Time-dependent upregulation of electron transport with concomitant induction of regulated excitation dissipation in Haslea diatoms.

PubMed

Perkins, R; Williamson, C; Lavaud, J; Mouget, J-L; Campbell, D A

2018-04-16

Photoacclimation by strains of Haslea "blue" diatom species H. ostrearia and H. silbo sp. nov. ined. was investigated with rapid light curves and induction-recovery curves using fast repetition rate fluorescence. Cultures were grown to exponential phase under 50 µmol m -2  s -1 photosynthetic available radiation (PAR) and then exposed to non-sequential rapid light curves where, once electron transport rate (ETR) had reached saturation, light intensity was decreased and then further increased prior to returning to near growth light intensity. The non-sequential rapid light curve revealed that ETR was not proportional to the instantaneously applied light intensity, due to rapid photoacclimation. Changes in the effective absorption cross sections for open PSII reaction centres (σ PSII ') or reaction centre connectivity (ρ) did not account for the observed increases in ETR under extended high light. σ PSII ' in fact decreased as a function of a time-dependent induction of regulated excitation dissipation Y(NPQ), once cells were at or above a PAR coinciding with saturation of ETR. Instead, the observed increases in ETR under extended high light were explained by an increase in the rate of PSII reopening, i.e. Q A - oxidation. This acceleration of electron transport was strictly light dependent and relaxed within seconds after a return to low light or darkness. The time-dependent nature of ETR upregulation and regulated NPQ induction was verified using induction-recovery curves. Our findings show a time-dependent induction of excitation dissipation, in parallel with very rapid photoacclimation of electron transport, which combine to make ETR independent of short-term changes in PAR. This supports a selective advantage for these diatoms when exposed to fluctuating light in their environment.

ISOFORM SPECIFIC REGULATION OF DIVALENT METAL (ION) TRANSPORTER (DMT1) BY PROTEASOMAL DEGRADATION

PubMed Central

Garrick, Michael D.; Zhao, Lin; Roth, Jerome A.; Jiang, Houbo; Feng, Jian; Foot, Natalie J.; Dalton, Hazel; Kumar, Sharad; Garrick, Laura M.

2012-01-01

DMT1 is the major transporter for iron entrance into mammalian cells and iron exit from endosomes during the transferrin cycle. Four major mRNA isoforms correspond to 4 protein isoforms, differing at 5'/3' and N-/C- termini, respectively. Isoforms are designated 1A vs. 1B reflecting where transcription starts or +IRE vs. −IRE reflecting the presence / absence of an iron responsive element in the 3' end of the mRNA. These differences imply regulation at transcriptional and posttranscriptional levels. Many proteins are degraded by a ubiquitination-dependent mechanism. Two different ubiquitin ligases (E3s) appear to be involved in DMT1 ubiquitination: Parkin or Nedd4 family E3s which often utilize Nedd4 family interacting protein-1 and -2 (Ndfip1 & 2) to ubiquitinate their substrate proteins. Prior data suggest that parkin ubiquitinates 1B DMT1 but not 1A DMT1 while Nedd4/Ndfips ligate ubiquitin to DMT1 in the duodenum where 1A/+IRE DMT1 predominates. Our assay for whether these systems target DMT1 depends on two HEK293 cell lines that express permanently transfected 1A/+IRE DMT1 or 1B/−IRE DMT1 after induction by doxycycline. Transient transfection with a parkin construct before induction diminishes 1B/−IRE DMT1 detected by immune-blots but not 1A/+IRE DMT1. Mutant parkin serves as a control that does not affect DMT1 levels. Thus DMT1 regulation in an isoform specific fashion can occur by ubiquitination and the events involved have implications for DMT1 function and disease processes. PMID:22310887

Characterization of Gonadotrope Secretoproteome Identifies Neurosecretory Protein VGF-derived Peptide Suppression of Follicle-stimulating Hormone Gene Expression.

PubMed

Choi, Soon Gang; Wang, Qian; Jia, Jingjing; Chikina, Maria; Pincas, Hanna; Dolios, Georgia; Sasaki, Kazuki; Wang, Rong; Minamino, Naoto; Salton, Stephen R J; Sealfon, Stuart C

2016-09-30

Reproductive function is controlled by the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates the expression of the gonadotropins luteinizing hormone and FSH in pituitary gonadotropes. Paradoxically, Fshb gene expression is maximally induced at lower frequency GnRH pulses, which provide a very low average concentration of GnRH stimulation. We studied the role of secreted factors in modulating gonadotropin gene expression. Inhibition of secretion specifically disrupted gonadotropin subunit gene regulation but left early gene induction intact. We characterized the gonadotrope secretoproteome and global mRNA expression at baseline and after Gα s knockdown, which has been found to increase Fshb gene expression (1). We identified 1077 secreted proteins or peptides, 19 of which showed mRNA regulation by GnRH or/and Gα s knockdown. Among several novel secreted factors implicated in Fshb gene regulation, we focused on the neurosecretory protein VGF. Vgf mRNA, whose gene has been implicated in fertility (2), exhibited high induction by GnRH and depended on Gα s In contrast with Fshb induction, Vgf induction occurred preferentially at high GnRH pulse frequency. We hypothesized that a VGF-derived peptide might regulate Fshb gene induction. siRNA knockdown or extracellular immunoneutralization of VGF augmented Fshb mRNA induction by GnRH. GnRH stimulated the secretion of the VGF-derived peptide NERP1. NERP1 caused a concentration-dependent decrease in Fshb gene induction. These findings implicate a VGF-derived peptide in selective regulation of the Fshb gene. Our results support the concept that signaling specificity from the cell membrane GnRH receptor to the nuclear Fshb gene involves integration of intracellular signaling and exosignaling regulatory motifs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of Gonadotrope Secretoproteome Identifies Neurosecretory Protein VGF-derived Peptide Suppression of Follicle-stimulating Hormone Gene Expression*

PubMed Central

Wang, Qian; Jia, Jingjing; Chikina, Maria; Pincas, Hanna; Dolios, Georgia; Sasaki, Kazuki; Wang, Rong; Minamino, Naoto; Sealfon, Stuart C.

2016-01-01

Reproductive function is controlled by the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates the expression of the gonadotropins luteinizing hormone and FSH in pituitary gonadotropes. Paradoxically, Fshb gene expression is maximally induced at lower frequency GnRH pulses, which provide a very low average concentration of GnRH stimulation. We studied the role of secreted factors in modulating gonadotropin gene expression. Inhibition of secretion specifically disrupted gonadotropin subunit gene regulation but left early gene induction intact. We characterized the gonadotrope secretoproteome and global mRNA expression at baseline and after Gαs knockdown, which has been found to increase Fshb gene expression (1). We identified 1077 secreted proteins or peptides, 19 of which showed mRNA regulation by GnRH or/and Gαs knockdown. Among several novel secreted factors implicated in Fshb gene regulation, we focused on the neurosecretory protein VGF. Vgf mRNA, whose gene has been implicated in fertility (2), exhibited high induction by GnRH and depended on Gαs. In contrast with Fshb induction, Vgf induction occurred preferentially at high GnRH pulse frequency. We hypothesized that a VGF-derived peptide might regulate Fshb gene induction. siRNA knockdown or extracellular immunoneutralization of VGF augmented Fshb mRNA induction by GnRH. GnRH stimulated the secretion of the VGF-derived peptide NERP1. NERP1 caused a concentration-dependent decrease in Fshb gene induction. These findings implicate a VGF-derived peptide in selective regulation of the Fshb gene. Our results support the concept that signaling specificity from the cell membrane GnRH receptor to the nuclear Fshb gene involves integration of intracellular signaling and exosignaling regulatory motifs. PMID:27466366

Substrate-specific regulation of ubiquitination by the anaphase-promoting complex

PubMed Central

Song, Ling

2011-01-01

By orchestrating the sequential degradation of a large number of cell cycle regulators, the ubiquitin ligase anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The correct timing of APC/C-dependent substrate degradation, a critical feature of progression through mitosis, was long known to be controlled by mechanisms targeting the core APC/C-machinery. Recent experiments, however have revealed an important contribution of substrate-specific regulation of the APC/C to achieve accurate cell division. In this perspective, we describe different mechanisms of substrate-specific APC/C-regulation and discuss their importance for cell division. PMID:21191176

Cross-talk between Smad and p38 MAPK signalling in transforming growth factor {beta} signal transduction in human glioblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dziembowska, Magdalena; Danilkiewicz, Malgorzata; Wesolowska, Aleksandra

2007-03-23

Transforming growth factor-beta (TGF-{beta}) is a multifunctional cytokine involved in the regulation of cell proliferation, differentiation, and survival. Malignant tumour cells often do not respond to TGF-{beta} by growth inhibition, but retain responsiveness to cytokine in regulating extracellular matrix deposition, cell adhesion, and migration. We demonstrated that TGF-{beta}1 does not affect viability or proliferation of human glioblastoma T98G, but increases transcriptional responses exemplified by induction of MMP-9 expression. TGF-{beta} receptors were functional in T98G glioblastoma cells leading to SMAD3/SMAD4 nuclear translocation and activation of SMAD-dependent promoter. In parallel, a selective activation of p38 MAPK, and phosphorylation of its substrates: ATF2more » and c-Jun proteins were followed by a transient activation of AP-1 transcription factor. Surprisingly, an inhibition of p38 MAPK with a specific inhibitor, SB202190, abolished TGF-inducible activation of Smad-dependent promoter and decreased Smad2 phosphorylation. It suggests an unexpected interaction between Smad and p38 MAPK pathways in TGF-{beta}1-induced signalling.« less

Interaction of Substrate Mechanics with Dental Pulp Stem Cells (DPSCs) differentiation to generate a scaffold for Bone regeneration

NASA Astrophysics Data System (ADS)

Rafailovich, Miriam; Bhatnagar, Divya; Bherwani, Aneel; Simon, Marcia

2012-02-01

This work investigates the interaction of the substrate mechanics with the differentiation in the absence of chemical induction and only resulting from the stimuli of the substrate mechanics and chemistry. We chose enzymatically cross-linked gelatin hydrogels substrates of different stiffness varying from 8KPa to 100Pa. DPSCs were cultured and differentiated on the substrates for 7, 14 and 21 days with and without dexamethasone induction media. SEM and EDX analysis after 21 days indicate that cells produced a sheet of biomineralized deposits, several tenths of mm thick on the hard substrate irrespective of chemical induction. Modulli of the cells was independent of the induction and stiffness of the hydrogels. RT-PCR assays indicated that cells expressed more osteocalcin when cultured in non-induction media and harder substrate. The shape of the deposits was more uniform and in close packing on the harder substrate with a higher Ca:P ratio. On soft substrate the deposits were more flat with less Ca:P ratio. Further experiments indicated that conformational change due to the crosslinking of gelatin could be the reason for biomineralization.

Top-level dynamics and the regulated gene response of feed-forward loop transcriptional motifs.

PubMed

Mayo, Michael; Abdelzaher, Ahmed; Perkins, Edward J; Ghosh, Preetam

2014-09-01

Feed-forward loops are hierarchical three-node transcriptional subnetworks, wherein a top-level protein regulates the activity of a target gene via two paths: a direct-regulatory path, and an indirect route, whereby the top-level proteins act implicitly through an intermediate transcription factor. Using a transcriptional network of the model bacterium Escherichia coli, we confirmed that nearly all types of feed-forward loop were significantly overrepresented in the bacterial network. We then used mathematical modeling to study their dynamics by manipulating the rise times of the top-level protein concentration, termed the induction time, through alteration of the protein destruction rates. Rise times of the regulated proteins exhibited two qualitatively different regimes, depending on whether top-level inductions were "fast" or "slow." In the fast regime, rise times were nearly independent of rapid top-level inductions, indicative of biological robustness, and occurred when RNA production rate-limits the protein yield. Alternatively, the protein rise times were dependent upon slower top-level inductions, greater than approximately one bacterial cell cycle. An equation is given for this crossover, which depends upon three parameters of the direct-regulatory path: transcriptional cooperation at the DNA-binding site, a protein-DNA dissociation constant, and the relative magnitude of the top-level protien concentration.

Regulation of rat heme oxygenase-1 expression by interleukin-6 via the Jak/STAT pathway in hepatocytes.

PubMed

Tron, Kyrylo; Samoylenko, Anatoly; Musikowski, Gernot; Kobe, Fritz; Immenschuh, Stephan; Schaper, Fred; Ramadori, Giuliano; Kietzmann, Thomas

2006-07-01

Heme oxygenase-1 (HO-1) can be induced by various stimuli, one of which is interleukin-6 (IL-6). Therefore, the aim of this study was to elucidate the molecular mechanisms responsible for IL-6-dependent HO-1 induction in the liver. The IL-6-dependent HO-1 regulation in rat primary hepatocytes and HepG2 hepatoma cells was studied by Northern and Western blot analyses, HO-1 promoter reporter gene assays and EMSA. The HO-1 expression was transcriptionally induced by IL-6 in a time- and dose-dependent manner. Activation of signal transducers and activators of transcription (STAT) factors by the IL-6 receptor was crucial for HO-1 induction. By contrast, negative regulation of HO-1 expression appeared to be mediated through the SH2-domain-containing tyrosine phosphatase-2 (SHP2)/ suppressors of cytokine signaling-3 (SOCS3) binding site within the gp130 IL-6 receptor subunit. Among the three putative STAT binding elements (SBE) in the HO-1 promoter, only the distal one was functional and when deleted, the remaining Luc induction was completely obliterated by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. The HO-1 SBE3 mediates HO-1 gene induction by IL-6 mainly via activation of the Jak/STAT pathway.

Dual regulation of skin sensitizer-induced HMOX1 expression by Bach1 and Nrf2: Comparison to regulation of the AKR1C2-ARE element in the KeratinoSens cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Emter, Roger; Natsch, Andreas, E-mail: andreas.natsch@givaudan.com

2015-11-01

Heme oxygenase (decycling) 1 (HMOX1) is the most consistently found genetic marker induced by skin sensitizers. HMOX1 is often referred to as typical gene regulated by nuclear factor erythroid 2-related factor 2 (Nrf2), however, it is also regulated by other DNA-binding factors, including BTB and CNC homolog 1 (Bach1). The KeratinoSens™ assay is the first validated in vitro assay for sensitizers that measures gene induction. It is based on luciferase expression regulated by the antioxidant response element (ARE) of the aldoketoreductase 1C2 (AKR1C2) gene. Luciferase upregulation is dependent on Nrf2, while HMOX1 upregulation is only partially Nrf2-dependent. Thus, sensitizer-dependent activationmore » of HMOX1 may integrate multiple signals thereby providing additional information. We constructed reporter cell lines containing the full HMOX1 regulatory region or the HMOX1-ARE sequence and compared them with the construct containing the AKR1C2-ARE sequence. Induction of the AKR1C2-ARE depends on Nrf2, but not on the repressor Bach1. Results obtained with HMOX1-ARE and the full HMOX1 promoter indicate that, within the HMOX1 promoter, the HMOX1-ARE is sufficient to explain the induction by sensitizers and that (i) inhibiting Bach1 leads to strong basal expression, (ii) fold-induction by sensitizers above this level is reduced in the absence of Bach1 and (iii) these constructs are less dependent on Nrf2 as compared to the AKR1C2-ARE. Nevertheless, congruent dose response curves for luciferase activity were obtained with all constructs. Thus, while sensitizer-induced HMOX1 activation is dependent on Nrf2 and Bach1, all constructs give identical information for the in vitro prediction of the sensitization potential. - Highlights: • HMOX1 is a key genetic marker up-regulated by skin sensitizers. • HMOX1-, but not AKR1C2-upregulation, is dependent on both Nrf2 and Bach1. • AKR1C2 and HMOX1-dependent reporter constructs yield congruent dose response curves. • Combining both constructs offers no advantage over either construct used alone.« less

Identification of heparin-binding EGF-like growth factor as a target in intercellular regulation of epidermal basal cell growth by suprabasal retinoic acid receptors.

PubMed Central

Xiao, J H; Feng, X; Di, W; Peng, Z H; Li, L A; Chambon, P; Voorhees, J J

1999-01-01

The role of retinoic acid receptors (RARs) in intercellular regulation of cell growth was assessed by targeting a dominant-negative RARalpha mutant (dnRARalpha) to differentiated suprabasal cells of mouse epidermis. dnRARalpha lacks transcriptional activation but not DNA-binding and receptor dimerization functions. Analysis of transgenic mice revealed that dnRARalpha dose-dependently impaired induction of basal cell proliferation and epidermal hyperplasia by all-trans RA (tRA). dnRARalpha formed heterodimers with endogenous retinoid X receptor-alpha (RXRalpha) over RA response elements in competition with remaining endogenous RARgamma-RXRalpha heterodimers, and dose-dependently impaired retinoid-dependent gene transcription. To identify genes regulated by retinoid receptors and involved in cell growth control, we analyzed the retinoid effects on expression of the epidermal growth factor (EGF) receptor, EGF, transforming growth factor-alpha, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin genes. In normal epidermis, tRA rapidly and selectively induced expression of HB-EGF but not the others. This induction occurred exclusively in suprabasal cells. In transgenic epidermis, dnRARalpha dose-dependently inhibited tRA induction of suprabasal HB-EGF and subsequent basal cell hyperproliferation. Together, our observations suggest that retinoid receptor heterodimers located in differentiated suprabasal cells mediate retinoid induction of HB-EGF, which in turn stimulates basal cell growth via intercellular signaling. These events may underlie retinoid action in epidermal regeneration during wound healing. PMID:10075925

Induction of autophagy by ARHI (DIRAS3) alters fundamental metabolic pathways in ovarian cancer models.

PubMed

Ornelas, Argentina; McCullough, Christopher R; Lu, Zhen; Zacharias, Niki M; Kelderhouse, Lindsay E; Gray, Joshua; Yang, Hailing; Engel, Brian J; Wang, Yan; Mao, Weiqun; Sutton, Margie N; Bhattacharya, Pratip K; Bast, Robert C; Millward, Steven W

2016-10-26

Autophagy is a bulk catabolic process that modulates tumorigenesis, therapeutic resistance, and dormancy. The tumor suppressor ARHI (DIRAS3) is a potent inducer of autophagy and its expression results in necroptotic cell death in vitro and tumor dormancy in vivo. ARHI is down-regulated or lost in over 60 % of primary ovarian tumors yet is dramatically up-regulated in metastatic disease. The metabolic changes that occur during ARHI induction and their role in modulating death and dormancy are unknown. We employed Nuclear Magnetic Resonance (NMR)-based metabolomic strategies to characterize changes in key metabolic pathways in both cell culture and xenograft models of ARHI expression and autophagy. These pathways were further interrogated by cell-based immunofluorescence imaging, tracer uptake studies, targeted metabolic inhibition, and in vivo PET/CT imaging. Induction of ARHI in cell culture models resulted in an autophagy-dependent increase in lactate production along with increased glucose uptake and enhanced sensitivity to glycolytic inhibitors. Increased uptake of glutamine was also dependent on autophagy and dramatically sensitized cultured ARHI-expressing ovarian cancer cell lines to glutaminase inhibition. Induction of ARHI resulted in a reduction in mitochondrial respiration, decreased mitochondrial membrane potential, and decreased Tom20 staining suggesting an ARHI-dependent loss of mitochondrial function. ARHI induction in mouse xenograft models resulted in an increase in free amino acids, a transient increase in [ 18 F]-FDG uptake, and significantly altered choline metabolism. ARHI expression has previously been shown to trigger autophagy-associated necroptosis in cell culture. In this study, we have demonstrated that ARHI expression results in decreased cellular ATP/ADP, increased oxidative stress, and decreased mitochondrial function. While this bioenergetic shock is consistent with programmed necrosis, our data indicates that the accompanying up-regulation of glycolysis and glutaminolysis is autophagy-dependent and serves to support cell viability rather than facilitate necroptotic cell death. While the mechanistic basis for metabolic up-regulation following ARHI induction is unknown, our preliminary data suggest that decreased mitochondrial function and increased metabolic demand may play a role. These alterations in fundamental metabolic pathways during autophagy-associated necroptosis may provide the basis for new therapeutic strategies for the treatment of dormant ovarian tumors.

Excitation-transcription coupling via calcium/calmodulin-dependent protein kinase/ERK1/2 signaling mediates the coordinate induction of VGLUT2 and Narp triggered by a prolonged increase in glutamatergic synaptic activity.

PubMed

Doyle, Sukhjeevan; Pyndiah, Slovénie; De Gois, Stéphanie; Erickson, Jeffrey D

2010-05-07

Homeostatic scaling of glutamatergic and GABAergic transmission is triggered by prolonged alterations in synaptic neuronal activity. We have previously described a presynaptic mechanism for synaptic homeostasis and plasticity that involves scaling the level of vesicular glutamate (VGLUT1) and gamma-aminobutyric acid (GABA) (VGAT) transporter biosynthesis. These molecular determinants of vesicle filling and quantal size are regulated by neuronal activity in an opposite manner and bi-directionally. Here, we report that a striking induction of VGLUT2 mRNA and synaptic protein is triggered by a prolonged increase in glutamatergic synaptic activity in mature neocortical neuronal networks in vitro together with two determinants of inhibitory synaptic strength, the neuronal activity-regulated pentraxin (Narp), and glutamate decarboxylase (GAD65). Activity-dependent induction of VGLUT2 and Narp exhibits a similar intermediate-early gene response that is blocked by actinomycin D and tetrodotoxin, by inhibitors of ionotropic glutamate receptors and L-type voltage-gated calcium channels, and is dependent on downstream signaling via calmodulin, calcium/calmodulin-dependent protein kinase (CaMK) and extracellular signal-regulated kinase 1/2 (ERK1/2). The co-induction of VGLUT2 and Narp triggered by prolonged gamma-aminobutyric acid type A receptor blockade is independent of brain-derived nerve growth factor and TrkB receptor signaling. VGLUT2 protein induction occurs on a subset of cortically derived synaptic vesicles in excitatory synapses on somata and dendritic processes of multipolar GABAergic interneurons, recognized sites for the clustering of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptors by Narp. We propose that VGLUT2 and Narp induction by excitation-transcription coupling leads to increased glutamatergic transmission at synapses on GABAergic inhibitory feedback neurons as part of a coordinated program of Ca(2+)-signal transcription involved in mechanisms of homeostatic plasticity after prolonged hyperactivity.

Checkpoint-dependent RNR induction promotes fork restart after replicative stress.

PubMed

Morafraile, Esther C; Diffley, John F X; Tercero, José Antonio; Segurado, Mónica

2015-01-20

The checkpoint kinase Rad53 is crucial to regulate DNA replication in the presence of replicative stress. Under conditions that interfere with the progression of replication forks, Rad53 prevents Exo1-dependent fork degradation. However, although EXO1 deletion avoids fork degradation in rad53 mutants, it does not suppress their sensitivity to the ribonucleotide reductase (RNR) inhibitor hydroxyurea (HU). In this case, the inability to restart stalled forks is likely to account for the lethality of rad53 mutant cells after replication blocks. Here we show that Rad53 regulates replication restart through the checkpoint-dependent transcriptional response, and more specifically, through RNR induction. Thus, in addition to preventing fork degradation, Rad53 prevents cell death in the presence of HU by regulating RNR-expression and localization. When RNR is induced in the absence of Exo1 and RNR negative regulators, cell viability of rad53 mutants treated with HU is increased and the ability of replication forks to restart after replicative stress is restored.

p21 induction plays a dual role in anti-cancer activity of ursolic acid

PubMed Central

Zhang, Xudong; Song, Xinhua; Yin, Shutao; Zhao, Chong; Fan, Lihong

2015-01-01

Previous studies have shown that induction of G1 arrest and apoptosis by ursolic acid is associated with up-regulation of cyclin-dependent kinase inhibitor (CDKI) protein p21 in multiple types of cancer cells. However, the functional role of p21 induction in G1 cell cycle arrest and apoptosis, and the mechanisms of p21 induction by ursolic acid have not been critically addressed. In the current study, we demonstrated that p21 played a mediator role in G1 cell cycle arrest by ursolic acid, whereas p21-mediated up-regulation of Mcl-1 compromised apoptotic effect of ursolic acid. These results suggest that p21 induction plays a dual role in the anti-cancer activity of ursolic acid in terms of cell cycle and apoptosis regulation. p21 induction by ursolic acid was attributed to p53 transcriptional activation. Moreover, we found that ursolic acid was able to inhibit murine double minute-2 protein (MDM2) and T-LAK cell-originated protein kinase (TOPK), the two negative regulator of p53, which in turn contributed to ursolic acid-induced p53 activation. Our findings provided novel insights into understanding of the mechanisms involved in cell cycle arrest and apoptosis induction in response to ursolic acid exposure. PMID:26582056

A novel regulation of IRS1 (insulin receptor substrate-1) expression following short term insulin administration

PubMed Central

2005-01-01

Reduced insulin-mediated glucose transport in skeletal muscle is a hallmark of the pathophysiology of T2DM (Type II diabetes mellitus). Impaired intracellular insulin signalling is implicated as a key underlying mechanism. Attention has focused on early signalling events such as defective tyrosine phosphorylation of IRS1 (insulin receptor substrate-1), a major target for the insulin receptor tyrosine kinase. This is required for normal induction of signalling pathways key to many of the metabolic actions of insulin. Conversely, increased serine/threonine phosphorylation of IRS1 following prolonged insulin exposure (or in obesity) reduces signalling capacity, partly by stimulating IRS1 degradation. We now show that IRS1 levels in human muscle are actually increased 3-fold following 1 h of hyperinsulinaemic euglycaemia. Similarly, transient induction of IRS1 (3-fold) in the liver or muscle of rodents occurs following feeding or insulin injection respectively. The induction by insulin is also observed in cell culture systems, although to a lesser degree, and is not due to reduced proteasomal targeting, increased protein synthesis or gene transcription. Elucidation of the mechanism by which insulin promotes IRS1 stability will permit characterization of the importance of this novel signalling event in insulin regulation of liver and muscle function. Impairment of this process would reduce IRS1 signalling capacity, thereby contributing to the development of hyperinsulinaemia/insulin resistance prior to the appearance of T2DM. PMID:16128672

Interferon-gamma regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways.

PubMed Central

Soler, Concepció; Felipe, Antonio; García-Manteiga, José; Serra, Maria; Guillén-Gómez, Elena; Casado, F Javier; MacLeod, Carol; Modolell, Manuel; Pastor-Anglada, Marçal; Celada, Antonio

2003-01-01

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-gamma (interferon-gamma). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-gamma was studied using macrophages from STAT1 knockout mice. IFN-gamma triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-gamma is required for DNA synthesis. Interestingly, IFN-gamma led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-gamma up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-gamma is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-gamma in macrophages. PMID:12868960

A Genetic Approach to Promoter Recognition during Trans Induction of Viral Gene Expression

NASA Astrophysics Data System (ADS)

Coen, Donald M.; Weinheimer, Steven P.; McKnight, Steven L.

1986-10-01

Viral infection of mammalian cells entails the regulated induction of viral gene expression. The induction of many viral genes, including the herpes simplex virus gene encoding thymidine kinase (tk), depends on viral regulatory proteins that act in trans. Because recognition of the tk promoter by cellular transcription factors is well understood, its trans induction by viral regulatory proteins may serve as a useful model for the regulation of eukaryotic gene expression. A comprehensive set of mutations was therefore introduced into the chromosome of herpes simplex virus at the tk promoter to directly analyze the effects of promoter mutations on tk transcription. The promoter domains required for efficient tk expression under conditions of trans induction corresponded to those important for recognition by cellular transcription factors. Thus, trans induction of tk expression may be catalyzed initially by the interaction of viral regulatory proteins with cellular transcription factors.

The translational repressor Crc controls the Pseudomonas putida benzoate and alkane catabolic pathways using a multi-tier regulation strategy.

PubMed

Hernández-Arranz, Sofía; Moreno, Renata; Rojo, Fernando

2013-01-01

Metabolically versatile bacteria usually perceive aromatic compounds and hydrocarbons as non-preferred carbon sources, and their assimilation is inhibited if more preferable substrates are available. This is achieved via catabolite repression. In Pseudomonas putida, the expression of the genes allowing the assimilation of benzoate and n-alkanes is strongly inhibited by catabolite repression, a process controlled by the translational repressor Crc. Crc binds to and inhibits the translation of benR and alkS mRNAs, which encode the transcriptional activators that induce the expression of the benzoate and alkane degradation genes respectively. However, sequences similar to those recognized by Crc in benR and alkS mRNAs exist as well in the translation initiation regions of the mRNA of several structural genes of the benzoate and alkane pathways, which suggests that Crc may also regulate their translation. The present results show that some of these sites are functional, and that Crc inhibits the induction of both pathways by limiting not only the translation of their transcriptional activators, but also that of genes coding for the first enzyme in each pathway. Crc may also inhibit the translation of a gene involved in benzoate uptake. This multi-tier approach probably ensures the rapid regulation of pathway genes, minimizing the assimilation of non-preferred substrates when better options are available. A survey of possible Crc sites in the mRNAs of genes associated with other catabolic pathways suggested that targeting substrate uptake, pathway induction and/or pathway enzymes may be a common strategy to control the assimilation of non-preferred compounds. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Quantitative regulation of bone-mimetic, oriented collagen/apatite matrix structure depends on the degree of osteoblast alignment on oriented collagen substrates.

PubMed

Matsugaki, Aira; Isobe, Yoshihiro; Saku, Taro; Nakano, Takayoshi

2015-02-01

Bone tissue has a specific anisotropic morphology derived from collagen fiber alignment and the related apatite crystal orientation as a bone quality index. However, the precise mechanism of cellular regulation of the crystallographic orientation of apatite has not been clarified. In this study, anisotropic construction of cell-produced mineralized matrix in vitro was established by initiating organized cellular alignment and subsequent oriented bone-like matrix (collagen/apatite) production. The oriented collagen substrates with three anisotropic levels were prepared by a hydrodynamic method. Primary osteoblasts were cultured on the fabricated substrates until mineralized matrix formation is confirmed. Osteoblast alignment was successfully regulated by the level of substrate collagen orientation, with preferential alignment along the direction of the collagen fibers. Notably, both fibrous orientation of newly synthesized collagen matrix and c-axis of produced apatite crystals showed preferential orientation along the cell direction. Because the degree of anisotropy of the deposited apatite crystals showed dependency on the directional distribution of osteoblasts cultured on the oriented collagen substrates, the cell orientation determines the crystallographic anisotropy of produced apatite crystals. To the best of our knowledge, this is the first report demonstrating that bone tissue anisotropy, even the alignment of apatite crystals, is controllable by varying the degree of osteoblast alignment via regulating the level of substrate orientation. © 2014 Wiley Periodicals, Inc.

Optimal Design of Litz Wire Coils With Sandwich Structure Wirelessly Powering an Artificial Anal Sphincter System.

PubMed

Ke, Lei; Yan, Guozheng; Yan, Sheng; Wang, Zhiwu; Li, Xiaoyang

2015-07-01

Transcutaneous energy transfer system (TETS) is widely used to energize implantable biomedical devices. As a key part of the TETS, a pair of applicable coils with low losses, high unloaded Q factor, and strong coupling is required to realize an efficient TETS. This article presents an optimal design methodology of planar litz wire coils sandwiched between two ferrite substrates wirelessly powering a novel mechanical artificial anal sphincter system for treating severe fecal incontinence, with focus on the main parameters of the coils such as the wire diameter, number of turns, geometry, and the properties of the ferrite substrate. The theoretical basis of optimal power transfer efficiency in an inductive link was analyzed. A set of analytical expressions are outlined to calculate the winding resistance of a litz wire coil on ferrite substrate, taking into account eddy-current losses, including conduction losses and induction losses. Expressions that describe the geometrical dimension dependence of self- and mutual inductance are derived. The influence of ferrite substrate relative permeability and dimensions is also considered. We have used this foundation to devise an applicable coil design method that starts with a set of realistic constraints and ends with the optimal coil pair geometries. All theoretical predictions are verified with measurements using different types of fabricated coils. The results indicate that the analysis is useful for optimizing the geometry design of windings and the ferrite substrate in a sandwich structure as part of which, in addition to providing design insight, allows speeding up the system efficiency-optimizing design process. Copyright © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Modification of the surface properties of glass-ceramic materials at low-pressure RF plasma stream

NASA Astrophysics Data System (ADS)

Tovstopyat, Alexander; Gafarov, Ildar; Galeev, Vadim; Azarova, Valentina; Golyaeva, Anastasia

2018-05-01

The surface roughness has a huge effect on the mechanical, optical, and electronic properties of materials. In modern optical systems, the specifications for the surface accuracy and smoothness of substrates are becoming even more stringent. Commercially available pre-polished glass-ceramic substrates were treated with the radio frequency (RF) inductively coupled (13.56 MHz) low-pressure plasma to clean the surface of the samples and decrease the roughness. Optical emission spectroscopy was used to investigate the plasma stream parameters and phase-shifted interferometry to investigate the surface of the specimen. In this work, the dependence of RF inductively coupled plasma on macroscopic parameters was investigated with the focus on improving the surfaces. The ion energy, sputtering rate, and homogeneity were investigated. The improvements of the glass-ceramic surfaces from 2.6 to 2.2 Å root mean square by removing the "waste" after the previous operations had been achieved.

Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP.

PubMed

Matheny, Sharon A; Chen, Chiyuan; Kortum, Robert L; Razidlo, Gina L; Lewis, Robert E; White, Michael A

2004-01-15

The signal transduction cascade comprising Raf, mitogen-activated protein (MAP) kinase kinase (MEK) and MAP kinase is a Ras effector pathway that mediates diverse cellular responses to environmental cues and contributes to Ras-dependent oncogenic transformation. Here we report that the Ras effector protein Impedes Mitogenic signal Propagation (IMP) modulates sensitivity of the MAP kinase cascade to stimulus-dependent activation by limiting functional assembly of the core enzymatic components through the inactivation of KSR, a scaffold/adaptor protein that couples activated Raf to its substrate MEK. IMP is a Ras-responsive E3 ubiquitin ligase that, on activation of Ras, is modified by auto-polyubiquitination, which releases the inhibition of Raf-MEK complex formation. Thus, Ras activates the MAP kinase cascade through simultaneous dual effector interactions: induction of Raf kinase activity and derepression of Raf-MEK complex formation. IMP depletion results in increased stimulus-dependent MEK activation without alterations in the timing or duration of the response. These observations suggest that IMP functions as a threshold modulator, controlling sensitivity of the cascade to stimulus and providing a mechanism to allow adaptive behaviour of the cascade in chronic or complex signalling environments.

Substrate micropatterns produced by polymer demixing regulate focal adhesions, actin anisotropy, and lineage differentiation of stem cells.

PubMed

Vega, Sebastián L; Arvind, Varun; Mishra, Prakhar; Kohn, Joachim; Sanjeeva Murthy, N; Moghe, Prabhas V

2018-06-12

Stem cells are adherent cells whose multipotency and differentiation can be regulated by numerous microenvironmental signals including soluble growth factors and surface topography. This study describes a simple method for creating distinct micropatterns via microphase separation resulting from polymer demixing of poly(desaminotyrosyl-tyrosine carbonate) (PDTEC) and polystyrene (PS). Substrates with co-continuous (ribbons) or discontinuous (islands and pits) PDTEC regions were obtained by varying the ratio of PDTEC and sacrificial PS. Human mesenchymal stem cells (MSCs) cultured on co-continuous PDTEC substrates for 3 days in bipotential adipogenic/osteogenic (AD/OS) induction medium showed no change in cell morphology but exhibited increased anisotropic cytoskeletal organization and larger focal adhesions when compared to MSCs cultured on discontinuous micropatterns. After 14 days in bipotential AD/OS induction medium, MSCs cultured on co-continuous micropatterns exhibited increased expression of osteogenic markers, whereas MSCs on discontinuous PDTEC substrates showed a low expression of adipogenic and osteogenic differentiation markers. Substrates with graded micropatterns were able to reproduce the influence of local underlying topography on MSC differentiation, thus demonstrating their potential for high throughput analysis. This work presents polymer demixing as a simple, non-lithographic technique to produce a wide range of micropatterns on surfaces with complex geometries to influence cellular and tissue regenerative responses. Gaining a better understanding of how engineered microenvironments influence stem cell differentiation is integral to increasing the use of stem cells and materials in a wide range of tissue engineering applications. In this study, we show the range of topography obtained by polymer demixing is sufficient for investigating how surface topography affects stem cell morphology and differentiation. Our findings show that co-continuous topographies favor early (3-day) cytoskeletal anisotropy and focal adhesion maturation as well as long-term (14-day) expression of osteogenic differentiation markers. Taken together, this study presents a simple approach to pattern topographies that induce divergent responses in stem cell morphology and differentiation. Copyright © 2018. Published by Elsevier Ltd.

Hyperosmolarity leads to an increase in derepressed system A activity in the renal epithelial cell line NBL-1.

PubMed Central

Soler, C; Felipe, A; Casado, F J; McGivan, J D; Pastor-Anglada, M

1993-01-01

Hyperosmolarity induced an increase in Na(+)-dependent L-alanine uptake in confluent monolayers of the established renal epithelial cell line NBL-1. This induction was attributable to system A and was only seen when the cells had been previously deprived of amino acids in the culture medium to derepress system A activity. It was additive to the adaptive regulation induction, and both were inhibited by cycloheximide. However, the hyperosmolarity effect was inhibited by colcemid (an inhibitor of microtubular function), but adaptive regulation was not. Otherwise, when cell monolayers were incubated in a control medium, basal Na(+)-dependent L-alanine uptake mediated by system B0 decreased. The results of this study show that: (i) system A activity was not induced by cell shrinkage and subsequent swelling due to extracellular hyperosmolarity when cells were incubated in control medium; (ii) previous expression of system A activity induced by amino acid starvation seems to be a prerequisite for further induction due to hyperosmolarity; and (iii) the effects of adaptive regulation and hyperosmotic stress are mediated by different mechanisms. PMID:8435065

Bactericidal effects of plasma-modified surface chemistry of silicon nanograss

NASA Astrophysics Data System (ADS)

Ostrikov, Kola; Macgregor-Ramiasa, Melanie; Cavallaro, Alex; (Ken Ostrikov, Kostya; Vasilev, Krasimir

2016-08-01

The surface chemistry and topography of biomaterials regulate the adhesion and growth of microorganisms in ways that are still poorly understood. Silicon nanograss structures prepared via inductively coupled plasma etching were coated with plasma deposited nanometer-thin polymeric films to produce substrates with controlled topography and defined surface chemistry. The influence of surface properties on Staphylococcus aureus proliferation is demonstrated and explained in terms of nanograss substrate wetting behaviour. With the combination of the nanograss topography; hydrophilic plasma polymer coatings enhanced antimicrobial activity while hydrophobic coatings reduced it. This study advances the understanding of the effects of surface wettability on the bactericidal properties of reactive nano-engineered surfaces.

New insights into the interplay between the lysine transporter LysP and the pH sensor CadC in Escherichia coli.

PubMed

Rauschmeier, Martina; Schüppel, Valentina; Tetsch, Larissa; Jung, Kirsten

2014-01-09

The coordination of signal transduction and substrate transport represents a sophisticated way to integrate information on metabolite fluxes into transcriptional regulation. This widely distributed process involves protein-protein interactions between two integral membrane proteins. Here we report new insights into the molecular mechanism of the regulatory interplay between the lysine-specific permease LysP and the membrane-integrated pH sensor CadC, which together induce lysine-dependent adaptation of E. coli under acidic stress. In vivo analyses revealed that, in the absence of either stimulus, the two proteins form a stable association, which is modulated by lysine and low pH. In addition to its transmembrane helix, the periplasmic domain of CadC also participated in the interaction. Site-directed mutagenesis pinpointed Arg265 and Arg268 in CadC as well as Asp275 and Asp278 in LysP as potential periplasmic interaction sites. Moreover, a systematic analysis of 100 LysP variants with single-site replacements indicated that the lysine signal is transduced from co-sensor to sensor via lysine-dependent conformational changes (upon substrate binding and/or transport) of LysP. Our results suggest a scenario in which CadC is inhibited by LysP via intramembrane and periplasmic contacts under non-inducing conditions. Upon induction, lysine-dependent conformational changes in LysP transduce the lysine signal via a direct conformational coupling to CadC without resolving the interaction completely. Moreover, concomitant pH-dependent protonation of periplasmic amino acids in both proteins dissolves their electrostatic connections resulting in further destabilization of the CadC/LysP interaction. © 2013.

A simulation study on the constancy of cardiac energy metabolites during workload transition.

PubMed

Saito, Ryuta; Takeuchi, Ayako; Himeno, Yukiko; Inagaki, Nobuya; Matsuoka, Satoshi

2016-12-01

The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant during physiological cardiac workload transition. How this is accomplished is not yet clarified, though Ca 2+ has been suggested to be one of the possible mechanisms. We constructed a detailed mathematical model of cardiac mitochondria based on experimental data and studied whether known Ca 2+ -dependent regulation mechanisms play roles in the metabolite constancy. Model simulations revealed that the Ca 2+ -dependent regulation mechanisms have important roles under the in vitro condition of isolated mitochondria where malate and glutamate were mitochondrial substrates, while they have only a minor role and the composition of substrates has marked influence on the metabolite constancy during workload transition under the simulated in vivo condition where many substrates exist. These results help us understand the regulation mechanisms of cardiac energy metabolism during physiological cardiac workload transition. The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant over a wide range of cardiac workload, though the mechanisms are not yet clarified. One possible regulator of mitochondrial metabolism is Ca 2+ , because it activates several mitochondrial enzymes and transporters. Here we constructed a mathematical model of cardiac mitochondria, including oxidative phosphorylation, substrate metabolism and ion/substrate transporters, based on experimental data, and studied whether the Ca 2+ -dependent activation mechanisms play roles in metabolite constancy. Under the in vitro condition of isolated mitochondria, where malate and glutamate were used as mitochondrial substrates, the model well reproduced the Ca 2+ and inorganic phosphate (P i ) dependences of oxygen consumption, NADH level and mitochondrial membrane potential. The Ca 2+ -dependent activations of the aspartate/glutamate carrier and the F 1 F o -ATPase, and the P i -dependent activation of Complex III were key factors in reproducing the experimental data. When the mitochondrial model was implemented in a simple cardiac cell model, simulation of workload transition revealed that cytoplasmic Ca 2+ concentration ([Ca 2+ ] cyt ) within the physiological range markedly increased NADH level. However, the addition of pyruvate or citrate attenuated the Ca 2+ dependence of NADH during the workload transition. Under the simulated in vivo condition where malate, glutamate, pyruvate, citrate and 2-oxoglutarate were used as mitochondrial substrates, the energy metabolites were more stable during the workload transition and NADH level was almost insensitive to [Ca 2+ ] cyt . It was revealed that mitochondrial substrates have a significant influence on metabolite constancy during cardiac workload transition, and Ca 2+ has only a minor role under physiological conditions. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

A protein phosphatase feedback mechanism regulates the basal phosphorylation of Chk2 kinase in the absence of DNA damage.

PubMed

Carlessi, Luigi; Buscemi, Giacomo; Fontanella, Enrico; Delia, Domenico

2010-10-01

The checkpoint kinase Chk2 is an effector component of the ATM-dependent DNA damage response (DDR) pathway. The activation of Chk2 by genotoxic stress involves its phosphorylation on T68 by ATM and additional auto/transphosphorylations. Here we demonstrate that in unperturbed cells, chemical inhibition of Chk2 by VRX0466617 (VRX) enhances the phosphorylation of Chk2-T68 throughout the cell cycle phases. This event, dependent on the presence of ATM and catalytically functional Chk2, is not consequential to DNA damage, as neither gamma-H2AX nuclear foci nor increased ATM activation is detected in VRX-treated cells, suggesting the involvement of other regulatory proteins. As serine/threonine protein phosphatases (PPs) regulate the phosphorylation and deactivation of proteins of the DDR pathway, we analyzed their role in phospho-T68-Chk2 regulation. We found that intracellular inhibition of PP1 and PP2A-like activities by okadaic acid markedly raised the accumulation of Chk2-pT68 without DNA damage induction, and this phenomenon was also seen when PP1-C, PP2A-C, and Wip1/PPM1D were simultaneously knockdown by siRNA. Altogether, these data indicate a novel mechanism in undamaged cells where PPs function to maintain the balance between ATM and its direct substrate Chk2 through a regulatory circuit. Copyright © 2010 Elsevier B.V. All rights reserved.

Cyclin K dependent regulation of Aurora B affects apoptosis and proliferation by induction of mitotic catastrophe in prostate cancer.

PubMed

Schecher, Sabrina; Walter, Britta; Falkenstein, Michael; Macher-Goeppinger, Stephan; Stenzel, Philipp; Krümpelmann, Kristina; Hadaschik, Boris; Perner, Sven; Kristiansen, Glen; Duensing, Stefan; Roth, Wilfried; Tagscherer, Katrin E

2017-10-15

Cyclin K plays a critical role in transcriptional regulation as well as cell development. However, the role of Cyclin K in prostate cancer is unknown. Here, we describe the impact of Cyclin K on prostate cancer cells and examine the clinical relevance of Cyclin K as a biomarker for patients with prostate cancer. We show that Cyclin K depletion in prostate cancer cells induces apoptosis and inhibits proliferation accompanied by an accumulation of cells in the G2/M phase. Moreover, knockdown of Cyclin K causes mitotic catastrophe displayed by multinucleation and spindle multipolarity. Furthermore, we demonstrate a Cyclin K dependent regulation of the mitotic kinase Aurora B and provide evidence for an Aurora B dependent induction of mitotic catastrophe. In addition, we show that Cyclin K expression is associated with poor biochemical recurrence-free survival in patients with prostate cancer treated with an adjuvant therapy. In conclusion, targeting Cyclin K represents a novel, promising anti-cancer strategy to induce cell cycle arrest and apoptotic cell death through induction of mitotic catastrophe in prostate cancer cells. Moreover, our results indicate that Cyclin K is a putative predictive biomarker for clinical outcome and therapy response for patients with prostate cancer. © 2017 UICC.

Induction of apoptosis by ethanolic extract of Corchorus olitorius leaf in human hepatocellular carcinoma (HepG2) cells via a mitochondria-dependent pathway.

PubMed

Li, Chia-Jung; Huang, Shang-Yu; Wu, Meng-Yu; Chen, Yu-Ching; Tsang, Shih-Fang; Chyuan, Jong-Ho; Hsu, Hsue-Yin

2012-08-03

Corchorus olitorius L.,is a culinary and medicinal herb, widely used as a vegetable in several countries in Asia. Many studies have shown that C. olitorius contains several antioxidants and exhibits anti-inflammatory and anti-proliferative activities in various in vitro and in vivo settings. Recently, C. olitorius has been approved for its antitumor activity; however, the underlying molecular mechanisms remain unclear. The goal of this study was to investigate the effects of ethanol extract of C. olitorius (ECO) on the growth of human hepatocellular carcinoma (HepG2) cells and gain some insights into the underlying mechanisms of its action. We found that HepG2 cells, treated with ECO for 24 h at a concentration higher than 12.5 μg/mL, displayed a strong reduction in cell viability, whereas normal FL83B hepatocytes were not affected. DNA fragmentation and nuclear condensation were evidenced by the increased subG1 population of ECO-treated HepG2 cells. ECO triggered the activation of procaspases-3 and -9 and caused the cleavage of downstream substrate, poly ADP-ribose polymerase (PARP), followed by down-regulation of the inhibitor of caspase-activated DNase (ICAD) signaling. Moreover, the increased release of cytochrome c from mitochondria with decreased membrane potential demonstrated the apoptosis induced through the caspases cascade. Our findings indicated that ECO might be effective against hepatocellular carcinoma through induction of apoptosis via mitochondria-dependent pathway.

Quantitative framework for ordered degradation of APC/C substrates.

PubMed

Lu, Dan; Girard, Juliet R; Li, Weihan; Mizrak, Arda; Morgan, David O

2015-11-16

During cell-cycle progression, substrates of a single master regulatory enzyme can be modified in a specific order. Here, we used experimental and computational approaches to dissect the quantitative mechanisms underlying the ordered degradation of the substrates of the ubiquitin ligase APC/C(Cdc20), a key regulator of chromosome segregation in mitosis. We show experimentally that the rate of catalysis varies with different substrates of APC/C(Cdc20). Using a computational model based on multi-step ubiquitination, we then show how changes in the interaction between a single substrate and APC/C(Cdc20) can alter the timing of degradation onset relative to APC/C(Cdc20) activation, while ensuring a fast degradation rate. Degradation timing and dynamics depend on substrate affinity for the enzyme as well as the catalytic rate at which the substrate is modified. When two substrates share the same pool of APC/C(Cdc20), their relative enzyme affinities and rates of catalysis influence the partitioning of APC/C(Cdc20) among substrates, resulting in substrate competition. Depending on how APC/C(Cdc20) is partitioned among its substrates, competition can have minor or major effects on the degradation of certain substrates. We show experimentally that increased expression of the early APC/C(Cdc20) substrate Clb5 does not delay the degradation of the later substrate securin, arguing against a role for competition with Clb5 in establishing securin degradation timing. The degradation timing of APC/C(Cdc20) substrates depends on the multi-step nature of ubiquitination, differences in substrate-APC/C(Cdc20) interactions, and competition among substrates. Our studies provide a conceptual framework for understanding how ordered modification can be established among substrates of the same regulatory enzyme, and facilitate our understanding of how precise temporal control is achieved by a small number of master regulators to ensure a successful cell division cycle.

Prostaglandin E2-induced up-regulation of c-fos messenger ribonucleic acid is primarily mediated by 3',5'-cyclic adenosine monophosphate in MC3T3-E1 osteoblasts

NASA Technical Reports Server (NTRS)

Fitzgerald, J.; Dietz, T. J.; Hughes-Fulford, M.

2000-01-01

The mechanism by which the proto-oncogene, c-fos, is up-regulated in response to PGE2 in the mouse osteoblastic (MC3T3-E1) cell line was investigated using RT-PCR. c-fos messenger RNA up-regulation by dmPGE2 is rapid, starting 10 min post stimulation, and transient. The specific protein kinase A (PKA) inhibitor, H89, inhibited c-fos induction. Moreover, down-regulation of protein kinase C (PKC) activity by chronic TPA treatment had no effect on the induction of c-fos by dmPGE2. We conclude that up-regulation of c-fos by dmPGE2 is primarily dependent on PKA in MC3T3-E1 osteoblasts. In S49 lymphoma wild-type but not S49 cyc- cells, which are deficient in cAMP signaling, dmPGE2 up-regulates c-fos and increases cell growth compared with unstimulated cells. Thus in S49 lymphoma cells, c-fos induction by PGE2 is also dependent on cAMP signaling. The minimal c-fos promoter region required for dmPGE2-induced expression was identified by transfecting c-fos promoter deletion constructs coupled to the chloramphenicol acetyltransferase (CAT) reporter gene into Vero cells. Transfection of a plasmid containing 99 bp c-fos proximal promoter was sufficient to direct c-fos/CAT expression following stimulation with dmPGE2. Because induction of c-fos is mediated by cAMP, these data are consistent with activation of c-fos via the CRE/ATF cis element.

Cellular volume regulation and substrate stiffness modulate the detachment dynamics of adherent cells

NASA Astrophysics Data System (ADS)

Yang, Yuehua; Jiang, Hongyuan

2018-03-01

Quantitative characterizations of cell detachment are vital for understanding the fundamental mechanisms of cell adhesion. Experiments have found that cell detachment shows strong rate dependence, which is mostly attributed to the binding-unbinding kinetics of receptor-ligand bond. However, our recent study showed that the cellular volume regulation can significantly regulate the dynamics of adherent cell and cell detachment. How this cellular volume regulation contributes to the rate dependence of cell detachment remains elusive. Here, we systematically study the role of cellular volume regulation in the rate dependence of cell detachment by investigating the cell detachments of nonspecific adhesion and specific adhesion. We find that the cellular volume regulation and the bond kinetics dominate the rate dependence of cell detachment at different time scales. We further test the validity of the traditional Johnson-Kendall-Roberts (JKR) contact model and the detachment model developed by Wyart and Gennes et al (W-G model). When the cell volume is changeable, the JKR model is not appropriate for both the detachments of convex cells and concave cells. The W-G model is valid for the detachment of convex cells but is no longer applicable for the detachment of concave cells. Finally, we show that the rupture force of adherent cells is also highly sensitive to substrate stiffness, since an increase in substrate stiffness will lead to more associated bonds. These findings can provide insight into the critical role of cell volume in cell detachment and might have profound implications for other adhesion-related physiological processes.

A role for calcium in the regulation of ATP-binding cassette, sub-family C, member 3 (ABCC3) gene expression in a model of epidermal growth factor-mediated breast cancer epithelial-mesenchymal transition.

PubMed

Stewart, Teneale A; Azimi, Iman; Thompson, Erik W; Roberts-Thomson, Sarah J; Monteith, Gregory R

2015-03-13

Epithelial-mesenchymal transition (EMT), a process implicated in cancer metastasis, is associated with the transcriptional regulation of members of the ATP-binding cassette superfamily of efflux pumps, and drug resistance in breast cancer cells. Epidermal growth factor (EGF)-induced EMT in MDA-MB-468 breast cancer cells is calcium signal dependent. In this study induction of EMT was shown to result in the transcriptional up-regulation of ATP-binding cassette, subfamily C, member 3 (ABCC3), a member of the ABC transporter superfamily, which has a recognized role in multidrug resistance. Buffering of cytosolic free calcium inhibited EGF-mediated ABCC3 increases, indicating a calcium-dependent mode of regulation. Silencing of TRPM7 (an ion channel involved in EMT associated vimentin induction) did not inhibit ABCC3 up-regulation. Silencing of the store operated calcium entry (SOCE) pathway components ORAI1 and STIM1 also did not alter ABCC3 induction by EGF. However, the calcium permeable ion channel transient receptor potential cation channel, subfamily C, member 1 (TRPC1) appears to contribute to the regulation of both basal and EGF-induced ABCC3 mRNA. Improved understanding of the relationship between calcium signaling, EMT and the regulation of genes important in therapeutic resistance may help identify novel therapeutic targets for breast cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

Exposure of LS-180 Cells to Drugs of Diverse Physicochemical and Therapeutic Properties Up-regulates P-glycoprotein Expression and Activity

PubMed Central

Abuznait, Alaa H.; Patrick, Shawn G.; Kaddoumi, Amal

2011-01-01

Purpose Drug transporters are increasingly recognized as important determinants of variability in drug disposition and therapeutic response, both in pre-clinical and clinical stages of drug development process. The role P-glycoprotein (P-gp) plays in drug interactions via its inhibition is well established. However, much less knowledge is available about drugs effect on P-gp up-regulation. The objective of this work was to in vitro investigate and rank commonly used drugs according to their potencies to up-regulate P-gp activity utilizing the same experimental conditions. Methods The in vitro potencies of several drugs of diverse physicochemical and therapeutic properties including rifampicin, dexamethasone, caffeine, verapamil, pentylenetetrazole, hyperforin, and β-estradiol over broad concentration range to up-regulate P-gp expression and activity were examined. For dose-response studies, LS-180 cells were treated with different concentrations of the selected drugs followed by P-gp protein and gene expressions analyses. P-gp functionality was determined by uptake studies with rhodamine 123 as a P-gp substrate, followed by Emax/EC50 evaluation. Results The results demonstrated a dose-dependent increase in P-gp expression and activity following treatments. At 50 μM concentration (hyperforin, 0.1 μM), examined drugs increased P-gp protein and gene expressions by up to 5.5 and 6.2-fold, respectively, while enhanced P-gp activity by 1.8–4-fold. The rank order of these drugs potencies to up-regulate P-gp activity was as following: hyperforin ⋙ dexamethasone ≈ β-estradiol > caffeine > rifampicin ≈ pentylenetetrazole > verapamil. Conclusions These drugs have the potential to be involved in drug interactions when administered with other drugs that are P-gp substrates. Further studies are needed to in vivo evaluate these drugs and verify the consequences of such induction on P-gp activity for in vitro-in vivo correlation purposes. PMID:21733412

Time-dependent pharmacokinetics of dexamethasone and its efficacy in human breast cancer xenograft mice: a semi-mechanism-based pharmacokinetic/pharmacodynamic model.

PubMed

Li, Jian; Chen, Rong; Yao, Qing-Yu; Liu, Sheng-Jun; Tian, Xiu-Yun; Hao, Chun-Yi; Lu, Wei; Zhou, Tian-Yan

2018-03-01

Dexamethasone (DEX) is the substrate of CYP3A. However, the activity of CYP3A could be induced by DEX when DEX was persistently administered, resulting in auto-induction and time-dependent pharmacokinetics (pharmacokinetics with time-dependent clearance) of DEX. In this study we investigated the pharmacokinetic profiles of DEX after single or multiple doses in human breast cancer xenograft nude mice and established a semi-mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model for characterizing the time-dependent PK of DEX as well as its anti-cancer effect. The mice were orally given a single or multiple doses (8 mg/kg) of DEX, and the plasma concentrations of DEX were assessed using LC-MS/MS. Tumor volumes were recorded daily. Based on the experimental data, a two-compartment model with first order absorption and time-dependent clearance was established, and the time-dependence of clearance was modeled by a sigmoid E max equation. Moreover, a semi-mechanism-based PK/PD model was developed, in which the auto-induction effect of DEX on its metabolizing enzyme CYP3A was integrated and drug potency was described using an E max equation. The PK/PD model was further used to predict the drug efficacy when the auto-induction effect was or was not considered, which further revealed the necessity of adding the auto-induction effect into the final PK/PD model. This study established a semi-mechanism-based PK/PD model for characterizing the time-dependent pharmacokinetics of DEX and its anti-cancer effect in breast cancer xenograft mice. The model may serve as a reference for DEX dose adjustments or optimization in future preclinical or clinical studies.

Rapid transporter regulation prevents substrate flow traffic jams in boron transport

PubMed Central

Sotta, Naoyuki; Duncan, Susan; Tanaka, Mayuki; Sato, Takafumi

2017-01-01

Nutrient uptake by roots often involves substrate-dependent regulated nutrient transporters. For robust uptake, the system requires a regulatory circuit within cells and a collective, coordinated behaviour across the tissue. A paradigm for such systems is boron uptake, known for its directional transport and homeostasis, as boron is essential for plant growth but toxic at high concentrations. In Arabidopsis thaliana, boron uptake occurs via diffusion facilitators (NIPs) and exporters (BORs), each presenting distinct polarity. Intriguingly, although boron soil concentrations are homogenous and stable, both transporters manifest strikingly swift boron-dependent regulation. Through mathematical modelling, we demonstrate that slower regulation of these transporters leads to physiologically detrimental oscillatory behaviour. Cells become periodically exposed to potentially cytotoxic boron levels, and nutrient throughput to the xylem becomes hampered. We conclude that, while maintaining homeostasis, swift transporter regulation within a polarised tissue context is critical to prevent intrinsic traffic-jam like behaviour of nutrient flow. PMID:28870285

Rapid transporter regulation prevents substrate flow traffic jams in boron transport.

PubMed

Sotta, Naoyuki; Duncan, Susan; Tanaka, Mayuki; Sato, Takafumi; Marée, Athanasius Fm; Fujiwara, Toru; Grieneisen, Verônica A

2017-09-05

Nutrient uptake by roots often involves substrate-dependent regulated nutrient transporters. For robust uptake, the system requires a regulatory circuit within cells and a collective, coordinated behaviour across the tissue. A paradigm for such systems is boron uptake, known for its directional transport and homeostasis, as boron is essential for plant growth but toxic at high concentrations. In Arabidopsis thaliana , boron uptake occurs via diffusion facilitators (NIPs) and exporters (BORs), each presenting distinct polarity. Intriguingly, although boron soil concentrations are homogenous and stable, both transporters manifest strikingly swift boron-dependent regulation. Through mathematical modelling, we demonstrate that slower regulation of these transporters leads to physiologically detrimental oscillatory behaviour. Cells become periodically exposed to potentially cytotoxic boron levels, and nutrient throughput to the xylem becomes hampered. We conclude that, while maintaining homeostasis, swift transporter regulation within a polarised tissue context is critical to prevent intrinsic traffic-jam like behaviour of nutrient flow.

SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2

NASA Astrophysics Data System (ADS)

Naudin, Cécile; Sirvent, Audrey; Leroy, Cédric; Larive, Romain; Simon, Valérie; Pannequin, Julie; Bourgaux, Jean-François; Pierre, Josiane; Robert, Bruno; Hollande, Frédéric; Roche, Serge

2014-01-01

The adaptor SLAP is a negative regulator of receptor signalling in immune cells but its role in human cancer is ill defined. Here we report that SLAP is abundantly expressed in healthy epithelial intestine but strongly downregulated in 50% of colorectal cancer. SLAP overexpression suppresses cell tumorigenicity and invasiveness while SLAP silencing enhances these transforming properties. Mechanistically, SLAP controls SRC/EPHA2/AKT signalling via destabilization of the SRC substrate and receptor tyrosine kinase EPHA2. This activity is independent from CBL but requires SLAP SH3 interaction with the ubiquitination factor UBE4A and SLAP SH2 interaction with pTyr594-EPHA2. SRC phosphorylates EPHA2 on Tyr594, thus creating a feedback loop that promotes EPHA2 destruction and thereby self-regulates its transforming potential. SLAP silencing enhances SRC oncogenicity and sensitizes colorectal tumour cells to SRC inhibitors. Collectively, these data establish a tumour-suppressive role for SLAP in colorectal cancer and a mechanism of SRC oncogenic induction through stabilization of its cognate substrates.

RIP2: A novel player in the regulation of keratinocyte proliferation and cutaneous wound repair?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adams, Stephanie; Valchanova, Ralitsa S.; Munz, Barbara, E-mail: barbara.munz@charite.de

2010-03-10

We could recently demonstrate an important role of receptor interacting protein 4 (RIP4) in the regulation of keratinocyte differentiation. Now, we analyzed a potential role of the RIP4 homolog RIP2 in keratinocytes. Specifically, we demonstrate here that rip2 expression is induced by scratch-wounding and after the induction of differentiation in these cells. Furthermore, serum growth factors and cytokines can induce rip2, with TNF-{alpha}-dependent induction being dependent on p38 MAPK. In addition, we demonstrate that scratch-induced upregulation of rip2 expression is completely blocked by the steroid dexamethasone. Since we also show that RIP2 is an important player in the regulation ofmore » keratinocyte proliferation, these data suggest that inhibition of rip2 upregulation after wounding might contribute to the reduced and delayed wound re-epithelialization phenotype seen in glucocorticoid-treated patients.« less

P-glycoprotein induction in Caco-2 cells by newly synthetized thioxanthones prevents paraquat cytotoxicity.

PubMed

Silva, Renata; Palmeira, Andreia; Carmo, Helena; Barbosa, Daniel José; Gameiro, Mariline; Gomes, Ana; Paiva, Ana Mafalda; Sousa, Emília; Pinto, Madalena; Bastos, Maria de Lourdes; Remião, Fernando

2015-10-01

The induction of P-glycoprotein (P-gp), an ATP-dependent efflux pump, has been proposed as a strategy against the toxicity induced by P-gp substrates such as the herbicide paraquat (PQ). The aim of this study was to screen five newly synthetized thioxanthonic derivatives, a group known to interact with P-gp, as potential inducers of the pump's expression and/or activity and to evaluate whether they would afford protection against PQ-induced toxicity in Caco-2 cells. All five thioxanthones (20 µM) caused a significant increase in both P-gp expression and activity as evaluated by flow cytometry using the UIC2 antibody and rhodamine 123, respectively. Additionally, it was demonstrated that the tested compounds, when present only during the efflux of rhodamine 123, rapidly induced an activation of P-gp. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, indicating that all derivatives acted as P-gp substrates. PQ cytotoxicity was significantly reduced in the presence of four thioxanthone derivatives, and this protective effect was reversed upon incubation with a specific P-gp inhibitor. In silico studies showed that all the tested thioxanthones fitted onto a previously described three-feature P-gp induction pharmacophore. Moreover, in silico interactions between thioxanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. Based on the in vitro activation results, a pharmacophore model for P-gp activation was built, which will be of further use in the screening for new P-gp activators. In conclusion, the study demonstrated the potential of the tested thioxanthonic compounds in protecting against toxic effects induced by P-gp substrates through P-gp induction and activation.

microRNA-206 in Rat Medial Prefrontal Cortex Regulates BDNF Expression and Alcohol Drinking

PubMed Central

Barbier, Estelle; Flanigan, Meghan; Solomon, Matthew; Pincus, Alexandra; Pilling, Andrew; Sun, Hui; Schank, Jesse R.; King, Courtney; Heilig, Markus

2014-01-01

Escalation of voluntary alcohol consumption is a hallmark of alcoholism, but its neural substrates remain unknown. In rats, escalation occurs following prolonged exposure to cycles of alcohol intoxication, and is associated with persistent, wide-ranging changes in gene expression within the medial prefrontal cortex (mPFC). Here, we examined whether induction of microRNA (miR) 206 in mPFC contributes to escalated alcohol consumption. Following up on a microarray screen, quantitative real-time reverse transcription PCR (qPCR) confirmed that a history of dependence results in persistent (>3weeks) up-regulation of miR-206 expression in the mPFC, but not in the ventral tegmental area, amygdala, or nucleus accumbens. Viral-mediated overexpression of miR-206 in the mPFC of nondependent rats reproduced the escalation of alcohol self-administration seen following a history of dependence and significantly inhibited BDNF expression. Bioinformatic analysis identified three conserved target sites for miR-206 in the 3′-UTR of the rat BDNF transcript. Accordingly, BDNF was downregulated in post-dependent rats on microarray analysis, and this was confirmed by qPCR. In vitro, BDNF expression was repressed by miR-206 but not miR-9 in a 3′-UTR reporter assay, confirming BDNF as a functional target of miR-206. Mutation analysis showed that repression was dependent on the presence of all three miR-206 target sites in the BDNF 3′-UTR. Inhibition of miR-206 expression in differentiated rat cortical primary neurons significantly increased secreted levels of BDNF. In conclusion, recruitment of miR-206 in the mPFC contributes to escalated alcohol consumption following a history of dependence, with BDNF as a possible mediator of its action. PMID:24672003

microRNA-206 in rat medial prefrontal cortex regulates BDNF expression and alcohol drinking.

PubMed

Tapocik, Jenica D; Barbier, Estelle; Flanigan, Meghan; Solomon, Matthew; Pincus, Alexandra; Pilling, Andrew; Sun, Hui; Schank, Jesse R; King, Courtney; Heilig, Markus

2014-03-26

Escalation of voluntary alcohol consumption is a hallmark of alcoholism, but its neural substrates remain unknown. In rats, escalation occurs following prolonged exposure to cycles of alcohol intoxication, and is associated with persistent, wide-ranging changes in gene expression within the medial prefrontal cortex (mPFC). Here, we examined whether induction of microRNA (miR) 206 in mPFC contributes to escalated alcohol consumption. Following up on a microarray screen, quantitative real-time reverse transcription PCR (qPCR) confirmed that a history of dependence results in persistent (>3weeks) up-regulation of miR-206 expression in the mPFC, but not in the ventral tegmental area, amygdala, or nucleus accumbens. Viral-mediated overexpression of miR-206 in the mPFC of nondependent rats reproduced the escalation of alcohol self-administration seen following a history of dependence and significantly inhibited BDNF expression. Bioinformatic analysis identified three conserved target sites for miR-206 in the 3'-UTR of the rat BDNF transcript. Accordingly, BDNF was downregulated in post-dependent rats on microarray analysis, and this was confirmed by qPCR. In vitro, BDNF expression was repressed by miR-206 but not miR-9 in a 3'-UTR reporter assay, confirming BDNF as a functional target of miR-206. Mutation analysis showed that repression was dependent on the presence of all three miR-206 target sites in the BDNF 3'-UTR. Inhibition of miR-206 expression in differentiated rat cortical primary neurons significantly increased secreted levels of BDNF. In conclusion, recruitment of miR-206 in the mPFC contributes to escalated alcohol consumption following a history of dependence, with BDNF as a possible mediator of its action.

Microtubule-dependent regulation of mitotic protein degradation

PubMed Central

Song, Ling; Craney, Allison; Rape, Michael

2014-01-01

Accurate cell division depends on tightly regulated ubiquitylation events catalyzed by the anaphase-promoting complex. Among its many substrates, the APC/C triggers the degradation of proteins that stabilize the mitotic spindle, and loss or accumulation of such spindle assembly factors can result in aneuploidy and cancer. Although critical for cell division, it has remained poorly understood how the timing of spindle assembly factor degradation is established during mitosis. Here, we report that active spindle assembly factors are protected from APC/C-dependent degradation by microtubules. In contrast, those molecules that are not bound to microtubules are highly susceptible to proteolysis and turned over immediately after APC/C-activation. The correct timing of spindle assembly factor degradation, as achieved by this regulatory circuit, is required for accurate spindle structure and function. We propose that the localized stabilization of APC/C-substrates provides a mechanism for the selective disposal of cell cycle regulators that have fulfilled their mitotic roles. PMID:24462202

Uniform modeling of bacterial colony patterns with varying nutrient and substrate

NASA Astrophysics Data System (ADS)

Schwarcz, Deborah; Levine, Herbert; Ben-Jacob, Eshel; Ariel, Gil

2016-04-01

Bacteria develop complex patterns depending on growth condition. For example, Bacillus subtilis exhibit five different patterns depending on substrate hardness and nutrient concentration. We present a unified integro-differential model that reproduces the entire experimentally observed morphology diagram at varying nutrient concentrations and substrate hardness. The model allows a comprehensive and quantitative comparison between experimental and numerical variables and parameters, such as colony growth rate, nutrient concentration and diffusion constants. As a result, the role of the different physical mechanisms underlying and regulating the growth of the colony can be evaluated.

Elk-3 is a transcriptional repressor of nitric-oxide synthase 2.

PubMed

Chen, Yen-Hsu; Layne, Matthew D; Chung, Su Wol; Ejima, Kuniaki; Baron, Rebecca M; Yet, Shaw-Fang; Perrella, Mark A

2003-10-10

The inducible isoform of nitric-oxide synthase (NOS2), a key enzyme catalyzing the dramatic increase in nitric oxide by lipopolysaccharide (LPS), plays an important role in the pathophysiology of endotoxemia and sepsis. Recent evidence suggests that Ets transcription factors may contribute to NOS2 induction by inflammatory stimuli. In this study, we investigated the role of Ets transcription factors in the regulation of NOS2 by LPS and transforming growth factor (TGF)-beta 1. Transient transfection assays in macrophages showed that Ets-2 produced an increase in NOS2 promoter activity, whereas the induction by Ets-1 was modest and NERF2 had no effect. Elk-3 (Net/Erp/Sap-2a) markedly repressed NOS2 promoter activity in a dose-dependent fashion, and overexpression of Elk-3 blunted the induction of endogenous NOS2 message. Mutation of the Net inhibitory domain of Elk-3, but not the C-terminal-binding protein interaction domain, partially alleviated this repressive effect. We also found that deletion of the Ets domain of Elk-3 completely abolished its repressive effect on the NOS2 promoter. LPS administration to macrophages led to a dose-dependent decrease in endogenous Elk-3 mRNA levels, and this decrease in Elk-3 preceded the induction of NOS2 mRNA. In a mouse model of endotoxemia, the expression of Elk-3 in kidney, lung, and heart was significantly down-regulated after systemic administration of LPS, and this down-regulation also preceded NOS2 induction. Moreover, TGF-beta 1 significantly increased endogenous Elk-3 mRNA levels that had been down-regulated by LPS in macrophages. This increase in Elk-3 correlated with a TGF-beta 1-induced down-regulation of NOS2. Taken together, our data suggest that Elk-3 is a strong repressor of NOS2 promoter activity and mRNA levels and that endogenous expression of Elk-3 inversely correlates with NOS2. Thus, Elk-3 may serve as an important mediator of NOS2 gene expression.

Light Regulation of the Arabidopsis Respiratory Chain. Multiple Discrete Photoreceptor Responses Contribute to Induction of Type II NAD(P)H Dehydrogenase Genes1

PubMed Central

Escobar, Matthew A.; Franklin, Keara A.; Svensson, Å. Staffan; Salter, Michael G.; Whitelam, Garry C.; Rasmusson, Allan G.

2004-01-01

Controlled oxidation reactions catalyzed by the large, proton-pumping complexes of the respiratory chain generate an electrochemical gradient across the mitochondrial inner membrane that is harnessed for ATP production. However, several alternative respiratory pathways in plants allow the maintenance of substrate oxidation while minimizing the production of ATP. We have investigated the role of light in the regulation of these energy-dissipating pathways by transcriptional profiling of the alternative oxidase, uncoupling protein, and type II NAD(P)H dehydrogenase gene families in etiolated Arabidopsis seedlings. Expression of the nda1 and ndc1 NAD(P)H dehydrogenase genes was rapidly up-regulated by a broad range of light intensities and qualities. For both genes, light induction appears to be a direct transcriptional effect that is independent of carbon status. Mutant analyses demonstrated the involvement of two separate photoreceptor families in nda1 and ndc1 light regulation: the phytochromes (phyA and phyB) and an undetermined blue light photoreceptor. In the case of the nda1 gene, the different photoreceptor systems generate distinct kinetic induction profiles that are integrated in white light response. Primary transcriptional control of light response was localized to a 99-bp region of the nda1 promoter, which contains an I-box flanked by two GT-1 elements, an arrangement prevalent in the promoters of photosynthesis-associated genes. Light induction was specific to nda1 and ndc1. The only other substantial light effect observed was a decrease in aox2 expression. Overall, these results suggest that light directly influences the respiratory electron transport chain via photoreceptor-mediated transcriptional control, likely for supporting photosynthetic metabolism. PMID:15333756

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palmieri, D.; Valli, M.; Viglio, S.

2010-03-10

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase ofmore » maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.« less

Ethanol Induction of CYP2A5: Role of CYP2E1-ROS-Nrf2 Pathway

PubMed Central

Lu, Yongke; Zhang, Xu Hannah

2012-01-01

Chronic ethanol consumption was previously shown to induce CYP2A5 in mice, and this induction of CYP2A5 by ethanol was CYP2E1 dependent. In this study, the mechanisms of CYP2E1-dependent ethanol induction of CYP2A5 were investigated. CYP2E1 was induced by chronic ethanol consumption to the same degree in wild-type (WT) mice and CYP2A5 knockout (Cyp2a5 –/–) mice, suggesting that unlike the CYP2E1-dependent ethanol induction of CYP2A5, ethanol induction of CYP2E1 is not CYP2A5 dependent. Microsomal ethanol oxidation was about 25% lower in Cyp2a5 –/– mice compared with that in WT mice, suggesting that CYP2A5 can oxidize ethanol although to a lesser extent than CYP2E1 does. CYP2A5 was induced by short-term ethanol consumption in human CYP2E1 transgenic knockin (Cyp2e1 –/– KI) mice but not in CYP2E1 knockout (Cyp2e1 –/–) mice. The redox-sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) was also induced by acute ethanol in Cyp2e1 –/– KI mice but not in Cyp2e1 –/– mice. Ethanol induction of CYP2A5 in Nrf2 knockout (Nrf2 –/–) mice was lower compared with that in WT mice, whereas CYP2E1 induction by ethanol was comparable in WT and Nrf2 –/– mice. Antioxidants (N-acetyl-cysteine and vitamin C), which blocked oxidative stress induced by chronic ethanol in WT mice and acute ethanol in Cyp2e1 –/– KI mice, also blunted the induction of CYP2A5 and Nrf2 by ethanol but not the induction of CYP2E1 by ethanol. These results suggest that oxidative stress induced by ethanol via induction of CYP2E1 upregulates Nrf2 activity, which in turn regulates ethanol induction of CYP2A5. Results obtained from primary hepatocytes, mice gavaged with binge ethanol or fed chronic ethanol, show that Nrf2-regulated ethanol induction of CYP2A5 protects against ethanol-induced steatosis. PMID:22552773

Tilapia (Oreochromis mossambicus) brain cells respond to hyperosmotic challenge by inducing myo-inositol biosynthesis

PubMed Central

Gardell, Alison M.; Yang, Jun; Sacchi, Romina; Fangue, Nann A.; Hammock, Bruce D.; Kültz, Dietmar

2013-01-01

SUMMARY This study aimed to determine the regulation of the de novo myo-inositol biosynthetic (MIB) pathway in Mozambique tilapia (Oreochromis mossambicus) brain following acute (25 ppt) and chronic (30, 60 and 90 ppt) salinity acclimations. The MIB pathway plays an important role in accumulating the compatible osmolyte, myo-inositol, in cells in response to hyperosmotic challenge and consists of two enzymes, myo-inositol phosphate synthase and inositol monophosphatase. In tilapia brain, MIB enzyme transcriptional regulation was found to robustly increase in a time (acute acclimation) or dose (chronic acclimation) dependent manner. Blood plasma osmolality and Na+ and Cl− concentrations were also measured and significantly increased in response to both acute and chronic salinity challenges. Interestingly, highly significant positive correlations were found between MIB enzyme mRNA and blood plasma osmolality in both acute and chronic salinity acclimations. Additionally, a mass spectrometry assay was established and used to quantify total myo-inositol concentration in tilapia brain, which closely mirrored the hyperosmotic MIB pathway induction. Thus, myo-inositol is a major compatible osmolyte that is accumulated in brain cells when exposed to acute and chronic hyperosmotic challenge. These data show that the MIB pathway is highly induced in response to environmental salinity challenge in tilapia brain and that this induction is likely prompted by increases in blood plasma osmolality. Because the MIB pathway uses glucose-6-phosphate as a substrate and large amounts of myo-inositol are being synthesized, our data also illustrate that the MIB pathway likely contributes to the high energetic demand posed by salinity challenge. PMID:24072790

Mechanically induced c-fos expression is mediated by cAMP in MC3T3-E1 osteoblasts

NASA Technical Reports Server (NTRS)

Fitzgerald, J.; Hughes-Fulford, M.

1999-01-01

In serum-deprived MC3T3-E1 osteoblasts, mechanical stimulation caused by mild (287 x g) centrifugation induced a 10-fold increase in mRNA levels of the proto-oncogene, c-fos. Induction of c-fos was abolished by the cAMP-dependent protein kinase inhibitor H-89, suggesting that the transient c-fos mRNA increase is mediated by cAMP. Down-regulation of protein kinase C (PKC) activity by chronic TPA treatment failed to significantly reduce c-fos induction, suggesting that TPA-sensitive isoforms of PKC are not responsible for c-fos up-regulation. In addition, 287 x g centrifugation increased intracellular prostaglandin E2 (PGE2) levels 2.8-fold (P<0. 005). Since we have previously shown that prostaglandin E2 (PGE2) can induce c-fos expression via a cAMP-mediated mechanism, we asked whether the increase in c-fos mRNA was due to centrifugation-induced PGE2 release. Pretreatment with the cyclooxygenase inhibitors indomethacin and flurbiprofen did not hinder the early induction of c-fos by mechanical stimulation. We conclude that c-fos expression induced by mild mechanical loading is dependent primarily on cAMP, not PKC, and initial induction of c-fos is not necessarily dependent on the action of newly synthesized PGE2.

