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Astrocytes are essential for proper central nervous system (CNS) function and are intricately involved in neuroinflammation. Despite evidence that immune-activated astrocytes contribute to many CNS pathologies, little is known about the inflammatory pathways controlling gene expression. Our laboratory identified altered levels of tissue inhibitor of metalloproteinase (TIMP)-1 in brain lysates from human immunodeficiency virus (HIV)-1 infected patients, compared to age-matched controls, and interleukin (IL)-1β as a key regulator of astrocyte TIMP-1. Additionally, CCAAT enhancer binding protein (C/EBP)β levels are elevated in brain specimens from HIV-1 patients and the transcription factor contributes to astrocyte TIMP-1 expression. In this report we sought to identify key signaling pathways necessary for IL-1β-mediated astrocyte TIMP-1 expression and their interaction with C/EBPβ. Primary human astrocytes were cultured and treated with mitogen activated protein kinase-selective small molecule inhibitors, and IL-1β. TIMP-1 and C/EBPβ mRNA and protein expression were evaluated at 12 and 24 h post-treatment, respectively. TIMP-1 promoter-driven luciferase plasmids were used to evaluate TIMP-1 promoter activity in inhibitor-treated astrocytes. These data show that extracellular regulated kinase (ERK) 1/2-selective inhibitors block IL-1β-induced astrocyte TIMP-1 expression, but did not decrease C/EBPβ expression in parallel. The p38 kinase (p38K) inhibitors partially blocked both IL-1β-induced astrocyte TIMP-1 expression and C/EBPβ expression. The ERK1/2-selective inhibitor abrogated IL-1β-mediated increases in TIMP-1 promoter activity. Our data demonstrate that ERK1/2 activation is critical for IL-1β-mediated astrocyte TIMP-1 expression. ERK1/2-selective inhibition may elicit a compensatory response in the form of enhanced IL-1β-mediated astrocyte C/EBPβ expression, or, alternatively, ERK1/2 signaling may function to moderate IL-1β-mediated astrocyte C/EBPβ expression. Furthermore, p38K activation contributes to IL-1β-induced astrocyte TIMP-1 and C/EBPβ expression. These data suggest that ERK1/2 signals downstream of C/EBPβ to facilitate IL-1β-induced astrocyte TIMP-1 expression. Astrocyte ERK1/2 and p38K signaling may serve as therapeutic targets for manipulating CNS TIMP-1 and C/EBPβ levels, respectively.
Citation: Fields J, Cisneros IE, Borgmann K, Ghorpade A (2013) Extracellular Regulated Kinase 1/2 Signaling Is a Critical Regulator of Interleukin-1β-Mediated Astrocyte Tissue Inhibitor of Metalloproteinase-1 Expression. PLoS ONE 8(2): e56891. doi:10.1371/journal.pone.0056891
Editor: Emilio Hirsch, University of Torino, Italy
Received: May 24, 2012; Accepted: January 17, 2013; Published: February 14, 2013
Copyright: © 2013 Fields et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by 2R01NS048837 from National Institute of Neurological Disorders and Stroke (NINDS) to A. Ghorpade; a fellowship from National Institute on Aging (NIA) T32 AG020494 and 1F31NS072006-01A1 to J. Fields. The authors appreciate the assistance of the Laboratory of Developmental Biology for providing them with human brain tissues; also supported by National Institutes of Health award number 5R24HD0008836 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Astrocytes are essential cells of the central nervous system (CNS) and are subject to the perturbations coinciding with neural pathologies, including human immunodeficiency virus (HIV)-1-associated neurocognitive disorders (HAND) [1], [2], [3]. During HAND, HIV-1-infected monocytes infiltrate the CNS where they disseminate viral particles, cytokines and other stimulatory molecules [4]. Cytokines and viral toxins produced in this inflamed environment may bring about deleterious changes in astrocyte gene expression [4], [5]. Dysfunctional astrocytes compromise optimal maintenance of the blood brain barrier, glutamate reuptake and the matrix metalloproteinase (MMP): tissue inhibitor of metalloproteinase (TIMP) balance [6], [7], [8], [9], [10], [11]. In the CNS astrocytes are major producers of TIMP-1 [5], [12], [13], a multifunctional glycoprotein that regulates extracellular matrix processing and cell growth/apoptosis [14], [15], [16]. TIMP-1 is expressed in multiple tissues, by various cell types and plays roles in angiogenesis, neurogenesis, metastasis and other physiological processes by binding MMPs to inhibit their function [17], [18], [19], [20]. TIMP-1 displays antiapoptotic activity independent of MMP-binding function; this phenomenon has led to a search for a definite TIMP-1 receptor [21]. TIMP-1 affects neuronal development by altering dendrite outgrowth [16]. These intriguing functions, along with TIMP-1 being the inducible form and highly prevalent in disease, are currently being studied in the context of cancer, ischemia, Alzheimer's disease and HIV-1-associated neurocognitive disorders (HAND) [17], [22], [23], [24].
