States)

    Makled, Mirhan N; El-Awady, Mohammed S; Abdelaziz, Rania R; Atwan, Nadia; Guns, Emma T; Gameil, Nariman M; Shehab El-Din, Ahmed B; Ammar, Elsayed M

    2016-04-01

    Acute liver injury secondary to sepsis is a major challenge in intensive care unit. This study was designed to investigate potential protective effects of pomegranate against sepsis-induced acute liver injury in rats and possible underlying mechanisms. Pomegranate was orally given (800mg/kg/day) for two weeks before sepsis induction by cecal ligation and puncture (CLP). Pomegranate improved survival and attenuated liver inflammatory response, likely related to downregulation of mRNA expression of toll like recptor-4, reduced nuclear translocation and DNA binding activity of proinflammatory transcription factor NF-κB subunit p65, decreased mRNA and protein expression of tumor necrosis factor-alpha and reduction in myeloperoxidase activity and mRNA expression. Pomegranate also decreased CLP-induced oxidative stress as reflected by decreased malondialdehyde content, and increased reduced glutathione level and superoxide dismutase activity. These results confirm the antiinflammatory and antioxidant effects of pomegranate in CLP-induced acute liver injury mediated through inhibiting TLR4/NF-κB pathway, lipid peroxidation and neutrophil infiltration. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions.

    Science.gov (United States)

    Römling, Ute; Galperin, Michael Y

    2015-09-01

    Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Allelic variation of HMW glutenin subunits of Ethiopian bread wheat ...

    African Journals Online (AJOL)

    High molecular weight glutenins are often effective in identifying wheat (Triticum aestivum) genotypes with good baking quality. The high molecular weight glutenin subunit composition of Ethiopian cultivars and advanced lines was investigated to determine their influence on quality. Three alleles at Glu-A1, five at Glu-B1 ...

  15. Cholera toxin B subunit induces local curvature on lipid bilayers

    DEFF Research Database (Denmark)

    Pezeshkian, Weria; Nåbo, Lina J.; Ipsen, John H.

    2017-01-01

    The B subunit of the bacterial cholera toxin (CTxB) is responsible for the toxin binding to the cell membrane and its intracellular trafficking. CTxB binds to the monosialotetrahexosyl ganglioside at the plasma membrane of the target cell and mediates toxin internalization by endocytosis. CTx...

  16. Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

    Science.gov (United States)

    Römling, Ute; Galperin, Michael Y.

    2015-01-01

    Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

  17. Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

    Science.gov (United States)

    Saha, Debarchana; Koli, Swanand; Patgaonkar, Mandar; Reddy, Kudumula Venkata Rami

    2017-01-01

    Hemoglobin (Hb) is a major protein involved in transport of oxygen (O2). It consists of Hb-α and Hb-β subunits, which are normally expressed by cells of erythroid lineage. However, till recently, it was not known whether non-erythroid cells like vaginal cells synthesize Hb and whether it has any functional significance. Therefore, we designed the following objectives: (1) to establish in-vitro culture system of human primary vaginal epithelial cells (hPVECs), (2) to determine whether Hb-α and Hb-β proteins are truly synthesized by hPVECs, (3) to evaluate the effect of LPS (lipopolysaccharide) on the expression of Hb-α and Hb-β proteins (4) to decipher the significance of the Hb-α and Hb-β expression in hPVECs and (5) to determine the molecular mechanism regulating the expression of Hb-α in hPVECs. To accomplish these studies, we applied a battery of assays such as RT-PCR, qRT-PCR, Flow cytometry, western blot, and immunofluorescence, Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The results revealed the expression of Hb-α and Hb-β at both mRNA and protein level in hPVECs. The expression was significantly upregulated following LPS treatment (10μg/ml for 6 hrs) and these results are comparable with the expression induced by LPS in human vaginal epithelial cell line (VK2/E6E7). These cells constitutively produced low levels of pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines. Also, the response of phosphorylated (p65)-NF-κB to LPS was upregulated with increased expression of IL-6, Toll-like receptor-4 (TLR4) and human beta defensin-1 (hBD-1) in hPVECs and VK2/E6E7 cells. However, Bay 11–7082 treatment (5μM for 24 hrs) could neutralize the effect of LPS-induced p65-NF-κB activity and represses the production`of Hb-α and Hb-β. The results of EMSA revealed the presence of putative binding sites of NF-κB in the human Hb-α promoter region (nt-115 to -106). ChIP analysis confirmed the binding of NF

  18. Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance.

    Directory of Open Access Journals (Sweden)

    Debarchana Saha

    Full Text Available Hemoglobin (Hb is a major protein involved in transport of oxygen (O2. It consists of Hb-α and Hb-β subunits, which are normally expressed by cells of erythroid lineage. However, till recently, it was not known whether non-erythroid cells like vaginal cells synthesize Hb and whether it has any functional significance. Therefore, we designed the following objectives: (1 to establish in-vitro culture system of human primary vaginal epithelial cells (hPVECs, (2 to determine whether Hb-α and Hb-β proteins are truly synthesized by hPVECs, (3 to evaluate the effect of LPS (lipopolysaccharide on the expression of Hb-α and Hb-β proteins (4 to decipher the significance of the Hb-α and Hb-β expression in hPVECs and (5 to determine the molecular mechanism regulating the expression of Hb-α in hPVECs. To accomplish these studies, we applied a battery of assays such as RT-PCR, qRT-PCR, Flow cytometry, western blot, and immunofluorescence, Electrophoretic mobility shift assay (EMSA and chromatin immunoprecipitation (ChIP. The results revealed the expression of Hb-α and Hb-β at both mRNA and protein level in hPVECs. The expression was significantly upregulated following LPS treatment (10μg/ml for 6 hrs and these results are comparable with the expression induced by LPS in human vaginal epithelial cell line (VK2/E6E7. These cells constitutively produced low levels of pro-inflammatory (IL-6 and anti-inflammatory (IL-10 cytokines. Also, the response of phosphorylated (p65-NF-κB to LPS was upregulated with increased expression of IL-6, Toll-like receptor-4 (TLR4 and human beta defensin-1 (hBD-1 in hPVECs and VK2/E6E7 cells. However, Bay 11-7082 treatment (5μM for 24 hrs could neutralize the effect of LPS-induced p65-NF-κB activity and represses the production`of Hb-α and Hb-β. The results of EMSA revealed the presence of putative binding sites of NF-κB in the human Hb-α promoter region (nt-115 to -106. ChIP analysis confirmed the binding of NF

  19. Inducible but not constitutive expression of PD-L1 in human melanoma cells is dependent on activation of NF-κB.

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    Kavitha Gowrishankar

    Full Text Available Monoclonal antibodies against immune checkpoint blockade have proven to be a major success in the treatment of melanoma. The programmed death receptor-1 ligand-1 (PD-L1 expression on melanoma cells is believed to have an inhibitory effect on T cell responses and to be an important escape mechanism from immune attack. Previous studies have shown that PD-L1 can be expressed constitutively or can be induced by IFN-γ secreted by infiltrating lymphocytes. In the present study we have investigated the mechanism underlying these two modes of PD-L1 expression in melanoma cells including cells that had acquired resistance to the BRAF inhibitor vemurafenib. PD-L1 expression was examined by flow cytometry and immunoblotting. Specific inhibitors and siRNA knockdown approaches were used to examine the roles of the RAF/ MEK, PI3K, NF-κB, STAT3 and AP1/ c-Jun pathways. IFN-γ inducible expression of PD-L1 was dependent on NF-κB as shown by inhibition with BMS-345541, an inhibitor of IκB and the BET protein inhibitor I-BET151, as well as by siRNA knockdown of NF-κB subunits. We were unable to implicate the BRAF/MEK pathway as major regulators in PD-L1 expression on vemurafenib resistant cells. Similarly the PI3K/AKT pathway and the transcription factors STAT3 and c-Jun had only minor roles in IFN-γ induced expression of PD-L1. The mechanism underlying constitutive expression remains unresolved. We suggest these results have significance in selection of treatments that can be used in combination with monoclonal antibodies against PD1, to enhance their effectiveness and to reduce inhibitory effects melanoma cells have against cytotoxic T cell activity.

  20. Kukoamine A Prevents Radiation-Induced Neuroinflammation and Preserves Hippocampal Neurogenesis in Rats by Inhibiting Activation of NF-κB and AP-1.

    Science.gov (United States)

    Zhang, Yaqiong; Gao, Lingyue; Cheng, Zhihua; Cai, Jiayi; Niu, Yixuan; Meng, Weihong; Zhao, Qingchun

    2017-02-01

    Impaired hippocampal neurogenesis and neuroinflammation are involved in the pathogenesis of radiation-induced brain injury. Kukoamine A (KuA) was demonstrated to have neuroprotective effects through inhibiting oxidative stress and apoptosis after whole-brain irradiation (WBI) in rats. The aim of this study was to investigate whether administration of KuA would prevent radiation-induced neuroinflammation and the detrimental effect on hippocampal neurogenesis. For this study, male Wistar rats received either sham irradiation or WBI (30 Gy single dose of X-rays) followed by the immediate injection of either KuA or vehicle intravenously. The dose of KuA was 5, 10, and 20 mg/kg, respectively. The levels of pro-inflammatory cytokines were assayed by ELISA kits. The newborn neurons were detected by 5-bromo-2-deoxyuridine (BrdU)/neuronal nuclei (NeuN) double immunofluorescence. Microglial activation was measured by Iba-1 immunofluorescence. The expression of Cox-2 and the activation of nuclear factor κB (NF-κB), activating protein 1(AP-1), and PPARδ were evaluated by western blot. WBI led to a significant increase in the level of TNF-α, IL-1β, and Cox-2, and it was alleviated by KuA administration. KuA attenuated microglial activation in rat hippocampus after WBI. Neurogenesis impairment induced by WBI was ameliorated by KuA. Additionally, KuA alleviated the increased translocation of NF-κB p65 subunit and phosphorylation of c-jun induced by WBI and elevated the expression of PPARδ. These data indicate that KuA could ameliorate the neuroinflammatory response and protect neurogenesis after WBI, partially through regulating the activation of NF-κB, AP-1, and PPARδ.

  1. Renoprotective effect of paricalcitol via a modulation of the TLR4-NF-κB pathway in ischemia/reperfusion-induced acute kidney injury

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jae-Won, E-mail: maestro97@hanmail.net; Kim, Sun Chul, E-mail: linefe99@hanmail.net; Ko, Yoon Sook, E-mail: rainboweyes@hanmail.net; Lee, Hee Young, E-mail: cell1023@hanmail.net; Cho, Eunjung, E-mail: icdej@naver.com; Kim, Myung-Gyu, E-mail: gyu219@hanmail.net; Jo, Sang-Kyung, E-mail: sang-kyung@korea.ac.kr; Cho, Won Yong, E-mail: wonyong@korea.ac.kr; Kim, Hyoung Kyu, E-mail: hyoung@korea.ac.kr

    2014-02-07

    Highlights: • Paricalcitol. • Attenuation of renal inflammation. • Modulation of TLR4-NF-κB signaling. - Abstract: Background: The pathophysiology of ischemic acute kidney injury (AKI) is thought to include a complex interplay between vascular endothelial cell dysfunction, inflammation, and tubular cell damage. Several lines of evidence suggest a potential anti-inflammatory effect of vitamin D in various kidney injury models. In this study, we investigated the effect of paricalcitol, a synthetic vitamin D analog, on renal inflammation in a mouse model of ischemia/reperfusion (I/R) induced acute kidney injury (AKI). Methods: Paricalcitol was administered via intraperitoneal (IP) injection at 24 h before ischemia, and then I/R was performed through bilateral clamping of the renal pedicles. Twenty-four hours after I/R, mice were sacrificed for the evaluation of injury and inflammation. Additionally, an in vitro experiment using HK-2 cells was also performed to examine the direct effect of paricalcitol on tubular cells. Results: Pre-treatment with paricalcitol attenuated functional deterioration and histological damage in I/R induced AKI, and significantly decreased tissue neutrophil and macrophage infiltration and the levels of chemokines, the pro-inflammatory cytokine interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). It also decreased IR-induced upregulation of Toll-like receptor 4 (TLR4), and nuclear translocation of p65 subunit of NF-κB. Results from the in vitro study showed pre-treatment with paricalcitol suppressed the TNF-α-induced depletion of cytosolic IκB in HK-2 cells. Conclusion: These results demonstrate that pre-treatment with paricalcitol has a renoprotective effect in ischemic AKI, possibly by suppressing TLR4-NF-κB mediated inflammation.

  2. Stevia and stevioside protect against cisplatin nephrotoxicity through inhibition of ERK1/2, STAT3, and NF-κB activation.

    Science.gov (United States)

    Potočnjak, Iva; Broznić, Dalibor; Kindl, Marija; Kropek, Matija; Vladimir-Knežević, Sanda; Domitrović, Robert

    2017-09-01

    We investigated the effect of natural sweetener Stevia rebaudiana and its constituent stevioside in cisplatin (CP)-induced kidney injury. Male BALB/cN mice were orally administered 10, 20, and 50 mg/kg body weight of Stevia rebaudiana ethanol extract (SE) or stevioside 50 mg/kg, 48 h after intraperitoneal administration of CP (13 mg/kg). Two days later, CP treatment resulted in histopathological changes showing kidney injury. Increased expression of 4-hydroxynonenal (4-HNE), 3-nitrotyrosine (3-NT), and heme oxygenase-1 (HO-1) in mice kidneys suggested oxidative stress. CP treatment also increased renal expression of nuclear factor-kappaB (NF-κB) p65 subunit and phosphorylated inhibitor of NF-κB (IκBα), as well as expression of pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α). Induction of apoptosis and inhibition of the cell cycle in kidneys was evidenced by increased expression of p53, Bax, caspase-9, and p21, proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), with concomitant suppression of Bcl-2 and cyclin D1 expression. The number of apoptotic cells in kidneys was also assessed. CP administration resulted in activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3). Both SE and stevioside attenuated CP nephrotoxicity by suppressing oxidative stress, inflammation, and apoptosis through mechanism involving ERK1/2, STAT3, and NF-κB suppression. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Cordyceps militaris fruit body extract ameliorates membranous glomerulonephritis by attenuating oxidative stress and renal inflammation via the NF-κB pathway.

    Science.gov (United States)

    Song, Jingjing; Wang, Yingwu; Liu, Chungang; Huang, Yan; He, Liying; Cai, Xueying; Lu, Jiahui; Liu, Yan; Wang, Di

    2016-04-01

    Membranous glomerulonephritis (MGN) is a common pathogenesis of nephritic syndrome in adult patients. Nuclear factor kappa B (NF-κB) serves as the main transcription factor for the inflammatory response mediated nephropathy. Cordyceps militaris, containing various pharmacological components, has been used as a kind of crude drug and folk tonic food for improving immunity and reducing inflammation. The current study aims to investigate the renoprotective activity of Cordyceps militaris aqueous extract (CM) in the cationic bovine serum albumin (C-BSA)-induced rat model of membranous glomerulonephritis. Significant renal dysfunction was observed in MGN rats; comparatively, 4-week CM administration strongly decreased the levels of 24 h urine protein, total cholesterol, triglyceride, blood urea nitrogen and serum creatinine, and increased the levels of serum albumin and total serum protein. Strikingly, recovery of the kidney histological architecture was noted in CM-treated MGN rats. A significant improvement in the glutathione peroxidase and superoxide dismutase levels, and a reduced malondialdehyde concentration were observed in the serum and kidney of CM-treated rats. Altered levels of inflammatory cytokines including interleukins, monocyte chemoattractant protein-1, intercellular adhesion molecule 1, vascular adhesion molecule 1, tumor necrosis factor-α, 6-keto-prostaglandin F1α, and nuclear transcriptional factor subunit NF-κB p65 reverted to normal levels upon treatment with CM. The present data suggest that CM protects rats against membranous glomerulonephritis via the normalization of NF-κB activity, thereby inhibiting oxidative damage and reducing inflammatory cytokine levels, which further provide experimental evidence in support of the clinical use of CM as an effective renoprotective agent.