Current opinion in Microbiology Roles of adaptor proteins in regulation of bacterial proteolysis

PubMed Central

Battesti, Aurelia; Gottesman, Susan

2013-01-01

Elimination of non-functional or unwanted proteins is critical for cell growth and regulation. In bacteria, ATP-dependent proteases target cytoplasmic proteins for degradation, contributing to both protein quality control and regulation of specific proteins, thus playing roles parallel to that of the proteasome in eukaryotic cells. Adaptor proteins provide a way to modulate the substrate specificity of the proteases and allow regulated proteolysis. Advances over the past few years have provided new insight into how adaptor proteins interact with both substrates and proteases and how adaptor functions are regulated. An important advance has come with the recognition of the critical roles of anti-adaptor proteins in regulating adaptor availability. PMID:23375660

Initial reactions involved in the dissimilation of mandelate by Rhodotorula graminis.

PubMed Central

Durham, D R

1984-01-01

Rhodotorula graminis utilized DL-mandelate, L(+)-mandelate, and D(-)-mandelate as sole sources of carbon and energy. Growth on these aromatic substrates resulted in the induction of an NAD-dependent D(-)-mandelate dehydrogenase and a dye-linked L(+)-mandelate dehydrogenase, each catalyzing the stereospecific conversion of its respective enantiomer of mandelate to benzoylformate. Benzoylformate was oxidized to benzaldehyde, which was dehydrogenated to benzoate by an NAD-dependent benzaldehyde dehydrogenase. Benzoate was further metabolized through p-hydroxybenzoate and the protocatechuate branch of the beta-ketoadipate pathway. PMID:6389497

The role of carbon starvation in the induction of enzymes that degrade plant-derived carbohydrates in Aspergillus niger

PubMed Central

van Munster, Jolanda M.; Daly, Paul; Delmas, Stéphane; Pullan, Steven T.; Blythe, Martin J.; Malla, Sunir; Kokolski, Matthew; Noltorp, Emelie C.M.; Wennberg, Kristin; Fetherston, Richard; Beniston, Richard; Yu, Xiaolan; Dupree, Paul; Archer, David B.

2014-01-01

Fungi are an important source of enzymes for saccharification of plant polysaccharides and production of biofuels. Understanding of the regulation and induction of expression of genes encoding these enzymes is still incomplete. To explore the induction mechanism, we analysed the response of the industrially important fungus Aspergillus niger to wheat straw, with a focus on events occurring shortly after exposure to the substrate. RNA sequencing showed that the transcriptional response after 6 h of exposure to wheat straw was very different from the response at 24 h of exposure to the same substrate. For example, less than half of the genes encoding carbohydrate active enzymes that were induced after 24 h of exposure to wheat straw, were also induced after 6 h exposure. Importantly, over a third of the genes induced after 6 h of exposure to wheat straw were also induced during 6 h of carbon starvation, indicating that carbon starvation is probably an important factor in the early response to wheat straw. The up-regulation of the expression of a high number of genes encoding CAZymes that are active on plant-derived carbohydrates during early carbon starvation suggests that these enzymes could be involved in a scouting role during starvation, releasing inducing sugars from complex plant polysaccharides. We show, using proteomics, that carbon-starved cultures indeed release CAZymes with predicted activity on plant polysaccharides. Analysis of the enzymatic activity and the reaction products, indicates that these proteins are enzymes that can degrade various plant polysaccharides to generate both known, as well as potentially new, inducers of CAZymes. PMID:24792495

The role of carbon starvation in the induction of enzymes that degrade plant-derived carbohydrates in Aspergillus niger.

PubMed

van Munster, Jolanda M; Daly, Paul; Delmas, Stéphane; Pullan, Steven T; Blythe, Martin J; Malla, Sunir; Kokolski, Matthew; Noltorp, Emelie C M; Wennberg, Kristin; Fetherston, Richard; Beniston, Richard; Yu, Xiaolan; Dupree, Paul; Archer, David B

2014-11-01

Fungi are an important source of enzymes for saccharification of plant polysaccharides and production of biofuels. Understanding of the regulation and induction of expression of genes encoding these enzymes is still incomplete. To explore the induction mechanism, we analysed the response of the industrially important fungus Aspergillus niger to wheat straw, with a focus on events occurring shortly after exposure to the substrate. RNA sequencing showed that the transcriptional response after 6h of exposure to wheat straw was very different from the response at 24h of exposure to the same substrate. For example, less than half of the genes encoding carbohydrate active enzymes that were induced after 24h of exposure to wheat straw, were also induced after 6h exposure. Importantly, over a third of the genes induced after 6h of exposure to wheat straw were also induced during 6h of carbon starvation, indicating that carbon starvation is probably an important factor in the early response to wheat straw. The up-regulation of the expression of a high number of genes encoding CAZymes that are active on plant-derived carbohydrates during early carbon starvation suggests that these enzymes could be involved in a scouting role during starvation, releasing inducing sugars from complex plant polysaccharides. We show, using proteomics, that carbon-starved cultures indeed release CAZymes with predicted activity on plant polysaccharides. Analysis of the enzymatic activity and the reaction products, indicates that these proteins are enzymes that can degrade various plant polysaccharides to generate both known, as well as potentially new, inducers of CAZymes. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors

PubMed Central

Whisenant, Thomas C.; Ho, David T.; Benz, Ryan W.; Rogers, Jeffrey S.; Kaake, Robyn M.; Gordon, Elizabeth A.; Huang, Lan; Baldi, Pierre; Bardwell, Lee

2010-01-01

In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new ‘D-site’ class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates. PMID:20865152

Gallium nitride heterostructures on 3D structured silicon.

PubMed

Fündling, Sönke; Sökmen, Unsal; Peiner, Erwin; Weimann, Thomas; Hinze, Peter; Jahn, Uwe; Trampert, Achim; Riechert, Henning; Bakin, Andrey; Wehmann, Hergo-Heinrich; Waag, Andreas

2008-10-08

We investigated GaN-based heterostructures grown on three-dimensionally patterned Si(111) substrates by metal organic vapour phase epitaxy, with the goal of fabricating well controlled high quality, defect reduced GaN-based nanoLEDs. The high aspect ratios of such pillars minimize the influence of the lattice mismatched substrate and improve the material quality. In contrast to other approaches, we employed deep etched silicon substrates to achieve a controlled pillar growth. For that a special low temperature inductively coupled plasma etching process has been developed. InGaN/GaN multi-quantum-well structures have been incorporated into the pillars. We found a pronounced dependence of the morphology of the GaN structures on the size and pitch of the pillars. Spatially resolved optical properties of the structures are analysed by cathodoluminescence.

Cellular Links between Neuronal Activity and Energy Homeostasis.

PubMed

Shetty, Pavan K; Galeffi, Francesca; Turner, Dennis A

2012-01-01

Neuronal activity, astrocytic responses to this activity, and energy homeostasis are linked together during baseline, conscious conditions, and short-term rapid activation (as occurs with sensory or motor function). Nervous system energy homeostasis also varies during long-term physiological conditions (i.e., development and aging) and with adaptation to pathological conditions, such as ischemia or low glucose. Neuronal activation requires increased metabolism (i.e., ATP generation) which leads initially to substrate depletion, induction of a variety of signals for enhanced astrocytic function, and increased local blood flow and substrate delivery. Energy generation (particularly in mitochondria) and use during ATP hydrolysis also lead to considerable heat generation. The local increases in blood flow noted following neuronal activation can both enhance local substrate delivery but also provides a heat sink to help cool the brain and removal of waste by-products. In this review we highlight the interactions between short-term neuronal activity and energy metabolism with an emphasis on signals and factors regulating astrocyte function and substrate supply.

Induction of a feed forward pro-apoptotic mechanistic loop by nitric oxide in a human breast cancer model.

PubMed

Sen, Suvajit; Kawahara, Brian; Fukuto, Jon; Chaudhuri, Gautam

2013-01-01

We have previously demonstrated that relatively high concentrations of NO [Nitric Oxide] as produced by activated macrophages induced apoptosis in the human breast cancer cell line, MDA-MB-468. More recently, we also demonstrated the importance of endogenous H2O2 in the regulation of growth in human breast cancer cells. In the present study we assessed the interplay between exogenously administered NO and the endogenously produced reactive oxygen species [ROS] in human breast cancer cells and evaluated the mechanism[s] in the induction of apoptosis. To this end we identified a novel mechanism by which NO down regulated endogenous hydrogen peroxide [H2O2] formation via the down-regulation of superoxide [O2 (.-)] and the activation of catalase. We further demonstrated the existence of a feed forward mechanistic loop involving protein phosphatase 2A [PP2A] and its downstream substrate FOXO1 in the induction of apoptosis and the synthesis of catalase. We utilized gene silencing of PP2A, FOXO1 and catalase to assess their relative importance and key roles in NO mediated apoptosis. This study provides the potential for a therapeutic approach in treating breast cancer by targeted delivery of NO where NO donors and activators of downstream players could initiate a self sustaining apoptotic cascade in breast cancer cells.

Induction of a Feed Forward Pro-Apoptotic Mechanistic Loop by Nitric Oxide in a Human Breast Cancer Model

PubMed Central

Sen, Suvajit; Kawahara, Brian; Fukuto, Jon; Chaudhuri, Gautam

2013-01-01

We have previously demonstrated that relatively high concentrations of NO [Nitric Oxide] as produced by activated macrophages induced apoptosis in the human breast cancer cell line, MDA-MB-468. More recently, we also demonstrated the importance of endogenous H2O2 in the regulation of growth in human breast cancer cells. In the present study we assessed the interplay between exogenously administered NO and the endogenously produced reactive oxygen species [ROS] in human breast cancer cells and evaluated the mechanism[s] in the induction of apoptosis. To this end we identified a novel mechanism by which NO down regulated endogenous hydrogen peroxide [H2O2] formation via the down-regulation of superoxide [O2 .−] and the activation of catalase. We further demonstrated the existence of a feed forward mechanistic loop involving protein phosphatase 2A [PP2A] and its downstream substrate FOXO1 in the induction of apoptosis and the synthesis of catalase. We utilized gene silencing of PP2A, FOXO1 and catalase to assess their relative importance and key roles in NO mediated apoptosis. This study provides the potential for a therapeutic approach in treating breast cancer by targeted delivery of NO where NO donors and activators of downstream players could initiate a self sustaining apoptotic cascade in breast cancer cells. PMID:23950968

CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes.

PubMed

Price, R J; Surry, D; Renwick, A B; Meneses-Lorente, G; Lake, B G; Evans, D C

2000-08-01

1. In this study, 7-benzyloxy-4-trifluoromethylcoumarin (BFC) was evaluated as a substrate to assess the induction of cytochrome P450 (CYP) isoform enzyme activities in rat hepatocytes using a 96-well plate format. 2. BFC was metabolized by both untreated and sodium phenobarbitone (NaPB)-treated rat hepatocytes in a time- and concentration-dependent manner to the highly fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC). 3. HFC was extensively conjugated with D-glucuronic acid and/or sulphate in both untreated and NaPB-treated rat hepatocytes, thus necessitating the inclusion of an enzymatic deconjugation step in the assay procedure. 4. The time-course of induction of 7-ethoxyresorufin metabolism by the CYP1A inducer beta-naphthoflavone (BNF), 7-benzyloxyresorufin metabolism by the CYP2B inducer NaPB and BFC metabolism b both BNF and NaPB was studied in rat hepatocytes treated for 24-96 h. The optimal time for induction of metabolism of all three substrates was 72 h, with no medium changes being necessary during this period. 5. The effect of treatment with 0.5-20 microM BNF, 50-2000 microM NaPB, 2-20 microM dexamethasone (DEX), 20-100 microM methylclofenapate (MCP), and 50 and 200 microM isoniazid (ISN) for 72 h on BFC metabolism in cultured rat hepatocytes was studied. BFC metabolism was induced by treatment with BNF, NaPB and MCP, but not with either DEX or ISN. 6. The metabolism of BFC in liver microsomes from the control rat and rat treated with CYP isoform inducers was also studied. BFC metabolism was induced by treatment with NaPB, BNF and DEX. 7. The metabolism of BFC was also studied using microsomes from baculovirus-infected insect cells containing rat cDNA-expressed CYP1A, CYP2B, CYP2C and CYP3A isoforms. Whereas BFC was metabolized to some extent by all the rat cDNA-expressed CYP isoforms examined, at a substrate concentration of 2.5 microM the greatest rates of BFC metabolism were observed with the CYP1A1, CYP1A2 and CYP2B1 preparations. 8. In summary, the results demonstrate that BFC is a good substrate for assessing the induction of CYP1A and CYP2B isoforms in rat hepatocytes in a 96-well plate format.

PECVD Growth of Carbon Nanotubes

NASA Technical Reports Server (NTRS)

McAninch, Ian; Arnold, James O. (Technical Monitor)

2001-01-01

Plasma enhanced chemical vapor deposition (PECVD), using inductively coupled plasma, has been used to grow carbon nanotubes (CNTs) and graphitic carbon fibers (GCF) on substrates sputtered with aluminum and iron catalyst. The capacitive plasma's power has been shown to cause a transition from nanotubes to nanofibers, depending on the strength of the plasma. The temperature, placement, and other factors have been shown to affect the height and density of the tube and fiber growth.

SILAC-based phosphoproteomics reveals new PP2A-Cdc55-regulated processes in budding yeast.

PubMed

Baro, Barbara; Játiva, Soraya; Calabria, Inés; Vinaixa, Judith; Bech-Serra, Joan-Josep; de LaTorre, Carolina; Rodrigues, João; Hernáez, María Luisa; Gil, Concha; Barceló-Batllori, Silvia; Larsen, Martin R; Queralt, Ethel

2018-05-01

Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A-related processes in mitotic arrested cells. We identified 62 statistically significant PP2ACdc55 substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2ACdc55 substrates such as Slk19 and Lte1, involved in early and late anaphase pathways, and Zeo1, a component of the cell wall integrity pathway. Finally, we constructed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction. We used phosphoproteomics of Cdc55-deficient cells to uncover new PP2ACdc55 substrates and functions in mitosis. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases' consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome.

Redox-Regulated Pathway of Tyrosine Phosphorylation Underlies NF-κB Induction by an Atypical Pathway Independent of the 26S Proteasome

PubMed Central

Cullen, Sarah; Ponnappan, Subramaniam; Ponnappan, Usha

2015-01-01

Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated. PMID:25671697

Hydrophobically stabilized open state for the lateral gate of the Sec translocon

PubMed Central

Zhang, Bin; Miller, Thomas F.

2010-01-01

The Sec translocon is a central component of cellular pathways for protein translocation and membrane integration. Using both atomistic and coarse-grained molecular simulations, we investigate the conformational landscape of the translocon and explore the role of peptide substrates in the regulation of the translocation and integration pathways. Inclusion of a hydrophobic peptide substrate in the translocon stabilizes the opening of the lateral gate for membrane integration, whereas a hydrophilic peptide substrate favors the closed lateral gate conformation. The relative orientation of the plug moiety and a peptide substrate within the translocon channel is similarly dependent on whether the substrate is hydrophobic or hydrophilic in character, and the energetics of the translocon lateral gate opening in the presence of a peptide substrate is governed by the energetics of the peptide interface with the membrane. Implications of these results for the regulation of Sec-mediated pathways for protein translocation vs. membrane integration are discussed. PMID:20203009

Stable Isotope Metabolic Labeling-based Quantitative Phosphoproteomic Analysis of Arabidopsis Mutants Reveals Ethylene-regulated Time-dependent Phosphoproteins and Putative Substrates of Constitutive Triple Response 1 Kinase*

PubMed Central

Yang, Zhu; Guo, Guangyu; Zhang, Manyu; Liu, Claire Y.; Hu, Qin; Lam, Henry; Cheng, Han; Xue, Yu; Li, Jiayang; Li, Ning

2013-01-01

Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on 15N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a protein–protein interaction filter revealed a total of 14 kinase–substrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light harvesting chlorophyll A/B binding protein 1.1, and flowering bHLH 3 proteins in a dual-and-opposing fashion. PMID:24043427

Dynamically Babinet-invertible metasurface: a capacitive-inductive reconfigurable filter for terahertz waves using vanadium-dioxide metal-insulator transition

NASA Astrophysics Data System (ADS)

Urade, Yoshiro; Nakata, Yosuke; Okimura, Kunio; Nakanishi, Toshihiro; Miyamaru, Fumiaki; Takeda, Mitsuo W.; Kitano, Masao

2016-03-01

This paper proposes a reconfigurable planar metamaterial that can be switched between capacitive and inductive responses using local changes in the electrical conductivity of its constituent material. The proposed device is based on Babinet's principle and exploits the singular electromagnetic responses of metallic checkerboard structures, which are dependent on the local electrical conductivity. Utilizing the heating-induced metal-insulator transition of vanadium dioxide ($\\mathrm{VO}_2$), the proposed metamaterial is designed to compensate for the effect of the substrate and is experimentally characterized in the terahertz regime. This reconfigurable metamaterial can be utilized as a switchable filter and as a switchable phase shifter for terahertz waves.

Dynamically Babinet-invertible metasurface: a capacitive-inductive reconfigurable filter for terahertz waves using vanadium-dioxide metal-insulator transition.

PubMed

Urade, Yoshiro; Nakata, Yosuke; Okimura, Kunio; Nakanishi, Toshihiro; Miyamaru, Fumiaki; Takeda, Mitsuo W; Kitano, Masao

2016-03-07

This paper proposes a reconfigurable planar metamaterial that can be switched between capacitive and inductive responses using local changes in the electrical conductivity of its constituent material. The proposed device is based on Babinet's principle and exploits the singular electromagnetic responses of metallic checkerboard structures, which are dependent on the local electrical conductivity. Utilizing the heating-induced metal-insulator transition of vanadium dioxide (VO 2 ), the proposed meta-material is designed to compensate for the effect of the substrate and is experimentally characterized in the terahertz regime. This reconfigurable metamaterial can be utilized as a switchable filter and as a switchable phase shifter for terahertz waves.

Myostatin induces insulin resistance via Casitas B-lineage lymphoma b (Cblb)-mediated degradation of insulin receptor substrate 1 (IRS1) protein in response to high calorie diet intake.

PubMed

Bonala, Sabeera; Lokireddy, Sudarsanareddy; McFarlane, Craig; Patnam, Sreekanth; Sharma, Mridula; Kambadur, Ravi

2014-03-14

To date a plethora of evidence has clearly demonstrated that continued high calorie intake leads to insulin resistance and type-2 diabetes with or without obesity. However, the necessary signals that initiate insulin resistance during high calorie intake remain largely unknown. Our results here show that in response to a regimen of high fat or high glucose diets, Mstn levels were induced in muscle and liver of mice. High glucose- or fat-mediated induction of Mstn was controlled at the level of transcription, as highly conserved carbohydrate response and sterol-responsive (E-box) elements were present in the Mstn promoter and were revealed to be critical for ChREBP (carbohydrate-responsive element-binding protein) or SREBP1c (sterol regulatory element-binding protein 1c) regulation of Mstn expression. Further molecular analysis suggested that the increased Mstn levels (due to high glucose or fatty acid loading) resulted in increased expression of Cblb in a Smad3-dependent manner. Casitas B-lineage lymphoma b (Cblb) is an ubiquitin E3 ligase that has been shown to specifically degrade insulin receptor substrate 1 (IRS1) protein. Consistent with this, our results revealed that elevated Mstn levels specifically up-regulated Cblb, resulting in enhanced ubiquitin proteasome-mediated degradation of IRS1. In addition, over expression or knock down of Cblb had a major impact on IRS1 and pAkt levels in the presence or absence of insulin. Collectively, these observations strongly suggest that increased glucose levels and high fat diet, both, result in increased circulatory Mstn levels. The increased Mstn in turn is a potent inducer of insulin resistance by degrading IRS1 protein via the E3 ligase, Cblb, in a Smad3-dependent manner.

Deciphering how LIP2 and POX2 promoters can optimally regulate recombinant protein production in the yeast Yarrowia lipolytica.

PubMed

Sassi, Hosni; Delvigne, Frank; Kar, Tambi; Nicaud, Jean-Marc; Coq, Anne-Marie Crutz-Le; Steels, Sebastien; Fickers, Patrick

2016-09-20

In recent years, the non-conventional model yeast species Yarrowia lipolytica has received much attention because it is a useful cell factory for producing recombinant proteins. In this species, expression vectors involving LIP2 and POX2 promoters have been developed and used successfully for protein production at yields similar to or even higher than those of other cell factories, such as Pichia pastoris. However, production processes involving these promoters can be difficult to manage, especially if carried out at large scales in fed-batch bioreactors, because they require hydrophobic inducers, such as oleic acid or methyl oleate. Thus, the challenge has become to reduce loads of hydrophobic substrates while simultaneously promoting recombinant protein production. One possible solution is to replace a portion of the inducer with a co-substrate that can serve as an alternative energy source. However, implementing such an approach would require detailed knowledge of how carbon sources impact promoter regulation, which is surprisingly still lacking for the LIP2 and POX2 promoters. This study's aim was thus to better characterize promoter regulation and cell metabolism in Y. lipolytica cultures grown in media supplemented with different carbon sources. pPOX2 induction could be detected when glucose or glycerol was used as sole carbon source, which meant these carbon source could not prevent promoter induction. In addition, when a mixture of glucose and oleic acid was used in complex medium, pPOX2 induction level was lower that that of pLIP2. In contrast, pLIP2 induction was absent when glucose was present in the culture medium, which meant that cell growth could occur without any recombinant gene expression. When a 40/60 mixture of glucose and oleic acid (w/w) was used, a tenfold increase in promoter induction, as compared to when an oleic-acid-only medium was observed. It was also clear that individual cells were adapting metabolically to use both glucose and oleic acid. Indeed, no distinct subpopulations that specialized on glucose versus oleic acid were observed; such an outcome would have led to producer and non-producer phenotypes. In medium containing both glucose and oleic acid, cells tended to directly metabolize oleic acid instead of storing it in lipid bodies. This study found that pLIP2 is a promoter of choice as compared to pPOX2 to drive gene expression for recombinant protein production by Y. lipolytica used as cell factory.

Analysis of laser-induction hybrid cladding processing conditions

NASA Astrophysics Data System (ADS)

Huang, Yongjun; Zeng, Xiaoyan; Hu, Qianwu

2007-12-01

A new cladding approach based on laser-induction hybrid technique on flat sheets is presented in this paper. Coating is produced by means of 5kw cw CO II laser equipped with 100kw high frequent inductor, and the experiments set-up, involving a special machining-head, which can provide laser-induction hybrid heat resources simultaneously. The formation of thick NiCrSiB coating on a steel substrate by off-axial powder feeding is studied from an experimental point of view. A substrate melting energy model is developed to describe the energy relationship between laser-induction hybrid cladding and laser cladding alone quantitatively. By comparing the experimental results with the calculational ones, it is shown that the tendency of fusion zone height of theoretical calculation is in agreement with that of tests in laser-induction hybrid cladding. Via analyses and tests, the conclusions can be lead to that the fusion zone height can be increased easily and the good bond of cladding track can be achieved within wide cladding processing window in laser-induction hybrid processing. It shows that the induction heating has an obvious effect on substrate melting and metallurgical bond.

Surface design for controlled crystallization: the role of surface chemistry and nanoscale pores in heterogeneous nucleation.

PubMed

Diao, Ying; Myerson, Allan S; Hatton, T Alan; Trout, Bernhardt L

2011-05-03

Current industrial practice for control of primary nucleation (nucleation from a system without pre-existing crystalline matter) during crystallization from solution involves control of supersaturation generation, impurity levels, and solvent composition. Nucleation behavior remains largely unpredictable, however, due to the presence of container surfaces, dust, dirt, and other impurities that can provide heterogeneous nucleation sites, thus making the control and scale-up of processes that depend on primary nucleation difficult. To develop a basis for the rational design of surfaces to control nucleation during crystallization from solution, we studied the role of surface chemistry and morphology of various polymeric substrates on heterogeneous nucleation using aspirin as a model compound. Nucleation induction time statistics were utilized to investigate and quantify systematically the effectiveness of polymer substrates in inducing nucleation. The nucleation induction time study revealed that poly(4-acryloylmorpholine) and poly(2-carboxyethyl acrylate), each cross-linked by divinylbenzene, significantly lowered the nucleation induction time of aspirin while the other polymers were essentially inactive. In addition, we found the presence of nanoscopic pores on certain polymer surfaces led to order-of-magnitude faster aspirin nucleation rates when compared with surfaces without pores. We studied the preferred orientation of aspirin crystals on polymer films and found the nucleation-active polymer surfaces preferentially nucleated the polar facets of aspirin, guided by hydrogen bonds. A model based on interfacial free energies was also developed which predicted the same trend of polymer surface nucleation activities as indicated by the nucleation induction times.

Predictions of ion energy distributions and radical fluxes in radio frequency biased inductively coupled plasma etching reactors

NASA Astrophysics Data System (ADS)

Hoekstra, Robert J.; Kushner, Mark J.

1996-03-01

Inductively coupled plasma (ICP) reactors are being developed for low gas pressure (<10s mTorr) and high plasma density ([e]≳1011 cm-3) microelectronics fabrication. In these reactors, the plasma is generated by the inductively coupled electric field while an additional radio frequency (rf) bias is applied to the substrate. One of the goals of these systems is to independently control the magnitude of the ion flux by the inductively coupled power deposition, and the acceleration of ions into the substrate by the rf bias. In high plasma density reactors the width of the sheath above the wafer may be sufficiently thin that ions are able to traverse it in approximately 1 rf cycle, even at 13.56 MHz. As a consequence, the ion energy distribution (IED) may have a shape typically associated with lower frequency operation in conventional reactive ion etching tools. In this paper, we present results from a computer model for the IED incident on the wafer in ICP etching reactors. We find that in the parameter space of interest, the shape of the IED depends both on the amplitude of the rf bias and on the ICP power. The former quantity determines the average energy of the IED. The latter quantity controls the width of the sheath, the transit time of ions across the sheath and hence the width of the IED. In general, high ICP powers (thinner sheaths) produce wider IEDs.

Genetic variation of temperature-regulated curd induction in cauliflower: elucidation of floral transition by genome-wide association mapping and gene expression analysis

PubMed Central

Matschegewski, Claudia; Zetzsche, Holger; Hasan, Yaser; Leibeguth, Lena; Briggs, William; Ordon, Frank; Uptmoor, Ralf

2015-01-01

Cauliflower (Brassica oleracea var. botrytis) is a vernalization-responsive crop. High ambient temperatures delay harvest time. The elucidation of the genetic regulation of floral transition is highly interesting for a precise harvest scheduling and to ensure stable market supply. This study aims at genetic dissection of temperature-dependent curd induction in cauliflower by genome-wide association studies and gene expression analysis. To assess temperature-dependent curd induction, two greenhouse trials under distinct temperature regimes were conducted on a diversity panel consisting of 111 cauliflower commercial parent lines, genotyped with 14,385 SNPs. Broad phenotypic variation and high heritability (0.93) were observed for temperature-related curd induction within the cauliflower population. GWA mapping identified a total of 18 QTL localized on chromosomes O1, O2, O3, O4, O6, O8, and O9 for curding time under two distinct temperature regimes. Among those, several QTL are localized within regions of promising candidate flowering genes. Inferring population structure and genetic relatedness among the diversity set assigned three main genetic clusters. Linkage disequilibrium (LD) patterns estimated global LD extent of r2 = 0.06 and a maximum physical distance of 400 kb for genetic linkage. Transcriptional profiling of flowering genes FLOWERING LOCUS C (BoFLC) and VERNALIZATION 2 (BoVRN2) was performed, showing increased expression levels of BoVRN2 in genotypes with faster curding. However, functional relevance of BoVRN2 and BoFLC2 could not consistently be supported, which probably suggests to act facultative and/or might evidence for BoVRN2/BoFLC-independent mechanisms in temperature-regulated floral transition in cauliflower. Genetic insights in temperature-regulated curd induction can underpin genetically informed phenology models and benefit molecular breeding strategies toward the development of thermo-tolerant cultivars. PMID:26442034

Transgenic HFE-dependent induction of hepcidin in mice does not require transferrin receptor-2.

PubMed

Schmidt, Paul J; Fleming, Mark D

2012-06-01

Hereditary hemochomatosis (HH) is caused by mutations in several genes, including HFE and transferrin receptor-2 (TFR2). Loss of either protein decreases expression of the iron regulatory hormone hepcidin by the liver, leading to inappropriately high iron uptake from the diet, and resulting in systemic iron overload. In tissue culture, overexpressed HFE and TFR2 physically interact. Hepatocellular overexpression of Hfe in vivo increases hepcidin expression, despite an associated decrease in Tfr2. On this basis, we hypothesized that Tfr2 would not be required for Hfe-dependent up-regulation of hepcidin. We show that hepatocellular overexpression of Hfe in Tfr2(Y245X/Y245X) mice leads to hepcidin induction eventuating in iron deficiency and a hypochromic, microcytic anemia. Furthermore, coimmunoprecipitation studies using liver lysates did not provide evidence for physical interaction between Hfe and Tfr2 in vivo. In conclusion, we demonstrate that Tfr2 is not essential for Hfe-mediated induction of hepcidin expression, supporting the possibility that TFR2 may regulate iron metabolism in an HFE-independent manner. Copyright © 2012 Wiley Periodicals, Inc.

Transgenic HFE-dependent induction of hepcidin in mice does not require transferrin receptor-2

PubMed Central

Schmidt, Paul J.; Fleming, Mark D.

2012-01-01

Hereditary hemochomatosis (HH) is caused by mutations in several genes, including HFE and transferrin receptor-2 (TFR2). Loss of either protein decreases expression of the iron regulatory hormone hepcidin by the liver, leading to inappropriately high iron uptake from the diet, and resulting in systemic iron overload. In tissue culture, overexpressed HFE and TFR2 physically interact. Hepatocellular overexpression of Hfe in vivo increases hepcidin expression, despite an associated decrease in Tfr2. On this basis, we hypothesized that Tfr2 would not be required for Hfe-dependent up-regulation of hepcidin. We show that hepatocellular overexpression of Hfe in Tfr2Y245X/Y245X mice leads to hepcidin induction eventuating in iron deficiency and a hypochromic, microcytic anemia. Furthermore, co-immunoprecipitation studies using liver lysates did not provide evidence for physical interaction between Hfe and Tfr2 in vivo. In conclusion, we demonstrate that Tfr2 is not essential for Hfe-mediated induction of hepcidin expression, supporting the possibility that TFR2 may regulate iron metabolism in an HFE-independent manner. PMID:22460705

Silencing of Tuberin Enhances Photoreceptor Survival and Function in a Preclinical Model of Retinitis Pigmentosa (An American Ophthalmological Society Thesis)

PubMed Central

Tsang, Stephen H.; Chan, Lawrence; Tsai, Yi-Ting; Wu, Wen-Hsuan; Hsu, Chun-Wei; Yang, Jin; Tosi, Joaquin; Wert, Katherine J.; Davis, Richard J.; Mahajan, Vinit B.

2014-01-01

Purpose: To assess the functional consequences of silencing of tuberin, an inhibitor of the mTOR signaling pathway, in a preclinical model of retinitis pigmentosa (RP) in order to test the hypothesis that insufficient induction of the protein kinase B (PKB)-regulated tuberin/mTOR self-survival pathway initiates apoptosis. Methods: In an unbiased genome-scale approach, kinase peptide substrate arrays were used to analyze self-survival pathways at the onset of photoreceptor degeneration. The mutant Pde6bH620Q/Pde6bH620Q at P14 and P18 photoreceptor outer segment (OS) lysates were labeled with P-ATP and hybridized to an array of 1,164 different synthetic peptide substrates. At this stage, OS of Pde6bH620Q/Pde6bH620Q rods are morphologically normal. In vitro kinase assays and immunohistochemistry were used to validate phosphorylation. Short hairpin RNA (shRNA) gene silencing was used to validate tuberin’s role in regulating survival. Results: At the onset of degeneration, 162 peptides were differentially phosphorylated. Protein kinases A, G, C (AGC kinases), and B exhibited increased activity in both peptide array and in vitro kinase assays. Immunohistochemical data confirmed altered phosphorylation patterns for phosphoinositide-dependent kinase-1 (PDK1), ribosomal protein S6 (RPS6), and tuberin. Tuberin gene silencing rescued photoreceptors from degeneration. Conclusions: Phosphorylation of tuberin and RPS6 is due to the upregulated activity of PKB. PKB/tuberin cell growth/survival signaling is activated before the onset of degeneration. Substrates of the AGC kinases in the PKB/tuberin pathway are phosphorylated to promote cell survival. Knockdown of tuberin, the inhibitor of the mTOR pathway, increased photoreceptor survival and function in a preclinical model of RP. PMID:25646031

Microbead-regulated surface wrinkling patterns in a film-substrate system

NASA Astrophysics Data System (ADS)

Zhang, Cheng; Wang, Jiawen; Cao, Yan-Ping; Lu, Conghua; Li, Bo; Feng, Xi-Qiao

2017-10-01

The control of surface wrinkling patterns at the microscale is a concern in many applications. In this letter, we regulate surface wrinkling patterns on a film-substrate system by introducing microbeads atop the film. Both experiments and theoretical analysis reveal the changes in surface wrinkles induced by microbeads. Under equibiaxial compression, the film-substrate system without microbeads bonded on its upper surface often buckles into global, uniform labyrinths, whereas the labyrinthine pattern locally gives way to radial stripes emanating from the microbeads. This regulation of surface wrinkles depends on the sizes and spacing of microbeads. We combine the finite element method and the Fourier spectral method to explore the physical mechanisms underlying the phenomena. This study offers a viable technique for engineering surfaces with tunable functions.

Regulatory role of calpain in neuronal death

PubMed Central

Cheng, Si-ying; Wang, Shu-chao; Lei, Ming; Wang, Zhen; Xiong, Kun

2018-01-01

Calpains are a group of calcium-dependent proteases that are over activated by increased intracellular calcium levels under pathological conditions. A wide range of substrates that regulate necrotic, apoptotic and autophagic pathways are affected by calpain. Calpain plays a very important role in neuronal death and various neurological disorders. This review introduces recent research progress related to the regulatory mechanisms of calpain in neuronal death. Various neuronal programmed death pathways including apoptosis, autophagy and regulated necrosis can be divided into receptor interacting protein-dependent necroptosis, mitochondrial permeability transition-dependent necrosis, pyroptosis and poly (ADP-ribose) polymerase 1-mediated parthanatos. Calpains cleave series of key substrates that may lead to cell death or participate in cell death. Regarding the investigation of calpain-mediated programed cell death, it is necessary to identify specific inhibitors that inhibit calpain mediated neuronal death and nervous system diseases. PMID:29623944

Striatal-enriched Protein-tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity*

PubMed Central

Xu, Jian; Kurup, Pradeep; Bartos, Jason A.; Patriarchi, Tommaso; Hell, Johannes W.; Lombroso, Paul J.

2012-01-01

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr1472. Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr402. In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr402. STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr402 and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP. PMID:22544749

Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murano, Tatsuro; Okamoto, Ryuichi, E-mail: rokamoto.gast@tmd.ac.jp; Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo

Highlights: •Hes1 enhances IL-22-STAT3 signaling in human intestinal epithelial cells. •Hes1 enhances REG family gene induction by IL-22-STAT3 signaling. •Protein level of Hes1 restricts the response to IL-22. •Present regulation of a cytokine signal represents a new mode of Hes1 function. -- Abstract: Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated inmore » the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.« less

Interactive Effects of Jasmonic Acid, Salicylic Acid, and Gibberellin on Induction of Trichomes in Arabidopsis1

PubMed Central

Traw, M. Brian; Bergelson, Joy

2003-01-01

Leaf trichomes protect plants from attack by insect herbivores and are often induced following damage. Hormonal regulation of this plant induction response has not been previously studied. In a series of experiments, we addressed the effects of artificial damage, jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis. Artificial damage and jasmonic acid caused significant increases in trichome production of leaves. The jar1-1 mutant exhibited normal trichome induction following treatment with jasmonic acid, suggesting that adenylation of jasmonic acid is not necessary. Salicylic acid had a negative effect on trichome production and consistently reduced the effect of jasmonic acid, suggesting negative cross-talk between the jasmonate and salicylate-dependent defense pathways. Interestingly, the effect of salicylic acid persisted in the nim1-1 mutant, suggesting that the Npr1/Nim1 gene is not downstream of salicylic acid in the negative regulation of trichome production. Last, we found that gibberellin and jasmonic acid had a synergistic effect on the induction of trichomes, suggesting important interactions between these two compounds. PMID:14551332

Inhibiting Insulin-Mediated β2-Adrenergic Receptor Activation Prevents Diabetes-Associated Cardiac Dysfunction.

PubMed

Wang, Qingtong; Liu, Yongming; Fu, Qin; Xu, Bing; Zhang, Yuan; Kim, Sungjin; Tan, Ruensern; Barbagallo, Federica; West, Toni; Anderson, Ethan; Wei, Wei; Abel, E Dale; Xiang, Yang K

2017-01-03

Type 2 diabetes mellitus (DM) and obesity independently increase the risk of heart failure by incompletely understood mechanisms. We propose that hyperinsulinemia might promote adverse consequences in the hearts of subjects with type-2 DM and obesity. High-fat diet feeding was used to induce obesity and DM in wild-type mice or mice lacking β 2 -adrenergic receptor (β 2 AR) or β-arrestin2. Wild-type mice fed with high-fat diet were treated with a β-blocker carvedilol or a GRK2 (G-protein-coupled receptor kinase 2) inhibitor. We examined signaling and cardiac contractile function. High-fat diet feeding selectively increases the expression of phosphodiesterase 4D (PDE4D) in mouse hearts, in concert with reduced protein kinase A phosphorylation of phospholamban, which contributes to systolic and diastolic dysfunction. The expression of PDE4D is also elevated in human hearts with DM. The induction of PDE4D expression is mediated by an insulin receptor, insulin receptor substrate, and GRK2 and β-arrestin2-dependent transactivation of a β 2 AR-extracellular regulated protein kinase signaling cascade. Thus, pharmacological inhibition of β 2 AR or GRK2, or genetic deletion of β 2 AR or β-arrestin2, all significantly attenuate insulin-induced phosphorylation of extracellular regulated protein kinase and PDE4D induction to prevent DM-related contractile dysfunction. These studies elucidate a novel mechanism by which hyperinsulinemia contributes to heart failure by increasing PDE4D expression and identify β 2 AR or GRK2 as plausible therapeutic targets for preventing or treating heart failure in subjects with type 2 DM. © 2016 American Heart Association, Inc.

Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation.

PubMed

Scott, Daniel C; Rhee, David Y; Duda, David M; Kelsall, Ian R; Olszewski, Jennifer L; Paulo, Joao A; de Jong, Annemieke; Ovaa, Huib; Alpi, Arno F; Harper, J Wade; Schulman, Brenda A

2016-08-25

Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation. Copyright © 2016 Elsevier Inc. All rights reserved.

Temporal profiling of gene networks associated with the late phase of long-term potentiation in vivo.

PubMed

Ryan, Margaret M; Ryan, Brigid; Kyrke-Smith, Madeleine; Logan, Barbara; Tate, Warren P; Abraham, Wickliffe C; Williams, Joanna M

2012-01-01

Long-term potentiation (LTP) is widely accepted as a cellular mechanism underlying memory processes. It is well established that LTP persistence is strongly dependent on activation of constitutive and inducible transcription factors, but there is limited information regarding the downstream gene networks and controlling elements that coalesce to stabilise LTP. To identify these gene networks, we used Affymetrix RAT230.2 microarrays to detect genes regulated 5 h and 24 h (n = 5) after LTP induction at perforant path synapses in the dentate gyrus of awake adult rats. The functional relationships of the differentially expressed genes were examined using DAVID and Ingenuity Pathway Analysis, and compared with our previous data derived 20 min post-LTP induction in vivo. This analysis showed that LTP-related genes are predominantly upregulated at 5 h but that there is pronounced downregulation of gene expression at 24 h after LTP induction. Analysis of the structure of the networks and canonical pathways predicted a regulation of calcium dynamics via G-protein coupled receptors, dendritogenesis and neurogenesis at the 5 h time-point. By 24 h neurotrophin-NFKB driven pathways of neuronal growth were identified. The temporal shift in gene expression appears to be mediated by regulation of protein synthesis, ubiquitination and time-dependent regulation of specific microRNA and histone deacetylase expression. Together this programme of genomic responses, marked by both homeostatic and growth pathways, is likely to be critical for the consolidation of LTP in vivo.

Investigation of the Biochemical Mechanism for Cell-Substrate Mechanical Sensing

NASA Astrophysics Data System (ADS)

Ricotta, Vincent Anthony

Advancements in stem cell biology and materials science have enabled the development of new treatments for tissue repair. Dental pulp stem cells (DPSCs), which are highly proliferative and can be induced to differentiate along several mesenchymal cell lineages, offer the possibility for pulpal regeneration and treatment of injured dentition. Polybutadiene (PB) may be used as a substrate for these cells. This elastomer can be spun casted into films of different thicknesses with different moduli. DPSCs grown on PB films, which are relatively hard (less than 1500 A thick), biomineralize depositing crystalline calcium phosphate without a requirement for the typical induction factor, dexamethasone (Dex). The moduli of cells track with the moduli of the surface suggesting that mechanics controls mineralization. The purpose of this study was to determine whether the major effect of Dex on biomineralization is the result of its ability to alter cell mechanics or its ability to induce osteogenesis/odontogenesis. DPSCs sense substrate mechanics through the focal adhesions, whose function is in part regulated by the Ras homolog gene (Rho) and its downstream effectors Rho associated kinases (ROCKs). ROCKs control actin filament polymerization and interactions with myosin light chain. Because cells sense substrate mechanics through focal adhesion proteins whose function is regulated by ROCKs, the impact of a ROCK inhibitor, Y-27632, was monitored. Blocking this pathway with Y-27632 suppressed the ability of DPSCs to sense the PB substrate. The cell modulus, plasma membrane stiffness, and cytosol stiffness were all lowered and biomineralization was suppressed in all cultures independent of substrate modulus or the presence of Dex. In other words, the inability of DPSCs to sense mechanical cues suppressed their ability to promote mineralization. On the other hand the expression of osteogenic/odontogenic markers (alkaline phosphatase and osteocalcin) was enhanced, perhaps due to Y-27632 induced changes in Wnt signaling as seen in other mesenchymal stem cells. How mechanical sensing regulates matrix proteins to promote their mineralization remains an open question.

Multilayered Regulation of Ethylene Induction Plays a Positive Role in Arabidopsis Resistance against Pseudomonas syringae1[OPEN

PubMed Central

Guan, Rongxia; Su, Jianbin; Meng, Xiangzong; Li, Sen; Liu, Yidong; Xu, Juan; Zhang, Shuqun

2015-01-01

Ethylene, a key phytohormone involved in plant-pathogen interaction, plays a positive role in plant resistance against fungal pathogens. However, its function in plant bacterial resistance remains unclear. Here, we report a detailed analysis of ethylene induction in Arabidopsis (Arabidopsis thaliana) in response to Pseudomonas syringae pv tomato DC3000 (Pst). Ethylene biosynthesis is highly induced in both pathogen/microbe-associated molecular pattern (PAMP)-triggered immunity and effector-triggered immunity (ETI), and the induction is potentiated by salicylic acid (SA) pretreatment. In addition, Pst actively suppresses PAMP-triggered ethylene induction in a type III secretion system-dependent manner. SA potentiation of ethylene induction is dependent mostly on MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and MPK3 and their downstream ACS2 and ACS6, two type I isoforms of 1-aminocyclopropane-1-carboxylic acid synthases (ACSs). ACS7, a type III ACS whose expression is enhanced by SA pretreatment, is also involved. Pst expressing the avrRpt2 effector gene (Pst-avrRpt2), which is capable of triggering ETI, induces a higher level of ethylene production, and the elevated portion is dependent on SALICYLIC ACID INDUCTION DEFICIENT2 and NONEXPRESSER OF PATHOGENESIS-RELATED GENE1, two key players in SA biosynthesis and signaling. High-order ACS mutants with reduced ethylene induction are more susceptible to both Pst and Pst-avrRpt2, demonstrating a positive role of ethylene in plant bacterial resistance mediated by both PAMP-triggered immunity and ETI. PMID:26265775

Active cell-matrix coupling regulates cellular force landscapes of cohesive epithelial monolayers

NASA Astrophysics Data System (ADS)

Zhao, Tiankai; Zhang, Yao; Wei, Qiong; Shi, Xuechen; Zhao, Peng; Chen, Long-Qing; Zhang, Sulin

2018-03-01

Epithelial cells can assemble into cohesive monolayers with rich morphologies on substrates due to competition between elastic, edge, and interfacial effects. Here we present a molecularly based thermodynamic model, integrating monolayer and substrate elasticity, and force-mediated focal adhesion formation, to elucidate the active biochemical regulation over the cellular force landscapes in cohesive epithelial monolayers, corroborated by microscopy and immunofluorescence studies. The predicted extracellular traction and intercellular tension are both monolayer size and substrate stiffness dependent, suggestive of cross-talks between intercellular and extracellular activities. Our model sets a firm ground toward a versatile computational framework to uncover the molecular origins of morphogenesis and disease in multicellular epithelia.

Analysis of the Transcriptional Regulator GlpR, Promoter Elements, and Posttranscriptional Processing Involved in Fructose-Induced Activation of the Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System in Haloferax mediterranei

PubMed Central

Cai, Lei; Cai, Shuangfeng; Zhao, Dahe; Wu, Jinhua; Wang, Lei; Liu, Xiaoqing; Li, Ming; Hou, Jing; Zhou, Jian; Liu, Jingfang; Han, Jing

2014-01-01

Among all known archaeal strains, the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for fructose utilization is used primarily by haloarchaea, which thrive in hypersaline environments, whereas the molecular details of the regulation of the archaeal PTS under fructose induction remain unclear. In this study, we present a comprehensive examination of the regulatory mechanism of the fructose PTS in the haloarchaeon Haloferax mediterranei. With gene knockout and complementation, microarray analysis, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), we revealed that GlpR is the indispensable activator, which specifically binds to the PTS promoter (PPTS) during fructose induction. Further promoter-scanning mutation indicated that three sites located upstream of the H. mediterranei PPTS, which are conserved in most haloarchaeal PPTSs, are involved in this induction. Interestingly, two PTS transcripts (named T8 and T17) with different lengths of 5′ untranslated region (UTR) were observed, and promoter or 5′ UTR swap experiments indicated that the shorter 5′ UTR was most likely generated from the longer one. Notably, the translation efficiency of the transcript with this shorter 5′ UTR was significantly higher and the ratio of T8 (with the shorter 5′ UTR) to T17 increased during fructose induction, implying that a posttranscriptional mechanism is also involved in PTS activation. With these insights into the molecular regulation of the haloarchaeal PTS, we have proposed a working model for haloarchaea in response to environmental fructose. PMID:24334671

Numerical investigation of melting and solidification processes in modified surface layers of metal at induction heating

NASA Astrophysics Data System (ADS)

Shchukin, V. G.; Popov, V. N.

2017-10-01

One of the perspective ways to improve the operational properties of parts of machines during induction treatment of their surfaces is the modification of the melt by specially prepared nanoscale particles of refractory compounds (carbides, nitrides, carbonitrides, etc.). This approach allows us to increase the number of crystallization centers and to refine the structural components of the solidified metal. The resulting high dispersity and homogeneity of crystalline grains favorably affect the quality of the treated surfaces. 3D numerical simulation of thermophysical processes in the modification of the surface layer of metal in a moving substrate was carried out. It is assumed that the surface of the substrate is covered with a layer of specially prepared nanoscale particles of a refractory compound, which, upon penetration into the melt, are uniformly distributed in it. The possibility of applying a high-frequency electromagnetic field of high power for heating and melting of a metal (iron) for the purpose of its subsequent modification is investigated. The distribution of electromagnetic energy in the metal is described by empirical formulas. Melting of the metal is considered in the Stefan approximation, and upon solidification it is assumed that all nanoparticles serve as centers for volume-sequential crystallization. Calculations were carried out with the following parameters: specific power p0 = 35 and 40 kW/cm2 at frequency f = 440 and 1200 kHz, the substrate velocity V = 0.5-2.5 cm/s, the nanoparticles' size is 50 nm and concentration Np = 2.0 . 109 cm-3. Based on the results obtained in a quasi-stationary formulation, the distribution of the temperature field, the dimensions of the melting and crystallization zones, the change in the solid fraction in the two-phase zone, the area of the treated substrate surface, depending on the speed of its movement and induction heating characteristics were estimated.

CSN-associated USP48 confers stability to nuclear NF-κB/RelA by trimming K48-linked Ub-chains.

PubMed

Schweitzer, Katrin; Naumann, Michael

2015-02-01

Diligent balance of nuclear factor kappa B (NF-κB) activity is essential owing to NF-κB's decisive role in cellular processes including inflammation, immunity and cell survival. Ubiquitin/proteasome-system (UPS)-dependent degradation of activated NF-κB/RelA involves the cullin-RING-ubiquitin-ligase (CRL) ECS(SOCS1). The COP9 signalosome (CSN) controls ubiquitin (Ub) ligation by CRLs through the removal of the CRL-activating Ub-like modifier NEDD8 from their cullin subunits and through deubiquitinase (DUB) activity of associated DUBs. However, knowledge about DUBs involved in the regulation of NF-κB activity within the nucleus is scarce. In this study we observed that USP48, a DUB of hitherto ill-defined function identified through a siRNA screen, associates with the CSN and RelA in the nucleus. We show that USP48 trims rather than completely disassembles long K48-linked free and substrate-anchored Ub-chains, a catalytic property only shared with ataxin-3 (Atx3) and otubain-1 (OTU1), and that USP48 Ub-chain-trimming activity is regulated by casein-kinase-2 (CK2)-mediated phosphorylation in response to cytokine-stimulation. Functionally, we demonstrate for the first time the CSN and USP48 to cooperatively stabilize the nuclear pool of RelA, thereby facilitating timely induction and shutoff of NF-κB target genes. In summary, this study demonstrates that USP48, a nuclear DUB regulated by CK2, controls the UPS-dependent turnover of activated NF-κB/RelA in the nucleus together with the CSN. Thereby USP48 contributes to a timely control of immune responses. Copyright © 2014 Elsevier B.V. All rights reserved.

Electrogenic Binding of Intracellular Cations Defines a Kinetic Decision Point in the Transport Cycle of the Human Serotonin Transporter.

PubMed

Hasenhuetl, Peter S; Freissmuth, Michael; Sandtner, Walter

2016-12-09

The plasmalemmal monoamine transporters clear the extracellular space from their cognate substrates and sustain cellular monoamine stores even during neuronal activity. In some instances, however, the transporters enter a substrate-exchange mode, which results in release of intracellular substrate. Understanding what determines the switch between these two transport modes demands time-resolved measurements of intracellular (co-)substrate binding and release. Here, we report an electrophysiological investigation of intracellular solute-binding to the human serotonin transporter (SERT) expressed in HEK-293 cells. We measured currents induced by rapid application of serotonin employing varying intracellular (co-)substrate concentrations and interpreted the data using kinetic modeling. Our measurements revealed that the induction of the substrate-exchange mode depends on both voltage and intracellular Na + concentrations because intracellular Na + release occurs before serotonin release and is highly electrogenic. This voltage dependence was blunted by electrogenic binding of intracellular K + and, notably, also H + In addition, our data suggest that Cl - is bound to SERT during the entire catalytic cycle. Our experiments, therefore, document an essential role of electrogenic binding of K + or of H + to the inward-facing conformation of SERT in (i) cancelling out the electrogenic nature of intracellular Na + release and (ii) in selecting the forward-transport over the substrate-exchange mode. Finally, the kinetics of intracellular Na + release and K + (or H + ) binding result in a voltage-independent rate-limiting step where SERT may return to the outward-facing state in a KCl- or HCl-bound form. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Process for depositing Cr-bearing layer

DOEpatents

Ellis, Timothy W.; Lograsso, Thomas A.; Eshelman, Mark A.

1995-05-09

A method of applying a Cr-bearing layer to a substrate, comprises introducing an organometallic compound, in vapor or solid powder form entrained in a carrier gas to a plasma of an inductively coupled plasma torch or device to thermally decompose the organometallic compound and contacting the plasma and the substrate to be coated so as to deposit the Cr-bearing layer on the substrate. A metallic Cr, Cr alloy or Cr compound such as chromium oxide, nitride and carbide can be provided on the substrate. Typically, the organometallic compound is introduced to an inductively coupled plasma torch that is disposed in ambient air so to thermally decompose the organometallic compound in the plasma. The plasma is directed at the substrate to deposit the Cr-bearing layer or coating on the substrate.

Process for depositing Cr-bearing layer

DOEpatents

Ellis, T.W.; Lograsso, T.A.; Eshelman, M.A.

1995-05-09

A method of applying a Cr-bearing layer to a substrate, comprises introducing an organometallic compound, in vapor or solid powder form entrained in a carrier gas to a plasma of an inductively coupled plasma torch or device to thermally decompose the organometallic compound and contacting the plasma and the substrate to be coated so as to deposit the Cr-bearing layer on the substrate. A metallic Cr, Cr alloy or Cr compound such as chromium oxide, nitride and carbide can be provided on the substrate. Typically, the organometallic compound is introduced to an inductively coupled plasma torch that is disposed in ambient air so to thermally decompose the organometallic compound in the plasma. The plasma is directed at the substrate to deposit the Cr-bearing layer or coating on the substrate. 7 figs.

Different patterns of extracellular proteolytic activity in W303a and BY4742 Saccharomyces cerevisiae strains.

PubMed

Seredyński, Rafał; Wolna, Dorota; Kędzior, Mateusz; Gutowicz, Jan

2017-01-01

Protease secretion in Saccharomyces cerevisiae cultures is a complex process, important for the application of this organism in the food industry and biotechnology. Previous studies provide rather quantitative data, yielding no information about the number of enzymes involved in proteolysis and their individual biochemical properties. Here we demonstrate that W303a and BY4742 S. cerevisiae strains reveal different patterns of spontaneous and gelatin-induced extracellular proteolytic activity. We applied the gelatin zymography assay to track changes of the proteolytic profile in time, finding the protease secretion dependent on the growth phase and the presence of the protein inducer. Detected enzymes were characterized regarding their substrate specificity, pH tolerance, and susceptibility to inhibitors. In case of the W303a strain, only one type of gelatin-degrading secretory protease (presumably metalloproteinase) was observed. However, the BY4742 strain secreted different proteases of the various catalytic types, depending on the substrate availability. Our study brings the evidence that S. cerevisiae strains secrete several kinds of proteases depending on the presence and type of the substrate. Protein induction may cause not only quantitative but also qualitative changes in the extracellular proteolytic patterns. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein–Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP)*

PubMed Central

Narayan, Vikram; Landré, Vivien; Ning, Jia; Hernychova, Lenka; Muller, Petr; Verma, Chandra; Walkinshaw, Malcolm D.; Blackburn, Elizabeth A.; Ball, Kathryn L.

2015-01-01

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. PMID:26330542

Light-dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5'-triphosphate.

PubMed

Horton, P; Black, M T

1981-03-12

Addition of ATP to chloroplasts causes a reversible 25-30% decrease in chlorophyll fluorescence. This quenching is light-dependent, uncoupler insensitive but inhibited by DCMU and electron acceptors and has a half-time of 3 minutes. Electron donors to Photosystem I can not overcome the inhibitory effect of DCMU, suggesting that light activation depends on the reduced state of plastoquinone. Fluorescence emission spectra recorded at -196 degrees C indicate that ATP treatment increases the amount of excitation energy transferred to Photosystem I. Examination of fluorescence induction curves indicate that ATP treatment decreases both the initial (F0) and variable (Fv) fluorescence such that the ratio of Fv to the maximum (Fm) yield is unchanged. The initial sigmoidal phase of induction is slowed down by ATP treatment and is quenched 3-fold more than the exponential slow phase, the rate of which is unchanged. A plot of Fv against area above the induction curve was identical plus or minus ATP. Thus ATP treatment can alter quantal distribution between Photosystems II and I without altering Photosystem II-Photosystem II interaction. The effect of ATP strongly resembles in its properties the phosphorylation of the light-harvesting complex by a light activated, ATP-dependent protein kinase found in chloroplast membranes and could be the basis of physiological mechanisms which contribute to slow fluorescence quenching in vivo and regulate excitation energy distribution between Photosystem I and II. It is suggested that the sensor for this regulation is the redox state of plastoquinone.

Matrix Rigidity Regulates Cancer Cell Growth by Modulating Cellular Metabolism and Protein Synthesis

PubMed Central

Tilghman, Robert W.; Blais, Edik M.; Cowan, Catharine R.; Sherman, Nicholas E.; Grigera, Pablo R.; Jeffery, Erin D.; Fox, Jay W.; Blackman, Brett R.; Tschumperlin, Daniel J.; Papin, Jason A.; Parsons, J. Thomas

2012-01-01

Background Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy. Methodology/Principal Findings This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates. Compared to cells growing on more rigid/stiff substrates (>10,000 Pa), cells on soft substrates (150–300 Pa) exhibited a longer cell cycle, due predominantly to an extension of the G1 phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis. Using stable isotope labeling of amino acids in culture (SILAC) and mass spectrometry, we measured the rates of protein synthesis of over 1200 cellular proteins under growth conditions on soft and rigid/stiff substrates. We identified cellular proteins whose syntheses were either preferentially inhibited or preserved on soft matrices. The former category included proteins that regulate cytoskeletal structures (e.g., tubulins) and glycolysis (e.g., phosphofructokinase-1), whereas the latter category included proteins that regulate key metabolic pathways required for survival, e.g., nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway. Conclusions/Significance The cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of dormant cancer cells, e.g., slow growth rate and reduced metabolism. We suggest that the use of relatively soft gels as cell culture substrates would allow molecular pathways to be studied under conditions that reflect the different mechanical environments encountered by cancer cells upon metastasis to distant sites. PMID:22623999

Phosphoproteomics analyses show subnetwork systems in T-cell receptor signaling.

PubMed

Hatano, Atsushi; Matsumoto, Masaki; Nakayama, Keiichi I

2016-10-01

A key issue in the study of signal transduction is how multiple signaling pathways are systematically integrated into the cell. We have now performed multiple phosphoproteomics analyses focused on the dynamics of the T-cell receptor (TCR) signaling network and its subsystem mediated by the Ca 2+ signaling pathway. Integration of these phosphoproteomics data sets and extraction of components of the TCR signaling network dependent on Ca 2+ signaling showed unexpected phosphorylation kinetics for candidate substrates of the Ca 2+ -dependent phosphatase calcineurin (CN) during TCR stimulation. Detailed characterization of the TCR-induced phosphorylation of a novel CN substrate, Itpkb, showed that phosphorylation of this protein is regulated by both CN and the mitogen-activated protein kinase Erk in a competitive manner. Phosphorylation of additional CN substrates was also found to be regulated by Erk and CN in a similar manner. The combination of multiple phosphoproteomics approaches thus showed two major subsystems mediated by Erk and CN in the TCR signaling network, with these subsystems regulating the phosphorylation of a group of proteins in a competitive manner. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Allosteric regulation of rhomboid intramembrane proteolysis.

PubMed

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-09-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. © 2014 The Authors.

Allosteric regulation of rhomboid intramembrane proteolysis

PubMed Central

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-01-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. PMID:25009246

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wieneke, N.; Neuschaefer-Rube, F.; Bode, L.M.

Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feedback-controlled regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase II enzymes of xenobiotic metabolism. We have recently shown, that PPAR{alpha} agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR{alpha} agonist could enhance the phenobarbital-dependentmore » acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPAR{alpha} agonist WY14643 to a larger extent than after induction with either compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UGT1A10. The PPAR{alpha}-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting-induced reduction in energy expenditure by fatty acids as natural PPAR{alpha} ligands. The synergism of the PPAR{alpha} agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics.« less

15-Deoxy-{delta}{sup 12,14}-prostaglandin J{sub 2}-induced down-regulation of endothelial nitric oxide synthase in association with HSP70 induction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Jinah; Lee, Hyun-Il; Chang, Young-Sun

2007-05-25

A natural ligand of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), 15-deoxy-{delta}{sup 12,14}-prostaglandin J{sub 2} (15d-PGJ{sub 2}), decreases endothelial nitric oxide synthase (eNOS) expression by an unknown mechanism. Here we found that 15d-PGJ{sub 2}-induced eNOS reduction is inversely associated with heat shock protein 70 (HSP70) induction in endothelial cells. Treatment of cells with 15d-PGJ{sub 2} decreased eNOS protein expression in a concentration- and time-dependent manner, but independently of PPAR{gamma} with no effect on mRNA levels. Although 15d-PGJ{sub 2} elicited endothelial apoptosis, inhibition of both pan-caspases and cathepsins failed to reverse reduction of eNOS protein. Interestingly, we observed that 15d-PGJ{sub 2} induced HSP70more » in a dose-dependent manner. Immunoprecipitation and heat shock treatment demonstrated that eNOS reduction was strongly related to HSP70 induction. Cellular fractionation revealed that treatment with 15d-PGJ{sub 2} increased eNOS distribution 2.5-fold from soluble to insoluble fractions. These findings provide new insights into mechanisms whereby eNOS regulation by 15d-PGJ{sub 2} is related to HSP70 induction.« less

Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators.

PubMed

Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone; Dai, Jie; Rogowska-Wrzesinska, Adelina; Mandrup, Susanne; Jensen, Ole N

2017-03-01

Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipid-laden mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein PTMs, in particular phosphorylation, play a major role in activating and propagating signals within TR networks upon induction of adipogenesis by extracellular stimulus. We applied MS-based quantitative proteomics and phosphoproteomics to monitor the alteration of nuclear proteins during the early stages (4 h) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation levels of proteins involved in gene expression, cell organization, and oxidation-reduction pathways. Furthermore, proteins acting as negative modulators involved in negative regulation of gene expression, insulin stimulated glucose uptake, and cytoskeletal organization showed a decrease in their nuclear abundance and/or phosphorylation levels during the first 4 h of adipogenesis. Among 288 identified TRs, 49 were regulated within 4 h of adipogenic stimulation including several known and many novel potential adipogenic regulators. We created a kinase-substrate database for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase family and the mitogen-activated protein kinase family including P38 and c-Jun N-terminal kinases, suggesting that these kinases act as orchestrators of early adipogenesis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transcription factor Sox4 is required for PUMA-mediated apoptosis induced by histone deacetylase inhibitor, TSA.

PubMed

Jang, Sang-Min; Kang, Eun-Jin; Kim, Jung-Woong; Kim, Chul-Hong; An, Joo-Hee; Choi, Kyung-Hee

2013-08-23

PUMA is a crucial regulator of apoptotic cell death mediated by p53-dependent and p53-independent mechanisms. In many cancer cells, PUMA expression is induced in response to DNA-damaging reagent in a p53-dependent manner. However, few studies have investigated transcription factors that lead to the induction of PUMA expression via p53-independent apoptotic signaling. In this study, we found that the transcription factor Sox4 increased PUMA expression in response to trichostatin A (TSA), a histone deacetylase inhibitor in the p53-null human lung cancer cell line H1299. Ectopic expression of Sox4 led to the induction of PUMA expression at the mRNA and protein levels, and TSA-mediated up-regulation of PUMA transcription was repressed by the knockdown of Sox4. Using luciferase assays and chromatin immunoprecipitation, we also determined that Sox4 recruits p300 on the PUMA promoter region and increases PUMA gene expression in response to TSA treatment. Taken together, these results suggest that Sox4 is required for p53-independent apoptotic cell death mediated by PUMA induction via TSA treatment. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Silencing of DNase Colicin E8 Gene Expression by a Complex Nucleoprotein Assembly Ensures Timely Colicin Induction.

PubMed

Kamenšek, Simona; Browning, Douglas F; Podlesek, Zdravko; Busby, Stephen J W; Žgur-Bertok, Darja; Butala, Matej

2015-06-01

Colicins are plasmid-encoded narrow spectrum antibiotics that are synthesized by strains of Escherichia coli and govern intraspecies competition. In a previous report, we demonstrated that the global transcriptional factor IscR, co dependently with the master regulator of the DNA damage response, LexA, delays induction of the pore forming colicin genes after SOS induction. Here we show that IscR is not involved in the regulation of nuclease colicins, but that the AsnC protein is. We report that AsnC, in concert with LexA, is the key controller of the temporal induction of the DNA degrading colicin E8 gene (cea8), after DNA damage. We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine. We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation. Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression. We propose that selection pressure has "chosen" highly conserved regulators to control colicin expression in E. coli strains, enabling similar colicin gene silencing among bacteria upon exchange of colicinogenic plasmids.

Timescales and bottlenecks in miRNA-dependent gene regulation.

PubMed

Hausser, Jean; Syed, Afzal Pasha; Selevsek, Nathalie; van Nimwegen, Erik; Jaskiewicz, Lukasz; Aebersold, Ruedi; Zavolan, Mihaela

2013-12-03

MiRNAs are post-transcriptional regulators that contribute to the establishment and maintenance of gene expression patterns. Although their biogenesis and decay appear to be under complex control, the implications of miRNA expression dynamics for the processes that they regulate are not well understood. We derived a mathematical model of miRNA-mediated gene regulation, inferred its parameters from experimental data sets, and found that the model describes well time-dependent changes in mRNA, protein and ribosome density levels measured upon miRNA transfection and induction. The inferred parameters indicate that the timescale of miRNA-dependent regulation is slower than initially thought. Delays in miRNA loading into Argonaute proteins and the slow decay of proteins relative to mRNAs can explain the typically small changes in protein levels observed upon miRNA transfection. For miRNAs to regulate protein expression on the timescale of a day, as miRNAs involved in cell-cycle regulation do, accelerated miRNA turnover is necessary.

Neuro-Hypnotism: Prospects for Hypnosis and Neuroscience

PubMed Central

Kihlstrom, John F.

2012-01-01

The neurophysiological substrates of hypnosis have been subject to speculation since the phenomenon got its name. Until recently, much of this research has been geared toward understanding hypnosis itself, including the biological bases of individual differences in hypnotizability, state-dependent changes in cortical activity occurring with the induction of hypnosis, and the neural correlates of response to particular hypnotic suggestions (especially the clinically useful hypnotic analgesia). More recently, hypnosis has begun to be employed as a method for manipulating subjects' mental states, both cognitive and affective, to provide information about the neural substrates of experience, thought, and action. This instrumental use of hypnosis is particularly well-suited for identifying the neural correlates of conscious and unconscious perception and memory, and of voluntary and involuntary action. PMID:22748566

ADAM13 Induces Cranial Neural Crest by Cleaving Class B Ephrins and Regulating Wnt Signaling

PubMed Central

Wei, Shuo; Xu, Guofeng; Bridges, Lance C.; Williams, Phoebe; White, Judith M.; DeSimone, Douglas W.

2010-01-01

SUMMARY The cranial neural crest (CNC) are multipotent embryonic cells that contribute to craniofacial structures and other cells and tissues of the vertebrate head. During embryogenesis, CNC is induced at the neural plate boundary through the interplay of several major signaling pathways. Here we report that the metalloproteinase activity of ADAM13 is required for early induction of CNC in Xenopus. In both cultured cells and X. tropicalis embryos, membrane-bound Ephrins (Efns) B1 and B2 were identified as substrates for ADAM13. ADAM13 upregulates canonical Wnt signaling and early expression of the transcription factor snail2, whereas EfnB1 inhibits the canonical Wnt pathway and snail2 expression. We propose that by cleaving class B Efns, ADAM13 promotes canonical Wnt signaling and early CNC induction. PMID:20708595

IL-15 regulates Bcl-2 family members Bim and Mcl-1 through JAK/STAT and PI3K/AKT pathways in T cells.

PubMed

Shenoy, Aparna R; Kirschnek, Susanne; Häcker, Georg

2014-08-01

Maintenance of T cells is determined by their survival capacity, which is regulated by Bcl-2 proteins. Cytokines signalling through the common gamma chains such as IL-2, IL-7 and IL-15 are important for T-cell survival but how these cytokines determine the expression of Bcl-2-family proteins is not clear. We report signalling events of cytokines that regulate expression of two key Bcl-2 proteins, pro-apoptotic Bim and anti-apoptotic Mcl-1, in resting C57BL/6 mouse T cells. IL-2, IL-7 and IL-15 inhibited apoptosis but paradoxically induced the expression of Bim, countered by concomitant induction of Mcl-1. Bim induction by IL-15 was found at the mRNA and protein levels and depended on both JAK/STAT and PI3K signals. A new STAT5-binding site was identified in the Bim promoter, which was occupied by STAT5 upon IL-15 stimulation. Although it also depended on JAK/STAT- and PI3K signalling, Mcl-1 regulation was independent of Mcl-1 mRNA levels and of regulation of protein stability, suggesting translational regulation. Concurrent CD3 signals inhibited some of the IL-7 effect but not the IL-15 effect on Bcl-2 proteins. The data suggest that cytokines induce Bim and prime T cells for apoptosis, but also inhibit apoptosis by stabilising Mcl-1. Later downregulation of short-lived Mcl-1 may induce efficient, Bim-dependent apoptosis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biosynthesis of the phytoalexin pisatin. [Pisum sativum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Preisig, C.L.; Bell, J.N.; Matthews, D.E.

1990-11-01

NADPH-dependent reduction of 2{prime},7-dihydroxy-4{prime},5{prime}-methylenedioxyisoflavone to the isoflavanone sophorol, a proposed intermediate step in pisatin biosynthesis, was detected in extracts of Pisum sativum. This isoflavone reductase activity was inducible by treatment of pea seedlings with CuCl{sub 2}. The timing of induction coincided with that of the 6a-hydroxymaackiain 3-O-methyltransferase, which catalyzes the terminal biosynthetic step. Neither enzyme was light inducible. Further NADPH-dependent metabolism of sophorol by extracts of CuCl{sub 2}-treated seedlings was also observed; three products were radiolabeled when ({sup 3}H)sophorol was the substrate, one of which is tentatively identified as maackiain.

Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals

PubMed Central

Mckenzie, Grahame; Ward, George; Stallwood, Yvette; Briend, Emmanuel; Papadia, Sofia; Lennard, Andrew; Turner, Martin; Champion, Brian; Hardingham, Giles E

2006-01-01

Background Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways. Results We show that cellular responsiveness to Notch signals depends on the activity of the PI3K-Akt pathway in cells as diverse as CHO cells, primary T-cells and hippocampal neurons. Induction of the endogenous PI3K-Akt pathway in CHO cells (by the insulin pathway), in T-cells (via TCR activation) or in neurons (via TrKB activation) potentiates Notch-dependent responses. We propose that the PI3K-Akt pathway exerts its influence on Notch primarily via inhibition of GSK3-beta, a kinase known to phosphorylate and regulate Notch signals. Conclusion The PI3K-Akt pathway acts as a "gain control" for Notch signal responses. Since physiological levels of intracellular Notch are often low, coincidence with PI3K-activation may be crucial for induction of Notch-dependent responses. PMID:16507111

HOG MAP kinase regulation of alternariol biosynthesis in Alternaria alternata is important for substrate colonization.

PubMed

Graf, Eva; Schmidt-Heydt, Markus; Geisen, Rolf

2012-07-16

Strains of the genus Alternaria are ubiquitously present and frequently found on fruits, vegetables and cereals. One of the most commonly found species from this genus is A. alternata which is able to produce the mycotoxin alternariol among others. To date only limited knowledge is available about the regulation of the biosynthesis of alternariol, especially under conditions relevant to food. Tomatoes are a typical substrate of A. alternata and have a high water activity. On the other hand cereals with moderate water activity are also frequently colonized by A. alternata. In the current analysis it was demonstrated that even minor changes in the osmotic status of the substrate affect the alternariol biosynthesis of strains from vegetables resulting in nearly complete inhibition. High osmolarity in the environment is usually transmitted to the transcriptional level of downstream regulated genes by the HOG signal cascade (high osmolarity glycerol cascade) which is a MAP kinase transduction pathway. The phosphorylation status of the A. alternata HOG (AaHOG) was determined. Various concentrations of NaCl induce the phosphorylation of AaHOG in a concentration, time and strain dependent manner. A strain with a genetically inactivated aahog gene was no longer able to produce alternariol indicating that the activity of the aahog gene is required for alternariol biosynthesis. Further experiments revealed that the biosynthesis of alternariol is important for the fungus to colonize tomato tissue. The tight water activity dependent regulation of alternariol biosynthesis ensures alternariol biosynthesis at conditions which indicate an optimal colonization substrate for the fungus. Copyright © 2012 Elsevier B.V. All rights reserved.

Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis.

PubMed

Li, Guojing; Meng, Xiangzong; Wang, Ruigang; Mao, Guohong; Han, Ling; Liu, Yidong; Zhang, Shuqun

2012-06-01

Plants under pathogen attack produce high levels of ethylene, which plays important roles in plant immunity. Previously, we reported the involvement of ACS2 and ACS6, two Type I ACS isoforms, in Botrytis cinerea-induced ethylene biosynthesis and their regulation at the protein stability level by MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs). The residual ethylene induction in the acs2/acs6 double mutant suggests the involvement of additional ACS isoforms. It is also known that a subset of ACS genes, including ACS6, is transcriptionally induced in plants under stress or pathogen attack. However, the importance of ACS gene activation and the regulatory mechanism(s) are not clear. In this report, we demonstrate using genetic analysis that ACS7 and ACS11, two Type III ACS isoforms, and ACS8, a Type II ACS isoform, also contribute to the B. cinerea-induced ethylene production. In addition to post-translational regulation, transcriptional activation of the ACS genes also plays a critical role in sustaining high levels of ethylene induction. Interestingly, MPK3 and MPK6 not only control the stability of ACS2 and ACS6 proteins via direct protein phosphorylation but also regulate the expression of ACS2 and ACS6 genes. WRKY33, another MPK3/MPK6 substrate, is involved in the MPK3/MPK6-induced ACS2/ACS6 gene expression based on genetic analyses. Furthermore, chromatin-immunoprecipitation assay reveals the direct binding of WRKY33 to the W-boxes in the promoters of ACS2 and ACS6 genes in vivo, suggesting that WRKY33 is directly involved in the activation of ACS2 and ACS6 expression downstream of MPK3/MPK6 cascade in response to pathogen invasion. Regulation of ACS activity by MPK3/MPK6 at both transcriptional and protein stability levels plays a key role in determining the kinetics and magnitude of ethylene induction.

The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch.

PubMed

Oberst, Andrew; Malatesta, Martina; Aqeilan, Rami I; Rossi, Mario; Salomoni, Paolo; Murillas, Rodolfo; Sharma, Prashant; Kuehn, Michael R; Oren, Moshe; Croce, Carlo M; Bernassola, Francesca; Melino, Gerry

2007-07-03

Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin-protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73 alpha, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73 alpha for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73 alpha and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1(-/-) cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73 alpha and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy.

STING-Dependent Interferon-λ1 Induction in HT29 Cells, a Human Colorectal Cancer Cell Line, After Gamma-Radiation.

PubMed

Chen, Jianzhou; Markelc, Bostjan; Kaeppler, Jakob; Ogundipe, Vivian M L; Cao, Yunhong; McKenna, W Gillies; Muschel, Ruth J

2018-05-01

To investigate the induction of type III interferons (IFNs) in human cancer cells by gamma-rays. Type III IFN expression in human cancer cell lines after gamma-ray irradiation in vitro was assessed by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Signaling pathways mediating type III IFN induction were examined by a variety of means, including immunoblotting, flow cytometry, confocal imaging, and reverse transcription-quantitative polymerase chain reaction. Key mediators in these pathways were further explored and validated using gene CRISPR knockout or short hairpin RNA knockdown. Exposure to gamma-rays directly induced type III IFNs (mainly IFNL1) in human cancer cell lines in dose- and time-dependent fashions. The induction of IFNL1 was primarily mediated by the cytosolic DNA sensors-STING-TBK1-IRF1 signaling axis, with a lesser contribution from the nuclear factor kappa b signaling in HT29 cells. In addition, type III IFN signaling through its receptors serves as a positive feedback loop, further enhancing IFN expression via up-regulation of the kinases in the STING-TBK1 signaling axis. Our results suggest that IFNL1 can be up-regulated in human cancer cell lines after gamma-ray treatment. In HT29 cells this induction occurs via the STING pathway, adding another layer of complexity to the understanding of radiation-induced antitumor immunity, and may provide novel insights into IFN-based cancer treatment. Copyright © 2018 Elsevier Inc. All rights reserved.

Multilayered Regulation of Ethylene Induction Plays a Positive Role in Arabidopsis Resistance against Pseudomonas syringae.

PubMed

Guan, Rongxia; Su, Jianbin; Meng, Xiangzong; Li, Sen; Liu, Yidong; Xu, Juan; Zhang, Shuqun

2015-09-01

Ethylene, a key phytohormone involved in plant-pathogen interaction, plays a positive role in plant resistance against fungal pathogens. However, its function in plant bacterial resistance remains unclear. Here, we report a detailed analysis of ethylene induction in Arabidopsis (Arabidopsis thaliana) in response to Pseudomonas syringae pv tomato DC3000 (Pst). Ethylene biosynthesis is highly induced in both pathogen/microbe-associated molecular pattern (PAMP)-triggered immunity and effector-triggered immunity (ETI), and the induction is potentiated by salicylic acid (SA) pretreatment. In addition, Pst actively suppresses PAMP-triggered ethylene induction in a type III secretion system-dependent manner. SA potentiation of ethylene induction is dependent mostly on MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and MPK3 and their downstream ACS2 and ACS6, two type I isoforms of 1-aminocyclopropane-1-carboxylic acid synthases (ACSs). ACS7, a type III ACS whose expression is enhanced by SA pretreatment, is also involved. Pst expressing the avrRpt2 effector gene (Pst-avrRpt2), which is capable of triggering ETI, induces a higher level of ethylene production, and the elevated portion is dependent on SALICYLIC ACID INDUCTION DEFICIENT2 and NONEXPRESSER OF PATHOGENESIS-RELATED GENE1, two key players in SA biosynthesis and signaling. High-order ACS mutants with reduced ethylene induction are more susceptible to both Pst and Pst-avrRpt2, demonstrating a positive role of ethylene in plant bacterial resistance mediated by both PAMP-triggered immunity and ETI. © 2015 American Society of Plant Biologists. All Rights Reserved.

p18(Hamlet) mediates different p53-dependent responses to DNA-damage inducing agents.