Recent studies have expanded a diverse list of cell- and tissue-specific TIMP-1 functions [21], [25]. However, knowledge of specific signal transduction pathways regulating TIMP-1 remains scant and, where present, appears to depend upon the stimuli and expressing cell type. Transforming growth factor-β induces activator protein-1 (AP-1) to promote fibroblast TIMP-1 expression [26]. Histone deacetylase and extracellular regulated kinase (ERK) signaling may also be required for fibroblast TIMP-1 expression [27], [28]. ERK1/2 or p38 kinase (p38K), but not c-jun N-terminal kinase (JNK), are required for oncostatin M-induced murine fibroblast TIMP-1 expression [29]. In rat granulosa cells, protein kinase A-, p38K- and ERK1/2-selective inhibitors blocked human chorionic gonadotropin-induced TIMP-1 expression [30]. In the brain, TIMP-1 is regulated in a time- and cell-dependent manner [31]. Recent studies, using human astrocytes suggest that p38K pathway activity is required for maximal IL-1β-induced astrocyte TIMP-1 expression; however, p38K inhibition was not sufficient to totally block TIMP-1 production [32]. Expectedly, knockdown of the p65 unit of nuclear factor (NF)κB transcription machinery blocked IL-1β-induced astrocyte TIMP-1 [32]. Taken together, these data suggest p38K functions in conjunction with other signaling pathways during IL-1β-induced astrocyte TIMP-1 expression. Our group reported that CCAAT enhancer binding protein (C/EBP)β is expressed in brains of HIV-1 associated dementia (HAD) patients and contributes to regulating IL-1β-induced astrocyte TIMP-1 expression [33].
In the context of HIV-1 infection, TIMP-1 levels are reduced in HAD patients when compared with age-matched controls [5], [34]. Additionally, human astrocytes treated with inflammatory cytokines [interleukin (IL)-1β or tumor necrosis factor-α] initially demonstrated increased TIMP-1 expression; however, production regresses during chronic stimulation with IL-1β [34]. Identifying the key pathways initiating IL-1β-induced astrocyte TIMP-1 expression may provide a target for restoring CNS TIMP-1. In this study, we used a combination of small molecule mitogen activated protein kinase (MAPK)-selective inhibitors and IL-1β to explore the role of p38K and ERK1/2 signal transduction pathways in IL-1β-induced astrocyte TIMP-1 expression. We employed reporter and overexpression plasmids along with MAPK-selective inhibitors to study how the IL-1β pathway connects C/EBPβ to astrocyte TIMP-1 expression. Herein, we report ERK1/2 may provide a therapeutic target to restore CNS TIMP-1 levels. Furthermore, manipulating C/EBPβ expression, along with that of its binding partners, NFκB, AP-1, or others, may be a promising route to manipulating CNS TIMP-1 levels.
Isolation, cultivation, and activation of human astrocytes
Human astrocytes were isolated from first- and early second-trimester aborted specimens obtained from the Birth Defects Laboratory, University of Washington, Seattle, in full compliance with the ethical guidelines of the NIH. The institutional review boards of both the Universities of Washington and North Texas Health Science Center approved the collection of human tissues for research. The Birth Defects Laboratory obtained written consent from all tissue donors.
Astrocytes were isolated from specimens as originally described by Gardner [34]. Activation of astrocytes was achieved by applying IL-1β for various time intervals. IL-1β is a prototypical inflammatory cytokine expressed during HAD; making IL-1β an excellent choice for studying human astrocyte function during neuroinflammation [35]. We empirically tested IL-1β dosage to determine a concentration that promotes maximal activation of astrocytes, 20 ng/ml. In mouse models infected with IL-1β-expressing adenovirus, IL-1β levels reach 10 ng/mg total protein 7 days post-injection and a mean of 41 ng/mg throughout the striatum [36], [37]. Accordingly, these data provide relevant implications for astrocyte function in many pathologies involving neuroinflammation. All experimental conditions were analyzed in multiple replicates, and all results were confirmed in at least two astrocyte donors.
Pharmacological inhibitors
Monolayers of astrocytes were treated with twice the final dose of the MAPK-selective inhibitors, SB203580, SB202190, U0126 and PD98059 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h before IL-1β treatment (12 h hours for mRNA or 24 h for protein isolation). Multiple doses of SB203580 (sc-353), and U0126 (sc-202374, 0.1-20 µM), and a single dose of SB202190 (sc-222294, 20 µM) and PD98059 (sc-3532, 20 µM) were tested as described in similar signaling studies [38], [39], [40], [41].
RNA Isolation and real-time reverse transcription polymerase chain reaction (RT2PCR)
Astrocyte RNA was extracted (RNeasy plus mini kit, Qiagen, Alameda, CA) and reverse-transcribed into cDNA as per the manufacturer's instructions (High Capacity cDNA Reverse Transcription Kit, Life Technologies Corp., Carlsbad, CA). TaqMan® 5' nuclease RT2PCR assays were performed using a StepOnePlus system (Life Tech). The following TaqMan® gene expression assay primers were used: TIMP-1 (Life Tech, C/N: Hs99999139_m1), C/EBPβ (C/N: Hs00270923_s1) and glyceraldehyde phosphate dehydrogenase (GAPDH; C/N: 4310859). The reactions were carried out at 48°C for 30 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Transcripts were quantified by the comparative CT method, and are represented as fold-change of control [42]. Samples were analyzed in triplicate.
Western Blot
Astrocytes were cultured as adherent monolayers in 75 cm2 flasks at a density of 8×106 cells per flask. The following day, cells were treated with MAPK-selective inhibitors (SB203580 and U0126) for one h and then IL-1β (20 ng/ml) for 24 h. Alternately, for western blot detection of protein phosphorylation status, astrocytes were activated for 30 min with IL-1β. Cells were lysed; total cell extracts were isolated using mammalian protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) or nuclear extracts were isolated using nuclear extraction reagent (Sigma-Aldrich Inc., St. Louis, MO). Equal amounts of protein (15 µg/lane) were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinyl diflouride membrane using i-Blot (Life Tech). The membrane was incubated in anti-C/EBPβ (C-19, Santa Cruz), p38K, phosphorylated (P)-p38K, ERK and P-ERK1/2 at a dilution of 1:200 (Cell Signaling Inc., Boston, MA, USA). Blots were then incubated in secondary antibody at a dilution of 1:5,000. β-actin (Sigma) or lamin A/C (Cell Signaling), as a loading control. The western blot was visualized with supersignal chemiluminescent substrate (Thermo Fisher) and band intensities were quantified by densitometry analysis (ProteinSimple, Santa Clara, CA, USA).