  4. Suppressive Effect on Lipopolysaccharide-Induced Proinflammatory Mediators by Citrus aurantium L. in Macrophage RAW 264.7 Cells via NF-κB Signal Pathway

    Directory of Open Access Journals (Sweden)

    Sang-Rim Kang

    2011-01-01

    Full Text Available Citrus fruits have been used as an edible fruit and a traditional medicine since ancient times. In particular, the peels of immature citrus fruits are used widely in traditional herbal medicine in Korea, as they are believed to contain bioactive components exerting anti-inflammatory activity. This study examined whether the crude methanol extract of Citrus aurantium L. (CME has a suppressive effect on inducible enzymes and proinflammatory cytokines by inhibiting the NF-κB pathway in LPS-stimulated macrophage RAW 264.7 cells. The cells were pretreated with the indicated concentrations of CME (5, 10, 20, and 50 μg/mL and then treated with LPS (1 μg/mL. The results showed that CME (10, 20, and 50 μg/mL inhibited the LPS- (1 μg/mL induced mRNA and protein expression of iNOS in macrophage Raw 264.7 cells. In addition, the expression of COX-2 was inhibited at the mRNA and protein levels by CME in a dose-dependent manner. The mRNA expression of proinflammatory cytokines, such as TNF-α and IL-6, were markedly reduced by CME (10, 20, and 50 μg/mL. Moreover, CME clearly suppressed the nuclear translocation of the NF-κB p65 subunits, which was correlated with its inhibitory effect on I-κB phosphorylation. These results suggest that CME has anti-inflammatory properties by modulating the expression of COX-2, iNOS, and proinflammatory cytokines, such as TNF-α and IL-6, in macrophage RAW 264.7 cells via the NF-κB pathway.

  5. TAK1 ubiquitination regulates Doxorubicin-induced NF-κB activation

    OpenAIRE

    Liang, Li; Fan, Yihui; Cheng, Jin; Cheng, Da; Zhao, Yanling; Baoshan CAO; Ma,LiWen; An, Lei; Jia, Wei; Su, Xu; Yang, Jianhua; Zhang, Hong

    2012-01-01

    Chemotherapeutic agents- and radiation therapy-induced NF-κB activation in cancer cells contributes to aggressive tumor growth and resistance to chemotherapy and ionizing radiation during cancer treatment. TAK1 has been shown to be required for genotoxic stress-induced NF-κB activation. However, whether TAK1 ubiquitination is involved in genotoxic stress-induced NF-κB activation remains unknown. Herein, we demonstrate that TAK1 ubiquitination plays an important role in the positive and negati...

  6. NF1 Is an Effector and Regulator of the GPCR Signaling in the Nervous System

    Science.gov (United States)

    2015-04-01

    of NF1 by infecting differentiated cultures with AAV carrying Cre recombinase (Task 2 A and B). We used these cultures along with uninfected...activation and regulation of its signaling to downstream protein kinases (Task 3). We infected neurons with the AAV virus encoding Cre-recombinase...cultured striatal neurons on MAPK signaling. Cultured neurons from NF1flx/flx mice were infected with either AAV -Cre deleting NF1 or control AAV

  7. A Transgenic Model for Learning Defects: Role of NF1 in Drosophila Visual-Spatial Learning

    Science.gov (United States)

    2008-12-01

    segments of different orientations and elevations, prior to assaying NF1 mutants. We will begin to prepare a manuscript for publication that describes...Role of NF1 in Drosophila Visual- Spatial Learning PRINCIPAL INVESTIGATOR: Frances Hannan, Ph.D. CONTRACTING...Model for Learning Defects: Role of NF1 in Drosophila Visual-Spatial Learnings 5b. GRANT NUMBER W81XWH-07-1-0436 5c. PROGRAM ELEMENT NUMBER 6

  8. DMPD: New insights into NF-kappaB regulation and function. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18775672 New insights into NF-kappaB regulation and function. Sun SC, Ley SC. Trend...ction. PubmedID 18775672 Title New insights into NF-kappaB regulation and function....s Immunol. 2008 Oct;29(10):469-78. Epub 2008 Sep 3. (.png) (.svg) (.html) (.csml) Show New insights into NF-kappaB regulation and fun

  9. Porcine arterivirus activates the NF-kappaB pathway through IkappaB degradation.

    Science.gov (United States)

    Lee, Sang-Myeong; Kleiboeker, Steven B

    2005-11-10

    Nuclear factor-kappaB (NF-kappaB) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-kappaB in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-kappaB activation was characterized by translocation of NF-kappaB from the cytoplasm to the nucleus, increased DNA binding activity, and NF-kappaB-regulated gene expression. NF-kappaB activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-kappaB activation. Degradation of IkappaB protein was detected late in PRRSV infection, and overexpression of the dominant negative form of IkappaBalpha (IkappaBalphaDN) significantly suppressed NF-kappaB activation induced by PRRSV. However, IkappaBalphaDN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-kappaB DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-kappaB was activated by PRRSV infection. Moreover, NF-kappaB-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-kappaB activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV.

  10. microRNA-146a inhibits G protein-coupled receptor-mediated activation of NF-κB by targeting CARD10 and COPS8 in gastric cancer

    Directory of Open Access Journals (Sweden)

    Crone Stephanie

    2012-09-01

    Full Text Available Abstract Background Gastric cancer is the second most common cause of cancer-related death in the world. Inflammatory signals originating from gastric cancer cells are important for recruiting inflammatory cells and regulation of metastasis of gastric cancer. Several microRNAs (miRNA have been shown to be involved in development and progression of gastric cancer. miRNA-146a (miR-146a is a modulator of inflammatory signals, but little is known about its importance in gastric cancer. We therefore wanted to identify targets of miR-146a in gastric cancer and examine its biological roles. Results The expression of miR-146a was evaluated by quantitative PCR (qPCR and found up-regulated in the gastrin knockout mice, a mouse model of gastric cancer, and in 73% of investigated human gastric adenocarcinomas. Expression of miR-146a by gastric cancer cells was confirmed by in situ hybridization. Global analysis of changes in mRNA levels after miR-146a transfection identified two transcripts, caspase recruitment domain-containing protein 10 (CARD10 and COP9 signalosome complex subunit 8 (COPS8, as new miR-146a targets. qPCR, Western blotting and luciferase assays confirmed these transcripts as direct miR-146a targets. CARD10 and COPS8 were shown to be part of the G protein-coupled receptor (GPCR pathway of nuclear factor-kappaB (NF-kappaB activation. Lysophosphatidic acid (LPA induces NF-kappaB activation via this pathway and over-expression of miR-146a inhibited LPA-induced NF-kappaB activation, reduced LPA-induced expression of tumor-promoting cytokines and growth factors and inhibited monocyte attraction. Conclusions miR-146a expression is up-regulated in a majority of gastric cancers where it targets CARD10 and COPS8, inhibiting GPCR-mediated activation of NF-kappaB, thus reducing expression of NF-kappaB-regulated tumor-promoting cytokines and growth factors. By targeting components of several NF-kappaB-activating pathways, miR-146a is a key component in

  11. Inhibition of NF-kB 1 (NF-kBp50 by RNA interference in chicken macrophage HD11 cell line challenged with Salmonella enteritidis

    Directory of Open Access Journals (Sweden)

    Hsin-I Chiang

    2009-01-01

    Full Text Available The NF-kB pathway plays an important role in regulating the immunity response in animals. In this study, small interfering RNAs (siRNA were used to specifically inhibit NF-kB 1 expression and to elucidate the role of NF-kB in the signal transduction pathway of the Salmonella challenge in the chicken HD11 cell line. The cells were transfected with either NF-kB 1 siRNA, glyceraldehyde 3-phosphate dehydrogenase siRNA (positive control or the negative control siRNA for 24 h, followed by Salmonella enteritidis (SE challenge or non-challenge for 1 h and 4 h. Eight candidate genes related to the signal pathway of SE challenge were selected to examine the effect of NF-kB 1 inhibition on their expressions by mRNA quantification. The results showed that, with a 36% inhibition of NF-kB 1 expression, gene expression of both Toll-like receptor (TLR 4 and interleukin (IL-6 was consistently and significantly increased at both 1 h and 4 h following SE challenge, whereas the gene expression of MyD88 and IL-1β was increased at 1 h and 4 h, respectively. These findings suggest a likely inhibitory regulation by NF-kB 1, and could lay the foundation for studying the gene network of the innate immune response of SE infection in chickens.

  12. The garlic NF-YC gene, AsNF-YC8, positively regulates non-ionic hyperosmotic stress tolerance in tobacco.

    Science.gov (United States)

    Sun, Xiudong; Lian, Haifeng; Liu, Xingchen; Zhou, Shumei; Liu, Shiqi

    2017-05-01

    To investigate the relationship between nuclear factor Y (NF-Y) and stress tolerance in garlic, we cloned a NF-Y family gene AsNF-YC8 from garlic, which was largely upregulated at dehydrate stage. Expression pattern analyses in garlic revealed that AsNF-YC8 is induced through abscisic acid (ABA) and abiotic stresses, such as NaCl and PEG. Compared with wild-type plants, the overexpressing-AsNF-YC8 transgenic tobacco plants showed higher seed germination rates, longer root length and better plant growth under salt and drought stresses. Under drought stress, the transgenic plants maintained higher relative water content (RWC), net photosynthesis, lower levels of malondialdehyde (MDA), and less ion leakage (IL) than wild-type control plants. These results indicate the high tolerance of the transgenic plants to drought stress compared to the WT. The transgenic tobacco lines accumulated less reactive oxygen species (ROS) and exhibited higher antioxidative enzyme activities compared with wild-type (WT) plants under drought stress, which suggested that the overexpression of AsNF-YC8 improves the antioxidant defense system by regulating the activities of these antioxidant enzymes, which in turn protect transgenic lines against drought stress. These results suggest that AsNF-YC8 plays an important role in tolerance to drought and salt stresses.

  13. Review: NF-κB pathways in the pathogenesis of Multiple Sclerosis and the therapeutic implications

    Directory of Open Access Journals (Sweden)

    Saskia Michaela Leibowitz

    2016-09-01

    Full Text Available Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB signalling pathways are involved in cell immune responses, apoptosis and infections. In Multiple Sclerosis (MS, NF-κB pathways are changed, leading to increased levels of NF-κB activation in cells. This may indicate a key role for NF-κB in MS pathogenesis. NF-κB signalling is complex, with many elements involved in its activation and regulation. Interestingly, current MS treatments are found to be directly or indirectly linked to NF-κB pathways and act to adjust the innate and adaptive immune system in patients. In this review, we will first focus on the intricacies of NF-κB signalling, including the activating pathways and regulatory elements. Next, we will theorise about the role of NF-κB in MS pathogenesis, based on current research findings, and discuss some of the associated therapeutic implications. Lastly, we will review four new MS treatments which interrupt NF-κB pathways - fingolimod, teriflunomide, dimethyl fumarate and laquinimod - and explain their mechanisms, and the possible strategy for MS treatments in the future.

  14. Overexpression of the transcription factor NF-YC9 confers abscisic acid hypersensitivity in Arabidopsis.

    Science.gov (United States)

    Bi, Chao; Ma, Yu; Wang, Xiao-Fang; Zhang, Da-Peng

    2017-11-01

    Nuclear factor Y (NF-Y) family proteins are involved in many developmental processes and responses to environmental cues in plants, but whether and how they regulate phytohormone abscisic acid (ABA) signaling need further studies. In the present study, we showed that over-expression of the NF-YC9 gene confers ABA hypersensitivity in both the early seedling growth and stomatal response, while down-regulation of NF-YC9 does not affect ABA response in these processes. We also showed that over-expression of the NF-YC9 gene confers salt and osmotic hypersensitivity in early seedling growth, which is likely to be directly associated with the ABA hypersensitivity. Further, we observed that NF-YC9 physically interacts with the ABA-responsive bZIP transcription factor ABA-INSENSITIVE5 (ABI5), and facilitates the function of ABI5 to bind and activate the promoter of a target gene EM6. Additionally, NF-YC9 up-regulates expression of the ABI5 gene in response to ABA. These findings show that NF-YC9 may be involved in ABA signaling as a positive regulator and likely functions redundantly together with other NF-YC members, and support the model that the NF-YC9 mediates ABA signaling via targeting to and aiding the ABA-responsive transcription factors such as ABI5.

  15. NF2/Merlin is required for the axial pattern formation in the Xenopus laevis embryo.

    Science.gov (United States)

    Zhu, Xuechen; Min, Zheying; Tan, Renbo; Tao, Qinghua

    2015-11-01

    The NF2 gene product Merlin is a FERM-domain protein possessing a broad tumor-suppressing function. NF2/Merlin has been implicated in regulating multiple signaling pathways critical for cell growth and survival. However, it remains unknown whether NF2/Merlin regulates Wnt/β-catenin signaling during vertebrate embryogenesis. Here we demonstrate that NF2/Merlin is required for body pattern formation in the Xenopus laevis embryo. Depletion of the maternal NF2/Merlin enhances organizer gene expression dependent on the presence of β-catenin, and causes dorsanteriorized development; Morpholino antisense oligo-mediated knockdown of the zygotic NF2/Merlin shifts posterior genes anteriorwards and reduces the anterior development. We further demonstrate that targeted depletion of NF2 in the presumptive dorsal tissues increases the levels of nuclear β-catenin in the neural epithelial cells. Biochemical analyses reveal that NF2 depletion promotes the production of active β-catenin and concurrently decreases the level of N-terminally phosphorylated β-catenin under the stimulation of the endogenous Wnt signaling. Our findings suggest that NF2/Merlin negatively regulates the Wnt/β-catenin signaling activity during the pattern formation in early X. laevis embryos. Copyright © 2015. Published by Elsevier Ireland Ltd.

  16. Oligodendrocyte Nf1 Controls Aberrant Notch Activation and Regulates Myelin Structure and Behavior

    Directory of Open Access Journals (Sweden)

    Alejandro López-Juárez

    2017-04-01

    Full Text Available The RASopathy neurofibromatosis type 1 (NF1 is one of the most common autosomal dominant genetic disorders. In NF1 patients, neurological issues may result from damaged myelin, and mice with a neurofibromin gene (Nf1 mutation show white matter (WM defects including myelin decompaction. Using mouse genetics, we find that altered Nf1 gene-dose in mature oligodendrocytes results in progressive myelin defects and behavioral abnormalities mediated by aberrant Notch activation. Blocking Notch, upstream mitogen-activated protein kinase (MAPK, or nitric oxide signaling rescues myelin defects in hemizygous Nf1 mutants, and pharmacological gamma secretase inhibition rescues aberrant behavior with no effects in wild-type (WT mice. Concomitant pathway inhibition rescues myelin abnormalities in homozygous mutants. Notch activation is also observed in Nf1+/− mouse brains, and cells containing active Notch are increased in NF1 patient WM. We thus identify Notch as an Nf1 effector regulating myelin structure and behavior in a RASopathy and suggest that inhibition of Notch signaling may be a therapeutic strategy for NF1.

  17. CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line

    Science.gov (United States)

    Sharina, Iraida G.; Martin, Emil; Thomas, Anthony; Uray, Karen L.; Murad, Ferid

    2003-01-01

    Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. We investigated human beta1 sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, our study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression.

  18. B-physics computations from Nf=2 tmQCD

    CERN Document Server

    Carrasco, N.; Dimopoulos, P.; Frezzotti, R.; Gimenez, V.; Herdoiza, G.; Lubicz, V.; Michael, C.; Picca, E.; Rossi, G.C.; Sanfilippo, F.; Shindler, A.; Silvestrini, L.; Simula, S.; Tarantino, C.

    2014-01-01

    We present an accurate lattice QCD computation of the b-quark mass, the B and Bs decay constants, the B-mixing bag-parameters for the full four-fermion operator basis, as well as estimates for \\xi and f_{Bq}\\sqrt{B_q} extrapolated to the continuum limit and the physical pion mass. We have used Nf = 2 dynamical quark gauge configurations at four values of the lattice spacing generated by ETMC. Extrapolation in the heavy quark mass from the charm to the bottom quark region has been carried out using ratios of physical quantities computed at nearby quark masses, having an exactly known infinite mass limit.