PubMed

Lafarga, Vanesa; Cuadrado, Ana; Nebreda, Angel R

2007-10-01

Cells organize appropriate responses to environmental cues by activating specific signaling networks. Two proteins that play key roles in coordinating stress responses are the kinase p38alpha (MAPK14) and the transcription factor p53 (TP53). Depending on the nature and the extent of the stress-induced damage, cells may respond by arresting the cell cycle or by undergoing cell death, and these responses are usually associated with the phosphorylation of particular substrates by p38alpha as well as the activation of specific target genes by p53. We recently characterized a new p38alpha substrate, named p18(Hamlet) (ZNHIT1), which mediates p53-dependent responses to different genotoxic stresses. Thus, cisplatin or UV light induce stabilization of the p18(Hamlet) protein, which then enhances the ability of p53 to bind to and activate the promoters of pro-apoptotic genes such as NOXA and PUMA leading to apoptosis induction. In a similar way, we report here that p18(Hamlet) can also mediate the cell cycle arrest induced in response to gamma-irradiation, by participating in the p53-dependent upregulation of the cell cycle inhibitor p21(Cip1) (CDKN1A).

Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate.

PubMed

Emanuele, Michael J; Ciccia, Alberto; Elia, Andrew E H; Elledge, Stephen J

2011-06-14

The anaphase-promoting complex/cyclosome (APC/C) is a cell cycle-regulated E3 ubiquitin ligase that controls the degradation of substrate proteins at mitotic exit and throughout the G1 phase. We have identified an APC/C substrate and cell cycle-regulated protein, KIAA0101/PAF15. PAF15 protein levels peak in the G2/M phase of the cell cycle and drop rapidly at mitotic exit in an APC/C- and KEN-box-dependent fashion. PAF15 associates with proliferating cell nuclear antigen (PCNA), and depletion of PAF15 decreases the number of cells in S phase, suggesting a role for it in cell cycle regulation. Following irradiation, PAF15 colocalized with γH2AX foci at sites of DNA damage through its interaction with PCNA. Finally, PAF15 depletion led to an increase in homologous recombination-mediated DNA repair, and overexpression caused sensitivity to UV-induced DNA damage. We conclude that PAF15 is an APC/C-regulated protein involved in both cell cycle progression and the DNA damage response.

An ortholog of farA of Aspergillus nidulans is implicated in the transcriptional activation of genes involved in fatty acid utilization in the yeast Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poopanitpan, Napapol; Kobayashi, Satoshi; Fukuda, Ryouichi

2010-11-26

Research highlights: {yields} POR1 is a Yarrowia lipolytica ortholog of farA involved in fatty acid response in A. nidulans. {yields} Deletion of POR1 caused growth defects on fatty acids. {yields} {Delta}por1 strain exhibited defects in the induction of genes involved in fatty acid utilization. -- Abstract: The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomyces cerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y.more » lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in {beta}-oxidation and peroxisome proliferation by oleate was distinctly diminished in the {Delta}por1 strains. These data suggest that POR1 encodes a transcriptional activator widely regulating fatty acid metabolism in Y. lipolytica.« less

REDD1 induction regulates the skeletal muscle gene expression signature following acute aerobic exercise.

PubMed

Gordon, Bradley S; Steiner, Jennifer L; Rossetti, Michael L; Qiao, Shuxi; Ellisen, Leif W; Govindarajan, Subramaniam S; Eroshkin, Alexey M; Williamson, David L; Coen, Paul M

2017-12-01

The metabolic stress placed on skeletal muscle by aerobic exercise promotes acute and long-term health benefits in part through changes in gene expression. However, the transducers that mediate altered gene expression signatures have not been completely elucidated. Regulated in development and DNA damage 1 (REDD1) is a stress-induced protein whose expression is transiently increased in skeletal muscle following acute aerobic exercise. However, the role of this induction remains unclear. Because REDD1 altered gene expression in other model systems, we sought to determine whether REDD1 induction following acute exercise altered the gene expression signature in muscle. To do this, wild-type and REDD1-null mice were randomized to remain sedentary or undergo a bout of acute treadmill exercise. Exercised mice recovered for 1, 3, or 6 h before euthanization. Acute exercise induced a transient increase in REDD1 protein expression within the plantaris only at 1 h postexercise, and the induction occurred in both cytosolic and nuclear fractions. At this time point, global changes in gene expression were surveyed using microarray. REDD1 induction was required for the exercise-induced change in expression of 24 genes. Validation by RT-PCR confirmed that the exercise-mediated changes in genes related to exercise capacity, muscle protein metabolism, neuromuscular junction remodeling, and Metformin action were negated in REDD1-null mice. Finally, the exercise-mediated induction of REDD1 was partially dependent upon glucocorticoid receptor activation. In all, these data show that REDD1 induction regulates the exercise-mediated change in a distinct set of genes within skeletal muscle. Copyright © 2017 the American Physiological Society.

Deletion analysis of Streptococcus pneumoniae late competence genes distinguishes virulence determinants that are dependent or independent of competence induction

PubMed Central

Zhu, Luchang; Lin, Jingjun; Kuang, Zhizhou; Vidal, Jorge E.; Lau, Gee W.

2015-01-01

Summary The competence regulon of Streptococcus pneumoniae (pneumococcus) is crucial for genetic transformation. During competence development, the alternative sigma factor ComX is activated, which in turn, initiates transcription of 80 “late” competence genes. Interestingly, only 16 late genes are essential for genetic transformation. We hypothesized that these late genes that are dispensable for competence are beneficial to pneumococcal fitness during infection. These late genes were systematically deleted, and the resulting mutants were examined for their fitness during mouse models of bacteremia and acute pneumonia. Among these, 14 late genes were important for fitness in mice. Significantly, deletion of some late genes attenuated pneumococcal fitness to the same level in both wild-type and ComX-null genetic backgrounds, suggesting that the constitutive baseline expression of these genes was important for bacterial fitness. In contrast, some mutants were attenuated only in the wild-type genetic background but not in the ComX-null background, suggesting that specific expression of these genes during competence state contributed to pneumococcal fitness. Increased virulence during competence state was partially caused by the induction of allolytic enzymes that enhanced pneumolysin release. These results distinguish the role of basal expression versus competence induction in virulence functions encoded by ComX-regulated late competence genes. Graphical abstract During genetic transformation of pneumococcus, the alternative sigma factor ComX regulates expression of 14 late competence genes important for virulence. The constitutive baseline expression of some of these genes is important for bacteremia and acute pneumonia infections. In contrast, elevated expression of DprA, CbpD, CibAB, and Cinbox are dependent on competence development, enhancing the release of pneumolysin. These results distinguish the role of basal expression versus competence induction in virulence determinants regulated by ComX. PMID:25846124

Fatty Acid-binding Protein 4, a Point of Convergence for Angiogenic and Metabolic Signaling Pathways in Endothelial Cells*

PubMed Central

Harjes, Ulrike; Bridges, Esther; McIntyre, Alan; Fielding, Barbara A.; Harris, Adrian L.

2014-01-01

Fatty acid-binding protein 4 (FABP4) is an adipogenic protein and is implicated in atherosclerosis, insulin resistance, and cancer. In endothelial cells, FABP4 is induced by VEGFA, and inhibition of FABP4 blocks most of the VEGFA effects. We investigated the DLL4-NOTCH-dependent regulation of FABP4 in human umbilical vein endothelial cells by gene/protein expression and interaction analyses following inhibitor treatment and RNA interference. We found that FABP4 is directly induced by NOTCH. Stimulation of NOTCH signaling with human recombinant DLL4 led to FABP4 induction, independently of VEGFA. FABP4 induction by VEGFA was reduced by blockade of DLL4 binding to NOTCH or inhibition of NOTCH signal transduction. Chromatin immunoprecipitation of the NOTCH intracellular domain showed increased binding to two specific regions in the FABP4 promoter. The induction of FABP4 gene expression was dependent on the transcription factor FOXO1, which was essential for basal expression of FABP4, and FABP4 up-regulation following stimulation of the VEGFA and/or the NOTCH pathway. Thus, we show that the DLL4-NOTCH pathway mediates endothelial FABP4 expression. This indicates that induction of the angiogenesis-restricting DLL4-NOTCH can have pro-angiogenic effects via this pathway. It also provides a link between DLL4-NOTCH and FOXO1-mediated regulation of endothelial gene transcription, and it shows that DLL4-NOTCH is a nodal point in the integration of pro-angiogenic and metabolic signaling in endothelial cells. This may be crucial for angiogenesis in the tumor environment. PMID:24939870

EPAS-1 mediates SP-1-dependent FBI-1 expression and regulates tumor cell survival and proliferation.

PubMed

Wang, Xiaogang; Cao, Peng; Li, Zhiqing; Wu, Dongyang; Wang, Xi; Liang, Guobiao

2014-09-04

Factor binding IST-1 (FBI-1) plays an important role in oncogenic transformation and tumorigenesis. As FBI-1 is over-expressed in multiple human cancers, the regulation of itself would provide new effective options for cancer intervention. In this work, we aimed to study the role that EPAS-1 plays in regulating FBI-1. We use the fact that specificity protein-1 (SP-1) is one of the crucial transcription factors of FBI-1, and that SP-1 can interact with the endothelial pas domain protein-1 (EPAS-1) for the induction of hypoxia related genes. The study showed that EPAS-1 plays an indispensible role in SP-1 transcription factor-mediated FBI-1 induction, and participated in tumor cell survival and proliferation. Thus, EPAS-1 could be a novel target for cancer therapeutics.

Substrate stiffness regulates cadherin-dependent collective migration through myosin-II contractility

PubMed Central

Ng, Mei Rosa; Besser, Achim

2012-01-01

The mechanical microenvironment is known to influence single-cell migration; however, the extent to which mechanical cues affect collective migration of adherent cells is not well understood. We measured the effects of varying substrate compliance on individual cell migratory properties in an epithelial wound-healing assay. Increasing substrate stiffness increased collective cell migration speed, persistence, and directionality as well as the coordination of cell movements. Dynamic analysis revealed that wounding initiated a wave of motion coordination from the wound edge into the sheet. This was accompanied by a front-to-back gradient of myosin-II activation and establishment of cell polarity. The propagation was faster and farther reaching on stiff substrates, indicating that substrate stiffness affects the transmission of directional cues. Manipulation of myosin-II activity and cadherin–catenin complexes revealed that this transmission is mediated by coupling of contractile forces between neighboring cells. Thus, our findings suggest that the mechanical environment integrates in a feedback with cell contractility and cell–cell adhesion to regulate collective migration. PMID:23091067

Gadd45a, the gene induced by the mood stabilizer valproic acid, regulates neurite outgrowth through JNK and the substrate paxillin in N1E-115 neuroblastoma cells.

PubMed

Yamauchi, Junji; Miyamoto, Yuki; Murabe, Mayu; Fujiwara, Yoko; Sanbe, Atsushi; Fujita, Yuko; Murase, Shoko; Tanoue, Akito

2007-05-15

Valproic acid (VPA), a mood stabilizer and anticonvulsant, has a variety of neurotrophic functions; however, less is known about how VPA regulates neurite outgrowth. Here, using N1E-115 neuroblastoma cells as the model, we show that VPA upregulates Gadd45a to trigger activation of the downstream JNK cascade controlling neurite outgrowth. VPA induces the phosphorylation of c-Jun N-terminal kinase (JNK) and the substrate paxillin, while VPA induction of neurite outgrowth is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) or a paxillin construct harboring a Ser 178-to-Ala mutation at the JNK phosphorylation. Transfection of Gadd45a, acting through the effector MEKK4, leads to the phosphorylation of the JNK cascade. Conversely, knockdown of Gadd45a with siRNA reduces the effect of VPA. Taken together, these results suggest that upregulation of Gadd45a explains one of the mechanisms whereby VPA induces the neurotrophic effect, providing a new role of Gadd45a in neurite outgrowth.

Hedgehog-mediated regulation of PPARγ controls metabolic patterns in neural precursors and shh-driven medulloblastoma.

PubMed

Bhatia, Bobby; Potts, Chad R; Guldal, Cemile; Choi, SunPhil; Korshunov, Andrey; Pfister, Stefan; Kenney, Anna M; Nahlé, Zaher A

2012-04-01

Sonic hedgehog (Shh) signaling is critical during development and its aberration is common across the spectrum of human malignancies. In the cerebellum, excessive activity of the Shh signaling pathway is associated with the devastating pediatric brain tumor medulloblastoma. We previously demonstrated that exaggerated de novo lipid synthesis is a hallmark of Shh-driven medulloblastoma and that hedgehog signaling inactivates the Rb/E2F tumor suppressor complex to promote lipogenesis. Indeed, such Shh-mediated metabolic reprogramming fuels tumor progression, in an E2F1- and FASN-dependent manner. Here, we show that the nutrient sensor PPARγ is a key component of the Shh metabolic network, particularly its regulation of glycolysis. Our data show that in primary cerebellar granule neural precursors (CGNPs), proposed medulloblastoma cells-of-origin, Shh stimulation elicits a marked induction of PPARγ alongside major glycolytic markers. This is also documented in the actively proliferating Shh-responsive CGNPs in the developing cerebellum, and PPARγ expression is strikingly elevated in Shh-driven medulloblastoma in vivo. Importantly, pharmacological blockade of PPARγ and/or Rb inactivation inhibits CGNP proliferation, drives medulloblastoma cell death and extends survival of medulloblastoma-bearing animals in vivo. This coupling of mitogenic Shh signaling to a major nutrient sensor and metabolic transcriptional regulator define a novel mechanism through which Shh signaling engages the nutrient sensing machinery in brain cancer, controls the cell cycle, and regulates the glycolytic index. This also reveals a dominant role of Shh in the etiology of glucose metabolism in medulloblastoma and underscores the function of the Shh → E2F1 → PPARγ axis in altering substrate utilization patterns in brain cancers in favor of tumor growth. These findings emphasize the value of PPARγ downstream of Shh as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors.

Regulated Formation of lncRNA-DNA Hybrids Enables Faster Transcriptional Induction and Environmental Adaptation.

PubMed

Cloutier, Sara C; Wang, Siwen; Ma, Wai Kit; Al Husini, Nadra; Dhoondia, Zuzer; Ansari, Athar; Pascuzzi, Pete E; Tran, Elizabeth J

2016-02-04

Long non-coding (lnc)RNAs, once thought to merely represent noise from imprecise transcription initiation, have now emerged as major regulatory entities in all eukaryotes. In contrast to the rapidly expanding identification of individual lncRNAs, mechanistic characterization has lagged behind. Here we provide evidence that the GAL lncRNAs in the budding yeast S. cerevisiae promote transcriptional induction in trans by formation of lncRNA-DNA hybrids or R-loops. The evolutionarily conserved RNA helicase Dbp2 regulates formation of these R-loops as genomic deletion or nuclear depletion results in accumulation of these structures across the GAL cluster gene promoters and coding regions. Enhanced transcriptional induction is manifested by lncRNA-dependent displacement of the Cyc8 co-repressor and subsequent gene looping, suggesting that these lncRNAs promote induction by altering chromatin architecture. Moreover, the GAL lncRNAs confer a competitive fitness advantage to yeast cells because expression of these non-coding molecules correlates with faster adaptation in response to an environmental switch. Copyright © 2016 Elsevier Inc. All rights reserved.

JIP1-Mediated JNK Activation Negatively Regulates Synaptic Plasticity and Spatial Memory.

PubMed

Morel, Caroline; Sherrin, Tessi; Kennedy, Norman J; Forest, Kelly H; Avcioglu Barutcu, Seda; Robles, Michael; Carpenter-Hyland, Ezekiel; Alfulaij, Naghum; Standen, Claire L; Nichols, Robert A; Benveniste, Morris; Davis, Roger J; Todorovic, Cedomir

2018-04-11

The c-Jun N-terminal kinase (JNK) signal transduction pathway is implicated in learning and memory. Here, we examined the role of JNK activation mediated by the JNK-interacting protein 1 (JIP1) scaffold protein. We compared male wild-type mice with a mouse model harboring a point mutation in the Jip1 gene that selectively blocks JIP1-mediated JNK activation. These male mutant mice exhibited increased NMDAR currents, increased NMDAR-mediated gene expression, and a lower threshold for induction of hippocampal long-term potentiation. The JIP1 mutant mice also displayed improved hippocampus-dependent spatial memory and enhanced associative fear conditioning. These results were confirmed using a second JIP1 mutant mouse model that suppresses JNK activity. Together, these observations establish that JIP1-mediated JNK activation contributes to the regulation of hippocampus-dependent, NMDAR-mediated synaptic plasticity and learning. SIGNIFICANCE STATEMENT The results of this study demonstrate that c-Jun N-terminal kinase (JNK) activation induced by the JNK-interacting protein 1 (JIP1) scaffold protein negatively regulates the threshold for induction of long-term synaptic plasticity through the NMDA-type glutamate receptor. This change in plasticity threshold influences learning. Indeed, mice with defects in JIP1-mediated JNK activation display enhanced memory in hippocampus-dependent tasks, such as contextual fear conditioning and Morris water maze, indicating that JIP1-JNK constrains spatial memory. This study identifies JIP1-mediated JNK activation as a novel molecular pathway that negatively regulates NMDAR-dependent synaptic plasticity and memory. Copyright © 2018 the authors 0270-6474/18/383708-21$15.00/0.

Activation of PPAR-α in the early stage of heart failure maintained myocardial function and energetics in pressure-overload heart failure.

PubMed

Kaimoto, Satoshi; Hoshino, Atsushi; Ariyoshi, Makoto; Okawa, Yoshifumi; Tateishi, Shuhei; Ono, Kazunori; Uchihashi, Motoki; Fukai, Kuniyoshi; Iwai-Kanai, Eri; Matoba, Satoaki

2017-02-01

Failing heart loses its metabolic flexibility, relying increasingly on glucose as its preferential substrate and decreasing fatty acid oxidation (FAO). Peroxisome proliferator-activated receptor α (PPAR-α) is a key regulator of this substrate shift. However, its role during heart failure is complex and remains unclear. Recent studies reported that heart failure develops in the heart of myosin heavy chain-PPAR-α transgenic mice in a manner similar to that of diabetic cardiomyopathy, whereas cardiac dysfunction is enhanced in PPAR-α knockout mice in response to chronic pressure overload. We created a pressure-overload heart failure model in mice through transverse aortic constriction (TAC) and activated PPAR-α during heart failure using an inducible transgenic model. After 8 wk of TAC, left ventricular (LV) function had decreased with the reduction of PPAR-α expression in wild-type mice. We examined the effect of PPAR-α induction during heart failure using the Tet-Off system. Eight weeks after the TAC operation, LV construction was preserved significantly by PPAR-α induction with an increase in PPAR-α-targeted genes related to fatty acid metabolism. The increase of expression of fibrosis-related genes was significantly attenuated by PPAR-α induction. Metabolic rates measured by isolated heart perfusions showed a reduction in FAO and glucose oxidation in TAC hearts, but the rate of FAO preserved significantly owing to the induction of PPAR-α. Myocardial high-energy phosphates were significantly preserved by PPAR-α induction. These results suggest that PPAR-α activation during pressure-overloaded heart failure improved myocardial function and energetics. Thus activating PPAR-α and modulation of FAO could be a promising therapeutic strategy for heart failure. NEW & NOTEWORTHY The present study demonstrates the role of PPAR-α activation in the early stage of heart failure using an inducible transgenic mouse model. Induction of PPAR-α preserved heart function, and myocardial energetics. Activating PPAR-α and modulation of fatty acid oxidation could be a promising therapeutic strategy for heart failure. Copyright © 2017 the American Physiological Society.

Interactions between Casein Kinase Iε (CKIε) and Two Substrates from Disparate Signaling Pathways Reveal Mechanisms for Substrate-Kinase Specificity

PubMed Central

Dahlberg, Caroline Lund; Nguyen, Elizabeth Z.; Goodlett, David; Kimelman, David

2009-01-01

Background Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIε and two substrates from different signaling pathways. Methodology/Principal Findings CKIε, but not CKIα, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIα's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIε does not determine Dishevelled's and Period's preference for CKIε nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIε with its substrates. We demonstrate that autophosphorylation of CKIε's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. Conclusions/Significance The biochemical interactions between CKIε and Disheveled, Period, and its own C-terminus lead to models that explain CKIε's specificity and regulation. PMID:19274088

Induction of cyclooxygenase-2 expression by allergens in lymphocytes from allergic patients.

PubMed

Chacón, Pedro; Vega, Antonio; Monteseirín, Javier; El Bekay, Rajaa; Alba, Gonzalo; Pérez-Formoso, José Luis; Msartínez, Alberto; Asturias, Juan A; Pérez-Cano, Ramón; Sobrino, Francisco; Conde, José

2005-08-01

Cyclooxygenase (COX) is a key enzyme in prostaglandin (PG) synthesis. Up-regulation of COX-2 expression is responsible for increased PG release during inflammatory conditions and is thought to be also involved in allergic states. In this study, we demonstrate that in human T, B and natural killer lymphocytes from allergic patients, COX-2 expression became induced upon cell challenge with specific allergens and that this process is presumably IgE dependent and occurs after CD23 receptor ligation. This induction took place at both mRNA and protein levels and was accompanied by PGD2 release. IgE-dependent lymphocyte treatment elicited, in parallel, an activation of the MAPK p38 and extracellular signal-regulated kinase 1/2, an enhancement of calcineurin (CaN) activity, and an increase of the DNA-binding activity of the nuclear factor of activated T cells and of NF-kappaB, with a concomitant decrease in the levels of the cytosolic inhibitor of kappaB, IkappaB. In addition, specific chemical inhibitors of MAPK, such as PD098059 and SB203580, as well as MG-132, an inhibitor of proteasomal activity, abolished allergen-induced COX-2 up-regulation, suggesting that this process is mediated by MAPK and NF-kappaB. However, induction of COX-2 expression was not hampered by the CaN inhibitor cyclosporin A. We also examined the effect of a selective COX-2 inhibitor, NS-398, on cytokine production by human lymphocytes. Treatment with NS-398 severely diminished the IgE-dependently induced production of IL-8 and TNF-alpha. These results underscore the relevant role of lymphocyte COX-2 in allergy and suggest that COX-2 inhibitors may contribute to the improvement of allergic inflammation through the reduction of inflammatory mediator production by human lymphocytes.

Differential regulation of p65 and c-Rel NF-kappaB transactivating activity by Cot, protein kinase C zeta and NIK protein kinases in CD3/CD28 activated T cells.

PubMed

Sánchez-Valdepeñas, Carmen; Punzón, Carmen; San-Antonio, Belén; Martin, Angel G; Fresno, Manuel

2007-03-01

It has been shown that phosphorylation of p65/RelA and c-Rel plays a role in the regulation of transcriptional activity of NF-kappaB independent on IkappaB degradation. In this study, we show that anti CD3/CD28 activation induces the transactivation activity of both p65/RelA and c-Rel in T cells using Gal4 dependent assays. Moreover, protein kinase C (PKC)zeta, Cot kinase and NF-kappaB-inducing kinase (NIK) seem to be involved in those processes in a different manner. Thus, transfection of dominant negative forms of Cot and PKCzeta inhibits CD3/CD28 induction of Gal4-p65 transactivation, whereas the kinase inactive versions of the 3 kinases inhibit induction of Gal4-c-Rel. Cot induction of Gal4-c-Rel transactivating activity seems to be mediated sequentially through PKCzeta and NIK activation, since dominant negative form of NIK blocks Cot and PKCzeta induction, whereas kinase inactive PKCzeta only blocks Cot activity. In contrast, the contribution of NIK to the transactivation function of p65/RelA seems to be negligible and more importantly NIK-KD did not inhibit induction by Cot and PKCzeta. Besides, the enhancing effect of Cot on Gal4-p65 was not decreased in mouse embryo fibroblasts from NIK deficient aly/aly mice in contrast with a greatest reduction on Gal4-c-Rel. By using Ser to Ala mutants in p65 and c-Rel transactivation domains, PKCzeta and NIK activities seem to be dependent of a restricted set of Ser in both proteins. In contrast, the enhancing effect of Cot seems to be less dependent of a particular set of Ser residues being partially abrogated by mutation of several Ser residues.

Regulating Anger under Stress via Cognitive Reappraisal and Sadness.

PubMed

Zhan, Jun; Wu, Xiaofei; Fan, Jin; Guo, Jianyou; Zhou, Jianshe; Ren, Jun; Liu, Chang; Luo, Jing

2017-01-01

Previous studies have reported the failure of cognitive emotion regulation (CER), especially in regulating unpleasant emotions under stress. The underlying reason for this failure was the application of CER depends heavily on the executive function of the prefrontal cortex (PFC), but this function can be impaired by stress-related neuroendocrine hormones. This observation highlights the necessity of developing self-regulatory strategies that require less top-down cognitive control. Based on traditional Chinese philosophy and medicine, which examine how different types of emotions promote or counteract one another, we have developed a novel emotion regulation strategy whereby one emotion is used to alter another. For example, our previous experiment showed that sadness induction (after watching a sad film) could reduce aggressive behavior associated with anger [i.e., "sadness counteracts anger" (SCA)] (Zhan et al., 2015). Relative to the CER strategy requiring someone to think about certain cognitive reappraisals to reinterpret the meaning of an unpleasant situation, watching a film or listening to music and experiencing the emotion contained therein seemingly requires less cognitive effort and control; therefore, this SCA strategy may be an alternative strategy that compensates for the limitations of cognitive regulation strategies, especially in stressful situations. The present study was designed to directly compare the effects of the CER and SCA strategy in regulating anger and anger-related aggression in stressful and non-stressful conditions. Participants' subjective feeling of anger, anger-related aggressive behavior, skin conductance, and salivary cortisol and alpha-amylase levels were measured. Our findings revealed that acute stress impaired one's ability to use CR to control angry responses provoked by others, whereas stress did not influence the efficiency of the SCA strategy. Compared with sadness or neutral emotion induction, CER induction was found to reduce the level of subjective anger more, but this difference only existed in non-stressful conditions. By contrast, irrespective of stress, the levels of aggressive behavior and related skin conductance after sadness induction were both significantly lower than those after CER induction or neutral emotion induction, thus suggesting the immunity of the regulatory effect of SCA strategy to the stress factor.

Regulating Anger under Stress via Cognitive Reappraisal and Sadness

PubMed Central

Zhan, Jun; Wu, Xiaofei; Fan, Jin; Guo, Jianyou; Zhou, Jianshe; Ren, Jun; Liu, Chang; Luo, Jing

2017-01-01

Previous studies have reported the failure of cognitive emotion regulation (CER), especially in regulating unpleasant emotions under stress. The underlying reason for this failure was the application of CER depends heavily on the executive function of the prefrontal cortex (PFC), but this function can be impaired by stress-related neuroendocrine hormones. This observation highlights the necessity of developing self-regulatory strategies that require less top-down cognitive control. Based on traditional Chinese philosophy and medicine, which examine how different types of emotions promote or counteract one another, we have developed a novel emotion regulation strategy whereby one emotion is used to alter another. For example, our previous experiment showed that sadness induction (after watching a sad film) could reduce aggressive behavior associated with anger [i.e., “sadness counteracts anger” (SCA)] (Zhan et al., 2015). Relative to the CER strategy requiring someone to think about certain cognitive reappraisals to reinterpret the meaning of an unpleasant situation, watching a film or listening to music and experiencing the emotion contained therein seemingly requires less cognitive effort and control; therefore, this SCA strategy may be an alternative strategy that compensates for the limitations of cognitive regulation strategies, especially in stressful situations. The present study was designed to directly compare the effects of the CER and SCA strategy in regulating anger and anger-related aggression in stressful and non-stressful conditions. Participants’ subjective feeling of anger, anger-related aggressive behavior, skin conductance, and salivary cortisol and alpha-amylase levels were measured. Our findings revealed that acute stress impaired one’s ability to use CR to control angry responses provoked by others, whereas stress did not influence the efficiency of the SCA strategy. Compared with sadness or neutral emotion induction, CER induction was found to reduce the level of subjective anger more, but this difference only existed in non-stressful conditions. By contrast, irrespective of stress, the levels of aggressive behavior and related skin conductance after sadness induction were both significantly lower than those after CER induction or neutral emotion induction, thus suggesting the immunity of the regulatory effect of SCA strategy to the stress factor. PMID:28855881

Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2–mediated heparin-binding EGF signaling in endothelial cells

PubMed Central

Svensson, Katrin J.; Kucharzewska, Paulina; Christianson, Helena C.; Sköld, Stefan; Löfstedt, Tobias; Johansson, Maria C.; Mörgelin, Matthias; Bengzon, Johan; Ruf, Wolfram; Belting, Mattias

2011-01-01

Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein–coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2–mediated activation of hypoxic ECs. We show that PAR-2–dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2–dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa–dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2–mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors. PMID:21788507

Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells.

PubMed

Svensson, Katrin J; Kucharzewska, Paulina; Christianson, Helena C; Sköld, Stefan; Löfstedt, Tobias; Johansson, Maria C; Mörgelin, Matthias; Bengzon, Johan; Ruf, Wolfram; Belting, Mattias

2011-08-09

Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein-coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2-mediated activation of hypoxic ECs. We show that PAR-2-dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2-dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa-dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2-mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors.

Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol-dependent detoxification pathway upon formaldehyde stress.

PubMed

Müller, Jonas E N; Meyer, Fabian; Litsanov, Boris; Kiefer, Patrick; Vorholt, Julia A

2015-12-01

Bacillus methanolicus MGA3 is a model facultative methylotroph of interest for fundamental research and biotechnological applications. Previous research uncovered a number of pathways potentially involved in one-carbon substrate utilization. Here, we applied dynamic (13) C labeling to elucidate which of these pathways operate during growth on methanol and to uncover potentially new ones. B. methanolicus MGA3 uses the assimilatory and dissimilatory ribulose monophosphate (RuMP) cycles for conversion of the central but toxic intermediate formaldehyde. Additionally, the operation of two cofactor-dependent formaldehyde oxidation pathways with distinct roles was revealed. One is dependent on tri- and tetraglutamylated tetrahydrofolate (THF) and is involved in formaldehyde oxidation during growth on methanol. A second pathway was discovered that is dependent on bacillithiol, a thiol cofactor present also in other Bacilli where it is known to function in redox-homeostasis. We show that bacillithiol-dependent formaldehyde oxidation is activated upon an upshift in formaldehyde induced by a substrate switch from mannitol to methanol. The genes and the corresponding enzymes involved in the biosynthesis of bacillithiol were identified by heterologous production of bacillithiol in Escherichia coli. The presented results indicate metabolic plasticity of the methylotroph allowing acclimation to fluctuating intracellular formaldehyde concentrations. © 2015 John Wiley & Sons Ltd.

Calcium-dependent protein kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates.

PubMed

Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D; Li, Ying; Romanowsky, Shawn; Cushman, John C; Gribskov, Michael; Harmon, Alice C; Harper, Jeffrey F

2011-01-01

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca(2+)-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with K(M) ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

Calcium-Dependent Protein Kinases from Arabidopsis Show Substrate Specificity Differences in an Analysis of 103 Substrates

PubMed Central

Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D.; Li, Ying; Romanowsky, Shawn; Cushman, John C.; Gribskov, Michael; Harmon, Alice C.; Harper, Jeffrey F.

2011-01-01

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies. PMID:22645532

Rare earth elements concentration in mushroom cultivation substrates affects the production process and fruit-bodies content of Pleurotus ostreatus and Cyclocybe cylindracea.

PubMed

Koutrotsios, Georgios; Danezis, Georgios P; Georgiou, Constantinos A; Zervakis, Georgios I

2018-04-20

Concentrations of 16 rare earth elements (REEs) and two actinides were determined for the first time both in cultivated mushrooms and in their production substrates by inductively coupled plasma mass spectroscopy. Moreover, the effect of REEs on cultivation parameters and composition of the final product was assessed, together with their potential use for authentication purposes. The concentrations of REEs varied greatly among seven cultivation substrates and correlated with measurements in Cyclocybe cylindracea mushrooms; no such correlation was established in Pleurotus ostreatus. Reduction of hemicellulose, cellulose, and lignin in substrates during P. ostreatus cultivation was positively correlated with REE concentrations, which also affected the production performance depending on the species examined. In all cases, a negative correlation was established between bioconcentration factors (BCF) in mushrooms and REE content in substrates, while the effect of substrate composition on BCF values varied according to the element studied. The estimated daily intake values of REEs through mushroom consumption was at much lower levels than those reported as potentially harmful for human health. The content of REEs in cultivation substrates and in mushrooms revealed that the bioaccumulation of elements differed in each fungus. The nature/origin of substrates seemed to affect the concentration of REEs in mushrooms to a considerable extent. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

p53-Regulated Apoptosis Is Differentiation Dependent in Ultraviolet B-Irradiated Mouse Keratinocytes

PubMed Central

Tron, Victor A.; Trotter, Martin J.; Tang, Liren; Krajewska, Maryla; Reed, John C.; Ho, Vincent C.; Li, Gang

1998-01-01

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (−/−) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53−/− mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m2 UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53−/− cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53−/− cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction. PMID:9708817

Nanoengineered Polystyrene Surfaces with Nanopore Array Pattern Alters Cytoskeleton Organization and Enhances Induction of Neural Differentiation of Human Adipose-Derived Stem Cells.

PubMed

Jung, Ae Ryang; Kim, Richard Y; Kim, Hyung Woo; Shrestha, Kshitiz Raj; Jeon, Seung Hwan; Cha, Kyoung Je; Park, Yong Hyun; Kim, Dong Sung; Lee, Ji Youl

2015-07-01

Human adipose-derived stem cells (hADSCs) can differentiate into various cell types depending on chemical and topographical cues. One topographical cue recently noted to be successful in inducing differentiation is the nanoengineered polystyrene surface containing nanopore array-patterned substrate (NP substrate), which is designed to mimic the nanoscale topographical features of the extracellular matrix. In this study, efficacies of NP and flat substrates in inducing neural differentiation of hADSCs were examined by comparing their substrate-cell adhesion rates, filopodia growth, nuclei elongation, and expression of neural-specific markers. The polystyrene nano Petri dishes containing NP substrates were fabricated by a nano injection molding process using a nickel electroformed nano-mold insert (Diameter: 200 nm. Depth of pore: 500 nm. Center-to-center distance: 500 nm). Cytoskeleton and filopodia structures were observed by scanning electron microscopy and F-actin staining, while cell adhesion was tested by vinculin staining after 24 and 48 h of seeding. Expression of neural specific markers was examined by real-time quantitative polymerase chain reaction and immunocytochemistry. Results showed that NP substrates lead to greater substrate-cell adhesion, filopodia growth, nuclei elongation, and expression of neural specific markers compared to flat substrates. These results not only show the advantages of NP substrates, but they also suggest that further study into cell-substrate interactions may yield great benefits for biomaterial engineering.

Influence of surface topology and electrostatic potential on water/electrode systems

NASA Astrophysics Data System (ADS)

Siepmann, J. Ilja; Sprik, Michiel

1995-01-01

We have used the classical molecular dynamics technique to simulate the ordering of a water film adsorbed on an atomic model of a tip of a scanning tunneling microscope approaching a planar metal surface. For this purpose, we have developed a classical model for the water-substrate interactions that solely depends on the coordinates of the particles and does not require the definition of geometrically smooth boundary surfaces or image planes. The model includes both an electrostatic induction for the metal atoms (determined by means of an extended Lagrangian technique) and a site-specific treatment of the water-metal chemisorption. As a validation of the model we have investigated the structure of water monolayers on metal substrates of various topology [the (111), (110), and (100) crystallographic faces] and composition (Pt, Ag, Cu, and Ni), and compared the results to experiments. The modeling of the electrostatic induction is compatible with a finite external potential imposed on the metal. This feature is used to investigate the structural rearrangements of the water bilayer between the pair of scanning tunneling microscope electrodes in response to an applied external voltage difference. We find significant asymmetry in the dependence on the sign of the applied voltage. Another result of the calculation is an estimate of the perturbation to the work function caused by the wetting film. For the conditions typical for operation of a scanning tunneling microscope probe, the change in the work function is found to be comparable to the applied voltage (a few hundred millivolts).

Peroxisome Proliferator-Activated Receptor γ Target Gene Encoding a Novel Angiopoietin-Related Protein Associated with Adipose Differentiation

PubMed Central

Yoon, J. Cliff; Chickering, Troy W.; Rosen, Evan D.; Dussault, Barry; Qin, Yubin; Soukas, Alexander; Friedman, Jeffrey M.; Holmes, William E.; Spiegelman, Bruce M.

2000-01-01

The nuclear receptor peroxisome proliferator-activated receptor γ regulates adipose differentiation and systemic insulin signaling via ligand-dependent transcriptional activation of target genes. However, the identities of the biologically relevant target genes are largely unknown. Here we describe the isolation and characterization of a novel target gene induced by PPARγ ligands, termed PGAR (for PPARγ angiopoietin related), which encodes a novel member of the angiopoietin family of secreted proteins. The transcriptional induction of PGAR follows a rapid time course typical of immediate-early genes and occurs in the absence of protein synthesis. The expression of PGAR is predominantly localized to adipose tissues and placenta and is consistently elevated in genetic models of obesity. Hormone-dependent adipocyte differentiation coincides with a dramatic early induction of the PGAR transcript. Alterations in nutrition and leptin administration are found to modulate the PGAR expression in vivo. Taken together, these data suggest a possible role for PGAR in the regulation of systemic lipid metabolism or glucose homeostasis. PMID:10866690

Regulation of Proteolysis by Human Deubiquitinating Enzymes

PubMed Central

Eletr, Ziad M.; Wilkinson, Keith D.

2013-01-01

The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein-protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USP) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN+/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase. PMID:23845989

Erythropoietin activates two distinct signaling pathways required for the initiation and the elongation of c-myc

NASA Technical Reports Server (NTRS)

Chen, C.; Sytkowski, A. J.

2001-01-01

Erythropoietin (Epo) stimulation of erythroid cells results in the activation of several kinases and a rapid induction of c-myc expression. Protein kinase C is necessary for Epo up-regulation of c-myc by promoting elongation at the 3'-end of exon 1. PKCepsilon mediates this signal. We now show that Epo triggers two signaling pathways to c-myc. Epo rapidly up-regulated Myc protein in BaF3-EpoR cells. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 blocked Myc up-regulation in a concentration-dependent manner but had no effect on the Epo-induced phosphorylation of ERK1 and ERK2. LY294002 also had no effect on Epo up-regulation of c-fos. MEK1 inhibitor PD98059 blocked both the c-myc and the c-fos responses to Epo. PD98059 and the PKC inhibitor H7 also blocked the phosphorylation of ERK1 and ERK2. PD98059 but not LY294002 inhibited Epo induction of ERK1 and ERK2 phosphorylation in normal erythroid cells. LY294002 blocked transcription of c-myc at exon 1. PD98059 had no effect on transcription from exon 1 but, rather, blocked Epo-induced c-myc elongation at the 3'-end of exon 1. These results identify two Epo signaling pathways to c-myc, one of which is PI3K-dependent operating on transcriptional initiation, whereas the other is mitogen-activated protein kinase-dependent operating on elongation.

Substrate stiffness governs the initiation of B cell activation by the concerted signaling of PKCβ and focal adhesion kinase

PubMed Central

Shaheen, Samina; Wan, Zhengpeng; Li, Zongyu; Chau, Alicia; Li, Xinxin; Zhang, Shaosen; Liu, Yang; Yi, Junyang; Zeng, Yingyue; Wang, Jing; Chen, Xiangjun; Xu, Liling; Chen, Wei; Wang, Fei; Lu, Yun; Zheng, Wenjie; Shi, Yan; Sun, Xiaolin; Li, Zhanguo; Xiong, Chunyang; Liu, Wanli

2017-01-01

The mechanosensing ability of lymphocytes regulates their activation in response to antigen stimulation, but the underlying mechanism remains unexplored. Here, we report that B cell mechanosensing-governed activation requires BCR signaling molecules. PMA-induced activation of PKCβ can bypass the Btk and PLC-γ2 signaling molecules that are usually required for B cells to discriminate substrate stiffness. Instead, PKCβ-dependent activation of FAK is required, leading to FAK-mediated potentiation of B cell spreading and adhesion responses. FAK inactivation or deficiency impaired B cell discrimination of substrate stiffness. Conversely, adhesion molecules greatly enhanced this capability of B cells. Lastly, B cells derived from rheumatoid arthritis (RA) patients exhibited an altered BCR response to substrate stiffness in comparison with healthy controls. These results provide a molecular explanation of how initiation of B cell activation discriminates substrate stiffness through a PKCβ-mediated FAK activation dependent manner. DOI: http://dx.doi.org/10.7554/eLife.23060.001 PMID:28755662

Induction of P450 genes in Nilaparvata lugens and Sogatella furcifera by two neonicotinoid insecticides.

PubMed

Yang, Yuan-Xue; Yu, Na; Zhang, Jian-Hua; Zhang, Yi-Xi; Liu, Ze-Wen

2018-06-01

Nilaparvata lugens and Sogatella furcifera are two primary planthoppers on rice throughout Asian countries and areas. Neonicotinoid insecticides, such as imidacloprid (IMI), have been extensively used to control rice planthoppers and IMI resistance consequently occurred with an important mechanism from the over-expression of P450 genes. The induction of P450 genes by IMI may increase the ability to metabolize this insecticide in planthoppers and increase the resistance risk. In this study, the induction of P450 genes was compared in S. furcifera treated with IMI and nitromethyleneimidazole (NMI), in two planthopper species by IMI lethal dose that kills 85% of the population (LD 85 ), and in N. lugens among three IMI doses (LD 15 , LD 50 and LD 85 ). When IMI and NMI at the LD 85 dose were applied to S. furcifera, the expression changes in most P450 genes were similar, including the up-regulation of nine genes and down-regulation of three genes. In terms of the expression changes in 12 homologous P450 genes between N. lugens and S. furcifera treated with IMI at the LD 85 dose, 10 genes had very similar patterns, such as up-regulation in seven genes, down-regulation in one gene and no significant changes in two genes. When three different IMI doses were applied to N. lugens, the changes in P450 gene expression were much different, such as up-regulation in four genes at all doses and dose-dependent regulation of the other nine genes. For example, CYP6AY1 could be induced by all IMI doses, while CYP6ER1 was only up-regulated by the LD 50 dose, although both genes were reported important in IMI resistance. In conclusion, P450 genes in two planthopper species showed similar regulation patterns in responding to IMI, and the two neonicotinoid insecticides had similar effects on P450 gene expression, although the regulation was often dose-dependent. © 2017 Institute of Zoology, Chinese Academy of Sciences.

Substrate stiffness-modulated registry phase correlations in cardiomyocytes map structural order to coherent beating

NASA Astrophysics Data System (ADS)

Dasbiswas, K.; Majkut, S.; Discher, D. E.; Safran, Samuel A.

2015-01-01

Recent experiments show that both striation, an indication of the structural registry in muscle fibres, as well as the contractile strains produced by beating cardiac muscle cells can be optimized by substrate stiffness. Here we show theoretically how the substrate rigidity dependence of the registry data can be mapped onto that of the strain measurements. We express the elasticity-mediated structural registry as a phase-order parameter using a statistical physics approach that takes the noise and disorder inherent in biological systems into account. By assuming that structurally registered myofibrils also tend to beat in phase, we explain the observed dependence of both striation and strain measurements of cardiomyocytes on substrate stiffness in a unified manner. The agreement of our ideas with experiment suggests that the correlated beating of heart cells may be limited by the structural order of the myofibrils, which in turn is regulated by their elastic environment.

Serotonin is an endogenous regulator of intestinal CYP1A1 via AhR.

PubMed

Manzella, Christopher; Singhal, Megha; Alrefai, Waddah A; Saksena, Seema; Dudeja, Pradeep K; Gill, Ravinder K

2018-04-17

Aryl hydrocarbon receptor (AhR) is a nuclear receptor that controls xenobiotic detoxification via induction of cytochrome P450 1A1 (CYP1A1) and regulates immune responses in the intestine. Metabolites of L-tryptophan activate AhR, which confers protection against intestinal inflammation. We tested the hypothesis that serotonin (5-HT) is an endogenous activator of AhR in intestinal epithelial cells. Treatment of Caco-2 monolayers with 5-HT induced CYP1A1 mRNA in a time- and concentration-dependent manner and also stimulated CYP1A1 activity. CYP1A1 induction by 5-HT was dependent upon uptake via serotonin transporter (SERT). Antagonism of AhR and knockdown of AhR and its binding partner aryl hydrocarbon receptor nuclear translocator (ARNT) attenuated CYP1A1 induction by 5-HT. Activation of AhR was evident by its nuclear translocation after 5-HT treatment and by induction of an AhR-responsive luciferase reporter. In vivo studies showed a dramatic decrease in CYP1A1 expression and other AhR target genes in SERT KO ileal mucosa by microarray analysis. These results suggest that intracellular accumulation of 5-HT via SERT induces CYP1A1 expression via AhR in intestinal epithelial cells, and SERT deficiency in vivo impairs activation of AhR. Our studies provide a novel link between the serotonergic and AhR pathways which has implications in xenobiotic metabolism and intestinal inflammation.

Aryl hydrocarbon receptor-dependent up-regulation of the heterodimeric amino acid transporter LAT1 (SLC7A5)/CD98hc (SLC3A2) by diesel exhaust particle extract in human bronchial epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Le Vee, Marc; Jouan, Elodie; Lecureur, Valérie

The heterodimeric L-type amino acid transporter (LAT) 1/CD98hc is overexpressed in lung cancers with a poor prognosis factor. Factors that contribute to LAT1/CD98hc overexpression in lung cells remain however to be determined, but the implication of atmospheric pollution can be suspected. The present study was therefore designed to analyze the effects of diesel exhaust particle (DEP) extract (DEPe) on LAT1/CD98hc expression in bronchial epithelial BEAS-2B cells. Exposure to DEPe up-regulated LAT1 and CD98hc mRNA levels in a concentration-dependent manner, with DEPe EC{sub 50} values (around 0.2 μg/mL) relevant to environmental situations. DEPe concomitantly induced LAT1/CD98hc protein expression and LAT1-mediated leucinemore » accumulation in BEAS-2B cells. Inhibition of the aryl hydrocarbon receptor (AhR) pathway through the use of a chemical AhR antagonist or the siRNA-mediated silencing of AhR expression was next found to prevent DEPe-mediated induction of LAT1/CD98hc, indicating that this regulation depends on AhR, known to be activated by major chemical DEP components like polycyclic aromatic hydrocarbons. DEPe exposure was finally shown to induce mRNA expression and activity of matrix metalloproteinase (MMP)-2 in BEAS-2B cells, in a CD98hc/focal adhesion kinase (FAK)/extracellular regulated kinase (ERK) manner, thus suggesting that DEPe-mediated induction of CD98hc triggers activation of the integrin/FAK/ERK signaling pathway known to be involved in MMP-2 regulation. Taken together, these data demonstrate that exposure to DEPe induces functional overexpression of the amino acid transporter LAT1/CD98hc in lung cells. Such a regulation may participate to pulmonary carcinogenic effects of DEPs, owing to the well-documented contribution of LAT1 and CD98hc to cancer development. - Highlights: • The amino acid transporter LAT1/CD98hc is up-regulated in DEPe-treated lung cells. • The aryl hydrocarbon receptor is involved in DEPe-triggered induction of LAT1/CD98hc. • DEPe enhances LAT1-mediated accumulation of leucine in lung cells. • DEPe increases MMP-2 expression and activity in a CD98hc/FAK/ERK-dependent manner.« less

p53-dependent inhibition of TrxR1 contributes to the tumor-specific induction of apoptosis by RITA.

PubMed

Hedström, Elisabeth; Eriksson, Sofi; Zawacka-Pankau, Joanna; Arnér, Elias S J; Selivanova, Galina

2009-11-01

Thioredoxin reductase 1 (TrxR1) is a key regulator in many redox-dependent cellular pathways, and is often overexpressed in cancer. Several studies have identified TrxR1 as a potentially important target for anticancer therapy. The low molecular weight compound RITA (NSC 652287) binds p53 and induces p53-dependent apoptosis. Here we found that RITA also targets TrxR1 by non-covalent binding, followed by inhibition of its activity in vitro and by inhibition of TrxR activity in cancer cells. Interestingly, a novel approximately 130 kDa form of TrxR1, presumably representing a stable covalently linked dimer, and an increased generation of reactive oxygen species (ROS) were induced by RITA in cancer cells in a p53-dependent manner. Similarly, the gold-based TrxR inhibitor auranofin induced apoptosis related to oxidative stress, but independently of p53 and without apparent induction of the approximately 130 kDa form of TrxR1. In contrast to the effects observed in cancer cells, RITA did not inhibit TrxR or ROS formation in normal fibroblasts (NHDF). The inhibition of TrxR1 can sensitize tumor cells to agents that induce oxidative stress and may directly trigger cell death. Thus, our results suggest that a unique p53-dependent effect of RITA on TrxR1 in cancer cells might synergize with p53-dependent induction of pro-apoptotic genes and oxidative stress, thereby leading to a robust induction of cancer cell death, without affecting non-transformed cells.

Clinical Drug-Drug Interactions Through Cytochrome P450 3A (CYP3A) for the Selective ALK Inhibitor Alectinib.

PubMed

Morcos, Peter N; Cleary, Yumi; Guerini, Elena; Dall, Georgina; Bogman, Katrijn; De Petris, Luigi; Viteri, Santiago; Bordogna, Walter; Yu, Li; Martin-Facklam, Meret; Phipps, Alex

2017-05-01

The efficacy and safety of alectinib, a central nervous system-active and selective anaplastic lymphoma kinase (ALK) inhibitor, has been demonstrated in patients with ALK-positive (ALK+) non-small cell lung cancer (NSCLC) progressing on crizotinib. Alectinib is mainly metabolized by cytochrome P450 3A (CYP3A) to a major similarly active metabolite, M4. Alectinib and M4 show evidence of weak time-dependent inhibition and small induction of CYP3A in vitro. We present results from 3 fixed-sequence studies evaluating drug-drug interactions for alectinib through CYP3A. Studies NP28990 and NP29042 enrolled 17 and 24 healthy subjects, respectively, and investigated potent CYP3A inhibition with posaconazole and potent CYP3A induction through rifampin, respectively, on the single oral dose pharmacokinetics (PK) of alectinib. A substudy of the global phase 2 NP28673 study enrolled 15 patients with ALK+ NSCLC to determine the effect of multiple doses of alectinib on the single oral dose PK of midazolam, a sensitive substrate of CYP3A. Potent CYP3A inhibition or induction resulted in only minor effects on the combined exposure of alectinib and M4. Multiple doses of alectinib did not influence midazolam exposure. These results suggest that dose adjustments may not be needed when alectinib is coadministered with CYP3A inhibitors or inducers or for coadministered CYP3A substrates. © 2016, The American College of Clinical Pharmacology.

Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and Reregulates Catabolism.

PubMed

He, Amanda; Penix, Stephanie R; Basting, Preston J; Griffith, Jessie M; Creamer, Kaitlin E; Camperchioli, Dominic; Clark, Michelle W; Gonzales, Alexandra S; Chávez Erazo, Jorge Sebastian; George, Nadja S; Bhagwat, Arvind A; Slonczewski, Joan L

2017-06-15

Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, 2015, https://doi.org/10.1128/AEM.03494-14). Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS 5 insertion or IS-mediated deletion in cadC , while population B11 had a point mutation affecting the arginine activator adiY The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR , yhiM , and Gad). Other strains showed downregulation of H 2 consumption mediated by hydrogenases ( hya and hyb ) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes ( dmsABC , frdABCD , hybABO , nikABCDE , and nrfAC ). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism. IMPORTANCE Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain. Copyright © 2017 American Society for Microbiology.

Pannexin 1 regulates bidirectional hippocampal synaptic plasticity in adult mice.

PubMed

Ardiles, Alvaro O; Flores-Muñoz, Carolina; Toro-Ayala, Gabriela; Cárdenas, Ana M; Palacios, Adrian G; Muñoz, Pablo; Fuenzalida, Marco; Sáez, Juan C; Martínez, Agustín D

2014-01-01

The threshold for bidirectional modification of synaptic plasticity is known to be controlled by several factors, including the balance between protein phosphorylation and dephosphorylation, postsynaptic free Ca(2+) concentration and NMDA receptor (NMDAR) composition of GluN2 subunits. Pannexin 1 (Panx1), a member of the integral membrane protein family, has been shown to form non-selective channels and to regulate the induction of synaptic plasticity as well as hippocampal-dependent learning. Although Panx1 channels have been suggested to play a role in excitatory long-term potentiation (LTP), it remains unknown whether these channels also modulate long-term depression (LTD) or the balance between both types of synaptic plasticity. To study how Panx1 contributes to excitatory synaptic efficacy, we examined the age-dependent effects of eliminating or blocking Panx1 channels on excitatory synaptic plasticity within the CA1 region of the mouse hippocampus. By using different protocols to induce bidirectional synaptic plasticity, Panx1 channel blockade or lack of Panx1 were found to enhance LTP, whereas both conditions precluded the induction of LTD in adults, but not in young animals. These findings suggest that Panx1 channels restrain the sliding threshold for the induction of synaptic plasticity and underlying brain mechanisms of learning and memory.

Pannexin 1 regulates bidirectional hippocampal synaptic plasticity in adult mice

PubMed Central

Ardiles, Alvaro O.; Flores-Muñoz, Carolina; Toro-Ayala, Gabriela; Cárdenas, Ana M.; Palacios, Adrian G.; Muñoz, Pablo; Fuenzalida, Marco; Sáez, Juan C.; Martínez, Agustín D.

2014-01-01

The threshold for bidirectional modification of synaptic plasticity is known to be controlled by several factors, including the balance between protein phosphorylation and dephosphorylation, postsynaptic free Ca2+ concentration and NMDA receptor (NMDAR) composition of GluN2 subunits. Pannexin 1 (Panx1), a member of the integral membrane protein family, has been shown to form non-selective channels and to regulate the induction of synaptic plasticity as well as hippocampal-dependent learning. Although Panx1 channels have been suggested to play a role in excitatory long-term potentiation (LTP), it remains unknown whether these channels also modulate long-term depression (LTD) or the balance between both types of synaptic plasticity. To study how Panx1 contributes to excitatory synaptic efficacy, we examined the age-dependent effects of eliminating or blocking Panx1 channels on excitatory synaptic plasticity within the CA1 region of the mouse hippocampus. By using different protocols to induce bidirectional synaptic plasticity, Panx1 channel blockade or lack of Panx1 were found to enhance LTP, whereas both conditions precluded the induction of LTD in adults, but not in young animals. These findings suggest that Panx1 channels restrain the sliding threshold for the induction of synaptic plasticity and underlying brain mechanisms of learning and memory. PMID:25360084

EPAS-1 Mediates SP-1-Dependent FBI-1 Expression and Regulates Tumor Cell Survival and Proliferation

PubMed Central

Wang, Xiaogang; Cao, Peng; Li, Zhiqing; Wu, Dongyang; Wang, Xi; Liang, Guobiao

2014-01-01

Factor binding IST-1 (FBI-1) plays an important role in oncogenic transformation and tumorigenesis. As FBI-1 is over-expressed in multiple human cancers, the regulation of itself would provide new effective options for cancer intervention. In this work, we aimed to study the role that EPAS-1 plays in regulating FBI-1. We use the fact that specificity protein-1 (SP-1) is one of the crucial transcription factors of FBI-1, and that SP-1 can interact with the endothelial pas domain protein-1 (EPAS-1) for the induction of hypoxia related genes. The study showed that EPAS-1 plays an indispensible role in SP-1 transcription factor-mediated FBI-1 induction, and participated in tumor cell survival and proliferation. Thus, EPAS-1 could be a novel target for cancer therapeutics. PMID:25192290

Dynamic regulation of VEGF-inducible genes by an ERK/ERG/p300 transcriptional network.

PubMed

Fish, Jason E; Cantu Gutierrez, Manuel; Dang, Lan T; Khyzha, Nadiya; Chen, Zhiqi; Veitch, Shawn; Cheng, Henry S; Khor, Melvin; Antounians, Lina; Njock, Makon-Sébastien; Boudreau, Emilie; Herman, Alexander M; Rhyner, Alexander M; Ruiz, Oscar E; Eisenhoffer, George T; Medina-Rivera, Alejandra; Wilson, Michael D; Wythe, Joshua D

2017-07-01

The transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis. © 2017. Published by The Company of Biologists Ltd.

Cell Death Control by Matrix Metalloproteinases1[OPEN

PubMed Central

Zimmermann, Dirk; Sieferer, Elke; Pfannstiel, Jens

2016-01-01

In contrast to mammalian matrix metalloproteinases (MMPs) that play important roles in the remodeling of the extracellular matrix in animals, the proteases responsible for dynamic modifications of the plant cell wall are largely unknown. A possible involvement of MMPs was addressed by cloning and functional characterization of Sl2-MMP and Sl3-MMP from tomato (Solanum lycopersicum). The two tomato MMPs were found to resemble mammalian homologs with respect to gelatinolytic activity, substrate preference for hydrophobic amino acids on both sides of the scissile bond, and catalytic properties. In transgenic tomato seedlings silenced for Sl2/3-MMP expression, necrotic lesions were observed at the base of the hypocotyl. Cell death initiated in the epidermis and proceeded to include outer cortical cell layers. In later developmental stages, necrosis spread, covering the entire stem and extending into the leaves of MMP-silenced plants. The subtilisin-like protease P69B was identified as a substrate of Sl2- and Sl3-MMP. P69B was shown to colocalize with Sl-MMPs in the apoplast of the tomato hypocotyl, it exhibited increased stability in transgenic plants silenced for Sl-MMP activity, and it was cleaved and inactivated by Sl-MMPs in vitro. The induction of cell death in Sl2/3-MMP-silenced plants depended on P69B, indicating that Sl2- and Sl3-MMP act upstream of P69B in an extracellular proteolytic cascade that contributes to the regulation of cell death in tomato. PMID:27208293

Xenopus Pkdcc1 and Pkdcc2 Are Two New Tyrosine Kinases Involved in the Regulation of JNK Dependent Wnt/PCP Signaling Pathway

PubMed Central

Vitorino, Marta; Silva, Ana Cristina; Inácio, José Manuel; Ramalho, José Silva; Gur, Michal; Fainsod, Abraham; Steinbeisser, Herbert; Belo, José António

2015-01-01

Protein Kinase Domain Containing, Cytoplasmic (PKDCC) is a protein kinase which has been implicated in longitudinal bone growth through regulation of chondrocytes formation. Nevertheless, the mechanism by which this occurs remains unknown. Here, we identified two new members of the PKDCC family, Pkdcc1 and Pkdcc2 from Xenopus laevis. Interestingly, our knockdown experiments revealed that these two proteins are both involved on blastopore and neural tube closure during gastrula and neurula stages, respectively. In vertebrates, tissue polarity and cell movement observed during gastrulation and neural tube closure are controlled by Wnt/Planar Cell Polarity (PCP) molecular pathway. Our results showed that Pkdcc1 and Pkdcc2 promote the recruitment of Dvl to the plasma membrane. But surprisingly, they revealed different roles in the induction of a luciferase reporter under the control of Atf2 promoter. While Pkdcc1 induces Atf2 expression, Pkdcc2 does not, and furthermore inhibits its normal induction by Wnt11 and Wnt5a. Altogether our data show, for the first time, that members of the PKDCC family are involved in the regulation of JNK dependent Wnt/PCP signaling pathway. PMID:26270962

CD14 Signaling Restrains Chronic Inflammation through Induction of p38-MAPK/SOCS-Dependent Tolerance

PubMed Central

Sahay, Bikash; Patsey, Rebeca L.; Eggers, Christian H.; Salazar, Juan C.; Radolf, Justin D.; Sellati, Timothy J.

2009-01-01

Current thinking emphasizes the primacy of CD14 in facilitating recognition of microbes by certain TLRs to initiate pro-inflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein, this paradigm is challenged by demonstrating that recognition of live Borrelia burgdorferi not only triggers an inflammatory response in the absence of CD14, but one that is, in part, a consequence of altered PI3K/AKT/p38-MAPK signaling and impaired negative regulation of TLR2. CD14 deficiency results in increased localization of PI3K to lipid rafts, hyperphosphorylation of AKT, and reduced activation of p38. Such aberrant signaling leads to decreased negative regulation by SOCS1, SOCS3, and CIS, thereby compromising the induction of tolerance in macrophages and engendering more severe and persistent inflammatory responses to B. burgdorferi. Importantly, these altered signaling events and the higher cytokine production observed can be mimicked through shRNA and pharmacological inhibition of p38 activity in CD14-expressing macrophages. Perturbation of this CD14/p38-MAPK-dependent immune regulation may underlie development of infectious chronic inflammatory syndromes. PMID:20011115

Dendritic small conductance calcium-activated potassium channels activated by action potentials suppress EPSPs and gate spike-timing dependent synaptic plasticity.

PubMed

Jones, Scott L; To, Minh-Son; Stuart, Greg J

2017-10-23

Small conductance calcium-activated potassium channels (SK channels) are present in spines and can be activated by backpropagating action potentials (APs). This suggests they may play a critical role in spike-timing dependent synaptic plasticity (STDP). Consistent with this idea, EPSPs in both cortical and hippocampal pyramidal neurons were suppressed by preceding APs in an SK-dependent manner. In cortical pyramidal neurons EPSP suppression by preceding APs depended on their precise timing as well as the distance of activated synapses from the soma, was dendritic in origin, and involved SK-dependent suppression of NMDA receptor activation. As a result SK channel activation by backpropagating APs gated STDP induction during low-frequency AP-EPSP pairing, with both LTP and LTD absent under control conditions but present after SK channel block. These findings indicate that activation of SK channels in spines by backpropagating APs plays a key role in regulating both EPSP amplitude and STDP induction.

Mutation of the 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) Substrate-Docking Site in the Developing Brain Causes Microcephaly with Abnormal Brain Morphogenesis Independently of Akt, Leading to Impaired Cognition and Disruptive Behaviors

PubMed Central

Cordón-Barris, Lluís; Pascual-Guiral, Sònia; Yang, Shaobin; Giménez-Llort, Lydia; Lope-Piedrafita, Silvia; Niemeyer, Carlota; Claro, Enrique; Lizcano, Jose M.

2016-01-01

The phosphoinositide (PI) 3-kinase/Akt signaling pathway plays essential roles during neuronal development. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) coordinates the PI 3-kinase signals by activating 23 kinases of the AGC family, including Akt. Phosphorylation of a conserved docking site in the substrate is a requisite for PDK1 to recognize, phosphorylate, and activate most of these kinases, with the exception of Akt. We exploited this differential mechanism of regulation by generating neuron-specific conditional knock-in mice expressing a mutant form of PDK1, L155E, in which the substrate-docking site binding motif, termed the PIF pocket, was disrupted. As a consequence, activation of all the PDK1 substrates tested except Akt was abolished. The mice exhibited microcephaly, altered cortical layering, and reduced circuitry, leading to cognitive deficits and exacerbated disruptive behavior combined with diminished motivation. The abnormal patterning of the adult brain arises from the reduced ability of the embryonic neurons to polarize and extend their axons, highlighting the essential roles that the PDK1 signaling beyond Akt plays in mediating the neuronal responses that regulate brain development. PMID:27644329

Temperature sensing in Yersinia pestis: regulation of yopE transcription by lcrF.

PubMed Central

Hoe, N P; Minion, F C; Goguen, J D

1992-01-01

In Escherichia coli, a yopE::lacZ fusion was found to be regulated by temperature in the presence of the cloned BamHI G fragment of Yersinia pestis plasmid pCD1, which contains the lcrF locus. Increasing the copy number of lcrF relative to that of the yopE reporter had a negligible effect on the induction ratio (26 versus 37 degrees C) but caused large reductions in the absolute levels of yopE transcription. We localized the lcrF gene by monitoring the induction phenotype of BamHI G deletion derivatives. Sequencing revealed an open reading frame capable of encoding a protein of 30.8 kDa. A protein product of this size was detected in a T7 expression system, and LcrF-dependent yopE-specific DNA binding activity was observed. As expected, LcrF exhibited 98% homology to VirF of Yersinia enterocolitica and significant homology to the carboxy termini of other members of the AraC family of transcriptional regulatory proteins. These proteins could be divided into two classes according to function: those regulating operons involved in catabolism of carbon and energy sources and those involved in regulating virulence genes. lcrF::lacZ transcriptional fusions were constructed and analyzed in Y. pestis and E. coli. The activity of the fusions was not affected by the native pCD1 virulence plasmid, an intact lcrF gene, or temperature. Thus, induction of lcrF transcription is not essential for temperature-dependent activation of yopE transcription. A portion of LcrF was found associated with the membrane fraction in E. coli; however, pulse-chase experiments indicated that this result is an artifact of fractionation. Images PMID:1624422

Substrate rigidity regulates Ca2+ oscillation via RhoA pathway in stem cells

PubMed Central

Kim, Tae-Jin; Seong, Jihye; Ouyang, Mingxing; Sun, Jie; Lu, Shaoying; Hong, Jun Pyu; Wang, Ning; Wang, Yingxiao

2008-01-01

Substrate rigidity plays crucial roles in regulating cellular functions, such as cell spreading, traction forces, and stem cell differentiation. However, it is not clear how substrate rigidity influences early cell signaling events such as calcium in living cells. Using highly-sensitive Ca2+ biosensors based on fluorescence resonance energy transfer (FRET), we investigated the molecular mechanism by which substrate rigidity affects calcium signaling in human mesenchymal stem cells (HMSCs). Spontaneous Ca2+ oscillations were observed inside the cytoplasm and the endoplasmic reticulum (ER) using the FRET biosensors targeted at subcellular locations in cells plated on rigid dishes. Lowering the substrate stiffness to 1 kPa significantly inhibited both the magnitudes and frequencies of the cytoplasmic Ca2+ oscillation in comparison to stiffer or rigid substrate. This Ca2+ oscillation was shown to be dependent on ROCK, a downstream effector molecule of RhoA, but independent of actin filaments, microtubules, myosin light chain kinase, or myosin activity. Lysophosphatidic acid, which activates RhoA, also inhibited the frequency of the Ca2+ oscillation. Consistently, either a constitutive active mutant of RhoA (RhoA-V14) or a dominant negative mutant of RhoA (RhoA-N19) inhibited the Ca2+ oscillation. Further experiments revealed that HMSCs cultured on gels with low elastic moduli displayed low RhoA activities. Therefore, our results demonstrate that RhoA and its downstream molecule ROCK may mediate the substrate rigidity-regulated Ca2+ oscillation, which determines the physiological functions of HMSCs. PMID:18844232

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

PubMed

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.

A culture system to study oligodendrocyte myelination-processes using engineered nanofibers

PubMed Central

Lee, Seonok; Leach, Michelle K.; Redmond, Stephanie A.; Chong, S.Y. Christin; Mellon, Synthia H.; Tuck, Samuel J.; Feng, Zhang-Qi; Corey, Joseph M.; Chan, Jonah R.

2012-01-01

Current methods for studying central nervous system myelination necessitate permissive axonal substrates conducive for myelin wrapping by oligodendrocytes. We have developed a neuron-free culture system in which electron-spun nanofibers of varying sizes substitute for axons as a substrate for oligodendrocyte myelination, thereby allowing manipulation of the biophysical elements of axonal-oligodendroglial interactions. To investigate axonal regulation of myelination, this system effectively uncouples the role of molecular (inductive) cues from that of biophysical properties of the axon. We use this method to uncover the causation and sufficiency of fiber diameter in the initiation of concentric wrapping by rat oligodendrocytes. We also show that oligodendrocyte precursor cells display sensitivity to the biophysical properties of fiber diameter and initiate membrane ensheathment prior to differentiation. The use of nanofiber scaffolds will enable screening for potential therapeutic agents that promote oligodendrocyte differentiation and myelination as well as provide valuable insight into the processes involved in remyelination. PMID:22796663

Tumor hypoxia induces nuclear paraspeckle formation through HIF-2α dependent transcriptional activation of NEAT1 leading to cancer cell survival

PubMed Central

Choudhry, H; Albukhari, A; Morotti, M; Haider, S; Moralli, D; Smythies, J; Schödel, J; Green, C M; Camps, C; Buffa, F; Ratcliffe, P; Ragoussis, J; Harris, A L; Mole, D R

2015-01-01

Activation of cellular transcriptional responses, mediated by hypoxia-inducible factor (HIF), is common in many types of cancer, and generally confers a poor prognosis. Known to induce many hundreds of protein-coding genes, HIF has also recently been shown to be a key regulator of the non-coding transcriptional response. Here, we show that NEAT1 long non-coding RNA (lncRNA) is a direct transcriptional target of HIF in many breast cancer cell lines and in solid tumors. Unlike previously described lncRNAs, NEAT1 is regulated principally by HIF-2 rather than by HIF-1. NEAT1 is a nuclear lncRNA that is an essential structural component of paraspeckles and the hypoxic induction of NEAT1 induces paraspeckle formation in a manner that is dependent upon both NEAT1 and on HIF-2. Paraspeckles are multifunction nuclear structures that sequester transcriptionally active proteins as well as RNA transcripts that have been subjected to adenosine-to-inosine (A-to-I) editing. We show that the nuclear retention of one such transcript, F11R (also known as junctional adhesion molecule 1, JAM1), in hypoxia is dependent upon the hypoxic increase in NEAT1, thereby conferring a novel mechanism of HIF-dependent gene regulation. Induction of NEAT1 in hypoxia also leads to accelerated cellular proliferation, improved clonogenic survival and reduced apoptosis, all of which are hallmarks of increased tumorigenesis. Furthermore, in patients with breast cancer, high tumor NEAT1 expression correlates with poor survival. Taken together, these results indicate a new role for HIF transcriptional pathways in the regulation of nuclear structure and that this contributes to the pro-tumorigenic hypoxia-phenotype in breast cancer. PMID:25417700

Regulation by magnesium of potato tuber mitochondrial respiratory activities.

PubMed

Vicente, Joaquim A F; Madeira, Vítor M C; Vercesi, Anibal E

2004-12-01

Dehydrogenase activities of potato tuber mitochondria and corresponding phosphorylation rates were measured for the dependence on external and mitochondrial matrix Mg2+. Magnesium stimulated state 3 and state 4 respiration, with significantly different concentrations of matrix Mg2+ required for optimal activities of the several substrates. Maximal stimulation of respiration with all substrates was obtained at 2-mM external Mg2+. However, respiration of malate, citrate, and alpha-ketoglutarate requires at least 4-mM Mg2+ inside mitochondria for maximization of dehydrogenase activities. The phosphorylation system, requires a low level of internal Mg2+ (0.25 mM) to reach high activity, as judged by succinate-dependent respiration. However, mitochondria respiring on citrate or alpha-ketoglutarate only sustain high levels of phosphorylation with at least 4-mM matrix Mg2+. Respiration of succinate is active without external and matrix Mg2+, although stimulated by the cation. Respiration of alpha-ketoglutarate was strictly dependent on external Mg2+ required for substrate transport into mitochondria, and internal Mg2+ is required for dehydrogenase activity. Respiration of citrate and malate also depend on internal Mg2+ but, unlike alpha-ketoglutarate, some activity still remains without external Mg2+. All the substrates revealed insensitive to external and internal mitochondrial Ca2+, except the exogenous NADH dehydrogenase, which requires either external Ca2+ or Mg2+ for detectable activity. Calcium is more efficient than Mg2+, both having cumulative stimulation. Unlike Ca2+, Mn2+ could substitute for Mg2+, before and after addition of A23, showing its ability to regulate phosphorylation and succinate dehydrogenase activities, with almost the same efficiency as Mg2+.

Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression

PubMed Central

Megger, Dominik A.; Philipp, Jos; Le-Trilling, Vu Thuy Khanh; Sitek, Barbara; Trilling, Mirko

2017-01-01

Interferons (IFNs) are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction). In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus. PMID:28959263

Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression.

PubMed

Megger, Dominik A; Philipp, Jos; Le-Trilling, Vu Thuy Khanh; Sitek, Barbara; Trilling, Mirko

2017-01-01

Interferons (IFNs) are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction). In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus.

Acinus integrates AKT1 and subapoptotic caspase activities to regulate basal autophagy.

PubMed

Nandi, Nilay; Tyra, Lauren K; Stenesen, Drew; Krämer, Helmut

2014-10-27

How cellular stresses up-regulate autophagy is not fully understood. One potential regulator is the Drosophila melanogaster protein Acinus (Acn), which is necessary for autophagy induction and triggers excess autophagy when overexpressed. We show that cell type-specific regulation of Acn depends on proteolysis by the caspase Dcp-1. Basal Dcp-1 activity in developing photoreceptors is sufficient for this cleavage without a need for apoptosis to elevate caspase activity. On the other hand, Acn was stabilized by loss of Dcp-1 function or by the presence of a mutation in Acn that eliminates its conserved caspase cleavage site. Acn stability also was regulated by AKT1-mediated phosphorylation. Flies that expressed stabilized forms of Acn, either the phosphomimetic Acn(S641,731D) or the caspase-resistant Acn(D527A), exhibited enhanced basal autophagy. Physiologically, these flies showed improvements in processes known to be autophagy dependent, including increased starvation resistance, reduced Huntingtin-induced neurodegeneration, and prolonged life span. These data indicate that AKT1 and caspase-dependent regulation of Acn stability adjusts basal autophagy levels. © 2014 Nandi et al.

Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

NASA Technical Reports Server (NTRS)

Liu, Z.; Xia, M.; Poovaiah, B. W.

1998-01-01

cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

Synthetic polymeric substrates as potent pro-oxidant versus anti-oxidant regulators of cytoskeletal remodeling and cell apoptosis.

PubMed

Sung, Hak-Joon; Chandra, Prafulla; Treiser, Matthew D; Liu, Er; Iovine, Carmine P; Moghe, Prabhas V; Kohn, Joachim

2009-03-01

The role of reactive oxygen species (ROS)-mediated cell signal transduction pathways emanating from engineered cell substrates remains unclear. To elucidate the role, polymers derived from the amino acid L-tyrosine were used as synthetic matrix substrates. Variations in their chemical properties were created by co-polymerizing hydrophobic L-tyrosine derivatives with uncharged hydrophilic poly(ethylene glycol) (PEG, Mw = 1,000 Da), and negatively charged desaminotyrosyl-tyrosine (DT). These substrates were characterized for their intrinsic ability to generate ROS, as well as their ability to elicit Saos-2 cell responses in terms of intracellular ROS production, actin remodeling, and apoptosis. PEG-containing substrates induced both exogenous and intracellular ROS production, whereas the charged substrates reduced production of both types, indicating a coupling of exogenous ROS generation and intracellular ROS production. Furthermore, PEG-mediated ROS induction caused nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase and an increase in caspase-3 activity, confirming a link with apoptosis. PEG-rich pro-oxidant substrates caused cytoskeletal actin remodeling through beta-actin cleavage by caspase-3 into fractins. The fractins co-localized to the mitochondria and reduced the mitochondrial membrane potential. The remnant cytosolic beta-actin was polymerized and condensed, events consistent with apoptotic cell shrinkage. The cytoskeletal remodeling was integral to the further augmentation of intracellular ROS production. Conversely, the anti-oxidant DT-containing charged substrates suppressed the entire cascade of apoptotic progression. We demonstrate that ROS activity serves an important role in "outside-in" signaling for cells grown on substrates: the ROS activity couples exogenous stress, driven by substrate composition, to changes in intracellular signaling. This signaling causes cell apoptosis, which is mediated by actin remodeling.

Developmental and light regulation of tumor suppressor protein PP2A in the retina

PubMed Central

Rajala, Ammaji; Wang, Yuhong; Abcouwer, Steven F.; Gardner, Thomas W.; Rajala, Raju V.S.

2018-01-01

Protein phosphatases are a group of universal enzymes that are responsible for the dephosphorylation of various proteins and enzymes in cells. Cellular signal transduction events are largely governed by the phosphorylation of key proteins. The length of cellular response depends on the activation of protein phosphatase that dephosphorylates the phosphate groups to halt a biological response, and fine-tune the defined cellular outcome. Dysregulation of these phosphatase(s) results in various disease phenotypes. The retina is a post-mitotic tissue, and oncogenic tyrosine and serine/ threonine kinase activities are important for retinal neuron survival. Aberrant activation of protein phosphatase(s) may have a negative effect on retinal neurons. In the current study, we characterized tumor suppressor protein phosphatase 2 (PP2A), a major serine/ threonine kinase with a broad substrate specificity. Our data suggest that PP2A is developmentally regulated in the retina, localized predominantly in the inner retina, and expressed in photoreceptor inner segments. Our findings indicate that PKCα and mTOR may serve as PP2A substrates. We found that light regulates PP2A activity. Our studies also suggest that rhodopsin regulates PP2A and its substrate(s) dephosphorylation. PP2A substrate phosphorylation is increased in mice lacking the A-subunit of PP2A. However, there is no accompanying effect on retina structure and function. Together, our findings suggest that controlling the activity of PP2A in the retina may be neuroprotective. PMID:29416710

PKA, novel PKC isoforms, and ERK is mediating PACAP auto-regulation via PAC1R in human neuroblastoma NB-1 cells.

PubMed

Georg, Birgitte; Falktoft, Birgitte; Fahrenkrug, Jan

2016-12-01

The neuropeptide PACAP is expressed throughout the central and peripheral nervous system where it modulates diverse physiological functions including neuropeptide gene expression. We here report that in human neuroblastoma NB-1 cells PACAP transiently induces its own expression. Maximal PACAP mRNA expression was found after stimulation with PACAP for 3h. PACAP auto-regulation was found to be mediated by activation of PACAP specific PAC 1 Rs as PACAP had >100-fold higher efficacy than VIP, and the PAC 1 R selective agonist Maxadilan potently induced PACAP gene expression. Experiments with pharmacological kinase inhibitors revealed that both PKA and novel but not conventional PKC isozymes were involved in the PACAP auto-regulation. Inhibition of MAPK/ERK kinase (MEK) also impeded the induction, and we found that PKA, novel PKC and ERK acted in parallel and were thus not part of the same pathways. The expression of the transcription factor EGR1 previously ascribed as target of PACAP signalling was found to be transiently induced by PACAP and pharmacological inhibition of either PKC or MEK1/2 abolished PACAP mediated EGR1 induction. In contrast, inhibition of PKA mediated increased PACAP mediated EGR1 induction. Experiments using siRNA against EGR1 to lower the expression did however not affect the PACAP auto-regulation indicating that this immediate early gene product is not part of PACAP auto-regulation in NB-1 cells. We here reveal that in NB-1 neuroblastoma cells, PACAP induces its own expression by activation of PAC 1 R, and that the signalling is different from the PAC 1 R signalling mediating induction of VIP in the same cells. PACAP auto-regulation depends on parallel activation of PKA, novel PKC isoforms, and ERK, while EGR1 does not seem to be part of the PACAP auto-regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

PubMed

Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Eichler, Duane C

2012-07-27

Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness.