Measurement of TIMP-1 protein and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay
TIMP-1 levels in astrocyte supernatants were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). The MTT assay was performed at appropriate time points according to the method originally described by Manthrope [43]. The ELISA determinations yielded quantities of protein in units of ng/ml and were normalized to MTT values.
Transfection of primary human astrocytes with luciferase reporter constructs
Three TIMP-1-luc promoter plasmid (pTIMP-1-luc) constructs were used to measure TIMP-1 promoter activity in transfected astrocytes. The −4200/+96 and −1718/+96 portions of the TIMP-1 sequence were cloned into the pGL3-basic reporter vector (Promega, Madison, WI) and were kindly provided by Dr. Ian Clark at the University of East Anglia, UK [44].
Astrocytes were cultured as adherent monolayers in a 48-well plate at a density of 0.15×106 cells per well. The following day, cells were transfected with 1.5 µg of total DNA using Lipofectamine™ reagent as per the manufacturer's instructions (Life Tech). Total DNA consisted of a mixture of the pTIMP-1-luc and the pGL3-driven Renilla luciferase (pGL3-RL, Promega) plasmids. Twenty-four h post-transfection media was aspirated from selected wells and replaced with media supplemented with MAPK-selective inhibitors at 20 µM for 24 h with or without 20 ng/ml IL-1β. At two days post-transfection promoter activity was measured as luciferase activity in cell extracts using the Dual-Glo® luciferase assay system per the manufacturers instructions (Promega). Luciferase activity was determined as a ratio of firefly to Renilla luciferase using the a Glomax luminimeter (Promega). Renilla luciferase activity was used as an internal control. All experimental conditions were analyzed in multiple replicates, and all results were confirmed in at least two astrocyte donors.
Astrocytes were cultured as adherent monolayers in a 48-well plate at a density of 0.1×106 cells per well. The following day, cells were treated with 20 µM concentration of SB203580 or U0126 for 1 h and then with IL-1β for 24 h. Cells were fixed with cold acetone:methanol (1:1) for 30 mins at −20°C. Cells were then blocked in phosphate buffered saline (PBS) with 2% bovine serum albumin for 1 h at room temperature. Cells were incubated in PBS (2% bovine serum albumin, 0.1% triton X) plus C/EBPβantibody at 1:500 and glial fibrillary acidic protein (GFAP) at 1:600 for 8 h at 4°C. Cells were washed with PBS and then incubated with Alexa Fluor secondary antibody (Life Tech, 1:800) for 1 h at room temperature. Micrographs were taken on a Nikon Eclipse Ti. (Nikon Inc., Melville, NY, USA).
Statistical Analyses
Results were analyzed with GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA) using one-way analysis of variance with Newman Keul's post-test for multiple comparisons. Significance was set at p<0.05 and data represent mean values ± standard error of the mean. Data presented are representative of a minimum of three independent experiments with two or more independent donors. In all figures, statistical significance is represented as *p<0.05, **p<0.01, and ***p<0.001, significance indicates versus control unless indicated by bar.
p38K- and ERK1/2-selective inhibitors block IL-1β-mediated primary human astrocyte TIMP-1 expression
Reports suggest that p38K is involved in IL-1β-induced astrocyte TIMP-1 expression, but the role of the ERK1/2 signaling pathway remains unexplored [32]. In the following studies we tested the efficacy of increasing doses of p38K- and ERK1/2-selective inhibitors in blocking IL-1β-induced astrocyte TIMP-1 expression. Pretreatment with SB203580 or U0126 prevented IL-1β-induced p38K and ERK1/2 phosphorylation, respectively (Figure S1). Consistent with previous findings [5], [11], IL-1β led to a 5-fold increase in TIMP-1 mRNA levels 12 h post-treatment (p<0.001, Figure 1A and B). TIMP-1 mRNA levels were elevated in astrocytes treated with increasing doses of SB203580; however, 1, 5 and 20 µM concentrations of the inhibitor reduced the TIMP-1 response by 40, 60 and 65%, respectively (p<0.001, Figure 1A). IL-1β-induced astroctye TIMP-1 mRNA levels were reduced by 25% in astrocytes treated 0.1 µM U0126, 60% in astrocytes treated with 1 µM U0126, and 5 µM U0126 resulted in basal TIMP-1 levels (p<0.001, Figure 1B). Analysis of cell supernatants, 24 h post treatment, showed that TIMP-1 protein production was statistically significantly greater (~200-fold) in cells treated with IL-1β as compared to control cells (Figure 1C). Furthermore, p38K- and ERK1/2-selective inhibitors significantly reduced TIMP-1 protein levels in IL-1β-treated cells (SB203580: 3-fold versus IL-1β, p<0.001, and U0126: 13-fold versus IL-1β, p<0.05, Figure 1C); with U0126 exhibiting the highest quantitative change both at mRNA and at protein level. Two additional p38K- and ERK1/2-selective inhibitors, SB202190 and PD98059, were also effective at inhibiting IL-1β-induced astrocyte TIMP-1 expression (Figure S2). These data suggest that p38K-selective inhibitors affect IL-1β-induced astrocyte TIMP-1 expression; however, the ERK1/2-selective inhibitor is most effective.