  19. Bernsteinov algoritam za vertikalnu 3NF normalizaciju sintezom

    OpenAIRE

    Varga, Matija

    2013-01-01

    Ovim radom se prikazuje korištenje Bernsteinovog algoritma za vertikalnu 3NF normalizaciju sintezom. Cilj ovog rada je ponuditi algoritam za normalizaciju baze podataka te prikazati skup koraka koji vode na svođenje redundantnosti na najmanju moguću mjeru u cilju povećanja učinkovitosti rada s bazom podataka te navesti testove i algoritme za ispitivanje i dokazivanje reverzibilnosti (tj. dokazivanje da tijekom normalizacije nije došlo do gubitka informacije). U radu su prikazani primjeri kroz...

  20. Effect of high and low molecular weight glutenin subunits, and subunits of gliadin on physicochemical parameters of different wheat genotypes

    Directory of Open Access Journals (Sweden)

    Mariana Souza Costa

    2013-02-01

    Full Text Available Identification of functional properties of wheat flour by specific tests allows genotypes with appropriate characteristics to be selected for specific industrial uses. The objective of wheat breeding programs is to improve the quality of germplasm bank in order to be able to develop wheat with suitable gluten strength and extensibility for bread making. The aim of this study was to evaluate 16 wheat genotypes by correlating both glutenin subunits of high and low molecular weight and gliadin subunits with the physicochemical characteristics of the grain. Protein content, sedimentation volume, sedimentation index, and falling number values were analyzed after the grains were milled. Hectoliter weight and mass of 1000 seeds were also determined. The glutenin and gliadin subunits were separated using polyacrylamide gel in the presence of sodium dodecyl sulfate. The data were evaluated using variance analysis, Pearson's correlation, principal component analysis, and cluster analysis. The IPR 85, IPR Catuara TM, T 091015, and T 091069 genotypes stood out from the others, which indicate their possibly superior grain quality with higher sedimentation volume, higher sedimentation index, and higher mass of 1000 seeds; these genotypes possessed the subunits 1 (Glu-A1, 5 + 10 (Glu-D1, c (Glu-A3, and b (Glu-B3, with exception of T 091069 genotype that possessed the g allele instead of b in the Glu-B3.

  1. Lyn regulates inflammatory responses in Klebsiella pneumoniae infection via the p38/NF-κB pathway.

    Science.gov (United States)

    Li, Xuefeng; Zhou, Xikun; Ye, Yan; Li, Yi; Li, Jiaxin; Privratsky, Breanna; Wu, Erxi; Gao, Hongwei; Huang, Canhua; Wu, Min

    2014-03-01

    Klebsiella pneumoniae (Kp) is one of the most common pathogens in nosocomial infections and is becoming increasingly multidrug resistant. However, the underlying molecular pathogenesis of this bacterium remains elusive, limiting the therapeutic options. Understanding the mechanism of its pathogenesis may facilitate the development of anti-bacterial therapeutics. Here, we show that Lyn, a pleiotropic Src tyrosine kinase, is involved in host defense against Kp by regulating phagocytosis process and simultaneously downregulating inflammatory responses. Using acute infection mouse models, we observed that lyn(-/-) mice were more susceptible to Kp with increased mortality and severe lung injury compared with WT mice. Kp infected-lyn(-/-) mice exhibited elevated inflammatory cytokines (IL-6 and TNF-α), and increased superoxide in the lung and other organs. In addition, the phosphorylation of p38 and NF-κB p65 subunit increased markedly in response to Kp infection in lyn(-/-) mice. We also demonstrated that the translocation of p65 from cytoplasm to nuclei increased in cultured murine lung epithelial cells by Lyn siRNA knockdown. Furthermore, lipid rafts clustered with activated Lyn and accumulated in the site of Kp invasion. Taken together, these findings revealed that Lyn may participate in host defense against Kp infection through the negative modulation of inflammatory cytokines. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Cocaine promotes both initiation and elongation phase of HIV-1 transcription by activating NF-κB and MSK1 and inducing selective epigenetic modifications at HIV-1 LTR

    Science.gov (United States)

    Sahu, Geetaram; Farley, Kalamo; El-Hage, Nazira; Aiamkitsumrit, Benjamas; Fassnacht, Ryan; Kashanchi, Fatah; Ochem, Alex; Simon, Gary L.; Karn, Jonathan; Hauser, Kurt F.; Tyagi, Mudit

    2015-01-01

    Cocaine accelerates human immunodeficiency virus (HIV-1) replication by altering specific cell-signaling and epigenetic pathways. We have elucidated the underlying molecular mechanisms through which cocaine exerts its effect in myeloid cells, a major target of HIV-1 in central nervous system (CNS). We demonstrate that cocaine treatment promotes HIV-1 gene expression by activating both nuclear factor-kappa B (NF-κB) and mitogen- and stress-activated kinase 1 (MSK1). MSK1 subsequently catalyzes the phosphorylation of histone H3 at serine 10, and p65 subunit of NF-κB at 276th serine residue. These modifications enhance the interaction of NF-κB with P300 and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the HIV-1 LTR, supporting the development of an open/relaxed chromatin configuration, and facilitating the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication. PMID:25980739

  3. Extractable and non-extractable polyphenols from blueberries modulate LPS-induced expression of iNOS and COX-2 in RAW264.7 macrophages via the NF-κB signalling pathway.

    Science.gov (United States)

    Cheng, Anwei; Han, Caijing; Fang, Xixiu; Sun, Jinyue; Chen, Xiangyan; Wan, Fachun

    2016-08-01

    Plant polyphenols are rich in blueberries that have a wide range of properties beneficial to human health. There are two types, according to the solubility of polyphenols, which were defined as extractable polyphenols (EPP) and non-extractable polyphenols (NEPP), respectively. At present, in most of reports, 'total polyphenol' refers only to EPP excluding NEPP. In this paper, the effects of EPP and NEPP on lipopolysaccharides (LPS) induced production of nitric oxide (NO) and gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW264.7 cells via nuclear factor-κB (NF-κB) signalling pathway were compared. The results showed that EPP and NEPP from blueberries significantly inhibited the LPS-induced production of NO and gene expression of iNOS and COX-2 in cells. The constitutive level of p65 sub-unit of NF-κB was obviously detected after the treatments with EPP or NEPP. By contrast, the level of phosphorylated p65 (P-p65) was strongly inhibited by EPP or NEPP. EPP had a stronger inhibition on the gene expression of iNOS and COX-2 than that of NEPP. These findings of inhibition of iNOS and COX-2 mRNA expression through the suppression of NF-κB suggest that EPP and ENPP from blueberries have significant anti-inflammatory effect and may be a potential medicine. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  4. Purification of the NF2 tumor suppressor protein from human erythrocytes.

    Science.gov (United States)

    Jindal, Hitesh K; Yoshinaga, Kazumi; Seo, Pil-Soo; Lutchman, Mohini; Dion, Patrick A; Rouleau, Guy A; Hanada, Toshihiko; Chishti, Athar H

    2006-11-01

    Neurofibromatosis type 2 (NF2) is an autosomal dominant disease predisposing individuals to the risk of developing tumors of cranial and spinal nerves. The NF2 tumor suppressor protein, known as Merlin/Schwanomin, is a member of the protein 4.1 superfamily that function as links between the cytoskeleton and the plasma membrane. Upon selective extraction of membrane-associated proteins from erythrocyte plasma membrane (ghosts) using low ionic strength solution, the bulk of NF2 protein remains associated with the spectrin-actin depleted inside-out-vesicles. Western blot analysis showed a approximately 70 kDa polypeptide in the erythrocyte plasma membrane. Furthermore, quantitative removal of NF2 protein from the inside-out-vesicles was achieved using 1.0 M potassium iodide, a treatment known to remove tightly-bound peripheral membrane proteins. These results suggest a novel mode of NF2 protein association with the erythrocyte membrane that is distinct from the known membrane interactions of protein 4.1. Based on these biochemical properties, several purification strategies were devised to isolate native NF2 protein from human erythrocyte ghosts. Using purified and recombinant NF2 protein as internal standards, we quantified approximately 41-65,000 molecules of NF2 protein per erythrocyte. We provide evidence for the presence of NF2 protein in the human erythrocyte membrane. The identification of NF2 protein in the human erythrocyte membrane will make it feasible to discover novel interactions of NF2 protein utilizing powerful techniques of erythrocyte biochemistry and genetics in mammalian cells.

  5. Borna disease virus possesses an NF-ĸB inhibitory sequence in the nucleoprotein gene.

    Science.gov (United States)

    Makino, Akiko; Fujino, Kan; Parrish, Nicholas F; Honda, Tomoyuki; Tomonaga, Keizo

    2015-03-03

    Borna disease virus (BDV) has a non-segmented, negative-stranded RNA genome and causes persistent infection in many animal species. Previous study has shown that the activation of the IκB kinase (IKK)/NF-κB pathway is reduced by BDV infection even in cells expressing constitutively active mutant IKK. This result suggests that BDV directly interferes with the IKK/NF-κB pathway. To elucidate the mechanism for the inhibition of NF-κB activation by BDV infection, we evaluated the cross-talk between BDV infection and the NF-κB pathway. Using Multiple EM for Motif Elicitation analysis, we found that the nucleoproteins of BDV (BDV-N) and NF-κB1 share a common ankyrin-like motif. When THP1-CD14 cells were pre-treated with the identified peptide, NF-κB activation by Toll-like receptor ligands was suppressed. The 20S proteasome assay showed that BDV-N and BDV-N-derived peptide inhibited the processing of NF-κB1 p105 into p50. Furthermore, immunoprecipitation assays showed that BDV-N interacted with NF-κB1 but not with NF-κB2, which shares no common motif with BDV-N. These results suggest BDV-N inhibits NF-κB1 processing by the 20S proteasome through its ankyrin-like peptide sequence, resulting in the suppression of IKK/NF-κB pathway activation. This inhibitory effect of BDV on the induction of the host innate immunity might provide benefits against persistent BDV infection.

  6. Molecular insights into NF2/Merlin tumor suppressor function.

    Science.gov (United States)

    Cooper, Jonathan; Giancotti, Filippo G

    2014-08-19

    The FERM domain protein Merlin, encoded by the NF2 tumor suppressor gene, regulates cell proliferation in response to adhesive signaling. The growth inhibitory function of Merlin is induced by intercellular adhesion and inactivated by joint integrin/receptor tyrosine kinase signaling. Merlin contributes to the formation of cell junctions in polarized tissues, activates anti-mitogenic signaling at tight-junctions, and inhibits oncogenic gene expression. Thus, inactivation of Merlin causes uncontrolled mitogenic signaling and tumorigenesis. Merlin's predominant tumor suppressive functions are attributable to its control of oncogenic gene expression through regulation of Hippo signaling. Notably, Merlin translocates to the nucleus where it directly inhibits the CRL4(DCAF1) E3 ubiquitin ligase, thereby suppressing inhibition of the Lats kinases. A dichotomy in NF2 function has emerged whereby Merlin acts at the cell cortex to organize cell junctions and propagate anti-mitogenic signaling, whereas it inhibits oncogenic gene expression through the inhibition of CRL4(DCAF1) and activation of Hippo signaling. The biochemical events underlying Merlin's normal function and tumor suppressive activity will be discussed in this Review, with emphasis on recent discoveries that have greatly influenced our understanding of Merlin biology. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. Single-dose monomeric HA subunit vaccine generates full protection from influenza challenge

    CSIR Research Space (South Africa)

    Mallajosyula, JK

    2014-03-01

    Full Text Available Recombinant subunit vaccines are an efficient strategy to meet the demands of a possible influenza pandemic, because of rapid and scalable production. However, vaccines made from recombinant hemagglutinin (HA) subunit protein are often of low...

  8. Regulation of NF-kappaB activity and inducible nitric oxide synthase by regulatory particle non-ATPase subunit 13 (Rpn13)

    DEFF Research Database (Denmark)

    Mazumdar, Tuhina; Gorgun, F Murat; Sha, Youbao

    2010-01-01

    activity. The specific substrates for the Rpn13/UCH37 complex have not been determined. Because of a previous discovery of an interaction between Rpn13 and inducible nitric oxide synthase (iNOS), we hypothesized that iNOS is one of the substrates for the Rpn13/UCH37 complex. In this study, we show that Rpn...

  9. Membrane invagination induced by Shiga toxin B-subunit

    DEFF Research Database (Denmark)

    Pezeshkian, W.; Hansen, Allan Grønhøj; Johannes, Ludger

    2016-01-01

    The bacterial Shiga toxin is composed of an enzymatically active A-subunit, and a receptor-binding homopentameric B-subunit (STxB) that mediates intracellular toxin trafficking. Upon STxB-mediated binding to the glycolipid globotriaosylceramide (Gb3) at the plasma membrane of target cells, Shiga...... toxin is internalized by clathrin-dependent and independent endocytosis. The formation of tubular membrane invaginations is an essential step in the clathrin-independent STxB uptake process. However, the mechanism by which STxB induces these invaginations has remained unclear. Using a combination of all...... toxin molecules then creates a tubular membrane invagination that drives toxin entry into the cell. This mechanism requires: (1) a precise molecular architecture of the STxB binding sites; (2) a fluid bilayer in order for the tubular invagination to form. Although, STxB binding to the membrane requires...

  10. Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

    Directory of Open Access Journals (Sweden)

    Cooper John A

    2000-07-01

    Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

  11. Testing experimental subunit furunculosis vaccines for rainbow trout

    DEFF Research Database (Denmark)

    Marana, Moonika H.; Chettri, Jiwan Kumar; Skov, Jakob

    2016-01-01

    Aeromonas salmonicida subsp. salmonicida (AS) is the etiological agent of typical furunculosis in salmonid fish. The disease causes bacterial septicemia and is a major fish health problem in salmonid aquaculture worldwide, inducing high morbidity and mortality. In this study we vaccinated rainbow...... trout with subunit vaccines containing protein antigens that were selected based on an in silico antigen discovery approach. Thus, the proteome of AS strain A449 was analyzed by an antigen discovery platform and its proteins consequently ranked by their predicted ability to evoke protective immune...... response against AS. Fourteen proteins were prepared in 3 different experimental subunit vaccine combinations and used to vaccinate rainbow trout by intraperitoneal (i.p.) injection. We tested the proteins for their ability to elicit antibody production and protection. Thus, fish were exposed to virulent...

  12. TNF-α expression, not iNOS expression, is correlated with NF-κB ...

    African Journals Online (AJOL)

    Previous studies have shown that pro-inflammatory cytokines were involved in the genesis and persistence of neuropathic pain. Nuclear factor kappa B (NF-κB) plays a crucial role in regulating proinflammatory cytokine gene expression. In this study, we examined the hypothesis that NF-κB would regulate the expression of ...

  13. Data of evolutionary structure change: 1AD0C-2V7NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1AD0C-2V7NF 1AD0 2V7N C F QTVLTQSPSSLSVSVGDRVTITCRASSSV---TYIHWYQ...QKPGLAPKSLIYATS-----NLASGVPSRFSGSGS--GTDYTFTISSLQPEDIATYYCQHWSSKPP---TFGQGTKVEVKR-TVAAPSVFIFPPSDEQLKSGTASVVCLLNNF...E ------EEEEEEEEEEEEE EEEEE-- EEE-- EEE EEEEEEEEEEEE - EEEEEEE --EEEEEE - ...bChain> 2V7NF TSGVH--TFPAV EEE-- E 3.7732269763946533 4.904972076416016

  14. Data of evolutionary structure change: 1AD0A-2V7NF [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1AD0A-2V7NF 1AD0 2V7N A F QTVLTQSPSSLSVSVGDRVTITCRASSSV---TYIHWYQ...QKPGLAPKSLIYATS-----NLASGVPSRFSGSGS--GTDYTFTISSLQPEDIATYYCQHWSSKPP---TFGQGTKVEVKR-TVAAPSVFIFPPSDEQLKSGTASVVCLLNNF...E ------EEEEEEEEEEEEE EEEEE-- EEE-- EEE EEEEEEEEEEEE - EEEEEEE --EEEEEE - ...hain> 2V7N F 2V7NF T.../rmsd> 2.2184600830078125 EVID> 1

  15. Macrophage-specific inhibition of NF-κB activation reduces foam-cell formation

    NARCIS (Netherlands)

    Ferreira, V.; Dijk, K.W. van; Groen, A.K.; Vos, R.M.; Kaa, J. van der; Gijbels, M.J.J.; Havekes, L.M.; Pannekoek, H.