PubMed

Kong, Hyun Joon; Polte, Thomas R; Alsberg, Eben; Mooney, David J

2005-03-22

The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and/or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, we describe a FRET technique that can be used to evaluate the mechanics of cell-material interactions at the molecular level and simultaneously quantify the cell-based nanoscale rearrangement of the material itself. We found that these events depended on the mechanical rigidity of the adhesion substrate. Furthermore, both the proliferation and differentiation of preosteoblasts (MC3T3-E1) correlated to the magnitude of force that cells generate to cluster the cell adhesion ligands, but not the extent of ligand clustering. Together, these data demonstrate the utility of FRET in analyzing cell-material interactions, and suggest that regulation of phenotype with substrate stiffness is related to alterations in cellular traction forces.

Aquatide Activation of SIRT1 Reduces Cellular Senescence through a SIRT1-FOXO1-Autophagy Axis.

PubMed

Lim, Chae Jin; Lee, Yong-Moon; Kang, Seung Goo; Lim, Hyung W; Shin, Kyong-Oh; Jeong, Se Kyoo; Huh, Yang Hoon; Choi, Suin; Kor, Myungho; Seo, Ho Seong; Park, Byeong Deog; Park, Keedon; Ahn, Jeong Keun; Uchida, Yoshikazu; Park, Kyungho

2017-09-01

Ultraviolet (UV) irradiation is a relevant environment factor to induce cellular senescence and photoaging. Both autophagy- and silent information regulator T1 (SIRT1)-dependent pathways are critical cellular processes of not only maintaining normal cellular functions, but also protecting cellular senescence in skin exposed to UV irradiation. In the present studies, we investigated whether modulation of autophagy induction using a novel synthetic SIRT1 activator, heptasodium hexacarboxymethyl dipeptide-12 (named as Aquatide), suppresses the UVB irradiation-induced skin aging. Treatment with Aquatide directly activates SIRT1 and stimulates autophagy induction in cultured human dermal fibroblasts. Next, we found that Aquatide-mediated activation of SIRT1 increases autophagy induction via deacetylation of forkhead box class O (FOXO) 1. Finally, UVB irradiation-induced cellular senescence measured by SA-β-gal staining was significantly decreased in cells treated with Aquatide in parallel to occurring SIRT1 activation-dependent autophagy. Together, Aquatide modulates autophagy through SIRT1 activation, contributing to suppression of skin aging caused by UV irradiation.

The spread of Ras activity triggered by activation of a single dendritic spine.

PubMed

Harvey, Christopher D; Yasuda, Ryohei; Zhong, Haining; Svoboda, Karel

2008-07-04

In neurons, individual dendritic spines isolate N-methyl-d-aspartate (NMDA) receptor-mediated calcium ion (Ca2+) accumulations from the dendrite and other spines. However, the extent to which spines compartmentalize signaling events downstream of Ca2+ influx is not known. We combined two-photon fluorescence lifetime imaging with two-photon glutamate uncaging to image the activity of the small guanosine triphosphatase Ras after NMDA receptor activation at individual spines. Induction of long-term potentiation (LTP) triggered robust Ca2+-dependent Ras activation in single spines that decayed in approximately 5 minutes. Ras activity spread over approximately 10 micrometers of dendrite and invaded neighboring spines by diffusion. The spread of Ras-dependent signaling was necessary for the local regulation of the threshold for LTP induction. Thus, Ca2+-dependent synaptic signals can spread to couple multiple synapses on short stretches of dendrite.

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level.

PubMed

Prielhofer, Roland; Cartwright, Stephanie P; Graf, Alexandra B; Valli, Minoska; Bill, Roslyn M; Mattanovich, Diethard; Gasser, Brigitte

2015-03-11

The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.

Inducing autophagy

PubMed Central

Harder, Lea M; Bunkenborg, Jakob; Andersen, Jens S

2014-01-01

Autophagy is a lysosomal-mediated catabolic process, which through degradation of different cytoplasmic components aids in maintaining cellular homeostasis and survival during exposure to extra- or intracellular stresses. Ammonia is a potential toxic and stress-inducing byproduct of glutamine catabolism, which has recently been found to induce autophagy in an MTOR independent way and support cancer cell survival. In this study, quantitative phosphoproteomics was applied to investigate the initial signaling events linking ammonia to the induction of autophagy. The MTOR inhibitor rapamycin was used as a reference treatment to emphasize the differences between an MTOR-dependent and -independent autophagy-induction. By this means 5901 phosphosites were identified of which 626 were treatment-specific regulated and 175 were coregulated. Investigation of the ammonia-specific regulated sites supported that MTOR activity was not affected, but indicated increased MAPK3 activity, regulation of proteins involved in Rho signal transduction, and a novel phosphorylation motif, serine-proline-threonine (SPT), which could be linked to cytoskeleton-associated proteins. MAPK3 could not be identified as the primary driver of ammonia-induced autophagy but instead the data suggested an upregulation of AMPK and the unfolded protein response (UPR), which might link ammonia to autophagy induction. Support of UPR induction was further obtained from the finding of increased protein levels of the ER stress markers DDIT3/CHOP and HSPA5 during ammonia treatment. The large-scale data set presented here comprises extensive high-quality quantitative information on phosphoprotein regulation in response to 2 very different autophagy inducers and should therefore be considered a general resource for the community. PMID:24300666

The attenuated inflammation of MPL is due to the lack of CD14-dependent tight dimerization of the TLR4/MD2 complex at the plasma membrane.

PubMed

Tanimura, Natsuko; Saitoh, Shin-Ichiroh; Ohto, Umeharu; Akashi-Takamura, Sachiko; Fujimoto, Yukari; Fukase, Koichi; Shimizu, Toshiyuki; Miyake, Kensuke

2014-06-01

TLR4/MD-2 senses lipid A, activating the MyD88-signaling pathway on the plasma membrane and the TRIF-signaling pathway after CD14-mediated TLR4/MD-2 internalization into endosomes. Monophosphoryl lipid A (MPL), a detoxified derivative of lipid A, is weaker than lipid A in activating the MyD88-dependent pathway. Little is known, however, about mechanisms underlying the attenuated activation of MyD88-dependent pathways. We here show that MPL was impaired in induction of CD14-dependent TLR4/MD-2 dimerization compared with lipid A. Impaired TLR4/MD-2 dimerization decreased CD14-mediated TNFα production. In contrast, MPL was comparable to lipid A in CD14-independent MyD88-dependent TNFα production and TRIF-dependent responses including cell surface CD86 up-regulation and IFNβ induction. Although CD86 up-regulation is dependent on TRIF signaling, it was induced by TLR4/MD-2 at the plasma membrane. These results revealed that the attenuated MPL responses were due to CD14-initiated responses at the plasma membrane, but not just to responses initiated by MyD88, that is, MPL was specifically unable to induce CD14-dependent TLR4/MD-2 dimerization that selectively enhances MyD88-mediated responses at the plasma membrane. © The Japanese Society for Immunology. 2013. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Transcriptional Regulation of S Phase Kinase-associated Protein 2 by NR4A Orphan Nuclear Receptor NOR1 in Vascular Smooth Muscle Cells*

PubMed Central

Gizard, Florence; Zhao, Yue; Findeisen, Hannes M.; Qing, Hua; Cohn, Dianne; Heywood, Elizabeth B.; Jones, Karrie L.; Nomiyama, Takashi; Bruemmer, Dennis

2011-01-01

Members of the NR4A subgroup of the nuclear hormone receptor superfamily have emerged as key transcriptional regulators of proliferation and inflammation. NOR1 constitutes a ligand-independent transcription factor of this subgroup and induces cell proliferation; however, the transcriptional mechanisms underlying this mitogenic role remain to be defined. Here, we demonstrate that the F-box protein SKP2 (S phase kinase-associated protein 2), the substrate-specific receptor of the ubiquitin ligase responsible for the degradation of p27KIP1 through the proteasome pathway, constitutes a direct transcriptional target for NOR1. Mitogen-induced Skp2 expression is silenced in vascular smooth muscle cells (VSMC) isolated from Nor1-deficient mice or transfected with Nor1 siRNA. Conversely, adenovirus-mediated overexpression of NOR1 induces Skp2 expression in VSMC and decreases protein abundance of its target p27. Transient transfection experiments establish that NOR1 transactivates the Skp2 promoter through a nerve growth factor-induced clone B response element (NBRE). Electrophoretic mobility shift and chromatin immunoprecipitation assays further revealed that NOR1 is recruited to this NBRE site in the Skp2 promoter in response to mitogenic stimulation. In vivo Skp2 expression is increased during the proliferative response underlying neointima formation, and this transcriptional induction depends on the expression of NOR1. Finally, we demonstrate that overexpression of Skp2 rescues the proliferative arrest of Nor1-deficient VSMC. Collectively, these results characterize Skp2 as a novel NOR1-regulated target gene and detail a previously unrecognized transcriptional cascade regulating mitogen-induced VSMC proliferation. PMID:21868379

Diindolylmethane, a naturally occurring compound, induces CYP3A4 and MDR1 gene expression by activating human PXR

PubMed Central

Pondugula, Satyanarayana R.; Flannery, Patrick C.; Abbott, Kodye L.; Coleman, Elaine S.; Mani, Sridhar; Samuel, Temesgen; Xie, Wen

2015-01-01

Activation of human pregnane X receptor (hPXR)-regulated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1) plays an important role in mediating adverse drug interactions. Given the common use of natural products as part of adjunct human health behavior, there is a growing concern about natural products for their potential to induce undesired drug interactions through the activation of hPXR-regulated CYP3A4 and MDR1. Here, we studied whether 3,3′-diindolylmethane (DIM), a natural health supplement, could induce hPXR-mediated regulation of CYP3A4 and MDR1 in human hepatocytes and intestinal cells. DIM, at its physiologically relevant concentrations, not only induced hPXR transactivation of CYP3A4 promoter activity but also induced gene expression of CYP3A4 and MDR1. DIM decreased intracellular accumulation of MDR1 substrate rhodamine 123, suggesting that DIM induces the functional expression of MDR1. Pharmacologic inhibition or genetic knockdown of hPXR resulted in attenuation of DIM induced CYP3A4 and MDR1 gene expression, suggesting that DIM induces CYP3A4 and MDR1 in an hPXR-dependent manner. Together, these results support our conclusion that DIM induces hPXR-regulated CYP3A4 and MDR1 gene expression. The inductive effects of DIM on CYP3A4 and MDR1 expression caution the use of DIM in conjunction with other medications metabolized and transported via CYP3A4 and MDR1, respectively. PMID:25542144

CD11b regulates antibody class switching via induction of AID.

PubMed

Park, Seohyun; Sim, Hyunsub; Kim, Hye-In; Jeong, Daecheol; Wu, Guang; Cho, Soo Young; Lee, Young Seek; Kwon, Hyung-Joo; Lee, Keunwook

2017-07-01

The integrin CD11b, which is encoded by the integrin subunit alpha M (ITGAM), is primarily expressed on the surface of innate immune cells. Genetic variations in ITGAM are among the strongest risk factors for systemic lupus erythematosus, an autoimmune disease characterized by the presence of autoantibodies. However, the regulatory function of CD11b in the antibody responses remains unclear. Here, we report the induction of CD11b in activated B2 B cells and define its unexpected role in immunoglobulin heavy chain class switch recombination (CSR). LPS-activated B cells lacking CD11b yielded fewer IgG subtypes such as IgG1 and IgG2a in vitro, and immunization-dependent CSR and affinity maturation of antibodies were severely impaired in CD11b-deficient mice. Notably, we observed the reduced expression of activation-induced cytidine deaminase (AID), an enzyme that initiates CSR and somatic hypermutation, and ectopic expression of AID was sufficient to rescue the defective CSR of CD11b-deficient B cells. LPS-induced phosphorylation of NF-κB p65 and IκBα was attenuated in CD11b-deficient B cells, and hyperactivation of IκB kinase 2 restored the defective AID expression and CSR, which implied that CD11b regulates the NF-κB-dependent induction of AID. Overall, our experimental evidence emphasized the function of CD11b in antibody responses and the role of CD11b as a vital regulator of CSR. Copyright © 2017 Elsevier Ltd. All rights reserved.

RsbV-Independent Induction of the SigB-Dependent General Stress Regulon of Bacillus subtilis during Growth at High Temperature

PubMed Central

Holtmann, Gudrun; Brigulla, Matthias; Steil, Leif; Schütz, Alexandra; Barnekow, Karsta; Völker, Uwe; Bremer, Erhard

2004-01-01

General stress proteins protect Bacillus subtilis cells against a variety of environmental insults. This adaptive response is particularly important for nongrowing cells, to which it confers a multiple, nonspecific, and preemptive stress resistance. Induction of the general stress response relies on the alternative transcription factor, SigB, whose activity is controlled by a partner switching mechanism that also involves the anti-sigma factor, RsbW, and the antagonist protein, RsbV. Recently, the SigB regulon has been shown to be continuously induced and functionally important in cells actively growing at low temperature. With the exception of this chill induction, all SigB-activating stimuli identified so far trigger a transient expression of the SigB regulon that depends on RsbV. Through a proteome analysis and Northern blot and gene fusion experiments, we now show that the SigB regulon is continuously induced in cells growing actively at 51°C, close to the upper growth limit of B. subtilis. This heat induction of SigB-dependent genes requires the environmental stress-responsive phosphatase RsbU, but not the metabolic stress-responsive phosphatase RsbP. RsbU dependence of SigB activation by heat is overcome in mutants that lack RsbV. In addition, loss of RsbV alone or in combination with RsbU triggers a hyperactivation of the general stress regulon exclusively at high temperatures detrimental for cell growth. These new facets of heat induction of the SigB regulon indicate that the current view of the complex genetic and biochemical regulation of SigB activity is still incomplete and that SigB perceives signals independent of the RsbV-mediated signal transduction pathways under heat stress conditions. PMID:15342585

A histone H3K36 chromatin switch coordinates DNA double-strand break repair pathway choice.

PubMed

Pai, Chen-Chun; Deegan, Rachel S; Subramanian, Lakxmi; Gal, Csenge; Sarkar, Sovan; Blaikley, Elizabeth J; Walker, Carol; Hulme, Lydia; Bernhard, Eric; Codlin, Sandra; Bähler, Jürg; Allshire, Robin; Whitehall, Simon; Humphrey, Timothy C

2014-06-09

DNA double-strand break (DSB) repair is a highly regulated process performed predominantly by non-homologous end joining (NHEJ) or homologous recombination (HR) pathways. How these pathways are coordinated in the context of chromatin is unclear. Here we uncover a role for histone H3K36 modification in regulating DSB repair pathway choice in fission yeast. We find Set2-dependent H3K36 methylation reduces chromatin accessibility, reduces resection and promotes NHEJ, while antagonistic Gcn5-dependent H3K36 acetylation increases chromatin accessibility, increases resection and promotes HR. Accordingly, loss of Set2 increases H3K36Ac, chromatin accessibility and resection, while Gcn5 loss results in the opposite phenotypes following DSB induction. Further, H3K36 modification is cell cycle regulated with Set2-dependent H3K36 methylation peaking in G1 when NHEJ occurs, while Gcn5-dependent H3K36 acetylation peaks in S/G2 when HR prevails. These findings support an H3K36 chromatin switch in regulating DSB repair pathway choice.

Influence of coil current modulation on polycrystalline diamond film deposition by irradiation of Ar/CH4/H2 inductively coupled thermal plasmas

NASA Astrophysics Data System (ADS)

Betsuin, Toshiki; Tanaka, Yasunori; Arai, T.; Uesugi, Y.; Ishijima, T.

2018-03-01

This paper describes the application of an Ar/CH4/H2 inductively coupled thermal plasma with and without coil current modulation to synthesise diamond films. Induction thermal plasma with coil current modulation is referred to as modulated induction thermal plasma (M-ITP), while that without modulation is referred to as non-modulated ITP (NM-ITP). First, spectroscopic observations of NM-ITP and M-ITP with different modulation waveforms were made to estimate the composition in flux from the thermal plasma by measuring the time evolution in the spectral intensity from the species. Secondly, we studied polycrystalline diamond film deposition tests on a Si substrate, and we studied monocrystalline diamond film growth tests using the irradiation of NM-ITP and M-ITP. From these tests, diamond nucleation effects by M-ITP were found. Finally, following the irradiation results, we attempted to use a time-series irradiation of M-ITP and NM-ITP for polycrystalline diamond film deposition on a Si substrate. The results indicated that numerous larger diamond particles were deposited with a high population density on the Si substrate by time-series irradiation.

p53 and metabolism: from mechanism to therapeutics

PubMed Central

Simabuco, Fernando M.; Morale, Mirian G.; Pavan, Isadora C.B.; Morelli, Ana P.; Silva, Fernando R.; Tamura, Rodrigo E.

2018-01-01

The tumor cell changes itself and its microenvironment to adapt to different situations, including action of drugs and other agents targeting tumor control. Therefore, metabolism plays an important role in the activation of survival mechanisms to keep the cell proliferative potential. The Warburg effect directs the cellular metabolism towards an aerobic glycolytic pathway, despite the fact that it generates less adenosine triphosphate than oxidative phosphorylation; because it creates the building blocks necessary for cell proliferation. The transcription factor p53 is the master tumor suppressor; it binds to more than 4,000 sites in the genome and regulates the expression of more than 500 genes. Among these genes are important regulators of metabolism, affecting glucose, lipids and amino acids metabolism, oxidative phosphorylation, reactive oxygen species (ROS) generation and growth factors signaling. Wild-type and mutant p53 may have opposing effects in the expression of these metabolic genes. Therefore, depending on the p53 status of the cell, drugs that target metabolism may have different outcomes and metabolism may modulate drug resistance. Conversely, induction of p53 expression may regulate differently the tumor cell metabolism, inducing senescence, autophagy and apoptosis, which are dependent on the regulation of the PI3K/AKT/mTOR pathway and/or ROS induction. The interplay between p53 and metabolism is essential in the decision of cell fate and for cancer therapeutics. PMID:29805774

Hormonal regulation of gluconeogenesis in cereal aleurone is strongly cultivar-dependent and gibberellin action involves SLENDER1 but not GAMYB.

PubMed

Eastmond, Peter J; Jones, Russell L

2005-11-01

Storage oil is a major constituent in the cereal aleurone layer. The aim of this study was to investigate how gibberellin (GA) and abscisic acid (ABA) regulate conversion of oil to sugar in barley aleurone. The activity of the glyoxylate cycle enzyme isocitrate lyase (ICL) was surveyed in eight barley cultivars. Surprisingly, some cultivars do not require GA for the induction of ICL (e.g. Himalaya), whereas some do (e.g. Golden Promise). Furthermore, in Golden Promise, GA also stimulates triacylglycerol breakdown and enhances the net flux of carbon from acetate to sugar. In contrast, ABA strongly represses ICL activity and the flux of carbon from oil to sugar in both Golden Promise and Himalaya. Biolistics using a promoter reporter showed that GA and ABA regulate ICL at the level of transcription. Studies using barley and rice mutants and pharmacological agents show that GA-dependent induction of ICL activity is mediated by SLENDER1 and requires cGMP, but does not involve the transcription factor GAMYB. Gibberellin and ABA therefore act antagonistically to regulate gluconeogenesis in the aleurone layer as well as controlling the production and secretion of hydrolases into the starchy endosperm. We suggest that the variation between different barley cultivars might be a result of selective breeding to alter seed dormancy.

Influence of the DNA structure on the free radical induction due to proflavine and light treatment.

PubMed

Piette, J; Calberg-Bacq, C M; Van de Vorst, A

1979-04-30

Induction of peroxide free radicals (detected by Electron Paramagnetic Resonance at 77 K) due to the photodynamic activity of proflavine was measured on bacteriophage phi X174 DNA either single-stranded (ss) as isolated from the virion, or double-stranded supercoiled (RFI) as isolated from the infected bacteria. Comparison was made with calf thymus DNA photosensitization. In order to use equivalent DNA-proflavine complexes, binding of the dye to the three DNA's was first determined under those conditions of high ionic strength favourable to the photodynamic reaction. Free radical induction was maximal for definite amounts of bound proflavine (which varied depending upon the DNA substrate) and at an ionic strength value of 0.5. The level of the maximal reaction increased in the following order: from phi Xss DNA to calf thymus DNA and finally to phi XRFI DNA. The conformation of the proflavine-DNA complex was thus a determinant for the efficiency of the photodynamic process. The ionic strength effect could not be explained by the evolution of the proflavine triplet state in irradiated proflavine-calf thymus DNA complexes.

Global impact of Salmonella type III secretion effector SteA on host cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cardenal-Muñoz, Elena, E-mail: e_cardenal@us.es; Gutiérrez, Gabriel, E-mail: ggpozo@us.es; Ramos-Morales, Francisco, E-mail: framos@us.es

Highlights: • We analyzed HeLa cells transcriptome in response to Salmonella SteA. • Significant differential expression was detected for 58 human genes. • They are involved in ECM organization and regulation of some signaling pathways. • Cell death, cell adhesion and cell migration were decreased in SteA-expressing cells. • These results contribute to understand the role of SteA during infections. - Abstract: Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. Thesemore » systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell–cell adhesion and migration.« less

ROCK mediates phorbol ester-induced apoptosis in prostate cancer cells via p21Cip1 up-regulation and JNK.

PubMed

Xiao, Liqing; Eto, Masumi; Kazanietz, Marcelo G

2009-10-23

It is established that androgen-dependent prostate cancer cells undergo apoptosis upon treatment with phorbol esters and related analogs, an effect primarily mediated by PKCdelta. Treatment of LNCaP prostate cancer cells with phorbol 12-myristate 13-acetate (PMA) causes a strong and sustained activation of RhoA and its downstream effector ROCK (Rho kinase) as well as the formation of stress fibers. These effects are impaired in cells subjected to PKCdelta RNA interference depletion. Functional studies revealed that expression of a dominant negative RhoA mutant or treatment with the ROCK inhibitor Y-27632 inhibits the apoptotic effect of PMA in LNCaP cells. Remarkably, the cytoskeleton inhibitors cytochalasin B and blebbistatin blocked not only PMA-induced apoptosis but also the activation of JNK, a mediator of the cell death effect by the phorbol ester. In addition, we found that up-regulation of the cell cycle inhibitor p21(Cip1) is required for PMA-induced apoptosis and that inhibitors of ROCK or the cytoskeleton organization prevent p21(Cip1) induction. Real time PCR analysis and reporter gene assay revealed that PMA induces p21(Cip1) transcriptionally in a ROCK- and cytoskeleton-dependent manner. p21(Cip1) promoter analysis revealed that PMA induction is dependent on Sp1 elements in the p21(Cip1) promoter but independent of p53. Taken together, our studies implicate ROCK-mediated up-regulation of p21(Cip1) and the cytoskeleton in PKCdelta-dependent apoptosis in prostate cancer cells.

Growth phase-dependent induction of stationary-phase promoters of Escherichia coli in different gram-negative bacteria.

PubMed Central

Miksch, G; Dobrowolski, P

1995-01-01

RSF1010-derived plasmids carrying a fusion of a promoterless lacZ gene with the sigma s-dependent growth phase-regulated promoters of Escherichia coli, bolAp1 and fic, were constructed. The plasmids were mobilized into the gram-negative bacterial species Acetobacter methanolicus, Xanthomonas campestris, Pseudomonas putida, and Rhizobium meliloti. The beta-galactosidase activities of bacterial cultures were determined during exponential and stationary growth phases. Transcriptional activation of the fic promoter in the different bacteria was growth phase dependent as in E. coli and was initiated generally during the transition to stationary phase. The induction of the bolA promoter was also growth phase dependent in the bacteria tested. While the expression in E. coli and R. meliloti was initiated during the transition from exponential to stationary phase, the induction in A. methanolicus, P. putida, and X. campestris started some hours after stationary growth phase was reached. In all the species tested, DNA fragments hybridizing with the rpoS gene of E. coli were detected. The results show that in different gram-negative bacteria, stationary-phase-specific sigma factors which are structurally and functionally homologous to sigma s and are able to recognize the promoter sequences of both bolA and fic exist. PMID:7665531

Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infection

PubMed Central

Alandijany, Thamir; Conn, Kristen L.; McFarlane, Steven; Orr, Anne

2018-01-01

Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2’-deoxyuridine (EdU) labelling of herpes simplex virus 1 (HSV-1) DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs) rapidly entrapped viral DNA (vDNA) leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16) and the induction of interferon stimulated gene (ISG) expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK)-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote vDNA release from PML-NBs and the onset of HSV-1 lytic replication. PMID:29309427

Bidirectional regulation of adenosine-to-inosine (A-to-I) RNA editing by DEAH box helicase 9 (DHX9) in cancer.

PubMed

Hong, HuiQi; An, Omer; Chan, Tim H M; Ng, Vanessa H E; Kwok, Hui Si; Lin, Jaymie S; Qi, Lihua; Han, Jian; Tay, Daryl J T; Tang, Sze Jing; Yang, Henry; Song, Yangyang; Bellido Molias, Fernando; Tenen, Daniel G; Chen, Leilei

2018-05-18

Adenosine-to-inosine (A-to-I) RNA editing entails the enzymatic deamination of adenosines to inosines by adenosine deaminases acting on RNA (ADARs). Dysregulated A-to-I editing has been implicated in various diseases, including cancers. However, the precise factors governing the A-to-I editing and their physiopathological implications remain as a long-standing question. Herein, we unravel that DEAH box helicase 9 (DHX9), at least partially dependent of its helicase activity, functions as a bidirectional regulator of A-to-I editing in cancer cells. Intriguingly, the ADAR substrate specificity determines the opposing effects of DHX9 on editing as DHX9 silencing preferentially represses editing of ADAR1-specific substrates, whereas augments ADAR2-specific substrate editing. Analysis of 11 cancer types from The Cancer Genome Atlas (TCGA) reveals a striking overexpression of DHX9 in tumors. Further, tumorigenicity studies demonstrate a helicase-dependent oncogenic role of DHX9 in cancer development. In sum, DHX9 constitutes a bidirectional regulatory mode in A-to-I editing, which is in part responsible for the dysregulated editome profile in cancer.

Rebamipide induces the gastric mucosal protective factor, cyclooxygenase-2, via activation of 5'-AMP-activated protein kinase.

PubMed

Lee, Sunyoung; Jeong, Seongkeun; Kim, Wooseong; Kim, Dohoon; Yang, Yejin; Yoon, Jeong-Hyun; Kim, Byung Joo; Min, Do Sik; Jung, Yunjin

2017-01-29

Rebamipide, an amino acid derivative of 2(1H)-quinolinone, has been used for mucosal protection, healing of gastroduodenal ulcers, and treatment of gastritis. Induction of cyclooxygenase (COX)-2, a gastric mucosal protective factor, by rebamipide has been suggested as the major mechanism of the drug action. However, how rebamipide induces COX-2 at the molecular level needs further investigation. In this study, the molecular mechanism underlying the induction of COX-2 by rebamipide was investigated. In gastric carcinoma cells and macrophage cells, rebamipide induced phosphorylation of AMP-activated protein kinase (AMPK), leading to phosphorylation of acetyl-CoA carboxylase (ACC), a substrate of AMPK. The induction of COX-2 by rebamipide was dependent on AMPK activation because compound C, an AMPK inhibitor, abolished COX-2 induction by rebamipide. In a mouse ulcer model, rebamipide protected against hydrochloric acid/ethanol-induced gastric ulcer, and these protective effects were deterred by co-administration of compound C. In parallel, in the gastric tissues, rebamipide increased the phosphorylation AMPK, whereas compound C reduced the levels of COX-2 and phosphorylated ACC, which were increased by rebamipide. Taken together, the activation of AMPK by rebamipide may be a molecular mechanism that contributes to induction of COX-2, probably resulting in protection against gastric ulcers. Copyright © 2016 Elsevier Inc. All rights reserved.

Pause, play, repeat

PubMed Central

Sansó, Miriam; Fisher, Robert P

2013-01-01

Cyclin-dependent kinases (CDKs) play a central role in governing eukaryotic cell division. It is becoming clear that the transcription cycle of RNA polymerase II (RNAP II) is also regulated by CDKs; in metazoans, the cell cycle and transcriptional CDK networks even share an upstream activating kinase, which is itself a CDK. From recent chemical-genetic analyses we know that CDKs and their substrates control events both early in transcription (the transition from initiation to elongation) and late (3′ end formation and transcription termination). Moreover, mutual dependence on CDK activity might couple the “beginning” and “end” of the cycle, to ensure the fidelity of mRNA maturation and the efficient recycling of RNAP II from sites of termination to the transcription start site (TSS). As is the case for CDKs involved in cell cycle regulation, different transcriptional CDKs act in defined sequence on multiple substrates. These phosphorylations are likely to influence gene expression by several mechanisms, including direct, allosteric effects on the transcription machinery, co-transcriptional recruitment of proteins needed for mRNA-capping, splicing and 3′ end maturation, dependent on multisite phosphorylation of the RNAP II C-terminal domain (CTD) and, perhaps, direct regulation of RNA-processing or histone-modifying machinery. Here we review these recent advances, and preview the emerging challenges for transcription-cycle research. PMID:23756342

Elicitor induction of a microsomal 5-O-(4-coumaroyl)shikimate 3'-hydroxylase in parsley cell suspension cultures.

PubMed

Heller, W; Kühnl, T

1985-09-01

Microsomal preparations from parsley cell suspension cultures challenged with an elicitor from Phytophthora megasperma f.sp. glycinea (Pmg) catalyze the formation of trans-5-O-caffeoylshikimate from trans-5-O-(4-coumaroyl)shikimate. Neither the cis isomer nor free 4-coumarate, 4-coumaroyl-CoA, or 5-O-(4-coumaroyl)quinate are substrates for this enzyme. The reaction is strictly dependent on NADPH as a reducing cofactor and on molecular oxygen. NADH, ascorbic acid, and 6,7-dimethyl-5,6,7,8-tetrahydropterine cannot substitute for NADPH. However, NADH enhances enzyme activity observed in the presence of NADPH. Cytochrome c and carbon monoxide inhibit the hydroxylation reaction, suggesting a cytochrome P-450-dependent mixed-function monooxygenase.

PHYTOCHROME KINASE SUBSTRATE 1 is a phototropin 1 binding protein required for phototropism

PubMed Central

Lariguet, Patricia; Schepens, Isabelle; Hodgson, Daniel; Pedmale, Ullas V.; Trevisan, Martine; Kami, Chitose; de Carbonnel, Matthieu; Alonso, José M.; Ecker, Joseph R.; Liscum, Emmanuel; Fankhauser, Christian

2006-01-01

Phototropism, or plant growth in response to unidirectional light, is an adaptive response of crucial importance. Lateral differences in low fluence rates of blue light are detected by phototropin 1 (phot1) in Arabidopsis. Only NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and root phototropism 2, both belonging to the same family of proteins, have been previously identified as phototropin-interacting signal transducers involved in phototropism. PHYTOCHROME KINASE SUBSTRATE (PKS) 1 and PKS2 are two phytochrome signaling components belonging to a small gene family in Arabidopsis (PKS1–PKS4). The strong enhancement of PKS1 expression by blue light and its light induction in the elongation zone of the hypocotyl prompted us to study the function of this gene family during phototropism. Photobiological experiments show that the PKS proteins are critical for hypocotyl phototropism. Furthermore, PKS1 interacts with phot1 and NPH3 in vivo at the plasma membrane and in vitro, indicating that the PKS proteins may function directly with phot1 and NPH3 to mediate phototropism. The phytochromes are known to influence phototropism but the mechanism involved is still unclear. We show that PKS1 induction by a pulse of blue light is phytochrome A-dependent, suggesting that the PKS proteins may provide a molecular link between these two photoreceptor families. PMID:16777956

PHYTOCHROME KINASE SUBSTRATE 1 is a phototropin 1 binding protein required for phototropism.

PubMed

Lariguet, Patricia; Schepens, Isabelle; Hodgson, Daniel; Pedmale, Ullas V; Trevisan, Martine; Kami, Chitose; de Carbonnel, Matthieu; Alonso, José M; Ecker, Joseph R; Liscum, Emmanuel; Fankhauser, Christian

2006-06-27

Phototropism, or plant growth in response to unidirectional light, is an adaptive response of crucial importance. Lateral differences in low fluence rates of blue light are detected by phototropin 1 (phot1) in Arabidopsis. Only NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and root phototropism 2, both belonging to the same family of proteins, have been previously identified as phototropin-interacting signal transducers involved in phototropism. PHYTOCHROME KINASE SUBSTRATE (PKS) 1 and PKS2 are two phytochrome signaling components belonging to a small gene family in Arabidopsis (PKS1-PKS4). The strong enhancement of PKS1 expression by blue light and its light induction in the elongation zone of the hypocotyl prompted us to study the function of this gene family during phototropism. Photobiological experiments show that the PKS proteins are critical for hypocotyl phototropism. Furthermore, PKS1 interacts with phot1 and NPH3 in vivo at the plasma membrane and in vitro, indicating that the PKS proteins may function directly with phot1 and NPH3 to mediate phototropism. The phytochromes are known to influence phototropism but the mechanism involved is still unclear. We show that PKS1 induction by a pulse of blue light is phytochrome A-dependent, suggesting that the PKS proteins may provide a molecular link between these two photoreceptor families.

AUDITORY ASSOCIATIVE MEMORY AND REPRESENTATIONAL PLASTICITY IN THE PRIMARY AUDITORY CORTEX

PubMed Central

Weinberger, Norman M.

2009-01-01

Historically, the primary auditory cortex has been largely ignored as a substrate of auditory memory, perhaps because studies of associative learning could not reveal the plasticity of receptive fields (RFs). The use of a unified experimental design, in which RFs are obtained before and after standard training (e.g., classical and instrumental conditioning) revealed associative representational plasticity, characterized by facilitation of responses to tonal conditioned stimuli (CSs) at the expense of other frequencies, producing CS-specific tuning shifts. Associative representational plasticity (ARP) possesses the major attributes of associative memory: it is highly specific, discriminative, rapidly acquired, consolidates over hours and days and can be retained indefinitely. The nucleus basalis cholinergic system is sufficient both for the induction of ARP and for the induction of specific auditory memory, including control of the amount of remembered acoustic details. Extant controversies regarding the form, function and neural substrates of ARP appear largely to reflect different assumptions, which are explicitly discussed. The view that the forms of plasticity are task-dependent is supported by ongoing studies in which auditory learning involves CS-specific decreases in threshold or bandwidth without affecting frequency tuning. Future research needs to focus on the factors that determine ARP and their functions in hearing and in auditory memory. PMID:17344002

Sphingoid Bases and the Serine Catabolic Enzyme CHA1 Define a Novel Feedforward/Feedback Mechanism in the Response to Serine Availability*

PubMed Central

Montefusco, David J.; Newcomb, Benjamin; Gandy, Jason L.; Brice, Sarah E.; Matmati, Nabil; Cowart, L. Ashley; Hannun, Yusuf A.

2012-01-01

Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation. PMID:22277656

Saint John's wort: An in vitro analysis of P-glycoprotein induction due to extended exposure

PubMed Central

Perloff, Michael D; von Moltke, Lisa L; Störmer, Elke; Shader, Richard I; Greenblatt, David J

2001-01-01

Chronic use of Saint John's wort (SJW) has been shown to lower the bioavailability for a variety of co-administered drugs including indinavir, cyclosporin, and digoxin. Decreases in intestinal absorption through induction of the multidrug resistance transporter, P-glycoprotein (P-gp), may explain decreased bioavailability. The present study characterized the response of P-gp to chronic and acute exposure of SJW and hypericin (HYP, a presumed active moiety within SJW) in an in vitro system. Experiments were performed with 3 to 300 μg ml−1 of methanol-extracted SJW and 0.03 to 3 μM HYP, representing low to high estimates of intestinal concentrations. In induction experiments, LS-180 intestinal carcinoma cells were exposed for 3 days to SJW, HYP, vehicle or a positive control (ritonavir). P-gp was quantified using Western blot analysis. P-gp expression was strongly induced by SJW (400% increase at 300 μg ml−1) and by HYP (700% at 3 μM) in a dose-dependent fashion. Cells chronically treated with SJW had decreased accumulation of rhodamine 123, a P-gp substrate, that was reversed with acute verapamil, a P-gp inhibitor. Fluorescence microscopy of intact cells validated these findings. In Caco-2 cell monolayers, SJW and HYP caused moderate inhibition of P-gp-attributed transport at the maximum concentrations tested. SJW and HYP significantly induced P-gp expression at low, clinically relevant concentrations. Similar effects occurring in vivo may explain the decreased bioavailability of P-gp substrate drugs when co-administered with SJW. PMID:11739235

Endothelial HO-1 induction by model TG-rich lipoproteins is regulated through a NOX4-Nrf2 pathway1[S

PubMed Central

Latham Birt, Sally H.; Purcell, Robert; Botham, Kathleen M.; Wheeler-Jones, Caroline P. D.

2016-01-01

Circulating levels of chylomicron remnants (CMRs) increase postprandially and their composition directly reflects dietary lipid intake. These TG-rich lipoproteins likely contribute to the development of endothelial dysfunction, albeit via unknown mechanisms. Here, we investigated how the FA composition of CMRs influences their actions on human aortic endothelial cells (HAECs) by comparing the effects of model CMRs—artificial TG-rich CMR-like particles (A-CRLPs)—containing TGs extracted from fish, DHA-rich algal, corn, or palm oils. HAECs responded with distinct transcriptional programs according to A-CRLP TG content and oxidation status, with genes involved in antioxidant defense and cytoprotection most prominently affected by n-3 PUFA-containing A-CRLPs. These particles were significantly more efficacious inducers of heme oxygenase-1 (HO-1) than n-6 PUFA corn or saturated FA-rich palm CRLPs. Mechanistically, HO-1 induction by all CRLPs requires NADPH oxidase 4, with PUFA-containing particles additionally dependent upon mitochondrial reactive oxygen species. Activation of both p38 MAPK and PPARβ/δ culminates in increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression/nuclear translocation and HO-1 induction. These studies define new molecular pathways coupling endothelial cell activation by model CMRs with adaptive regulation of Nrf2-dependent HO-1 expression and may represent key mechanisms through which dietary FAs differentially impact progression of endothelial dysfunction. PMID:27185859

Regulated Eukaryotic DNA Replication Origin Firing with Purified Proteins

PubMed Central

Yeeles, Joseph T.P.; Deegan, Tom D.; Janska, Agnieszka; Early, Anne; Diffley, John F. X.

2016-01-01

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric MCM complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45, MCM, GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4 dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication. PMID:25739503

Regulated eukaryotic DNA replication origin firing with purified proteins.

PubMed

Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

2015-03-26

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

A Compact Inductive Position Sensor Made by Inkjet Printing Technology on a Flexible Substrate

PubMed Central

Jeranče, Nikola; Vasiljević, Dragana; Samardžić, Nataša; Stojanović, Goran

2012-01-01

This paper describes the design, simulation and fabrication of an inductive angular position sensor on a flexible substrate. The sensor is composed of meandering silver coils printed on a flexible substrate (Kapton film) using inkjet technology. The flexibility enables that after printing in the plane, the coils could be rolled and put inside each other. By changing the angular position of the internal coil (rotor) related to the external one (stator), the mutual inductance is changed and consequently the impedance. It is possible to determine the angular position from the measured real and imaginary part of the impedance, in our case in the frequency range from 1 MHz to 10 MHz. Experimental results were compared with simulation results obtained by in-house developed software tool, and very good agreement has been achieved. Thanks to the simple design and fabrication, smaller package space requirements and weight, the presented sensor represents a cost-effective alternative to the other sensors currently used in series production applications. PMID:22438710

The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch

PubMed Central

Oberst, Andrew; Malatesta, Martina; Aqeilan, Rami I.; Rossi, Mario; Salomoni, Paolo; Murillas, Rodolfo; Sharma, Prashant; Kuehn, Michael R.; Oren, Moshe; Croce, Carlo M.; Bernassola, Francesca; Melino, Gerry

2007-01-01

Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin–protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73α, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73α for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73α and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1−/− cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73α and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy. PMID:17592138

The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene.

PubMed Central

Schüller, C; Brewster, J L; Alexander, M R; Gustin, M C; Ruis, H

1994-01-01

The HOG signal pathway of the yeast Saccharomyces cerevisiae is defined by the PBS2 and HOG1 genes encoding members of the MAP kinase kinase and of the MAP kinase family, respectively. Mutations in this pathway (deletions of PBS2 or HOG1, or point mutations in HOG1) almost completely abolish the induction of transcription by osmotic stress that is mediated by stress response elements (STREs). We have demonstrated previously that STREs also mediate induction of transcription by heat shock, nitrogen starvation and oxidative stress. This study shows that they are also activated by low external pH, sorbate, benzoate or ethanol stress. Induction by these other stress signals appears to be HOG pathway independent. HOG1-dependent osmotic induction of transcription of the CTT1 gene encoding the cytosolic catalase T occurs in the presence of a protein synthesis inhibitor and can be detected rapidly after an increase of tyrosine phosphorylation of Hog1p triggered by high osmolarity. Consistent with a role of STREs in the induction of stress resistance, a number of other stress protein genes (e.g. HSP104) are regulated like CTT1. Furthermore, catalase T was shown to be important for viability under severe osmotic stress, and heat shock was demonstrated to provide cross-protection against osmotic stress. Images PMID:7523111

The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene.

PubMed

Schüller, C; Brewster, J L; Alexander, M R; Gustin, M C; Ruis, H

1994-09-15

The HOG signal pathway of the yeast Saccharomyces cerevisiae is defined by the PBS2 and HOG1 genes encoding members of the MAP kinase kinase and of the MAP kinase family, respectively. Mutations in this pathway (deletions of PBS2 or HOG1, or point mutations in HOG1) almost completely abolish the induction of transcription by osmotic stress that is mediated by stress response elements (STREs). We have demonstrated previously that STREs also mediate induction of transcription by heat shock, nitrogen starvation and oxidative stress. This study shows that they are also activated by low external pH, sorbate, benzoate or ethanol stress. Induction by these other stress signals appears to be HOG pathway independent. HOG1-dependent osmotic induction of transcription of the CTT1 gene encoding the cytosolic catalase T occurs in the presence of a protein synthesis inhibitor and can be detected rapidly after an increase of tyrosine phosphorylation of Hog1p triggered by high osmolarity. Consistent with a role of STREs in the induction of stress resistance, a number of other stress protein genes (e.g. HSP104) are regulated like CTT1. Furthermore, catalase T was shown to be important for viability under severe osmotic stress, and heat shock was demonstrated to provide cross-protection against osmotic stress.

Ube2w and ataxin-3 coordinately regulate the ubiquitin ligase CHIP

PubMed Central

Scaglione, K. Matthew; Zavodszky, Eszter; Todi, Sokol V.; Patury, Srikanth; Xu, Ping; Rodríguez-Lebrón, Edgardo; Fischer, Svetlana; Konen, John; Djarmati, Ana; Peng, Junmin; Gestwicki, Jason E.; Paulson, Henry L.

2011-01-01

Summary The mechanisms by which ubiquitin ligases are regulated remain poorly understood. Here we describe a series of molecular events that coordinately regulate CHIP, a neuroprotective E3 implicated in protein quality control. Through their opposing activities, the initiator E2, Ube2w, and the specialized deubiquitinating enzyme (DUB), ataxin-3, participate in initiating, regulating and terminating the CHIP ubiquitination cycle. Monoubiquitination of CHIP by Ube2w stabilizes the interaction between CHIP and ataxin-3, which through its DUB activity limits the length of chains attached to CHIP substrates. Upon completion of substrate ubiquitination ataxin-3 deubiquitinates CHIP, effectively terminating the reaction. Our results suggest that functional pairing of E3s with ataxin-3 or similar DUBs represents an important point of regulation in ubiquitin-dependent protein quality control. In addition, the results shed light on disease pathogenesis in SCA3, a neurodegenerative disorder caused by polyglutamine expansion in ataxin-3. PMID:21855799

A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1.

PubMed

Prattes, Michael; Loibl, Mathias; Zisser, Gertrude; Luschnig, Daniel; Kappel, Lisa; Rössler, Ingrid; Grassegger, Manuela; Hromic, Altijana; Krieger, Elmar; Gruber, Karl; Pertschy, Brigitte; Bergler, Helmut

2017-03-17

AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD (inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia). Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family.

Microelectrode array recordings of cultured hippocampal networks reveal a simple model for transcription and protein synthesis-dependent plasticity

PubMed Central

Arnold, Fiona JL; Hofmann, Frank; Bengtson, C. Peter; Wittmann, Malte; Vanhoutte, Peter; Bading, Hilmar

2005-01-01

A simplified cell culture system was developed to study neuronal plasticity. As changes in synaptic strength may alter network activity patterns, we grew hippocampal neurones on a microelectrode array (MEA) and monitored their collective behaviour with 60 electrodes simultaneously. We found that exposure of the network for 15 min to the GABAA receptor antagonist bicuculline induced an increase in synaptic efficacy at excitatory synapses that was associated with an increase in the frequency of miniature AMPA receptor-mediated EPSCs and a change in network activity from uncoordinated firing of neurones (lacking any recognizable pattern) to a highly organized, periodic and synchronous burst pattern. Induction of recurrent synchronous bursting was dependent on NMDA receptor activation and required extracellular signal-regulated kinase (ERK)1/2 signalling and translation of pre-existing mRNAs. Once induced, the burst pattern persisted for several days; its maintenance phase (> 4 h) was dependent on gene transcription taking place in a critical period of 120 min following induction. Thus, cultured hippocampal neurones display a simple, transcription and protein synthesis-dependent form of plasticity. The non-invasive nature of MEA recordings provides a significant advantage over traditional assays for synaptic connectivity (i.e. long-term potentiation in brain slices) and facilitates the search for activity-regulated genes critical for late-phase plasticity. PMID:15618268

Microelectrode array recordings of cultured hippocampal networks reveal a simple model for transcription and protein synthesis-dependent plasticity.

PubMed

Arnold, Fiona J L; Hofmann, Frank; Bengtson, C Peter; Wittmann, Malte; Vanhoutte, Peter; Bading, Hilmar

2005-04-01

A simplified cell culture system was developed to study neuronal plasticity. As changes in synaptic strength may alter network activity patterns, we grew hippocampal neurones on a microelectrode array (MEA) and monitored their collective behaviour with 60 electrodes simultaneously. We found that exposure of the network for 15 min to the GABA(A) receptor antagonist bicuculline induced an increase in synaptic efficacy at excitatory synapses that was associated with an increase in the frequency of miniature AMPA receptor-mediated EPSCs and a change in network activity from uncoordinated firing of neurones (lacking any recognizable pattern) to a highly organized, periodic and synchronous burst pattern. Induction of recurrent synchronous bursting was dependent on NMDA receptor activation and required extracellular signal-regulated kinase (ERK)1/2 signalling and translation of pre-existing mRNAs. Once induced, the burst pattern persisted for several days; its maintenance phase (> 4 h) was dependent on gene transcription taking place in a critical period of 120 min following induction. Thus, cultured hippocampal neurones display a simple, transcription and protein synthesis-dependent form of plasticity. The non-invasive nature of MEA recordings provides a significant advantage over traditional assays for synaptic connectivity (i.e. long-term potentiation in brain slices) and facilitates the search for activity-regulated genes critical for late-phase plasticity.

Microstructural Evolution of NiCrBSi Coatings Fabricated by Stationary Local Induction Cladding

NASA Astrophysics Data System (ADS)

Chen, Xuliang; Qin, Xunpeng; Gao, Kai; Zhu, Zhenhua; Huang, Feng

2018-04-01

The development of induction cladding has been restricted by the complicated geometric characteristics of workpieces and the large heat-affected zone in the cladded workpieces. In this paper, three-dimensional continual local induction cladding (3D-CLIC) was proposed as a potential process to clad coating over a substrate with curved surface, and a stationary local induction cladding (SLIC) experiment was conducted as an exploratory study of 3D-CLIC. The microstructures and microhardness in the coatings were measured by SEM, EDS, XRD and microsclerometer, respectively. The results indicate that the coating is metallurgically bonded with the substrate without any defects. A compositional gradient exists in the diffusion transfer belt (DTB), and it decreases with the increase in induction heating time. The coating is mainly composed of (Fe, Ni), CrB, M7C3, Ni3B, Ni3Si and M23C6 (M = Cr, Ni, Fe). Among the carbides, M7C3 presents several morphologies and M23C6 is always attached to the DTB. A special phenomenon of texture was found in the SLIC coatings. The preferred orientation in (200) crystal plane or the restrained orientation in (111) (200) crystal plane becomes more obvious as the scanning speed increases. The maximum average microhardness is 721 HV when the coating is heated for 5 s. The wear loss of different samples increases with increasing induction heating time. The longer heating time would result in higher dilution in the SLIC coatings due to the complete mixing with the substrate, thus leading to the decrease in microhardness and wear loss.

Microstructural Evolution of NiCrBSi Coatings Fabricated by Stationary Local Induction Cladding

NASA Astrophysics Data System (ADS)

Chen, Xuliang; Qin, Xunpeng; Gao, Kai; Zhu, Zhenhua; Huang, Feng

2018-05-01

The development of induction cladding has been restricted by the complicated geometric characteristics of workpieces and the large heat-affected zone in the cladded workpieces. In this paper, three-dimensional continual local induction cladding (3D-CLIC) was proposed as a potential process to clad coating over a substrate with curved surface, and a stationary local induction cladding (SLIC) experiment was conducted as an exploratory study of 3D-CLIC. The microstructures and microhardness in the coatings were measured by SEM, EDS, XRD and microsclerometer, respectively. The results indicate that the coating is metallurgically bonded with the substrate without any defects. A compositional gradient exists in the diffusion transfer belt (DTB), and it decreases with the increase in induction heating time. The coating is mainly composed of (Fe, Ni), CrB, M7C3, Ni3B, Ni3Si and M23C6 (M = Cr, Ni, Fe). Among the carbides, M7C3 presents several morphologies and M23C6 is always attached to the DTB. A special phenomenon of texture was found in the SLIC coatings. The preferred orientation in (200) crystal plane or the restrained orientation in (111) (200) crystal plane becomes more obvious as the scanning speed increases. The maximum average microhardness is 721 HV when the coating is heated for 5 s. The wear loss of different samples increases with increasing induction heating time. The longer heating time would result in higher dilution in the SLIC coatings due to the complete mixing with the substrate, thus leading to the decrease in microhardness and wear loss.

Time-evolving genetic networks reveal a NAC troika that negatively regulates leaf senescence in Arabidopsis.

PubMed

Kim, Hyo Jung; Park, Ji-Hwan; Kim, Jingil; Kim, Jung Ju; Hong, Sunghyun; Kim, Jeongsik; Kim, Jin Hee; Woo, Hye Ryun; Hyeon, Changbong; Lim, Pyung Ok; Nam, Hong Gil; Hwang, Daehee

2018-05-22

Senescence is controlled by time-evolving networks that describe the temporal transition of interactions among senescence regulators. Here, we present time-evolving networks for NAM/ATAF/CUC (NAC) transcription factors in Arabidopsis during leaf aging. The most evident characteristic of these time-dependent networks was a shift from positive to negative regulation among NACs at a presenescent stage. ANAC017, ANAC082, and ANAC090, referred to as a "NAC troika," govern the positive-to-negative regulatory shift. Knockout of the NAC troika accelerated senescence and the induction of other NAC s, whereas overexpression of the NAC troika had the opposite effects. Transcriptome and molecular analyses revealed shared suppression of senescence-promoting processes by the NAC troika, including salicylic acid (SA) and reactive oxygen species (ROS) responses, but with predominant regulation of SA and ROS responses by ANAC090 and ANAC017, respectively. Our time-evolving networks provide a unique regulatory module of presenescent repressors that direct the timely induction of senescence-promoting processes at the presenescent stage of leaf aging. Copyright © 2018 the Author(s). Published by PNAS.

Time-evolving genetic networks reveal a NAC troika that negatively regulates leaf senescence in Arabidopsis

PubMed Central

Kim, Hyo Jung; Park, Ji-Hwan; Kim, Jingil; Kim, Jung Ju; Hong, Sunghyun; Kim, Jin Hee; Woo, Hye Ryun; Lim, Pyung Ok; Nam, Hong Gil; Hwang, Daehee

2018-01-01

Senescence is controlled by time-evolving networks that describe the temporal transition of interactions among senescence regulators. Here, we present time-evolving networks for NAM/ATAF/CUC (NAC) transcription factors in Arabidopsis during leaf aging. The most evident characteristic of these time-dependent networks was a shift from positive to negative regulation among NACs at a presenescent stage. ANAC017, ANAC082, and ANAC090, referred to as a “NAC troika,” govern the positive-to-negative regulatory shift. Knockout of the NAC troika accelerated senescence and the induction of other NACs, whereas overexpression of the NAC troika had the opposite effects. Transcriptome and molecular analyses revealed shared suppression of senescence-promoting processes by the NAC troika, including salicylic acid (SA) and reactive oxygen species (ROS) responses, but with predominant regulation of SA and ROS responses by ANAC090 and ANAC017, respectively. Our time-evolving networks provide a unique regulatory module of presenescent repressors that direct the timely induction of senescence-promoting processes at the presenescent stage of leaf aging. PMID:29735710

Characterization of AcrD, a resistance-nodulation-cell division-type multidrug efflux pump from the fire blight pathogen Erwinia amylovora.

PubMed

Pletzer, Daniel; Weingart, Helge

2014-01-21

Multidrug efflux pumps are membrane translocases that have the ability to extrude a variety of structurally unrelated compounds from the cell. AcrD, a resistance-nodulation-cell division (RND) transporter, was shown to be involved in efflux of highly hydrophilic aminoglycosides and a limited number of amphiphilic compounds in E. coli. Here, a homologue of AcrD in the plant pathogen and causal agent of fire blight disease Erwinia amylovora was identified. The substrate specificity of AcrD was studied by overexpression of the corresponding gene from a high-copy plasmid in E. amylovora Ea1189-3, which is hypersensitive to many drugs due to a deficiency of the major multidrug pump AcrB. AcrD mediated resistance to several amphiphilic compounds including clotrimazole and luteolin, two compounds hitherto not described as substrates of AcrD in enterobacteria. However, AcrD was not able to expel aminoglycosides. An acrD mutant exhibited full virulence on apple rootstock and immature pear fruits. RT-PCR analysis revealed an induction of acrD expression in infected apple tissue but not on pear fruits. Moreover, a direct binding of BaeR, the response regulator of the two-component regulatory system BaeSR, to the acrD promoter was observed as has already been shown in other enterobacteria. AcrD from E. amylovora is involved in resistance to a limited number of amphiphilic compounds, but in contrast to AcrD of E. coli, it is not involved in resistance to aminoglycosides. The expression of acrD was up-regulated by addition of the substrates deoxycholate, naringenin, tetracycline and zinc. AcrD appears to be regulated by the BaeSR two-component system, an envelope stress signal transduction pathway.

Characterization of AcrD, a Resistance-Nodulation-Cell Division-type multidrug efflux pump from the fire blight pathogen Erwinia amylovora

PubMed Central

2014-01-01

Background Multidrug efflux pumps are membrane translocases that have the ability to extrude a variety of structurally unrelated compounds from the cell. AcrD, a resistance-nodulation-cell division (RND) transporter, was shown to be involved in efflux of highly hydrophilic aminoglycosides and a limited number of amphiphilic compounds in E. coli. Here, a homologue of AcrD in the plant pathogen and causal agent of fire blight disease Erwinia amylovora was identified. Results The substrate specificity of AcrD was studied by overexpression of the corresponding gene from a high-copy plasmid in E. amylovora Ea1189-3, which is hypersensitive to many drugs due to a deficiency of the major multidrug pump AcrB. AcrD mediated resistance to several amphiphilic compounds including clotrimazole and luteolin, two compounds hitherto not described as substrates of AcrD in enterobacteria. However, AcrD was not able to expel aminoglycosides. An acrD mutant exhibited full virulence on apple rootstock and immature pear fruits. RT-PCR analysis revealed an induction of acrD expression in infected apple tissue but not on pear fruits. Moreover, a direct binding of BaeR, the response regulator of the two-component regulatory system BaeSR, to the acrD promoter was observed as has already been shown in other enterobacteria. Conclusions AcrD from E. amylovora is involved in resistance to a limited number of amphiphilic compounds, but in contrast to AcrD of E. coli, it is not involved in resistance to aminoglycosides. The expression of acrD was up-regulated by addition of the substrates deoxycholate, naringenin, tetracycline and zinc. AcrD appears to be regulated by the BaeSR two-component system, an envelope stress signal transduction pathway. PMID:24443882

Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability

PubMed Central

Murchie, Ryan; Guo, Cong-Hui; Persaud, Avinash; Muise, Aleixo; Rotin, Daniela

2014-01-01

Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ−/− mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ−/− mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ−/− mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper–tyrosine-phosphorylated in the PTPσ−/− mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ−/− mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD. PMID:24385580

Epigenetic Regulation of the NR4A Orphan Nuclear Receptor NOR1 By Histone Acetylation

PubMed Central

Zhao, Yue; Nomiyama, Takashi; Findeisen, Hannes M.; Qing, Hua; Aono, Jun; Jones, Karrie L.; Heywood, Elizabeth B.; Bruemmer, Dennis

2014-01-01

The nuclear receptor NOR1 is an immediate-early response gene implicated in the transcriptional control of proliferation. Since the expression level of NOR1 is rapidly induced through cAMP response element binding (CREB) protein-dependent promoter activation, we investigated the contribution of histone acetylation to this transient induction. We demonstrate that NOR1 transcription is induced by histone deacetylase (HDAC) inhibition and by depletion of HDAC1 and HDAC3. HDAC inhibition activated the NOR1 promoter, increased histone acetylation and augmented the recruitment of phosphorylated CREB to the promoter. Furthermore, HDAC inhibition increased Ser133 phosphorylation of CREB and augmented NOR1 protein stability. These data outline previously unrecognized mechanisms of NOR1 regulation and illustrate a key role for histone acetylation in the rapid induction of NOR1. PMID:25451221

Axl is a novel target of withaferin A in the induction of apoptosis and the suppression of invasion.

PubMed

Woo, Seon Min; Min, Kyoung-jin; Kim, Shin; Park, Jong-Wook; Kim, Dong Eun; Kim, Sang Hyun; Choi, Yung Hyun; Kwon, Taeg Kyu

2014-08-29

Withaferin A, a withanolide derived from the medicinal plant Withania somnifera, has been reported to exhibit anti-tumorigenic activity against various cancer cells. In this study, we show that withaferin A inhibits the constitutive and recombinant human growth-arrest-specific protein 6 (rhGas6)-induced phosphorylation of Axl and STAT3. In addition, withaferin A also induces the down-regulation of Axl protein expression in a lysosome-dependent manner and inhibits rhGas6-induced wound healing and cell migration. Furthermore, the overexpression of Axl attenuates withaferin A-induced apoptosis. Taken together, the data from the present study indicate that the withaferin A-mediated down-regulation of the Gas6/Axl signaling pathway mediates the inhibition of cell migration and the induction of apoptosis. Copyright © 2014 Elsevier Inc. All rights reserved.

Orphan Nuclear Receptor Small Heterodimer Partner Negatively Regulates Growth Hormone-mediated Induction of Hepatic Gluconeogenesis through Inhibition of Signal Transducer and Activator of Transcription 5 (STAT5) Transactivation*

PubMed Central

Kim, Yong Deuk; Li, Tiangang; Ahn, Seung-Won; Kim, Don-Kyu; Lee, Ji-Min; Hwang, Seung-Lark; Kim, Yong-Hoon; Lee, Chul-Ho; Lee, In-Kyu; Chiang, John Y. L.; Choi, Hueng-Sik

2012-01-01

Growth hormone (GH) is a key metabolic regulator mediating glucose and lipid metabolism. Ataxia telangiectasia mutated (ATM) is a member of the phosphatidylinositol 3-kinase superfamily and regulates cell cycle progression. The orphan nuclear receptor small heterodimer partner (SHP: NR0B2) plays a pivotal role in regulating metabolic processes. Here, we studied the role of ATM on GH-dependent regulation of hepatic gluconeogenesis in the liver. GH induced phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase gene expression in primary hepatocytes. GH treatment and adenovirus-mediated STAT5 overexpression in hepatocytes increased glucose production, which was blocked by a JAK2 inhibitor, AG490, dominant negative STAT5, and STAT5 knockdown. We identified a STAT5 binding site on the PEPCK gene promoter using reporter assays and point mutation analysis. Up-regulation of SHP by metformin-mediated activation of the ATM-AMP-activated protein kinase pathway led to inhibition of GH-mediated induction of hepatic gluconeogenesis, which was abolished by an ATM inhibitor, KU-55933. Immunoprecipitation studies showed that SHP physically interacted with STAT5 and inhibited STAT5 recruitment on the PEPCK gene promoter. GH-induced hepatic gluconeogenesis was decreased by either metformin or Ad-SHP, whereas the inhibition by metformin was abolished by SHP knockdown. Finally, the increase of hepatic gluconeogenesis following GH treatment was significantly higher in the liver of SHP null mice compared with that of wild-type mice. Overall, our results suggest that the ATM-AMP-activated protein kinase-SHP network, as a novel mechanism for regulating hepatic glucose homeostasis via a GH-dependent pathway, may be a potential therapeutic target for insulin resistance. PMID:22977252

Mechanosensing regulates virulence in Escherichia coli O157:H7.

PubMed

Islam, Md Shahidul; Krachler, Anne Marie

2016-01-01

Enterohemorrhagic Escherichia coli O157:H7 is a food-borne pathogen transmitted via the fecal-oral route, and can cause bloody diarrhea and hemolytic uremic syndrome (HUS) in the human host. Although a range of colonization factors, Shiga toxins and a type III secretion system (T3SS) all contribute to disease development, the locus of enterocyte effacement (LEE) encoded T3SS is responsible for the formation of lesions in the intestinal tract. While a variety of chemical cues in the host environment are known to up-regulate LEE expression, we recently demonstrated that changes in physical forces at the site of attachment are required for localized, full induction of the system and thus spatial regulation of virulence in the intestinal tract. Here, we discuss our findings in the light of other recent studies describing mechanosensing of the host and force-dependent induction of virulence mechanisms. We discuss potential mechanisms of mechanosensing and mechanotransduction, and the level of conservation across bacterial species.

Coffee induces breast cancer resistance protein expression in Caco-2 cells.

PubMed

Isshiki, Marina; Umezawa, Kazuo; Tamura, Hiroomi

2011-01-01

Coffee is a beverage that is consumed world-wide on a daily basis and is known to induce a series of metabolic and pharmacological effects, especially in the digestive tract. However, little is known concerning the effects of coffee on transporters in the gastrointestinal tract. To elucidate the effect of coffee on intestinal transporters, we investigated its effect on expression of the breast cancer resistance protein (BCRP/ABCG2) in a human colorectal cancer cell line, Caco-2. Coffee induced BCRP gene expression in Caco-2 cells in a coffee-dose dependent manner. Coffee treatment of Caco-2 cells also increased the level of BCRP protein, which corresponded to induction of gene expression, and also increased cellular efflux activity, as judged by Hoechst33342 accumulation. None of the major constituents of coffee tested could induce BCRP gene expression. The constituent of coffee that mediated this induction was extractable with ethyl acetate and was produced during the roasting process. Dehydromethylepoxyquinomicin (DHMEQ), an inhibitor of nuclear factor (NF)-κB, inhibited coffee-mediated induction of BCRP gene expression, suggesting involvement of NF-κB in this induction. Our data suggest that daily consumption of coffee might induce BCRP expression in the gastrointestinal tract and may affect the bioavailability of BCRP substrates.

Phosphorylation of 1-Aminocyclopropane-1-Carboxylic Acid Synthase by MPK6, a Stress-Responsive Mitogen-Activated Protein Kinase, Induces Ethylene Biosynthesis in ArabidopsisW⃞

PubMed Central

Liu, Yidong; Zhang, Shuqun

2004-01-01

Mitogen-activated protein kinases (MAPKs) are implicated in regulating plant growth, development, and response to the environment. However, the underlying mechanisms are unknown because of the lack of information about their substrates. Using a conditional gain-of-function transgenic system, we demonstrated that the activation of SIPK, a tobacco (Nicotiana tabacum) stress-responsive MAPK, induces the biosynthesis of ethylene. Here, we report that MPK6, the Arabidopsis thaliana ortholog of tobacco SIPK, is required for ethylene induction in this transgenic system. Furthermore, we found that selected isoforms of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), the rate-limiting enzyme of ethylene biosynthesis, are substrates of MPK6. Phosphorylation of ACS2 and ACS6 by MPK6 leads to the accumulation of ACS protein and, thus, elevated levels of cellular ACS activity and ethylene production. Expression of ACS6DDD, a gain-of-function ACS6 mutant that mimics the phosphorylated form of ACS6, confers constitutive ethylene production and ethylene-induced phenotypes. Increasing numbers of stress stimuli have been shown to activate Arabidopsis MPK6 or its orthologs in other plant species. The identification of the first plant MAPK substrate in this report reveals one mechanism by which MPK6/SIPK regulates plant stress responses. Equally important, this study uncovers a signaling pathway that modulates the biosynthesis of ethylene, an important plant hormone, in plants under stress. PMID:15539472

Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhdanov, Alexander V., E-mail: a.zhdanov@ucc.ie; Waters, Alicia H.C.; Golubeva, Anna V.

2015-01-01

Changes in availability and utilisation of O{sub 2} and metabolic substrates are common in ischemia and cancer. We examined effects of substrate deprivation on HIF signalling in PC12 cells exposed to different atmospheric O{sub 2}. Upon 2–4 h moderate hypoxia, HIF-α protein levels were dictated by the availability of glutamine and glucose, essential for deep cell deoxygenation and glycolytic ATP flux. Nuclear accumulation of HIF-1α dramatically decreased upon inhibition of glutaminolysis or glutamine deprivation. Elevation of HIF-2α levels was transcription-independent and associated with the activation of Akt and Erk1/2. Upon 2 h anoxia, HIF-2α levels strongly correlated with cellular ATP,more » produced exclusively via glycolysis. Without glucose, HIF signalling was suppressed, giving way to other regulators of cell adaptation to energy crisis, e.g. AMPK. Consequently, viability of cells deprived of O{sub 2} and glucose decreased upon inhibition of AMPK with dorsomorphin. The capacity of cells to accumulate HIF-2α decreased after 24 h glucose deprivation. This effect, associated with increased AMPKα phosphorylation, was sensitive to dorsomorphin. In chronically hypoxic cells, glutamine played no major role in HIF-2α accumulation, which became mainly glucose-dependent. Overall, the availability of O{sub 2} and metabolic substrates intricately regulates HIF signalling by affecting cell oxygenation, ATP levels and pathways involved in production of HIF-α. - Highlights: • Gln and Glc regulate HIF levels in hypoxic cells by maintaining low O{sub 2} and high ATP. • HIF-α levels under anoxia correlate with cellular ATP and critically depend on Glc. • Gln and Glc modulate activity of Akt, Erk and AMPK, regulating HIF production. • HIF signalling is differentially inhibited by prolonged Glc and Gln deprivation. • Unlike Glc, Gln plays no major role in HIF signalling in chronically hypoxic cells.« less

Glucose deprivation increases monocarboxylate transporter 1 (MCT1) expression and MCT1-dependent tumor cell migration.

PubMed

De Saedeleer, C J; Porporato, P E; Copetti, T; Pérez-Escuredo, J; Payen, V L; Brisson, L; Feron, O; Sonveaux, P

2014-07-31

The glycolytic end-product lactate is a pleiotropic tumor growth-promoting factor. Its activities primarily depend on its uptake, a process facilitated by the lactate-proton symporter monocarboxylate transporter 1 (MCT1). Therefore, targeting the transporter or its chaperon protein CD147/basigin, itself involved in the aggressive malignant phenotype, is an attractive therapeutic option for cancer, but basic information is still lacking regarding the regulation of the expression, interaction and activities of both proteins. In this study, we found that glucose deprivation dose-dependently upregulates MCT1 and CD147 protein expression and their interaction in oxidative tumor cells. While this posttranslational induction could be recapitulated using glycolysis inhibition, hypoxia, oxidative phosphorylation (OXPHOS) inhibitor rotenone or hydrogen peroxide, it was blocked with alternative oxidative substrates and specific antioxidants, pointing out at a mitochondrial control. Indeed, we found that the stabilization of MCT1 and CD147 proteins upon glucose removal depends on mitochondrial impairment and the associated generation of reactive oxygen species. When glucose was a limited resource (a situation occurring naturally or during the treatment of many tumors), MCT1-CD147 heterocomplexes accumulated, including in cell protrusions of the plasma membrane. It endowed oxidative tumor cells with increased migratory capacities towards glucose. Migration increased in cells overexpressing MCT1 and CD147, but it was inhibited in glucose-starved cells provided with an alternative oxidative fuel, treated with an antioxidant, lacking MCT1 expression, or submitted to pharmacological MCT1 inhibition. While our study identifies the mitochondrion as a glucose sensor promoting tumor cell migration, MCT1 is also revealed as a transducer of this response, providing a new rationale for the use of MCT1 inhibitors in cancer.

Novel targets for Huntington’s disease in an mTOR-independent autophagy pathway

PubMed Central

Williams, Andrea; Sarkar, Sovan; Cuddon, Paul; Ttofi, Evangelia K.; Saiki, Shinji; Siddiqi, Farah H.; Jahreiss, Luca; Fleming, Angeleen; Pask, Dean; Goldsmith, Paul; O’Kane, Cahir J.; Floto, R. Andres; Rubinsztein, David C.

2009-01-01

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases like Huntington’s disease. Autophagy induction with the mTOR inhibitor, rapamycin, accelerates clearance of these toxic substrates. As rapamycin has non-trivial side effects, we screened FDA-approved drugs to identify novel autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the Gi signaling activator clonidine, induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, where cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating Gsα, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced and we provide proof-of-principle for therapeutic relevance in Huntington’s disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+, like excitotoxicity, will inhibit autophagy, thus retarding clearance of aggregate-prone proteins. PMID:18391949

Mechanism of enhanced hematopoietic response by soluble beta-glucan SCG in cyclophosphamide-treated mice.

PubMed

Harada, Toshie; Kawaminami, Hiromi; Miura, Noriko N; Adachi, Yoshiyuki; Nakajima, Mitsuhiro; Yadomae, Toshiro; Ohno, Naohito

2006-01-01

SCG is a major 6-branched 1,3-beta-D-glucan in Sparassis crispa Fr. SCG shows antitumor activity and also enhances the hematopoietic response in cyclophosphamide (CY)-treated mice. In the present study, the molecular mechanism of the enhancement of the hematopoietic response was investigated. The levels of interferon-(IFN-)gamma, tumor necrosis factor-(TNF-)alpha, granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin-(IL-) 6 and IL-12p70 were significantly increased by SCG in CY-treated mice. GM-CSF production in the splenocytes from the CY-treated mice was higher than that in normal mice regardless of SCG stimulation. Neutralizing GM-CSF significantly inhibited the induction of IFN-gamma, TNF-alpha and IL-12p70 by SCG. The level of cytokine induction by SCG was regulated by the amount of endogenous GM-CSF produced in response to CY treatment in a dose-dependent manner. The expression of beta-glucan receptors, such as CR3 and dectin-1, was up-regulated by CY treatment. Blocking dectin-1 significantly inhibited the induction of TNF-alpha and IL-12p70 production by SCG. Taken together, these results suggest that the key factors in the cytokine induction in CY-treated mice were the enhanced levels of both endogenous GM-CSF production and dectin-1 expression.

Nutrient sensing by the mitochondrial transcription machinery dictates oxidative phosphorylation

PubMed Central

Liu, Lijun; Nam, Minwoo; Fan, Wei; Akie, Thomas E.; Hoaglin, David C.; Gao, Guangping; Keaney, John F.; Cooper, Marcus P.

2014-01-01

Sirtuin 3 (SIRT3), an important regulator of energy metabolism and lipid oxidation, is induced in fasted liver mitochondria and implicated in metabolic syndrome. In fasted liver, SIRT3-mediated increases in substrate flux depend on oxidative phosphorylation (OXPHOS), but precisely how OXPHOS meets the challenge of increased substrate oxidation in fasted liver remains unclear. Here, we show that liver mitochondria in fasting mice adapt to the demand of increased substrate oxidation by increasing their OXPHOS efficiency. In response to cAMP signaling, SIRT3 deacetylated and activated leucine-rich protein 130 (LRP130; official symbol, LRPPRC), promoting a mitochondrial transcriptional program that enhanced hepatic OXPHOS. Using mass spectrometry, we identified SIRT3-regulated lysine residues in LRP130 that generated a lysine-to-arginine (KR) mutant of LRP130 that mimics deacetylated protein. Compared with wild-type LRP130 protein, expression of the KR mutant increased mitochondrial transcription and OXPHOS in vitro. Indeed, even when SIRT3 activity was abolished, activation of mitochondrial transcription and OXPHOS by the KR mutant remained robust, further highlighting the contribution of LRP130 deacetylation to increased OXPHOS in fasted liver. These data establish a link between nutrient sensing and mitochondrial transcription that regulates OXPHOS in fasted liver and may explain how fasted liver adapts to increased substrate oxidation. PMID:24430182

Mechanism of phytoestrogen puerarin-mediated cytoprotection following oxidative injury: Estrogen receptor-dependent up-regulation of PI3K/Akt and HO-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Yong Pil; Jeong, Hye Gwang

2008-12-15

Phytoestrogens are polyphenolic non-steroidal plant compounds with estrogen-like biological activity. The phytoestrogen puerarin, the main isoflavone glycoside found in the root of Pueraria lobata, has been used for various medicinal purposes in traditional Chinese medicines for thousands of years. Recent studies have indicated that the estrogen receptor (ER), through interaction with p85, regulates phosphoinositide 3-kinase (PI3K) activity, revealing a physiologic, non-nuclear function of ER that may be relevant in cytoprotection. In this study, we demonstrate that the phytoestrogen puerarin inhibits tert-butyl hydroperoxide (t-BHP)-induced oxidative injury via an ER-dependent G{beta}1/PI3K/Akt and heme oxygenase-1 (HO-1) pathway. Pretreatment of Hepa1c1c7 and HepG2 cellsmore » with puerarin significantly reduced t-BHP-induced caspase-3 activation and subsequent cell death. Also, puerarin up-regulated HO-1 expression and this expression conferred cytoprotection against oxidative injury induced by t-BHP. Moreover, puerarin induced Nrf2 nuclear translocation, which is upstream of puerarin-induced HO-1 expression, and PI3K activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection. Puerarin-induced up-regulation of HO-1 and cytoprotection against t-BHP were abolished by silencing Nrf2 expression with specific siRNA. Also, puerarin-mediated increases in PI3K activation and HO-1 induction were reversed by co-treatment with ICI 182,780 and pertussis toxin. Taken together, these results suggest that puerarin augments cellular antioxidant defense capacity through ER-dependent HO-1 induction via the G{beta}1/PI3K/Akt-Nrf2 signaling pathway, thereby protecting cells from oxidative stress.« less

Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

PubMed Central

Cargnello, Marie; Roux, Philippe P.

2011-01-01

Summary: The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries. PMID:21372320

Cell Cycle Regulators during Human Atrial Development

PubMed Central

Kim, Won Ho; Joo, Chan Uhng; Ku, Ja Hong; Ryu, Chul Hee; Koh, Keum Nim; Koh, Gou Young; Ko, Jae Ki

1998-01-01

Objectives The molecular mechanisms that regulate cardiomyocyte cell cycle and terminal differentiation in humans remain largely unknown. To determine which cyclins, cyclin dependent kinases (CDKs) and cyclin kinase inhibitors (CKIs) are important for cardiomyocyte proliferation, we have examined protein levels of cyclins, CDKs and CKIs during normal atrial development in humans. Methods Atrial tissues were obtained in the fetus from inevitable abortion and in the adult during surgery, Cyclin and CDK proteins were determined by Western blot analysis, CDK activities were determined by phosphorylation amount using specific substrate. Results Most cyclins and CDKs were high during the fetal period and their levels decreased at different rates during the adult period. While the protein levels of cyclin D1, cyclin D3, CDK4, CDK6 and CDK2 were still detectable in adult atria, the protein levels of cyclin E, cyclin A, cyclin B, cdc2 and PCNA were not detectable. Interestingly, p27KIP 1 protein increased markedly in the adult period, while p21C IP 1 protein in atria was detectable only in the fetal period. While the activities of CDK6, CDK2 and cdc2 decreased markedly, the activity of CDK4 did not change from the fetal period to the adult period. Conclusion These findings indicate that marked reduction of protein levels and activities of cyclins and CDKs, and marked induction of p27KIP 1 in atria, are associated with the withdrawal of cardiac cell cycle in adult humans. PMID:9735660

Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation.

PubMed

Fuchs, Christiane; Rosner, Margit; Dolznig, Helmut; Mikula, Mario; Kramer, Nina; Hengstschläger, Markus

2012-03-01

Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.

The RhoA Activator GEF-H1/Lfc Is a Transforming Growth Factor-β Target Gene and Effector That Regulates α-Smooth Muscle Actin Expression and Cell Migration

PubMed Central

Tsapara, Anna; Luthert, Phillip; Greenwood, John; Hill, Caroline S.