Figure 1. p38K- and ERK1/2-selective small molecule inhibitors block IL-1β-mediated astrocyte TIMP-1.
(A-B) Astrocytes were treated with increasing doses of p38K- and ERK1/2-selective inhibitors (SB203580 and U0126, respectively) for 1 h, and then IL-1β (20 ng/ml) for 12 h. Total RNA was collected and reverse transcribed. TIMP-1 and GAPDH transcripts were quantified by real-time PCR. (C) In parallel, supernatants from 48 well cultures were collected after 24 h IL-1β treatment. Supernatants were analyzed for TIMP-1 expression by ELISA and normalized via MTT assay. Data presented are representative of a minimum of three independent experiments with two or more independent donors. (*p<0.05, **p<0.01, ***p<0.001; significance indicates versus untreated unless indicated by bar).doi:10.1371/journal.pone.0056891.g001
p38K and ERK1/2 pathways differentially regulate C/EBPβ mRNA and nuclear protein levels
Previously, we reported that C/EBPβ is expressed in brain lysates of HIV-1-infected individuals, and that C/EBPβ contributes to IL-1β-induced astrocyte TIMP-1 expression [33]. C/EBPβ knockdown partially blocked IL-1β-induced astrocyte TIMP-1 expression; however, blocking ERK1/2 activation resulted in >90% reduction in IL-1β-induced astrocyte TIMP-1 expression (Figure 1B). These studies suggest that ERK1/2 is a critical pathway for IL-1β-induced astrocyte TIMP-1 expression. In the current studies, we aimed to elucidate the signal transduction pathways controlling IL-1β-induced astrocyte C/EBPβ expression. IL-1β induced astrocyte C/EBPβ mRNA levels by ~5-fold compared to control (p<0.001, Figure 2A). The p38K-selective inhibitor, SB203580, showed a dose-dependent effect on IL-1β-induced astrocyte C/EBPβ mRNA expression. SB203580 at 0.1 µM reduced IL-1β-induced astrocyte C/EBPβ mRNA expression by 15%, and by ~55% at concentrations >1 µM (p<0.001, Figure 2A). IL-1β induced astrocyte C/EBPβ mRNA expression at doses of SB203580 through 20 µM. A low 1 µM dose of U0126 reduced IL-1β-induced astrocyte C/EBPβ mRNA expression but had lesser inhibitory effects at higher doses 10–20 µM (p<0.001, Figure 2B). Two additional p38K- and ERK1/2-selective inhibitors, SB202190 and PD98059, were used to assess their effectiveness at inhibiting IL-1β-induced C/EBPβ mRNA expression (Figure S2). The p38K inhibitor (SB202190) inhibited IL-1β-induced astrocyte C/EBPβ mRNA expression; however, the ERK1/2 inhibitor (PD98059) increased the expression of C/EBPβ during inflammation.
Figure 2. p38K- and ERK1/2-selective small molecule inhibitors block and enhance IL-1β-mediated astrocyte C/EBPβ expression, respectively.
(A-B) Astrocytes were treated with increasing doses of p38K- and ERK1/2-selective inhibitors (SB203580 and U0126, respectively) for 1 h, and then IL-1β (20 ng/ml) for 12 h. Total RNA was collected and reverse transcribed. C/EBPβ and GAPDH transcripts were quantified by real-time PCR. (C) Nuclear extracts were isolated 24 h post-IL-1β treatment, resolved by SDS-PAGE and then immunoblotted for C/EBPβ and lamin A/C protein. (D) C/EBPβ and lamin A/C band intensity was analyzed by densitometry analysis. Data presented are representative of a minimum of three independent experiments with two or more independent donors (*p<0.05, **p<0.01, ***p<0.001; significance indicates versus untreated unless indicated by bar).doi:10.1371/journal.pone.0056891.g002
C/EBPβ isoforms were undetectable in nuclear lysates from control astrocytes; however, increased C/EBPβ, 42 and 40 kDa isoforms, were detected in IL-1β-treated astrocytes (Figure 2C). C/EBPβ isoforms were decreased in SB203580-treated astrocytes, but increased in U0126-treated astrocytes (Figure 2C). C/EBPβ band intensity from IL-1β-treated astrocytes was increased by 2.5-fold compared to control cells (p<0.05, Figure 2D). IL-1β-induced C/EBPβ band intensity from SB203580-treated astrocytes was increased by 1.5-fold compared to control cells, whereas IL-1β-induced C/EBPβ band intensity from U0126-treated astrocytes was increased by 3-fold compared to control astrocytes (p<0.001; Figure 2B). These data were corroborated in experiments using additional p38K- and ERK1/2-selective inhibitors, each of which produced similar results at the mRNA level (Figure S2). Overall, these data show that p38K activation is required for full IL-1β-induced astrocyte TIMP-1 and C/EBPβ expression. ERK1/2 activation is critical for IL-1β-induced astrocyte TIMP-1 expression; however, ERK1/2 inhibition may enhance IL-1β-induced astrocyte C/EBPβ expression.