    2007-01-01

    Accumulation of lipid-laden macrophages is a hallmark of atherosclerosis. The relevance of the key transcription factor nuclear factor κB (NF-κB) for macrophage-derived foam-cell formation has not been unequivocally resolved. Transgenic mice lines were generated in which NF-κB activation is

  16. NF-Y dependent epigenetic modifications discriminate between proliferating and postmitotic tissue

    DEFF Research Database (Denmark)

    Gurtner, Aymone; Fuschi, Paola; Magi, Fiorenza

    2008-01-01

    and differentiation. Compelling evidence suggests that the transcription factor NF-Y acts as a master regulator of cell cycle progression, activating the transcription of many cell cycle regulatory genes. However, the underlying molecular mechanisms are not yet completely understood. Here we show that NF-Y exerts its...

  17. NF135.C10: a new Plasmodium falciparum clone for controlled human malaria infections

    NARCIS (Netherlands)

    Teirlinck, A.C.; Roestenberg, M.; Vegte-Bolmer, M.G. van de; Scholzen, A.; Heinrichs, M.J.; Siebelink-Stoter, R.; Graumans, W.; Gemert, G.J.A. van; Teelen, K.A.E.M.; Vos, M.W.; Nganou Makamdop, C.K.; Borrmann, S.; Rozier, Y.P.; Erkens, M.A.; Luty, A.J.F.; Hermsen, C.C.; Sim, B.K.; Lieshout, L. van; Hoffman, S.L.; Visser, L.G.; Sauerwein, R.W.

    2013-01-01

    We established a new field clone of Plasmodium falciparum for use in controlled human malaria infections and vaccine studies to complement the current small portfolio of P. falciparum strains, primarily based on NF54. The Cambodian clone NF135.C10 consistently produced gametocytes and generated

  18. Screening for AMPA receptor auxiliary subunit specific modulators.

    Directory of Open Access Journals (Sweden)

    Caleigh M Azumaya

    Full Text Available AMPA receptors (AMPAR are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM, respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R-CNIH3 and GluA2(Q complexes and PAM activity in GluA2(Q-GSG1L complexes.

  19. Amyloid-related biomarkers and axonal damage proteins in parkinsonian syndromes

    DEFF Research Database (Denmark)

    Bech, Sara; Hjermind, Lena E; Salvesen, Lisette

    2012-01-01

    Clinical differentiation between parkinsonian syndromes (PS) remains a challenge despite well-established clinical diagnostic criteria. Specific diagnostic biomarkers have yet to be identified, though in recent years, studies have been published on the aid of certain brain related proteins (BRP......) in the diagnosing of PS. We investigated the levels of the light subunit of neurofilament triplet protein (NF-L), total tau and phosphorylated tau, amyloid-ß(1-42), and the soluble a- and ß-cleaved fragments of amyloid precursor proteins in a cohort of patients with various PS....

  20. Chloroflexus aurantiacus has 30S ribosomal subunits of the eubacterial type.

    Science.gov (United States)

    Henderson, E; Pierson, B K; Lake, J A

    1983-01-01

    Ribosomal subunits from Chloroflexus aurantiacus were isolated and examined by sucrose gradient sedimentation, gel electrophoresis, and electron microscopy. The 30S subunits had all the characteristic structural features of other eubacterial 30S subunits. The data support the proposal that the absence of the archaebacterial bill is a valid phylogenetic marker of the eubacterial lineage. Images PMID:6348030

  1. Chloroflexus aurantiacus has 30S ribosomal subunits of the eubacterial type.

    OpenAIRE

    Henderson, E; Pierson, B. K.; Lake, J A

    1983-01-01

    Ribosomal subunits from Chloroflexus aurantiacus were isolated and examined by sucrose gradient sedimentation, gel electrophoresis, and electron microscopy. The 30S subunits had all the characteristic structural features of other eubacterial 30S subunits. The data support the proposal that the absence of the archaebacterial bill is a valid phylogenetic marker of the eubacterial lineage.

  2. Restoration of the orbital aesthetic subunit in complex midface defects.

    Science.gov (United States)

    Chepeha, Douglas B; Wang, Steven J; Marentette, Lawrence J; Bradford, Carol R; Boyd, Charles M; Prince, Mark E; Teknos, Theodoros N

    2004-10-01

    Although various options exist for restoration of the orbital defect in complex craniofacial resections, the aesthetic appearance and functional result of the orbit are optimized when the bony orbital architecture, orbital volume, and facial contour are specifically addressed. The study describes an approach using free tissue transfer for restoration of the native orbital aesthetic subunit. Retrospective case series. Nineteen patients (male-to-female ratio, 14:5; mean age, 52 y [age range, 8-79 y]) in the study period between 1997 and 2001 had orbital defects that could be classified into one of the following categories: 1) orbital exenteration cavities only, 2) orbital exenteration cavities with resection of less than 30% of the bony orbital rim, or 3) radical orbital exenteration cavities with resection of overlying skin and bony malar eminence. Group 1 had reconstructions with fasciocutaneous forearm flaps; group 2, with osseocutaneous forearm flaps; and group 3, with osseocutaneous scapula flaps. Eighteen of 19 patients achieved a closed orbital reconstruction with restoration of the orbital aesthetic subunit. Among 16 patients with more than 4 months of follow-up, 10 patients had minimal or no resulting facial contour deformity and 8 patients engaged in social activities outside the home on a frequent basis. Five of the nine patients who were working before their surgery were able to return to work. Patients with complex midface defects involving the orbit can undergo free tissue transfer and have successful restoration of the native orbital aesthetic subunit without an orbital prosthesis.

  3. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation.

    Science.gov (United States)

    Ferreira, R M; Franco, E; Teixeira, A R

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a +5 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photo-synthetic tissues.

  4. Specific Mutations in Mammalian P4-ATPase ATP8A2 Catalytic Subunit Entail Differential Glycosylation of the Accessory CDC50A Subunit

    DEFF Research Database (Denmark)

    Vestergaard, Anna L.; Mikkelsen, Stine A.; Coleman, Jonathan A.

    2015-01-01

    P4-ATPases, or flippases, translocate phospholipids between the two leaflets of eukaryotic biological membranes. They are essential to the physiologically crucial phospholipid asymmetry and involved in severe diseases, but their molecular structure and mechanism are still unresolved. Here, we sho...... that in an extensive mutational alanine screening of the mammalian flippase ATP8A2 catalytic subunit, five mutations stand out by leading to reduced glycosylation of the accessory subunit CDC50A. These mutations may disturb the interaction between the subunits....

  5. DMPD: Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16982211 Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Wullaer...vg) (.html) (.csml) Show Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. PubmedID 16982211 Title Ubiqui

  6. Transcriptional Control of Synaptic Plasticity by Transcription Factor NF-κB

    Science.gov (United States)

    Engelmann, Christian; Haenold, Ronny

    2016-01-01

    Activation of nuclear factor kappa B (NF-κB) transcription factors is required for the induction of synaptic plasticity and memory formation. All components of this signaling pathway are localized at synapses, and transcriptionally active NF-κB dimers move to the nucleus to translate synaptic signals into altered gene expression. Neuron-specific inhibition results in altered connectivity of excitatory and inhibitory synapses and functionally in selective learning deficits. Recent research on transgenic mice with impaired or hyperactivated NF-κB gave important insights into plasticity-related target gene expression that is regulated by NF-κB. In this minireview, we update the available data on the role of this transcription factor for learning and memory formation and comment on cross-sectional activation of NF-κB in the aged and diseased brain that may directly or indirectly affect κB-dependent transcription of synaptic genes. PMID:26881128

  7. Inhibition of NLRP3 Inflammasome Prevents LPS-Induced Inflammatory Hyperalgesia in Mice: Contribution of NF-κB, Caspase-1/11, ASC, NOX, and NOS Isoforms.

    Science.gov (United States)

    Dolunay, Abdurrahman; Senol, Sefika Pinar; Temiz-Resitoglu, Meryem; Guden, Demet Sinem; Sari, Ayse Nihal; Sahan-Firat, Seyhan; Tunctan, Bahar

    2017-04-01

    The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3), an intracellular signaling molecule that senses many environmental- and pathogen/host-derived factors, has been implicated in the pathogenesis of several diseases associated with inflammation. It has been suggested that NLRP3 inflammasome inhibitors may have a therapeutic potential in the treatment of NLRP3-related inflammatory diseases. The aim of this study was to determine whether inhibition of NLRP3 inflammasome prevents inflammatory hyperalgesia induced by lipopolysaccharide (LPS) in mice as well as changes in expression/activity of nuclear factor κB (NF-κB), caspase-1/11, nicotinamide adenine dinucleotide phosphate oxidase (NOX), and endothelial/neuronal/inducible nitric oxide synthase (eNOS/nNOS/iNOS) that may regulate NLRP3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/pro-caspase-1 inflammasome formation and activity by using a selective NLRP3 inflammasome inhibitor, MCC950. Male mice received saline (10 ml/kg; i.p.), LPS (10 mg/kg; i.p.), and/or MCC950 (3 mg/kg; i.p.). Reaction time to thermal stimuli within 1 min was evaluated after 6 h. The mice were killed and the brains, hearts, and lungs were collected for measurement of NF-κB, caspase-1, caspase-11, NLRP3, ASC, NOX subunits (gp91 phox ; NOX2), and p47 phox ; NOXO2), nitrotyrosine, eNOS, nNOS, iNOS, and β-actin protein expression, NOS activity, and interleukin (IL)-1β levels. LPS-induced hyperalgesia was associated with a decrease in eNOS, nNOS, and iNOS protein expression and activity as well as an increase in expression of NF-κB p65, caspase-1 p20, caspase-11 p20, NLRP3, ASC, gp91 phox , p47 phox , and nitrotyrosine proteins in addition to elevated IL-1β levels. The LPS-induced changes were prevented by MCC950. The results suggest that inhibition of NLRP3/ASC/pro-caspase-1 inflammasome formation and activity prevents inflammatory hyperalgesia induced by LPS in mice as well as

  8. Anti-neuroinflammatory effects of grossamide from hemp seed via suppression of TLR-4-mediated NF-κB signaling pathways in lipopolysaccharide-stimulated BV2 microglia cells.

    Science.gov (United States)

    Luo, Qian; Yan, Xiaoli; Bobrovskaya, Larisa; Ji, Mei; Yuan, Huiqing; Lou, Hongxiang; Fan, Peihong

    2017-04-01

    Grossamide, a representative lignanamide in hemp seed, has been reported to possess potential anti-inflammatory effects. However, the potential anti-neuroinflammatory effects and underlying mechanisms of action of grossamide are still unclear. Therefore, the present study investigated the possible effects and underlying mechanisms of grossamide against lipopolysaccharide (LPS)-induced inflammatory response in BV2 microglia cells. BV2 microglia cells were pre-treated with various concentrations of grossamide before being stimulated with LPS to induce inflammation. The levels of pro-inflammatory cytokines were determined using the enzyme-linked immunoassay (ELISA) and mRNA expression levels were measured by real-time PCR. The translocation of nuclear factor-kappa B (NF-κB) and contribution of TLR4-mediated NF-κB activation on inflammatory effects were evaluated by immunostaining and Western blot analysis. This study demonstrated that grossamide significantly inhibited the secretion of pro-inflammatory mediators such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), and decreased the level of LPS-mediated IL-6 and TNF-α mRNA. In addition, it significantly reduced the phosphorylation levels of NF-κB subunit p65 in a concentration-dependent manner and suppressed translocation of NF-κB p65 into the nucleus. Furthermore, grossamide markedly attenuated the LPS-induced expression of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). Taken together, these data suggest that grossamide could be a potential therapeutic candidate for inhibiting neuroinflammation in neurodegenerative diseases.

  9. DMPD: NF-kappaB activation by reactive oxygen species: fifteen years later. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16723122 NF-kappaB activation by reactive oxygen species: fifteen years later. Gloi...svg) (.html) (.csml) Show NF-kappaB activation by reactive oxygen species: fifteen years later. PubmedID 167...23122 Title NF-kappaB activation by reactive oxygen species: fifteen years later.

  10. On the multiple roles of the voltage gated sodium channel β1 subunit in genetic diseases

    Directory of Open Access Journals (Sweden)

    Debora eBaroni

    2015-05-01

    Full Text Available Voltage-gated sodium channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are composed of a pore-forming α-subunit and associated β-subunits. The β1-subunit was the first accessory subunit to be cloned. It can be important for controlling cell excitability and modulating multiple aspects of sodium channel physiology. Mutations of β1 are implicated in a wide variety of inherited pathologies, including epilepsy and cardiac conduction diseases. This review summarizes β1-subunit related channelopathies pointing out the current knowledge concerning their genetic background and their underlying molecular mechanisms.

  11. Roberge-Weiss endpoint in Nf=2 QCD

    Science.gov (United States)

    Bonati, Claudio; Cossu, Guido; D'Elia, Massimo; Sanfilippo, Francesco

    2011-03-01

    We present the results of extensive simulations regarding the critical behavior at the endpoint of the Roberge-Weiss transition for Nf=2 QCD. We confirm early evidence, presented in Ref. [M. D’Elia and F. Sanfilippo, Phys. Rev. DPRVDAQ1550-799810.1103/PhysRevD.80.111501 80, 111501(R) (2009).], according to which the Roberge-Weiss endpoint is first order in the limit of large or small quark masses, and second order for intermediate masses. A systematic study of the transition strength as a function of the quark mass in the first order regions, permits us to estimate the tricritical values of the quark mass separating the second order region from the first order ones.

  12. Proteomic Analyses of NF1-Interacting Proteins in Keratinocytes

    Science.gov (United States)

    2015-04-01

    TBP W T Im iq ui m od tr ea te d #1 Im iq ui m od...expression relative to TBP W T Im iq ui m od tre at ed #1 Im iq ui m od tre at ed #2 0.0 0.5 1.0 1.5 re la tiv e va lu e W T Sn ail T g 0.0 0.5 1.0 1.5 N...F1 r el at iv e to a ct in Figure 3. Expression of corneodesmin (CDSN) in Snail transgenic skin ! P7 skin, CDSN relative to TBP W T Sn ail T g

  13. Neuronal RING finger protein 11 (RNF11 regulates canonical NF-κB signaling

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    Pranski Elaine L

    2012-04-01

    Full Text Available Abstract Background The RING domain-containing protein RING finger protein 11 (RNF11 is a member of the A20 ubiquitin-editing protein complex and modulates peripheral NF-κB signaling. RNF11 is robustly expressed in neurons and colocalizes with a population of α-synuclein-positive Lewy bodies and neurites in Parkinson disease patients. The NF-κB pathway has an important role in the vertebrate nervous system, where the absence of NF-κB activity during development can result in learning and memory deficits, whereas chronic NF-κB activation is associated with persistent neuroinflammation. We examined the functional role of RNF11 with respect to canonical NF-κB signaling in neurons to gain understanding of the tight association of inflammatory pathways, including NF-κB, with the pathogenesis of neurodegenerative diseases. Methods and results Luciferase assays were employed to assess NF-κB activity under targeted short hairpin RNA (shRNA knockdown of RNF11 in human neuroblastoma cells and murine primary neurons, which suggested that RNF11 acts as a negative regulator of canonical neuronal NF-κB signaling. These results were further supported by analyses of p65 translocation to the nucleus following depletion of RNF11. Coimmunoprecipitation experiments indicated that RNF11 associates with members of the A20 ubiquitin-editing protein complex in neurons. Site-directed mutagenesis of the myristoylation domain, which is necessary for endosomal targeting of RNF11, altered the impact of RNF11 on NF-κB signaling and abrogated RNF11’s association with the A20 ubiquitin-editing protein complex. A partial effect on canonical NF-κB signaling and an association with the A20 ubiquitin-editing protein complex was observed with mutagenesis of the PPxY motif, a proline-rich region involved in Nedd4-like protein interactions. Last, shRNA-mediated reduction of RNF11 in neurons and neuronal cell lines elevated levels of monocyte chemoattractant protein 1 and

  14. Allotopic Expression of a Gene Encoding FLAG Tagged-subunit 8 of Yeast Mitochondrial ATP Synthase

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    I MADE ARTIKA

    2006-03-01

    Full Text Available Subunit 8 of yeast mitochondrial ATP synthase is a polypeptide of 48 amino acids encoded by the mitochondrial ATP8 gene. A nuclear version of subunit 8 gene has been designed to encode FLAG tagged-subunit 8 fused with a mitochondrial signal peptide. The gene has been cloned into a yeast expression vector and then expressed in a yeast strain lacking endogenous subunit 8. Results showed that the gene was successfully expressed and the synthesized FLAG tagged-subunit 8 protein was imported into mitochondria. Following import, the FLAG tagged-subunit 8 protein assembled into functional mitochondrial ATP synthase complex. Furthermore, the subunit 8 protein could be detected using anti-FLAG tag monoclonal antibody.