2010-01-01

Maintenance of the epithelial phenotype is crucial for tissue homeostasis. In the retina, dedifferentiation and loss of integrity of the retinal pigment epithelium (RPE) leads to retinal dysfunction and fibrosis. Transforming growth factor (TGF)-β critically contributes to RPE dedifferentiation and induces various responses, including increased Rho signaling, up-regulation of α-smooth muscle actin (SMA), and cell migration and dedifferentiation. Cellular TGF-β responses are stimulated by different signal transduction pathways: some are Smad dependent and others Smad independent. Alterations in Rho signaling are crucial to both types of TGF-β signaling, but how TGF-β-stimulates Rho signaling is poorly understood. Here, we show that primary RPE cells up-regulated GEF-H1 in response to TGF-β. GEF-H1 was the only detectable Rho exchange factor increased by TGF-β1 in a genome-wide expression analysis. GEF-H1 induction was Smad4-dependant and led to Rho activation. GEF-H1 inhibition counteracted α-SMA up-regulation and cell migration. In patients with retinal detachments and fibrosis, migratory RPE cells exhibited increased GEF-H1 expression, indicating that induction occurs in diseased RPE in vivo. Our data indicate that GEF-H1 is a target and functional effector of TGF-β by orchestrating Rho signaling to regulate gene expression and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases. PMID:20089843

Fragile X Mental Retardation Protein Is Required to Maintain Visual Conditioning-Induced Behavioral Plasticity by Limiting Local Protein Synthesis

PubMed Central

Liu, Han-Hsuan

2016-01-01

Fragile X mental retardation protein (FMRP) is thought to regulate neuronal plasticity by limiting dendritic protein synthesis, but direct demonstration of a requirement for FMRP control of local protein synthesis during behavioral plasticity is lacking. Here we tested whether FMRP knockdown in Xenopus optic tectum affects local protein synthesis in vivo and whether FMRP knockdown affects protein synthesis-dependent visual avoidance behavioral plasticity. We tagged newly synthesized proteins by incorporation of the noncanonical amino acid azidohomoalanine and visualized them with fluorescent noncanonical amino acid tagging (FUNCAT). Visual conditioning and FMRP knockdown produce similar increases in FUNCAT in tectal neuropil. Induction of visual conditioning-dependent behavioral plasticity occurs normally in FMRP knockdown animals, but plasticity degrades over 24 h. These results indicate that FMRP affects visual conditioning-induced local protein synthesis and is required to maintain the visual conditioning-induced behavioral plasticity. SIGNIFICANCE STATEMENT Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Exaggerated dendritic protein synthesis resulting from loss of fragile X mental retardation protein (FMRP) is thought to underlie cognitive deficits in FXS, but no direct evidence has demonstrated that FMRP-regulated dendritic protein synthesis affects behavioral plasticity in intact animals. Xenopus tadpoles exhibit a visual avoidance behavior that improves with visual conditioning in a protein synthesis-dependent manner. We showed that FMRP knockdown and visual conditioning dramatically increase protein synthesis in neuronal processes. Furthermore, induction of visual conditioning-dependent behavioral plasticity occurs normally after FMRP knockdown, but performance rapidly deteriorated in the absence of FMRP. These studies show that FMRP negatively regulates local protein synthesis and is required to maintain visual conditioning-induced behavioral plasticity in vivo. PMID:27383604

Fragile X Mental Retardation Protein Is Required to Maintain Visual Conditioning-Induced Behavioral Plasticity by Limiting Local Protein Synthesis.

PubMed

Liu, Han-Hsuan; Cline, Hollis T

2016-07-06

Fragile X mental retardation protein (FMRP) is thought to regulate neuronal plasticity by limiting dendritic protein synthesis, but direct demonstration of a requirement for FMRP control of local protein synthesis during behavioral plasticity is lacking. Here we tested whether FMRP knockdown in Xenopus optic tectum affects local protein synthesis in vivo and whether FMRP knockdown affects protein synthesis-dependent visual avoidance behavioral plasticity. We tagged newly synthesized proteins by incorporation of the noncanonical amino acid azidohomoalanine and visualized them with fluorescent noncanonical amino acid tagging (FUNCAT). Visual conditioning and FMRP knockdown produce similar increases in FUNCAT in tectal neuropil. Induction of visual conditioning-dependent behavioral plasticity occurs normally in FMRP knockdown animals, but plasticity degrades over 24 h. These results indicate that FMRP affects visual conditioning-induced local protein synthesis and is required to maintain the visual conditioning-induced behavioral plasticity. Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Exaggerated dendritic protein synthesis resulting from loss of fragile X mental retardation protein (FMRP) is thought to underlie cognitive deficits in FXS, but no direct evidence has demonstrated that FMRP-regulated dendritic protein synthesis affects behavioral plasticity in intact animals. Xenopus tadpoles exhibit a visual avoidance behavior that improves with visual conditioning in a protein synthesis-dependent manner. We showed that FMRP knockdown and visual conditioning dramatically increase protein synthesis in neuronal processes. Furthermore, induction of visual conditioning-dependent behavioral plasticity occurs normally after FMRP knockdown, but performance rapidly deteriorated in the absence of FMRP. These studies show that FMRP negatively regulates local protein synthesis and is required to maintain visual conditioning-induced behavioral plasticity in vivo. Copyright © 2016 the authors 0270-6474/16/367325-15$15.00/0.

Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

PubMed

Bunik, Victoria I; Tylicki, Adam; Lukashev, Nikolay V

2013-12-01

Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed. © 2013 FEBS.

Prioritizing plant defence over growth through WRKY regulation facilitates infestation by non-target herbivores.

PubMed

Li, Ran; Zhang, Jin; Li, Jiancai; Zhou, Guoxin; Wang, Qi; Bian, Wenbo; Erb, Matthias; Lou, Yonggen

2015-06-17

Plants generally respond to herbivore attack by increasing resistance and decreasing growth. This prioritization is achieved through the regulation of phytohormonal signaling networks. However, it remains unknown how this prioritization affects resistance against non-target herbivores. In this study, we identify WRKY70 as a specific herbivore-induced, mitogen-activated protein kinase-regulated rice transcription factor that physically interacts with W-box motives and prioritizes defence over growth by positively regulating jasmonic acid (JA) and negatively regulating gibberellin (GA) biosynthesis upon attack by the chewing herbivore Chilo suppressalis. WRKY70-dependent JA biosynthesis is required for proteinase inhibitor activation and resistance against C. suppressalis. In contrast, WRKY70 induction increases plant susceptibility against the rice brown planthopper Nilaparvata lugens. Experiments with GA-deficient rice lines identify WRKY70-dependent GA signaling as the causal factor in N. lugens susceptibility. Our study shows that prioritizing defence over growth leads to a significant resistance trade-off with important implications for the evolution and agricultural exploitation of plant immunity.

Characterization of the Regulation and Function of Zinc-Dependent Histone Deacetylases During Mouse Liver Regeneration

PubMed Central

Huang, Jiansheng; Barr, Emily; Rudnick, David A.

2013-01-01

The studies reported here were undertaken to define the regulation and functional importance of zinc-dependent histone deacetylase (Zn-HDAC) activity during liver regeneration using the mouse partial hepatectomy (PH) model. The results showed that hepatic HDAC activity was significantly increased in nuclear and cytoplasmic fractions following PH. Further analyses showed isoform-specific effects of PH on HDAC mRNA and protein expression, with increased expression of the class I HDACs, 1 and 8, and class II HDAC4 in regenerating liver. Hepatic expression of (class II) HDAC5 was unchanged after PH; however HDAC5 exhibited transient nuclear accumulation in regenerating liver. These changes in hepatic HDAC expression, subcellular localization, and activity coincided with diminished histone acetylation in regenerating liver. The significance of these events was investigated by determining the effects of suberoylanilide hydroxyamic acid (SAHA, a specific inhibitor of Zn-HDAC activity) on hepatic regeneration. The results showed that SAHA-treatment suppressed the effects of PH on histone deacetylation and hepatocellular BrdU incorporation. Further examination showed that SAHA blunted hepatic expression and activation of cell cycle signals downstream of induction of cyclin D1 expression in mice subjected to PH. Conclusion The data reported here demonstrate isoform-specific regulation of Zn-HDAC expression, subcellular localization, and activity in regenerating liver. These studies also indicate that HDAC activity promotes liver regeneration by regulating hepatocellular cell cycle progression at a step downstream of cyclin D1 induction. PMID:23258575

Tyrosine kinase oncogenes abrogate interleukin-3 dependence of murine myeloid cells through signaling pathways involving c-myc: conditional regulation of c-myc transcription by temperature-sensitive v-abl.

PubMed Central

Cleveland, J L; Dean, M; Rosenberg, N; Wang, J Y; Rapp, U R

1989-01-01

Retroviral expression vectors carrying the tyrosine kinase oncogenes abl, fms, src, and trk abrogate the requirements of murine myeloid FDC-P1 cells for interleukin-3 (IL-3). Factor-independent clones constitutively express c-myc in the absence of IL-3, whereas in parental cultures c-myc transcription requires the presence of the ligand. To directly test the effect of a tyrosine kinase oncogene on c-myc expression, retroviral constructs containing three different temperature-sensitive mutants of v-abl were introduced into myeloid IL-3-dependent FDC-P1 and 32D cells. At the permissive temperature, clones expressing temperature-sensitive abl behaved like wild-type abl-containing cells in their growth properties and expressed c-myc constitutively. Temperature shift experiments demonstrated that both IL-3 abrogation and the regulation of c-myc expression correlated with the presence of functional v-abl. Induction of c-myc expression by reactivation of temperature-sensitive v-abl mimicked c-myc induction by IL-3 in that it did not require protein synthesis and occurred at the level of transcription, with effects on both initiation and a transcription elongation block. However, v-abl-regulated FDC-P1 cell growth differed from IL-3-regulated growth in that c-fos and junB, which are normally induced by IL-3, were not induced by activation of v-abl. Images PMID:2555703

Drug transport by reconstituted P-glycoprotein in proteoliposomes. Effect of substrates and modulators, and dependence on bilayer phase state.

PubMed

Lu, P; Liu, R; Sharom, F J

2001-03-01

The P-glycoprotein multidrug transporter (Pgp) is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Pgp is envisaged as a 'hydrophobic vacuum cleaner', and drugs are believed to gain access to the substrate binding sites from within the membrane, rather than from the aqueous phase. The intimate association of both Pgp and its substrates with the membrane suggests that its function may be regulated by the biophysical properties of the lipid bilayer. Using the high affinity fluorescent substrate tetramethylrosamine (TMR), we have monitored, in real time, transport in proteoliposomes containing reconstituted Pgp. The TMR concentration gradient generated by Pgp was collapsed by the addition of either the ATPase inhibitor, vanadate, or Pgp modulators. TMR transport by Pgp obeyed Michaelis--Menten kinetics with respect to both of its substrates. The Km for ATP was 0.48 mM, close to the K(m) for ATP hydrolysis, and the K(m) for TMR was 0.3 microM. TMR transport was inhibited in a concentration-dependent fashion by verapamil and cyclosporin A, and activated (probably by a positive allosteric effect) by the transport substrate colchicine. TMR transport by Pgp reconstituted into proteoliposomes composed of two synthetic phosphatidylcholines showed a highly unusual biphasic temperature dependence. The rate of TMR transport was relatively high in the rigid gel phase, reached a maximum at the melting temperature of the bilayer, and then decreased in the fluid liquid crystalline phase. This pattern of temperature dependence suggests that the rate of drug transport by Pgp may be dominated by partitioning of drug into the bilayer.

Stimulation of phagocytosis by sulforaphane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suganuma, Hiroyuki, E-mail: hsuganu1@jhmi.edu; Fahey, Jed W., E-mail: jfahey@jhmi.edu; Bryan, Kelley E., E-mail: kbryanm1@jhmi.edu

2011-02-04

Research highlights: {yields} Sulforaphane stimulates the phagocytosis of RAW 264.7 macrophages under conditions of serum deprivation. {yields} This effect does not require Nrf2-dependent induction of phase 2 genes. {yields} Inactivation of macrophage migration inhibitory factor (MIF) by sulforaphane may be involved in stimulation of phagocytosis by sulforaphane. -- Abstract: Sulforaphane, a major isothiocyanate derived from cruciferous vegetables, protects living systems against electrophile toxicity, oxidative stress, inflammation, and radiation. A major protective mechanism is the induction of a network of endogenous cytoprotective (phase 2) genes that are regulated by transcription factor Nrf2. To obtain a more detailed understanding of the anti-inflammatorymore » and immunomodulatory effects of sulforaphane, we evaluated its effect on the phagocytosis activity of RAW 264.7 murine macrophage-like cells by measuring the uptake of 2-{mu}m diameter polystyrene beads. Sulforaphane raised the phagocytosis activity of RAW 264.7 cells but only in the absence or presence of low concentrations (1%) of fetal bovine serum. Higher serum concentrations depressed phagocytosis and abolished its stimulation by sulforaphane. This stimulation did not depend on the induction of Nrf2-regulated genes since it occurred in peritoneal macrophages of nrf2{sup -/-} mice. Moreover, a potent triterpenoid inducer of Nrf2-dependent genes did not stimulate phagocytosis, whereas sulforaphane and another isothiocyanate (benzyl isothiocyanate) had comparable inducer potencies. It has been shown recently that sulforaphane is a potent and direct inactivator of macrophage migration inhibitory factor (MIF), an inflammatory cytokine. Moreover, the addition of recombinant MIF to RAW 264.7 cells attenuated phagocytosis, but sulforaphane-inactivated MIF did not affect phagocytosis. The inactivation of MIF may therefore be involved in the phagocytosis-enhancing activity of sulforaphane.« less

Enhanced osteogenic differentiation of rat bone marrow mesenchymal stem cells on titanium substrates by inhibiting Notch3.

PubMed

Wang, Huiming; Jiang, Zhiwei; Zhang, Jing; Xie, Zhijian; Wang, Ying; Yang, Guoli

2017-08-01

The role of the Notch pathway has already been identified as a crucial regulator of bone development. However, the Notch signaling pathway has gone largely unexplored during osseointegration. This study aims to investigate the role of Notch signaling on osteogenic differentiation of rat derived bone marrow mesenchymal stem cells (BMSCs) on sandblasted, large-grit, acid-etched (SLA) treated Ti disks. The involved target genes in Notch pathways were identified by in vitro microarray and bioinformatics analyses with or without osteogenic induction. Adhesion, proliferation, and osteogenic related assay were subsequently conducted with target gene shRNA treatment. We found that 11 genes in the Notch signaling pathway were differentially expressed after osteogenic induction on SLA-treated Ti disks, which included up-regulated genes (Notch2, Dll1, Dll3, Ncstn, Ncor2, and Hes5) and down-regulated genes (Notch3, Lfng, Mfng, Jag2 and Maml2). With Notch3 shRNA treatment, the adhesion and proliferation of BMSCs on SLA-treated Ti disks were inhibited. Moreover, the expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), calcium deposition, BMP2 and Runx2 increased significantly compared with that observed in control groups, suggesting that the function of Notch3 was inhibitory in the osteogenic differentiation of BMSCs on SLA-treated titanium. Inhibition Notch3 can enhance osteogenic differentiation of BMSCs on SLA-treated Ti disks, which potentially provides a gene target for improving osseointegration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Investigation of drug-drug interactions caused by human pregnane X receptor-mediated induction of CYP3A4 and CYP2C subfamilies in chimeric mice with a humanized liver.

PubMed

Hasegawa, Maki; Tahara, Harunobu; Inoue, Ryo; Kakuni, Masakazu; Tateno, Chise; Ushiki, Junko

2012-03-01

The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4.

Prolonged Fasting Identifies Heat Shock Protein 10 as a Sirtuin 3 Substrate

PubMed Central

Lu, Zhongping; Chen, Yong; Aponte, Angel M.; Battaglia, Valentina; Gucek, Marjan; Sack, Michael N.

2015-01-01

Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function. PMID:25505263

Adaptation of Candida albicans to Reactive Sulfur Species

PubMed Central

Chebaro, Yasmin; Lorenz, Michael; Fa, Alice; Zheng, Rui; Gustin, Michael

2017-01-01

Candida albicans is an opportunistic fungal pathogen that is highly resistant to different oxidative stresses. How reactive sulfur species (RSS) such as sulfite regulate gene expression and the role of the transcription factor Zcf2 and the sulfite exporter Ssu1 in such responses are not known. Here, we show that C. albicans specifically adapts to sulfite stress and that Zcf2 is required for that response as well as induction of genes predicted to remove sulfite from cells and to increase the intracellular amount of a subset of nitrogen metabolites. Analysis of mutants in the sulfate assimilation pathway show that sulfite conversion to sulfide accounts for part of sulfite toxicity and that Zcf2-dependent expression of the SSU1 sulfite exporter is induced by both sulfite and sulfide. Mutations in the SSU1 promoter that selectively inhibit induction by the reactive nitrogen species (RNS) nitrite, a previously reported activator of SSU1, support a model for C. albicans in which Cta4-dependent RNS induction and Zcf2-dependent RSS induction are mediated by parallel pathways, different from S. cerevisiae in which the transcription factor Fzf1 mediates responses to both RNS and RSS. Lastly, we found that endogenous sulfite production leads to an increase in resistance to exogenously added sulfite. These results demonstrate that C. albicans has a unique response to sulfite that differs from the general oxidative stress response, and that adaptation to internal and external sulfite is largely mediated by one transcription factor and one effector gene. PMID:28235888

The role of polyamine catabolism in anti-tumour drug response.

PubMed

Casero, R A; Wang, Y; Stewart, T M; Devereux, W; Hacker, A; Wang, Y; Smith, R; Woster, P M

2003-04-01

Interest in polyamine catabolism has increased since it has been directly associated with the cytotoxic response of multiple tumour types to exposure to specific anti-tumour polyamine analogues. Human polyamine catabolism was considered to be a two-step pathway regulated by the rate-limiting enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (APAO). Further, the super-induction of SSAT by several anti-tumour polyamine analogues has been implicated in the cytotoxic response of specific solid-tumour phenotypes to these agents. This high induction of SSAT has been correlated with cellular response to the anti-tumour polyamine analogues in several systems and considerable progress has been made in understanding the molecular mechanisms that regulate the analogue-induced expression of SSAT. A polyamine response element has been identified and the transacting transcription factors that bind and stimulate transcription of SSAT have been cloned and characterized. The link between SSAT activity and cellular toxicity is thought to be based on the production of H(2)O(2) by the activity of the constitutive APAO that uses the SSAT-produced acetylated polyamines. The high induction of SSAT and the subsequent activity of APAO are linked to the cytotoxic response of some tumour cell types to specific polyamine analogues. However, we have recently cloned a variably spliced human polyamine oxidase (PAOh1) that is inducible by specific polyamine analogues, efficiently uses unacetylated spermine as a substrate, and also produces toxic H(2)O(2) as a product. The results of studies with PAOh1 suggest that it is an additional enzyme in polyamine catabolism that has the potential to significantly contribute to polyamine homoeostasis and drug response. Most importantly, PAOh1 is induced by specific polyamine analogues in a tumour-phenotype-specific manner in cell lines representative of the major forms of solid tumours, including lung, breast, colon and prostate. The sensitivity to these anti-tumour polyamine analogues can be significantly reduced if the tumour cells are co-treated with 250 microM of the polyamine oxidase inhibitor N (1), N (4)-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527), suggesting that the H(2)O(2) produced by PAOh1 does in fact play a direct role in the observed cytotoxicity. These results strongly implicate PAOh1 as a new target that, in combination with SSAT, may be exploited for therapeutic advantage. The current understanding of the role and regulation of these two important polyamine catabolic enzymes are discussed.

Collagen remodeling by phagocytosis is determined by collagen substrate topology and calcium-dependent interactions of gelsolin with nonmuscle myosin IIA in cell adhesions

PubMed Central

Arora, P. D.; Wang, Y.; Bresnick, A.; Dawson, J.; Janmey, P. A.; McCulloch, C. A.

2013-01-01

We examine how collagen substrate topography, free intracellular calcium ion concentration ([Ca2+]i, and the association of gelsolin with nonmuscle myosin IIA (NMMIIA) at collagen adhesions are regulated to enable collagen phagocytosis. Fibroblasts plated on planar, collagen-coated substrates show minimal increase of [Ca2+]i, minimal colocalization of gelsolin and NMMIIA in focal adhesions, and minimal intracellular collagen degradation. In fibroblasts plated on collagen-coated latex beads there are large increases of [Ca2+]i, time- and Ca2+-dependent enrichment of NMMIIA and gelsolin at collagen adhesions, and abundant intracellular collagen degradation. NMMIIA knockdown retards gelsolin recruitment to adhesions and blocks collagen phagocytosis. Gelsolin exhibits tight, Ca2+-dependent binding to full-length NMMIIA. Gelsolin domains G4–G6 selectively require Ca2+ to interact with NMMIIA, which is restricted to residues 1339–1899 of NMMIIA. We conclude that cell adhesion to collagen presented on beads activates Ca2+ entry and promotes the formation of phagosomes enriched with NMMIIA and gelsolin. The Ca2+ -dependent interaction of gelsolin and NMMIIA in turn enables actin remodeling and enhances collagen degradation by phagocytosis. PMID:23325791

HOXB9 induction of mesenchymal-to-epithelial transition in gastric carcinoma is negatively regulated by its hexapeptide motif

PubMed Central

He, Changyu; Zhang, Baogui; Zhang, Jun; Liu, Bingya; Zeng, Naiyan; Zhu, Zhenggang

2015-01-01

HOXB9, a transcription factor, plays an important role in development. While HOXB9 has been implicated in tumorigenesis and metastasis, its mechanisms are variable and its role in gastric carcinoma (GC) remains unclear. In the present study, we demonstrated that the expression of HOXB9 decreased in gastric carcinoma and was associated with malignancy and metastasis. Re-expression of HOXB9 in gastric cell lines resulted in the suppression of cell proliferation, migration, and invasion, which was accompanied by the induction of mesenchymal-to-epithelial transition (MET). Comparative sequence analysis and examination of a HOXB9 structural model indicated that three sites might possibly be involved in MET regulation. The in vitro study of HOXB9 mutants showed that these were unable to inhibit MET induction. However, when overexpressing a HOXB9 mutant lacking the hexapeptide motif, a more potent MET induction and tumor suppression was observed compared to that of the wild-type, indicating that the presence of the hexapeptide motif reduced HOXB9 MET induction and tumor suppression activity. Therefore, the results of the present study suggested that HOXB9 is a tumor suppressor in gastric carcinoma, and its activity was controlled by different regulatory mechanisms such as the hexapeptide motif as a “brake” in this case. The results of these regulatory effects could lead to either oncogenic or tumor suppressive roles of HOXB9, depending on the context of the particular type of cancer involved. PMID:26536658

Acute myotube protein synthesis regulation by IL-6-related cytokines.

PubMed

Gao, Song; Durstine, J Larry; Koh, Ho-Jin; Carver, Wayne E; Frizzell, Norma; Carson, James A

2017-11-01

IL-6 and leukemia inhibitory factor (LIF), members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mammalian target of rapamycin complex 1 (mTORC1)-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of glycoprotein 130 (gp130) receptor and downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)-Akt-mTORC1 and signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3), was investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation, and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knockdown and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6- or LIF-induced SOCS3 negatively regulates the activation of myotube protein synthesis. Copyright © 2017 the American Physiological Society.

Epigenetic regulation of the NR4A orphan nuclear receptor NOR1 by histone acetylation.

PubMed

Zhao, Yue; Nomiyama, Takashi; Findeisen, Hannes M; Qing, Hua; Aono, Jun; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

2014-12-20

The nuclear receptor NOR1 is an immediate-early response gene implicated in the transcriptional control of proliferation. Since the expression level of NOR1 is rapidly induced through cAMP response element binding (CREB) protein-dependent promoter activation, we investigated the contribution of histone acetylation to this transient induction. We demonstrate that NOR1 transcription is induced by histone deacetylase (HDAC) inhibition and by depletion of HDAC1 and HDAC3. HDAC inhibition activated the NOR1 promoter, increased histone acetylation and augmented the recruitment of phosphorylated CREB to the promoter. Furthermore, HDAC inhibition increased Ser133 phosphorylation of CREB and augmented NOR1 protein stability. These data outline previously unrecognized mechanisms of NOR1 regulation and illustrate a key role for histone acetylation in the rapid induction of NOR1. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Ubiquitination of Cdc20 by the APC occurs through an intramolecular mechanism

PubMed Central

Foe, Ian T.; Foster, Scott A.; Cheung, Stephanie K.; DeLuca, Steven Z.; Morgan, David O.; Toczyski, David P.

2012-01-01

SUMMARY Background Cells control progression through late mitosis by regulating Cdc20 and Cdh1, the two mitotic activators of the Anaphase Promoting Complex (APC). The control of Cdc20 protein levels during the cell cycle is not well understood. Results Here, we demonstrate that Cdc20 is degraded in budding yeast by multiple APC-dependent mechanisms. We find that the majority of Cdc20 turnover does not involve a second activator molecule, but instead depends on in cis Cdc20 autoubiquitination while it is bound to its activator-binding site on the APC core. Unlike in trans ubiquitination of Cdc20 substrates, the APC ubiquitinates Cdc20 independent of APC activation by Cdc20’s C-box. Cdc20 turnover by this intramolecular mechanism is cell cycle-regulated, contributing to the decline in Cdc20 levels that occurs after anaphase. Interestingly, high substrate levels in vitro significantly reduce Cdc20 autoubiquitination. Conclusion We show here that Cdc20 fluctuates through the cell cycle via a distinct form of APC-mediated ubiquitination. This in cis autoubiquitination may preferentially occur in early anaphase, following depletion of Cdc20 substrates. This suggests that distinct mechanisms are able to target Cdc20 for ubiquitination at different points during the cell cycle. PMID:22079111

Ras-Induced and Extracellular Signal-Regulated Kinase 1 and 2 Phosphorylation-Dependent Isomerization of Protein Tyrosine Phosphatase (PTP)-PEST by PIN1 Promotes FAK Dephosphorylation by PTP-PEST ▿

PubMed Central

Zheng, Yanhua; Yang, Weiwei; Xia, Yan; Hawke, David; Liu, David X.; Lu, Zhimin

2011-01-01

Protein tyrosine phosphatase (PTP)-PEST is a critical regulator of cell adhesion and migration. However, the mechanism by which PTP-PEST is regulated in response to oncogenic signaling to dephosphorylate its substrates remains unclear. Here, we demonstrate that activated Ras induces extracellular signal-regulated kinase 1 and 2-dependent phosphorylation of PTP-PEST at S571, which recruits PIN1 to bind to PTP-PEST. Isomerization of the phosphorylated PTP-PEST by PIN1 increases the interaction between PTP-PEST and FAK, which leads to the dephosphorylation of FAK Y397 and the promotion of migration, invasion, and metastasis of v-H-Ras-transformed cells. These findings uncover an important mechanism for the regulation of PTP-PEST in activated Ras-induced tumor progression. PMID:21876001

JBP485 improves gentamicin-induced acute renal failure by regulating the expression and function of Oat1 and Oat3 in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Xinjin; Meng, Qiang; Liu, Qi

2013-09-01

We investigated the effects of JBP485 (an anti-inflammatory dipeptide and a substrate of OAT) on regulation of the expression and function of renal Oat1 and Oat3, which can accelerate the excretion of accumulated uremic toxins (e.g. indoxyl sulfate) in the kidney to improve gentamicin-induced ARF in rats. JBP485 caused a significant decrease in the accumulation of endogenous substances (creatinine, blood urea nitrogen and indoxyl sulfate) in vivo, an increase in the excretion of exogenous compounds (lisinopril and inulin) into urine, and up-regulation of the expressions of renal Oat1 and Oat3 in the kidney tissues and slices via substrate induction. Tomore » determine the effect of JBP485 on the accelerated excretion of uremic toxins mediated by Oat1 and Oat3, the mRNA and protein expression levels of renal basolateral Oats were assessed by quantitative real-time PCR, western blot, immunohistochemical analysis and an immunofluorescence method. Gentamicin down-regulated the expression of Oats mRNA and protein in rat kidney, and these effects were reversed after administration of JBP485. In addition, JBP485 caused a significant decrease in MPO and MDA levels in the kidney, and improved the pathological condition of rat kidney. These results indicated that JBP485 improved acute renal failure by increasing the expression and function of Oat1 and Oat3, and by decreasing overoxidation of the kidney in gentamicin-induced ARF rats. - Highlights: • JBP485 could up-regulate function and expression of Oat1 and Oat3 in kidney. • Effects of JBP485 on ARF are mediated by stimulating excretion of uremic toxins. • JBP485 protected against gentamicin-induced ARF by decreasing MPO and MDA.« less

Interleukin 4 signals through two related pathways.

PubMed

Pernis, A; Witthuhn, B; Keegan, A D; Nelms, K; Garfein, E; Ihle, J N; Paul, W E; Pierce, J H; Rothman, P

1995-08-15

The interleukin 4 (IL-4) signaling pathway involves activation, by tyrosine phosphorylation, of two distinct substrates, a signal-transducing factor (STF-IL4) and the IL-4-induced phosphotyrosine substrate (4PS). It is not known whether the IL-4-mediated activation of these substrates occurs via related or distinct signaling pathways. We report that 32D cells, an IL-3-dependent myeloid progenitor cell line in which no phosphorylated 4PS is found, activate high levels of STF-IL4 in response to IL-4. Consistent with the known requirement for 4PS or insulin receptor substrate 1 (IRS-1) in IL-4-mediated mitogenesis, activation of STF-IL4 in 32D cells is not sufficient for IL-4-inducible c-myc expression. In addition, we have examined the ability of 32D cells transfected with different truncation mutants of the human IL-4 receptor to activate Jak-3 kinase and STF-IL4 in response to human IL-4. As in the case of 4PS/IRS-1, we have found that activation of both Jak-3 and STF-IL4 requires the presence of the IL-4 receptor region comprising aa 437-557. The finding that the same region of the IL-4 receptor is required for the induction of both 4PS/IRS-1 and STF-IL4 suggests that the IL-4-stimulated activation of these two substrates might involve common factors.

Induction heating to trigger the nickel surface modification by in situ generated 4-carboxybenzene diazonium

NASA Astrophysics Data System (ADS)

Arrotin, Bastien; Jacques, Amory; Devillers, Sébastien; Delhalle, Joseph; Mekhalif, Zineb

2016-05-01

Nickel is commonly used in numerous applications and is one of the few materials that present strong ferromagnetic properties. These make it a suitable material for induction heating which can be used to activate the grafting of organic species such as diazonium salts onto the material. Diazonium compounds are often used for the modification of metals and alloys thanks to their easy chemical reduction onto the substrates and the possibility to apply a one-step in situ generation process of the diazonium species. This work focuses on the grafting of 4-aminocarboxybenzene on nickel substrates in the context of a spontaneous grafting conducted either at room temperature or by thermal assistance through conventional heating and induction heating. These modifications are also carried out with the goal of maintaining the oxides layer as much as possible unaffected. The benefits of using induction heating with respect to conventional heating are an increase of the grafting rate, a better control of the reaction and a slighter impact on the oxides layer.

Hexavalent Chromium Cr(VI) Up-Regulates COX-2 Expression through an NFκB/c-Jun/AP-1–Dependent Pathway

PubMed Central

Zuo, Zhenghong; Cai, Tongjian; Li, Jingxia; Zhang, Dongyun; Yu, Yonghui

2012-01-01

Background: Hexavalent chromium [Cr(VI)] is recognized as a human carcinogen via inhalation. However, the molecular mechanisms by which Cr(VI) causes cancers are not well understood. Objectives: We evaluated cyclooxygenase-2 (COX-2) expression and the signaling pathway leading to this induction due to Cr(VI) exposure in cultured cells. Methods: We used the luciferase reporter assay and Western blotting to determine COX-2 induction by Cr(VI). We used dominant negative mutant, genetic knockout, gene knockdown, and chromatin immunoprecipitation approaches to elucidate the signaling pathway leading to COX-2 induction. Results: We found that Cr(VI) exposure induced COX-2 expression in both normal human bronchial epithelial cells and mouse embryonic fibroblasts in a concentration- and time-dependent manner. Deletion of IKKβ [inhibitor of transcription factor NFκB (IκB) kinase β; an upstream kinase responsible for nuclear factor κB (NFκB) activation] or overexpression of TAM67 (a dominant-negative mutant of c-Jun) dramatically inhibited the COX-2 induction due to Cr(VI), suggesting that both NFκB and c-Jun/AP-1 pathways were required for Cr(VI)-induced COX-2 expression. Our results show that p65 and c-Jun are two major components involved in NFκB and AP-1 activation, respectively. Moreover, our studies suggest crosstalk between NFκB and c-Jun/AP-1 pathways in cellular response to Cr(VI) exposure for COX-2 induction. Conclusion: We demonstrate for the first time that Cr(VI) is able to induce COX-2 expression via an NFκB/c-Jun/AP-1–dependent pathway. Our results provide novel insight into the molecular mechanisms linking Cr(VI) exposure to lung inflammation and carcinogenesis. PMID:22472290

Biological Effects of the Pim Kinase Inhibitor, SGI-1776, in Multiple Myeloma

PubMed Central

Cervantes-Gomez, Fabiola; Chen, Lisa S.; Orlowski, Robert Z.; Gandhi, Varsha

2013-01-01

Pim kinases are constitutively active serine/threonine/tyrosine kinases that are overexpressed in hematological malignancies such as multiple myeloma. Pim kinase substrates are involved in transcription, protein translation, cell proliferation, and apoptosis. SGI-1776 is a potent Pim kinase inhibitor that has proven to be cytotoxic to leukemia and lymphoma cells. Based on this background, we hypothesized that SGI-1776 treatment would result in myeloma cytotoxicity. To test this, myeloma cell lines and primary CD138+ cells from myeloma patients were treated with SGI-1776 in a dose- and time-dependent manner and effect on cell death and proliferation, induction of autophagy, as wells as changes in cell cycle profile were measured. SGI-1776 treatment resulted in limited apoptosis in cell lines (mean 30%) and CD138+ cells (<10%) as assessed by Annexin-V/PI. Limited effect was observed in cell cycle profile or growth in cell lines. However, DNA synthesis was decreased by 70% at 3 μM (all time points) in U266 though this was not observed in MM.1S. In accordance, immunoblot analyses revealed no change in transcription (c-Myc and H3), or apoptotic (Bad) proteins that are substrates of Pim kinases. In contrast, autophagy, as assessed by acridine orange staining, was induced with SGI-1776 treatment in both cell lines (U266 25-70%; MM.1S 8-52%) and CD138+ cells (19-21%). Immunoblot analyses of autophagy LC3b marker and translation initiation proteins (phospho p70S6K and 4E-BP1) corroborated autophagy induction. These data indicate that SGI-1776 treatment in myeloma cell lines and CD138+ myeloma cells elicits its deleterious effects through inhibition of translation and induction of autophagy. PMID:23988451

Biological effects of the Pim kinase inhibitor, SGI-1776, in multiple myeloma.

PubMed

Cervantes-Gomez, Fabiola; Chen, Lisa S; Orlowski, Robert Z; Gandhi, Varsha

2013-09-01

Pim kinases are constitutively active serine/threonine/tyrosine kinases that are overexpressed in hematological malignancies such as multiple myeloma. Pim kinase substrates are involved in transcription, protein translation, cell proliferation, and apoptosis. SGI-1776 is a potent Pim kinase inhibitor that has proven to be cytotoxic to leukemia and lymphoma cells. Based on this background, we hypothesized that SGI-1776 treatment would result in myeloma cytotoxicity. To test this, myeloma cell lines and primary CD138(+) cells from myeloma patients were treated with SGI-1776 in a dose- and time-dependent manner, and effect on cell death and proliferation, induction of autophagy, and changes in cell cycle profile were measured. SGI-1776 treatment resulted in limited apoptosis in cell lines (mean 30%) and CD138(+) cells (< 10%) assessed using Annexin-V/propidium iodide. Limited effect was observed in cell cycle profile or growth in cell lines. However, DNA synthesis was decreased by 70% at 3 μM (all time points) in U266 though this was not observed in MM.1S. In accordance, immunoblot analyses revealed no change in transcription (c-Myc and H3), or apoptotic (Bad) proteins that are substrates of Pim kinases. In contrast, autophagy, assessed using acridine orange staining, was induced with SGI-1776 treatment in both cell lines (U266, 25%-70%; MM.1S, 8%-52%) and CD138(+) cells (19%-21%). Immunoblot analyses of the autophagy LC3b marker and translation initiation proteins (phospho-p70S6K and 4E-BP1) corroborated autophagy induction. These data indicate that SGI-1776 treatment in myeloma cell lines and CD138(+) myeloma cells elicits its deleterious effects through inhibition of translation and induction of autophagy. Copyright © 2013 Elsevier Inc. All rights reserved.

ATG3-dependent autophagy mediates mitochondrial homeostasis in pluripotency acquirement and maintenance

PubMed Central

Liu, Kun; Zhao, Qian; Liu, Pinglei; Cao, Jiani; Gong, Jiaqi; Wang, Chaoqun; Wang, Weixu; Li, Xiaoyan; Sun, Hongyan; Zhang, Chao; Li, Yufei; Jiang, Minggui; Zhu, Shaohua; Sun, Qingyuan; Jiao, Jianwei; Hu, Baoyang; Zhao, Xiaoyang; Li, Wei; Chen, Quan; Zhou, Qi; Zhao, Tongbiao

2016-01-01

ABSTRACT Pluripotent stem cells, including induced pluripotent and embryonic stem cells (ESCs), have less developed mitochondria than somatic cells and, therefore, rely more heavily on glycolysis for energy production.1-3 However, how mitochondrial homeostasis matches the demands of nuclear reprogramming and regulates pluripotency in ESCs is largely unknown. Here, we identified ATG3-dependent autophagy as an executor for both mitochondrial remodeling during somatic cell reprogramming and mitochondrial homeostasis regulation in ESCs. Dysfunctional autophagy by Atg3 deletion inhibited mitochondrial removal during pluripotency induction, resulting in decreased reprogramming efficiency and accumulation of abnormal mitochondria in established iPSCs. In Atg3 null mouse ESCs, accumulation of aberrant mitochondria was accompanied by enhanced ROS generation, defective ATP production and attenuated pluripotency gene expression, leading to abnormal self-renewal and differentiation. These defects were rescued by reacquisition of wild-type but not lipidation-deficient Atg3 expression. Taken together, our findings highlight a critical role of ATG3-dependent autophagy for mitochondrial homeostasis regulation in both pluripotency acquirement and maintenance. PMID:27575019

ATG3-dependent autophagy mediates mitochondrial homeostasis in pluripotency acquirement and maintenance.

PubMed

Liu, Kun; Zhao, Qian; Liu, Pinglei; Cao, Jiani; Gong, Jiaqi; Wang, Chaoqun; Wang, Weixu; Li, Xiaoyan; Sun, Hongyan; Zhang, Chao; Li, Yufei; Jiang, Minggui; Zhu, Shaohua; Sun, Qingyuan; Jiao, Jianwei; Hu, Baoyang; Zhao, Xiaoyang; Li, Wei; Chen, Quan; Zhou, Qi; Zhao, Tongbiao

2016-11-01

Pluripotent stem cells, including induced pluripotent and embryonic stem cells (ESCs), have less developed mitochondria than somatic cells and, therefore, rely more heavily on glycolysis for energy production. 1-3 However, how mitochondrial homeostasis matches the demands of nuclear reprogramming and regulates pluripotency in ESCs is largely unknown. Here, we identified ATG3-dependent autophagy as an executor for both mitochondrial remodeling during somatic cell reprogramming and mitochondrial homeostasis regulation in ESCs. Dysfunctional autophagy by Atg3 deletion inhibited mitochondrial removal during pluripotency induction, resulting in decreased reprogramming efficiency and accumulation of abnormal mitochondria in established iPSCs. In Atg3 null mouse ESCs, accumulation of aberrant mitochondria was accompanied by enhanced ROS generation, defective ATP production and attenuated pluripotency gene expression, leading to abnormal self-renewal and differentiation. These defects were rescued by reacquisition of wild-type but not lipidation-deficient Atg3 expression. Taken together, our findings highlight a critical role of ATG3-dependent autophagy for mitochondrial homeostasis regulation in both pluripotency acquirement and maintenance.

Direct repeat sequences in the Streptomyces chitinase-63 promoter direct both glucose repression and chitin induction

PubMed Central

Ni, Xiangyang; Westpheling, Janet

1997-01-01

The chi63 promoter directs glucose-sensitive, chitin-dependent transcription of a gene involved in the utilization of chitin as carbon source. Analysis of 5′ and 3′ deletions of the promoter region revealed that a 350-bp segment is sufficient for wild-type levels of expression and regulation. The analysis of single base changes throughout the promoter region, introduced by random and site-directed mutagenesis, identified several sequences to be important for activity and regulation. Single base changes at −10, −12, −32, −33, −35, and −37 upstream of the transcription start site resulted in loss of activity from the promoter, suggesting that bases in these positions are important for RNA polymerase interaction. The sequences centered around −10 (TATTCT) and −35 (TTGACC) in this promoter are, in fact, prototypical of eubacterial promoters. Overlapping the RNA polymerase binding site is a perfect 12-bp direct repeat sequence. Some base changes within this direct repeat resulted in constitutive expression, suggesting that this sequence is an operator for negative regulation. Other base changes resulted in loss of glucose repression while retaining the requirement for chitin induction, suggesting that this sequence is also involved in glucose repression. The fact that cis-acting mutations resulted in glucose resistance but not inducer independence rules out the possibility that glucose repression acts exclusively by inducer exclusion. The fact that mutations that affect glucose repression and chitin induction fall within the same direct repeat sequence module suggests that the direct repeat sequence facilitates both chitin induction and glucose repression. PMID:9371809

Quetiapine and aripiprazole signal differently to ERK, p90RSK and c-Fos in mouse frontal cortex and striatum: role of the EGF receptor

PubMed Central

2014-01-01

Background Signaling pathways outside dopamine D2 receptor antagonism may govern the variable clinical profile of antipsychotic drugs (APD) in schizophrenia. One postulated mechanism causal to APD action may regulate synaptic plasticity and neuronal connectivity via the extracellular signal-regulated kinase (ERK) cascade that links G-protein coupled receptors (GPCR) and ErbB growth factor signaling, systems disturbed in schizophrenia. This was based upon our finding that the low D2 receptor affinity APD clozapine induced initial down-regulation and delayed epidermal growth factor receptor (EGFR or ErbB1) mediated activation of the cortical and striatal ERK response in vivo distinct from olanzapine or haloperidol. Here we map whether the second generation atypical APDs aripiprazole and quetiapine affect the EGFR-ERK pathway and its substrates p90RSK and c-Fos in mouse brain, given their divergent agonist and antagonist properties on dopaminergic transmission, respectively. Results In prefrontal cortex, aripiprazole triggered triphasic ERK phosphorylation that was EGFR-independent but had no significant effect in striatum. Conversely quetiapine did not alter cortical ERK signaling but elevated striatal ERK levels in an EGFR-dependent manner. Induction of ERK by aripiprazole did not affect p90RSK signaling but quetiapine decreased RSK phosphorylation within 1-hour of administration. The transcription factor c-Fos by comparison was a direct target of ERK phosphorylation induced by aripiprazole in cortex and quetiapine in striatum with protein levels in temporal alignment with that of ERK. Conclusions These data indicate that aripiprazole and quetiapine signal to specific nuclear targets of ERK, which for quetiapine occurs via an EGFR-linked mechanism, possibly indicating involvement of this system in its action. PMID:24552586

Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis.

PubMed

Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K

2014-01-01

Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic diseases associated with increased energy combustion in liver.

From the Cover: Adipose tissue mass can be regulated through the vasculature

NASA Astrophysics Data System (ADS)

Rupnick, Maria A.; Panigrahy, Dipak; Zhang, Chen-Yu; Dallabrida, Susan M.; Lowell, Bradford B.; Langer, Robert; Judah Folkman, M.

2002-08-01

Tumor growth is angiogenesis dependent. We hypothesized that nonneoplastic tissue growth also depends on neovascularization. We chose adipose tissue as an experimental system because of its remodeling capacity. Mice from different obesity models received anti-angiogenic agents. Treatment resulted in dose-dependent, reversible weight reduction and adipose tissue loss. Marked vascular remodeling was evident in adipose tissue sections, which revealed decreased endothelial proliferation and increased apoptosis in treated mice compared with controls. Continuous treatment maintained mice near normal body weights for age without adverse effects. Metabolic adaptations in food intake, metabolic rate, and energy substrate utilization were associated with anti-angiogenic weight loss. We conclude that adipose tissue mass is sensitive to angiogenesis inhibitors and can be regulated by its vasculature.

Myosin IIb-dependent Regulation of Actin Dynamics Is Required for N-Methyl-D-aspartate Receptor Trafficking during Synaptic Plasticity.

PubMed

Bu, Yunfei; Wang, Ning; Wang, Shaoli; Sheng, Tao; Tian, Tian; Chen, Linlin; Pan, Weiwei; Zhu, Minsheng; Luo, Jianhong; Lu, Wei

2015-10-16

N-Methyl-d-aspartate receptor (NMDAR) synaptic incorporation changes the number of NMDARs at synapses and is thus critical to various NMDAR-dependent brain functions. To date, the molecules involved in NMDAR trafficking and the underlying mechanisms are poorly understood. Here, we report that myosin IIb is an essential molecule in NMDAR synaptic incorporation during PKC- or θ burst stimulation-induced synaptic plasticity. Moreover, we demonstrate that myosin light chain kinase (MLCK)-dependent actin reorganization contributes to NMDAR trafficking. The findings from additional mutual occlusion experiments demonstrate that PKC and MLCK share a common signaling pathway in NMDAR-mediated synaptic regulation. Because myosin IIb is the primary substrate of MLCK and can regulate actin dynamics during synaptic plasticity, we propose that the MLCK- and myosin IIb-dependent regulation of actin dynamics is required for NMDAR trafficking during synaptic plasticity. This study provides important insights into a mechanical framework for understanding NMDAR trafficking associated with synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cofactor-dependent specificity of a DEAD-box protein.

PubMed

Young, Crystal L; Khoshnevis, Sohail; Karbstein, Katrin

2013-07-16

DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.

Active Site Metal Identity Alters Histone Deacetylase 8 Substrate Selectivity: A Potential Novel Regulatory Mechanism.

PubMed

Castaneda, Carol Ann; Lopez, Jeffrey E; Joseph, Caleb G; Scholle, Michael D; Mrksich, Milan; Fierke, Carol A

2017-10-24

Histone deacetylase 8 (HDAC8) is a well-characterized member of the class I acetyl-lysine deacetylase (HDAC) family. Previous work has shown that the efficiency of HDAC8-catalyzed deacetylation of a methylcoumarin peptide varies depending on the identity of the divalent metal ion in the HDAC8 active site. Here we demonstrate that both HDAC8 activity and substrate selectivity for a diverse range of peptide substrates depend on the identity of the active site metal ion. Varied deacetylase activities of Fe(II)- and Zn(II)-HDAC8 toward an array of peptide substrates were identified using self-assembled monolayers for matrix-assisted laser desorption ionization (SAMDI) mass spectrometry. Subsequently, the metal dependence of deacetylation of peptides of biological interest was measured using an in vitro peptide assay. While Fe(II)-HDAC8 is generally more active than Zn(II)-HDAC8, the Fe(II)/Zn(II) HDAC8 activity ratio varies widely (from 2 to 150) among the peptides tested. These data provide support for the hypothesis that HDAC8 may undergo metal switching in vivo that, in turn, may regulate its activity. However, future studies are needed to explore the identity of the metal ion bound to HDAC8 in cells under varied conditions.

Korean red ginseng extract induces apoptosis and decreases telomerase activity in human leukemia cells.

PubMed

Park, Sang Eun; Park, Cheol; Kim, Sun Hee; Hossain, Mohammad Akbar; Kim, Min Young; Chung, Hae Young; Son, Woo Sung; Kim, Gi-Young; Choi, Yung Hyun; Kim, Nam Deuk

2009-01-21

Korean red ginseng (KRG, Panax ginseng C.A. Meyer Radix rubra) has been used to treat various diseases including cancer. However, the molecular mechanisms responsible for KRG extract induced apoptosis and telomerase inhibition remain unclear. The hot water extract from KRG was used to evaluate the mechanism of induction of apoptosis in U937 human leukemia cells and its effects on cyclooxgenase-2 (COX-2) and telomerase activity. KRG extract treatment to U937 cells resulted in growth inhibition and induction of apoptosis in a concentration-dependent manner as measured by hemacytometer counts, MTT assay, fluorescence microscopy, agarose gel electrophoresis and flow cytometry analysis. The increase in apoptosis was associated with the down-regulation of antiapoptotic Bcl-2, Bcl-X(L), and IAPs family members, and the activation of caspase-3. KRG extract treatment also decreased the expression levels of COX-2 and inducible nitric oxide synthase. Furthermore, KRG extract treatment progressively down-regulated the expression of human telomerase reverse transcriptase, a main determinant of the telomerase enzymatic activity, with inhibiting the expression of c-Myc in a concentration-dependent manner. These results provide important new insights into the possible molecular mechanisms of the anticancer activity of KRG extract.

Metabolic gene regulation in a dynamically changing environment.

PubMed

Bennett, Matthew R; Pang, Wyming Lee; Ostroff, Natalie A; Baumgartner, Bridget L; Nayak, Sujata; Tsimring, Lev S; Hasty, Jeff

2008-08-28

Natural selection dictates that cells constantly adapt to dynamically changing environments in a context-dependent manner. Gene-regulatory networks often mediate the cellular response to perturbation, and an understanding of cellular adaptation will require experimental approaches aimed at subjecting cells to a dynamic environment that mimics their natural habitat. Here we monitor the response of Saccharomyces cerevisiae metabolic gene regulation to periodic changes in the external carbon source by using a microfluidic platform that allows precise, dynamic control over environmental conditions. We show that the metabolic system acts as a low-pass filter that reliably responds to a slowly changing environment, while effectively ignoring fast fluctuations. The sensitive low-frequency response was significantly faster than in predictions arising from our computational modelling, and this discrepancy was resolved by the discovery that two key galactose transcripts possess half-lives that depend on the carbon source. Finally, to explore how induction characteristics affect frequency response, we compare two S. cerevisiae strains and show that they have the same frequency response despite having markedly different induction properties. This suggests that although certain characteristics of the complex networks may differ when probed in a static environment, the system has been optimized for a robust response to a dynamically changing environment.

Phytochemical regulation of the tumor suppressive microRNA, miR-34a, by p53-dependent and independent responses in human breast cancer cells

PubMed Central

Hargraves, Kris G.; He, Lin; Firestone, Gary L.

2016-01-01

The tumor suppressive microRNA miR-34a is transcriptionally regulated by p53 and shown to inhibit breast cancer cell proliferation as well as being a marker of increased disease free survival. Indole-3-carbinol (I3C) derived from cruciferous vegetables, artemisinin, extracted from the sweet wormwood plant, and artesunate, a semi-synthetic derivative of artemisinin, are phytochemicals with anti-tumorigenic properties however, little is known about the role of microRNAs in their mechanism of action. Human breast cancer cells expressing wild-type (MCF-7) or mutant p53 (T47D) were treated with a concentration range and time course of each phytochemical under conditions of cell cycle arrest as detected by flow cytometry to examine the potential connection between miR-34a expression and their anti-proliferative responses. Real-time PCR and western blot analysis of extracted RNA and total protein revealed artemsinin and artesunate increased miR-34a expression in a dose-dependent manner correlating with down-regulation of the miR-34a target gene, CDK4. I3C stimulation of miR-34a expression required functional p53, whereas, both artemisinin and artesunate up-regulated miR-34a expression regardless of p53 mutational status or in the presence of dominant negative p53. Phytochemical treatments inhibited the luciferase activity of a construct containing the wild-type 3′UTR of CDK4, but not those with a mutated miR-34a binding site, whereas, transfection of miR-34a inhibitors ablated the phytochemical mediated down-regulation of CDK4 and induction of cell cycle arrest. Our results suggest that miR-34a is an essential component of the anti-proliferative activities of I3C, artemisinin and artesunate and demonstrate that both wild-type p53 dependent and independent pathways are responsible for miR-34a induction. PMID:25789847

Modelling the CDK-dependent transcription cycle in fission yeast.

PubMed

Sansó, Miriam; Fisher, Robert P

2013-12-01

CDKs (cyclin-dependent kinases) ensure directionality and fidelity of the eukaryotic cell division cycle. In a similar fashion, the transcription cycle is governed by a conserved subfamily of CDKs that phosphorylate Pol II (RNA polymerase II) and other substrates. A genetic model organism, the fission yeast Schizosaccharomyces pombe, has yielded robust models of cell-cycle control, applicable to higher eukaryotes. From a similar approach combining classical and chemical genetics, fundamental principles of transcriptional regulation by CDKs are now emerging. In the present paper, we review the current knowledge of each transcriptional CDK with respect to its substrate specificity, function in transcription and effects on chromatin modifications, highlighting the important roles of CDKs in ensuring quantity and quality control over gene expression in eukaryotes.

Cell-fate determination by ubiquitin-dependent regulation of translation.

PubMed

Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen A; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T; Rape, Michael

2015-09-24

Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3(KBTBD8)) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3(KBTBD8) monoubiquitylates NOLC1 and its paralogue TCOF1, the mutation of which underlies the neurocristopathy Treacher Collins syndrome. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination.

Distinct signalling properties of insulin receptor substrate (IRS)-1 and IRS-2 in mediating insulin/IGF-1 action.

PubMed

Rabiee, Atefeh; Krüger, Marcus; Ardenkjær-Larsen, Jacob; Kahn, C Ronald; Emanuelli, Brice

2018-07-01

Insulin/IGF-1 action is driven by a complex and highly integrated signalling network. Loss-of-function studies indicate that the major insulin/IGF-1 receptor substrate (IRS) proteins, IRS-1 and IRS-2, mediate different biological functions in vitro and in vivo, suggesting specific signalling properties despite their high degree of homology. To identify mechanisms contributing to the differential signalling properties of IRS-1 and IRS-2 in the mediation of insulin/IGF-1 action, we performed comprehensive mass spectrometry (MS)-based phosphoproteomic profiling of brown preadipocytes from wild type, IRS-1 -/- and IRS-2 -/- mice in the basal and IGF-1-stimulated states. We applied stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of changes in protein phosphorylation. We found ~10% of the 6262 unique phosphorylation sites detected to be regulated by IGF-1. These regulated sites included previously reported substrates of the insulin/IGF-1 signalling pathway, as well as novel substrates including Nuclear Factor I X and Semaphorin-4B. In silico prediction suggests the protein kinase B (PKB), protein kinase C (PKC), and cyclin-dependent kinase (CDK) as the main mediators of these phosphorylation events. Importantly, we found preferential phosphorylation patterns depending on the presence of either IRS-1 or IRS-2, which was associated with specific sets of kinases involved in signal transduction downstream of these substrates such as PDHK1, MAPK3, and PKD1 for IRS-1, and PIN1 and PKC beta for IRS-2. Overall, by generating a comprehensive phosphoproteomic profile from brown preadipocyte cells in response to IGF-1 stimulation, we reveal both common and distinct insulin/IGF-1 signalling events mediated by specific IRS proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise.

PubMed

Chiba, K; Alderton, J M; Hoshi, M; Steinhardt, R A

1999-05-01

The mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low. V0 measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited by clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome. There has been in vitro evidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700-proteasome complex. Since both intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]i and pHi were varied independently during activation. When the pHi of unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater, V0 increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]i elevation without pHi change, induced by calcium ionophore in sodium-free seawater, had no effect on V0 in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi, V0 increased in a pH-dependent manner. These results indicate that the pHi rise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle. Copyright 1999 Academic Press.

Substrate specificity of the ubiquitin and Ubl proteases

PubMed Central

Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

2016-01-01

Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize[W

PubMed Central

Juranić, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanić, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

2012-01-01

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle–dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s). PMID:23250449

A simple biosynthetic pathway for large product generation from small substrate amounts

NASA Astrophysics Data System (ADS)

Djordjevic, Marko; Djordjevic, Magdalena

2012-10-01

A recently emerging discipline of synthetic biology has the aim of constructing new biosynthetic pathways with useful biological functions. A major application of these pathways is generating a large amount of the desired product. However, toxicity due to the possible presence of toxic precursors is one of the main problems for such production. We consider here the problem of generating a large amount of product from a potentially toxic substrate. To address this, we propose a simple biosynthetic pathway, which can be induced in order to produce a large number of the product molecules, by keeping the substrate amount at low levels. Surprisingly, we show that the large product generation crucially depends on fast non-specific degradation of the substrate molecules. We derive an optimal induction strategy, which allows as much as three orders of magnitude increase in the product amount through biologically realistic parameter values. We point to a recently discovered bacterial immune system (CRISPR/Cas in E. coli) as a putative example of the pathway analysed here. We also argue that the scheme proposed here can be used not only as a stand-alone pathway, but also as a strategy to produce a large amount of the desired molecules with small perturbations of endogenous biosynthetic pathways.

Identification of a Dehydrogenase Required for Lactose Metabolism in Caulobacter crescentus▿ †‡

PubMed Central

Arellano, Benjamin H.; Ortiz, Janett D.; Manzano, Janet; Chen, Joseph C.

2010-01-01

Caulobacter crescentus, which thrives in freshwater environments with low nutrient levels, serves as a model system for studying bacterial cell cycle regulation and organelle development. We examined its ability to utilize lactose (i) to gain insight into the metabolic capacities of oligotrophic bacteria and (ii) to obtain an additional genetic tool for studying this model organism, aiming to eliminate the basal enzymatic activity that hydrolyzes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). Using a previously isolated transposon mutant, we identified a gene, lacA, that is required for growth on lactose as the sole carbon source and for turning colonies blue in the presence of X-gal. LacA, which contains a glucose-methanol-choline (GMC) oxidoreductase domain, has homology to the flavin subunit of Pectobacterium cypripedii's gluconate dehydrogenase. Sequence comparisons indicated that two genes near lacA, lacB and lacC, encode the other subunits of the membrane-bound dehydrogenase. In addition to lactose, all three lac genes are involved in the catabolism of three other β-galactosides (lactulose, lactitol, and methyl-β-d-galactoside) and two glucosides (salicin and trehalose). Dehydrogenase assays confirmed that the lac gene products oxidize lactose, salicin, and trehalose. This enzymatic activity is inducible, and increased lac expression in the presence of lactose and salicin likely contributes to the induction. Expression of lacA also depends on the presence of the lac genes, implying that the dehydrogenase participates in induction. The involvement of a dehydrogenase suggests that degradation of lactose and other sugars in C. crescentus may resemble a proposed pathway in Agrobacterium tumefaciens. PMID:20190087

Dipeptidyl peptidase IV (DPPIV) enzyme activity on immature T-cell line R1.1 is down-regulated by dynorphin-A(1-17) as a non-substrate inhibitor.

PubMed

Gabrilovac, Jelka; Abramić, Marija; Uzarević, Branka; Andreis, Ana; Poljak, Ljiljana

2003-05-30

In this study we examined surface expression of CD26 and the corresponding enzyme activity of dipeptidyl peptidase IV (DPPIV) on the cells of immature murine T-cell line, R1.1. The data obtained have shown that R1.1 cells express high density of surface CD26 as compared to normal thymus cells. This was associated with strong enzyme activity, which, based on substrates and inhibitor specificity, corresponded to DPPIV. The DPPIV enzyme activity of R1.1 cells was 10 times stronger than that found on normal murine thymus cells (V(max) = 39 micromol/min/10(6) cells, vs 3.7 micromol/min/10(6) cells, respectively). Upon activation with anti-CD3, up-regulation of both membrane CD26, as well as of DPPIV enzyme activity on R1.1 cells were observed. The finding of strong DPPIV on R1.1 cells makes them suitable model for testing putative substrates/inhibitors of the enzyme in its natural microenvironment. Since in addition to strong DPPIV, R1.1 cells also express kappa opioid receptors (KOR) [European Journal of Pharmacology 227 (1992) 257], we tested the effect of dynorphin-A(1-17), an endogenous opioid peptide with KOR selectivity, on DPPIV of R1.1 cells. Dynorphin-A(1-17) down-regulated DPPIV in a dose-dependent manner, with the potency similar to that of substance P, a known natural DPPIV substrate [Journal of Pharmacology and Experimental Therapeutics 260 (1992) 1257]. DPPIV down-regulation was resistant to bestatin and thiorphan, the inhibitors of two cell surface peptidases (APN and NEP, respectively) with potential of dynorphin-A(1-17) degradation, suggesting that the mechanism underlying the observed effect does not involve degradative products of dynorphin-A(1-17). DPPIV down-regulation was also resistent to KOR antagonist, NBI, suggesting that the mechanism underlying the observed phenomenon involves neither cointernalization of KOR and DPPIV. Collectively, cells of immature T cell line, R1.1 exert strong DPPIV enzyme activity, which could be down-regulated in the presence of dynorphin-A(1-17) by mechanism that presumably includes non-substrate inhibition. By down-regulating DPPIV, dynorphin-A(1-17) may indirectly affect activity and/or specificity of natural substrates of DPPIV, such as substance P, RANTES, and endomorphins.

CDODA-Me decreases specificity protein transcription factors and induces apoptosis in bladder cancer cells through induction of reactive oxygen species.

PubMed

Takeuchi, Hisashi; Taoka, Rikiya; Mmeje, Chinedu O; Jinesh, Goodwin G; Safe, Stephen; Kamat, Ashish M

2016-08-01

The objective is to determine whether methyl 2-cyano-3,11-dioxo-18b-olean-1,12-dien-30-oate (CDODA-Me) has therapeutic potential in bladder cancer. We investigated the effects of CDODA-Me on the growth and survival of bladder cancer cells, and expression of specificity protein (Sp) transcription factors that regulate genes associated with cancer cell proliferation and survival. J82, RT4P, and 253JB-V bladder cancer cell lines were treated with vehicle alone or with CDODA-Me with or without the antioxidant l-glutathione. Cell viability and DNA fragmentation were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide-fluorescence-activated cell sorting (FACS) analysis, respectively. Intracellular reactive oxygen species (ROS) were measured by 2',7'-dichlorofluorescin diacetate-FACS analysis. We assessed CDODA's effects on the levels of Sp and Sp-regulated proteins and induction of apoptosis in bladder cancer cells by Western blotting. We also assessed the anticancer effects of CDODA-Me in nude mice bearing RT4v6 bladder cancer. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and FACS analysis revealed that CDODA-Me inhibited the proliferation and survival of the 3 bladder cancer cell lines in a dose-dependent manner. FACS analysis also indicated that CDODA-Me-induced intracellular ROS, and Western blot analysis indicated that CDODA-Me decreased levels of Sp and Sp-regulated proteins and induced apoptosis in a dose-dependent and time-dependent manner. l-Glutathione attenuated CDODA-Me's down-regulation of Sp and Sp-regulated proteins. Compared with the control treatment, CDODA-Me substantially inhibited tumor growth in vivo. CDODA-Me has antineoplastic activity in bladder cancer cells by inducing ROS, which down-regulate Sp and Sp-regulated proteins. Thus, CDODA-Me has therapeutic potential in bladder cancer, and additional studies of the agent's efficacy and mode of action are warranted. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of She3 as an SCFGrr1 Substrate in Budding Yeast

PubMed Central

Wang, Ruiwen; Solomon, Mark J.

2012-01-01

The highly orchestrated progression of the cell cycle depends on the degradation of many regulatory proteins at different cell cycle stages. One of the key cell cycle ubiquitin ligases is the Skp1-cullin-F-box (SCF) complex. Acting in concert with the substrate-binding F-box protein Grr1, SCFGrr1 promotes the degradation of cell cycle regulators as well as various metabolic enzymes. Using a yeast two-hybrid assay with a Grr1 derivative as the bait, we identified She3, which is an adaptor protein in the asymmetric mRNA transport system, as a novel Grr1 substrate. We generated stabilized She3 mutants, which no longer bound to Grr1, and found that the degradation of She3 is not required for regulating asymmetric mRNA transport. However, She3 stabilization leads to slower growth compared to wild-type cells in a co-culture assay, demonstrating that the degradation of She3 by Grr1 is required for optimal cell growth. PMID:23144720

Synaptic Synthesis, Dephosphorylation, and Degradation

PubMed Central

La Montanara, Paolo; Rusconi, Laura; Locarno, Albina; Forti, Lia; Barbiero, Isabella; Tramarin, Marco; Chandola, Chetan; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

2015-01-01

Mutations in the X-linked CDKL5 (cyclin-dependent kinase-like 5) gene have been associated with several forms of neurodevelopmental disorders, including atypical Rett syndrome, autism spectrum disorders, and early infantile epileptic encephalopathy. Accordingly, loss of CDKL5 in mice results in autistic-like features and impaired neuronal communication. Although the biological functions of CDKL5 remain largely unknown, recent pieces of evidence suggest that CDKL5 is involved in neuronal plasticity. Herein, we show that, at all stages of development, neuronal depolarization induces a rapid increase in CDKL5 levels, mostly mediated by extrasomatic synthesis. In young neurons, this induction is prolonged, whereas in more mature neurons, NMDA receptor stimulation induces a protein phosphatase 1-dependent dephosphorylation of CDKL5 that is mandatory for its proteasome-dependent degradation. As a corollary, neuronal activity leads to a prolonged induction of CDKL5 levels in immature neurons but to a short lasting increase of the kinase in mature neurons. Recent results demonstrate that many genes associated with autism spectrum disorders are crucial components of the activity-dependent signaling networks regulating the composition, shape, and strength of the synapse. Thus, we speculate that CDKL5 deficiency disrupts activity-dependent signaling and the consequent synapse development, maturation, and refinement. PMID:25555910

Induction of calcium-dependent nitric oxide synthases by sex hormones.

PubMed

Weiner, C P; Lizasoain, I; Baylis, S A; Knowles, R G; Charles, I G; Moncada, S

1994-05-24

We have examined the effects of pregnancy and sex hormones on calcium-dependent and calcium-independent nitric oxide synthases (NOSs) in the guinea pig. Pregnancy (near term) caused a > 4-fold increase in the activity of calcium-dependent NOS in the uterine artery and at least a doubling in the heart, kidney, skeletal muscle, esophagus, and cerebellum. The increase in NOS activity in the cerebellum during pregnancy was inhibited by the estrogen-receptor antagonist tamoxifen. Treatment with estradiol (but not progesterone) also increased calcium-dependent NOS activity in the tissues examined from both females and males. Testosterone increased calcium-dependent NOS only in the cerebellum. No significant change in calcium-independent NOS activity was observed either during pregnancy or after the administration of any sex hormone. Both pregnancy and estradiol treatment increased the amount of mRNAs for NOS isozymes eNOS and nNOS in skeletal muscle, suggesting that the increases in NOS activity result from enzyme induction. Thus both eNOS and nNOS are subject to regulation by estrogen, an action that could explain some of the changes that occur during pregnancy and some gender differences in physiology and pathophysiology.

Cancer dormancy and cell signaling: Induction of p21waf1 initiated by membrane IgM engagement increases survival of B lymphoma cells

PubMed Central

Marches, Radu; Hsueh, Robert; Uhr, Jonathan W.

1999-01-01

The p21WAF1 (p21) cyclin-dependent kinase inhibitor plays a major role in regulating cell cycle arrest. It was recently reported that the p53-independent elevation of p21 protein levels is essential in mediating the G1 arrest resulting from signal transduction events initiated by the crosslinking of membrane IgM on Daudi Burkitt lymphoma cells. Although the role of p21 in cell cycle regulation is well documented, there is little information concerning its role in antibody-mediated apoptosis. In the present study, we examined the involvement of p21 in the regulation of apoptosis by suppressing its induction in anti-IgM-treated Daudi cells through a p21 antisense expression construct approach. Reduction in induced p21 protein levels resulted in diminished G1 arrest and increased apoptosis. The increased susceptibility to anti-IgM-mediated apoptosis was associated with increased caspase-3-like activity and poly-(ADP)ribose polymerase cleavage. These data suggest that p21 may directly interfere with the caspase cascade, thus playing a dual role in regulating both cell cycle progression and apoptosis. PMID:10411940

GPATCH3 negatively regulates RLR-mediated innate antiviral responses by disrupting the assembly of VISA signalosome.

PubMed

Nie, Ying; Ran, Yong; Zhang, Hong-Yan; Huang, Zhe-Fu; Pan, Zhao-Yi; Wang, Su-Yun; Wang, Yan-Yi

2017-04-01

Upon viral infection, retinoic acid-inducible gene I-like receptors (RLRs) recognize viral RNA and trigger a series of signaling events, leading to the induction of type I interferons (IFNs). These processes are delicately regulated to prevent excessive and harmful immune responses. In this study, we identified G patch domain-containing protein 3 (GPATCH3) as a negative regulator of RLR-mediated antiviral signaling pathways. Overexpression of GPATCH3 impaired RNA virus- triggered induction of downstream antiviral genes, whereas its knockdown had opposite effects and attenuated viral replication. In addition, GPATCH3-deficient cells had higher IFNB1 mRNA level compared with control cells after RNA virus infection. Mechanistically, GPATCH3 was recruited to VISA in a viral infection dependent manner and the assembly of VISA/TRAF6/TBK1 signalosome was impaired in GPATCH3-overexpressing cells. In contrast, upon viral infection, the recruitment of TRAF6 and TBK1 to VISA was enhanced in GPATCH3 deficient cells. Taking together, our findings demonstrate that GPATCH3 interacts with VISA and disrupts the assembly of virus-induced VISA signalosome therefore acts as a negative regulator of RLR-mediated innate antiviral immune responses.

GPATCH3 negatively regulates RLR-mediated innate antiviral responses by disrupting the assembly of VISA signalosome

PubMed Central

Nie, Ying; Ran, Yong; Zhang, Hong-Yan; Huang, Zhe-Fu; Wang, Su-Yun

2017-01-01

Upon viral infection, retinoic acid–inducible gene I–like receptors (RLRs) recognize viral RNA and trigger a series of signaling events, leading to the induction of type I interferons (IFNs). These processes are delicately regulated to prevent excessive and harmful immune responses. In this study, we identified G patch domain-containing protein 3 (GPATCH3) as a negative regulator of RLR-mediated antiviral signaling pathways. Overexpression of GPATCH3 impaired RNA virus- triggered induction of downstream antiviral genes, whereas its knockdown had opposite effects and attenuated viral replication. In addition, GPATCH3-deficient cells had higher IFNB1 mRNA level compared with control cells after RNA virus infection. Mechanistically, GPATCH3 was recruited to VISA in a viral infection dependent manner and the assembly of VISA/TRAF6/TBK1 signalosome was impaired in GPATCH3-overexpressing cells. In contrast, upon viral infection, the recruitment of TRAF6 and TBK1 to VISA was enhanced in GPATCH3 deficient cells. Taking together, our findings demonstrate that GPATCH3 interacts with VISA and disrupts the assembly of virus-induced VISA signalosome therefore acts as a negative regulator of RLR-mediated innate antiviral immune responses. PMID:28414768

A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit

PubMed Central

Cundell, Michael J.; Holder, James

2016-01-01

PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit. These substrates share a bipartite polybasic recognition determinant (BPR) flanking a Cdk1 phosphorylation site. Experiments and modeling show that dephosphorylation rate is encoded into B55 substrates, including its inhibitor ENSA, by cooperative action of basic residues within the BPR. A complementary acidic surface on B55 decodes this signal, supporting a cooperative electrostatic mechanism for substrate selection. A further level of specificity is encoded into B55 substrates because B55 displays selectivity for phosphothreonine. These simple biochemical properties, combined with feedback control of B55 activity by the phosphoserine-containing substrate/inhibitor ENSA, can help explain the temporal sequence of events during exit from mitosis. PMID:27551054

Inflammasome and Fas-Mediated IL-1β Contributes to Th17/Th1 Cell Induction in Pathogenic Bacterial Infection In Vivo.

PubMed

Uchiyama, Ryosuke; Yonehara, Shin; Taniguchi, Shun'ichiro; Ishido, Satoshi; Ishii, Ken J; Tsutsui, Hiroko

2017-08-01

CD4 + Th cells play crucial roles in orchestrating immune responses against pathogenic microbes, after differentiating into effector subsets. Recent research has revealed the importance of IFN-γ and IL-17 double-producing CD4 + Th cells, termed Th17/Th1 cells, in the induction of autoimmune and inflammatory diseases. In addition, Th17/Th1 cells are involved in the regulation of infection caused by the intracellular bacterium Mycobacterium tuberculosis in humans. However, the precise mechanism of Th17/Th1 induction during pathogen infection is unclear. In this study, we showed that the inflammasome and Fas-dependent IL-1β induces Th17/Th1 cells in mice, in response to infection with the pathogenic intracellular bacterium Listeria monocytogenes In the spleens of infected wild-type mice, Th17/Th1 cells were induced, and expressed T-bet and Rorγt. In Pycard -/- mice, which lack the adaptor molecule of the inflammasome (apoptosis-associated speck-like protein containing a caspase recruitment domain), Th17/Th1 induction was abolished. In addition, the Fas-mediated IL-1β production was required for Th17/Th1 induction during bacterial infection: Th17/Th1 induction was abolished in Fas -/- mice, whereas supplementation with recombinant IL-1β restored Th17/Th1 induction via IL-1 receptor 1 (IL-1R1), and rescued the mortality of Fas -/- mice infected with Listeria IL-1R1, but not apoptosis-associated speck-like protein containing a caspase recruitment domain or Fas on T cells, was required for Th17/Th1 induction, indicating that IL-1β stimulates IL-1R1 on T cells for Th17/Th1 induction. These results indicate that IL-1β, produced by the inflammasome and Fas-dependent mechanisms, contributes cooperatively to the Th17/Th1 induction during bacterial infection. This study provides a deeper understanding of the molecular mechanisms underlying Th17/Th1 induction during pathogenic microbial infections in vivo. Copyright © 2017 by The American Association of Immunologists, Inc.

[Induction and regulation of cellulase expression in filamentous fungi: a review].

PubMed

Zhang, Fei; Bai, Fengwu; Zhao, Xinqing

2016-11-25

Production of bioenergy and bio-based chemicals by using fermentable sugars released from low-cost renewable lignocellulosic biomass has received great attention. Efficient cellulolytic enzymes are crucial for lignocellulose bioconversion, but high cellulase production cost is limiting the bioconversion efficiency of cellulosic biomass and industrial applications of lignocellulose biorefinery. Studies on induction and regulation of cellulase in filamentous fungi will help to further develop superior fungal strains for efficient cellulase production and reduce cellulase production cost. With the advances in high-throughput sequencing and gene manipulation technology using fungal strains, an in-depth understanding of cellulase induction and regulation mechanisms of enzyme expression has been achieved. We reviewed recent progresses in the induction and regulation of cellulase expression in several model filamentous fungi, emphasizing sugar transporters, transcription factors and chromatin remodeling. Future prospects in application of artificial zinc finger proteins for cellulase induction and regulation in filamentous fungi were discussed.

Usp5 links suppression of p53 and FAS levels in melanoma to the BRAF pathway

PubMed Central

Potu, Harish; Peterson, Luke F.; Pal, Anupama; Verhaegen, Monique; Cao, Juxiang; Talpaz, Moshe; Donato, Nicholas J.

2014-01-01

Usp5 is a deubiquitinase (DUB) previously shown to regulate unanchored polyubiquitin (Ub) chains, p53 transcriptional activity and double-strand DNA repair. In BRAF mutant melanoma cells, Usp5 activity was suppressed by BRAF inhibitor (vemurafenib) in sensitive but not in acquired or intrinsically resistant cells. Usp5 knockdown overcame acquired vemurafenib resistance and sensitized BRAF and NRAS mutant melanoma cells to apoptosis initiated by MEK inhibitor, cytokines or DNA-damaging agents. Knockdown and overexpression studies demonstrated that Usp5 regulates p53 (and p73) levels and alters cell growth and cell cycle distribution associated with p21 induction. Usp5 also regulates the intrinsic apoptotic pathway by modulating p53-dependent FAS expression. A small molecule DUB inhibitor (EOAI3402143) phenocopied the FAS induction and apoptotic sensitization of Usp5 knockdown and fully blocked melanoma tumor growth in mice. Overall, our results demonstrate that BRAF activates Usp5 to suppress cell cycle checkpoint control and apoptosis by blocking p53 and FAS induction; all of which can be restored by small molecule-mediated Usp5 inhibition. These results suggest that Usp5 inhibition can provide an alternate approach in recovery of diminished p53 (or p73) function in melanoma and can add to the targeted therapies already used in the treatment of melanoma. PMID:24980819

Fluorescent sensors for activity and regulation of the nitrate transceptor CHL1/NRT1.1 and oligopeptide transporters

PubMed Central

Ho, Cheng-Hsun; Frommer, Wolf B

2014-01-01

To monitor nitrate and peptide transport activity in vivo, we converted the dual-affinity nitrate transceptor CHL1/NRT1.1/NPF6.3 and four related oligopeptide transporters PTR1, 2, 4, and 5 into fluorescence activity sensors (NiTrac1, PepTrac). Substrate addition to yeast expressing transporter fusions with yellow fluorescent protein and mCerulean triggered substrate-dependent donor quenching or resonance energy transfer. Fluorescence changes were nitrate/peptide-specific, respectively. Like CHL1, NiTrac1 had biphasic kinetics. Mutation of T101A eliminated high-affinity transport and blocked the fluorescence response to low nitrate. NiTrac was used for characterizing side chains considered important for substrate interaction, proton coupling, and regulation. We observed a striking correlation between transport activity and sensor output. Coexpression of NiTrac with known calcineurin-like proteins (CBL1, 9; CIPK23) and candidates identified in an interactome screen (CBL1, KT2, WNKinase 8) blocked NiTrac1 responses, demonstrating the suitability for in vivo analysis of activity and regulation. The new technology is applicable in plant and medical research. DOI: http://dx.doi.org/10.7554/eLife.01917.001 PMID:24623305

Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells.

PubMed

Zhang, Chuanzhao; Zhi, Wanqing Iris; Lu, Haiquan; Samanta, Debangshu; Chen, Ivan; Gabrielson, Edward; Semenza, Gregg L

2016-10-04

Exposure of breast cancer cells to hypoxia increases the percentage of breast cancer stem cells (BCSCs), which are required for tumor initiation and metastasis, and this response is dependent on the activity of hypoxia-inducible factors (HIFs). We previously reported that exposure of breast cancer cells to hypoxia induces the ALKBH5-mediated demethylation of N6-methyladenosine (m6A) in NANOG mRNA leading to increased expression of NANOG, which is a pluripotency factor that promotes BCSC specification. Here we report that exposure of breast cancer cells to hypoxia also induces ZNF217-dependent inhibition of m6A methylation of mRNAs encoding NANOG and KLF4, which is another pluripotency factor that mediates BCSC specification. Although hypoxia induced the BCSC phenotype in all breast-cancer cell lines analyzed, it did so through variable induction of pluripotency factors and ALKBH5 or ZNF217. However, in every breast cancer line, the hypoxic induction of pluripotency factor and ALKBH5 or ZNF217 expression was HIF-dependent. Immunohistochemistry revealed that expression of HIF-1α and ALKBH5 was concordant in all human breast cancer biopsies analyzed. ALKBH5 knockdown in MDA-MB-231 breast cancer cells significantly decreased metastasis from breast to lungs in immunodeficient mice. Thus, HIFs stimulate pluripotency factor expression and BCSC specification by negative regulation of RNA methylation.

Dexamethasone Enhances 1α,25-Dihydroxyvitamin D3 Effects by Increasing Vitamin D Receptor Transcription*

PubMed Central

Hidalgo, Alejandro A.; Deeb, Kristin K.; Pike, J. Wesley; Johnson, Candace S.; Trump, Donald L.

2011-01-01

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner. PMID:21868377

Sustainable epoxy and oxetane thermosets from photo-initiated cationic polymerization

NASA Astrophysics Data System (ADS)

Ryu, Chang

A group of sustainable materials are proposed and produced from multifunctional epoxides and oxetanes obtained from renewable sources. Monomers are photopolymerized using diaryliodonium salts designed and synthesized by our group as initiator. A detailed investigation of the network formation of epoxidized linseed oil revealed that crosslinks is markedly dependent to the thickness and viscosity of substrate. Copolymerization studies of difunctional oxetane showed that limonene dioxide was effective in increasing the reaction rates and shorten the inherent induction period, also known as kick-starting effect. Such oxetane thermoset can achieve desirable curing rates and Tg compared to petroleum based epoxy used in applications such as large scale surface coatings.