As a transcription factor, C/EBPβ is localized to nuclei where it can affect gene transcription. We pretreated astrocytes with inhibitors and then IL-1β for 24 h, fixed and colocalized GFAP as an astrocyte-specific marker (green) with C/EBPβ (red) in control and activated human astrocytes. In control cells, GFAP is present throughout the cell body of the astrocytes. Control astrocytes have a larger cell body (Figure 3A), whereas activated astrocytes have extensive processes protruding from their soma and denser staining of GFAP (Figure 3B-D). Low levels of C/EBPβ are present in the nuclei of control human astrocytes (Figure 3A) compared with activated astrocytes (Figure 3B), where the red signal from the nuclei is markedly enhanced. Low levels of C/EBPβ are present in the nuclei of SB203580-pretreated astrocytes (Figure 3C) compared to those treated with IL-1β alone (Figure 3B) or U0126-pretreated astrocytes (Figure 3D). Intense red signal is detected in nuclei of U0126-pretreated astrocytes (Figure 3D). Combined with the mRNA and protein expression data represented earlier, these data suggest that blocking p38K activation decreases IL-1β-induced astrocyte C/EBPβ nuclear expression.
Figure 3. p38K-selective small molecule inhibitor blocks C/EBPβ localization to the nucleus in response to IL-1β.
(A-D) Astrocytes were cultured in 48-well plates for 24 h, treated with selective inhibitors (SB203580, 20 µM and U0126, 20 µM) for 1 h, and then IL-1β (20 ng/ml) for 24 h. Cells were fixed, blocked and then incubated in antibodies against GFAP (green) and C/EBPβ (red). All pictures were taken at X200 and data represent at least three independent experiments in at least two independent donors.doi:10.1371/journal.pone.0056891.g003
ERK1/2-selective inhibitor, U0126, overrides IL-1β- or C/EBPβ-mediated increase in TIMP-1 promoter activity
We previously demonstrated that overexpressing C/EBPβ increases transcription from −1718/+988 of the TIMP-1 promoter [33]. Here we used −4200/+96or −1718/+96 driven-pTIMP-luc expression in combination with selective inhibitors and IL-1β to investigate regulation of TIMP-1 transcription. The two TIMP-1 promoter regions, −4200/+96 and−1718/+96, each contain the CCAAT site near the transcription start site. However, the −4200/+96 TIMP-1 promoter region retains the IL-1β-responsive element, while the −1718/+96 TIMP-1 promoter region has the basal TIMP-1 promoter (Figure 4A)
Figure 4. ERK1/2-selective inhibitor, U0126, overrides IL-1β- and C/EBPβ-mediated increases in TIMP-1 promoter activity.
Astrocytes were cultured in 48-well plates for 24 h and then cotransfected with TIMP-1 promoter-driven luciferase plasmids (B, −4200/+96 or C −1718/+96) and internal control pGL3-RL. (A) The −4200/+96 promoter harbors an IL-1β responsive element, while the −1718/+96 is the basal TIMP-1 promoter. Both promoters possess a predicted CCAAT-binding site prior to the start site [44]. Cells were allowed to recover for 24 h and then treated with MAPK-selective inhibitors (SB203580, 20 µM; and U0126, 20 µM) for 1 h prior to activation with IL-1β (20 ng/ml) for 24 h. Total cell lysates were collected 24 h post-inhibitor treatment and assayed for luciferase expression. Data represent mean values ± standard error of the mean in at least three independent experiments in at least two independent donors (*p<0.05, **p<0.01, ***p<0.001; significance indicates versus untreated unless indicated by bar).doi:10.1371/journal.pone.0056891.g004
Astrocytes were cotransfected with pTIMP-luc and pGL3-RL and then treated with selective inhibitors and/or IL-1β. IL-1β induced a 2.5-fold (p<0.001) increase in luciferase activity from the −4200/+96 region, but U0126 treatment completely blocked this increase, as well as basal promoter activity (p<0.001, Figure 4B). SB203580 treatment reduced the IL-1β-mediated effect by 25% (p<0.001) (Figure 4B). These data corroborate our results from experiments using mRNA and protein assays. To better understand the relationship between ERK1/2 and C/EBPβ we assayed a promoter, lacking the IL-1β-responsive element, −1718/+96. IL-1β significantly reduced transcription from −1718/+96-driven pTIMP-luc, and U0126 treatment extended this reduction (p<0.05 and p<0.001, respectively; Figure 4C). These data suggest that ERK1/2 signaling is required for IL-1β- mediated transcription from −4200/+96 of the TIMP-1 promoter.
Currently many studies focus on TIMP-1 activity in many diseases, multiple tissues and cell types [16], [17], [45]; however, the regulation of TIMP-1 expression is as important as its activity in the tissue microenvironment. As previously mentioned, data suggest multiple signal transduction pathways may regulate TIMP-1 expression depending upon stimuli and the expressing cell [46]. In these studies we aimed to identify signaling pathways responsible for IL-1β-induced astrocyte TIMP-1 expression. Our data indicate, for the first time, that the ERK1/2 pathway is essential for IL-1β-mediated increases in astrocyte TIMP-1 and ERK1/2-targeted pharmacological inhibitors will prove sufficient in abrogating astrocyte-TIMP-1 upregulation. In these studies, we show that the p38K-selective inhibitors partially, but significantly, reduce IL-1β-mediated increases in astrocyte TIMP-1 mRNA in a dose-dependent manner up to 1 µM; at which point, it plateaus. p38K-selective inhibitors also reduced IL-1β-induced astrocyte TIMP-1 protein, suggesting the p38K pathway contributes to IL-1β-mediated increases in astrocyte TIMP-1. Concentrations of ERK1/2-selective inhibitor, U0126, as low as 0.1 µM significantly reduced IL-1β-mediated increases in astrocyte TIMP-1; however, 5–20 µM concentrations reduced TIMP-1 mRNA below basal levels. Inhibiting the ERK1/2 pathway is sufficient to block IL-1β-mediated increases in astrocyte TIMP-1 protein, indicating that the ERK1/2 pathway is critical for IL-1β-mediated increases in astrocyte TIMP-1. ERK1/2-selective inhibitors were most effective at blocking IL-1β-induced astrocyte TIMP-1 expression, at all doses tested. Blocking p38K activity diminishes IL-1β-mediated increases in astrocyte C/EBPβ. Interestingly, blocking ERK1/2 activation with 20 µM of U0126 may enhance IL-1β-induced C/EBPβ expression while at the same time blocking IL-1β-mediated increases in astrocyte TIMP-1 expression. Lastly, blocking ERK1/2 activation was shown to block IL-1β- or C/EBPβ-mediated increases in TIMP-1 promoter activity, suggesting ERK1/2 signals to key transcription factors necessary for C/EBPβ-mediated TIMP-1 promoter regulation. These data may shed light on how astrocytes regulate C/EBPβ and TIMP-1 expression during neuroinflammation by identifying essential and auxiliary pathways.