  15. Modulation of BK Channel Function by Auxiliary Beta and Gamma Subunits

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    Li, Q.; Yan, J.

    2016-01-01

    The large-conductance, Ca2+- and voltage-activated K+ (BK) channel is ubiquitously expressed in mammalian tissues and displays diverse biophysical or pharmacological characteristics. This diversity is in part conferred by channel modulation with different regulatory auxiliary subunits. To date, two distinct classes of BK channel auxiliary subunits have been identified: β subunits and γ subunits. Modulation of BK channels by the four auxiliary β (β1–β4) subunits has been well established and intensively investigated over the past two decades. The auxiliary γ subunits, however, were identified only very recently, which adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. This chapter will review the current understanding of BK channel modulation by auxiliary β and γ subunits, especially the latest findings. PMID:27238261

  16. Raf Kinase Inhibitor Protein Interacts with NF-κB-Inducing Kinase and TAK1 and Inhibits NF-κB Activation

    Science.gov (United States)

    Yeung, Kam C.; Rose, David W.; Dhillon, Amardeep S.; Yaros, Diane; Gustafsson, Marcus; Chatterjee, Devasis; McFerran, Brian; Wyche, James; Kolch, Walter; Sedivy, John M.

    2001-01-01

    The Raf kinase inhibitor protein (RKIP) acts as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by Raf-1. RKIP inhibits the phosphorylation of MAP/extracellular signal-regulated kinase 1 (MEK1) by Raf-1 by disrupting the interaction between these two kinases. We show here that RKIP also antagonizes the signal transduction pathways that mediate the activation of the transcription factor nuclear factor kappa B (NF-κB) in response to stimulation with tumor necrosis factor alpha (TNF-α) or interleukin 1 beta. Modulation of RKIP expression levels affected NF-κB signaling independent of the MAPK pathway. Genetic epistasis analysis involving the ectopic expression of kinases acting in the NF-κB pathway indicated that RKIP acts upstream of the kinase complex that mediates the phosphorylation and inactivation of the inhibitor of NF-κB (IκB). In vitro kinase assays showed that RKIP antagonizes the activation of the IκB kinase (IKK) activity elicited by TNF-α. RKIP physically interacted with four kinases of the NF-κB activation pathway, NF-κB-inducing kinase, transforming growth factor beta-activated kinase 1, IKKα, and IKKβ. This mode of action bears striking similarities to the interactions of RKIP with Raf-1 and MEK1 in the MAPK pathway. Emerging data from diverse organisms suggest that RKIP and RKIP-related proteins represent a new and evolutionarily highly conserved family of protein kinase regulators. Since the MAPK and NF-κB pathways have physiologically distinct roles, the function of RKIP may be, in part, to coordinate the regulation of these pathways. PMID:11585904

  17. 1,8-Cineol inhibits nuclear translocation of NF-κB p65 and NF-κB-dependent transcriptional activity.

    Science.gov (United States)

    Greiner, Johannes F-W; Müller, Janine; Zeuner, Marie-Theres; Hauser, Stefan; Seidel, Thorsten; Klenke, Christin; Grunwald, Lena-Marie; Schomann, Timo; Widera, Darius; Sudhoff, Holger; Kaltschmidt, Barbara; Kaltschmidt, Christian

    2013-12-01

    Natural plant-derived products are commonly applied to treat a broad range of human diseases, including cancer as well as chronic and acute airway inflammation. In this regard, the monoterpene oxide 1,8-cineol, the active ingredient of the clinically approved drug Soledum®, is well-established for the therapy of airway diseases, such as chronic sinusitis and bronchitis, chronic obstructive pulmonary disease and bronchial asthma. Although clinical trials underline the beneficial effects of 1,8-cineol in treating inflammatory diseases, the molecular mode of action still remains unclear. Here, we demonstrate for the first time a 1,8-cineol-depending reduction of NF-κB-activity in human cell lines U373 and HeLa upon stimulation using lipopolysaccharides (LPS). Immunocytochemistry further revealed a reduced nuclear translocation of NF-κB p65, while qPCR and western blot analyses showed strongly attenuated expression of NF-κB target genes. Treatment with 1,8-cineol further led to increased protein levels of IκBα in an IKK-independent matter, while FRET-analyses showed restoring of LPS-associated loss of interaction between NF-κB p65 and IκBα. We likewise observed reduced amounts of phosphorylated c-Jun N-terminal kinase 1/2 protein in U373 cells after exposure to 1,8-cineol. In addition, 1,8-cineol led to decreased amount of nuclear NF-κB p65 and reduction of its target gene IκBα at protein level in human peripheral blood mononuclear cells. Our findings suggest a novel mode of action of 1,8-cineol through inhibition of nuclear NF-κB p65 translocation via IκBα resulting in decreased levels of proinflammatory NF-κB target genes and may therefore broaden the field of clinical application of this natural drug for treating inflammatory diseases. © 2013.

  18. Suppression of NF-κB and NF-κB-Regulated Gene Expression by Apigenin through IκBα and IKK Pathway in TRAMP Mice.

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    Sanjeev Shukla

    Full Text Available Aberrant Nuclear Factor-κappaB (NF-κB activation due to rapid IκBα turnover and high basal IκBα kinase (IKK activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2, anti-apoptosis (Bcl-2 and Bcl-xL, and angiogenesis (vascular endothelial growth factor were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it's phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.

  19. Genotype-phenotype associations in neurofibromatosis type 1 (NF1: an increased risk of tumor complications in patients with NF1 splice-site mutations?

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    Alkindy Adila

    2012-08-01

    Full Text Available Abstract Neurofibromatosis type 1 (NF1 is a complex neurocutaneous disorder with an increased susceptibility to develop both benign and malignant tumors but with a wide spectrum of inter and intrafamilial clinical variability. The establishment of genotype-phenotype associations in NF1 is potentially useful for targeted therapeutic intervention but has generally been unsuccessful, apart from small subsets of molecularly defined patients. The objective of this study was to evaluate the clinical phenotype associated with the specific types of NF1 mutation in a retrospectively recorded clinical dataset comprising 149 NF1 mutation-known individuals from unrelated families. Each patient was assessed for ten NF1-related clinical features, including the number of café-au-lait spots, cutaneous and subcutaneous neurofibromas and the presence/absence of intertriginous skin freckling, Lisch nodules, plexiform and spinal neurofibromas, optic gliomas, other neoplasms (in particular CNS gliomas, malignant peripheral nerve sheath tumors (MPNSTs, juvenile myelomonocytic leukemia, rhabdomyosarcoma, phaechromocytoma, gastrointestinal stromal tumors, juvenile xanthogranuloma, and lipoma and evidence of learning difficulties. Gender and age at examination were also recorded. Patients were subcategorized according to their associated NF1 germ line mutations: frame shift deletions (52, splice-site mutations (23, nonsense mutations (36, missense mutations (32 and other types of mutation (6. A significant association was apparent between possession of a splice-site mutation and the presence of brain gliomas and MPNSTs (p = 0.006. If confirmed, these findings are likely to be clinically important since up to a third of NF1 patients harbor splice-site mutations. A significant influence of gender was also observed on the number of subcutaneous neurofibromas (females, p = 0.009 and preschool learning difficulties (females, p = 0.022.

  20. CD40-mediated Activation of the NF-B2 Pathway

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    Gail Abendroth Bishop

    2013-11-01

    Full Text Available CD40 is a critical stimulatory receptor on antigen-presenting cells of the immune system. CD40-mediated activation of B cells is particularly important for normal humoral immune function. Engagement of CD40 by its ligand, CD154, on activated T cell membranes initiates a variety of signals in B cells including activation of MAP kinases and NF-B. The transcriptional regulator NF-B is in reality a family of factors that can promote B cell activation, differentiation, and proliferation. Complex and only partially understood biochemical mechanisms allow CD40 to trigger two distinct NF-B activation pathways, canonical (NF-B1 and non-canonical (NF-B2 NF-B. This brief review provides a summary of mechanisms responsible for activation of the latter, which appears to be particularly important for enhancing B cell viability at various stages in their life cycle and may also contribute to development of B cell malignancies. CD40 is also expressed by various cell types in addition to B cells, including T cells, macrophages, dendritic cells, and certain non-hematopoietic cells. Here too, while perhaps less extensively studied than in B cells, the CD40-mediated activation of NF-B2 appears to have important roles in cellular physiology.

  1. Marine natural products as targeted modulators of the transcription factor NF-kappaB.

    Science.gov (United States)

    Folmer, Florence; Jaspars, Marcel; Dicato, Mario; Diederich, Marc

    2008-02-01

    The inducible transcription factor nuclear factor-kappaB (NF-kappaB) plays an important role in the regulation of immune, inflammatory and carcinogenic responses. While normal NF-kappaB activation is necessary for cell survival and immunity, deregulated NF-kappaB expression is a characteristic phenomenon in cancer development, as well as in several inflammatory diseases. Hence, NF-kappaB has become a major target in drug discovery, and several natural and synthetic compounds have been investigated for their potential to inhibit NF-kappaB. Here, we discuss the applications of marine natural products, in particular, as novel, potent NF-kappaB inhibitors. With the oceans covering two-thirds of the Earth's surface, and with the uniqueness of the environmental properties of marine habitats, it is easily understandable that organisms thriving in the oceans constitute a rich source of chemically unique and biomedically powerful secondary metabolites. Since the early 1960s, significant effort has been placed on the pharmacological evaluation of marine secondary metabolites. Noteworthy achievements of this field of biomedically guided marine exploration, a scientific endeavour often referred to as the search for "Drugs from the Sea", include the discovery of numerous potent anti-cancer, anti-inflammatory, antimicrobial, and analgesic compounds. The chemical characteristics and molecular targets of marine NF-kappaB inhibitors discovered to date are presented and discussed in the context of marine chemical ecology.

  2. Inhibition of SIRT2 in merlin/NF2-mutant Schwann cells triggers necrosis.

    Science.gov (United States)

    Petrilli, Alejandra; Bott, Marga; Fernández-Valle, Cristina

    2013-12-01

    Mutations in the NF2 gene cause Neurofibromatosis Type 2 (NF2), a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system. Merlin, a tumor suppressor encoded by the NF2 gene, modulates activity of many essential signaling pathways. Yet despite increasing knowledge of merlin function, there are no NF2 drug therapies. In a pilot high-throughput screen of the Library of Pharmacologically Active Compounds, we assayed for compounds capable of reducing viability of mouse Schwann cells (MSC) with Nf2 inactivation as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2 is one of seven mammalian sirtuins that are NAD+-dependent protein deacetylases. We show that merlin-mutant MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. Inhibition of SIRT2 activity in merlin-mutant MSC is accompanied by release of lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.

  3. Acidosis promotes invasiveness of breast cancer cells through ROS-AKT-NF-κB pathway.

    Science.gov (United States)

    Gupta, Subash C; Singh, Ramesh; Pochampally, Radhika; Watabe, Kounosuke; Mo, Yin-Yuan

    2014-12-15

    It is well known that acidic microenvironment promotes tumorigenesis, however, the underlying mechanism remains largely unknown. In the present study, we show that acidosis promotes invasiveness of breast cancer cells through a series of signaling events. First, our study indicates that NF-κB is a key factor for acidosis-induced cell invasion. Acidosis activates NF-κB without affecting STAT3 activity; knockdown of NF-κB p65 abrogates the acidosis-induced invasion activity. Next, we show that the activation of NF-κB is mediated through phosphorylation and degradation of IκBα; and phosphorylation and nuclear translocation of p65. Upstream to NF-κB signaling, AKT is activated under acidic conditions. Moreover, acidosis induces generation of reactive oxygen species (ROS) which can be suppressed by ROS scavengers, reversing the acidosis-induced activation of AKT and NF-κB, and invasiveness. As a negative regulator of AKT, PTEN is oxidized and inactivated by the acidosis-induced ROS. Finally, inhibition of NADPH oxidase (NOX) suppresses acidosis-induced ROS production, suggesting involvement of NOX in acidosis-induced signaling cascade. Of considerable interest, acidosis-induced ROS production and activation of AKT and NF-κB can be only detected in cancer cells, but not in non-malignant cells. Together, these results demonstrate a cancer specific acidosis-induced signaling cascade in breast cancer cells, leading to cell invasion.

  4. Cinnamaldehyde and 2-methoxycinnamaldehyde as NF-kappaB inhibitors from Cinnamomum cassia.

    Science.gov (United States)

    Reddy, Alavala Matta; Seo, Jee Hee; Ryu, Shi Yong; Kim, Yeong Shik; Kim, Young Sup; Min, Kyung Rak; Kim, Youngsoo

    2004-09-01

    Nuclear factor (NF)-cB is a transcription factor regulating the expression of inflammatory and immune genes. In the present study, an extract from stem bark of Cinnamomum cassia Blume(Lauraceae) was discovered to have an inhibitory effect on LPS-induced NF-KB transcriptional activity, which was determined using macrophages RAW 264.7 transfected stably with an alkaline phosphatase reporter construct containing four copies of the NF-KB binding KB sequence. Following activity-guided fractionation, trans-cinnamaldehyde and 2-methoxycinnamaldehyde were identified as the NF-KB inhibitors from C cassia with IC50 values of 43 MM and 31 pM, respectively. As a positive control, caffeic acid phenethyl ester (CAPE) showed an IC50 value of 2 uM on NF-KB transcriptional activity. Both trans-cinnamaldehyde and 2-methoxycinnamaldehyde inhibited LPS-induced DNA binding activity of NF-KB in addition to NF-KB transcription-al activity.

  5. Modulation of the NF-kappaB pathway by Bordetella pertussis filamentous hemagglutinin.

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    Tzvia Abramson

    Full Text Available Filamentous hemagglutinin (FHA is a cell-associated and secreted adhesin produced by Bordetella pertussis with pro-apoptotic and pro-inflammatory activity in host cells. Given the importance of the NF-kappaB transcription factor family in these host cell responses, we examined the effect of FHA on NF-kappaB activation in macrophages and bronchial epithelial cells, both of which are relevant cell types during natural infection.Exposure to FHA of primary human monocytes and transformed U-937 macrophages, but not BEAS-2B epithelial cells, resulted in early activation of the NF-kappaB pathway, as manifested by the degradation of cytosolic IkappaB alpha, by NF-kappaB DNA binding, and by the subsequent secretion of NF-kappaB-regulated inflammatory cytokines. However, exposure of macrophages and human monocytes to FHA for two hours or more resulted in the accumulation of cytosolic IkappaB alpha, and the failure of TNF-alpha to activate NF-kappaB. Proteasome activity was attenuated following exposure of cells to FHA for 2 hours, as was the nuclear translocation of RelA in BEAS-2B cells.These results reveal a complex temporal dynamic, and suggest that despite short term effects to the contrary, longer exposures of host cells to this secreted adhesin may block NF-kappaB activation, and perhaps lead to a compromised immune response to this bacterial pathogen.

  6. Signaling from p53 to NF-κB Determines the Chemotherapy Responsiveness of Neuroblastoma

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    Michael B. Armstrong

    2006-11-01

    Full Text Available Neuroblastic (N type neuroblastoma (NB is the predominant cell type in NB tumors. Previously, we determined that activated nuclear factor κB (NF-κB is required for doxorubicin and etoposide to kill N-type NB cells. This study was undertaken to determine how NF-κB is activated by these agents. The results show that p53 protein levels increase within 15 to 30 minutes of treatment. This increase occurs before the degradation of inhibitor of NF-κB (I-κB a and the NF-κB-dependent activation of gene transcription. Moreover, p53 is necessary for NF-κB activation because cells with inactive p53 were resistant to NF-κB-mediated cell death. This pathway was further defined to show that p53 leads to the activation of MAPK/ERK activity kinase (MEK 1 through a process that depends on protein synthesis and H-Ras. MEK1, in turn, mediates I-κB kinase activation. Together, these results demonstrate for the first time how NF-κB is activated in NB cells in response to conventional drugs. Furthermore, these findings provide an explanation as to why H-Ras expression correlates with a favorable prognosis in NB and identify intermediary signaling molecules that are targets for discovering treatments for NB that is resistant to conventional agents.