P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates

PubMed Central

Sansó, Miriam; Levin, Rebecca S.; Lipp, Jesse J.; Wang, Vivien Ya-Fan; Greifenberg, Ann Katrin; Quezada, Elizabeth M.; Ali, Akbar; Ghosh, Animesh; Larochelle, Stéphane; Rana, Tariq M.; Geyer, Matthias; Tong, Liang; Shokat, Kevan M.; Fisher, Robert P.

2016-01-01

The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5′-to-3′ “torpedo” exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2. PMID:26728557

Kinetically Defined Mechanisms and Positions of Action of Two New Modulators of Glucocorticoid Receptor-regulated Gene Induction*

PubMed Central

Pradhan, Madhumita A.; Blackford, John A.; Devaiah, Ballachanda N.; Thompson, Petria S.; Chow, Carson C.; Singer, Dinah S.; Simons, S. Stoney

2016-01-01

Most of the steps in, and many of the factors contributing to, glucocorticoid receptor (GR)-regulated gene induction are currently unknown. A competition assay, based on a validated chemical kinetic model of steroid hormone action, is now used to identify two new factors (BRD4 and negative elongation factor (NELF)-E) and to define their sites and mechanisms of action. BRD4 is a kinase involved in numerous initial steps of gene induction. Consistent with its complicated biochemistry, BRD4 is shown to alter both the maximal activity (Amax) and the steroid concentration required for half-maximal induction (EC50) of GR-mediated gene expression by acting at a minimum of three different kinetically defined steps. The action at two of these steps is dependent on BRD4 concentration, whereas the third step requires the association of BRD4 with P-TEFb. BRD4 is also found to bind to NELF-E, a component of the NELF complex. Unexpectedly, NELF-E modifies GR induction in a manner that is independent of the NELF complex. Several of the kinetically defined steps of BRD4 in this study are proposed to be related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR-controlled gene induction have been uncovered. The model-based competition assay is also unique in being able to order, for the first time, the sites of action of the various reaction components: GR < Cdk9 < BRD4 ≤ induced gene < NELF-E. This ability to order factor actions will assist efforts to reduce the side effects of steroid treatments. PMID:26504077

Autophagy induction under carbon starvation conditions is negatively regulated by carbon catabolite repression.

PubMed

Adachi, Atsuhiro; Koizumi, Michiko; Ohsumi, Yoshinori

2017-12-01

Autophagy is a conserved process in which cytoplasmic components are sequestered for degradation in the vacuole/lysosomes in eukaryotic cells. Autophagy is induced under a variety of starvation conditions, such as the depletion of nitrogen, carbon, phosphorus, zinc, and others. However, apart from nitrogen starvation, it remains unclear how these stimuli induce autophagy. In yeast, for example, it remains contentious whether autophagy is induced under carbon starvation conditions, with reports variously suggesting both induction and lack of induction upon depletion of carbon. We therefore undertook an analysis to account for these inconsistencies, concluding that autophagy is induced in response to abrupt carbon starvation when cells are grown with glycerol but not glucose as the carbon source. We found that autophagy under these conditions is mediated by nonselective degradation that is highly dependent on the autophagosome-associated scaffold proteins Atg11 and Atg17. We also found that the extent of carbon starvation-induced autophagy is positively correlated with cells' oxygen consumption rate, drawing a link between autophagy induction and respiratory metabolism. Further biochemical analyses indicated that maintenance of intracellular ATP levels is also required for carbon starvation-induced autophagy and that autophagy plays an important role in cell viability during prolonged carbon starvation. Our findings suggest that carbon starvation-induced autophagy is negatively regulated by carbon catabolite repression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Dynamic interplay and function of multiple noncoding genes governing X chromosome inactivation

PubMed Central

Yue, Minghui; Richard, John Lalith Charles

2015-01-01

There is increasing evidence for the emergence of long noncoding RNAs (IncRNAs) as important components, especially in the regulation of gene expression. In the event of X chromosome inactivation, robust epigenetic marks are established in a long noncoding Xist RNA-dependent manner, giving rise to a distinct epigenetic landscape on the inactive X chromosome (Xi). The X inactivation center (Xic is essential for induction of X chromosome inactivation and harbors two topologically associated domains (TADs) to regulate monoallelic Xist expression: one at the noncoding Xist gene and its upstream region, and the other at the antisense Tsix and its upstream region. The monoallelic expression of Xist is tightly regulated by these two functionally distinct TADs as well as their constituting IncRNAs and proteins. In this review, we summarize recent updates in our knowledge of IncRNAs found at the Xic and discuss their overall mechanisms of action. We also discuss our current understanding of the molecular mechanism behind Xist RNA-mediated induction of the repressive epigenetic landscape at the Xi. PMID:26260844

Cutting edge: A transcriptional repressor and corepressor induced by the STAT3-regulated anti-inflammatory signaling pathway.

PubMed

El Kasmi, Karim C; Smith, Amber M; Williams, Lynn; Neale, Geoffrey; Panopoulos, Athanasia D; Panopolous, Athanasia; Watowich, Stephanie S; Häcker, Hans; Foxwell, Brian M J; Murray, Peter J

2007-12-01

IL-10 regulates anti-inflammatory signaling via the activation of STAT3, which in turn controls the induction of a gene expression program whose products execute inhibitory effects on proinflammatory mediator production. In this study we show that IL-10 induces the expression of an ETS family transcriptional repressor, ETV3, and a helicase family corepressor, Strawberry notch homologue 2 (SBNO2), in mouse and human macrophages. IL-10-mediated induction of ETV3 and SBNO2 expression was dependent upon both STAT3 and a stimulus through the TLR pathway. We also observed that ETV3 expression was strongly induced by the STAT3 pathway regulated by IL-10 but not by STAT3 signaling activated by IL-6, which cannot activate the anti-inflammatory signaling pathway. ETV3 and SBNO2 repressed NF-kappaB- but not IFN regulatory factor 7 (IRF7)-activated transcriptional reporters. Collectively our data suggest that ETV3 and SBNO2 are components of the pathways that contribute to the downstream anti-inflammatory effects of IL-10.

Pharmacological induction of skin pigmentation unveils the neuroendocrine circuit regulated by light.

PubMed

Bertolesi, Gabriel E; Vazhappilly, Sherene T; Hehr, Carrie L; McFarlane, Sarah

2016-03-01

Light-regulated skin colour change is an important physiological process in invertebrates and lower vertebrates, and includes daily circadian variation and camouflage (i.e. background adaptation). The photoactivation of melanopsin-expressing retinal ganglion cells (mRGCs) in the eye initiates an uncharacterized neuroendocrine circuit that regulates melanin dispersion/aggregation through the secretion of alpha-melanocyte-stimulating hormone (α-MSH). We developed experimental models of normal or enucleated Xenopus embryos, as well as in situ cultures of skin of isolated dorsal head and tails, to analyse pharmacological induction of skin pigmentation and α-MSH synthesis. Both processes are triggered by a melanopsin inhibitor, AA92593, as well as chloride channel modulators. The AA9253 effect is eye-dependent, while functional data in vivo point to GABAA receptors expressed on pituitary melanotrope cells as the chloride channel blocker target. Based on the pharmacological data, we suggest a neuroendocrine circuit linking mRGCs with α-MSH secretion, which is used normally during background adaptation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Apoptosis induction in MV4-11 and K562 human leukemic cells by Pereskia sacharosa (Cactaceae) leaf crude extract.

PubMed

Asmaa, Mat Jusoh Siti; Al-Jamal, Hamid Ali Nagi; Ang, Cheng Yong; Asan, Jamaruddin Mat; Seeni, Azman; Johan, Muhammad Farid

2014-01-01

Pereskia sacharosa is a genus of cacti widely used in folk medicine for cancer-related treatment. Anti-proliferative effects have been studied in recent years against colon, breast, cervical and lung cancer cell lines, with promising results. We here extended study of anti-proliferative effects to a blood malignancy, leukemia. Two leukemic cell lines, MV4-11 (acute myeloid leukemia) and K562 (chronic myeloid leukemia), were studied. IC50 concentrations were determined and apoptosis and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell-cycle related regulatory proteins was assessed by Western blotting. P sacharosa inhibited growth of MV4-11 and K562 cells in a dose-dependent manner. The mode of cell death was via induction of intrinsic apoptotic pathways and cell cycle arrest. There was profound up-regulation of cytochrome c, caspases, p21 and p53 expression and repression of Akt and Bcl-2 expression in treated cells. These results suggest that P sacharosa induces leukemic cell death via apoptosis induction and changes in cell cycle checkpoint, thus deserves further study for anti-leukemic potential.

Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

PubMed

Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Sang-Hee; Kwon, Daeho; Shin, Ho-Joon

2012-08-01

Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

Isoform-specific PKA dynamics revealed by dye-triggered aggregation and DAKAP1alpha-mediated localization in living cells.

PubMed

Martin, Brent R; Deerinck, Thomas J; Ellisman, Mark H; Taylor, Susan S; Tsien, Roger Y

2007-09-01

The tetracysteine sequence YRECCPGCCMWR fused to the N terminus of green fluorescent protein (GFP) self-aggregates upon biarsenical labeling in living cells or in vitro. Such dye-triggered aggregates form temperature-dependent morphologies and are dispersed by photobleaching. Fusion of the biarsenical aggregating GFP to the regulatory (R) or catalytic (C) subunit of PKA traps intact holoenzyme in compact fluorescent puncta upon biarsenical labeling. Contrary to the classical model of PKA activation, elevated cAMP does not allow RIalpha and Calpha to diffuse far apart unless the pseudosubstrate inhibitor PKI or locally concentrated substrate is coexpressed. However, RIIalpha releases Calpha upon elevated cAMP alone, dependent on autophosphorylation of the RIIalpha inhibitory domain. DAKAP1alpha overexpression induced R and C outer mitochondrial colocalization and showed similar regulation. Overall, effective separation of type I PKA is substrate dependent, whereas type II PKA dissociation relies on autophosphorylation.

Plasmodium sporozoite motility is modulated by the turnover of discrete adhesion sites.

PubMed

Münter, Sylvia; Sabass, Benedikt; Selhuber-Unkel, Christine; Kudryashev, Mikhail; Hegge, Stephan; Engel, Ulrike; Spatz, Joachim P; Matuschewski, Kai; Schwarz, Ulrich S; Frischknecht, Friedrich

2009-12-17

Sporozoites are the highly motile stages of the malaria parasite injected into the host's skin during a mosquito bite. In order to navigate inside of the host, sporozoites rely on actin-dependent gliding motility. Although the major components of the gliding machinery are known, the spatiotemporal dynamics of the proteins and the underlying mechanism powering forward locomotion remain unclear. Here, we show that sporozoite motility is characterized by a continuous sequence of stick-and-slip phases. Reflection interference contrast and traction force microscopy identified the repeated turnover of discrete adhesion sites as the underlying mechanism of this substrate-dependent type of motility. Transient forces correlated with the formation and rupture of distinct substrate contact sites and were dependent on actin dynamics. Further, we show that the essential sporozoite surface protein TRAP is critical for the regulated formation and rupture of adhesion sites but is dispensable for retrograde capping.

A UHF RFID system with on-chip-antenna tag for short range communication

NASA Astrophysics Data System (ADS)

Qi, Peng; Chun, Zhang; Xijin, Zhao; Zhihua, Wang

2015-05-01

A UHF RF identification system based on the 0.18 μm CMOS process has been developed for short range and harsh size requirement applications, which is composed of a fully integrated tag and a special reader. The whole tag chip with the antenna takes up an area of 0.36 mm2, which is smaller than other reported tags with an on-chip antenna (OCA) using the standard CMOS process. A self-defined protocol is proposed to reduce the power consumption, and minimize the size of the tag. The specialized SOC reader system consists of the RF transceiver, digital baseband, MCU and host interface. Its power consumption is about 500 mW. Measurement results show that the system's reading range is 2 mm with 20 dBm reader output power. With an inductive antenna printed on a paper substrate around the OCA tag, the reading range can be extended from several centimeters to meters, depending on the shape and size of the inductive antenna.

Effects of a chirped bias voltage on ion energy distributions in inductively coupled plasma reactors

NASA Astrophysics Data System (ADS)

Lanham, Steven J.; Kushner, Mark J.

2017-08-01

The metrics for controlling reactive fluxes to wafers for microelectronics processing are becoming more stringent as feature sizes continue to shrink. Recent strategies for controlling ion energy distributions to the wafer involve using several different frequencies and/or pulsed powers. Although effective, these strategies are often costly or present challenges in impedance matching. With the advent of matching schemes for wide band amplifiers, other strategies to customize ion energy distributions become available. In this paper, we discuss results from a computational investigation of biasing substrates using chirped frequencies in high density, electronegative inductively coupled plasmas. Depending on the frequency range and chirp duration, the resulting ion energy distributions exhibit components sampled from the entire frequency range. However, the chirping process also produces transient shifts in the self-generated dc bias due to the reapportionment of displacement and conduction with frequency to balance the current in the system. The dynamics of the dc bias can also be leveraged towards customizing ion energy distributions.

Nanogranular soft magnetic material and on-package integrated inductors

NASA Astrophysics Data System (ADS)

Li, Liangliang

2007-12-01

Integrated inductors used in electronic circuits are mainly spiral-shaped aluminum devices fabricated on Si chip. They have several disadvantages---large silicon area consumption, high DC resistance and high cost. An attractive approach to address these issues is directly integrating inductors into package substrates, which provide plenty of usage area, low resistance and low cost. The goals of this dissertation are designing and fabricating magnetic and air-core inductors with characteristic low resistance and high quality factor on package substrates. The research work includes three parts which are summarized below. First, the CoFeHfO nanogranular magnetic material developed on Si wafers and package substrates by pulsed DC reactive sputtering were investigated. On Si wafers, the optimized CoFeHfO film has soft magnetic properties. On printed circuit board (PCB) substrates, these magnetic properties degrade due to the rough surface. Surface planarization such as chemical-mechanical polishing can be applied on PCB substrates to reduce the surface roughness and hence improve these properties. Second, on-package inductors with small resistances and high quality factors were designed, fabricated, measured and analyzed. Air-core and magnetic inductors (20 design variations) were built on 8-inch PCB substrates. The DC resistances of these inductors are less than 12 mO, one of the lowest values ever reported. The maximum quality factors can be as large as ˜80 at around 1 GHz for the air-core inductors and ˜25 at 200 MHz for the magnetic inductors. Third, inductor simulation was carried out to study the effects of magnetic materials on the properties of inductors using the Ansoft HFSS software package. The measurement data for the permeability spectra of the CoFeHfO film and the tensor nature of the permeability were taken into account in the simulation. The simulation results matched the experimental data for the inductances, resistances and quality factors. This established an accurate method for modeling high-frequency magnetic devices. Using this method, an inductor with a closed magnetic core was studied by varying the geometry of the core and copper coil. It has been found that the inductance of this inductor depends strongly on whether the permeability of the magnetic core is isotropic or anisotropic.

PTP1B is a negative regulator of interleukin 4–induced STAT6 signaling

PubMed Central

Lu, Xiaoqing; Malumbres, Raquel; Shields, Benjamin; Jiang, Xiaoyu; Sarosiek, Kristopher A.; Natkunam, Yasodha

2008-01-01

Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed enzyme shown to negatively regulate multiple tyrosine phosphorylation-dependent signaling pathways. PTP1B can modulate cytokine signaling pathways by dephosphorylating JAK2, TYK2, and STAT5a/b. Herein, we report that phosphorylated STAT6 may serve as a cytoplasmic substrate for PTP1B. Overexpression of PTP1B led to STAT6 dephosphorylation and the suppression of STAT6 transcriptional activity, whereas PTP1B knockdown or deficiency augmented IL-4–induced STAT6 signaling. Pretreatment of these cells with the PTK inhibitor staurosporine led to sustained STAT6 phosphorylation consistent with STAT6 serving as a direct substrate of PTP1B. Furthermore, PTP1B-D181A “substrate-trapping” mutants formed stable complexes with phosphorylated STAT6 in a cellular context and endogenous PTP1B and STAT6 interacted in an interleukin 4 (IL-4)–inducible manner. We delineate a new negative regulatory loop of IL-4–JAK-STAT6 signaling. We demonstrate that IL-4 induces PTP1B mRNA expression in a phosphatidylinositol 3-kinase–dependent manner and enhances PTP1B protein stability to suppress IL-4–induced STAT6 signaling. Finally, we show that PTP1B expression may be preferentially elevated in activated B cell–like diffuse large B-cell lymphomas. These observations identify a novel regulatory loop for the regulation of IL-4–induced STAT6 signaling that may have important implications in both neoplastic and inflammatory processes. PMID:18716132

A Ubiquitin-Proteasome Pathway for the Repair of Topoisomerase I-DNA Covalent Complexes*S⃞

PubMed Central

Lin, Chao-Po; Ban, Yi; Lyu, Yi Lisa; Desai, Shyamal D.; Liu, Leroy F.

2008-01-01

Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged DNA, UV-irradiated DNA, alkylated DNA, uracil-substituted DNA, mismatched DNA, gapped and nicked DNA, and DNA with abasic sites). Top1 cleavage complexes arrest transcription and trigger transcription-dependent degradation of Top1, a phenomenon termed Top1 down-regulation. In the current study, we have investigated the role of Top1 down-regulation in the repair of Top1 cleavage complexes. Using quiescent (serum-starved) human WI-38 cells, camptothecin (CPT) was shown to induce Top1 down-regulation, which paralleled the induction of DNA single-strand breaks (SSBs) (assayed by comet assays) and ATM autophosphorylation (at Ser-1981). Interestingly, Top1 down-regulation, induction of DNA SSBs and ATM autophosphorylation were all abolished by the proteasome inhibitor MG132. Furthermore, studies using immunoprecipitation and dominant-negative ubiquitin mutants have suggested a specific requirement for the assembly of Lys-48-linked polyubiquitin chains for CPT-induced Top1 down-regulation. In contrast to the effect of proteasome inhibition, inactivation of PARP1 was shown to increase the amount of CPT-induced SSBs and the level of ATM autophosphorylation. Together, these results support a model in which Top1 cleavage complexes arrest transcription and activate a ubiquitin-proteasome pathway leading to the degradation of Top1 cleavage complexes. Degradation of Top1 cleavage complexes results in the exposure of Top1-concealed SSBs for repair through a PARP1-dependent process. PMID:18515798

AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1

PubMed Central

Strappazzon, F; Nazio, F; Corrado, M; Cianfanelli, V; Romagnoli, A; Fimia, G M; Campello, S; Nardacci, R; Piacentini, M; Campanella, M; Cecconi, F

2015-01-01

Damaged mitochondria are eliminated by mitophagy, a selective form of autophagy whose dysfunction associates with neurodegenerative diseases. PINK1, PARKIN and p62/SQTMS1 have been shown to regulate mitophagy, leaving hitherto ill-defined the contribution by key players in ‘general' autophagy. In basal conditions, a pool of AMBRA1 – an upstream autophagy regulator and a PARKIN interactor – is present at the mitochondria, where its pro-autophagic activity is inhibited by Bcl-2. Here we show that, upon mitophagy induction, AMBRA1 binds the autophagosome adapter LC3 through a LIR (LC3 interacting region) motif, this interaction being crucial for regulating both canonical PARKIN-dependent and -independent mitochondrial clearance. Moreover, forcing AMBRA1 localization to the outer mitochondrial membrane unleashes a massive PARKIN- and p62-independent but LC3-dependent mitophagy. These results highlight a novel role for AMBRA1 as a powerful mitophagy regulator, through both canonical or noncanonical pathways. PMID:25215947

AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1.

PubMed

Strappazzon, F; Nazio, F; Corrado, M; Cianfanelli, V; Romagnoli, A; Fimia, G M; Campello, S; Nardacci, R; Piacentini, M; Campanella, M; Cecconi, F

2015-03-01

Damaged mitochondria are eliminated by mitophagy, a selective form of autophagy whose dysfunction associates with neurodegenerative diseases. PINK1, PARKIN and p62/SQTMS1 have been shown to regulate mitophagy, leaving hitherto ill-defined the contribution by key players in 'general' autophagy. In basal conditions, a pool of AMBRA1 - an upstream autophagy regulator and a PARKIN interactor - is present at the mitochondria, where its pro-autophagic activity is inhibited by Bcl-2. Here we show that, upon mitophagy induction, AMBRA1 binds the autophagosome adapter LC3 through a LIR (LC3 interacting region) motif, this interaction being crucial for regulating both canonical PARKIN-dependent and -independent mitochondrial clearance. Moreover, forcing AMBRA1 localization to the outer mitochondrial membrane unleashes a massive PARKIN- and p62-independent but LC3-dependent mitophagy. These results highlight a novel role for AMBRA1 as a powerful mitophagy regulator, through both canonical or noncanonical pathways.

Frontal EEG and emotion regulation: electrocortical activity in response to emotional film clips is associated with reduced mood induction and attention interference effects.

PubMed

Dennis, Tracy A; Solomon, Beylul

2010-12-01

Frontal EEG activity is thought to reflect affective dispositions, but may also reflect the emotional demands of a specific context combined with the capability to regulate emotions in that context. The present study examined this hypothesis by testing whether frontal EEG activity during mood inductions versus a resting baseline predicted emotion regulation. EEG was recorded while participants (N=66, 40 females) received a fearful, sad, or neutral mood induction. Emotion regulation was measured following the mood inductions as self-reported change in negative mood and as attention interference in a task with mood-congruent emotional distracters. Greater frontal EEG activity during the mood inductions versus baseline was associated with more effective emotion regulation: less post-induction sadness and anxiety and reduced mood-congruent attention interference effects. Effects did not differ between the left and right hemispheres. Results support the hypothesis that frontal EEG activity reflects both emotional context and emotion-regulatory capabilities. Copyright © 2010 Elsevier B.V. All rights reserved.

Angiotensin-I converting enzyme (ACE): structure, biological roles, and molecular basis for chloride ion dependence.

PubMed

Masuyer, Geoffrey; Yates, Christopher J; Sturrock, Edward D; Acharya, K Ravi

2014-10-01

Somatic angiotensin-I converting enzyme (sACE) has an essential role in the regulation of blood pressure and electrolyte fluid homeostasis. It is a zinc protease that cleaves angiotensin-I (AngI), bradykinin, and a broad range of other signalling peptides. The enzyme activity is provided by two homologous domains (N- and C-), which display clear differences in substrate specificities and chloride activation. The presence of chloride ions in sACE and its unusual role in activity was identified early on in the characterisation of the enzyme. The molecular mechanisms of chloride activation have been investigated thoroughly through mutagenesis studies and shown to be substrate-dependent. Recent results from X-ray crystallography structural analysis have provided the basis for the intricate interactions between ACE, its substrate and chloride ions. Here we describe the role of chloride ions in human ACE and its physiological consequences. Insights into the chloride activation of the N- and C-domains could impact the design of improved domain-specific ACE inhibitors.

Dynamic Localization of a Transcription Factor in Bacillus subtilis: the LicT Antiterminator Relocalizes in Response to Inducer Availability

PubMed Central

Rothe, Fabian M.; Wrede, Christoph; Lehnik-Habrink, Martin; Görke, Boris

2013-01-01

Bacillus subtilis transports β-glucosides such as salicin by a dedicated phosphotransferase system (PTS). The expression of the β-glucoside permease BglP is induced in the presence of the substrate salicin, and this induction requires the binding of the antiterminator protein LicT to a specific RNA target in the 5′ region of the bglP mRNA to prevent the formation of a transcription terminator. LicT is composed of an N-terminal RNA-binding domain and two consecutive PTS regulation domains, PRD1 and PRD2. In the absence of salicin, LicT is phosphorylated on PRD1 by BglP and thereby inactivated. In the presence of the inducer, the phosphate group from PRD1 is transferred back to BglP and consequently to the incoming substrate, resulting in the activation of LicT. In this study, we have investigated the intracellular localization of LicT. While the protein was evenly distributed in the cell in the absence of the inducer, we observed a subpolar localization of LicT if salicin was present in the medium. Upon addition or removal of the inducer, LicT rapidly relocalized in the cells. This dynamic relocalization did not depend on the binding of LicT to its RNA target sites, since the localization pattern was not affected by deletion of all LicT binding sites. In contrast, experiments with mutants affected in the PTS components as well as mutations of the LicT phosphorylation sites revealed that phosphorylation of LicT by the PTS components plays a major role in the control of the subcellular localization of this RNA-binding transcription factor. PMID:23475962

Characterization of the regulation and function of zinc-dependent histone deacetylases during rodent liver regeneration.

PubMed

Huang, Jiansheng; Barr, Emily; Rudnick, David A

2013-05-01

The studies reported here were undertaken to define the regulation and functional importance of zinc-dependent histone deacetylase (Zn-HDAC) activity during liver regeneration using the mouse partial hepatectomy (PH) model. The results showed that hepatic HDAC activity was significantly increased in nuclear and cytoplasmic fractions following PH. Further analyses showed isoform-specific effects of PH on HDAC messenger RNA (mRNA) and protein expression, with increased expression of the class I HDACs, 1 and 8, and class II HDAC4 in regenerating liver. Hepatic expression of (class II) HDAC5 was unchanged after PH; however, HDAC5 exhibited transient nuclear accumulation in regenerating liver. These changes in hepatic HDAC expression, subcellular localization, and activity coincided with diminished histone acetylation in regenerating liver. The significance of these events was investigated by determining the effects of suberoylanilide hydroxyamic acid (SAHA, a specific inhibitor of Zn-HDAC activity) on hepatic regeneration. The results showed that SAHA treatment suppressed the effects of PH on histone deacetylation and hepatocellular bromodeoxyuridine (BrdU) incorporation. Further examination showed that SAHA blunted hepatic expression and activation of cell cycle signals downstream of induction of cyclin D1 expression in mice subjected to PH. The data reported here demonstrate isoform-specific regulation of Zn-HDAC expression, subcellular localization, and activity in regenerating liver. These studies also indicate that HDAC activity promotes liver regeneration by regulating hepatocellular cell cycle progression at a step downstream of cyclin D1 induction. Copyright © 2012 American Association for the Study of Liver Diseases.

Zebrafish Foxi1 provides a neuronal ground state during inner ear induction preceding the Dlx3b/4b-regulated sensory lineage.

PubMed

Hans, Stefan; Irmscher, Anne; Brand, Michael

2013-05-01

Vertebrate inner ear development is a complex process that involves the induction of a common territory for otic and epibranchial precursors and their subsequent segregation into otic and epibranchial cell fates. In zebrafish, the otic-epibranchial progenitor domain (OEPD) is induced by Fgf signaling in a Foxi1- and Dlx3b/4b-dependent manner, but the functional differences of Foxi1 and Dlx3b/4b in subsequent cell fate specifications within the developing inner ear are poorly understood. Based on pioneer tracking (PioTrack), a novel Cre-dependent genetic lineage tracing method, and genetic data, we show that the competence to embark on a neuronal or sensory fate is provided sequentially and very early during otic placode induction. Loss of Foxi1 prevents neuronal precursor formation without affecting hair cell specification, whereas loss of Dlx3b/4b inhibits hair cell but not neuronal precursor formation. Consistently, in Dlx3b/4b- and Sox9a-deficient b380 mutants almost all otic epithelial fates are absent, including sensory hair cells, and the remaining otic cells adopt a neuronal fate. Furthermore, the progenitors of the anterior lateral line ganglia also arise from the OEPD in a Foxi1-dependent manner but are unaffected in the absence of Dlx3b/4b or in b380 mutants. Thus, in addition to otic fate Foxi1 provides neuronal competence during OEPD induction prior to and independently of the Dlx3b/4b-mediated sensory fate of the developing inner ear.

LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs.

PubMed

Hong, Sungki; Freeberg, Mallory A; Han, Ting; Kamath, Avani; Yao, Yao; Fukuda, Tomoko; Suzuki, Tsukasa; Kim, John K; Inoki, Ken

2017-06-26

The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5'TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5'TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5' and 3'UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5'UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3'UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.

Parkin regulation of CHOP modulates susceptibility to cardiac endoplasmic reticulum stress.

PubMed

Han, Kim; Hassanzadeh, Shahin; Singh, Komudi; Menazza, Sara; Nguyen, Tiffany T; Stevens, Mark V; Nguyen, An; San, Hong; Anderson, Stasia A; Lin, Yongshun; Zou, Jizhong; Murphy, Elizabeth; Sack, Michael N

2017-05-18

The regulatory control of cardiac endoplasmic reticulum (ER) stress is incompletely characterized. As ER stress signaling upregulates the E3-ubiquitin ligase Parkin, we investigated the role of Parkin in cardiac ER stress. Parkin knockout mice exposed to aortic constriction-induced cardiac pressure-overload or in response to systemic tunicamycin (TM) developed adverse ventricular remodeling with excessive levels of the ER regulatory C/EBP homologous protein CHOP. CHOP was identified as a Parkin substrate and its turnover was Parkin-dose and proteasome-dependent. Parkin depletion in cardiac HL-1 cells increased CHOP levels and enhanced susceptibility to TM-induced cell death. Parkin reconstitution rescued this phenotype and the contribution of excess CHOP to this ER stress injury was confirmed by reduction in TM-induced cell death when CHOP was depleted in Parkin knockdown cardiomyocytes. Isogenic Parkin mutant iPSC-derived cardiomyocytes showed exaggerated ER stress induced CHOP and apoptotic signatures and myocardium from subjects with dilated cardiomyopathy showed excessive Parkin and CHOP induction. This study identifies that Parkin functions to blunt excessive CHOP to prevent maladaptive ER stress-induced cell death and adverse cardiac ventricular remodeling. Additionally, Parkin is identified as a novel post-translational regulatory moderator of CHOP stability and uncovers an additional stress-modifying function of this E3-ubiquitin ligase.

Regulation of tumor cell migration by protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-, and threonine-rich sequence (PEST)

PubMed Central

Zheng, Yanhua; Lu, Zhimin

2013-01-01

Protein tyrosine phosphatase (PTP)–proline-, glutamate-, serine-, and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration. PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications, including phosphorylation, oxidation, and caspase-dependent cleavage. PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins. Dephosphorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process. PMID:23237212

Glucocorticoid-mediated BIM induction and apoptosis are regulated by Runx2 and c-Jun in leukemia cells

PubMed Central

Heidari, N; Miller, A V; Hicks, M A; Marking, C B; Harada, H

2012-01-01

Glucocorticoids (GCs) are common components of many chemotherapeutic regimens for lymphoid malignancies. GC-induced apoptosis involves an intrinsic mitochondria-dependent pathway. BIM (BCL-2-interacting mediator of cell death), a BCL-2 homology 3-only pro-apoptotic protein, is upregulated by dexamethasone (Dex) treatment in acute lymphoblastic leukemia cells and has an essential role in Dex-induced apoptosis. It has been indicated that Dex-induced BIM is regulated mainly by transcription, however, the molecular mechanisms including responsible transcription factors are unclear. In this study, we found that Dex treatment induced transcription factor Runx2 and c-Jun in parallel with BIM induction. Dex-induced BIM and apoptosis were decreased in cells harboring dominant-negative c-Jun and were increased in cells with c-Jun overexpression. Cells harboring short hairpin RNA for Runx2 also decreased BIM induction and apoptosis. On the Bim promoter, c-Jun bound to and activated the AP-1-binding site at about −2.7 kb from the transcription start site. Treatment with RU486, a GC receptor antagonist, blocked Dex-induced Runx2, c-Jun and BIM induction, as well as apoptosis. Furthermore, pretreatment with SB203580, a p38-mitogen-activated protein kinase (MAPK) inhibitor, decreased Dex-induced Runx2, c-Jun and BIM, suggesting that p38-MAPK activation is upstream of the induction of these molecules. In conclusion, we identified the critical signaling pathway for GC-induced apoptosis, and targeting these molecules may be an alternative approach to overcome GC-resistance in leukemia treatment. PMID:22825467

Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression.

PubMed

Franz, André; Pirson, Paul A; Pilger, Domenic; Halder, Swagata; Achuthankutty, Divya; Kashkar, Hamid; Ramadan, Kristijan; Hoppe, Thorsten

2016-02-04

The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging.

Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression

PubMed Central

Franz, André; Pirson, Paul A.; Pilger, Domenic; Halder, Swagata; Achuthankutty, Divya; Kashkar, Hamid; Ramadan, Kristijan; Hoppe, Thorsten

2016-01-01

The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging. PMID:26842564

Negative regulation of quorum-sensing systems in Pseudomonas aeruginosa by ATP-dependent Lon protease.

PubMed

Takaya, Akiko; Tabuchi, Fumiaki; Tsuchiya, Hiroko; Isogai, Emiko; Yamamoto, Tomoko

2008-06-01

Lon protease, a member of the ATP-dependent protease family, regulates numerous cellular systems by degrading specific substrates. Here, we demonstrate that Lon is involved in the regulation of quorum-sensing (QS) signaling systems in Pseudomonas aeruginosa, an opportunistic human pathogen. The organism has two acyl-homoserine lactone (HSL)-mediated QS systems, LasR/LasI and RhlR/RhlI. Many reports have demonstrated that these two systems are regulated and interconnected by global regulators. We found that lon-disrupted cells overproduce pyocyanin, the biosynthesis of which depends on the RhlR/RhlI system, and show increased levels of a transcriptional regulator, RhlR. The QS systems are organized hierarchically: the RhlR/RhlI system is subordinate to LasR/LasI. To elucidate the mechanism by which Lon negatively regulates RhlR/RhlI, we examined the effect of lon disruption on the LasR/LasI system. We found that Lon represses the expression of LasR/LasI by degrading LasI, an HSL synthase, leading to negative regulation of the RhlR/RhlI system. RhlR/RhlI was also shown to be regulated by Lon independently of LasR/LasI via regulation of RhlI, an HSL synthase. In view of these findings, it is suggested that Lon protease is a powerful negative regulator of both HSL-mediated QS systems in P. aeruginosa.

Non-coding RNA derived from the region adjacent to the human HO-1 E2 enhancer selectively regulates HO-1 gene induction by modulating Pol II binding

PubMed Central

Maruyama, Atsushi; Mimura, Junsei; Itoh, Ken

2014-01-01

Recent studies have disclosed the function of enhancer RNAs (eRNAs), which are long non-coding RNAs transcribed from gene enhancer regions, in transcriptional regulation. However, it remains unclear whether eRNAs are involved in the regulation of human heme oxygenase-1 gene (HO-1) induction. Here, we report that multiple nuclear-enriched eRNAs are transcribed from the regions adjacent to two human HO-1 enhancers (i.e. the distal E2 and proximal E1 enhancers), and some of these eRNAs are induced by the oxidative stress-causing reagent diethyl maleate (DEM). We demonstrated that the expression of one forward direction (5′ to 3′) eRNA transcribed from the human HO-1 E2 enhancer region (named human HO-1enhancer RNA E2-3; hereafter called eRNA E2-3) was induced by DEM in an NRF2-dependent manner in HeLa cells. Conversely, knockdown of BACH1, a repressor of HO-1 transcription, further increased DEM-inducible eRNA E2-3 transcription as well as HO-1 expression. In addition, we showed that knockdown of eRNA E2-3 selectively down-regulated DEM-induced HO-1 expression. Furthermore, eRNA E2-3 knockdown attenuated DEM-induced Pol II binding to the promoter and E2 enhancer regions of HO-1 without affecting NRF2 recruitment to the E2 enhancer. These findings indicate that eRNAE2-3 is functional and is required for HO-1 induction. PMID:25404134

Loss of Vacuolar H+-ATPase (V-ATPase) Activity in Yeast Generates an Iron Deprivation Signal That Is Moderated by Induction of the Peroxiredoxin TSA2 *

PubMed Central

Diab, Heba I.; Kane, Patricia M.

2013-01-01

Vacuolar H+-ATPases (V-ATPases) acidify intracellular organelles and help to regulate overall cellular pH. Yeast vma mutants lack V-ATPase activity and allow exploration of connections between cellular pH, iron, and redox homeostasis common to all eukaryotes. A previous microarray study in a vma mutant demonstrated up-regulation of multiple iron uptake genes under control of Aft1p (the iron regulon) and only one antioxidant gene, the peroxiredoxin TSA2 (Milgrom, E., Diab, H., Middleton, F., and Kane, P. M. (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J. Biol. Chem. 282, 7125–7136). Fluorescent biosensors placing GFP under transcriptional control of either an Aft1-dependent promoter (PFIT2-GFP) or the TSA2 promoter (PTSA2-GFP) were constructed to monitor transcriptional signaling. Both biosensors were up-regulated in the vma2Δ mutant, and acute V-ATPase inhibition with concanamycin A induced coordinate up-regulation from both promoters. PTSA2-GFP induction was Yap1p-dependent, indicating an oxidative stress signal. Total cell iron measurements indicate that the vma2Δ mutant is iron-replete, despite up-regulation of the iron regulon. Acetic acid up-regulated PFIT2-GFP expression in wild-type cells, suggesting that loss of pH control contributes to an iron deficiency signal in the mutant. Iron supplementation significantly decreased PFIT2-GFP expression and, surprisingly, restored PTSA2-GFP to wild-type levels. A tsa2Δ mutation induced both nuclear localization of Aft1p and PFIT2-GFP expression. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis. PMID:23457300

Regulation of mitosis-meiosis transition by the ubiquitin ligase β-TrCP in male germ cells.

PubMed

Nakagawa, Tadashi; Zhang, Teng; Kushi, Ryo; Nakano, Seiji; Endo, Takahiro; Nakagawa, Makiko; Yanagihara, Noriko; Zarkower, David; Nakayama, Keiko

2017-11-15

The mitosis-meiosis transition is essential for spermatogenesis. Specific and timely downregulation of the transcription factor DMRT1, and consequent induction of Stra8 expression, is required for this process in mammals, but the molecular mechanism has remained unclear. Here, we show that β-TrCP, the substrate recognition component of an E3 ubiquitin ligase complex, targets DMRT1 for degradation and thereby controls the mitosis-meiosis transition in mouse male germ cells. Conditional inactivation of β-TrCP2 in male germ cells of β-TrCP1 knockout mice resulted in sterility due to a lack of mature sperm. The β-TrCP-deficient male germ cells did not enter meiosis, but instead underwent apoptosis. The induction of Stra8 expression was also attenuated in association with the accumulation of DMRT1 at the Stra8 promoter in β-TrCP-deficient testes. DMRT1 contains a consensus β-TrCP degron sequence that was found to bind β-TrCP. Overexpression of β-TrCP induced the ubiquitylation and degradation of DMRT1. Heterozygous deletion of Dmrt1 in β-TrCP-deficient spermatogonia increased meiotic cells with a concomitant reduction of apoptosis. Collectively, our data indicate that β-TrCP regulates the transition from mitosis to meiosis in male germ cells by targeting DMRT1 for degradation. © 2017. Published by The Company of Biologists Ltd.

Relationship between hydrocarbon structure and induction of P450: effects on protein levels and enzyme activities.

PubMed

Backes, W L; Sequeira, D J; Cawley, G F; Eyer, C S

1993-12-01

1. Treatment of male rat with the small aromatic hydrocarbons, benzene, toluene, ethylbenzene, n-propylbenzene, m-xylene, and p-xylene increased several P450-dependent activities, with ethylbenzene, m-xylene, and n-propylbenzene producing the greatest response. Hydrocarbon treatment differentially affected toluene metabolism, producing a response dependent on the metabolite monitored. In untreated rats, benzyl alcohol was the major hydroxylation product of toluene metabolism, comprising > 99% of the total metabolites formed. Hydrocarbon treatment increased the overall rate of toluene metabolism by dramatically increasing the amount of aromatic hydroxylation. Ethylbenzene, n-propylbenzene and m-xylene were the most effective inducers of aromatic hydroxylation of toluene. In contrast, production of the major toluene metabolite benzyl alcohol was increased only after treatment with m-xylene. 2. P450 2B1/2B2 levels were induced by each of the hydrocarbons examined, with the magnitude of induction increasing with increasing hydrocarbon size. P450 1A1 was also induced after hydrocarbon exposure; however, the degree of induction was smaller than that observed for P450 2B1/2B2. P450 2C11 levels were suppressed after treatment with benzene, ethylbenzene and n-propylbenzene. 3. Taken together these results display two induction patterns. The first generally corresponds to changes in the P450 2B subfamily, where activities (e.g. the aromatic hydroxylations of toluene) were most effectively induced by ethylbenzene, n-propylbenzene and m-xylene. In the second, induction was observed only after m-xylene treatment, a pattern that was found when the metabolism of the substrate was catalysed by both the P450 2B subfamily and P450 2C11. Hydrocarbons that both induced P450 2B1/2B2 and suppressed P450 2C11 (such as ethylbenzene and n-propylbenzene) showed little change in activities catalysed by both isozymes (e.g. aliphatic hydroxylation of toluene, and aniline hydroxylation); however, m-xylene treatment led to elevated P450 2B1/2B2 levels without significantly suppressing P450 2C11. m-Xylene produced significant increases in activities efficiently catalysed by both isozymes. Therefore, the unique induction pattern observed after m-xylene treatment can be accounted for by induction of P450 2B1/2B2 without concomitant suppression of P450 2C11.

Prostacyclin induction by high-density lipoprotein (HDL) in vascular smooth muscle cells depends on sphingosine 1-phosphate receptors: effect of simvastatin.

PubMed

González-Díez, María; Rodríguez, Cristina; Badimon, Lina; Martínez-González, José

2008-07-01

Prostacyclin (PGI2) is an important regulator of vascular homeostasis. Our goal was to analyze the role of sphingosine 1-phosphate (S1P) and its receptors in the up-regulation of cyclooxygenase-2 (Cox-2) induced by HDL in human vascular smooth muscle cells (VSMC). S1P induces Cox-2 expression in a time-and dose-dependent manner at concentrations (0.02-1 microM) compatible with those present in physiological HDL levels. The effect was mimicked by dihydro-S1P (DhS1P), a S1P derivative that only acts through cell surface S1P receptors. Desensitization of S1P receptors with S1P (or DhS1P) abolished HDL-induced Cox-2 up-regulation and PGI2 release. Inhibition of S1P receptors by suramin (inhibitor of S1P3), JTE013 (inhibitor of S1P2) or VPC23019 (inhibitor of S1P1 and S1P3) reduced the up-regulation of Cox-2 induced by HDL and S1P. The combination of suramin and JTE013 increased the inhibitory effect compared to that observed in cells treated with each inhibitor alone. siRNA against S1P2 or S1P3 significantly reduced the ability of HDL and S1P to up-regulate Cox-2. Simvastatin induced over-expression of S1P3 and potentiated the induction of Cox-2 expression produced by HDL (or S1P). Finally, suramin, JTE013 and VPC23019 inhibited p38 MAPK and ERK1/2 signaling pathways activated by HDL (or S1P) and the downstream activation of CREB, a key transcription factor involved in Cox-2 transcriptional up-regulation. These results indicate that S1P receptors, in particular S1P2 and S1P3, are involved in the Cox-2-dependent effects of HDL on vascular cells. Strategies aimed to therapeutically modulate S1P or S1P receptors could be useful to improve cardiovascular protection.

The human estrogen receptor can regulate exogenous but not endogenous vitellogenin gene promoters in a Xenopus cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seiler-Tuyns, A.; Merillat, A.M.; Haefliger, D.N.

Transfection of a human estrogen receptor cDNA expression vector (HEO) into cultured Xenopus kidney cells confers estrogen responsiveness to the recipient cells as demonstrated by the hormone dependent expression of co-transfected Xenopus vitellogenin-CAT chimeric genes. The estrogen stimulation of these vit-CAT genes is dependent upon the presence of the vitellogenin estrogen responsive element (ERE) in their 5{prime} flanking region. Thus, functional human estrogen receptor (hER) can be synthesized in heterologous lower vertebrate cells and can act as a trans-acting regulatory factor that is necessary, together with estradiol, for the induction of the vit-CAT constructs in these cells. In addition, vitellogeninmore » minigenes co-transfected with the HEO expression vector also respond to hormonal stimulation. Their induction is not higher than that of the vit-CAT chimeric genes. It suggests that in the Xenopus kidney cell line B 3.2, the structural parts of the vitellogenin minigenes do not play a role in the induction process. Furthermore, no stabilizing effect of estrogen on vitellogenin mRNA is observed in these cells.« less

Prioritizing plant defence over growth through WRKY regulation facilitates infestation by non-target herbivores

PubMed Central

Li, Ran; Zhang, Jin; Li, Jiancai; Zhou, Guoxin; Wang, Qi; Bian, Wenbo; Erb, Matthias; Lou, Yonggen

2015-01-01

Plants generally respond to herbivore attack by increasing resistance and decreasing growth. This prioritization is achieved through the regulation of phytohormonal signaling networks. However, it remains unknown how this prioritization affects resistance against non-target herbivores. In this study, we identify WRKY70 as a specific herbivore-induced, mitogen-activated protein kinase-regulated rice transcription factor that physically interacts with W-box motifs and prioritizes defence over growth by positively regulating jasmonic acid (JA) and negatively regulating gibberellin (GA) biosynthesis upon attack by the chewing herbivore Chilo suppressalis. WRKY70-dependent JA biosynthesis is required for proteinase inhibitor activation and resistance against C. suppressalis. In contrast, WRKY70 induction increases plant susceptibility against the rice brown planthopper Nilaparvata lugens. Experiments with GA-deficient rice lines identify WRKY70-dependent GA signaling as the causal factor in N. lugens susceptibility. Our study shows that prioritizing defence over growth leads to a significant resistance trade-off with important implications for the evolution and agricultural exploitation of plant immunity. DOI: http://dx.doi.org/10.7554/eLife.04805.001 PMID:26083713

Single-Cell Analysis of Experience-Dependent Transcriptomic States in Mouse Visual Cortex

PubMed Central

Hrvatin, Sinisa; Hochbaum, Daniel R.; Nagy, M. Aurel; Cicconet, Marcelo; Robertson, Keiramarie; Cheadle, Lucas; Zilionis, Rapolas; Ratner, Alex; Borges-Monroy, Rebeca; Klein, Allon M.; Sabatini, Bernardo L.; Greenberg, Michael E.

2017-01-01

Activity-dependent transcriptional responses shape cortical function. However, we lack a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease. Here we applied high-throughput single-cell RNA-sequencing to investigate the breadth of transcriptional changes that occur across cell types in mouse visual cortex following exposure to light. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibit inter- and intra-laminar heterogeneity in the induction of stimulus responsive genes. Non-neuronal cells demonstrated clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of stimulus-dependent transcriptional changes that occur across cell types in visual cortex, which are likely critical for cortical function and may be sites of de-regulation in developmental brain disorders. PMID:29230054

Mechanotransductive cascade of Myo-II-dependent mesoderm and endoderm invaginations in embryo gastrulation

PubMed Central

Mitrossilis, Démosthène; Röper, Jens-Christian; Le Roy, Damien; Driquez, Benjamin; Michel, Aude; Ménager, Christine; Shaw, Gorky; Le Denmat, Simon; Ranno, Laurent; Dumas-Bouchiat, Frédéric; Dempsey, Nora M.; Farge, Emmanuel

2017-01-01

Animal development consists of a cascade of tissue differentiation and shape change. Associated mechanical signals regulate tissue differentiation. Here we demonstrate that endogenous mechanical cues also trigger biochemical pathways, generating the active morphogenetic movements shaping animal development through a mechanotransductive cascade of Myo-II medio-apical stabilization. To mimic physiological tissue deformation with a cell scale resolution, liposomes containing magnetic nanoparticles are injected into embryonic epithelia and submitted to time-variable forces generated by a linear array of micrometric soft magnets. Periodic magnetically induced deformations quantitatively phenocopy the soft mechanical endogenous snail-dependent apex pulsations, rescue the medio-apical accumulation of Rok, Myo-II and subsequent mesoderm invagination lacking in sna mutants, in a Fog-dependent mechanotransductive process. Mesoderm invagination then activates Myo-II apical accumulation, in a similar Fog-dependent mechanotransductive process, which in turn initiates endoderm invagination. This reveals the existence of a highly dynamic self-inductive cascade of mesoderm and endoderm invaginations, regulated by mechano-induced medio-apical stabilization of Myo-II. PMID:28112149

Mechanotransductive cascade of Myo-II-dependent mesoderm and endoderm invaginations in embryo gastrulation

NASA Astrophysics Data System (ADS)

Mitrossilis, Démosthène; Röper, Jens-Christian; Le Roy, Damien; Driquez, Benjamin; Michel, Aude; Ménager, Christine; Shaw, Gorky; Le Denmat, Simon; Ranno, Laurent; Dumas-Bouchiat, Frédéric; Dempsey, Nora M.; Farge, Emmanuel

2017-01-01

Animal development consists of a cascade of tissue differentiation and shape change. Associated mechanical signals regulate tissue differentiation. Here we demonstrate that endogenous mechanical cues also trigger biochemical pathways, generating the active morphogenetic movements shaping animal development through a mechanotransductive cascade of Myo-II medio-apical stabilization. To mimic physiological tissue deformation with a cell scale resolution, liposomes containing magnetic nanoparticles are injected into embryonic epithelia and submitted to time-variable forces generated by a linear array of micrometric soft magnets. Periodic magnetically induced deformations quantitatively phenocopy the soft mechanical endogenous snail-dependent apex pulsations, rescue the medio-apical accumulation of Rok, Myo-II and subsequent mesoderm invagination lacking in sna mutants, in a Fog-dependent mechanotransductive process. Mesoderm invagination then activates Myo-II apical accumulation, in a similar Fog-dependent mechanotransductive process, which in turn initiates endoderm invagination. This reveals the existence of a highly dynamic self-inductive cascade of mesoderm and endoderm invaginations, regulated by mechano-induced medio-apical stabilization of Myo-II.

The U6 snRNA m6A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention.

PubMed

Pendleton, Kathryn E; Chen, Beibei; Liu, Kuanqing; Hunter, Olga V; Xie, Yang; Tu, Benjamin P; Conrad, Nicholas K

2017-05-18

Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N 6 -adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

Persistence of fan-shaped keratocytes is a matrix-rigidity-dependent mechanism that requires α5β1 integrin engagement.

PubMed

Riaz, Maryam; Versaevel, Marie; Mohammed, Danahe; Glinel, Karine; Gabriele, Sylvain

2016-09-28

Despite the importance of matrix rigidity on cell functions, many aspects of the mechanosensing process in highly migratory cells remain elusive. Here, we studied the migration of highly motile keratocytes on culture substrates with similar biochemical properties and rigidities spanning the range between soft tissues (~kPa) and stiff culture substrates (~GPa). We show that morphology, polarization and persistence of motile keratocytes are regulated by the matrix stiffness over seven orders of magnitude, without changing the cell spreading area. Increasing the matrix rigidity leads to more F-actin in the lamellipodia and to the formation of mature contractile actomyosin fibers that control the cell rear retraction. Keratocytes remain rounded and form nascent adhesions on compliant substrates, whereas large and uniformly distributed focal adhesions are formed on fan-shaped keratocytes migrating on rigid surfaces. By combining poly-L-lysine, fibronectin and vitronectin coatings with selective blocking of α v β 3 or α 5 β 1 integrins, we show that α V β 3 integrins permit the spreading of keratocytes but are not sufficient for polarization and rigidity sensing that require the engagement of α 5 β 1 integrins. Our study demonstrates a matrix rigidity-dependent regulation of the directional persistence in motile keratocytes and refines the role of α v β 3 and α 5 β 1 integrins in the molecular clutch model.

Sodium-dependent bile salt transporters of the SCL10A Transporter Family: More than solute transporters

PubMed Central

Anwer, M. Sawkat; Stieger, Bruno

2013-01-01

Summary The SLC10A transporter gene family consists of seven members and substrates transported by three members (SLC10A1, SLC10A2 and SLC10A6) are Na+-dependent. SLC10A1 (sodium taurocholate cotransporting polypeptide or NTCP) and SLC10A2 (apical sodium-dependent bile salt transporter or ASBT) transport bile salts and play an important role in maintaining enterohepatic circulation of bile salts. Solutes other than bile salts are also transported by NTCP. However, ASBT has not been shown to be a transporter for non-bile salt substrates. While the transport function of NTCP can potentially be used as liver function test, interpretation of such a test may be complicated by altered expression of NTCP in diseases and presence of drugs that may inhibit NTCP function. Transport of bile salts by NTCP and ASBT is inhibited by a number of drugs and it appears that ASBT is more permissive to drug inhibition than NTCP. The clinical significance of this inhibition in drug disposition and drug-drug interaction remains to be determined. Both NCTP and ASBT undergo post-translational regulations that involve phosphorylation/dephosphorylation, translocation to and retrieval from the plasma membrane and degradation by the ubiquitin-proteasome system. These posttranslational regulations are mediated via signaling pathways involving cAMP, calcium, nitric oxide, phosphoinositide-3-kinase (PI3K), protein kinase C (PKC) and protein phosphatases. There appears to be species difference in the substrate specificity and the regulation of plasma membrane localization of human and rodent NTCP. These differences should be taken into account when extrapolating rodent data for human clinical relevance and developing novel therapies. NTCP has recently been shown to play an important role in HBV and HDV infection by serving as a receptor for entry of these viruses into hepatocytes. PMID:24196564

Zinc Regulation of Aminopeptidase B Involved in Neuropeptide Production

PubMed Central

Hwang, Shin-Rong; Hook, Vivian

2009-01-01

Aminopeptidase B (AP-B) is a metallopeptidase that removes basic residues from the N-termini of neuropeptide substrates in secretory vesicles. This study assessed zinc regulation of AP-B activity, since secretory vesicles contain endogenous zinc. AP-B was inhibited by zinc at concentrations typically present in secretory vesicles. Zinc effects were dependent on concentration, incubation time, and the molar ratio of zinc to enzyme. AP-B activity was recovered upon removal of zinc. AP-B with zinc became susceptible to degradation by trypsin, suggesting that zinc alters enzyme conformation. Zinc regulation demonstrates the metallopeptidase property of AP-B. PMID:18571504

Interleukin 4 signals through two related pathways.

PubMed Central

Pernis, A; Witthuhn, B; Keegan, A D; Nelms, K; Garfein, E; Ihle, J N; Paul, W E; Pierce, J H; Rothman, P

1995-01-01

The interleukin 4 (IL-4) signaling pathway involves activation, by tyrosine phosphorylation, of two distinct substrates, a signal-transducing factor (STF-IL4) and the IL-4-induced phosphotyrosine substrate (4PS). It is not known whether the IL-4-mediated activation of these substrates occurs via related or distinct signaling pathways. We report that 32D cells, an IL-3-dependent myeloid progenitor cell line in which no phosphorylated 4PS is found, activate high levels of STF-IL4 in response to IL-4. Consistent with the known requirement for 4PS or insulin receptor substrate 1 (IRS-1) in IL-4-mediated mitogenesis, activation of STF-IL4 in 32D cells is not sufficient for IL-4-inducible c-myc expression. In addition, we have examined the ability of 32D cells transfected with different truncation mutants of the human IL-4 receptor to activate Jak-3 kinase and STF-IL4 in response to human IL-4. As in the case of 4PS/IRS-1, we have found that activation of both Jak-3 and STF-IL4 requires the presence of the IL-4 receptor region comprising aa 437-557. The finding that the same region of the IL-4 receptor is required for the induction of both 4PS/IRS-1 and STF-IL4 suggests that the IL-4-stimulated activation of these two substrates might involve common factors. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7544011

JAK2-V617F-induced MAPK activity is regulated by PI3K and acts synergistically with PI3K on the proliferation of JAK2-V617F-positive cells

PubMed Central

Wolf, Alexandra; Eulenfeld, René; Gäbler, Karoline; Rolvering, Catherine; Haan, Serge; Behrmann, Iris; Denecke, Bernd; Haan, Claude; Schaper, Fred

2013-01-01

The identification of a constitutively active JAK2 mutant, namely JAK2-V617F, was a milestone in the understanding of Philadelphia chromosome-negative myeloproliferative neoplasms. The JAK2-V617F mutation confers cytokine hypersensitivity, constitutive activation of the JAK-STAT pathway, and cytokine-independent growth. In this study we investigated the mechanism of JAK2-V617F-dependent signaling with a special focus on the activation of the MAPK pathway. We observed JAK2-V617F-dependent deregulated activation of the multi-site docking protein Gab1 as indicated by constitutive, PI3K-dependent membrane localization and tyrosine phosphorylation of Gab1. Furthermore, we demonstrate that PI3K signaling regulates MAPK activation in JAK2-V617F-positve cells. This cross-regulation of the MAPK pathway by PI3K affects JAK2-V617F-specific target gene induction, erythroid colony formation, and regulates proliferation of JAK2-V617F-positive patient cells in a synergistically manner. PMID:24069558

MARCH2 regulates autophagy by promoting CFTR ubiquitination and degradation and PIK3CA-AKT-MTOR signaling.

PubMed

Xia, Dan; Qu, Liujing; Li, Ge; Hongdu, Beiqi; Xu, Chentong; Lin, Xin; Lou, Yaxin; He, Qihua; Ma, Dalong; Chen, Yingyu

2016-09-01

MARCH2 (membrane-associated RING-CH protein 2), an E3 ubiquitin ligase, is mainly associated with the vesicle trafficking. In the present study, for the first time, we demonstrated that MARCH2 negatively regulates autophagy. Our data indicated that overexpression of MARCH2 impaired autophagy, as evidenced by attenuated levels of LC3B-II and impaired degradation of endogenous and exogenous autophagic substrates. By contrast, loss of MARCH2 expression had the opposite effects. In vivo experiments demonstrate that MARCH2 knockout mediated autophagy results in an inhibition of tumorigenicity. Further investigation revealed that the induction of autophagy by MARCH2 deficiency was mediated through the PIK3CA-AKT-MTOR signaling pathway. Additionally, we found that MARCH2 interacts with CFTR (cystic fibrosis transmembrane conductance regulator), promotes the ubiquitination and degradation of CFTR, and inhibits CFTR-mediated autophagy in tumor cells. The functional PDZ domain of MARCH2 is required for the association with CFTR. Thus, our study identified a novel negative regulator of autophagy and suggested that the physical and functional connection between the MARCH2 and CFTR in different conditions will be elucidated in the further experiments.

The Quinone Methide Aurin Is a Heat Shock Response Inducer That Causes Proteotoxic Stress and Noxa-dependent Apoptosis in Malignant Melanoma Cells*

PubMed Central

Davis, Angela L.; Qiao, Shuxi; Lesson, Jessica L.; Rojo de la Vega, Montserrat; Park, Sophia L.; Seanez, Carol M.; Gokhale, Vijay; Cabello, Christopher M.; Wondrak, Georg T.

2015-01-01

Pharmacological induction of proteotoxic stress is rapidly emerging as a promising strategy for cancer cell-directed chemotherapeutic intervention. Here, we describe the identification of a novel drug-like heat shock response inducer for the therapeutic induction of proteotoxic stress targeting malignant human melanoma cells. Screening a focused library of compounds containing redox-directed electrophilic pharmacophores employing the Stress & Toxicity PathwayFinderTM PCR Array technology as a discovery tool, a drug-like triphenylmethane-derivative (aurin; 4-[bis(p-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one) was identified as an experimental cell stress modulator that causes (i) heat shock factor transcriptional activation, (ii) up-regulation of heat shock response gene expression (HSPA6, HSPA1A, DNAJB4, HMOX1), (iii) early unfolded protein response signaling (phospho-PERK, phospho-eIF2α, CHOP (CCAAT/enhancer-binding protein homologous protein)), (iv) proteasome impairment with increased protein-ubiquitination, and (v) oxidative stress with glutathione depletion. Fluorescence polarization-based experiments revealed that aurin displays activity as a geldanamycin-competitive Hsp90α-antagonist, a finding further substantiated by molecular docking and ATPase inhibition analysis. Aurin exposure caused caspase-dependent cell death in a panel of human malignant melanoma cells (A375, G361, LOX-IMVI) but not in non-malignant human skin cells (Hs27 fibroblasts, HaCaT keratinocytes, primary melanocytes) undergoing the aurin-induced heat shock response without impairment of viability. Aurin-induced melanoma cell apoptosis depends on Noxa up-regulation as confirmed by siRNA rescue experiments demonstrating that siPMAIP1-based target down-regulation suppresses aurin-induced cell death. Taken together, our data suggest feasibility of apoptotic elimination of malignant melanoma cells using the quinone methide-derived heat shock response inducer aurin. PMID:25477506

Genetic modification of alternative respiration in Nicotiana benthamiana affects basal and salicylic acid-induced resistance to potato virus X

PubMed Central

2011-01-01

Background Salicylic acid (SA) regulates multiple anti-viral mechanisms, including mechanism(s) that may be negatively regulated by the mitochondrial enzyme, alternative oxidase (AOX), the sole component of the alternative respiratory pathway. However, studies of this mechanism can be confounded by SA-mediated induction of RNA-dependent RNA polymerase 1, a component of the antiviral RNA silencing pathway. We made transgenic Nicotiana benthamiana plants in which alternative respiratory pathway capacity was either increased by constitutive expression of AOX, or decreased by expression of a dominant-negative mutant protein (AOX-E). N. benthamiana was used because it is a natural mutant that does not express a functional RNA-dependent RNA polymerase 1. Results Antimycin A (an alternative respiratory pathway inducer and also an inducer of resistance to viruses) and SA triggered resistance to tobacco mosaic virus (TMV). Resistance to TMV induced by antimycin A, but not by SA, was inhibited in Aox transgenic plants while SA-induced resistance to this virus appeared to be stronger in Aox-E transgenic plants. These effects, which were limited to directly inoculated leaves, were not affected by the presence or absence of a transgene constitutively expressing a functional RNA-dependent RNA polymerase (MtRDR1). Unexpectedly, Aox-transgenic plants infected with potato virus X (PVX) showed markedly increased susceptibility to systemic disease induction and virus accumulation in inoculated and systemically infected leaves. SA-induced resistance to PVX was compromised in Aox-transgenic plants but plants expressing AOX-E exhibited enhanced SA-induced resistance to this virus. Conclusions We conclude that AOX-regulated mechanisms not only play a role in SA-induced resistance but also make an important contribution to basal resistance against certain viruses such as PVX. PMID:21356081

Molecular resolution friction microscopy of Cu phthalocyanine thin films on dolomite (104) in water

NASA Astrophysics Data System (ADS)

Nita, Paweł; Pimentel, Carlos; Luo, Feng; Milián-Medina, Begoña; Gierschner, Johannes; Pina, Carlos M.; Gnecco, Enrico

2014-06-01

The reliability of ultrathin organic layers as active components for molecular electronic devices depends ultimately on an accurate characterization of the layer morphology and ability to withstand mechanical stresses on the nanoscale. To this end, since the molecular layers need to be electrically decoupled using thick insulating substrates, the use of AFM becomes mandatory. Here, we show how friction force microscopy (FFM) in water allows us to identify the orientation of copper(ii)phthalocyanine (CuPc) molecules previously self-assembled on a dolomite (104) mineral surface in ultra-high vacuum. The molecular features observed in the friction images show that the CuPc molecules are stacked in parallel rows with no preferential orientation with respect to the dolomite lattice, while the stacking features resemble well the single CuPc crystal structure. This proves that the substrate induction is low and makes friction force microscopy in water a suitable alternative to more demanding dynamic AFM techniques in ultra-high vacuum.

Luminogenic cytochrome P450 assays.

PubMed

Cali, James J; Ma, Dongping; Sobol, Mary; Simpson, Daniel J; Frackman, Susan; Good, Troy D; Daily, William J; Liu, David

2006-08-01

Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

Molecular resolution friction microscopy of Cu phthalocyanine thin films on dolomite (104) in water.

PubMed

Nita, Paweł; Pimentel, Carlos; Luo, Feng; Milián-Medina, Begoña; Gierschner, Johannes; Pina, Carlos M; Gnecco, Enrico

2014-07-21

The reliability of ultrathin organic layers as active components for molecular electronic devices depends ultimately on an accurate characterization of the layer morphology and ability to withstand mechanical stresses on the nanoscale. To this end, since the molecular layers need to be electrically decoupled using thick insulating substrates, the use of AFM becomes mandatory. Here, we show how friction force microscopy (FFM) in water allows us to identify the orientation of copper(ii)phthalocyanine (CuPc) molecules previously self-assembled on a dolomite (104) mineral surface in ultra-high vacuum. The molecular features observed in the friction images show that the CuPc molecules are stacked in parallel rows with no preferential orientation with respect to the dolomite lattice, while the stacking features resemble well the single CuPc crystal structure. This proves that the substrate induction is low and makes friction force microscopy in water a suitable alternative to more demanding dynamic AFM techniques in ultra-high vacuum.

Dissecting the Impact of Matrix Anchorage and Elasticity in Cell Adhesion

PubMed Central

Pompe, Tilo; Glorius, Stefan; Bischoff, Thomas; Uhlmann, Ina; Kaufmann, Martin; Brenner, Sebastian; Werner, Carsten

2009-01-01

Abstract Extracellular matrices determine cellular fate decisions through the regulation of intracellular force and stress. Previous studies suggest that matrix stiffness and ligand anchorage cause distinct signaling effects. We show herein how defined noncovalent anchorage of adhesion ligands to elastic substrates allows for dissection of intracellular adhesion signaling pathways related to matrix stiffness and receptor forces. Quantitative analysis of the mechanical balance in cell adhesion using traction force microscopy revealed distinct scalings of the strain energy imparted by the cells on the substrates dependent either on matrix stiffness or on receptor force. Those scalings suggested the applicability of a linear elastic theoretical framework for the description of cell adhesion in a certain parameter range, which is cell-type-dependent. Besides the deconvolution of biophysical adhesion signaling, site-specific phosphorylation of focal adhesion kinase, dependent either on matrix stiffness or on receptor force, also demonstrated the dissection of biochemical signaling events in our approach. Moreover, the net contractile moment of the adherent cells and their strain energy exerted on the elastic substrate was found to be a robust measure of cell adhesion with a unifying power-law scaling exponent of 1.5 independent of matrix stiffness. PMID:19843448

Regulated overexpression of interleukin 11 in the lung. Use to dissociate development-dependent and -independent phenotypes.

PubMed Central

Ray, P; Tang, W; Wang, P; Homer, R; Kuhn, C; Flavell, R A; Elias, J A

1997-01-01

Standard overexpression transgenic approaches are limited in their ability to model waxing and waning diseases and frequently superimpose development-dependent and -independent phenotypic manifestations. We used the clara cell 10-kD protein (CC10) promoter and the reverse tetracycline transactivator (rtTA) to create a lung-specific, externally regulatable, overexpression transgenic system and used this system to express human interleukin 11 (IL-11) in respiratory structures. Gene induction could be achieved in utero, in neonates and in adult animals. Moreover, gene expression could be turned off by removal of the inducing stimulus. When gene activation was initiated in utero and continued into adulthood, subepithelial airway fibrosis, peribronchiolar mononuclear nodules, and alveolar enlargement (emphysema) were noted. Induction in the mature lung caused airway remodeling and peribronchiolar nodules, but alveolar enlargement was not appreciated. In contrast, induction in utero and during the first 14 d of life caused alveolar enlargement without airway remodeling or peribronchiolar nodules. Thus, IL-11 overexpression causes abnormalities that are dependent (large alveoli) and independent (airway remodeling, peribronchiolar nodules) of lung growth and development, and the CC10-rtTA system can be used to differentiate among these effector functions. The CC10-rtTA transgenic system can be used to model waxing and waning, childhood and growth and development-related biologic processes with enhanced fidelity. PMID:9366564

Mechanics regulates ATP-stimulated collective calcium response in fibroblast cells

PubMed Central

Lembong, Josephine; Sabass, Benedikt; Sun, Bo; Rogers, Matthew E.; Stone, Howard A.

2015-01-01

Cells constantly sense their chemical and mechanical environments. We study the effect of mechanics on the ATP-induced collective calcium response of fibroblast cells in experiments that mimic various tissue environments. We find that closely packed two-dimensional cell cultures on a soft polyacrylamide gel (Young's modulus E = 690 Pa) contain more cells exhibiting calcium oscillations than cultures on a rigid substrate (E = 36 000 Pa). Calcium responses of cells on soft substrates show a slower decay of calcium level relative to those on rigid substrates. Actin enhancement and disruption experiments for the cell cultures allow us to conclude that actin filaments determine the collective Ca2+ oscillatory behaviour in the culture. Inhibition of gap junctions results in a decrease of the oscillation period and reduced correlation of calcium responses, which suggests additional complexity of signalling upon cell–cell contact. Moreover, the frequency of calcium oscillations is independent of the rigidity of the substrate but depends on ATP concentration. We compare our results with those from similar experiments on individual cells. Overall, our observations show that collective chemical signalling in cell cultures via calcium depends critically on the mechanical environment. PMID:26063818

Kinetic characterization of Vibrio cholerae ApbE: Substrate specificity and regulatory mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Xuan; Liang, Pingdong; Raba, Daniel Alexander

ApbE is a member of a novel family of flavin transferases that incorporates flavin mononucleotide (FMN) to subunits of diverse respiratory complexes, which fulfill important homeostatic functions. In this work a detailed characterization of Vibrio cholerae ApbE physiologic activity, substrate specificity and pH dependency was carried out. The data obtained show novel characteristics of the regulation and function of this family. For instance, our experiments indicate that divalent cations are essential for ApbE function, and that the selectivity depends largely on size and the coordination sphere of the cation. Our data also show that ApbE regulation by pH, ADP andmore » potassium is an important mechanism that enhances the adaptation, survival and colonization of V. cholerae in the small intestine. Moreover, studies of the pH-dependency of the activity show that the reaction is favored under alkaline conditions, with a pKa of 8.4. These studies, together with sequence and structure analysis allowed us to identify His257, which is absolutely conserved in the family, as a candidate for the residue whose deprotonation controls the activity. Remarkably, the mutant H257G abolished the flavin transfer activity, strongly indicating that this residue plays an important role in the catalytic mechanism of ApbE.« less

Evaluation of inductively heated ferromagnetic alloy implants for therapeutic interstitial hyperthermia.

PubMed

Paulus, J A; Richardson, J S; Tucker, R D; Park, J B

1996-04-01

Ferromagnetic alloys heated by magnetic induction have been investigated as interstitial hyperthermia delivery implants for over a decade, utilizing low Curie temperatures to provide thermal self-regulation. The minimally invasive method is attractive for fractionated thermal treatment of tumors which are not easily heated by focused microwave or ultrasound techniques. Past analyses of ferromagnetic seeds by other authors depict poor experimental correlation with theoretical heating predictions. Improvements in computer hardware and commercially available finite element analysis software have simplified the analysis of inductively heated thermal seeds considerably. This manuscript examines end effects of finite length implants and nonlinear magnetic material properties to account for previous inconsistencies. Two alloys, Ni-28 wt% Cu (NiCu) and Pd-6.15 wt% Co (PdCo), were used for comparison of theoretical and experimental calorimetric results. Length to diameter (L/d) ratios of over 20 for cylindrical seeds are necessary for minimization of end effects. Magnetic properties tested for alloys of NiCu and PdCo illustrate considerable nonlinearity of these materials in field strength ranges used for induction heating. Field strength dependent magnetic permeabilities and calorimetric data illustrate that more detailed material information must be included to accurately estimate induction power loss for these implants.

Clonorchis sinensis excretory-secretory products regulate migration and invasion in cholangiocarcinoma cells via extracellular signal-regulated kinase 1/2/nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

PubMed

Pak, Jhang Ho; Shin, Jimin; Song, In-Sung; Shim, Sungbo; Jang, Sung-Wuk

2017-01-01

Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory-secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory-secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory-secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory-secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory-secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory-secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Insulin receptor substrate-2 regulates aerobic glycolysis in mouse mammary tumor cells via glucose transporter 1.

PubMed

Pankratz, Shannon L; Tan, Ernest Y; Fine, Yumiko; Mercurio, Arthur M; Shaw, Leslie M

2009-01-23

The insulin receptor substrate (IRS) proteins are cytoplasmic adaptor molecules that function as signaling intermediates downstream of activated cell surface receptors. Based on data implicating IRS-2 but not IRS-1 in breast cancer invasion, survival, and metastasis, we assessed the contribution of IRS-1 and IRS-2 to aerobic glycolysis, which is known to impact tumor growth and progression. For this purpose, we used tumor cell lines derived from transgenic mice that express the polyoma virus middle T antigen (PyV-MT) in the mammary gland and that are wild-type (WT) or null for either Irs-1 (Irs-1-/-) or Irs-2 (Irs-2-/-). Aerobic glycolysis, as assessed by the rate of lactic acid production and glucose consumption, was diminished significantly in Irs-2-/- cells when compared with WT and Irs-1-/- cells. Expression of exogenous Irs-2 in Irs-2-/- cells restored the rate of glycolysis to that observed in WT cells. The transcription factor FoxO1 does not appear to be involved in Irs-2-mediated glycolysis. However, Irs-2 does regulate the surface expression of glucose transporter 1 (Glut1) as assessed by flow cytometry using a Glut1-specific ligand. Suppression of Glut1 expression inhibits Irs-2-dependent invasion, which links glycolysis to mammary tumor progression. Irs-2 was shown to be important for mammalian target of rapamycin (mTor) activation, and Irs-2-dependent regulation of Glut1 surface expression is rapamycin-sensitive. Collectively, our data indicate that Irs-2, but not Irs-1, promotes invasion by sustaining the aerobic glycolysis of mouse mammary tumor cells and that it does so by regulating the mTor-dependent surface expression of Glut1.

Zn2+-dependent Activation of the Trk Signaling Pathway Induces Phosphorylation of the Brain-enriched Tyrosine Phosphatase STEP

PubMed Central

Poddar, Ranjana; Rajagopal, Sathyanarayanan; Shuttleworth, C. William; Paul, Surojit

2016-01-01

Excessive release of Zn2+ in the brain is implicated in the progression of acute brain injuries. Although several signaling cascades have been reported to be involved in Zn2+-induced neurotoxicity, a potential contribution of tyrosine phosphatases in this process has not been well explored. Here we show that exposure to high concentrations of Zn2+ led to a progressive increase in phosphorylation of the striatal-enriched phosphatase (STEP), a component of the excitotoxic-signaling pathway that plays a role in neuroprotection. Zn2+-mediated phosphorylation of STEP61 at multiple sites (hyperphosphorylation) was induced by the up-regulation of brain-derived neurotropic factor (BDNF), tropomyosin receptor kinase (Trk) signaling, and activation of cAMP-dependent PKA (protein kinase A). Mutational studies further show that differential phosphorylation of STEP61 at the PKA sites, Ser-160 and Ser-221 regulates the affinity of STEP61 toward its substrates. Consistent with these findings we also show that BDNF/Trk/PKA mediated signaling is required for Zn2+-induced phosphorylation of extracellular regulated kinase 2 (ERK2), a substrate of STEP that is involved in Zn2+-dependent neurotoxicity. The strong correlation between the temporal profile of STEP61 hyperphosphorylation and ERK2 phosphorylation indicates that loss of function of STEP61 through phosphorylation is necessary for maintaining sustained ERK2 phosphorylation. This interpretation is further supported by the findings that deletion of the STEP gene led to a rapid and sustained increase in ERK2 phosphorylation within minutes of exposure to Zn2+. The study provides further insight into the mechanisms of regulation of STEP61 and also offers a molecular basis for the Zn2+-induced sustained activation of ERK2. PMID:26574547

Feedback regulated induction heater for a flowing fluid

DOEpatents

Migliori, Albert; Swift, Gregory W.

1985-01-01

A regulated induction heater for heating a stream of flowing fluid to a predetermined desired temperature. The heater includes a radiofrequency induction coil which surrounds a glass tube through which the fluid flows. A heating element consisting of a bundle of approximately 200 stainless steel capillary tubes located within the glass tube couples the output of the induction coil to the fluid. The temperature of the fluid downstream from the heating element is sensed with a platinum resistance thermometer, the output of which is applied to an adjustable proportional and integral feedback control circuit which regulates the power applied to the induction coil. The heater regulates the fluid temperature to within 0.005.degree. C. at a flow rate of 50 cm.sup.3 /second with a response time of less than 0.1 second, and can accommodate changes in heat load up to 1500 watts.

Feedback regulated induction heater for a flowing fluid

DOEpatents

Migliori, A.; Swift, G.W.

1984-06-13

A regulated induction heater for heating a stream of flowing fluid to a predetermined desired temperature. The heater includes a radiofrequency induction coil which surrounds a glass tube through which the fluid flows. A heating element consisting of a bundle of approximately 200 stainless steel capillary tubes located within the glass tube couples the output of the induction coil to the fluid. The temperature of the fluid downstream from the heating element is sensed with a platinum resistance thermometer, the output of which is applied to an adjustable porportional and integral feedback control circuit which regulates the power applied to the induction coil. The heater regulates the fluid temperature to within 0.005/sup 0/C at a flow rate of 50 cm/sup 3//sec with a response time of less than 0.1 second, and can accommodate changes in heat load up to 1500 watts.

TGFβ1-induced down-regulation of microRNA-138 contributes to epithelial-mesenchymal transition in primary lung cancer cells.

PubMed

Zhang, Fang; Li, Tiepeng; Han, Lu; Qin, Peng; Wu, Zhao; Xu, Benling; Gao, Quanli; Song, Yongping

2018-02-19

The existence of cancer stem cells within the tumor could lead to cancer therapy resistance. TGFβ1 is considered as one of the most powerful players in the generation of CSCs through induction of epithelial-mesenchymal transition in different types of cancer including lung cancer, however, the detailed mechanisms by which TGFβ1 contribute to EMT induction and CSC maintenance remains unclear. Here, we showed primary lung cancer cells treated by TGFβ1 exhibit mesenchymal features, including morphology and expression of mesenchymal marker in a time-dependent manner. We also observed long-term TGFβ1 exposure leads to an enrichment of a sub-population of CD44 + CD90 + cells which represent CSCs in lung cancer cells. Moreover, the differential expression microRNAs between CSCs and non-CSCs were identified using next-generation sequencing to screen key miRNAs which might contribute to TGFβ1-induced EMT and CSCs generation. Among those differentially expressed miRNAs, the expression of microRNA-138 was time-dependently down-regulated by TGFβ1 treatment. We further demonstrated primary lung cancer cells, in which we knockdown the expression of miR-138, exhibit mesenchymal phenotypes and stem cell properties. Taken together, these findings indicate TGFβ1-induced down-regulation of microRNA-138 contributes to EMT in primary lung cancer cells, and suggest that miR-138 might serve as a potential therapeutic target. Copyright © 2018 Elsevier Inc. All rights reserved.

Autophagy induced by AXL receptor tyrosine kinase alleviates acute liver injury via inhibition of NLRP3 inflammasome activation in mice.