Inflammatory stimuli lead to rapid phosphorylation of MAPK, p38K and ERK1/2 in astrocytes, initiating a cascade of signal transduction events that ultimately influence cellular function [47]. ERK1/2 and p38K regulate astrocyte morphology, cell death and extracellular matrix turnover [38], [39], [40]. Furthermore, small molecule inhibitors of signal transduction pathways are being explored as treatment options for multiple diseases [41]. At minimum, small molecule inhibitors are useful tools to identify pathways responsible for cellular processes [48]. The p38K-selective inhibitor, SB203580, inhibited IL-1β-mediated increases in astrocyte TIMP-1 mRNA by 50% at doses as low as 5 µM and as high as 20 µM. Pretreatment with the 0.1-20 µM concentrations of ERK1/2-selective inhibitor, U0126, reduced IL-1β-mediated increases in astrocyte TIMP-1 mRNA expression to basal levels. Additional p38K- (SB202190) and ERK1/2- (PD98059) selective inhibitors were similarly able to block IL-1β-mediated increases in astrocyte TIMP-1 expression (Figure S2). Crosstalk among MAPK proteins may account for multiple MAPK-selective inhibitors' effect on IL-1β-mediated increases in astrocyte TIMP-1. Alternatively p38K and ERK1/2 may phosphorylate, to different degrees, the same or different transcription factors that bind to astrocyte TIMP-1 promoter. In either case, we add TIMP-1, a multifunctional molecule of major importance to CNS health and disease, to the list of astrocyte genes critically regulated by the ERK1/2 pathway.
It is well established that NFκB mediates IL-1β-induced gene expression in many cell types, including astrocytes. However, NFκB activity alone may not account for the massive changes in gene expression following immune stimulation; indeed, p65 and p50 associate with multiple factors, including C/EBP's, to regulate gene transcription[49]. Wilczynska et al. showed that NFκB is involved in IL-1β-induced astrocyte TIMP-1 expression [32]. Here, we focused on elucidating the role of another transcription factor, C/EBPβ that is expressed in astrocytes and is associated with inflammatory responses. C/EBPβ is expressed in the brain of HIV-1-infected individuals and contributes to IL-1β-mediated increases in astrocyte TIMP-1 [33]. Therefore, it was surprising that ERK1/2-selective inhibitors did not reduce and may enhance IL-1β-mediated increases in astrocyte C/EBPβ expression while decreasing TIMP-1 expression. p38K-selective inhibitor, SB203580, decreased IL-1β-mediated increases in astrocyte C/EBPβ and TIMP-1 expression. The fact that ERK1/2 inhibition strongly blocks IL-1β-mediated increases in astrocyte TIMP-1 suggests ERK1/2 may activate one or more critical transcription factors that lay the framework for IL-1β-mediated increases in astrocyte TIMP-1, independent of C/EBPβ activity. Consistent with previous reports, IL-1β induces transcription from the TIMP-1 promoter region containing the upstream elements between −2200 and −2700 [32]. However, ERK1/2 inhibition was more effective than p38K inhibition at blocking IL-1β-induced astrocyte TIMP-1 expression; suggesting ERK1/2 may activate factors that bind the TIMP-1 promoter IL-1β-responsive element. Data suggest that ERK1/2 signaling can repress C/EBPβ activity through direct phosphorylation [49]. In control human astrocytes ERK1/2 activity may affect C/EBPβ activity indirectly through regulation of binding partners. C/EBPβ may utilize some, or all, of the TIMP-1 basal promoter, exon 1 and 2 and five CCAAT sites [44]. Our data show that IL-1β induces C/EBPβ nuclear localization in U0126-pretreated cells; suggesting other critical factors required for C/EBPβ increases in TIMP-1 promoter activity may be absent in these conditions. These data suggest that ERK1/2 activity is required for IL-1β- and C/EBPβ-mediated increases in astrocyte TIMP-1 expression.
TIMP-1 expression has been studied in several cell types, but astrocyte TIMP-1 regulation is not completely understood [14], [24], [50]. Wilczynska et al. showed NFκB and p38K are involved in IL-1β-induced astrocyte TIMP-1 expression; however, our studies suggest, though p38K may be involved, ERK1/2 is critical for basal and IL-1β-activated astrocyte TIMP-1 expression (Figure 5) [32]. Recently it was reported that TIMP-1 activates the AKT pathway in fibroblasts, indicating the molecule initiates further cascading of signal transduction, presumably playing an important role in response to inflammation [51]. Given the multiple functions of TIMP-1, restoring TIMP-1 levels in HAND patients may prevent or reverse deleterious effects of an MMP:TIMP imbalance and TIMP-1 deficiency. Knowledge of astrocyte TIMP-1 regulation may lead to identification of novel pharmacological targets, and ultimately therapies for HAND patients.