  7. Signaling from p53 to NF-κB Determines the Chemotherapy Responsiveness of Neuroblastoma1

    Science.gov (United States)

    Armstrong, Michael B; Bian, Xin; Liu, Yihong; Subramanian, Chitra; Ratanaproeksa, Anthony B; Shao, Feng; Yu, Victor C; Kwok, Roland P S; Opipari, Anthony W; Castle, Valerie P

    2006-01-01

    Abstract Neuroblastic (N) type neuroblastoma (NB) is the predominant cell type in NB tumors. Previously, we determined that activated nuclear factor κB (NF-κB) is required for doxorubicin and etoposide to kill N-type NB cells. This study was undertaken to determine how NF-κB is activated by these agents. The results show that p53 protein levels increase within 15 to 30 minutes of treatment. This increase occurs before the degradation of inhibitor of NF-κB (I-KB) α and the NF-κB-dependent activation of gene transcription. Moreover, p53 is necessary for NF-κB activation because cells with inactive p53 were resistant to NF-κB-mediated cell death. This pathway was further defined to show that p53 leads to the activation of MAPK/ERK activity kinase (MEK) 1 through a process that depends on protein synthesis and H-Ras. MEK1, in turn, mediates I-κB kinase activation. Together, these results demonstrate for the first time how NF-κB is activated in NB cells in response to conventional drugs. Furthermore, these findings provide an explanation as to why H-Ras expression correlates with a favorable prognosis in NB and identify intermediary signaling molecules that are targets for discovering treatments for NB that is resistant to conventional agents. PMID:17215959

  8. Role of the proteasome and NF-κB in streptococcal cell wall-induced polyarthritis

    Science.gov (United States)

    Palombella, Vito J.; Conner, Elaine M.; Fuseler, John W.; Destree, Antonia; Davis, Jonathan M.; Laroux, F. Stephen; Wolf, Robert E.; Huang, Jianqing; Brand, Stephen; Elliott, Peter J.; Lazarus, Douglas; McCormack, Teresa; Parent, Lana; Stein, Ross; Adams, Julian; Grisham, Matthew B.

    1998-01-01

    The transcription factor NF-κB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-κB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IκB is degraded by the ubiquitin–proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-κB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin–proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-κB and the subsequent transcription of genes that are regulated by NF-κB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IκBα degradation and NF-κB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin–proteasome pathway and NF-κB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect. PMID:9861028

  9. Human mediator subunit MED15 promotes transcriptional activation.

    Science.gov (United States)

    Nakatsubo, Takuya; Nishitani, Saori; Kikuchi, Yuko; Iida, Satoshi; Yamada, Kana; Tanaka, Aki; Ohkuma, Yoshiaki

    2014-10-01

    In eukaryotes, the Mediator complex is an essential transcriptional cofactor of RNA polymerase II (Pol II). In humans, it contains up to 30 subunits and consists of four modules: head, middle, tail, and CDK/Cyclin. One of the subunits, MED15, is located in the tail module, and was initially identified as Gal11 in budding yeast, where it plays an essential role in the transcriptional regulation of galactose metabolism with the potent transcriptional activator Gal4. For this reason, we investigated the function of the human MED15 subunit (hMED15) in transcriptional activation. First, we measured the effect of hMED15 knockdown on cell growth in HeLa cells. The growth rate was greatly reduced. By immunostaining, we observed the colocalization of hMED15 with the general transcription factors TFIIE and TFIIH in the nucleus. We measured the effects of siRNA-mediated knockdown of hMED15 on transcriptional activation using two different transcriptional activators, VP16 and SREBP1a. Treatment with siRNAs reduced transcriptional activation, and this reduction could be rescued by overexpression of HA/Flag-tagged, wild-type hMED15. To investigate hMED15 localization, we treated human MCF-7 cells with the MDM2 inhibitor Nutlin-3, thus inducing p21 transcription. We found that hMED15 localized to both the p53 binding site and the p21 promoter region, along with TFIIE and TFIIH. These results indicate that hMED15 promotes transcriptional activation.

  10. Characterisation of the tryptophan synthase alpha subunit in maize

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    Gierl Alfons

    2008-04-01

    Full Text Available Abstract Background In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA and β (TSB homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS complex in Arabidopsis. On the other hand maize (Zea mays expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. Results In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP, and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native α-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. Conclusion It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as α-subunit in this complex.

  11. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation

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    Ferreira, R.M.B. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior de Agronomia]|[Universidade Nova de Lisboa, Oeiras (Portugal). Instituto de Tecnologia Quimica e Biologica; Franco, E.; Teixeira, A.R.N. [Universidade Tecnica, Lisbon (Portugal). Inst. Superior de Agronomia

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a 65 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of {sup 35}S-labelled ribulose biphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose biphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose biphosphate carboxylase. For short periods of time (<1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose biphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photosynthetic tissues. (Author).

  12. Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly.

    Science.gov (United States)

    Hasek, Mary L; Maurer, Joshua B; Hendrix, Roger W; Duda, Robert L

    2017-08-04

    Viruses build icosahedral capsids of specific size and shape by regulating the spatial arrangement of the hexameric and pentameric protein capsomers in the growing shell during assembly. In the T=7 capsids of Escherichia coli bacteriophage HK97 and other phages, 60 capsomers are hexons, while the rest are pentons that are correctly positioned during assembly. Assembly of the HK97 capsid to the correct size and shape has been shown to depend on specific ionic contacts between capsomers. We now describe additional ionic interactions within capsomers that also regulate assembly. Each is between the long hairpin, the "E-loop," that extends from one subunit to the adjacent subunit within the same capsomer. Glutamate E153 on the E-loop and arginine R210 on the adjacent subunit's backbone alpha-helix form salt bridges in hexamers and pentamers. Mutations that disrupt these salt bridges were lethal for virus production, because the mutant proteins assembled into tubes or sheets instead of capsids. X-ray structures show that the E153-R210 links are flexible and maintained during maturation despite radical changes in capsomer shape. The E153-R210 links appear to form early in assembly to enable capsomers to make programmed changes in their shape during assembly. The links also prevent flattening of capsomers and premature maturation. Mutant phenotypes and modeling support an assembly model in which flexible E153-R210 links mediate capsomer shape changes that control where pentons are placed to create normal-sized capsids. The E-loop may be conserved in other systems in order to play similar roles in regulating assembly. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Airway epithelial NF-κB activation promotes Mycoplasma pneumoniae clearance in mice.

    Directory of Open Access Journals (Sweden)

    Di Jiang

    Full Text Available Respiratory infections including atypical bacteria Mycoplasma pneumoniae (Mp contribute to the pathobiology of asthma and chronic obstructive pulmonary disease (COPD. Mp infection mainly targets airway epithelium and activates various signaling pathways such as nuclear factor κB (NF-κB. We have shown that short palate, lung, and nasal epithelium clone 1 (SPLUNC1 serves as a novel host defense protein and is up-regulated upon Mp infection through NF-κB activation in cultured human and mouse primary airway epithelial cells. However, the in vivo role of airway epithelial NF-κB activation in host defense against Mp infection has not been investigated. In the current study, we investigated the effects of in vivo airway epithelial NF-κB activation on lung Mp clearance and its association with airway epithelial SPLUNC1 expression.Non-antimicrobial tetracycline analog 9-t-butyl doxycycline (9-TB was initially optimized in mouse primary tracheal epithelial cell culture, and then utilized to induce in vivo airway epithelial specific NF-κB activation in conditional NF-κB transgenic mice (CC10-(CAIKKβ with or without Mp infection. Lung Mp load and inflammation were evaluated, and airway epithelial SPLUNC1 protein was examined by immunohistochemistry. We found that 9-TB treatment in NF-κB transgene positive (Tg+, but not transgene negative (Tg- mice significantly reduced lung Mp load. Moreover, 9-TB increased airway epithelial SPLUNC1 protein expression in NF-κB Tg+ mice.By using the non-antimicrobial 9-TB, our study demonstrates that in vivo airway epithelial NF-κB activation promotes lung bacterial clearance, which is accompanied by increased epithelial SPLUNC1 expression.

  14. Activation of NF-kappa B signaling promotes growth of prostate cancer cells in bone.

    Directory of Open Access Journals (Sweden)

    Renjie Jin

    Full Text Available Patients with advanced prostate cancer almost invariably develop osseous metastasis. Although many studies indicate that the activation of NF-κB signaling appears to be correlated with advanced cancer and promotes tumor metastasis by influencing tumor cell migration and angiogenesis, the influence of altered NF-κB signaling in prostate cancer cells within boney metastatic lesions is not clearly understood. While C4-2B and PC3 prostate cancer cells grow well in the bone, LNCaP cells are difficult to grow in murine bone following intraskeletal injection. Our studies show that when compared to LNCaP, NF-κB activity is significantly higher in C4-2B and PC3, and that the activation of NF-κB signaling in prostate cancer cells resulted in the increased expression of the osteoclast inducing genes PTHrP and RANKL. Further, conditioned medium derived from NF-κB activated LNCaP cells induce osteoclast differentiation. In addition, inactivation of NF-κB signaling in prostate cancer cells inhibited tumor formation in the bone, both in the osteolytic PC3 and osteoblastic/osteoclastic mixed C4-2B cells; while the activation of NF-κB signaling in LNCaP cells promoted tumor establishment and proliferation in the bone. The activation of NF-κB in LNCaP cells resulted in the formation of an osteoblastic/osteoclastic mixed tumor with increased osteoclasts surrounding the new formed bone, similar to metastases commonly seen in patients with prostate cancer. These results indicate that osteoclastic reaction is required even in the osteoblastic cancer cells and the activation of NF-κB signaling in prostate cancer cells increases osteoclastogenesis by up-regulating osteoclastogenic genes, thereby contributing to bone metastatic formation.

  15. NF-kappaΒ-inducing kinase regulates stem cell phenotype in breast cancer

    OpenAIRE

    Karla Vazquez-Santillan; Jorge Melendez-Zajgla; Luis Enrique Jimenez-Hernandez; Javier Gaytan-Cervantes; Laura Muñoz-Galindo; Patricia Piña-Sanchez; Gustavo Martinez-Ruiz; Javier Torres; Patricia Garcia-Lopez; Carolina Gonzalez-Torres; Victor Ruiz; Federico Avila-Moreno; Marco Velasco-Velazquez; Mayra Perez-Tapia; Vilma Maldonado

    2016-01-01

    Breast cancer stem cells (BCSCs) overexpress components of the Nuclear factor-kappa B (NF-?B) signaling cascade and consequently display high NF-?B activity levels. Breast cancer cell lines with high proportion of CSCs exhibit high NF-?B-inducing kinase (NIK) expression. The role of NIK in the phenotype of cancer stem cell regulation is poorly understood. Expression of NIK was analyzed by quantitative RT-PCR in BCSCs. NIK levels were manipulated through transfection of specific shRNAs or an e...

  16. NF-kB activation and its downstream target genes expression after heavy ions exposure

    Science.gov (United States)

    Chishti, Arif Ali; Baumstark-Khan, Christa; Hellweg, Christine; Schmitz, Claudia; Koch, Kristina; Feles, Sebastian

    2016-07-01

    To enable long-term human space flight cellular radiation response to densely ionizing radiation needs to be better understood for developing appropriate countermeasures to mitigate acute effects and late radiation risks for the astronaut. The biological effectiveness of accelerated heavy ions (which constitute the most important radiation type in space) with high linear energy transfer (LET) for effecting DNA damage response pathways as a gateway to cell death or survival is of major concern not only for space missions but also for new regimes of tumor radiotherapy. In the current research study, the contribution of NF-κB in response to space-relevant radiation qualities was determined by a NF-κB reporter cell line (HEK-pNF-κB-d2EGFP/Neo L2). The NF-κB dependent reporter gene expression (d2EGFP) after ionizing radiation (X-rays and heavy ions) exposure was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after X-irradiation and heavy ions exposure, it was expected that radiation quality (LET) might play an important role in the cellular radiation response. In addition, the biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival was examined for heavy ions having a broad range of LET (˜0.3 - 9674 keV/µm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real time reverse transcriptase quantitative PCR (RT-qPCR). In this study it was proven that NF-κB activation and NF-κB dependent gene expression comprises an early step in cellular radiation response. Taken together, this study clearly demonstrates that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ˜50-200 keV/μupm. The up-regulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, IL-8 and TNF) might be important for cell-cell communication among hit as well as unhit cells (bystander effect). The results obtained suggest the NF-κB pathway to be a

  17. Familial spinal neurofibromatosis due to a multiexonic NF1 gene deletion.

    Science.gov (United States)

    Pizzuti, Antonio; Bottillo, Irene; Inzana, Francesca; Lanari, Valentina; Buttarelli, Francesca; Torrente, Isabella; Giallonardo, Anna Teresa; De Luca, Alessandro; Dallapiccola, Bruno

    2011-08-01

    We report the detailed clinical presentation and molecular features of a spinal neurofibromatosis familial case where a 40-year-old woman, presenting with multiple bilateral spinal neurofibromas and no other clinical feature of neurofibromatosis type 1 (NF1), inherited a paternal large multiexonic deletion (c.5944-?_7126+?del) which resulted in NF1 gene haploinsufficiency at the RNA level. In the clinically unaffected 73-year-old father, spinal cord MRI disclosed bilateral and symmetrical hypertrophy of spinal lumbosacral roots. Our study widens the phenotypic and mutational spectrum of NF1 and illustrates the difficulties of counseling patients with border-line or atypical presentation of this disorder.

  18. MicroRNA Roles in the NF-κB Signaling Pathway during Viral Infections

    Directory of Open Access Journals (Sweden)

    Zeqian Gao

    2014-01-01

    Full Text Available NF-κB signaling network is a crucial component of innate immunity. miRNAs are a subtype of small noncoding RNAs, involved in regulation of gene expression at the posttranscriptional level. Increasing evidence has emerged that miRNAs play an important role in regulation of NF-κB signaling pathway during viral infections. Both host and viral miRNAs are attributed to modulation of NF-κB activity, thus affecting viral infection and clearance. Understandings of the mechanisms of these miRNAs will open a direction for development of novel antivirus drugs.

  19. A role for NF-κB activity in skin hyperplasia and the development of keratoacanthomata in mice.

    Directory of Open Access Journals (Sweden)

    Brian Poligone

    Full Text Available BACKGROUND: Previous studies have implicated NF-κB signaling in both cutaneous development and oncogenesis. However, these studies have been limited in part by the lethality that results from extreme over- or under-expression of NF-κB in available mouse models. Even cre-driven tissue specific expression of transgenes, or targeted deletion of NF-κB can cause cell death. Therefore, the present study was undertaken to evaluate a novel mouse model of enhanced NF-κB activity in the skin. METHODS: A knock-in homologous recombination technique was utilized to develop a mouse model (referred to as PD mice with increased NF-κB activity. RESULTS: The data show that increased NF-κB activity leads to hyperproliferation and dysplasia of the mouse epidermis. Chemical carcinogenesis in the context of enhanced NF-κB activity promotes the development of keratoacanthomata. CONCLUSION: Our findings support an important role for NF-κB in keratinocyte dysplasia. We have found that enhanced NF-κB activity renders keratinocytes susceptible to hyperproliferation and keratoacanthoma (KA development but is not sufficient for transformation and SCC development. We therefore propose that NF-κB activation in the absence of additional oncogenic events can promote TNF-dependent, actinic keratosis-like dysplasia and TNF-independent, KAs upon chemical carcinogensis. These studies suggest that resolution of KA cannot occur when NF-κB activation is constitutively enforced.