PubMed

Han, Jihye; Bae, Joonbeom; Choi, Chang-Yong; Choi, Sang-Pil; Kang, Hyung-Sik; Jo, Eun-Kyeong; Park, Jongsun; Lee, Young Sik; Moon, Hyun-Seuk; Park, Chung-Gyu; Lee, Myung-Shik; Chun, Taehoon

2016-12-01

Severe hepatic inflammation is a common cause of acute or chronic liver disease. Macrophages are one of the key mediators which regulate the progress of hepatic inflammation. Increasing evidence shows that the TAM (TYRO3, AXL and MERTK) family of RTKs (receptor tyrosine kinases), which is expressed in macrophages, alleviates inflammatory responses through a negative feedback loop. However, the functional contribution of each TAM family member to the progression of hepatic inflammation remains elusive. In this study, we explore the role of individual TAM family proteins during autophagy induction and evaluate their contribution to hepatic inflammation. Among the TAM family of RTKs, AXL (AXL receptor tyrosine kinase) only induces autophagy in macrophages after interaction with its ligand, GAS6 (growth arrest specific 6). Based on our results, autophosphorylation of 2 tyrosine residues (Tyr815 and Tyr860) in the cytoplasmic domain of AXL in mice is required for autophagy induction and AXL-mediated autophagy induction is dependent on MAPK (mitogen-activated protein kinase)14 activity. Furthermore, induction of AXL-mediated autophagy prevents CASP1 (caspase 1)-dependent IL1B (interleukin 1, β) and IL18 (interleukin 18) maturation by inhibiting NLRP3 (NLR family, pyrin domain containing 3) inflammasome activation. In agreement with these observations, axl -/- mice show more severe symptoms than do wild-type (Axl +/+ ) mice following acute hepatic injury induced by administration of lipopolysaccharide (LPS) or carbon tetrachloride (CCl 4 ). Hence, GAS6-AXL signaling-mediated autophagy induction in murine macrophages ameliorates hepatic inflammatory responses by inhibiting NLRP3 inflammasome activation.

The hetC Gene Is a Direct Target of the NtcA Transcriptional Regulator in Cyanobacterial Heterocyst Development

PubMed Central

Muro-Pastor, Alicia M.; Valladares, Ana; Flores, Enrique; Herrero, Antonia

1999-01-01

The heterocyst is the site of nitrogen fixation in aerobically grown cultures of some filamentous cyanobacteria. Heterocyst development in Anabaena sp. strain PCC 7120 is dependent on the global nitrogen regulator NtcA and requires, among others, the products of the hetR and hetC genes. Expression of hetC, tested by RNA- DNA hybridization, was impaired in an ntcA mutant. A nitrogen-regulated, NtcA-dependent putative transcription start point was localized at nucleotide −571 with respect to the hetC translational start. Sequences upstream from this transcription start point exhibit the structure of the canonical cyanobacterial promoter activated by NtcA, and purified NtcA protein specifically bound to a DNA fragment containing this promoter. Activation of expression of hetC during heterocyst development appears thus to be directly operated by NtcA. NtcA-mediated activation of hetR expression was not impaired in a hetC mutant, indicating that HetC is not an NtcA-dependent element required for hetR induction. PMID:10542167

The induction of cytochrome P450 2E1 by ethanol leads to the loss of synaptic proteins via PPARα down-regulation.

PubMed

Na, Shufang; Li, Jie; Zhang, Huibo; Li, Yueran; Yang, Zheqiong; Zhong, Yanjun; Dong, Guicheng; Yang, Jing; Yue, Jiang

2017-06-15

Ethanol, one of the most commonly abused substances throughout history, is a substrate and potent inducer of cytochrome P450 2E1 (CYP2E1). Our previous study showed that brain CYP2E1 was induced by chronic ethanol treatment and was associated with ethanol-induced neurotoxicity in rats. We therefore investigated the possible mechanism of brain CYP2E1 involvement in ethanol-induced neurodegeneration. Compared with the controls, chronic ethanol treatment (3.0g/kg, i.g., 160days) significantly increased CYP2E1 mRNA levels in the rat cortex, but the mRNA levels of peroxisome proliferator-activated receptor α (PPARα) and the pre- and post-synaptic proteins (synaptophysin, SYP, and drebrin1, DBN1) were decreased. Ethanol treatment dose-dependently induced CYP2E1 mRNA expression, and CYP2E1 overexpression exacerbated the ethanol-induced neurotoxicity. Pretreatment with p38 inhibitor (SB202190) and ERK1/2 inhibitor (U0126) attenuated the induction of CYP2E1 mRNA and protein levels by ethanol; however, no change was observed with JNK inhibitor pretreatment. Ethanol exposure or CYP2E1 overexpression significantly decreased PPARα, SYP, and DBN1 expression as indicated by the data from real-time RT-PCR, Western blotting and immunocytochemistry. The activation of PPARα by WY14643 increased the activity of the SYP and DBN1 promoters and attenuated the inhibition of these genes by ethanol. The specific siRNA for CYP2E1 significantly attenuated the ethanol-induced inhibition of PPARα, SYP and DBN1 mRNA levels. These results suggest that the induction of CYP2E1 by ethanol may be mediated via the p38 and ERK1/2 signaling pathways in neurons but not via the JNK pathway. The CYP2E1-PPARα axis may play a role in ethanol-induced neurotoxicity via the alteration of the genes related with synaptic function. Copyright © 2017. Published by Elsevier B.V.

A Study of Gibberellin Homeostasis and Cryptochrome-Mediated Blue Light Inhibition of Hypocotyl Elongation1[W][OA

PubMed Central

Zhao, Xiaoying; Yu, Xuhong; Foo, Eloise; Symons, Gregory M.; Lopez, Javier; Bendehakkalu, Krishnaprasad T.; Xiang, Jing; Weller, James L.; Liu, Xuanming; Reid, James B.; Lin, Chentao

2007-01-01

Cryptochromes mediate blue light-dependent photomorphogenic responses, such as inhibition of hypocotyl elongation. To investigate the underlying mechanism, we analyzed a genetic suppressor, scc7-D (suppressors of cry1cry2), which suppressed the long-hypocotyl phenotype of the cry1cry2 (cryptochrome1/cryptochrome2) mutant in a light-dependent but wavelength-independent manner. scc7-D is a gain-of-expression allele of the GA2ox8 gene encoding a gibberellin (GA)-inactivating enzyme, GA 2-oxidase. Although scc7-D is hypersensitive to light, transgenic seedlings expressing GA2ox at a level higher than scc7-D showed a constitutive photomorphogenic phenotype, confirming a general role of GA2ox and GA in the suppression of hypocotyl elongation. Prompted by this result, we investigated blue light regulation of mRNA expression of the GA metabolic and catabolic genes. We demonstrated that cryptochromes are required for the blue light regulation of GA2ox1, GA20ox1, and GA3ox1 expression in transient induction, continuous illumination, and photoperiodic conditions. The kinetics of cryptochrome induction of GA2ox1 expression and cryptochrome suppression of GA20ox1 or GA3ox1 expression correlate with the cryptochrome-dependent transient reduction of GA4 in etiolated wild-type seedlings exposed to blue light. Therefore we propose that in deetiolating seedlings, cryptochromes mediate blue light regulation of GA catabolic/metabolic genes, which affect GA levels and hypocotyl elongation. Surprisingly, no significant change in the GA4 content was detected in the whole shoot samples of the wild-type or cry1cry2 seedlings grown in the dark or continuous blue light, suggesting that cryptochromes may also regulate GA responsiveness and/or trigger cell- or tissue-specific changes of the level of bioactive GAs. PMID:17644628

The RING-Type E3 Ligase XBAT35.2 Is Involved in Cell Death Induction and Pathogen Response1[OPEN

PubMed Central

Liu, Hongxia; Ravichandran, Sridhar; McVey, Sarah; Lilley, Carly; Teresinski, Howard J.; Gonzalez-Ferrer, Carmen; Prithiviraj, Balakrishnan

2017-01-01

XBAT35 belongs to a subfamily of Arabidopsis (Arabidopsis thaliana) RING-type E3s that are similar in domain architecture to the rice (Oryza sativa) XA21 Binding Protein3, a defense protein. The XBAT35 transcript undergoes alternative splicing to produce two protein isoforms, XBAT35.1 and XBAT35.2. Here, we demonstrate that XBAT35.2 localizes predominantly to the Golgi and is involved in cell death induction and pathogen response. XBAT35.2, but not XBAT35.1, was found to trigger cell death when overexpressed in tobacco (Nicotiana benthamiana) leaves and does so in a manner that requires its RING domain. Loss of XBAT35 gene function disrupts the plant’s ability to defend against pathogen attack, whereas overexpression of XBAT35.2 enhances resistance to pathogens. XBAT35.2 was found to be unstable and promotes its own degradation, suggesting self-regulation. Inoculation with virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv tomato DC3000 results in a drastic reduction in the levels of ubiquitinated XBAT35.2 and an increase in the abundance of the E3. This implies that pathogen infection prohibits XBAT35.2 self-regulation and stabilizes the E3. In agreement with a role in defending against pathogens, XBAT35.2 interacts with defense-related Accelerated Cell Death11 (ACD11) in planta and promotes the proteasome-dependent turnover of ACD11 in cell-free degradation assays. In accordance with regulation by a stabilized XBAT35.2, the levels of ubiquitinated ACD11 increased considerably, and the abundance of ACD11 was reduced following pathogen infection. In addition, treatment of transgenic seedlings with a proteasome inhibitor results in the accumulation of ACD11, confirming proteasome-dependent degradation. Collectively, these results highlight a novel role for XBAT35.2 in cell death induction and defense against pathogens. PMID:28951488

Cyclin-Dependent Kinase 5 Links Extracellular Cues to Actin Cytoskeleton During Dendritic Spine Development

PubMed Central

Fu, Amy KY

2007-01-01

Emerging evidence has indicated a regulatory role of cyclin-dependent kinase 5 (Cdk5) in synaptic plasticity as well as in higher brain functions, such as learning and memory. However, the molecular and cellular mechanisms underlying the actions of Cdk5 at synapses remain unclear. Recent findings demonstrate that Cdk5 regulates dendritic spine morphogenesis through modulating actin dynamics. Ephexin1 and WAVE-1, two important regulators of the actin cytoskeleton, have both been recently identified as substrates for Cdk5. Importantly, phosphorylation of these proteins by Cdk5 leads to dendritic spine loss, revealing a potential mechanism by which Cdk5 regulates synapse remodeling. Furthermore, Cdk5-dependent phosphorylation of ephexin1 is required for the ephrin-A1 mediated spine retraction, pointing to a critical role of Cdk5 in conveying signals from extracellular cues to actin cytoskeleton at synapses. Taken together, understanding the precise regulation of Cdk5 and its downstream targets at synapses would provide important insights into the multi-regulatory roles of Cdk5 in actin remodeling during dendritic spine development. PMID:19270534

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

2014-05-16

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

Apoptogenic effects of β-sitosterol glucoside from Castanopsis indica leaves.

PubMed

Dolai, Narayan; Kumar, Ashish; Islam, Aminul; Haldar, Pallab K

2016-01-01

β-Sitosterol glucoside (BSSG) is a natural biologically active substance isolated from the Castanopsis indica leaves. This study explored the apoptogenic mechanistic studies of BSSG against Ehrlich's ascites carcinoma (EAC) treated mice through morphological study, comet assay, flow cytometry (FACS) and Western blotting assay method. AO/EB staining and FACS analysis showed that BSSG possessed apoptosis induction activities on EAC cells. Dose dependent induction of DNA damage was observed after BSSG treatment. Increase the expression of apoptotic protein p53 and p21 in EAC, multiple downstream factors contributing to apoptosis pathway. The increase of caspase-9 and caspase-3 activities revealed that caspase was a key mediator of the apoptotic pathway induced by BSSG, and up-regulation of Bax and down-regulation of anti-apoptotic protein Bcl-2 resulted in the decrease of Bcl-2/Bax ratio. Owing to the combination of significant antitumour activity by inducing apoptosis, BSSG holds the promise of being an interesting chemo-preventive agent active in cancer therapy.

Discovery of cellular substrates for protein kinase A using a peptide array screening protocol.

PubMed

Smith, F Donelson; Samelson, Bret K; Scott, John D

2011-08-15

Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser(119) inside cells and that this mode of regulation may control its ability to affect cell growth. © The Authors Journal compilation © 2011 Biochemical Society

In vitro study of biocompatibility of a graphene composite with gold nanoparticles and hydroxyapatite on human osteoblasts.

PubMed

Crisan, Liana; Crisan, Bogdan; Soritau, Olga; Baciut, Mihaela; Biris, Alexandru Radu; Baciut, Grigore; Lucaciu, Ondine

2015-10-01

The purpose of this study was to evaluate the biocompatibility of some composites consisting of different proportions of graphene in combination with gold nanoparticles (AuNPs) and nanostructured hydroxyapatite (HA) on osteoblast viability, proliferation and differentiation. Au/HA@graphene composites synthesized by the catalytic chemical vapor deposition induction heating method with acetylene as the carbon source and over an Au/HA catalyst, were characterized by transmission electron microscopy, thermogravimetric analysis and Raman spectroscopy and showed that the few-layer graphene was grown over the Au/HA catalyst. The cytocompatibility study was performed using the fluorescein diacetate assay for assessment of the viability and proliferation of osteoblasts cultivated in the presence of HA, Au/HA and Au/HA@graphene composites as colloidal suspensions or as substrates. The most favorable composites for cell adhesion and proliferation were HA, Au/HA and Au/HA composites with 1.6% and 3.15% concentration of graphenes. Immunocytochemical staining performed after 19 days of osteoblasts cultivation on substrates showed that the graphene composites induced low expression of alkaline phosphatase compared to the control group and HA and Au/HA substrates. The presence of graphene in the substrate composition also induced an increased level of intracellular osteopontin and cytoskeleton reorganization (actin-F) depending on graphene concentration, suggesting cell activation, increased cellular adhesion and acquisition of a mechanosensorial osteocyte phenotype. Copyright © 2015 John Wiley & Sons, Ltd.

Understanding Artifacts in Impedance Spectroscopy

DOE PAGES

Veal, B. W.; Baldo, P. M.; Paulikas, A. P.; ...

2014-11-22

Four-terminal measurements of impedance spectra have long been troubled by the presence of high frequency artifacts that typically indicate unphysically large inductive behavior. In this paper, we follow up on the observation of Fleig et al., that voltage and current are necessarily measured in different locations of the potentiostat circuit, and that, typically, the electrometer input is a virtual ground. In this case, the capacitance of coaxial cables that connect sample electrodes to the potentiostat provides a high frequency conduction path to ground, so that some of the current that passes through the sample bypasses the electrometer. In four-electrode measurements,more » this mechanism produces the observed inductive artifacts. We examine a variety of simulated samples, with calculations compared to measurements of relevant circuits, to quantitatively investigate the nature of the artifacts. Model results agree with measurements when the leakage capacitances are properly included in the circuit analyses. With understanding of the origin of the inductive artifacts, the four-electrode method can be effectively utilized, enabling a combination of two-, three- and four-electrode measurements to be used to best advantage. Finally, using this combination of electrode configurations, temperature dependent measurements of SrTiO 3, Y 2O 3-stabilized ZrO 2, and In 2O 3 films deposited on YSZ substrates are presented.« less

A Novel Yeast Surface Display Method for Large-Scale Screen Inhibitors of Sortase A.

PubMed

Wu, Lin; Li, Huijun; Tang, Tianle

2017-01-24

Fluorescence resonance energy transfer substrates of sortase A are too expensive to be used to roughly screen high-throughput sortase A inhibitors. This makes therapeutic strategies difficult to realize in a clinical therapeutic use. Instead, we design here an LPETG-EGFP (leucine, proline, glutamic, threonine and glycine-enhanced green fluorescence) protein displayed on a yeast surface as a substrate by adaptively reducing the cost. We do this by optimizing the induction conditions of sortase A expression in Escherichia coli DE3(BL21) and catalyzing LPETG proteins, which are displayed on surface of Pichia pastoris . Different expression conditions of sortase A include: induction temperature (22 °C, 28 °C, 37 °C and 40 °C), induction time (4 h, 5 h, 6 h and 7 h) and induction concentration of isopropyl β-d-thiogalactoside IPTG (0.25 mmol/L, 0.5 mmol/L, 1 mmol/L, and 2 mmol/L). The fluorescence change of the LPETG-EGFP protein on the surface of P. pastoris over time was detected by flow cytometry and fluorescence spectrophotometry, and then the sensitivities of the two methods were compared. Using berberine chloride as an inhibitor, the activity of sortase A was investigated with the substrates of LPETG-EGFP protein, and compared to Dabcyl-QALPETGEE-Edans. A high yield of sortase A was achieved by inducing 1.0 mmol/L IPTG at 28 °C for 6 h. The intensity of green fluorescence of substrates displayed on the yeast surface was increased over time, while the stability was decreased slightly. Both fluorescence spectrophotometery and flow cytometry were fit for detection because of their high sensitivity. We utilized two different substrates of sortase A to investigate sortase A activity, which resulted in the increase of fluorescence intensity with respect to the increased time of growth. However, the method with Dabcyl-QALPETGEE-Edans as its substrate was more robust. Thus, the method described in this paper is a simple and cheap method which is very suitable for high-throughput analysis, but the conventional method is much more sensitive. The method described in this paper is expected to lead to large-scale screening of sortase A inhibitors which can be used to decrease the risk of drug resistance development.

Distinct DC subsets regulate adaptive Th1 and 2 responses during Trichuris muris infection.

PubMed

Demiri, M; Müller-Luda, K; Agace, W W; Svensson-Frej, M

2017-10-01

Low- and high-dose infections with the murine large intestinal nematode Trichuris muris are associated with induction of adaptive Th1 and Th2 responses, respectively, in mesenteric lymph nodes (MLN). Classical dendritic cells (cDC) accumulate in the large intestinal mucosa and MLN upon T. muris infection, yet their role in driving adaptive responses to infection remains largely unknown. We performed low- and high-dose T. muris infections of mice deficient in defined cDC subsets to investigate their role in induction of adaptive immune responses. Mice lacking IRF4-dependent cDC failed to clear a high-dose infection and displayed impaired Th2 responses. Conversely, mice lacking IRF8-dependent cDC cleared a low-dose infection and displayed an impaired Th1 response while increased production of Th2 cytokines. Finally, mice lacking both IRF4- and IRF8-dependent cDC were able to generate a Th2 response and clear a low-dose infection. Collectively, these results suggest that IRF4- and IRF8-dependent cDC act antagonistically during T. muris infection, and demonstrate that intestinal Th2 responses can be generated towards T. muris in the absence of IRF4-dependent cDC. © 2017 John Wiley & Sons Ltd.

Molecular and thermodynamic mechanisms of the chloride-dependent human angiotensin-I-converting enzyme (ACE).

PubMed

Yates, Christopher J; Masuyer, Geoffrey; Schwager, Sylva L U; Akif, Mohd; Sturrock, Edward D; Acharya, K Ravi

2014-01-17

Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg(522). Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu(403)-Lys(118) salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains.

Molecular and Thermodynamic Mechanisms of the Chloride-dependent Human Angiotensin-I-converting Enzyme (ACE)*

PubMed Central

Yates, Christopher J.; Masuyer, Geoffrey; Schwager, Sylva L. U.; Akif, Mohd; Sturrock, Edward D.; Acharya, K. Ravi

2014-01-01

Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg522. Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu403-Lys118 salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains. PMID:24297181

Apoptosis induction by silica nanoparticles mediated through reactive oxygen species in human liver cell line HepG2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahmad, Javed; Ahamed, Maqusood, E-mail: maqusood@gmail.com; Akhtar, Mohd Javed

Silica nanoparticles are increasingly utilized in various applications including agriculture and medicine. In vivo studies have shown that liver is one of the primary target organ of silica nanoparticles. However, possible mechanisms of hepatotoxicity caused by silica nanoparticles still remain unclear. In this study, we explored the reactive oxygen species (ROS) mediated apoptosis induced by well-characterized 14 nm silica nanoparticles in human liver cell line HepG2. Silica nanoparticles (25–200 μg/ml) induced a dose-dependent cytotoxicity in HepG2 cells. Silica nanoparticles were also found to induce oxidative stress in dose-dependent manner indicated by induction of ROS and lipid peroxidation and depletion ofmore » glutathione (GSH). Quantitative real-time PCR and immunoblotting results showed that both the mRNA and protein expressions of cell cycle checkpoint gene p53 and apoptotic genes (bax and caspase-3) were up-regulated while the anti-apoptotic gene bcl-2 was down-regulated in silica nanoparticles treated cells. Moreover, co-treatment of ROS scavenger vitamin C significantly attenuated the modulation of apoptotic markers along with the preservation of cell viability caused by silica nanoparticles. Our data demonstrated that silica nanoparticles induced apoptosis in human liver cells, which is ROS mediated and regulated through p53, bax/bcl-2 and caspase pathways. This study suggests that toxicity mechanisms of silica nanoparticles should be further investigated at in vivo level. -- Highlights: ► We explored the mechanisms of toxicity caused by silica NPs in human liver HepG2 cells. ► Silica NPs induced a dose-dependent cytotoxicity in HepG2 cells. ► Silica NPs induced ROS generation and oxidative stress in a dose-dependent manner. ► Silica NPs were also modulated apoptosis markers both at mRNA and protein levels. ► ROS mediated apoptosis induced by silica NPs was preserved by vitamin C.« less

Tumor Necrosis Factor-α– and Interleukin-1β–Dependent Matrix Metalloproteinase-3 Expression in Nucleus Pulposus Cells Requires Cooperative Signaling via Syndecan 4 and Mitogen-Activated Protein Kinase–NF-κB Axis

PubMed Central

Wang, Xin; Wang, Hua; Yang, Hao; Li, Jun; Cai, Qiqing; Shapiro, Irving M.; Risbud, Makarand V.

2015-01-01

Matrix metalloproteinase-3 (MMP-3) plays an important role in intervertebral disc degeneration, a ubiquitous condition closely linked to low back pain and disability. Elevated expression of syndecan 4, a cell surface heparan sulfate proteoglycan, actively controls disc matrix catabolism. However, the relationship between MMP-3 expression and syndecan 4 in the context of inflammatory disc disease has not been clearly defined. We investigated the mechanisms by which cytokines control MMP-3 expression in rat and human nucleus pulposus cells. Cytokine treatment increased MMP-3 expression and promoter activity. Stable silencing of syndecan 4 blocked cytokine-mediated MMP-3 expression; more important, syndecan 4 did not mediate its effects through NF-κB or mitogen-activated protein kinase (MAPK) pathways. However, treatment with MAPK and NF-κB inhibitors resulted in partial blocking of the inductive effect of cytokines on MMP-3 expression. Loss-of-function studies confirmed that NF-κB, p38α/β2/γ/δ, and extracellular signal–regulated kinase (ERK) 2, but not ERK1, contributed to cytokine-dependent induction of MMP3 promoter activity. Similarly, inhibitor treatments, lentiviral short hairpin-p65, and short hairpin-IκB kinase β significantly decreased cytokine-dependent up-regulation in MMP-3 expression. Finally, we show that transforming growth factor-β can block the up-regulation of MMP-3 induced by tumor necrosis factor (TNF)-α by counteracting the NF-κB pathway and syndecan 4 expression. Taken together, our results suggest that cooperative signaling through syndecan 4 and the TNF receptor 1–MAPK–NF-κB axis is required for TNF-α–dependent expression of MMP-3 in nucleus pulposus cells. Controlling these pathways may slow the progression of intervertebral disc degeneration and matrix catabolism. PMID:25063530

Identification of Putative Mek1 Substrates during Meiosis in Saccharomyces cerevisiae Using Quantitative Phosphoproteomics

PubMed Central

Suhandynata, Raymond T.; Wan, Lihong; Zhou, Huilin; Hollingsworth, Nancy M.

2016-01-01

Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast. PMID:27214570

Identification of Arabidopsis MYB56 as a novel substrate for CRL3(BPM) E3 ligases.

PubMed

Chen, Liyuan; Bernhardt, Anne; Lee, JooHyun; Hellmann, Hanjo

2015-02-01

Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

PubMed Central

Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

2015-01-01

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

PubMed

Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

2015-02-13

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A discrete mathematical model applied to genetic regulation and metabolic networks.

PubMed

Asenjo, A J; Ramirez, P; Rapaport, I; Aracena, J; Goles, E; Andrews, B A

2007-03-01

This paper describes the use of a discrete mathematical model to represent the basic mechanisms of regulation of the bacteria E. coli in batch fermentation. The specific phenomena studied were the changes in metabolism and genetic regulation when the bacteria use three different carbon substrates (glucose, glycerol, and acetate). The model correctly predicts the behavior of E. coli vis-à-vis substrate mixtures. In a mixture of glucose, glycerol, and acetate, it prefers glucose, then glycerol, and finally acetate. The model included 67 nodes; 28 were genes, 20 enzymes, and 19 regulators/biochemical compounds. The model represents both the genetic regulation and metabolic networks in an inrtegrated form, which is how they function biologically. This is one of the first attempts to include both of these networks in one model. Previously, discrete mathematical models were used only to describe genetic regulation networks. The study of the network dynamics generated 8 (2(3)) fixed points, one for each nutrient configuration (substrate mixture) in the medium. The fixed points of the discrete model reflect the phenotypes described. Gene expression and the patterns of the metabolic fluxes generated are described accurately. The activation of the gene regulation network depends basically on the presence of glucose and glycerol. The model predicts the behavior when mixed carbon sources are utilized as well as when there is no carbon source present. Fictitious jokers (Joker1, Joker2, and Repressor SdhC) had to be created to control 12 genes whose regulation mechanism is unknown, since glycerol and glucose do not act directly on the genes. The approach presented in this paper is particularly useful to investigate potential unknown gene regulation mechanisms; such a novel approach can also be used to describe other gene regulation situations such as the comparison between non-recombinant and recombinant yeast strain, producing recombinant proteins, presently under investigation in our group.

Effects of Inductively Coupled Plasma Hydrogen on Long-Wavelength Infrared HgCdTe Photodiodes

NASA Astrophysics Data System (ADS)

Boieriu, P.; Buurma, C.; Bommena, R.; Blissett, C.; Grein, C.; Sivananthan, S.

2013-12-01

Bulk passivation of semiconductors with hydrogen continues to be investigated for its potential to improve device performance. In this work, hydrogen-only inductively coupled plasma (ICP) was used to incorporate hydrogen into long-wavelength infrared HgCdTe photodiodes grown by molecular-beam epitaxy. Fully fabricated devices exposed to ICP showed statistically significant increases in zero-bias impedance values, improved uniformity, and decreased dark currents. HgCdTe photodiodes on Si substrates passivated with amorphous ZnS exhibited reductions in shunt currents, whereas devices on CdZnTe substrates passivated with polycrystalline CdTe exhibited reduced surface leakage, suggesting that hydrogen passivates defects in bulk HgCdTe and in CdTe.

TOR Complex 2-Regulated Protein Kinase Fpk1 Stimulates Endocytosis via Inhibition of Ark1/Prk1-Related Protein Kinase Akl1 in Saccharomyces cerevisiae.

PubMed

Roelants, Françoise M; Leskoske, Kristin L; Pedersen, Ross T A; Muir, Alexander; Liu, Jeffrey M-H; Finnigan, Gregory C; Thorner, Jeremy

2017-04-01

Depending on the stress, plasma membrane alterations activate or inhibit yeast target of rapamycin (TOR) complex 2, which, in turn, upregulates or downregulates the activity of its essential downstream effector, protein kinase Ypk1. Through phosphorylation of multiple substrates, Ypk1 controls many processes that restore homeostasis. One such substrate is protein kinase Fpk1, which is negatively regulated by Ypk1. Fpk1 phosphorylates and stimulates flippases that translocate aminoglycerophospholipids from the outer to the inner leaflet of the plasma membrane. Fpk1 has additional roles, but other substrates were uncharacterized. We show that Fpk1 phosphorylates and inhibits protein kinase Akl1, related to protein kinases Ark1 and Prk1, which modulate the dynamics of actin patch-mediated endocytosis. Akl1 has two Fpk1 phosphorylation sites (Ark1 and Prk1 have none) and is hypophosphorylated when Fpk1 is absent. Conversely, under conditions that inactivate TORC2-Ypk1 signaling, which alleviates Fpk1 inhibition, Akl1 is hyperphosphorylated. Monitoring phosphorylation of known Akl1 substrates (Sla1 and Ent2) confirmed that Akl1 is hyperactive when not phosphorylated by Fpk1. Fpk1-mediated negative regulation of Akl1 enhances endocytosis, because an Akl1 mutant immune to Fpk1 phosphorylation causes faster dissociation of Sla1 from actin patches, confers elevated resistance to doxorubicin (a toxic compound whose entry requires endocytosis), and impedes Lucifer yellow uptake (a marker of fluid phase endocytosis). Thus, TORC2-Ypk1, by regulating Fpk1-mediated phosphorylation of Akl1, adjusts the rate of endocytosis. Copyright © 2017 Roelants et al.

Myc-driven overgrowth requires unfolded protein response-mediated induction of autophagy and antioxidant responses in Drosophila melanogaster.

PubMed

Nagy, Péter; Varga, Agnes; Pircs, Karolina; Hegedűs, Krisztina; Juhász, Gábor

2013-01-01

Autophagy, a lysosomal self-degradation and recycling pathway, plays dual roles in tumorigenesis. Autophagy deficiency predisposes to cancer, at least in part, through accumulation of the selective autophagy cargo p62, leading to activation of antioxidant responses and tumor formation. While cell growth and autophagy are inversely regulated in most cells, elevated levels of autophagy are observed in many established tumors, presumably mediating survival of cancer cells. Still, the relationship of autophagy and oncogenic signaling is poorly characterized. Here we show that the evolutionarily conserved transcription factor Myc (dm), a proto-oncogene involved in cell growth and proliferation, is also a physiological regulator of autophagy in Drosophila melanogaster. Loss of Myc activity in null mutants or in somatic clones of cells inhibits autophagy. Forced expression of Myc results in cell-autonomous increases in cell growth, autophagy induction, and p62 (Ref2P)-mediated activation of Nrf2 (cnc), a transcription factor promoting antioxidant responses. Mechanistically, Myc overexpression increases unfolded protein response (UPR), which leads to PERK-dependent autophagy induction and may be responsible for p62 accumulation. Genetic or pharmacological inhibition of UPR, autophagy or p62/Nrf2 signaling prevents Myc-induced overgrowth, while these pathways are dispensable for proper growth of control cells. In addition, we show that the autophagy and antioxidant pathways are required in parallel for excess cell growth driven by Myc. Deregulated expression of Myc drives tumor progression in most human cancers, and UPR and autophagy have been implicated in the survival of Myc-dependent cancer cells. Our data obtained in a complete animal show that UPR, autophagy and p62/Nrf2 signaling are required for Myc-dependent cell growth. These novel results give additional support for finding future approaches to specifically inhibit the growth of cancer cells addicted to oncogenic Myc.

TP53-dependent autophagy links the ATR-CHEK1 axis activation to proinflammatory VEGFA production in human bronchial epithelial cells exposed to fine particulate matter (PM2.5).

PubMed

Xu, Xiuduan; Wang, Hongli; Liu, Shasha; Xing, Chen; Liu, Yang; Aodengqimuge; Zhou, Wei; Yuan, Xiaoyan; Ma, Yongfu; Hu, Meiru; Hu, Yongliang; Zou, Shuxian; Gu, Ye; Peng, Shuangqing; Yuan, Shengtao; Li, Weiping; Ma, Yuanfang; Song, Lun

2016-10-02

ABSTARCT Epidemiological and clinical studies have increasingly shown that fine particulate matter (PM2.5) is associated with a number of pathological respiratory diseases, such as bronchitis, asthma, and chronic obstructive pulmonary disease, which share the common feature of airway inflammation induced by particle exposure. Thus, understanding how PM2.5 triggers inflammatory responses in the respiratory system is crucial for the study of PM2.5 toxicity. In the current study, we found that exposing human bronchial epithelial cells (immortalized Beas-2B cells and primary cells) to PM2.5 collected in the winter in Wuhan, a city in southern China, induced a significant upregulation of VEGFA (vascular endothelial growth factor A) production, a signaling event that typically functions to control chronic airway inflammation and vascular remodeling. Further investigations showed that macroautophagy/autophagy was induced upon PM2.5 exposure and then mediated VEGFA upregulation by activating the SRC (SRC proto-oncogene, non-receptor tyrosine kinase)-STAT3 (signal transducer and activator of transcription 3) pathway in bronchial epithelial cells. By exploring the upstream signaling events responsible for autophagy induction, we revealed a requirement for TP53 (tumor protein p53) activation and the expression of its downstream target DRAM1 (DNA damage regulated autophagy modulator 1) for the induction of autophagy. These results thus extend the role of TP53-DRAM1-dependent autophagy beyond cell fate determination under genotoxic stress and to the control of proinflammatory cytokine production. Moreover, PM2.5 exposure strongly induced the activation of the ATR (ATR serine/threonine kinase)-CHEK1/CHK1 (checkpoint kinase 1) axis, which subsequently triggered TP53-dependent autophagy and VEGFA production in Beas-2B cells. Therefore, these findings suggest a novel link between processes regulating genomic integrity and airway inflammation via autophagy induction in bronchial epithelial cells under PM2.5 exposure.

Boldine enhances bile production in rats via osmotic and farnesoid X receptor dependent mechanisms.

PubMed

Cermanova, Jolana; Kadova, Zuzana; Zagorova, Marie; Hroch, Milos; Tomsik, Pavel; Nachtigal, Petr; Kudlackova, Zdenka; Pavek, Petr; Dubecka, Michaela; Ceckova, Martina; Staud, Frantisek; Laho, Tomas; Micuda, Stanislav

2015-05-15

Boldine, the major alkaloid from the Chilean Boldo tree, is used in traditional medicine to support bile production, but evidence to support this function is controversial. We analyzed the choleretic potential of boldine, including its molecular background. The acute- and long-term effects of boldine were evaluated in rats either during intravenous infusion or after 28-day oral treatment. Infusion of boldine instantly increased the bile flow 1.4-fold in healthy rats as well as in animals with Mrp2 deficiency or ethinylestradiol induced cholestasis. This effect was not associated with a corresponding increase in bile acid or glutathione biliary excretion, indicating that the effect is not related to stimulation of either bile acid dependent or independent mechanisms of bile formation and points to the osmotic activity of boldine itself. We subsequently analyzed bile production under conditions of changing biliary excretion of boldine after bolus intravenous administration and found strong correlations between both parameters. HPLC analysis showed that bile concentrations of boldine above 10 μM were required for induction of choleresis. Importantly, long-term pretreatment, when the bile collection study was performed 24-h after the last administration of boldine, also accelerated bile formation despite undetectable levels of the compound in bile. The effect paralleled upregulation of the Bsep transporter and increased biliary clearance of its substrates, bile acids. We consequently confirmed the ability of boldine to stimulate the Bsep transcriptional regulator, FXR receptor. In conclusion, our study clarified the mechanisms and circumstances surrounding the choleretic activity of boldine. Copyright © 2015 Elsevier Inc. All rights reserved.

Difference in luteal and placental P450(17) alpha: substrate preference and hormonal regulation in the rat.

PubMed

Eckstein, B; Khan, I; Gibori, G

1987-12-01

The purpose of this study was to assess the substrate specificity of P450(17) alpha in both the corpus luteum and placenta of pregnant rats, and to analyse the site at which LH/human chorionic gonadotrophin (hCG) regulates the activities of this enzyme. To distinguish the substrate preference, placentas and corpora lutea were obtained from rats on day 15 of pregnancy. Tissues were homogenized and the 10,000 g supernatants incubated in the presence of equimolar concentrations of [14C]progesterone and [3H]17 alpha-hydroxyprogesterone as substrate with either NADH or NADPH as cofactors for 2, 8, 16 and 24 min. The labelling pattern of both 17 alpha-hydroxyprogesterone and testosterone indicated that the corpus luteum produced testosterone preferentially from progesterone, whereas the placenta principally used 17 alpha-hydroxyprogesterone and synthesized six times as much testosterone from 17 alpha-hydroxyprogesterone than from progesterone. Addition of either NADPH or NADH as cofactors had no effect on substrate preference. The products of the two enzymatic activities were identified by recrystallization to constant 14C/3H ratios. The ratio of 14C/3H in testosterone produced by the corpus luteum was 16-fold higher than in that produced by the placenta. To explore which of the two activities of P450(17) alpha is regulated by the gonadotrophin, rats were treated with either 1.5 IU hCG or vehicle between days 13 and 15 of pregnancy. Hydroxylase and lyase activities were determined on day 15 after incubation for 2, 8, 16 or 24 min in the presence of either NADH or NADPH. Administration of hCG significantly inhibited NADH-dependent 17 alpha-hydroxylase in the placenta at each time-point studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of eddy current losses in inductive transmission systems with ferrite sheets.

PubMed

Maaß, Matthias; Griessner, Andreas; Steixner, Viktor; Zierhofer, Clemens

2017-01-05

Improvements in eddy current suppression are necessary to meet the demand for increasing miniaturization of inductively driven transmission systems in industrial and biomedical applications. The high magnetic permeability and the simultaneously low electrical conductivity of ferrite materials make them ideal candidates for shielding metallic surfaces. For systems like cochlear implants the transmission of data as well as energy over an inductive link is conducted within a well-defined parameter set. For these systems, the shielding can be of particular importance if the properties of the link can be preserved. In this work, we investigate the effect of single and double-layered substrates consisting of ferrite and/or copper on the inductance and coupling of planar spiral coils. The examined link systems represent realistic configurations for active implantable systems such as cochlear implants. Experimental measurements are complemented with analytical calculations and finite element simulations, which are in good agreement for all measured parameters. The results are then used to study the transfer efficiency of an inductive link in a series-parallel resonant topology as a function of substrate size, the number of coil turns and coil separation. We find that ferrite sheets can be used to shield the system from unwanted metallic surfaces and to retain the inductive link parameters of the unperturbed system, particularly its transfer efficiency. The required size of the ferrite plates is comparable to the size of the coils, which makes the setup suitable for practical implementations. Since the sizes and geometries chosen for the studied inductive links are comparable to those of cochlear implants, our conclusions apply in particular to these systems.

Crystal Structures of Human SIRT[subscript 3] Displaying Substrate-induced Conformational Changes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Lei; Wei, Wentao; Jiang, Yaobin

2009-11-04

SIRT3 is a major mitochondrial NAD{sup +}-dependent protein deacetylase playing important roles in regulating mitochondrial metabolism and energy production and has been linked to the beneficial effects of exercise and caloric restriction. SIRT3 is emerging as a potential therapeutic target to treat metabolic and neurological diseases. We report the first sets of crystal structures of human SIRT3, an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide, and a structure with the dethioacetylated peptide bound. These structures provide insights into themore » conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptide and NAD+. In addition, the binding study by isothermal titration calorimetry suggests that the acetylated peptide is the first substrate to bind to SIRT3, before NAD{sup +}. These structures and biophysical studies provide key insight into the structural and functional relationship of the SIRT3 deacetylation activity.« less

Erufosine simultaneously induces apoptosis and autophagy by modulating the Akt-mTOR signaling pathway in oral squamous cell carcinoma.

PubMed

Kapoor, Vaishali; Zaharieva, Maya M; Das, Satya N; Berger, Martin R

2012-06-01

We investigated the anticancer activity of erufosine in oral squamous carcinoma cell lines in terms of cell proliferation, colony formation, induction of autophagy/apoptosis, cell cycle and mTOR signaling pathway. Erufosine showed dose-dependent cytotoxicity in all cell lines, it induced autophagy as well as apoptosis, G2 cell cycle arrest and modulation of cyclin D1 expression. Further erufosine downregulated the phosphorylation of major components of mTOR pathway, like p-Akt at Ser473 and Thr308 residues, p-Raptor, p-mTOR, p-PRAS40 and its downstream substrates p-p70S6K and p-4EBP1 in a dose-dependent manner. The pre-treatment of tumor cells with p-mTOR siRNA increased cytotoxic effects of erufosine comparable to cisplatin but higher than rapamycin. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-α-prolyl-4-hydroxylases

PubMed Central

2004-01-01

An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive α-subunit (HIF-1α or HIF-2α) and a constitutively expressed β-subunit. In normoxia, HIF-1α is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1α. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1α expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-α. Using a VHL capture assay, we demonstrate that HIF-α prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1α after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1α in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia. PMID:15104534

Neuromodulation, development and synaptic plasticity.

PubMed

Foehring, R C; Lorenzon, N M

1999-03-01

We discuss parallels in the mechanisms underlying use-dependent synaptic plasticity during development and long-term potentiation (LTP) and long-term depression (LTD) in neocortical synapses. Neuromodulators, such as norepinephrine, serotonin, and acetylcholine have also been implicated in regulating both developmental plasticity and LTP/LTD. There are many potential levels of interaction between neuromodulators and plasticity. Ion channels are substrates for modulation in many cell types. We discuss examples of modulation of voltage-gated Ca2+ channels and Ca(2+)-dependent K+ channels and the consequences for neocortical pyramidal cell firing behaviour. At the time when developmental plasticity is most evident in rat cortex, the substrate for modulation is changing as the densities and relative proportions of various ion channels types are altered during ontogeny. We discuss examples of changes in K+ and Ca2+ channels and the consequence for modulation of neuronal activity.

COX-2 expression mediated by calcium-TonEBP signaling axis under hyperosmotic conditions serves osmoprotective function in nucleus pulposus cells.

PubMed

Choi, Hyowon; Chaiyamongkol, Weera; Doolittle, Alexandra C; Johnson, Zariel I; Gogate, Shilpa S; Schoepflin, Zachary R; Shapiro, Irving M; Risbud, Makarand V

2018-06-08

The nucleus pulposus (NP) of intervertebral discs experiences dynamic changes in tissue osmolarity because of diurnal loading of the spine. TonEBP/NFAT5 is a transcription factor that is critical in osmoregulation as well as survival of NP cells in the hyperosmotic milieu. The goal of this study was to investigate whether cyclooxygenase-2 (COX-2) expression is osmoresponsive and dependent on TonEBP, and whether it serves an osmoprotective role. NP cells up-regulated COX-2 expression in hyperosmotic media. The induction of COX-2 depended on elevation of intracellular calcium levels and p38 MAPK pathway, but independent of calcineurin signaling as well as MEK/ERK and JNK pathways. Under hyperosmotic conditions, both COX-2 mRNA stability and its proximal promoter activity were increased. The proximal COX-2 promoter (-1840/+123 bp) contained predicted binding sites for TonEBP, AP-1, NF-κB, and C/EBP-β. While COX-2 promoter activity was positively regulated by both AP-1 and NF-κB, AP-1 had no effect and NF-κB negatively regulated COX-2 protein levels under hyperosmotic conditions. On the other hand, TonEBP was necessary for both COX-2 promoter activity and protein up-regulation in response to hyperosmotic stimuli. Ex vivo disc organ culture studies using hypomorphic TonEBP +/- mice confirmed that TonEBP is required for hyperosmotic induction of COX-2. Importantly, the inhibition of COX-2 activity under hyperosmotic conditions resulted in decreased cell viability, suggesting that COX-2 plays a cytoprotective and homeostatic role in NP cells for their adaptation to dynamically loaded hyperosmotic niches. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Modulation of IgE-dependent COX-2 gene expression by reactive oxygen species in human neutrophils.

PubMed

Vega, Antonio; Chacón, Pedro; Alba, Gonzalo; El Bekay, Rajaa; Martín-Nieto, José; Sobrino, Francisco

2006-07-01

Cyclooxygenase (COX) is a key enzyme in prostaglandin (PG) synthesis. Up-regulation of its COX-2 isoform is responsible for the increased PG release, taking place under inflammatory conditions, and also, is thought to be involved in allergic and inflammatory diseases. In the present work, we demonstrate that COX-2 expression becomes highly induced by anti-immunoglobulin E (IgE) antibodies and by antigens in human neutrophils from allergic patients. This induction was detected at mRNA and protein levels and was accompanied by a concomitant PGE(2) and thromboxane A(2) release. We also show evidence that inhibitors of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, such as 4-(2-aminoethyl)benzenesulphonyl fluoride and 4-hydroxy-3-methoxyaceto-phenone, completely cancelled anti-IgE-induced COX-2 protein up-regulation, suggesting that this process is mediated by reactive oxygen species (ROS) derived from NADPH oxidase activity. Moreover, the mitogen-activated protein kinases (MAPKs), p38 and extracellular signal-regulated kinase, and also, the transcription factor, nuclear factor (NF)-kappaB, are involved in the up-regulation of COX-2 expression, as specific chemical inhibitors of these two kinases, such as SB203580 and PD098059, and of the NF-kappaB pathway, such as N(alpha)-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal, abolished IgE-dependent COX-2 induction. Evidence is also presented, using Fe(2)(+)/Cu(2)(+) ions, that hydroxyl radicals generated from hydrogen peroxide through Fenton reactions could constitute candidate modulators able to directly trigger anti-IgE-elicited COX-2 expression through MAPK and NF-kappaB pathways. Present results underscore a new role for ROS as second messengers in the modulation of COX-2 expression by human neutrophils in allergic conditions.

CD95/Fas Increases Stemness in Cancer Cells by Inducing a STAT1-Dependent Type I Interferon Response.

PubMed

Qadir, Abdul S; Ceppi, Paolo; Brockway, Sonia; Law, Calvin; Mu, Liang; Khodarev, Nikolai N; Kim, Jung; Zhao, Jonathan C; Putzbach, William; Murmann, Andrea E; Chen, Zhuo; Chen, Wenjing; Liu, Xia; Salomon, Arthur R; Liu, Huiping; Weichselbaum, Ralph R; Yu, Jindan; Peter, Marcus E

2017-03-07

Stimulation of CD95/Fas drives and maintains cancer stem cells (CSCs). We now report that this involves activation of signal transducer and activator of transcription 1 (STAT1) and induction of STAT1-regulated genes and that this process is inhibited by active caspases. STAT1 is enriched in CSCs in cancer cell lines, patient-derived human breast cancer, and CD95 high -expressing glioblastoma neurospheres. CD95 stimulation of cancer cells induced secretion of type I interferons (IFNs) that bind to type I IFN receptors, resulting in activation of Janus-activated kinases, activation of STAT1, and induction of a number of STAT1-regulated genes that are part of a gene signature recently linked to therapy resistance in five primary human cancers. Consequently, we identified type I IFNs as drivers of cancer stemness. Knockdown or knockout of STAT1 resulted in a strongly reduced ability of CD95L or type I IFN to increase cancer stemness. This identifies STAT1 as a key regulator of the CSC-inducing activity of CD95. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Infection by Toxoplasma gondii Specifically Induces Host c-Myc and the Genes This Pivotal Transcription Factor Regulates

PubMed Central

Franco, Magdalena; Shastri, Anjali J.

2014-01-01

Toxoplasma gondii infection has previously been described to cause dramatic changes in the host transcriptome by manipulating key regulators, including STATs, NF-κB, and microRNAs. Here, we report that Toxoplasma tachyzoites also mediate rapid and sustained induction of another pivotal regulator of host cell transcription, c-Myc. This induction is seen in cells infected with all three canonical types of Toxoplasma but not the closely related apicomplexan parasite Neospora caninum. Coinfection of cells with both Toxoplasma and Neospora still results in an increase in the level of host c-Myc, showing that c-Myc is actively upregulated by Toxoplasma infection (rather than repressed by Neospora). We further demonstrate that this upregulation may be mediated through c-Jun N-terminal protein kinase (JNK) and is unlikely to be a nonspecific host response, as heat-killed Toxoplasma parasites do not induce this increase and neither do nonviable parasites inside the host cell. Finally, we show that the induced c-Myc is active and that transcripts dependent on its function are upregulated, as predicted. Hence, c-Myc represents an additional way in which Toxoplasma tachyzoites have evolved to specifically alter host cell functions during intracellular growth. PMID:24532536

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

PubMed Central

Paulovich, A G; Armour, C D; Hartwell, L H

1998-01-01

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication. PMID:9725831

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

PubMed

Paulovich, A G; Armour, C D; Hartwell, L H

1998-09-01

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication.

Pure non-dioxin-like PCB congeners suppress induction of AhR-dependent endpoints in rat liver cells.

PubMed

Brenerová, Petra; Hamers, Timo; Kamstra, Jorke H; Vondráček, Jan; Strapáčová, Simona; Andersson, Patrik L; Machala, Miroslav

2016-02-01

The relative potencies of non-ortho-substituted coplanar polychlorinated biphenyl (PCB) congeners to activate the aryl hydrocarbon receptor (AhR) and to cause the AhR-dependent toxic events are essential for their risk assessment. Since some studies suggested that abundant non-dioxin-like PCB congeners (NDL-PCBs) may alter the AhR activation by PCB mixtures and possibly cause non-additive effects, we evaluated potential suppressive effects of NDL-PCBs on AhR activation, using a series of 24 highly purified NDL-PCBs. We investigated their impact on the model AhR agonist-induced luciferase reporter gene expression in rat hepatoma cells and on induction of CYP1A1/1B1 mRNAs and deregulation of AhR-dependent cell proliferation in rat liver epithelial cells. PCBs 128, 138, and 170 significantly suppressed AhR activation (with IC50 values from 1.4 to 5.6 μM), followed by PCBs 28, 47, 52, and 180; additionally, PCBs 122, 153, and 168 showed low but still significant potency to reduce luciferase activity. Detection of CYP1A1 mRNA levels in liver epithelial cells largely confirmed these results for the most abundant NDL-PCBs, whereas the other AhR-dependent events (CYP1B1 mRNA expression, induction of cell proliferation in confluent cells) were less sensitive to NDL-PCBs, thus indicating a more complex regulation of these endpoints. The present data suggest that some NDL-PCBs could modulate overall dioxin-like effects in complex mixtures.

Bul Proteins, a Nonredundant, Antagonistic Family of Ubiquitin Ligase Regulatory Proteins

PubMed Central

Novoselova, Tatiana V.; Zahira, Kiran; Rose, Ruth-Sarah

2012-01-01

Like other Nedd4 ligases, Saccharomyces cerevisiae E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of BUL gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using in vitro ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination. PMID:22307975

In vitro biofilm model for studying tongue flora and malodour.

PubMed

Spencer, P; Greenman, J; McKenzie, C; Gafan, G; Spratt, D; Flanagan, A

2007-10-01

To develop a perfusion biofilm system to model tongue biofilm microflora and their physiological response to sulfur-containing substrates (S-substrates) in terms of volatile sulfide compound (VSC) production. Tongue-scrape inocula were used to establish in vitro perfusion biofilms which were examined in terms of ecological composition using culture-dependent and independent (PCR-DGGE) approaches. VSC-specific activity of cells was measured by a cell suspension assay, using a portable industrial sulfide monitor which was also used to monitor VSC production from biofilms in situ. Quasi steady states were achieved by 48 h and continued to 96 h. The mean (+/-SEM) growth rate for 72-h biofilms (n=4) was micro=0.014 h(-1) (+/-0.005 h(-1)). Comparison of biofilms, perfusate and original inoculum showed their ecological composition to be similar (Pearson coefficient>0.64). Perfusate and biofilm cells derived from the same condition (co-sampled) were equivalent with regard to VSC-specific activities which were up-regulated in the presence of S-substrates. The model maintained a stable tongue microcosm suitable for studying VSC production; biofilm growth in the presence of S-substrates up-regulated VSC activity. The method is apt for studying ecological and physiological aspects of oral biofilms and could be useful for screening inhibitory agents.

A bioactive coating with submicron-sized titania crystallites fabricated by induction heating of titanium after tensile deformations.

PubMed

Li, Ning-Bo; Xu, Wen-Hua; Xiao, Gui-Yong; Zhao, Jun-Han; Lu, Yu-Peng

2017-11-01

Thermal oxidation technology was widely investigated as one of effective surface modification method for improving the bioactivity and biocompatibility of titanium and its alloys. In this work, the induction heat oxidization method, a fast, efficient, economical and environmental protective technology, was applied to prepare the submicron-morphological oxide coating with variable rutile TiO 2 equiaxed crystallites on the surface of pure Ti substrates after cold-drawing with 10-20% deformations. The results showed the plastic-deformed Ti cylinders recrystallized during induction heating treatment (IHT) for 10-20s which resulted in evolution of microstructures as well as slight improvement of microhardness. The surface characteristics of TiO 2 crystallites in oxidation layers were determined by the microstructural evolutions of Ti substrate in terms of the nucleation and growth of TiO 2 crystallites. Specially, the oxidized surface with 50-75nm roughness and more uniform and finer equiaxed oxide grains remarkablely improved the apatite deposition after bioactive evaluation in 1.5 × SBF for 7 days. This work provided a potential method to create controlled bioactive oxide coatings with submicro-/nano-scaled TiO 2 crystallites on titanium substrate in terms of the role of metallographic microstructure in the formation process of titanium oxides. Copyright © 2017 Elsevier Ltd. All rights reserved.

Δ(9)-THC modulation of fatty acid 2-hydroxylase (FA2H) gene expression: possible involvement of induced levels of PPARα in MDA-MB-231 breast cancer cells.

PubMed

Takeda, Shuso; Ikeda, Eriko; Su, Shengzhong; Harada, Mari; Okazaki, Hiroyuki; Yoshioka, Yasushi; Nishimura, Hajime; Ishii, Hiroyuki; Kakizoe, Kazuhiro; Taniguchi, Aya; Tokuyasu, Miki; Himeno, Taichi; Watanabe, Kazuhito; Omiecinski, Curtis J; Aramaki, Hironori

2014-12-04

We recently reported that Δ(9)-tetrahydrocannabinol (Δ(9)-THC), a major cannabinoid component in Cannabis Sativa (marijuana), significantly stimulated the expression of fatty acid 2-hydroxylase (FA2H) in human breast cancer MDA-MB-231 cells. Peroxisome proliferator-activated receptor α (PPARα) was previously implicated in this induction. However, the mechanisms mediating this induction have not been elucidated in detail. We performed a DNA microarray analysis of Δ(9)-THC-treated samples and showed the selective up-regulation of the PPARα isoform coupled with the induction of FA2H over the other isoforms (β and γ). Δ(9)-THC itself had no binding/activation potential to/on PPARα, and palmitic acid (PA), a PPARα ligand, exhibited no stimulatory effects on FA2H in MDA-MB-231 cells; thus, we hypothesized that the levels of PPARα induced were involved in the Δ(9)-THC-mediated increase in FA2H. In support of this hypothesis, we herein demonstrated that; (i) Δ(9)-THC activated the basal transcriptional activity of PPARα in a concentration-dependent manner, (ii) the concomitant up-regulation of PPARα/FA2H was caused by Δ(9)-THC, (iii) PA could activate PPARα after the PPARα expression plasmid was introduced, and (iv) the Δ(9)-THC-induced up-regulation of FA2H was further stimulated by the co-treatment with L-663,536 (a known PPARα inducer). Taken together, these results support the concept that the induced levels of PPARα may be involved in the Δ(9)-THC up-regulation of FA2H in MDA-MB-231 cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

MicroRNA-31 is a positive modulator of endothelial-mesenchymal transition and associated secretory phenotype induced by TGF-β.

PubMed

Katsura, Akihiro; Suzuki, Hiroshi I; Ueno, Toshihide; Mihira, Hajime; Yamazaki, Tomoko; Yasuda, Takahiko; Watabe, Tetsuro; Mano, Hiroyuki; Yamada, Yoshitsugu; Miyazono, Kohei

2016-01-01

Transforming growth factor-β (TGF-β) plays central roles in endothelial-mesenchymal transition (EndMT) involved in development and pathogenesis. Although EndMT and epithelial-mesenchymal transition are similar processes, roles of microRNAs in EndMT are largely unknown. Here, we report that constitutively active microRNA-31 (miR-31) is a positive regulator of TGF-β-induced EndMT. Although the expression is not induced by TGF-β, miR-31 is required for induction of mesenchymal genes including α-SMA, actin reorganization and MRTF-A activation during EndMT. We identified VAV3, a regulator of actin remodeling and MRTF-A activity, as a miR-31 target. Global transcriptome analysis further showed that miR-31 positively regulates EndMT-associated unique secretory phenotype (EndMT-SP) characterized by induction of multiple inflammatory chemokines and cytokines including CCL17, CX3CL1, CXCL16, IL-6 and Angptl2. As a mechanism for this phenomenon, TGF-β and miR-31 suppress Stk40, a negative regulator of NF-κB pathway. Interestingly, TGF-β induces alternative polyadenylation (APA)-coupled miR-31-dependent Stk40 suppression without concomitant miR-31 induction, and APA-mediated exclusion of internal poly(A) sequence in Stk40 3'UTR enhances target efficiency of Stk40. Finally, miR-31 functions as a molecular hub to integrate TGF-β and TNF-α signaling to enhance EndMT. These data confirm that constitutively active microRNAs, as well as inducible microRNAs, serve as phenotypic modifiers interconnected with transcriptome dynamics during EndMT. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Blunted activation of NF-{kappa}B and NF-{kappa}B-dependent gene expression by geranylgeranylacetone: Involvement of unfolded protein response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayakawa, Kunihiro; Hiramatsu, Nobuhiko; Okamura, Maro

2008-01-04

Geranylgeranylacetone (GGA), an anti-ulcer agent, has anti-inflammatory potential against experimental colitis and ischemia-induced renal inflammation. However, molecular mechanisms involved in its anti-inflammatory effects are largely unknown. We found that, in glomerular mesangial cells, GGA blocked activation of nuclear factor-{kappa}B and consequent induction of monocyte chemoattractant protein 1 (MCP-1) by inflammatory cytokines. It was inversely correlated with induction of unfolded protein response (UPR) evidenced by expression of 78 kDa glucose-regulated protein (GRP78) and suppression of endoplasmic reticulum stress-responsive alkaline phosphatase. Various inducers of UPR including tunicamycin, thapsigargin, A23187, 2-deoxyglucose, dithiothreitol, and AB{sub 5} subtilase cytotoxin reproduced the suppressive effects of GGA.more » Furthermore, attenuation of UPR by stable transfection with GRP78 diminished the anti-inflammatory effects of GGA. These results disclosed a novel, UPR-dependent mechanism underlying the anti-inflammatory potential of GGA.« less

miR-191 and miR-135 are required for long-lasting spine remodelling associated with synaptic long-term depression

NASA Astrophysics Data System (ADS)

Hu, Zhonghua; Yu, Danni; Gu, Qin-Hua; Yang, Yanqin; Tu, Kang; Zhu, Jun; Li, Zheng

2014-02-01

Activity-dependent modification of dendritic spines, subcellular compartments accommodating postsynaptic specializations in the brain, is an important cellular mechanism for brain development, cognition and synaptic pathology of brain disorders. NMDA receptor-dependent long-term depression (NMDAR-LTD), a prototypic form of synaptic plasticity, is accompanied by prolonged remodelling of spines. The mechanisms underlying long-lasting spine remodelling in NMDAR-LTD, however, are largely unclear. Here we show that LTD induction causes global changes in miRNA transcriptomes affecting many cellular activities. Specifically, we show that expression changes of miR-191 and miR-135 are required for maintenance but not induction of spine restructuring. Moreover, we find that actin depolymerization and AMPA receptor exocytosis are regulated for extended periods of time by miRNAs to support long-lasting spine plasticity. These findings reveal a miRNA-mediated mechanism and a role for AMPA receptor exocytosis in long-lasting spine plasticity, and identify a number of candidate miRNAs involved in LTD.

PXR-dependent induction of human CYP3A4 gene expression by organochlorine pesticides.

PubMed

Coumoul, Xavier; Diry, Monique; Barouki, Robert

2002-11-15

OCP are xenobiotics which display various toxic effects on animal and human health. One of their effects is to bind and activate estrogen receptor alpha (ERalpha). We have previously studied the down-regulation of induced CYP1A1 (cytochrome P450) expression by this class of molecules in mammary carcinoma cells and shown the importance of ERalpha in this process. However, an alternative mechanism was suggested by those experiments in hepatoma cells. In this study, we have performed Northern blot and transient transfection assays in various cell lines and shown that OCP activate human pregnane X receptor (PXR) and subsequent CYP3A4 mRNA expression. This effect is mediated by the distal xenobiotic responsive element modulator of the promoter. The induction of CYP3A4 by OCP was dose-dependent within the 1-10 microM range. The data suggest that chronic exposure to OCP could alter a major metabolite pathway in human liver and putatively modify the pharmacokinetics of drugs and pollutants.

Synaptic synthesis, dephosphorylation, and degradation: a novel paradigm for an activity-dependent neuronal control of CDKL5.

PubMed

La Montanara, Paolo; Rusconi, Laura; Locarno, Albina; Forti, Lia; Barbiero, Isabella; Tramarin, Marco; Chandola, Chetan; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

2015-02-13

Mutations in the X-linked CDKL5 (cyclin-dependent kinase-like 5) gene have been associated with several forms of neurodevelopmental disorders, including atypical Rett syndrome, autism spectrum disorders, and early infantile epileptic encephalopathy. Accordingly, loss of CDKL5 in mice results in autistic-like features and impaired neuronal communication. Although the biological functions of CDKL5 remain largely unknown, recent pieces of evidence suggest that CDKL5 is involved in neuronal plasticity. Herein, we show that, at all stages of development, neuronal depolarization induces a rapid increase in CDKL5 levels, mostly mediated by extrasomatic synthesis. In young neurons, this induction is prolonged, whereas in more mature neurons, NMDA receptor stimulation induces a protein phosphatase 1-dependent dephosphorylation of CDKL5 that is mandatory for its proteasome-dependent degradation. As a corollary, neuronal activity leads to a prolonged induction of CDKL5 levels in immature neurons but to a short lasting increase of the kinase in mature neurons. Recent results demonstrate that many genes associated with autism spectrum disorders are crucial components of the activity-dependent signaling networks regulating the composition, shape, and strength of the synapse. Thus, we speculate that CDKL5 deficiency disrupts activity-dependent signaling and the consequent synapse development, maturation, and refinement. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

PPARdelta inhibits IL-1beta-stimulated proliferation and migration of vascular smooth muscle cells via up-regulation of IL-1Ra.

PubMed

Kim, H J; Kim, M Y; Hwang, J S; Kim, H J; Lee, J H; Chang, K C; Kim, J-H; Han, C W; Kim, J-H; Seo, H G

2010-06-01

Activation of peroxisome proliferator-activated receptor (PPAR) delta by GW501516, a specific PPARdelta ligand, significantly inhibited interleukin (IL)-1beta-induced proliferation and migration of vascular smooth muscle cells (VSMCs). This effect of GW501516 was dependent on transforming growth factor-beta, and was mediated through the up-regulation of IL-1 receptor antagonist. The inhibitory effect of GW501516 on VSMC proliferation was associated with cell cycle arrest at the G1 to S phase transition, which was accompanied by the induction of p21 and p53 along with decreased cyclin-dependent kinase 4 expression. Inhibition of cell migration by GW501516 was associated with the down-regulation of matrix metalloproteinase (MMP)-2 and MMP-9 in IL-1beta-treated VSMCs. Inhibition of extracellular signal-regulated kinase significantly reduced the GW501516-mediated inhibition of IL-1beta-stimulated VSMC proliferation. These results suggest that PPARdelta plays an important role in the pathophysiology of diseases associated with the proliferation and migration of VSMCs.

Listeria monocytogenes differential transcriptome analysis reveals temperature-dependent Agr regulation and suggests overlaps with other regulons.

PubMed

Garmyn, Dominique; Augagneur, Yoann; Gal, Laurent; Vivant, Anne-Laure; Piveteau, Pascal

2012-01-01

Listeria monocytogenes is a ubiquitous, opportunistic pathogenic organism. Environmental adaptation requires constant regulation of gene expression. Among transcriptional regulators, AgrA is part of an auto-induction system. Temperature is an environmental cue critical for in vivo adaptation. In order to investigate how temperature may affect AgrA-dependent transcription, we compared the transcriptomes of the parental strain L. monocytogenes EGD-e and its ΔagrA mutant at the saprophytic temperature of 25°C and in vivo temperature of 37°C. Variations of transcriptome were higher at 37°C than at 25°C. Results suggested that AgrA may be involved in the regulation of nitrogen transport, amino acids, purine and pyrimidine biosynthetic pathways and phage-related functions. Deregulations resulted in a growth advantage at 37°C, but affected salt tolerance. Finally, our results suggest overlaps with PrfA, σB, σH and CodY regulons. These overlaps may suggest that through AgrA, Listeria monocytogenes integrates information on its biotic environment.

Listeria monocytogenes Differential Transcriptome Analysis Reveals Temperature-Dependent Agr Regulation and Suggests Overlaps with Other Regulons

PubMed Central

Garmyn, Dominique; Augagneur, Yoann; Gal, Laurent; Vivant, Anne-Laure; Piveteau, Pascal

2012-01-01

Listeria monocytogenes is a ubiquitous, opportunistic pathogenic organism. Environmental adaptation requires constant regulation of gene expression. Among transcriptional regulators, AgrA is part of an auto-induction system. Temperature is an environmental cue critical for in vivo adaptation. In order to investigate how temperature may affect AgrA-dependent transcription, we compared the transcriptomes of the parental strain L. monocytogenes EGD-e and its ΔagrA mutant at the saprophytic temperature of 25°C and in vivo temperature of 37°C. Variations of transcriptome were higher at 37°C than at 25°C. Results suggested that AgrA may be involved in the regulation of nitrogen transport, amino acids, purine and pyrimidine biosynthetic pathways and phage-related functions. Deregulations resulted in a growth advantage at 37°C, but affected salt tolerance. Finally, our results suggest overlaps with PrfA, σB, σH and CodY regulons. These overlaps may suggest that through AgrA, Listeria monocytogenes integrates information on its biotic environment. PMID:23024744

SIRT1 Activity Is Linked to Its Brain Region-Specific Phosphorylation and Is Impaired in Huntington’s Disease Mice

PubMed Central

Tulino, Raffaella; Benjamin, Agnesska C.; Jolinon, Nelly; Smith, Donna L.; Chini, Eduardo N.; Carnemolla, Alisia; Bates, Gillian P.

2016-01-01

Huntington’s disease (HD) is a neurodegenerative disorder for which there are no disease-modifying treatments. SIRT1 is a NAD+-dependent protein deacetylase that is implicated in maintaining neuronal health during development, differentiation and ageing. Previous studies suggested that the modulation of SIRT1 activity is neuroprotective in HD mouse models, however, the mechanisms controlling SIRT1 activity are unknown. We have identified a striatum-specific phosphorylation-dependent regulatory mechanism of SIRT1 induction under normal physiological conditions, which is impaired in HD. We demonstrate that SIRT1 activity is down-regulated in the brains of two complementary HD mouse models, which correlated with altered SIRT1 phosphorylation levels. This SIRT1 impairment could not be rescued by the ablation of DBC1, a negative regulator of SIRT1, but was linked to changes in the sub-cellular distribution of AMPK-α1, a positive regulator of SIRT1 function. This work provides insights into the regulation of SIRT1 activity with the potential for the development of novel therapeutic strategies. PMID:26815359

Rectification of Ion Current in Nanopipettes by External Substrates

PubMed Central

Shi, Wenqing; Baker, Lane A.

2014-01-01

We describe ion distribution and the current-voltage (i-V) response of nanopipettes at different probe-to-substrate distances (Dps) as simulated by finite-element methods. Results suggest electrostatic interactions between a charged substrate and the nanopipette dominate electrophoretic ion transport through the nanopipette when Dps is within one order of magnitude of the Debye length (~10 nm for a 1 mM solution as employed in the simulation). Ion current rectification (ICR) and permselectivity associated with a neutral or charged nanopipette can be reversibly enhanced or reduced dependent on Dps, charge polarity and charge density (σ) of the substrate. Regulation of nanopipette current is a consequence of the enrichment or depletion of ions within the nanopipette interior which influences conductivity of the nanopipette. When the external substrate is less negatively charged than the nanopipette, the substrate first reduces, and then enhances the ICR as Dps decreases. Surprisingly, both experimental and simulated data show that a neutral substrate was also able to reduce and reverse the ICR of a slightly negatively charged nanopipette. Simulated results ascribe such effects to the elimination of ion depletion within the nanopipette at positive potentials. PMID:24200344

Rectification of ion current in nanopipettes by external substrates.

PubMed

Sa, Niya; Lan, Wen-Jie; Shi, Wenqing; Baker, Lane A

2013-12-23

We describe ion distribution and the current-voltage (i-V) response of nanopipettes at different probe-to-substrate distances (Dps) as simulated by finite-element methods. Results suggest electrostatic interactions between a charged substrate and the nanopipette dominate electrophoretic ion transport through the nanopipette when Dps is within 1 order of magnitude of the Debye length (∼10 nm for a 1 mM solution as employed in the simulation). Ion current rectification (ICR) and permselectivity associated with a neutral or charged nanopipette can be reversibly enhanced or reduced dependent on Dps, charge polarity, and charge density (σ) of the substrate. Regulation of nanopipette current is a consequence of the enrichment or depletion of ions within the nanopipette interior, which influences conductivity of the nanopipette. When the external substrate is less negatively charged than the nanopipette, the substrate first reduces, and then enhances the ICR as Dps decreases. Surprisingly, both experimental and simulated data show that a neutral substrate was also able to reduce and reverse the ICR of a slightly negatively charged nanopipette. Simulated results ascribe such effects to the elimination of ion depletion within the nanopipette at positive potentials.

Method to determine transcriptional regulation pathways in organisms

DOEpatents

Gardner, Timothy S.; Collins, James J.; Hayete, Boris; Faith, Jeremiah

2012-11-06

The invention relates to computer-implemented methods and systems for identifying regulatory relationships between expressed regulating polypeptides and targets of the regulatory activities of such regulating polypeptides. More specifically, the invention provides a new method for identifying regulatory dependencies between biochemical species in a cell. In particular embodiments, provided are computer-implemented methods for identifying a regulatory interaction between a transcription factor and a gene target of the transcription factor, or between a transcription factor and a set of gene targets of the transcription factor. Further provided are genome-scale methods for predicting regulatory interactions between a set of transcription factors and a corresponding set of transcriptional target substrates thereof.

Activation of pregnane X receptor and induction of MDR1 by dietary phytochemicals.

PubMed

Satsu, Hideo; Hiura, Yuto; Mochizuki, Keiichi; Hamada, Mika; Shimizu, Makoto

2008-07-09

The pregnane X receptor (PXR) is understood to be the key regulator for gene expression of such drug-metabolizing enzymes and transporters as multidrug-resistant protein 1 (MDR1) and the cytochrome P450 (CYP) family. We examined the effect of dietary phytochemicals on the PXR-dependent transcriptional activity in human intestinal LS180 cells by using a reporter assay. Among approximately 40 kinds of phytochemicals, tangeretin and ginkgolides A and B markedly induced the PXR-dependent transcriptional activity and also the activity of the human MDR1 promoter. The expression levels of MDR1 mRNA as well as of CYP3A4 mRNA, another gene regulated by PXR, were significantly increased by these phytochemicals. Furthermore, an increase was observed of the MDR1 protein and its functional activity by tangeretin and by ginkgolides A and B. These findings strongly suggest that tangeretin and ginkgolides A and B activated PXR, thereby regulating detoxification enzymes and transporters in the intestines.

Hippo signaling regulates Microprocessor and links cell density-dependent miRNA biogenesis to cancer

PubMed Central

Mori, Masaki; Triboulet, Robinson; Mohseni, Morvarid; Schlegelmilch, Karin; Shrestha, Kriti; Camargo, Fernando D.; Gregory, Richard I.

2014-01-01

SUMMARY Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly understood. Here we show that YAP, the downstream target of the tumor-suppressive Hippo signaling pathway regulates miRNA biogenesis in a cell density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding post-transcriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer. PMID:24581491

Antimicrobial Treatment Improves Mycobacterial Survival in Nonpermissive Growth Conditions

PubMed Central

Turapov, Obolbek; Waddell, Simon J.; Burke, Bernard; Glenn, Sarah; Sarybaeva, Asel A.; Tudo, Griselda; Labesse, Gilles; Young, Danielle I.; Young, Michael; Andrew, Peter W.; Butcher, Philip D.; Cohen-Gonsaud, Martin

2014-01-01

Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically, we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases, such as tuberculosis. PMID:24590482

GABAB Receptor Positive Modulation Decreases Selective Molecular and Behavioral Effects of Cocaine

PubMed Central

Lhuillier, Loic; Mombereau, Cedric; Cryan, John F.; Kaupmann, Klemens

2006-01-01

Exposure to cocaine induces selective behavioral and molecular adaptations. In rodents, acute cocaine induces increased locomotor activity whereas prolonged drug exposure results in behavioral locomotor sensitization, which is thought to be a consequence of drug–induced neuroadaptive changes. Recent attention has been given to compounds activating GABAB receptors as potential anti-addictive therapies. In particular the principle of allosteric positive GABAB receptor modulators is very promising in this respect, as positive modulators lack the sedative and muscle relaxant properties of full GABAB receptor agonists such as baclofen. Here we investigated the effects of systemic application of the GABAB receptor positive modulator GS39783 in animals treated with acute and chronic cocaine administration. Both GS39783 and baclofen dose-dependently attenuated acute cocaine-induced hyperlocomotion. Furthermore, both compounds also efficiently blocked cocaine-induced Fos induction in the striatal complex. In chronic studies GS39783 induced a modest attenuation of cocaine-induced locomotor sensitization. Chronic cocaine induces the accumulation of the transcription factor ΔFosB and up regulates cAMP-response-element-binding-protein (CREB) and dopamine-and-cAMP-regulated-phosphoprotein of 32 kd (DARPP-32). GS39783 blocked the induction/activation of DARPP-32 and CREB in the nucleus accumbens and dorsal striatum and partially inhibited ΔFosB accumulation in the dorsal striatum. In summary our data provide evidence that GS39783 attenuates the acute behavioral effects of cocaine exposure in rodents and in addition prevents the induction of selective long-term adaptive changes in dopaminergic signaling pathways. Further investigation of GABAB receptor positive modulation as a novel therapeutic strategy for the treatment of cocaine dependence and possibly other drugs of abuse is therefore warranted. PMID:16710312

Ovarian Expression and Regulation of the Stromelysins During the Periovulatory Period in the Human and the Rat1

PubMed Central

McCord, Lauren A.; Li, Feixue; Rosewell, Katherine L.; Brännström, Mats; Curry, Thomas E.

2011-01-01

ABSTRACT The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization. PMID:22116802

Ovarian expression and regulation of the stromelysins during the periovulatory period in the human and the rat.

PubMed

McCord, Lauren A; Li, Feixue; Rosewell, Katherine L; Brännström, Mats; Curry, Thomas E

2012-03-01

The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization.

A novel pyrazolo[1,5-a]pyrimidine is a potent inhibitor of cyclin-dependent protein kinases 1, 2, and 9, which demonstrates antitumor effects in human tumor xenografts following oral administration.

PubMed

Heathcote, Dean A; Patel, Hetal; Kroll, Sebastian H B; Hazel, Pascale; Periyasamy, Manikandan; Alikian, Mary; Kanneganti, Seshu K; Jogalekar, Ashutosh S; Scheiper, Bodo; Barbazanges, Marion; Blum, Andreas; Brackow, Jan; Siwicka, Alekasandra; Pace, Robert D M; Fuchter, Matthew J; Snyder, James P; Liotta, Dennis C; Freemont, Paul S; Aboagye, Eric O; Coombes, R Charles; Barrett, Anthony G M; Ali, Simak

2010-12-23

Cyclin-dependent protein kinases (CDKs) are central to the appropriate regulation of cell proliferation, apoptosis, and gene expression. Abnormalities in CDK activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Here, we report the identification of a pyrazolo[1,5-a]pyrimidine derived compound, 4k (BS-194), as a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7, and CDK9 (IC₅₀= 3, 30, 30, 250, and 90 nmol/L, respectively). Cell-based studies showed inhibition of the phosphorylation of CDK substrates, Rb and the RNA polymerase II C-terminal domain, down-regulation of cyclins A, E, and D1, and cell cycle block in the S and G₂/M phases. Consistent with these findings, 4k demonstrated potent antiproliferative activity in 60 cancer cell lines tested (mean GI₅₀= 280 nmol/L). Pharmacokinetic studies showed that 4k is orally bioavailable, with an elimination half-life of 178 min following oral dosing in mice. When administered at a concentration of 25 mg/kg orally, 4k inhibited human tumor xenografts and suppressed CDK substrate phosphorylation. These findings identify 4k as a novel, potent CDK selective inhibitor with potential for oral delivery in cancer patients.

Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis.

PubMed

Barberon, Marie; Dubeaux, Guillaume; Kolb, Cornelia; Isono, Erika; Zelazny, Enric; Vert, Grégory

2014-06-03

In plants, the controlled absorption of soil nutrients by root epidermal cells is critical for growth and development. IRON-REGULATED TRANSPORTER 1 (IRT1) is the main root transporter taking up iron from the soil and is also the main entry route in plants for potentially toxic metals such as manganese, zinc, cobalt, and cadmium. Previous work demonstrated that the IRT1 protein localizes to early endosomes/trans-Golgi network (EE/TGN) and is constitutively endocytosed through a monoubiquitin- and clathrin-dependent mechanism. Here, we show that the availability of secondary non-iron metal substrates of IRT1 (Zn, Mn, and Co) controls the localization of IRT1 between the outer polar domain of the plasma membrane and EE/TGN in root epidermal cells. We also identify FYVE1, a phosphatidylinositol-3-phosphate-binding protein recruited to late endosomes, as an important regulator of IRT1-dependent metal transport and metal homeostasis in plants. FYVE1 controls IRT1 recycling to the plasma membrane and impacts the polar delivery of this transporter to the outer plasma membrane domain. This work establishes a functional link between the dynamics and the lateral polarity of IRT1 and the transport of its substrates, and identifies a molecular mechanism driving polar localization of a cell surface protein in plants.

Insulin receptor substrate signaling controls cardiac energy metabolism and heart failure.

PubMed

Guo, Cathy A; Guo, Shaodong

2017-06-01

The heart is an insulin-dependent and energy-consuming organ in which insulin and nutritional signaling integrates to the regulation of cardiac metabolism, growth and survival. Heart failure is highly associated with insulin resistance, and heart failure patients suffer from the cardiac energy deficiency and structural and functional dysfunction. Chronic pathological conditions, such as obesity and type 2 diabetes mellitus, involve various mechanisms in promoting heart failure by remodeling metabolic pathways, modulating cardiac energetics and impairing cardiac contractility. Recent studies demonstrated that insulin receptor substrates 1 and 2 (IRS-1,-2) are major mediators of both insulin and insulin-like growth factor-1 (IGF-1) signaling responsible for myocardial energetics, structure, function and organismal survival. Importantly, the insulin receptor substrates (IRS) play an important role in the activation of the phosphatidylinositide-3-dependent kinase (PI-3K) that controls Akt and Foxo1 signaling cascade, regulating the mitochondrial function, cardiac energy metabolism and the renin-angiotensin system. Dysregulation of this branch in signaling cascades by insulin resistance in the heart through the endocrine system promotes heart failure, providing a novel mechanism for diabetic cardiomyopathy. Therefore, targeting this branch of IRS→PI-3K→Foxo1 signaling cascade and associated pathways may provide a fundamental strategy for the therapeutic and nutritional development in control of metabolic and cardiovascular diseases. In this review, we focus on insulin signaling and resistance in the heart and the role energetics play in cardiac metabolism, structure and function. © 2017 Society for Endocrinology.