Figure 5. ERK1/2 is the predominant signaling pathway facilitating IL-1β-mediated astrocyte TIMP-1 expression.
Neuroinflammation activates bystander microglia and astrocytes to secrete IL-1β. Astrocytes respond to IL-1β by secreting TIMP-1. In this study we use a panel of small molecule pathway-selective inhibitors to delineate the signal transduction mechanism facilitating IL-1β-induced astrocyte TIMP-1 expression. p38K- and ERK1/2-selective inhibitors reduced and completely blocked IL-1β-induced astrocyte TIMP-1 mRNA and protein expression, respectively. U0126, the ERK1/2-selective inhibitor, also blocked TIMP-1 promoter activity.doi:10.1371/journal.pone.0056891.g005
Supporting Information
MAPK-selective inhibitors block IL-1β-induced astrocyte p38K and ERK1/2 phosphorylation. (A and C) Astrocytes were treated with of p38K- and ERK1/2-selective inhibitors (SB203580 and U0126, respectively) for 1 h, and then IL-1β (20 ng/ml). Total protein extracts were isolated 30 min post-IL-1β treatment, resolved by SDS-PAGE and then immunoblotted for p38K, P-p38K, ERK/2 and P-ERK1/2. (B and D) Phosphorylated and nonphosphorylated isoforms' band intensity was analyzed by densitometry analysis. Data presented are representative of a minimum of three independent experiments with two independent donors (n = 2), (*p<0.05; significance indicates versus untreated unless indicated by bar).
p38K- and ERK1/2-selective small molecule inhibitors alter IL-1β-mediated astrocyte TIMP-1 and C/EBPβ expression. (A and C) Astrocytes were treated with the p38K-selective inhibitor, SB202190, for 1 h, and then IL-1β (20 ng/ml) for 12 h. Total RNA was collected and reverse transcribed. TIMP-1, C/EBPβ and GAPDH transcripts were quantified by real-time PCR. (B and D) Astrocytes were pretreated with the ERK1/2-selective inhibitor, PD98059, for 1 h, and then IL-1β (20 ng/ml) for 12 h. Total RNA was collected and reverse transcribed. TIMP-1, C/EBPβ and GAPDH transcripts were quantified by real-time PCR. Data presented are representative of a minimum of three independent experiments with two or more independent donors (*p<0.05, ***p<0.001; significance indicates versus untreated unless indicated by bar).
We appreciate the assistance of the Laboratory of Developmental Biology for providing us with tissues and Lin Tang for graciously caring for primary human astrocyte cultures.
Author Contributions
Conceived and designed the experiments: JF AG. Performed the experiments: JF IC. Analyzed the data: JF KB LC AG. Contributed reagents/materials/analysis tools: JF AG. Wrote the paper: JF KB AG.
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text:
Grateful Dead
Technical Problems - Broken Links
i have IE and when i try to click on "trouble ahead, trouble behind" in forums, it goes straight to "grateful geezers". can't get to TATB. thanks
****NEVERMIND, the "broken link" is in between my ears somewhere.........all is ok here. :) sorry
I'm still using windows and ie. I figure once they get everything worked out with the new site, everything will work like it used to. At least I can dream.
We may have been down this road already, but what OS/browser?
...I get a 'Gateway Timeout' message, or 'HTTP 500 Internal Server Error' message:
1. Got Questions? Ask Here...
2. Technical Problems - Forums, Features, Etc.
3. Welcome!
P.S. The last two just started happening today, but number 1 has been happening a very long time. Strangely enough, I can access the pages if I'm logged out. I can see other people access the pages so I don't know what the dea-leo is...
will pass this on.
when I click on the Wine advert on the home page I get this message
We're sorry!
This offer is not available in your area.
You will be redirected shortly.
Presumably this is because I am not in the USA, but in France
I am then automatically redirected to a very suspicious and unwelcome half constructed messy page with the url
Please make this go away
posted a couple updates yesterday and not sure what happened
edited those post tonight and the expected happened
like new format!
as I see your posts just fine. Hm.
Just went back to recent posts and noticed this forum topic shows marye's 9:24 post as the last post here. Curious stuff
I was thinking of the My Posts button which was really helpful in keeping up with stuff I was involved in. Not that it belongs here, but, I paid extra for shipping items ordered from the 90's offering as they became available to avoid the order not being fulfilled until mid Sept. when "So Glad You Made It" became available. As of this morning they don't have any idea about what has happened to my black Indian Skull shirts that were supposed to ship on 8/15 other than those are "sold out". Arrrrggghhh!!!