  20. N-linked Glycans are Required on Epithelial Na+ Channel Subunits for Maturation and Surface Expression.

    Science.gov (United States)

    Kashlan, Ossama B; Kinlough, Carol L; Myerburg, Michael M; Shi, Shujie; Chen, Jingxin; Blobner, Brandon M; Buck, Teresa M; Brodsky, Jeffrey L; Hughey, Rebecca P; Kleyman, Thomas R

    2017-11-29

    Epithelial Na+ channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, thirteen and five consensus sites (Asn-x-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α, β and γ subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fisher rat thyroid cells with αβγENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na+ The lack of N-linked glycans on the β subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-link glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the β subunit had the largest effect on total levels, with the lack of N-linked glycans on the α and γ subunits having intermediate and modest effects, respectively. Finally, channels with wild type β subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression and function of the channel. Copyright © 2017, American Journal of Physiology-Renal Physiology.

  1. YC-1 BINDING TO THE BETA SUBUNIT OF SOLUBLE GUANYLYL CYCLASE OVERCOMES ALLOSTERIC INHIBITION BY THE ALPHA SUBUNIT

    Science.gov (United States)

    Purohit, Rahul; Fritz, Bradley G.; The, Juliana; Issaian, Aaron; Weichsel, Andrzej; David, Cynthia L.; Campbell, Eric; Hausrath, Andrew C.; Rassouli-Taylor, Leida; Garcin, Elsa D.; Gage, Matthew J.; Montfort, William R.

    2014-01-01

    Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remains unknown. Using linked-equilibria binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the beta subunit. In the absence of CO, YC-1 binds with Kd = 9–21 μM, depending on construct. In the presence of CO, these values decrease to 0.6–1.1 μM. Pfizer compound 25 bound ~10-fold weaker than YC-1 in the absence of CO whereas compound BAY 41–2272 bound particularly tightly in the presence of CO (Kd = 30–90 nM). Additionally, we found that CO binding is much weaker to heterodimeric sGC proteins (Kd = 50–100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca beta H-NOX/PAS). YC-1 greatly enhanced CO binding to heterodimeric sGC, as expected (Kd = ~1 μM). These data indicate the alpha subunit induces a heme pocket conformation with lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the alpha subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity. PMID:24328155

  2. Thermostable cross-protective subunit vaccine against Brucella species.

    Science.gov (United States)

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  3. Characterization of tryptophan synthase alpha subunit mutants of Arabidopsis thaliana.

    Science.gov (United States)

    Radwanski, E R; Barczak, A J; Last, R L

    1996-12-13

    Three mutations in the Arabidopsis thaliana gene encoding the alpha subunit of tryptophan synthase were isolated by selection for resistance to 5-methylanthranilate or 5-fluoroindole, toxic analogs of tryptophan pathway intermediates. Plants homozygous for trp3-1 and trp3-2 are light-conditional tryptophan auxotrophs, while trp3-100 is a more leaky mutant. Genetic complementation crosses demonstrated that the three mutations are allelic to each other, and define a new complementation group. All three mutants have decreased steady-state levels of tryptophan synthase alpha protein, and the trp3-100 polypeptide exhibits altered electrophoretic mobility. All three mutations were shown to be in the TSA1 (tryptophan synthase alpha subunit) structural gene by several criteria. Firstly, the trp3-1 mutation is linked to TSA1 on the bottom of chromosome 3. Secondly, the trp3-1 mutation was complemented when transformed with the wild-type TSA1 gene. Finally, DNA sequence analysis of the TSA1 gene revealed a single transition mutation in each trp3 mutant.

  4. Kinetic mechanism of luciferase subunit folding and assembly.

    Science.gov (United States)

    Clark, A C; Raso, S W; Sinclair, J F; Ziegler, M M; Chaffotte, A F; Baldwin, T O

    1997-02-18

    The kinetic mechanism in vitro of the folding and assembly of the heterodimeric flavin monooxygenase bacterial luciferase has been defined by a unique set of rate constants which describe both the productive refolding pathway and competing off-pathway reactions in 50 mM phosphate, pH 7.0 at 18 degrees C. The individual alpha and beta subunits fold independently to form heterodimerization-competent species, alpha i and beta i. The alpha i beta i species can interact to form an inactive heterodimeric intermediate, [alpha beta ]i, which isomerizes to form the active alpha beta structure; the structure of the enzyme has been determined to 1.5 A resolution [Fisher, A. J., Thompson, T. B., Thoden, J. B., Baldwin, T. O., & Rayment, I. (1996) J. Biol. Chem. 271, 21956-21968]. In the absence of alpha i, beta i can form a kinetically trapped homodimer, beta 2, with a second-order rate constant of about 180 M-1 s-1 [Sinclair, J. F., Ziegler, M. M., & Baldwin, T. O. (1994) Nat. Struct. Biol. 1, 320-326]; the structure of beta 2 has recently been reported [Thoden. J. B., Holden, H. M., Fisher, A. J., Sinclair. J. F., Wesenberg, G., Baldwin, T.O., & Rayment, I. (1997) Protein Sci. 6, 13-23]. The beta i species, or some other form that precedes beta i on the refolding pathway, can also undergo a first-order conversion into a form (designated beta x) that cannot associate with alpha i to form the native enzyme. The rate constant for this process, assigned here, accounts well for the previously observed dependence of final yield on concentration of refolding species [Ziegler, M.M., Goldberg, M.E., Chaffotte, A. F., & Baldwin, T. O. (1993) J. Biol. Chem. 268, 10760-10765]. In simulations of the refolding reaction, all processes associated with the refolding of the individual subunits were combined into single first-order rate constants for each subunit which were consistent with the rate constants determined from stopped-flow circular dichroism studies. The first-order rate constant

  5. Role of the beta subunit of casein kinase-2 on the stability and specificity of the recombinant reconstituted holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Marin, O

    1992-01-01

    Recombinant human alpha subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human beta subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the beta subunit could drastically reduce...... the loss of kinase activity, strongly suggesting a protective function for the beta subunit. Assaying different peptides for specificity toward the recombinant alpha subunit and the recombinant reconstituted enzyme, showed that the presence of the beta subunit could modify the specificity of the catalytic...... alpha subunit. Therefore, a dual function for the beta subunit is proposed which confers both specificity and stability to the catalytic alpha subunit within the CK-2 holoenzyme complex. The peptide DLEPDEELEDNPNQSDL, reproducing the highly acidic amino acid 55-71 segment of the human beta subunit...

  6. TOVS GLA 5 DAY GRIDS from NOAA-9 V01 (TOVSA5NF) at GES DISC

    Data.gov (United States)

    National Aeronautics and Space Administration — This dataset (TOVSA5NF) contains the TIROS Operational Vertical Sounder (TOVS) level 3 geophysical parameters derived using data from NOAA-9 and the physical...

  7. Health-Related Quality of Life for Pediatic NF1 Patients

    National Research Council Canada - National Science Library

    Bradlyn, Andrew

    2003-01-01

    ...) instrument for pediatric patients. Semi-structured interviews with children with NF1, their parents, health care professionals, and teachers will yield relevant domains and content that can be used to develop the first-generation instrument...

  8. Health-Related Quality of Life for Pediatric NF1 Patients

    National Research Council Canada - National Science Library

    Bradlyn, Andrew S; Harris, Carole V

    2006-01-01

    ...) instrument for use with pediatric patients. Semistructured interviews with children with NF1, their parents, and teachers were used to derive important domains and item content, and the first generation instrument was developed...

  9. Health-Related Quality of Life for Pediatric NF1 Patients

    National Research Council Canada - National Science Library

    Bradlyn, Andrew S

    2005-01-01

    ...) instrument for pediatric patients and their parents. Semi-structured interviews with children with NF1, their parents, and teachers yielded relevant domains and content that were used to develop the first-generation instrument...

  10. Health-Related Quality of Life for Pediatric NF1 Patients

    National Research Council Canada - National Science Library

    Bradlyn, Andrew S; Harris, Carole V

    2004-01-01

    ...) instrument for pediatric patients. Semi-structured interviews with children with NF-1, their parents, and teachers have yielded relevant domains and content that have been used to develop the first-generation instrument...

  11. Health-Related Quality of Life for Pediatric NF-1 Patients

    National Research Council Canada - National Science Library

    Bradlyn, Andrew S

    2007-01-01

    ...) instrument for use with pediatric patients. Semi. structured interviews with children with NF1, their parents, and teachers were used to derive important domains and item content, and the first generation instrument was developed...

  12. Modulation of Ras Signaling by NF1 and CRKL in Development

    National Research Council Canada - National Science Library

    Imamoto, Akira

    2003-01-01

    Mutations in the NF1 gene are the genetic basis of neurofibromatosis type I, a common genetic disorder which predisposes the patient to neoplasia in the peripheral nervous system as well as other tissues...

  13. Gastric schwannoma with adjacent external progression harbored aberrant NF2 gene.

    Science.gov (United States)

    Ogasawara, Naotaka; Sasaki, Makoto; Ishiguro, Hideyuki; Itoh, Yukimi; Nojiri, Syunsuke; Kubota, Eiji; Wada, Tsuneya; Kataoka, Hiromi; Kuwabara, Yoshiyuki; Joh, Takashi

    2009-07-01

    Gastric schwannomas are rare benign mesenchymal tumors. We describe a schwannoma of gastric origin with adjacent external progression. Sections showed a spindle cell tumor arranged in interlaced bundles and fascicles that was S-100 and CD34 positive but c-KIT protein negative. Histology and immunohistochemistry revealed the typical appearance of a gastric schwannoma. Genetic evaluation revealed that the tumor harbored a point mutation in exon 6 of the tumor suppressor neurofibromatosis 2 (NF2) gene, which resulted in an amino acid substitution of NF2 protein, and no mutation in exon 4b of the NF1 gene. In conclusion, we identified a rare mutation of the NF2 gene in gastric schwannoma. A diagnosis can only be definitive when based on histological and immunohistochemical findings. Digestive tract schwannomas are rare mesenchymal tumors that are differentiated from gastrointestinal stromal tumors by the absence of KIT protein. Follow up suggested that complete resection is an effective long-term treatment strategy.

  14. Advancing Venus Geophysics with the NF4 Venus Origins Explorer (VOX) Gravity Investigation

    Science.gov (United States)

    Mazarico, E.; Iess, L.; de Marchi, F.; Andrews-Hanna, J. C.; Smrekar, S. E.

    2017-11-01

    The proposed Venus Origins Explorer NF4 mission will obtain a high-resolution field to address Venus crust evolution, in particular the structure and origin of tesserae. We present comprehensive simulation results validating science requirements.

  15. Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls.

    Science.gov (United States)

    Baud, Véronique; Karin, Michael

    2009-01-01

    Nuclear factor kappaB (NF-kappaB) transcription factors have a key role in many physiological processes such as innate and adaptive immune responses, cell proliferation, cell death, and inflammation. It has become clear that aberrant regulation of NF-kappaB and the signalling pathways that control its activity are involved in cancer development and progression, as well as in resistance to chemotherapy and radiotherapy. This article discusses recent evidence from cancer genetics and cancer genome studies that support the involvement of NF-kappaB in human cancer, particularly in multiple myeloma. The therapeutic potential and benefit of targeting NF-kappaB in cancer, and the possible complications and pitfalls of such an approach, are explored.

  16. [Chromatographic and spectroscopic characterization of phycocyanin and its subunits purified from Anabaena variabilis CCC421].

    Science.gov (United States)

    Chakdar, N; Sakha, S; Pabbi, S

    2014-01-01

    Phycocyanin, a high value pigment was purified from diazotrophic cyanobacteria Anabaena variabilis CCC421 using a strategy involving ammonium sulfate precipitation, dialysis and anion exchange chromatography using DEAE-cellulose column. 36% phycocyanin with a purity of 2.75 was recovered finally after anion exchange chromatography. Purified phycocyanin was found to contain 2 subunits of 17 and 18 kDa which were identified as a-and (3 subunits by SDS-PAGE and MALDI-TOE HPLC method using a C5 column coupled with fluorescence or photodiode-based detection was also developed to separate and detect the A. variabilis CCC421 phycocyanin subunits. The fluorescence method was more sensitive than photodiode one. The purified phycocyanin from A. variabilis CCC421 as well as its subunits was characterized with respect to absorption and IR spectra. Spectral characterization of the subunits revealed that alpha and beta subunits contained one and two phycocyanobilin groups as chromophores, respectively.

  17. Bee venom ameliorates lipopolysaccharide-induced memory loss by preventing NF-kappaB pathway

    OpenAIRE

    Gu, Sun Mi; Park, Mi Hee; Hwang, Chul Ju; Song, Ho Sueb; Lee, Ung Soo; Han, Sang Bae; Oh, Ki Wan; Ham, Young Wan; Song, Min Jong; Son, Dong Ju; Hong, Jin Tae

    2015-01-01

    Background Accumulation of beta-amyloid and neuroinflammation trigger Alzheimer?s disease. We previously found that lipopolysaccharide (LPS) caused neuroinflammation with concomitant accumulation of beta-amyloid peptides leading to memory loss. A variety of anti-inflammatory compounds inhibiting nuclear factor kappaB (NF-?B) activation have showed efficacy to hinder neuroinflammation and amyloidogenesis. We also found that bee venom (BV) inhibits NF-?B. Methods A mouse model of LPS-induced me...

  18. Dichotomous actions of NF-kappaB signaling pathways in heart.

    Science.gov (United States)

    Dhingra, Rimpy; Shaw, James A; Aviv, Yaron; Kirshenbaum, Lorrie A

    2010-08-01

    Despite the substantial progress in heart research over the past two decades heart failure still remains a major cause of morbidity and mortality in North America and is reaching pandemic proportions worldwide. Though the underlying causes are varied, the functional loss of contractile myocytes through apoptosis, necrosis, and autophagy has emerged a central unifying theme to explain diminished cardiac performance in individuals with heart failure. At the molecular level, there has been considerable interest in understanding the signaling pathways that regulate cell death in the heart with specific interest in the extrinsic and intrinsic cell death pathways. The cellular factor nuclear factor-kappaB (NF-kappaB) is a key transcription factor involved in the regulation of a wide range of genes involved in cellular process including inflammation, immune cell maturation, cell proliferation, and, most recently, cell survival. NF-kappaB signaling is important for the normal cellular growth and is a major target of inflammatory cytokines. Several studies have highlighted a protective role of NF-kappaB in the heart under certain circumstances including hypoxic or ischemic myocardial injury. The diverse nature and involvement of NF-kappaB in regulation of vital cellular processes including cell survival notably in the post-mitotic heart has sparked considerable interest in understanding the signaling pathways involved in regulating NF-kappaB in the heart under normal and pathological conditions. However, whether NF-kappaB is adaptive, maladaptive or is a homeostatic response to cardiac injury may simply depend on the context and timing of its activation. In this forum we discuss NF-kappaB signaling pathways and therapeutic opportunities to modulate NF-kappaB activity in heart failure.

  19. Neuroadaptations in human chronic alcoholics: dysregulation of the NF-kappaB system.

    Directory of Open Access Journals (Sweden)

    Anna Okvist

    2007-09-01

    Full Text Available Alcohol dependence and associated cognitive impairments apparently result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. Here we investigated whether transcription factors of Nuclear Factor-kappaB (NF-kappaB family, controlling neuronal plasticity and neurodegeneration, are involved in these adaptations in human chronic alcoholics.Analysis of DNA-binding of NF-kappaB (p65/p50 heterodimer and the p50 homodimer as well as NF-kappaB proteins and mRNAs was performed in postmortem human brain samples from 15 chronic alcoholics and 15 control subjects. The prefrontal cortex involved in alcohol dependence and cognition was analyzed and the motor cortex was studied for comparison. The p50 homodimer was identified as dominant kappaB binding factor in analyzed tissues. NF-kappaB and p50 homodimer DNA-binding was downregulated, levels of p65 (RELA mRNA were attenuated, and the stoichiometry of p65/p50 proteins and respective mRNAs was altered in the prefrontal cortex of alcoholics. Comparison of a number of p50 homodimer/NF-kappaB target DNA sites, kappaB elements in 479 genes, down- or upregulated in alcoholics demonstrated that genes with kappaB elements were generally upregulated in alcoholics. No significant differences between alcoholics and controls were observed in the motor cortex.We suggest that cycles of alcohol intoxication/withdrawal, which may initially activate NF-kappaB, when repeated over years downregulate RELA expression and NF-kappaB and p50 homodimer DNA-binding. Downregulation of the dominant p50 homodimer, a potent inhibitor of gene transcription apparently resulted in derepression of kappaB regulated genes. Alterations in expression of p50 homodimer/NF-kappaB regulated genes may contribute to neuroplastic adaptation underlying alcoholism.