Expression-based clustering of CAZyme-encoding genes of Aspergillus niger.

PubMed

Gruben, Birgit S; Mäkelä, Miia R; Kowalczyk, Joanna E; Zhou, Miaomiao; Benoit-Gelber, Isabelle; De Vries, Ronald P

2017-11-23

The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains ΔxlnR, ΔaraR, ΔamyR, ΔrhaR and ΔgalX that were grown on their specific inducing compounds. The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far. Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In addition, the data provides additional evidence in favor of and against the similarity-based functions assigned to uncharacterized genes.

Exposure to rosiglitazone, a PPAR-γ agonist, in late gestation reduces the abundance of factors regulating cardiac metabolism and cardiomyocyte size in the sheep fetus

PubMed Central

Lie, Shervi; Hui, Melisa; McMillen, I. Caroline; Muhlhausler, Beverly S.; Posterino, Giuseppe S.; Dunn, Stacey L.; Wang, Kimberley C.; Botting, Kimberley J.

2014-01-01

It is unknown whether cardiomyocyte hypertrophy and the transition to fatty acid oxidation as the main source of energy after birth is dependent on the maturation of the cardiomyocytes' metabolic system, or on the limitation of substrate availability before birth. This study aimed to investigate whether intrafetal administration of a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, during late gestation can stimulate the expression of factors regulating cardiac growth and metabolism in preparation for birth, and the consequences of cardiac contractility in the fetal sheep at ∼140 days gestation. The mRNA expression and protein abundance of key factors regulating growth and metabolism were quantified using quantitative RT-PCR and Western blot analysis, respectively. Cardiac contractility was determined by measuring the Ca2+ sensitivity and maximum Ca2+-activated force of skinned cardiomyocyte bundles. Rosiglitazone-treated fetuses had a lower cardiac abundance of insulin-signaling molecules, including insulin receptor-β, insulin receptor substrate-1 (IRS-1), phospho-IRS-1 (Tyr-895), phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, PI3K catalytic subunit p110α, phospho-3-phosphoinositide-dependent protein kinase 1 (Ser-241), protein kinase B (Akt-1), phospho-Akt (Ser-273), PKCζ, phospho-PKCζ(Thr-410), Akt substrate 160 kDa (AS160), phospho-AS160 (Thr-642), and glucose transporter type-4. Additionally, cardiac abundance of regulators of fatty acid β-oxidation, including adiponectin receptor 1, AMPKα, phospho-AMPKα (Thr-172), phospho-acetyl CoA carboxylase (Ser-79), carnitine palmitoyltransferase-1, and PGC-1α was lower in the rosiglitazone-treated group. Rosiglitazone administration also resulted in a decrease in cardiomyocyte size. Rosiglitazone administration in the late-gestation sheep fetus resulted in a decreased abundance of factors regulating cardiac glucose uptake, fatty acid β-oxidation, and cardiomyocyte size. These findings suggest that activation of PPAR-γ using rosiglitazone does not promote the maturation of cardiomyocytes; rather, it may decrease cardiac metabolism and compromise cardiac health later in life. PMID:24477540

Exposure to rosiglitazone, a PPAR-γ agonist, in late gestation reduces the abundance of factors regulating cardiac metabolism and cardiomyocyte size in the sheep fetus.

PubMed

Lie, Shervi; Hui, Melisa; McMillen, I Caroline; Muhlhausler, Beverly S; Posterino, Giuseppe S; Dunn, Stacey L; Wang, Kimberley C; Botting, Kimberley J; Morrison, Janna L

2014-03-15

It is unknown whether cardiomyocyte hypertrophy and the transition to fatty acid oxidation as the main source of energy after birth is dependent on the maturation of the cardiomyocytes' metabolic system, or on the limitation of substrate availability before birth. This study aimed to investigate whether intrafetal administration of a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, during late gestation can stimulate the expression of factors regulating cardiac growth and metabolism in preparation for birth, and the consequences of cardiac contractility in the fetal sheep at ∼140 days gestation. The mRNA expression and protein abundance of key factors regulating growth and metabolism were quantified using quantitative RT-PCR and Western blot analysis, respectively. Cardiac contractility was determined by measuring the Ca(2+) sensitivity and maximum Ca(2+)-activated force of skinned cardiomyocyte bundles. Rosiglitazone-treated fetuses had a lower cardiac abundance of insulin-signaling molecules, including insulin receptor-β, insulin receptor substrate-1 (IRS-1), phospho-IRS-1 (Tyr-895), phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, PI3K catalytic subunit p110α, phospho-3-phosphoinositide-dependent protein kinase 1 (Ser-241), protein kinase B (Akt-1), phospho-Akt (Ser-273), PKCζ, phospho-PKCζ(Thr-410), Akt substrate 160 kDa (AS160), phospho-AS160 (Thr-642), and glucose transporter type-4. Additionally, cardiac abundance of regulators of fatty acid β-oxidation, including adiponectin receptor 1, AMPKα, phospho-AMPKα (Thr-172), phospho-acetyl CoA carboxylase (Ser-79), carnitine palmitoyltransferase-1, and PGC-1α was lower in the rosiglitazone-treated group. Rosiglitazone administration also resulted in a decrease in cardiomyocyte size. Rosiglitazone administration in the late-gestation sheep fetus resulted in a decreased abundance of factors regulating cardiac glucose uptake, fatty acid β-oxidation, and cardiomyocyte size. These findings suggest that activation of PPAR-γ using rosiglitazone does not promote the maturation of cardiomyocytes; rather, it may decrease cardiac metabolism and compromise cardiac health later in life.

KDM1 Class Flavin-Dependent Protein Lysine Demethylases

PubMed Central

Burg, Jonathan M.; Link, Jennifer E.; Morgan, Brittany S.; Heller, Frederick J.; Hargrove, Amanda E.; McCafferty, Dewey G.

2015-01-01

Flavin-dependent, lysine-specific protein demethylases (KDM1s) are a subfamily of amine oxidases that catalyze the selective posttranslational oxidative demethylation of methyllysine side chains within protein and peptide substrates. KDM1s participate in the widespread epigenetic regulation of both normal and disease state transcriptional programs. Their activities are central to various cellular functions, such as hematopoietic and neuronal differentiation, cancer proliferation and metastasis, and viral lytic replication and establishment of latency. Interestingly, KDM1s function as catalytic subunits within complexes with coregulatory molecules that modulate enzymatic activity of the demethylases and coordinate their access to specific substrates at distinct sites within the cell and chromatin. Although several classes of KDM1 -selective small molecule inhibitors have been recently developed, these pan-active site inhibition strategies lack the ability to selectively discriminate between KDM1 activity in specific, and occasionally opposing, functional contexts within these complexes. Here we review the discovery of this class of demethylases, their structures, chemical mechanisms, and specificity. Additionally, we review inhibition of this class of enzymes as well as emerging interactions with coregulatory molecules that regulate demethylase activity in highly specific functional contexts of biological and potential therapeutic importance. PMID:25787087

Different Expression Patterns of Genes from the Exo-Xis Region of Bacteriophage λ and Shiga Toxin-Converting Bacteriophage Ф24B following Infection or Prophage Induction in Escherichia coli

PubMed Central

Bloch, Sylwia; Nejman-Faleńczyk, Bożena; Dydecka, Aleksandra; Łoś, Joanna M.; Felczykowska, Agnieszka; Węgrzyn, Alicja; Węgrzyn, Grzegorz

2014-01-01

Lambdoid bacteriophages serve as useful models in microbiological and molecular studies on basic biological process. Moreover, this family of viruses plays an important role in pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, as they are carriers of genes coding for Shiga toxins. Efficient expression of these genes requires lambdoid prophage induction and multiplication of the phage genome. Therefore, understanding the mechanisms regulating these processes appears essential for both basic knowledge and potential anti-EHEC applications. The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. Recent report indicated that the Ea8.5 protein, encoded in this region, contains a newly discovered fused homeodomain/zinc-finger fold, suggesting its plausible regulatory role. Moreover, subsequent studies demonstrated that overexpression of the exo-xis region from a multicopy plasmid resulted in impaired lysogenization of E. coli and more effective induction of λ and Ф24B prophages. In this report, we demonstrate that after prophage induction, the increase in phage DNA content in the host cells is more efficient in E. coli bearing additional copies of the exo-xis region, while survival rate of such bacteria is lower, which corroborated previous observations. Importantly, by using quantitative real-time reverse transcription PCR, we have determined patterns of expressions of particular genes from this region. Unexpectedly, in both phages λ and Ф24B, these patterns were significantly different not only between conditions of the host cells infection by bacteriophages and prophage induction, but also between induction of prophages with various agents (mitomycin C and hydrogen peroxide). This may shed a new light on our understanding of regulation of lambdoid phage development, depending on the mode of lytic cycle initiation. PMID:25310402

Biphasic Somatic A-Type K+ Channel Downregulation Mediates Intrinsic Plasticity in Hippocampal CA1 Pyramidal Neurons

PubMed Central

Jung, Sung-Cherl; Hoffman, Dax A.

2009-01-01

Since its original description, the induction of synaptic long-term potentiation (LTP) has been known to be accompanied by a lasting increase in the intrinsic excitability (intrinsic plasticity) of hippocampal neurons. Recent evidence shows that dendritic excitability can be enhanced by an activity-dependent decrease in the activity of A-type K+ channels. In the present manuscript, we examined the role of A-type K+ channels in regulating intrinsic excitability of CA1 pyramidal neurons of the hippocampus after synapse-specific LTP induction. In electrophysiological recordings we found that LTP induced a potentiation of excitability which was accompanied by a two-phased change in A-type K+ channel activity recorded in nucleated patches from organotypic slices of rat hippocampus. Induction of LTP resulted in an immediate but short lasting hyperpolarization of the voltage-dependence of steady-state A-type K+ channel inactivation along with a progressive, long-lasting decrease in peak A-current density. Blocking clathrin-mediated endocytosis prevented the A-current decrease and most measures of intrinsic plasticity. These results suggest that two temporally distinct but overlapping mechanisms of A-channel downregulation together contribute to the plasticity of intrinsic excitability. Finally we show that intrinsic plasticity resulted in a global enhancement of EPSP-spike coupling. PMID:19662093

Induction of cap-independent BiP (hsp-3) and Bcl-2 (ced-9) translation in response to eIF4G (IFG-1) depletion in C. elegans

PubMed Central

Morrison, J Kaitlin; Friday, Andrew J; Henderson, Melissa A; Hao, Enhui; Keiper, Brett D

2014-01-01

During apoptosis, activated caspases cleave the translation initiation factor eIF4G. This cleavage disrupts cap-dependent mRNA translation initiation within the cell. However, a specific subset of mRNAs can still be recruited for protein synthesis in a cap-independent manner by the residual initiation machinery. Many of these mRNAs, including cell death related mRNAs, contain internal ribosome entry sites (IRESes) that promote their enhanced translation during apoptosis. Still other mRNAs have little dependence on the cap recognition mechanism. The expression of the encoded proteins, both anti- and pro-apoptotic, allows for an initial period of attempted cell survival, then commitment to cell death when damage is extensive. In this study we address the translational regulation of the stress and apoptosis-related mRNAs in C. elegans: BiP (hsp-3) (hsp-4), Hif-1 (hif-1), p53 (cep-1), Bcl-2 (ced-9) and Apaf-1 (ced-4). Altered translational efficiency of these messages was observed upon depletion of cap-dependent translation and induction of apoptosis within the C. elegans gonad. Our findings suggest a physiological link between the cap-independent mechanism and the enhanced translation of hsp-3 and ced-9. This increase in the efficiency of translation may be integral to the stress response during the induction of physiological apoptosis. PMID:26779406

Substrate specific effects of calcium on metabolism of rat heart mitochondria.

PubMed

Panov, A V; Scaduto, R C

1996-04-01

Oxidative metabolism in the heart is tightly coupled to mechanical work. Because this coupling process is believed to involve Ca2+, the roles of mitochondrial Ca2+ in the regulation of oxidative phosphorylation was studied in isolated rat heart mitochondria. The electrical component of the mitochondrial membrane potential (delta psi) and the redox state of the pyridine nucleotides were determined during the oxidation of various substrates under different metabolic states. In the absence of added adenine nucleotides, the NADP+ redox couple was almost completely reduced, regardless of the specific substrate and the presence of Ca2+, whereas NAD+ couple redox state was highly dependent on the substrate type and the presence of Ca2+. Titration of respiration with ADP, in the presence of excess hexokinase and glucose, showed that both respiration and NAD(P)+ reduction were very sensitive to ADP. The maximal enzyme reaction rate of ADP-stimulated respiration Michaelis constants (Km) for ADP were dependent on the particular substrate employed. delta psi was much less sensitive to ADP. With either alpha-ketoglutarate or glutamate as substrate, Ca2+ significantly increased reduction of NAD(P)+.Ca2+ did not influence NAD(P)+ reduction with either acetylcarnitine or pyruvate as substrate. In the presence of ADP, delta psi was increased by Ca2+ at all metabolic states with glutamate plus malate, 0.5 mM alpha-ketoglutarate plus malate, or pyruvate plus malate as substrates. The data presented support the hypothesis that cardiac respiration is controlled by the availability of both Ca2+ and ADP to mitochondria. The data indicate that an increase in substrate supply to mitochondria can increase mitochondrial respiration at given level of ADP. This effect can be produced by Ca2+ with substrates such as glutamate, which utilize alpha-ketoglutarate dehydrogenase activity for oxidation. Increases in respiration by Ca2+ may mitigate an increase in ADP during periods of increased cardiac work.

The Gene Regulatory Network of Lens Induction Is Wired through Meis-Dependent Shadow Enhancers of Pax6

PubMed Central

Antosova, Barbora; Smolikova, Jana; Klimova, Lucie; Lachova, Jitka; Bendova, Michaela; Kozmikova, Iryna; Machon, Ondrej; Kozmik, Zbynek

2016-01-01

Lens induction is a classical developmental model allowing investigation of cell specification, spatiotemporal control of gene expression, as well as how transcription factors are integrated into highly complex gene regulatory networks (GRNs). Pax6 represents a key node in the gene regulatory network governing mammalian lens induction. Meis1 and Meis2 homeoproteins are considered as essential upstream regulators of Pax6 during lens morphogenesis based on their interaction with the ectoderm enhancer (EE) located upstream of Pax6 transcription start site. Despite this generally accepted regulatory pathway, Meis1-, Meis2- and EE-deficient mice have surprisingly mild eye phenotypes at placodal stage of lens development. Here, we show that simultaneous deletion of Meis1 and Meis2 in presumptive lens ectoderm results in arrested lens development in the pre-placodal stage, and neither lens placode nor lens is formed. We found that in the presumptive lens ectoderm of Meis1/Meis2 deficient embryos Pax6 expression is absent. We demonstrate using chromatin immunoprecipitation (ChIP) that in addition to EE, Meis homeoproteins bind to a remote, ultraconserved SIMO enhancer of Pax6. We further show, using in vivo gene reporter analyses, that the lens-specific activity of SIMO enhancer is dependent on the presence of three Meis binding sites, phylogenetically conserved from man to zebrafish. Genetic ablation of EE and SIMO enhancers demostrates their requirement for lens induction and uncovers an apparent redundancy at early stages of lens development. These findings identify a genetic requirement for Meis1 and Meis2 during the early steps of mammalian eye development. Moreover, they reveal an apparent robustness in the gene regulatory mechanism whereby two independent "shadow enhancers" maintain critical levels of a dosage-sensitive gene, Pax6, during lens induction. PMID:27918583

Mineralization induction effects of osteopontin, bone sialoprotein, and dentin phosphoprotein on a biomimetic collagen substrate.

PubMed

Zurick, Kevin M; Qin, Chunlin; Bernards, Matthew T

2013-06-01

Native bone tissue is composed of a matrix of collagen, noncollagenous proteins, and calcium phosphate minerals, which are primarily hydroxyapatite. The SIBLING (small integrin-binding ligand, N-linked glycoprotein) family of proteins is the primary noncollagenous protein group found in mineralized tissues. In this work, the mineralization induction capabilities of three of the SIBLING members, bone sialoprotein (BSP), osteopontin (OPN), and the calcium-binding subdomain of dentin sialophosphoprotein, dentin phosphoprotein (DPP), are directly compared on a biomimetic collagen substrate. A self-assembled, loosely aligned collagen fibril substrate was prepared, and then (125) I-radiolabeled adsorption isotherms were developed for BSP, OPN, and DPP. The results showed that BSP exhibited the highest binding capacity for collagen at lower concentrations, followed by DPP and OPN. However, at the highest concentrations, all three proteins had similar adsorption levels. The adsorption isotherms were then used to identify conditions that resulted in identical amounts of adsorbed protein. These substrates were prepared and placed in simulated body fluid for 5, 10, and 24 h at 37°C. The resulting mineral morphology was assessed by atomic force microscopy, and the composition was determined using photochemical assays. Mineralization was seen in the presence of all the proteins. However, DPP was seen to be the only protein that formed individual mineral nodules similar to those seen in developing bone. This suggests that DPP plays a significant role in the biomineralization process and that the incorporation of DPP into tissue engineering constructs may facilitate the induction of biomimetic mineral formation. Copyright © 2012 Wiley Periodicals, Inc.

Mineralization Induction Effects of Osteopontin, Bone Sialoprotein, and Dentin Phosphoprotein on a Biomimetic Collagen Substrate

PubMed Central

Zurick, Kevin M.; Qin, Chunlin; Bernards, Matthew T.

2012-01-01

Native bone tissue is composed of a matrix of collagen, non-collagenous proteins, and calcium phosphate minerals, which are primarily hydroxyapatite (HA). The SIBLING (small integrin-binding ligand, N-linked glycoprotein) family of proteins is the primary non-collagenous protein group found in mineralized tissues. In this work, the mineralization induction capabilities of three of the SIBLING members, bone sialoprotein (BSP), osteopontin (OPN), and the calcium binding subdomain of dentin sialophosphoprotein, dentin phosphoprotein (DPP), are directly compared on a biomimetic collagen substrate. A self-assembled, loosely aligned collagen fibril substrate was prepared and then 125I radiolabeled adsorption isotherms were developed for BSP, OPN, and DPP. The results showed that BSP exhibited the highest binding capacity for collagen at lower concentrations, followed by DPP and OPN. However, at the highest concentrations all three proteins had similar adsorption levels. The adsorption isotherms were then used to identify conditions that resulted in identical amounts of adsorbed protein. These substrates were prepared and placed in simulated body fluid for 5 hours, 10 hours, and 24 hours at 37°C. The resulting mineral morphology was assessed by atomic force microscopy and the composition was determined using photochemical assays. Mineralization was seen in the presence of all of the proteins. However, DPP was seen to be the only protein that formed individual mineral nodules similar to those seen in developing bone. This suggests that DPP plays a significant role in the biomineralization process and that the incorporation of DPP into tissue engineering constructs may facilitate the induction of biomimetic mineral formation. PMID:23161527

Proinflammatory TLR signaling is regulated by a TRAF2-dependent proteolysis mechanism in macrophages

PubMed Central

Jin, Jin; Xiao, Yichuan; Hu, Hongbo; Zou, Qiang; Li, Yanchuan; Gao, Yanpan; Ge, Wei; Cheng, Xuhong; Sun, Shao-Cong

2014-01-01

Signal transduction from toll-like receptors (TLRs) is important for innate immunity against infections, but deregulated TLR signaling contributes to inflammatory disorders. Here we show that myeloid cell-specific ablation of TRAF2 greatly promotes TLR-stimulated proinflammatory cytokine expression in macrophages and exacerbates colitis in an animal model of inflammatory bowel disease. TRAF2 deficiency does not enhance upstream signaling events, but it causes accumulation of two transcription factors, c-Rel and IRF5, known to mediate proinflammatory cytokine induction. Interestingly, TRAF2 controls the fate of c-Rel and IRF5 via a proteasome-dependent mechanism that also requires TRAF3 and the E3 ubiquitin ligase cIAP. We further show that TRAF2 also regulates inflammatory cytokine production in tumor-associated macrophages and facilitates tumor growth. These findings demonstrate an unexpected anti-inflammatory function of TRAF2 and suggest a proteasome-dependent mechanism that limits the proinflammatory TLR signaling. PMID:25565375

Aptamer-Binding Directed DNA Origami Pattern for Logic Gates.

PubMed

Yang, Jing; Jiang, Shuoxing; Liu, Xiangrong; Pan, Linqiang; Zhang, Cheng

2016-12-14

In this study, an aptamer-substrate strategy is introduced to control programmable DNA origami pattern. Combined with DNA aptamer-substrate binding and DNAzyme-cutting, small DNA tiles were specifically controlled to fill into the predesigned DNA origami frame. Here, a set of DNA logic gates (OR, YES, and AND) are performed in response to the stimuli of adenosine triphosphate (ATP) and cocaine. The experimental results are confirmed by AFM imaging and time-dependent fluorescence changes, demonstrating that the geometric patterns are regulated in a controllable and programmable manner. Our approach provides a new platform for engineering programmable origami nanopatterns and constructing complex DNA nanodevices.

Mechanisms for Antagonistic Regulation of AMPA and NMDA-D1 Receptor Complexes at Postsynaptic Sites

NASA Technical Reports Server (NTRS)

Schumann, Johann; Scheler, Gabriele

2004-01-01

From the analysis of these pathways we conclude that postsynaptic processes that regulate synaptic transmission undergo significant cross-talk with respect to glutamatergic and neuromodulatory (dopamine) signals. The main hypothesis is that of a compensatory regulation, a competitive switch between the induction of increased AMPA conductance by CaMKII-dependent phosphorylation and reduced expression of PP2A, and increased D1 receptor sensitivity and expression by increased PKA, PP2A and decreased PP-1/calcineurin expression. Both types of plasticity are induced by NMDA receptor activation and increased internal calcium, they require different internal conditions to become expressed. Specifically we propose that AMPA regulation and D1 regulation are inversely coupled;The net result may be a bifurcation of synaptic state into predominantly AMPA or NMDA-D1 synapses. This could have functional consequences: stable connections for AMPA and conditional gating for NMDA-D1 synapses.

JACKDAW controls epidermal patterning in the Arabidopsis root meristem through a non-cell-autonomous mechanism.

PubMed

Hassan, Hala; Scheres, Ben; Blilou, Ikram

2010-05-01

In Arabidopsis, specification of the hair and non-hair epidermal cell types is position dependent, in that hair cells arise over clefts in the underlying cortical cell layer. Epidermal patterning is determined by a network of transcriptional regulators that respond to an as yet unknown cue from underlying tissues. Previously, we showed that JACKDAW (JKD), a zinc finger protein, localizes in the quiescent centre and the ground tissue, and regulates tissue boundaries and asymmetric cell division by delimiting SHORT-ROOT movement. Here, we provide evidence that JKD controls position-dependent signals that regulate epidermal-cell-type patterning. JKD is required for appropriately patterned expression of the epidermal cell fate regulators GLABRA2, CAPRICE and WEREWOLF. Genetic interaction studies indicate that JKD operates upstream of the epidermal patterning network in a SCRAMBLED (SCM)-dependent fashion after embryogenesis, but acts independent of SCM in embryogenesis. Tissue-specific induction experiments indicate non-cell-autonomous action of JKD from the underlying cortex cell layer to specify epidermal cell fate. Our findings are consistent with a model where JKD induces a signal in every cortex cell that is more abundant in the hair cell position owing to the larger surface contact of cells located over a cleft.

The Developmental Intestinal Regulator ELT-2 Controls p38-Dependent Immune Responses in Adult C. elegans

PubMed Central

Block, Dena H. S.; Twumasi-Boateng, Kwame; Kang, Hae Sung; Carlisle, Jolie A.; Hanganu, Alexandru; Lai, Ty Yu-Jen; Shapira, Michael

2015-01-01

GATA transcription factors play critical roles in cellular differentiation and development. However, their roles in mature tissues are less understood. In C. elegans larvae, the transcription factor ELT-2 regulates terminal differentiation of the intestine. It is also expressed in the adult intestine, where it was suggested to maintain intestinal structure and function, and where it was additionally shown to contribute to infection resistance. To study the function of elt-2 in adults we characterized elt-2-dependent gene expression following its knock-down specifically in adults. Microarray analysis identified two ELT-2-regulated gene subsets: one, enriched for hydrolytic enzymes, pointed at regulation of constitutive digestive functions as a dominant role of adult elt-2; the second was enriched for immune genes that are induced in response to Pseudomonas aeruginosa infection. Focusing on the latter, we used genetic analyses coupled to survival assays and quantitative RT-PCR to interrogate the mechanism(s) through which elt-2 contributes to immunity. We show that elt-2 controls p38-dependent gene induction, cooperating with two p38-activated transcription factors, ATF-7 and SKN-1. This demonstrates a mechanism through which the constitutively nuclear elt-2 can impact induced responses, and play a dominant role in C. elegans immunity. PMID:26016853

Hormonal regulation of platypus Beta-lactoglobulin and monotreme lactation protein genes.

PubMed

Enjapoori, Ashwantha Kumar; Lefèvre, Christophe M; Nicholas, Kevin R; Sharp, Julie A

2017-02-01

Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantitative prediction of repaglinide-rifampicin complex drug interactions using dynamic and static mechanistic models: delineating differential CYP3A4 induction and OATP1B1 inhibition potential of rifampicin.

PubMed

Varma, Manthena V S; Lin, Jian; Bi, Yi-An; Rotter, Charles J; Fahmi, Odette A; Lam, Justine L; El-Kattan, Ayman F; Goosen, Theunis C; Lai, Yurong

2013-05-01

Repaglinide is mainly metabolized by cytochrome P450 enzymes CYP2C8 and CYP3A4, and it is also a substrate to a hepatic uptake transporter, organic anion transporting polypeptide (OATP)1B1. The purpose of this study is to predict the dosing time-dependent pharmacokinetic interactions of repaglinide with rifampicin, using mechanistic models. In vitro hepatic transport of repaglinide, characterized using sandwich-cultured human hepatocytes, and intrinsic metabolic parameters were used to build a dynamic whole-body physiologically-based pharmacokinetic (PBPK) model. The PBPK model adequately described repaglinide plasma concentration-time profiles and successfully predicted area under the plasma concentration-time curve ratios of repaglinide (within ± 25% error), dosed (staggered 0-24 hours) after rifampicin treatment when primarily considering induction of CYP3A4 and reversible inhibition of OATP1B1 by rifampicin. Further, a static mechanistic "extended net-effect" model incorporating transport and metabolic disposition parameters of repaglinide and interaction potency of rifampicin was devised. Predictions based on the static model are similar to those observed in the clinic (average error ∼19%) and to those based on the PBPK model. Both the models suggested that the combined effect of increased gut extraction and decreased hepatic uptake caused minimal repaglinide systemic exposure change when repaglinide is dosed simultaneously or 1 hour after the rifampicin dose. On the other hand, isolated induction effect as a result of temporal separation of the two drugs translated to an approximate 5-fold reduction in repaglinide systemic exposure. In conclusion, both dynamic and static mechanistic models are instrumental in delineating the quantitative contribution of transport and metabolism in the dosing time-dependent repaglinide-rifampicin interactions.

Atomic layer deposition and characterization of hafnium oxide grown on silicon from tetrakis(diethylamino)hafnium and water vapor

NASA Astrophysics Data System (ADS)

Deshpande, Anand; Inman, Ronald; Jursich, Gregory; Takoudis, Christos

2004-09-01

In this work thin films of hafnium oxide are deposited on Si(100) substrates by means of atomic layer deposition (ALD) using tetrakis(diethylamino)hafnium and water vapor at substrate temperatures of 250-350ºC. Our system capabilities include fast transient delivery of reactive fluids, real-time vapor phase detection (in situ tunable diode laser hygrometer), precursor thermochemical capabilities, and ppt level elemental analysis by inductive coupling plasma mass spectrometry. The composition, purity, and other properties of the films and resulting interfaces are determined using x-ray and Fourier transform infrared spectroscopies, Z-contrast imaging and electron energy loss spectroscopy in a scanning transmission electron microscope with A˚ scale resolution, and spectroscopic ellipsometry. The observed ALD rate is ~1.4 A˚ per cycle. The nonuniformity across the film is less than 4%. Negligible carbon contamination is found in the resulting stoichiometric films under all conditions studied. The pulse sequence was optimized to prevent disastrous particulate problems while still minimizing purge times. The film deposition is investigated as a function of substrate temperature and reagent pulsing characteristics. A mild inverse temperature dependence of the ALD rate is observed. The initial stage of the HfO2 growth is investigated in detail.

Tumour necrosis factor-α-mediated disruption of cerebrovascular endothelial barrier integrity in vitro involves the production of proinflammatory interleukin-6.

PubMed

Rochfort, Keith D; Collins, Laura E; McLoughlin, Alisha; Cummins, Philip M

2016-02-01

The co-involvement of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during blood-brain barrier (BBB) injury has been reported in various models of neuroinflammation, although the precise functional interplay between these archetypal proinflammatory cytokines remains largely undefined within this context. In the current paper, we tested the hypothesis that TNF-α-mediated BBB disruption is measurably attributable in-part to induction of microvascular endothelial IL-6 production. In initial experiments, we observed that treatment of human brain microvascular endothelial cells (HBMvECs) with TNF-α (0-100 ng/mL, 0-24 h) robustly elicited both time- and dose-dependent induction of IL-6 expression and release, as well as expression of the IL-6 family receptor, GP130. Further experiments demonstrated that the TNF-α-dependent generation of reactive oxygen species, down-regulation of adherens/tight junction proteins, and concomitant elevation of HBMvEC permeability, were all significantly attenuated by blockade of IL-6 signalling using either an anti-IL-6 neutralizing antibody or an IL-6 siRNA. Based on these observations, we conclude that TNF-α treatment of HBMvECs in vitro activates IL-6 production and signalling, events that were shown to synergize with TNF-α actions to elicit HBMvEC permeabilization. These novel findings offer a constructive insight into the specific contribution of downstream cytokine induction to the injurious actions of TNF-α at the BBB microvascular endothelium interface. The co-involvement of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during blood-brain barrier (BBB) injury has been widely reported. Using human brain microvascular endothelial cells (HBMvEC), we show that TNF-α-mediated BBB disruption is measurably attributable in-part to induction of endothelial IL-6 production and signalling. We demonstrate that the TNF-α-dependent generation of reactive oxygen species (ROS), down-regulation of interendothelial junctions, and concomitant elevation of HBMvEC permeability, could be significantly attenuated by using either an IL-6 neutralizing antibody or an IL-6-specific siRNA. These findings provide insight into the complex nature of proinflammatory cytokine injury at the BBB microvascular endothelium interface. © 2015 International Society for Neurochemistry.

PUMA amplifies necroptosis signaling by activating cytosolic DNA sensors

PubMed Central

Tong, Jingshan; Yang, Liheng; Wei, Liang; Stolz, Donna B.; Yu, Jian; Zhang, Jianke; Zhang, Lin

2018-01-01

Necroptosis, a form of regulated necrotic cell death, is governed by RIP1/RIP3-mediated activation of MLKL. However, the signaling process leading to necroptotic death remains to be elucidated. In this study, we found that PUMA, a proapoptotic BH3-only Bcl-2 family member, is transcriptionally activated in an RIP3/MLKL-dependent manner following induction of necroptosis. The induction of PUMA, which is mediated by autocrine TNF-α and enhanced NF-κB activity, contributes to necroptotic death in RIP3-expressing cells with caspases inhibited. On induction, PUMA promotes the cytosolic release of mitochondrial DNA and activation of the DNA sensors DAI/Zbp1 and STING, leading to enhanced RIP3 and MLKL phosphorylation in a positive feedback loop. Furthermore, deletion of PUMA partially rescues necroptosis-mediated developmental defects in FADD-deficient embryos. Collectively, our results reveal a signal amplification mechanism mediated by PUMA and cytosolic DNA sensors that is involved in TNF-driven necroptotic death in vitro and in vivo. PMID:29581256

A novel nutrient sensing mechanism underlies substrate-induced regulation of monocarboxylate transporter-1

PubMed Central

Priyamvada, Shubha; Kumar, Anoop; Natarajan, Arivarasu A.; Gill, Ravinder K.; Alrefai, Waddah A.; Dudeja, Pradeep K.

2012-01-01

Monocarboxylate transporter isoform-1 (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFAs) in the colon. Butyrate, a major SCFA, serves as the primary energy source for the colonic mucosa, maintains epithelial integrity, and ameliorates intestinal inflammation. Previous studies have shown substrate (butyrate)-induced upregulation of MCT1 expression and function via transcriptional mechanisms. The present studies provide evidence that short-term MCT1 regulation by substrates could be mediated via a novel nutrient sensing mechanism. Short-term regulation of MCT1 by butyrate was examined in vitro in human intestinal C2BBe1 and rat intestinal IEC-6 cells and ex vivo in rat intestinal mucosa. Effects of pectin feeding on MCT1, in vivo, were determined in rat model. Butyrate treatment (30–120 min) of C2BBe1 cells increased MCT1 function {p-(chloromercuri) benzene sulfonate (PCMBS)-sensitive [14C]butyrate uptake} in a pertussis toxin-sensitive manner. The effects were associated with decreased intracellular cAMP levels, increased Vmax of butyrate uptake, and GPR109A-dependent increase in apical membrane MCT1 level. Nicotinic acid, an agonist for the SCFA receptor GPR109A, also increased MCT1 function and decreased intracellular cAMP. Pectin feeding increased apical membrane MCT1 levels and nicotinate-induced transepithelial butyrate flux in rat colon. Our data provide strong evidence for substrate-induced enhancement of MCT1 surface expression and function via a novel nutrient sensing mechanism involving GPR109A as a SCFA sensor. PMID:22982338

A VCP inhibitor substrate trapping approach (VISTA) enables proteomic profiling of endogenous ERAD substrates.

PubMed

Huang, Edmond Y; To, Milton; Tran, Erica; Dionisio, Lorraine T Ador; Cho, Hyejin J; Baney, Katherine L M; Pataki, Camille I; Olzmann, James A

2018-05-01

Endoplasmic reticulum (ER)-associated degradation (ERAD) mediates the proteasomal clearance of proteins from the early secretory pathway. In this process, ubiquitinated substrates are extracted from membrane-embedded dislocation complexes by the AAA ATPase VCP and targeted to the cytosolic 26S proteasome. In addition to its well-established role in the degradation of misfolded proteins, ERAD also regulates the abundance of key proteins such as enzymes involved in cholesterol synthesis. However, due to the lack of generalizable methods, our understanding of the scope of proteins targeted by ERAD remains limited. To overcome this obstacle, we developed a VCP inhibitor substrate trapping approach (VISTA) to identify endogenous ERAD substrates. VISTA exploits the small-molecule VCP inhibitor CB5083 to trap ERAD substrates in a membrane-associated, ubiquitinated form. This strategy, coupled with quantitative ubiquitin proteomics, identified previously validated (e.g., ApoB100, Insig2, and DHCR7) and novel (e.g., SCD1 and RNF5) ERAD substrates in cultured human hepatocellular carcinoma cells. Moreover, our results indicate that RNF5 autoubiquitination on multiple lysine residues targets it for ubiquitin and VCP--dependent clearance. Thus, VISTA provides a generalizable discovery method that expands the available toolbox of strategies to elucidate the ERAD substrate landscape.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qian, Qinyi; Zhou, Hao; Chen, Yan

Highlights: •This research confirmed VMP1 as a regulator of autophagy in colorectal cancer cell lines. •We proved the pro-survival role of VMP1-mediated autophagy in colorectal cancer cell lines. •We found the interaction between VMP1 and BECLIN1 also existing in colorectal cancer cell lines. -- Abstract: Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to addressmore » this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.« less

Dusty-Plasma Particle Accelerator

NASA Technical Reports Server (NTRS)

Foster, John E.

2005-01-01

A dusty-plasma apparatus is being investigated as means of accelerating nanometer- and micrometer-sized particles. Applications for the dusty-plasma particle accelerators fall into two classes: Simulation of a variety of rapidly moving dust particles and micrometeoroids in outer-space environments that include micrometeoroid streams, comet tails, planetary rings, and nebulae and Deposition or implantation of nanoparticles on substrates for diverse industrial purposes that could include hardening, increasing thermal insulation, altering optical properties, and/or increasing permittivities of substrate materials. Relative to prior apparatuses used for similar applications, dusty-plasma particle accelerators offer such potential advantages as smaller size, lower cost, less complexity, and increased particle flux densities. A dusty-plasma particle accelerator exploits the fact that an isolated particle immersed in plasma acquires a net electric charge that depends on the relative mobilities of electrons and ions. Typically, a particle that is immersed in a low-temperature, partially ionized gas, wherein the average kinetic energy of electrons exceeds that of ions, causes the particle to become negatively charged. The particle can then be accelerated by applying an appropriate electric field. A dusty-plasma particle accelerator (see figure) includes a plasma source such as a radio-frequency induction discharge apparatus containing (1) a shallow cup with a biasable electrode to hold the particles to be accelerated and (2) a holder for the substrate on which the particles are to impinge. Depending on the specific design, a pair of electrostatic-acceleration grids between the substrate and discharge plasma can be used to both collimate and further accelerate particles exiting the particle holder. Once exposed to the discharge plasma, the particles in the cup quickly acquire a negative charge. Application of a negative voltage pulse to the biasable electrode results in the initiation of a low-current, high-voltage cathode spot. Plasma pressure associated with the cathode spot as well as the large voltage drop at the cathode spot accelerates the charged particles toward the substrate. The ultimate kinetic energy attained by particles exiting the particle holder depends in part on the magnitude of the cathode spot sheath potential difference, which is proportional to the magnitude of the voltage pulse, and the on the electric charge on the dust. The magnitude of the voltage pulse can be controlled directly, whereas the particle s electric charge can be controlled indirectly by controlling the operating parameters of the plasma apparatus.

Targets of B-cell antigen receptor signaling: the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase-3 signaling pathway and the Rap1 GTPase.

PubMed

Gold, M R; Ingham, R J; McLeod, S J; Christian, S L; Scheid, M P; Duronio, V; Santos, L; Matsuuchi, L

2000-08-01

In this review, we discuss the role of phosphatidylinositol 3-kinase (PI3K) and Rap 1 in B-cell receptor (BCR) signaling. PI3K produces lipids that recruit pleckstrin homology domain-containing proteins to the plasma membrane. Akt is a kinase that the BCR activates in this manner. Akt phosphorylates several transcription factors as well as proteins that regulate apoptosis and protein synthesis. Akt also regulates glycogen synthase kinase-3, a kinase whose substrates include the nuclear factor of activated T cells (NF-AT)cl and beta-catenin transcriptional activators. In addition to Akt, PI3K-derived lipids also regulate the activity and localization of other targets of BCR signaling. Thus, a key event in BCR signaling is the recruitment of PI3K to the plasma membrane where its substrates are located. This is mediated by binding of the Src homology (SH) 2 domains in PI3K to phosphotyrosine-containing sequences on membrane-associated docking proteins. The docking proteins that the BCR uses to recruit PI3K include CD19, Cbl, Gab1, and perhaps Gab2. We have shown that Gab1 colocalizes PI3K with SH2 domain-containing inositol phosphatase (SHIP) and SHP2, two enzymes that regulate PI3K-dependent signaling. In contrast to PI3K, little is known about the Rap1 GTPase. We showed that the BCR activates Rap1 via phospholipase C-dependent production of diacylglycerol. Since Rap1 is thought to regulate cell adhesion and cell polarity, it may be involved in B-cell migration.

RIPK3 regulates p62-LC3 complex formation via the caspase-8-dependent cleavage of p62.

PubMed

Matsuzawa, Yu; Oshima, Shigeru; Nibe, Yoichi; Kobayashi, Masanori; Maeyashiki, Chiaki; Nemoto, Yasuhiro; Nagaishi, Takashi; Okamoto, Ryuichi; Tsuchiya, Kiichiro; Nakamura, Tetsuya; Watanabe, Mamoru

2015-01-02

RIPK3 is a key molecule for necroptosis, initially characterized by necrotic cell death morphology and the activation of autophagy. Cell death and autophagic signaling are believed to tightly regulate each other. However, the associated recruitment of signaling proteins remains poorly understood. p62/sequestosome-1 is a selective autophagy substrate and a selective receptor for ubiquitinated proteins. In this study, we illustrated that both mouse and human RIPK3 mediate p62 cleavage and that RIPK3 interacts with p62, resulting in complex formation. In addition, RIPK3-dependent p62 cleavage is restricted by the inhibition of caspases, especially caspase-8. Moreover, overexpression of A20, a ubiquitin-editing enzyme and an inhibitor of caspase-8 activity, inhibits RIPK3-dependent p62 cleavage. To further investigate the potential role of RIPK3 in selective autophagy, we analyzed p62-LC3 complex formation, revealing that RIPK3 prevents the localization of LC3 and ubiquitinated proteins to the p62 complex. In addition, RIPK3-dependent p62-LC3 complex disruption is regulated by caspase inhibition. Taken together, these results demonstrated that RIPK3 interacts with p62 and regulates p62-LC3 complex formation. These findings suggested that RIPK3 serves as a negative regulator of selective autophagy and provides new insights into the mechanism by which RIPK3 regulates autophagic signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

Atomically flat platinum films grown on synthetic mica

NASA Astrophysics Data System (ADS)

Tanaka, Hiroyuki; Taniguchi, Masateru

2018-04-01

Atomically flat platinum thin films were heteroepitaxially grown on synthetic fluorophlogopite mica [KMg3(AlSi3O10)F2] by van der Waals epitaxy. Platinum films deposited on a fluorophlogopite mica substrate by inductively coupled plasma-assisted sputtering with oxygen introduction on a synthetic mica substrate resulted in the growth of twin single-crystalline epitaxial Pt(111) films.

PDE4 and mAKAPβ are nodal organizers of β2-ARs nuclear PKA signaling in cardiac myocytes.

PubMed

Bedioune, Ibrahim; Lefebvre, Florence; Lechêne, Patrick; Varin, Audrey; Domergue, Valérie; Kapiloff, Michael S; Fischmeister, Rodolphe; Vandecasteele, Grégoire

2018-05-03

β1- and β2-adrenergic receptors (β-ARs) produce different acute contractile effects on the heart partly because they impact on different cytosolic pools of cAMP-dependent protein kinase (PKA). They also exert different effects on gene expression but the underlying mechanisms remain unknown. The aim of this study was to understand the mechanisms by which β1- and β2-ARs regulate nuclear PKA activity in cardiomyocytes. We used cytoplasmic and nuclear targeted biosensors to examine cAMP signals and PKA activity in adult rat ventricular myocytes upon selective β1- or β2-ARs stimulation. Both β1- and β2-AR stimulation increased cAMP and activated PKA in the cytoplasm. While the two receptors also increased cAMP in the nucleus, only β1-ARs increased nuclear PKA activity and up-regulated the PKA target gene and pro-apoptotic factor, inducible cAMP element repressor (ICER). Inhibition of PDE4, but not Gi, PDE3, GRK2 nor caveolae disruption disclosed nuclear PKA activation and ICER induction by β2-ARs. Both nuclear and cytoplasmic PKI prevented nuclear PKA activation and ICER induction by β1-ARs, indicating that PKA activation outside the nucleus is required for subsequent nuclear PKA activation and ICER mRNA expression. Cytoplasmic PKI also blocked ICER induction by β2-AR stimulation (with concomitant PDE4 inhibition). However, in this case nuclear PKI decreased ICER up-regulation by only 30%, indicating that other mechanisms are involved. Down-regulation of mAKAPβ partially inhibited nuclear PKA activation upon β1-AR stimulation, and drastically decreased nuclear PKA activation upon β2-AR stimulation in the presence of PDE4 inhibition. β1- and β2-ARs differentially regulate nuclear PKA activity and ICER expression in cardiomyocytes. PDE4 insulates a mAKAPβ-targeted PKA pool at the nuclear envelope that prevents nuclear PKA activation upon β2-AR stimulation.

SHORT VEGETATIVE PHASE Up-Regulates TEMPRANILLO2 Floral Repressor at Low Ambient Temperatures1[OPEN

PubMed Central

Marín-González, Esther; Matías-Hernández, Luis; Aguilar-Jaramillo, Andrea E.; Lee, Jeong Hwan; Ahn, Ji Hoon; Suárez-López, Paula; Pelaz, Soraya

2015-01-01

Plants integrate day length and ambient temperature to determine the optimal timing for developmental transitions. In Arabidopsis (Arabidopsis thaliana), the floral integrator FLOWERING LOCUS T (FT) and its closest homolog TWIN SISTER OF FT promote flowering in response to their activator CONSTANS under long-day inductive conditions. Low ambient temperature (16°C) delays flowering, even under inductive photoperiods, through repression of FT, revealing the importance of floral repressors acting at low temperatures. Previously, we have reported that the floral repressors TEMPRANILLO (TEM; TEM1 and TEM2) control flowering time through direct regulation of FT at 22°C. Here, we show that tem mutants are less sensitive than the wild type to changes in ambient growth temperature, indicating that TEM genes may play a role in floral repression at 16°C. Moreover, we have found that TEM2 directly represses the expression of FT and TWIN SISTER OF FT at 16°C. In addition, the floral repressor SHORT VEGETATIVE PHASE (SVP) directly regulates TEM2 but not TEM1 expression at 16°C. Flowering time analyses of svp tem mutants indicate that TEM may act in the same genetic pathway as SVP to repress flowering at 22°C but that SVP and TEM are partially independent at 16°C. Thus, TEM2 partially mediates the temperature-dependent function of SVP at low temperatures. Taken together, our results indicate that TEM genes are also able to repress flowering at low ambient temperatures under inductive long-day conditions. PMID:26243615

Lack of effect of tofacitinib (CP-690,550) on the pharmacokinetics of the CYP3A4 substrate midazolam in healthy volunteers: confirmation of in vitro data

PubMed Central

Gupta, Pankaj; Alvey, Christine; Wang, Rong; Dowty, Martin E; Fahmi, Odette A; Walsky, Robert L; Riese, Richard J; Krishnaswami, Sriram

2012-01-01

AIMS To investigate inhibitive and inductive effects of tofacitinib (CP-690,550), a Janus kinase inhibitor, on CYP3A4 function via in vitro and in vivo studies. METHODS In vitro experiments were conducted to assess the inhibition and induction potential of tofacitinib for major drug metabolizing enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4). A phase 1, randomized, open-label, two-way crossover study (NCT00902460) was conducted to confirm the lack of inhibitive/inductive effect on a sensitive CYP3A4 substrate, midazolam, in healthy subjects. Midazolam pharmacokinetics were assessed over 24 h following single dose 2 mg administration prior to administering tofacitinib and after twice daily dosing of tofacitinib 30 mg for 6 days. The primary endpoint was midazolam area under the concentration–time profile, from time 0 to infinity (AUC(0,∞)). RESULTS In vitro studies demonstrated low potential for CYP inhibition (IC50 estimates tofacitinib >30 µm), CYP3A4 mRNA induction (observed at tofacitinib concentrations ≥25 µm) and no effect on enzymatic activity of CYP substrates. In the human study, AUC(0,∞) adjusted geometric mean ratio for midazolam plus tofacitinib to midazolam alone was 103.97% [90% confidence interval (CI) 95.57, 113.12], wholly within the pre-specified acceptance region (80, 125). The 90% CI for the ratio of adjusted geometric means of maximum plasma concentration (Cmax) (95.98, 108.87) was also wholly within this acceptance region. CONCLUSIONS These data confirm a lack of an inhibitive or inductive effect of tofacitinib on CYP3A activity in humans and, in conjunction with in vitro data, support the conclusion that tofacitinib is unlikely to influence the CYP enzyme system as a whole. PMID:22233204

Tamoxifen induces the expression of maspin through estrogen receptor-alpha.

PubMed

Liu, Zesheng; Shi, Heidi Y; Nawaz, Zafar; Zhang, Ming

2004-06-08

Maspin (mammary serine protease inhibitor) is a tumor suppressor gene that plays an important role in inhibiting tumor growth, invasion and metastasis. Maspin expression is down regulated at transcription level in primary and metastatic breast tumor cells. Previous studies on hormonal regulation of maspin prompt us to test whether an estrogen antagonist tamoxifen (TAM) can exert its anti-tumor function by up regulating maspin gene expression. For this purpose, we first tested whether maspin promoter could be activated in normal and several breast tumor cells. We then carried out a series of promoter analysis in which estrogen receptors and TAM were reconstituted in an in vitro cell culture system. Here we report our new finding that tumor suppresser gene maspin is one of the TAM target genes. TAM induces a maspin/luciferase reporter in cell culture and this induction requires the presence of (estrogen receptor alpha) ERalpha but not estrogen receptor-beta (ERbeta). Maspin promoter deletion and mutation analysis showed that the cis element(s) within a region between -90and+87 bp but not the HRE site (-272 bp) was involved in TAM induction of maspin expression. TAM bound ERalpha may directly control maspin gene expression through the interaction with cofactor (s). Analysis using several ERalpha mutants showed that the N-terminal A/B motif (AF-1) was critical for maspin basal level transcription activation. An ERalpha mutant with point mutations at DNA binding domain abolished estrogen induction of an ERE-luciferase reporter but was still active in activating maspin promoter by TAM. LBD-AF2 domain was required for ERalpha-dependent TAM induction. Deletion of LBD-AF2 or a point mutation in the ERalpha LBD-AF2 region (LBDmtL539A) completely abolished the activation of maspin promoter, suggesting that TAM induction of maspin involves the recruitment of cofactor(s) by ERalpha to the maspin promoter region. This finding indicates that one of the pathways for cancer prevention and tumor inhibition by TAM is mediated through the activation of tumor suppressor gene maspin in breast cancer.

Quantitative analysis of the TNF-α-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKKβ substrate

PubMed Central

Krishnan, Ramesh K.; Nolte, Hendrik; Sun, Tianliang; Kaur, Harmandeep; Sreenivasan, Krishnamoorthy; Looso, Mario; Offermanns, Stefan; Krüger, Marcus; Swiercz, Jakub M.

2015-01-01

The inhibitor of the nuclear factor-κB (IκB) kinase (IKK) complex is a key regulator of the canonical NF-κB signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-κB activation; however, there is increasing evidence for NF-κB pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-α-IKKβ-induced phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which ∼1% are regulated up on TNF-α stimulation. We identify various potential novel IKKβ substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKKβ on serine 298. We provide evidence that IKKβ-mediated AEG-1 phosphorylation is essential for IκBα degradation as well as NF-κB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo. PMID:25849741

p205, a potential tumor suppressor, inhibits cell proliferation via multiple pathways of cell cycle regulation.

PubMed

Asefa, Benyam; Dermott, Jonathan M; Kaldis, Philipp; Stefanisko, Karen; Garfinkel, David J; Keller, Jonathan R

2006-02-20

p205 is a member of the interferon-inducible p200 family of proteins that regulate cell proliferation. Over-expression of p205 inhibits cell growth, although its mechanism of action is currently unknown. Therefore, we evaluated the effect of p205 on the p53 and Rb-dependent pathways of cell cycle regulation. p205 expression results in elevated levels of p21, and activates the p21 promoter in vitro in a p53-dependent manner. In addition, p205 induces increased expression of Rb, and binds directly to Rb and p53. Interestingly, p205 also induces growth inhibition independent of p53 and Rb by delaying G2/M progression in proliferating cells, and is a substrate for Cdk2 kinase activity. Finally, we have identified other binding partners of p205 by a yeast two-hybrid screen, including the paired homeodomain protein HoxB2. Taken together, our results indicate that p205 induces growth arrest by interaction with multiple transcription factors that regulate the cell cycle, including but not entirely dependent on the Rb- and p53-mediated pathways of growth inhibition.

The cytokine-protease connection: identification of a 96-kD THP-1 gelatinase and regulation by interleukin-1 and cytokine inducers.

PubMed

Van Ranst, M; Norga, K; Masure, S; Proost, P; Vandekerckhove, F; Auwerx, J; Van Damme, J; Opdenakker, G

1991-05-01

The induction of proteolytic enzymes is an important mechanism in the migration of monocytes into tissues and body fluids. The monocytic cell line THP-1 was used as a model system to study the production of a particular gelatinase. Upon stimulation with phorbol myristate acetate (PMA) the cells differentiated to the adherent phenotype and produced significant amounts of a 96-kD gelatinase in a dose-dependent way. The secretion rate was maximal between 12 and 24 h after induction. Study of gelatinase mRNA steady state levels showed that the synthesis of THP-1 gelatinase is regulated by PMA at transcriptional or posttranscriptional levels. Stimulation of signal transduction pathways with other substances, including calcium ionophore A 23187, dibutyryl cyclic AMP, and dexamethasone, were ineffective in inducing gelatinase mRNA or enzyme activity. However, THP-1 cells were responsive to the cytokine interleukin (IL)-1 beta, to bacterial lipopolysaccharide (LPS), and the lectin concanavalin A (Con A), the kinetics of gelatinase induction being similar to those of induction by PMA. The THP-1 cells did not synthesize and/or secrete detectable levels of IL-6 after stimulation with PMA, Con A, LPS, or IL-1 beta. The 96-kD monocytic THP-1 gelatinase was shown to be a neutral metalloproteinase that cross-reacted with hepatoma-derived and neutrophil gelatinases in immunoprecipitation experiments. The active enzyme produced by THP-1 cells consistently showed, however, a molecular mass different from that of normal granulocyte-, monocyte-, and tumor cell-derived gelatinases.(ABSTRACT TRUNCATED AT 250 WORDS)

The SARS coronavirus papain like protease can inhibit IRF3 at a post activation step that requires deubiquitination activity.

PubMed

Matthews, Krystal; Schäfer, Alexandra; Pham, Alissa; Frieman, Matthew

2014-12-07

The outcome of a viral infection is regulated by complex interactions of viral and host factors. SARS coronavirus (SARS-CoV) engages and regulates several innate immune response pathways during infection. We have previously shown that the SARS-CoV Papain-like Protease (PLpro) inhibits type I interferon (IFN) by inhibiting IRF3 phosphorylation thereby blocking downstream Interferon induction. This finding prompted us to identify other potential mechanisms of inhibition of PLpro on IFN induction. We have used plasmids expressing PLpro and IRF3 including an IRF3 mutant that is constitutively active, called IRF3(5D). In these experiments we utilize transfections, chromatin immunoprecipitation, Electro-mobility Shift Assays (EMSA) and protein localization to identify where IRF3 and IRF3(5D) are inhibited by PLpro. Here we show that PLpro also inhibits IRF3 activation at a step after phosphorylation and that this inhibition is dependent on the de-ubiquitination (DUB) activity of PLpro. We found that PLpro is able to block the type I IFN induction of a constitutively active IRF3, but does not inhibit IRF3 dimerization, nuclear localization or DNA binding. However, inhibition of PLpro's DUB activity by mutagenesis blocked the IRF3 inhibition activity of PLpro, suggesting a role for IRF3 ubiquitination in induction of a type I IFN innate immune response. These results demonstrate an additional mechanism that PLpro is able to inhibit IRF3 signaling. These data suggest novel innate immune antagonism activities of PLpro that may contribute to SARS-CoV pathogenesis.

The activation of c-Jun NH2-terminal kinase is required for dihydroartemisinin-induced autophagy in pancreatic cancer cells

PubMed Central

2014-01-01

Background c-Jun NH2-terminal kinases (JNKs) are strongly activated by a stressful cellular environment, such as chemotherapy and oxidative stress. Autophagy is a protein-degradation system in which double-membrane vacuoles called autophagosomes are formed. The autophagy-related gene Beclin 1 plays a key role in this process. We previously found that autophagy was induced by dihydroartemisinin (DHA) in pancreatic cancer cells. However, little is known about the complex relationship between ROS, JNK activation, autophagy induction, and Beclin 1 expression. Methods Cell viability and CCK-8 assays were carried out to determine the cell proliferation; small interfering RNAs (siRNAs) were used to knockdown c-Jun NH2-terminal kinases (JNK1/2) genes; western blot was performed to detect the protein expression of LC3, JNK, Beclin 1, caspase 3 and β-actin; production of intracellular ROS was analyzed using FACS flow cytometry; autophagy induction was confirmed by electron microscopy. Results In the present study, we explored the role of DHA and Beclin 1 expression in autophagy. DHA-treated cells showed autophagy characteristics, and DHA also activated the JNK pathway and up-regulated the expression of Beclin 1. Conversely, blocking JNK signaling inhibited Beclin 1 up-regulation. JNK activation was found to primarily depend on reactive oxygen species (ROS) resulting from the DHA treatment. Moreover, JNK pathway inhibition and Beclin 1 silencing prevented the induction of DHA-induced autophagy. Conclusions These results suggest that the induction of autophagy by DHA is required for JNK-mediated Beclin 1 expression. PMID:24438216

Loss of vacuolar H+-ATPase (V-ATPase) activity in yeast generates an iron deprivation signal that is moderated by induction of the peroxiredoxin TSA2.

PubMed

Diab, Heba I; Kane, Patricia M

2013-04-19

Vacuolar H(+)-ATPases (V-ATPases) acidify intracellular organelles and help to regulate overall cellular pH. Yeast vma mutants lack V-ATPase activity and allow exploration of connections between cellular pH, iron, and redox homeostasis common to all eukaryotes. A previous microarray study in a vma mutant demonstrated up-regulation of multiple iron uptake genes under control of Aft1p (the iron regulon) and only one antioxidant gene, the peroxiredoxin TSA2 (Milgrom, E., Diab, H., Middleton, F., and Kane, P. M. (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J. Biol. Chem. 282, 7125-7136). Fluorescent biosensors placing GFP under transcriptional control of either an Aft1-dependent promoter (P(FIT2)-GFP) or the TSA2 promoter (P(TSA2)-GFP) were constructed to monitor transcriptional signaling. Both biosensors were up-regulated in the vma2Δ mutant, and acute V-ATPase inhibition with concanamycin A induced coordinate up-regulation from both promoters. PTSA2-GFP induction was Yap1p-dependent, indicating an oxidative stress signal. Total cell iron measurements indicate that the vma2Δ mutant is iron-replete, despite up-regulation of the iron regulon. Acetic acid up-regulated P(FIT2)-GFP expression in wild-type cells, suggesting that loss of pH control contributes to an iron deficiency signal in the mutant. Iron supplementation significantly decreased P(FIT2)-GFP expression and, surprisingly, restored P(TSA2)-GFP to wild-type levels. A tsa2Δ mutation induced both nuclear localization of Aft1p and P(FIT2)-GFP expression. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis.

Regulation of plant cell wall degradation by light in Trichoderma.

PubMed

Schmoll, Monika

2018-01-01

Trichoderma reesei (syn. Hypocrea jecorina ) is the model organism for industrial production of plant cell wall degradating enzymes. The integration of light and nutrient signals for adaptation of enzyme production in T. reesei emerged as an important regulatory mechanism to be tackled for strain improvement. Gene regulation specific for cellulase inducing conditions is different in light and darkness with substantial regulation by photoreceptors. Genes regulated by light are clustered in the genome, with several of the clusters overlapping with CAZyme clusters. Major cellulase transcription factor genes and at least 75% of glycoside hydrolase encoding genes show the potential of light dependent regulation. Accordingly, light dependent protein complex formation occurs within the promoters of cellulases and their regulators. Additionally growth on diverse carbon sources is different between light and darkness and dependent on the presence of photoreceptors in several cases. Thereby, also light intensity plays a regulatory role, with cellulase levels dropping at higher light intensities dependent in the strain background. The heterotrimeric G-protein pathway is the most important nutrient signaling pathway in the connection with light response and triggers posttranscriptional regulation of cellulase expression. All G-protein alpha subunits impact cellulase regulation in a light dependent manner. The downstream cAMP pathway is involved in light dependent regulation as well. Connections between the regulatory pathways are mainly established via the photoreceptor ENV1. The effect of photoreceptors on plant cell wall degradation also occurs in the model filamentous fungus Neurospora crassa . In the currently proposed model, T. reesei senses the presence of plant biomass in its environment by detection of building blocks of cellulose and hemicellulose. Interpretation of the respective signals is subsequently adjusted to the requirements in light and darkness (or on the surface versus within the substrate) by an interconnection of nutrient signaling with light response. This review provides an overview on the importance of light, photoreceptors and related signaling pathways for formation of plant cell wall degrading enzymes in T. reesei . Additionally, the relevance of light dependent gene regulation for industrial fermentations with Trichoderma as well as strategies for exploitation of the observed effects are discussed.

Laminar distribution of cholinergic- and serotonergic-dependent plasticity within kitten visual cortex.

PubMed

Kojic, L; Gu, Q; Douglas, R M; Cynader, M S

2001-02-28

Both cholinergic and serotonergic modulatory projections to mammalian striate cortex have been demonstrated to be involved in the regulation of postnatal plasticity, and a striking alteration in the number and intracortical distribution of cholinergic and serotonergic receptors takes place during the critical period for cortical plasticity. As well, agonists of cholinergic and serotonergic receptors have been demonstrated to facilitate induction of long-term synaptic plasticity in visual cortical slices supporting their involvement in the control of activity-dependent plasticity. We recorded field potentials from layers 4 and 2/3 in visual cortex slices of 60--80 day old kittens after white matter stimulation, before and after a period of high frequency stimulation (HFS), in the absence or presence of either cholinergic or serotonergic agonists. At these ages, the HFS protocol alone almost never induced long-term changes of synaptic plasticity in either layers 2/3 or 4. In layer 2/3, agonist stimulation of m1 receptors facilitated induction of long-term potentiation (LTP) with HFS stimulation, while the activation of serotonergic receptors had only a modest effect. By contrast, a strong serotonin-dependent LTP facilitation and insignificant muscarinic effects were observed after HFS within layer 4. The results show that receptor-dependent laminar stratification of synaptic modifiability occurs in the cortex at these ages. This plasticity may underly a control system gating the experience-dependent changes of synaptic organization within developing visual cortex.

Stimulation of phagocytosis by sulforaphane.

PubMed

Suganuma, Hiroyuki; Fahey, Jed W; Bryan, Kelley E; Healy, Zachary R; Talalay, Paul

2011-02-04

Sulforaphane, a major isothiocyanate derived from cruciferous vegetables, protects living systems against electrophile toxicity, oxidative stress, inflammation, and radiation. A major protective mechanism is the induction of a network of endogenous cytoprotective (phase 2) genes that are regulated by transcription factor Nrf2. To obtain a more detailed understanding of the anti-inflammatory and immunomodulatory effects of sulforaphane, we evaluated its effect on the phagocytosis activity of RAW 264.7 murine macrophage-like cells by measuring the uptake of 2-μm diameter polystyrene beads. Sulforaphane raised the phagocytosis activity of RAW 264.7 cells but only in the absence or presence of low concentrations (1%) of fetal bovine serum. Higher serum concentrations depressed phagocytosis and abolished its stimulation by sulforaphane. This stimulation did not depend on the induction of Nrf2-regulated genes since it occurred in peritoneal macrophages of nrf2(-/-) mice. Moreover, a potent triterpenoid inducer of Nrf2-dependent genes did not stimulate phagocytosis, whereas sulforaphane and another isothiocyanate (benzyl isothiocyanate) had comparable inducer potencies. It has been shown recently that sulforaphane is a potent and direct inactivator of macrophage migration inhibitory factor (MIF), an inflammatory cytokine. Moreover, the addition of recombinant MIF to RAW 264.7 cells attenuated phagocytosis, but sulforaphane-inactivated MIF did not affect phagocytosis. The inactivation of MIF may therefore be involved in the phagocytosis-enhancing activity of sulforaphane. Copyright © 2011 Elsevier Inc. All rights reserved.

Mangiferin suppresses endoplasmic reticulum stress in perivascular adipose tissue and prevents insulin resistance in the endothelium.

PubMed

Xu, Xiaoshan; Chen, Yupeng; Song, Junna; Hou, Fangjie; Ma, Xuelian; Liu, Baolin; Huang, Fang

2018-06-01

Mangiferin is a naturally occurring glucosylxanthone with beneficial effects on glucose and lipid homeostasis. This study investigates the potential therapeutic effect of Mangiferin in perivascular adipose tissue (PVAT) and whether it contributes to regulating insulin action in the endothelium. Palmitate challenge evoked ROS-associated endoplasmic reticulum stress (ER stress) and NLRP3 inflammasome activation in PVAT. The conditioned medium from PA-stimulated PVAT was prepared to induce endothelial insulin resistance, and improved endothelium-dependent vasodilation in response to insulin was detected in vitro and in vivo. Mangiferin treatment enhanced LKB1-dependent AMPK activity and suppressed ER stress with downregulation of TXNIP induction, leading to the inhibition of NLRP3 inflammasome activation evidenced by attenuated NLRP3 and cleaved caspase-1 expression as well as reduced IL-1β secretion. Moreover, Mangiferin restored insulin-mediated Akt and eNOS phosphorylations with increased NO production, immunohistochemistry examination of adipocytes, and endothelial tissue in high-fat diet-fed mice also showed that oral administration of Mangiferin inhibited ER stress and NLRP3 induction in PVAT, and then effectively prevented insulin resistance in the vessel endothelium. Taken together, these results revealed that Mangiferin suppressed ER stress-associated NLRP3 inflammasome activation in PVAT through regulation of AMPK activity, which prevented endothelial insulin resistance. These findings suggested that the amelioration of PVAT dysfunction may be a therapeutic strategy for the prevention of endothelial insulin resistance.

Conduction in In 2O 3/YSZ heterostructures: Complex interplay between electrons and ions, mediated by interfaces

DOE PAGES

Veal, B. W.; Eastman, J. A.

2017-03-01

Thin film In 2O 3/YSZ heterostructures exhibit significant increases in electrical conductance with time when small in-plane electric fields are applied. Contact resistances between the current electrodes and film, and between current electrodes and substrate are responsible for the behavior. With an in-plane electric field, different field profiles are established in the two materials, with the result that oxygen ions can be driven across the heterointerface, altering the doping of the n-type In 2O 3. Furthermore, a low frequency inductive feature observed in AC impedance spectroscopy measurements under DC bias conditions was found to be due to frequency-dependent changes inmore » the contact resistance.« less

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

PubMed Central

Wang, Evelyn W.; Kessler, Benedikt M.; Borodovsky, Anna; Cravatt, Benjamin F.; Bogyo, Matthew; Ploegh, Hidde L.; Glas, Rickard

2000-01-01

Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability. PMID:10954757

COPPER RESPONSE REGULATOR1–Dependent and –Independent Responses of the Chlamydomonas reinhardtii Transcriptome to Dark Anoxia[W

PubMed Central

Hemschemeier, Anja; Casero, David; Liu, Bensheng; Benning, Christoph; Pellegrini, Matteo; Happe, Thomas; Merchant, Sabeeha S.

2013-01-01

Anaerobiosis is a stress condition for aerobic organisms and requires extensive acclimation responses. We used RNA-Seq for a whole-genome view of the acclimation of Chlamydomonas reinhardtii to anoxic conditions imposed simultaneously with transfer to the dark. Nearly 1.4 × 103 genes were affected by hypoxia. Comparing transcript profiles from early (hypoxic) with those from late (anoxic) time points indicated that cells activate oxidative energy generation pathways before employing fermentation. Probable substrates include amino acids and fatty acids (FAs). Lipid profiling of the C. reinhardtii cells revealed that they degraded FAs but also accumulated triacylglycerols (TAGs). In contrast with N-deprived cells, the TAGs in hypoxic cells were enriched in desaturated FAs, suggesting a distinct pathway for TAG accumulation. To distinguish transcriptional responses dependent on COPPER RESPONSE REGULATOR1 (CRR1), which is also involved in hypoxic gene regulation, we compared the transcriptomes of crr1 mutants and complemented strains. In crr1 mutants, ∼40 genes were aberrantly regulated, reaffirming the importance of CRR1 for the hypoxic response, but indicating also the contribution of additional signaling strategies to account for the remaining differentially regulated transcripts. Based on transcript patterns and previous results, we conclude that nitric oxide–dependent signaling cascades operate in anoxic C. reinhardtii cells. PMID:24014546

Modulation of anaerobic energy metabolism of Bacillus subtilis by arfM (ywiD).

PubMed

Marino, M; Ramos, H C; Hoffmann, T; Glaser, P; Jahn, D

2001-12-01

Bacillus subtilis grows under anaerobic conditions utilizing nitrate ammonification and various fermentative processes. The two-component regulatory system ResDE and the redox regulator Fnr are the currently known parts of the regulatory system for anaerobic adaptation. Mutation of the open reading frame ywiD located upstream of the respiratory nitrate reductase operon narGHJI resulted in elimination of the contribution of nitrite dissimilation to anaerobic nitrate respiratory growth. Significantly reduced nitrite reductase (NasDE) activity was detected, while respiratory nitrate reductase activity was unchanged. Anaerobic induction of nasDE expression was found to be significantly dependent on intact ywiD, while anaerobic narGHJI expression was ywiD independent. Anaerobic transcription of hmp, encoding a flavohemoglobin-like protein, and of the fermentative operons lctEP and alsSD, responsible for lactate and acetoin formation, was partially dependent on ywiD. Expression of pta, encoding phosphotransacetylase involved in fermentative acetate formation, was not influenced by ywiD. Transcription of the ywiD gene was anaerobically induced by the redox regulator Fnr via the conserved Fnr-box (TGTGA-6N-TCACT) centered 40.5 bp upstream of the transcriptional start site. Anaerobic induction of ywiD by resDE was found to be indirect via resDE-dependent activation of fnr. The ywiD gene is subject to autorepression and nitrite repression. These results suggest a ResDE --> Fnr --> YwiD regulatory cascade for the modulation of genes involved in the anaerobic metabolism of B. subtilis. Therefore, ywiD was renamed arfM for anaerobic respiration and fermentation modulator.

KDM4B/JMJD2B is a p53 target gene that modulates the amplitude of p53 response after DNA damage

PubMed Central

Moon, Eui Jung; Razorenova, Olga V.; Krieg, Adam J.; von Eyben, Rie

2017-01-01

Abstract The p53 tumor suppressor protein plays a critical role in orchestrating the genomic response to various stress signals by acting as a master transcriptional regulator. Differential gene activity is controlled by transcription factors but also dependent on the underlying chromatin structure, especially on covalent histone modifications. After screening different histone lysine methyltransferases and demethylases, we identified JMJD2B/KDM4B as a p53-inducible gene in response to DNA damage. p53 directly regulates JMJD2B gene expression by binding to a canonical p53-consensus motif in the JMJD2B promoter. JMJD2B induction attenuates the transcription of key p53 transcriptional targets including p21, PIG3 and PUMA, and this modulation is dependent on the catalytic capacity of JMJD2B. Conversely, JMJD2B silencing led to an enhancement of the DNA-damage driven induction of p21 and PIG3. These findings indicate that JMJD2B acts in an auto-regulatory loop by which p53, through JMJD2B activation, is able to influence its own transcriptional program. Functionally, exogenous expression of JMJD2B enhanced subcutaneous tumor growth of colon cancer cells in a p53-dependent manner, and genetic inhibition of JMJD2B impaired tumor growth in vivo. These studies provide new insights into the regulatory effect exerted by JMJD2B on tumor growth through the modulation of p53 target genes. PMID:28073943

Comparative energetics of carbon storage molecules in green algae

DOE Office of Scientific and Technical Information (OSTI.GOV)

McKie-Krisberg, Zaid M.; Laurens, Lieve M. L.; Huang, Andy

Several members of the green algae possess the ability to produce lipids and/or high value compounds in significant quantities. While for several of these green algal species induction of increased lipid production has been shown, and cultivation of species for high value molecules occurs at production scale, the molecular mechanisms governing over-accumulation of molecules synthesized from isoprenoid precursors, carotenoids, for example, have received far less attention. Here, we present a calculation of the required ATP equivalencies per carbon atom and reducing power equivalencies as NADH/NADPH (NAD(P)H) per carbon atom for the isoprenoid molecules ..beta..-carotene (C40), astaxanthin (C40), and squalene (C30).more » We compared energetic requirements of carbohydrates, triacylglycerol, and isoprenoid molecules under a gradient of conditions of cellular stress. Our calculations revealed slightly less ATP and NAD(P)H equivalency per carbon atom between triacylglycerol and the three isoprenoid molecules. Based on our results, we propose that the driving force for differences in accumulation patterns of carotenoids vs. triacylglycerols in algal cells under stress is largely dependent on the presence and regulation of bypass mechanisms at metabolic junction bottlenecks, like pyruvate dehydrogenase (PDH), within particular species. We provide a discussion of several molecular mechanisms that may influence carbon partitioning within different groups of green algae, including metabolic inhibition through accumulation of specific substrates related to ATP and reducing equivalent production (NAD(P)H) as well as cellular compartmentalization. This work contributes to the ongoing discussion of cellular homeostatic regulation during stress, as well as the potential mechanisms driving long-term carbon storage as it relates to energy and redox states within the algal cell.« less

Comparative energetics of carbon storage molecules in green algae

DOE PAGES

McKie-Krisberg, Zaid M.; Laurens, Lieve M. L.; Huang, Andy; ...

2018-02-28

Several members of the green algae possess the ability to produce lipids and/or high value compounds in significant quantities. While for several of these green algal species induction of increased lipid production has been shown, and cultivation of species for high value molecules occurs at production scale, the molecular mechanisms governing over-accumulation of molecules synthesized from isoprenoid precursors, carotenoids, for example, have received far less attention. Here, we present a calculation of the required ATP equivalencies per carbon atom and reducing power equivalencies as NADH/NADPH (NAD(P)H) per carbon atom for the isoprenoid molecules ..beta..-carotene (C40), astaxanthin (C40), and squalene (C30).more » We compared energetic requirements of carbohydrates, triacylglycerol, and isoprenoid molecules under a gradient of conditions of cellular stress. Our calculations revealed slightly less ATP and NAD(P)H equivalency per carbon atom between triacylglycerol and the three isoprenoid molecules. Based on our results, we propose that the driving force for differences in accumulation patterns of carotenoids vs. triacylglycerols in algal cells under stress is largely dependent on the presence and regulation of bypass mechanisms at metabolic junction bottlenecks, like pyruvate dehydrogenase (PDH), within particular species. We provide a discussion of several molecular mechanisms that may influence carbon partitioning within different groups of green algae, including metabolic inhibition through accumulation of specific substrates related to ATP and reducing equivalent production (NAD(P)H) as well as cellular compartmentalization. This work contributes to the ongoing discussion of cellular homeostatic regulation during stress, as well as the potential mechanisms driving long-term carbon storage as it relates to energy and redox states within the algal cell.« less

BPM-CUL3 E3 ligase modulates thermotolerance by facilitating negative regulatory domain-mediated degradation of DREB2A in Arabidopsis.

PubMed

Morimoto, Kyoko; Ohama, Naohiko; Kidokoro, Satoshi; Mizoi, Junya; Takahashi, Fuminori; Todaka, Daisuke; Mogami, Junro; Sato, Hikaru; Qin, Feng; Kim, June-Sik; Fukao, Yoichiro; Fujiwara, Masayuki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2017-10-03

DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) acts as a key transcription factor in both drought and heat stress tolerance in Arabidopsis and induces the expression of many drought- and heat stress-inducible genes. Although DREB2A expression itself is induced by stress, the posttranslational regulation of DREB2A, including protein stabilization, is required for its transcriptional activity. The deletion of a 30-aa central region of DREB2A known as the negative regulatory domain (NRD) transforms DREB2A into a stable and constitutively active form referred to as DREB2A CA. However, the molecular basis of this stabilization and activation has remained unknown for a decade. Here we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the Cullin3 (CUL3)-based E3 ligase, as DREB2A-interacting proteins. We observed that DREB2A and BPMs interact in the nuclei, and that the NRD of DREB2A is sufficient for its interaction with BPMs. BPM -knockdown plants exhibited increased DREB2A accumulation and induction of DREB2A target genes under heat and drought stress conditions. Genetic analysis indicated that the depletion of BPM expression conferred enhanced thermotolerance via DREB2A stabilization. Thus, the BPM-CUL3 E3 ligase is likely the long-sought factor responsible for NRD-dependent DREB2A degradation. Through the negative regulation of DREB2A stability, BPMs modulate the heat stress response and prevent an adverse effect of excess DREB2A on plant growth. Furthermore, we found the BPM recognition motif in various transcription factors, implying a general contribution of BPM-mediated proteolysis to divergent cellular responses via an accelerated turnover of transcription factors.

Acetate scavenging activity in Escherichia coli: interplay of acetyl-CoA synthetase and the PEP-glyoxylate cycle in chemostat cultures.

PubMed

Renilla, Sergio; Bernal, Vicente; Fuhrer, Tobias; Castaño-Cerezo, Sara; Pastor, José M; Iborra, José L; Sauer, Uwe; Cánovas, Manuel

2012-03-01

Impairment of acetate production in Escherichia coli is crucial for the performance of many biotechnological processes. Aerobic production of acetate (or acetate overflow) results from changes in the expression of central metabolism genes. Acetyl-CoA synthetase scavenges extracellular acetate in glucose-limited cultures. Once converted to acetyl-CoA, it can be catabolized by the tricarboxylic acid cycle or the glyoxylate pathway. In this work, we assessed the significance of these pathways on acetate overflow during glucose excess and limitation. Gene expression, enzyme activities, and metabolic fluxes were studied in E. coli knock-out mutants related to the glyoxylate pathway operon and its regulators. The relevance of post-translational regulation by AceK-mediated phosphorylation of isocitrate dehydrogenase for pathway functionality was underlined. In chemostat cultures performed at increasing dilution rates, acetate overflow occurs when growing over a threshold glucose uptake rate. This threshold was not affected in a glyoxylate-pathway-deficient strain (lacking isocitrate lyase, the first enzyme of the pathway), indicating that it is not relevant for acetate overflow. In carbon-limited chemostat cultures, gluconeogenesis (maeB, sfcA, and pck), the glyoxylate operon and, especially, acetyl-CoA synthetase are upregulated. A mutant in acs (encoding acetyl-CoA synthetase) produced acetate at all dilution rates. This work demonstrates that, in E. coli, acetate production occurs at all dilution rates and that overflow is the result of unbalanced synthesis and scavenging activities. The over-expression of acetyl-CoA synthetase by cAMP-CRP-dependent induction limits this phenomenon in cultures consuming glucose at low rate, ensuring the recycling of the acetyl-CoA and acetyl-phosphate pools, although establishing an energy-dissipating substrate cycle.