  20. Targeting the NF-κB Pathway as a Combination Therapy for Advanced Thyroid Cancer.

    Directory of Open Access Journals (Sweden)

    Nikita Pozdeyev

    Full Text Available NF-κB signaling plays an important role in tumor cell proliferation, cell survival, angiogenesis, invasion, metastasis and drug/radiation resistance. Combination therapy involving NF-κB pathway inhibition is an attractive strategy for the treatment of advanced forms of thyroid cancer. This study was designed to test the efficacy of NF-κB pathway inhibition in combination with cytotoxic chemotherapy, using docetaxel and ionizing radiation in in vitro models of thyroid cancer. We found that while both docetaxel and ionizing radiation activated NF-κB signaling in thyroid cancer cells, there was no synergistic effect on cell proliferation and/or programmed cell death with either genetic (transduction of a dominant negative mutant form of IκBα or pharmacologic (proteasome inhibitor bortezomib and IKKβ inhibitor GO-Y030 inhibition of the NF-κB pathway in thyroid cancer cell lines BCPAP, 8505C, THJ16T and SW1736. Docetaxel plus bortezomib synergistically decreased in vitro invasion of 8505C cells, but not in the other cell lines. Screening of a panel of clinically relevant targeted therapies for synergy with genetic NF-κB inhibition in a proliferation/cytotoxicity assay identified the histone deacetylase (HDAC inhibitor suberoylanilide hydroxamic acid (SAHA as a potential candidate. However, the synergistic effect was confirmed only in the BCPAP cells. These results indicate that NF-κB inhibitors are unlikely to be beneficial as combination therapy with taxane cytotoxic chemotherapy, external radiation therapy or radioiodine therapy. There may be unique circumstances where NF-κB inhibitors may be considered in combination with docetaxel to reduce tumor invasion or in combination with HDAC inhibitors to reduce tumor growth, but this does not appear to be a combination therapy that could be broadly applied to patients with advanced thyroid cancer. Further research may identify which subsets of patients/tumors may respond to this therapeutic

  1. Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay.

    Directory of Open Access Journals (Sweden)

    Vasundhara Sharma

    Full Text Available The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD, a chimeric construct containing the TAD derived from p65 was also generated (p50TAD to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

  2. Pre-folding IkappaBalpha alters control of NF-kappaB signaling.

    Science.gov (United States)

    Truhlar, Stephanie M E; Mathes, Erika; Cervantes, Carla F; Ghosh, Gourisankar; Komives, Elizabeth A

    2008-06-27

    Transcription complex components frequently show coupled folding and binding but the functional significance of this mode of molecular recognition is unclear. IkappaBalpha binds to and inhibits the transcriptional activity of NF-kappaB via its ankyrin repeat (AR) domain. The beta-hairpins in ARs 5-6 in IkappaBalpha are weakly-folded in the free protein, and their folding is coupled to NF-kappaB binding. Here, we show that introduction of two stabilizing mutations in IkappaBalpha AR 6 causes ARs 5-6 to fold cooperatively to a conformation similar to that in NF-kappaB-bound IkappaBalpha. Free IkappaBalpha is degraded by a proteasome-dependent but ubiquitin-independent mechanism, and this process is slower for the pre-folded mutants both in vitro and in cells. Interestingly, the pre-folded mutants bind NF-kappaB more weakly, as shown by both surface plasmon resonance and isothermal titration calorimetry in vitro and immunoprecipitation experiments from cells. One consequence of the weaker binding is that resting cells containing these mutants show incomplete inhibition of NF-kappaB activation; they have significant amounts of nuclear NF-kappaB. Additionally, the weaker binding combined with the slower rate of degradation of the free protein results in reduced levels of nuclear NF-kappaB upon stimulation. These data demonstrate clearly that the coupled folding and binding of IkappaBalpha is critical for its precise control of NF-kappaB transcriptional activity.

  3. The testis-specific Cα2 subunit of PKA is kinetically indistinguishable from the common Cα1 subunit of PKA

    Directory of Open Access Journals (Sweden)

    Herberg Friedrich W

    2011-08-01

    Full Text Available Abstract Background The two variants of the α-form of the catalytic (C subunit of protein kinase A (PKA, designated Cα1 and Cα2, are encoded by the PRKACA gene. Whereas Cα1 is ubiquitous, Cα2 expression is restricted to the sperm cell. Cα1 and Cα2 are encoded with different N-terminal domains. In Cα1 but not Cα2 the N-terminal end introduces three sites for posttranslational modifications which include myristylation at Gly1, Asp-specific deamidation at Asn2 and autophosphorylation at Ser10. Previous reports have implicated specific biological features correlating with these modifications on Cα1. Since Cα2 is not modified in the same way as Cα1 we tested if they have distinct biochemical activities that may be reflected in different biological properties. Results We show that Cα2 interacts with the two major forms of the regulatory subunit (R of PKA, RI and RII, to form cAMP-sensitive PKAI and PKAII holoenzymes both in vitro and in vivo as is also the case with Cα1. Moreover, using Surface Plasmon Resonance (SPR, we show that the interaction patterns of the physiological inhibitors RI, RII and PKI were comparable for Cα2 and Cα1. This is also the case for their potency to inhibit catalytic activities of Cα2 and Cα1. Conclusion We conclude that the regulatory complexes formed with either Cα1 or Cα2, respectively, are indistinguishable.

  4. Phylogenetic position of Placozoa based on large subunit (LSU and small subunit (SSU rRNA genes

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    Fernanda Britto da Silva

    2007-01-01

    Full Text Available The phylum Placozoa, represented by its single species Trichoplax adhaerens, has always been considered one of the most basal animal clades due to its low morphological complexity. However, despite its importance, the phylogenetic position of the Placozoa remains uncertain, as does the relationships between the basal metazoans. We sequenced the nearly complete large subunit (LSU of the T. adhaerens rRNA gene together with the small subunit (SSU rRNA and used several methods to infer the phylogenetic position of Placozoa and the relationships between the basal animal phyla. The phylogenetic trees obtained from the LSU, SSU and SSU + LSU datasets support monophyly of all basal metazoan phyla, except the Porifera. However, the relationships between all these phyla were not consistently resolved due to a strong conflict between the LSU and SSU phylogenetic signals. The hypotheses that Placozoa is a derived Cnidaria or the most basal metazoan were not supported by our data. Moreover, our analyses suggests that Placozoa is most likely the sister group of Cnidaria and/or Bilateria.

  5. The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

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    Wieczorek, Anna; McHenry, Charles S

    2006-05-05

    The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

  6. Oncogenic role of Merlin/NF2 in glioblastoma.

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    Guerrero, P A; Yin, W; Camacho, L; Marchetti, D

    2015-05-14

    Glioblastoma is the most common and aggressive primary brain tumor in adults, with a poor prognosis because of its resistance to radiotherapy and chemotherapy. Merlin/NF2 (moesin-ezrin-radixin-like protein/neurofibromatosis type 2) is a tumor suppressor found to be mutated in most nervous system tumors; however, it is not mutated in glioblastomas. Merlin associates with several transmembrane receptors and intracellular proteins serving as an anchoring molecule. Additionally, it acts as a key component of cell motility. By selecting sub-populations of U251 glioblastoma cells, we observed that high expression of phosphorylated Merlin at serine 518 (S518-Merlin), NOTCH1 and epidermal growth factor receptor (EGFR) correlated with increased cell proliferation and tumorigenesis. These cells were defective in cell-contact inhibition with changes in Merlin phosphorylation directly affecting NOTCH1 and EGFR expression, as well as downstream targets HES1 (hairy and enhancer of split-1) and CCND1 (cyclin D1). Of note, we identified a function for S518-Merlin, which is distinct from what has been reported when the expression of Merlin is diminished in relation to EGFR and NOTCH1 expression, providing first-time evidence that demonstrates that the phosphorylation of S518-Merlin in glioblastoma promotes oncogenic properties that are not only the result of inactivation of the tumor suppressor role of Merlin but also an independent process implicating a Merlin-driven regulation of NOTCH1 and EGFR.

  7. The PTB thermal neutron reference field at GeNF

    Energy Technology Data Exchange (ETDEWEB)

    Boettger, R.; Friedrich, H.; Janssen, H.

    2004-07-01

    The experimental setup and procedure for the characterization of the thermal neutron reference field established at the Geesthacht neutron facility (GeNF) of the GKSS is described. The neutron beam, free in air, with a maximum flux of 10{sup 6} s{sup -1}, can easily be reduced to less than 10{sup 4} s{sup -1} by using a diaphragm variable in size and without changing the beam divergence. Also, the spectral distribution with a mean energy of 45 meV, measured by time-of-flight over a 6.6 m long flight path, is independent of the beam current chosen. In the 2002/2003 experiments reported here, a {sup 6}Li glass detector was employed to determine the absolute beam current and to calibrate the {sup 3}He transmission beam monitor. In addition, activation measurements of gold foils were carried out at a reduced beam divergence. The results agree within {+-}2%. Furthermore, the beam is characterized by a low gamma background intensity and a negligible fraction of epithermal neutrons. Irradiations in combination with a scanner device to achieve a homogeneously illuminated scan field have shown that the thermal beam is well suited for dosemeter development and for the calibration of radiation protection instruments. (orig.)

  8. Efficient expression of functional (α6β22β3 AChRs in Xenopus oocytes from free subunits using slightly modified α6 subunits.

    Directory of Open Access Journals (Sweden)

    Carson Kai-Kwong Ley

    Full Text Available Human (α6β2(α4β2β3 nicotinic acetylcholine receptors (AChRs are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β22β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β22β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.

  9. A structural comparison of the A and B subunits of Griffonia simplicifolia I isolectins.

    Science.gov (United States)

    Lamb, J E; Goldstein, I J

    1984-02-15

    A structural comparison between the A and B subunits of the five tetrameric Griffonia simplicifolia I isolectins (A4, A3B, A2B2, AB3, B4) was undertaken to determine the extent of homology between the subunits. The first 25 N-terminal amino acids of both A and B subunits were determined following the enzymatic removal of N-terminal pyroglutamate blocking groups with pyroglutamate aminopeptidase. Although 21 amino acids were common to both subunits, there were four unique amino acids in the N-terminal sequence of A and B. Residues 8, 9, 17, and 19 were asparagine, leucine, lysine, and asparagine in subunit A and threonine, phenylalanine, glutamic acid, and serine in subunit B. The last six C-terminal amino acids, released by digestion with carboxypeptidase Y, were the same for both subunits: Arg-(Phe, Val)-Leu-Thr-Ser-COOH. Subunit B, which contains one methionyl residue, was cleaved by cyanogen bromide into two fragments, a large (Mr = 31,000) and a small (Mr = 2700) polypeptide. Failure of the small fragment to undergo manual Edman degradation indicated an N-terminal blocking group, presumably pyroglutamate. Both subunits were digested with trypsin and the tryptic peptides were analyzed using reverse-phase HPLC. Tryptic glycopeptides were identified by labeling the carbohydrate moiety of the A and B subunit using sodium [3H] borohydride. Cysteine-containing tryptic peptides were similarly identified by using [1-14C]iodoacetamide. Approximately 30% of the tryptic peptides were common to both subunits. Thus, although the N- and C-terminal regions of A and B are similar, the subunits each possess unique sequences.

  10. Genetics vs. entropy: longevity factors suppress the NF-kappaB-driven entropic aging process.

    Science.gov (United States)

    Salminen, Antero; Kaarniranta, Kai

    2010-07-01

    Molecular studies in model organisms have identified potent longevity genes which can delay the aging process and extend the lifespan. Longevity factors promote stress resistance and cellular survival. It seems that the aging process itself is not genetically programmed but a random process involving the loss of molecular fidelity and subsequent accumulation of waste products. This age-related increase in cellular entropy is compatible with the disposable soma theory of aging. A large array of host defence systems has been linked to the NF-kappaB system which is an ancient signaling pathway specialized to host defence, e.g. functioning in immune system. Emerging evidence demonstrates that the NF-kappaB system is activated during aging. Oxidative stress and DNA damage increase with aging and elicit a sustained activation of the NF-kappaB system which has negative consequences, e.g. chronic inflammatory response, increase in apoptotic resistance, decline in autophagic cleansing, and tissue atrophy, i.e. processes that enhance the aging process. We will discuss the role of NF-kappaB system in the pro-aging signaling and will emphasize that several longevity factors seem to be inhibitors of NF-kappaB signaling and in that way they can suppress the NF-kappaB-driven entropic host defence catastrophe. 2009 Elsevier Ireland Ltd. All rights reserved.

  11. Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes

    Science.gov (United States)

    Li, Y. P.; Atkins, C. M.; Sweatt, J. D.; Reid, M. B.; Hamilton, S. L. (Principal Investigator)

    1999-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

  12. RAGE Plays a Role in LPS-Induced NF-κB Activation and Endothelial Hyperpermeability.

    Science.gov (United States)

    Wang, Liqun; Wu, Jie; Guo, Xiaohua; Huang, Xuliang; Huang, Qiaobing

    2017-03-30

    Endothelial functional dysregulation and barrier disruption contribute to the initiation and development of sepsis. The receptor for advanced glycation end products (RAGE) has been demonstrated to be involved in the pathogenesis of sepsis. The present study aimed to investigate the role of RAGE in lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) activation in endothelial cells and the consequent endothelial hyperpermeability. LPS-induced upregulation of RAGE protein expression in human umbilical vein endothelial cells (HUVECs) was detected by western blotting. Activation of NF-κB was revealed using western blotting and immunofluorescent staining. LPS-elicited endothelial hyperpermeability was explored by transendothelial electrical resistance (TER) assay and endothelial monolayer permeability assay. The blocking antibody specific to RAGE was used to confirm the role of RAGE in LPS-mediated NF-κB activation and endothelial barrier disruption. We found that LPS upregulated the protein expression of RAGE in a dose- and time-dependent manner in HUVECs. Moreover, LPS triggered a significant phosphorylation and degradation of IκBα, as well as NF-κB p65 nuclear translocation. Moreover, we observed a significant increase in endothelial permeability after LPS treatment. However, the RAGE blocking antibody attenuated LPS-evoked NF-κB activation and endothelial hyperpermeability. Our results suggest that RAGE plays an important role in LPS-induced NF-κB activation and endothelial barrier dysfunction.

  13. NOTCH and NF-κB interplay in chronic lymphocytic leukemia is independent of genetic lesion.

    Science.gov (United States)

    Baldoni, Stefano; Sportoletti, Paolo; Del Papa, Beatrice; Aureli, Patrizia; Dorillo, Erica; Rosati, Emanuela; Ciurnelli, Raffaella; Marconi, Pierfrancesco; Falzetti, Franca; Di Ianni, Mauro

    2013-08-01

    The NOTCH and nuclear factor kappa B (NF-κB) pathways are both constitutively activated in Chronic Lymphocytic Leukemia (CLL). We first described the NOTCH1 PEST domain mutation in a CLL subgroup, but the activation of the NOTCH pathway in NOTCH1-unmutated cases remains unexplained. Here, we investigated whether genetic lesions in the NF-κB/NOTCH loop might support the NOTCH activation status by sequencing negative (TNFAIP3/A20) and positive (TRAF2, TRAF5, TNFRSF11A/RANK, MAP3K7/TAK1, and CARD11) regulators of NF-κB together with NF-κB targets on the NOTCH pathway, the NOTCH ligands Jagged1 and Jagged2, in CLL patients. The sequence analysis revealed four missense mutations for A20, TRAF2, TRAF5 and RANK1 genes, all causing a change in amino acid group from polar to non-polar, but functional domains were not involved. Specific predictive software analyses confirmed that the amino acid changes have a low-functional impact on the protein. Our results show that in CLL, NF-κB regulators and Jagged are both unmutated, suggesting that the Jagged-mediated interplay between NF-κB and NOTCH is independent of genetic lesions.

  14. Intrachromosomal mitotic nonallelic homologous recombination is the major molecular mechanism underlying type-2 NF1 deletions.

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