subset: commoncrawl


text:
LEXINGTON, KY. (WDRB) -- As part of the reunion of the 1972 U.S. Olympic men's basketball team at Georgetown College this weekend -- the first gathering of the entire roster since the team left the troubled and tragic Munich Games -- there was a seminar Friday titled, "The ethical debate about not accepting the silver medals."Somehow, I doubt that in Russia they're holding similar discussions about, "The ethical debate about accepting the gold."Every four years at Olympics time, the '72 team comes back into public memory, having refused all these years to accept silver medals for a game they won (twice) before the Soviet Union prevailed on a last-second shot given a third chance to make one.The sequence of events is one of the most infamous in Olympic history. Doug Collins coming up with a steal and being slammed into the basket support on the fast break. Rising up, bruised, to make two free throws -- shots still called by team members "the winning free throws."The horn that sounded before Collins' second shot. The chaos that ensued. The USSR claiming to have called a timeout. The Soviet coach storming onto the court -- illegally. The Soviets making a substitution -- illegally.It is as farcical today as it was the day it happened. Having twice won the game at the final buzzer under the rules of basketball at the time, the Americans voted not to accept the result that was eventually presented to them after the Soviets made their third try the charm."According to the rules that game was played under, we won," said Kenny Davis, the team captain and one of the organizers of last week's event at his alma mater just outside Lexington, with the backing of his employer, Converse. "So based on that, we felt like we certainly would not want that Silver Medal. If we had lost that game according to the rules, we would have proudly taken that Silver Medal, because losing gracefully, I believe, is much more important than winning gracefully. But to have gone out and played by the rules and won the gold, then had some bureaucrat say to us, 'We'd like you to take the silver,' I always felt like it was something we shouldn't have done."And they still feel that way. In a room together for the first time in 40 years, an ESPN documentary crew preparing to shoot for an upcoming program, the team was unanimous in another vote -- they want the silver medals no more now than they did then.Some, like Davis, have gone the extra step of stipulating in their wills that not even their families are to accept the medals.They've been asked several times by the International Olympic Committee. And every year when the Olympics roll around, they are asked again if they want the medals.Maybe it's an American curiosity. There are even those who believe it poor sportsmanship -- though no sportsmanship creed that I know of binds a man or team to abide by an outcome that is corrupt. Certainly, the IOC steps in all the time on issues when there is less evidence of foul play than exists still for this long ago basketball game. Nor am I aware of stories asking the Russian players if they feel they should give their medals back. Six of the players from that Soviet team have passed away.But all 12 Americans and one coach -- John Bach, who assisted Henry Iba and Don Haskins -- are alive and well. In fact, to a man, they look remarkably well."This was not a happy team the last time I saw them," said Bach, now 88 years old and still involved in coaching. "Forty years ago, we were the most disappointed, angry, upset team in basketball. All of them have transcended that, done very well in their lives, been very successful, and I see they've improved in every way. And frankly so have I. As you get older in coaching, you're quite willing to share more of yourself.""Transcend," is the operative word. It was a bitter loss. And the very claim of having been cheated implies anger or bitterness. But these men didn't seem angry to me. They seemed resolute. They even laughed about portions of the game, about how badly the Soviets beat them up, about bad calls and foul trouble.Collins is now a leading candidate to coach the next Olympic basketball team."I certainly would be honored if my name came up in that discussion and I would be honored to serve," he said.But on Saturday, after reliving those monumental free-throws, made not only amid distraction and Olympic-gold pressure, but Cold War pressure, as well, Collins smiled as he remembered the welt that rose up under his eye after he was fouled, and gathering himself to shoot the free throws. A bitter loss, yes. But bitter men? No."I think the thing is we have a tremendous amount of respect not only for each other but for the game, and for everything the game has given us, from a chance to do what we love to make a life, to create families, to be friends, leaders, teachers and all the things that really will be our legacy," Collins said. "It was my coming out party in 1972, from Illinois State University, to be a part of these guys. It was tough . . . But we've all said that we've grown from this experience. We're better men."They may not want anything to do with silver, but the legacy of this group is sterling. And not just because of its stand. Bitter though the loss was, none of those players left Munich feeling sorry for themselves -- in large part because of the nine Israeli athletes who were murdered by terrorists during the games.Billy Reed, the Hall of Fame Kentucky journalist who worked so hard to organize the event, asked Tom Burleson near the end of one press Q&A if he would recount his experience of the terrorist situation, because Burleson by happenstance wandered closer to it than anyone on the team, as close as anyone in Munich, perhaps. He was taking a train back to the Olympic Village when it stopped unexpectedly, and he pushed his way off with a couple of Italian basketball players. You can picture the 7-2 Burleson as he got out into the crowd of hundreds of athletes, looking above them and seeing the garage door across the way that they'd been using to get in and out of the athletes' compound. He and the Italians made their way through the crowd, past one German officer, then another, who flashed rifles and spoke German. Burleson told them they had nothing to worry about, he was going back to his room."By the time I got to the garage door, a German soldier came up to me and he spoke English as well as anybody here, and said, 'Son, you're in the wrong place at the wrong time. We're in the process of bringing the hostages out right now,'" Burleson remembered. "He said, 'I've got to have you stand against that wall, face it, with your hands on it, and let us bring out the hostages.' And I'm going, 'Oh man.' And as I looked to the left, the two Italian players were on the ground, with guns to their backs."I had a rifle in my back, and I had my hands to the wall, there at the garage opening. Then I could hear them start to bring the hostages out. The guy in the green leisure suit and black mask and stuff like that, he stepped around the corner and I looked at him, we were probably 60 feet away, and as I looked at him, the German soldier took the gun out of the small of my back and placed it in the back of my head and said, 'Face the wall.' At this time, my hands are there (in front of him) and I see the foundation wall, and I still see the blemishes today, of that wall, and I started praying to God to allow me to get out of this situation and get, you know, back to the room where I needed to be. So the terrorists started bringing the nine Israeli hostages by me, and I could hear the shuffling of the feet."At this, Burleson began to rub his hands against each other in a shuffling motion. And then he did something else. He teared up. And then he began to sob. And through those sobs, he said, "And I could hear them crying." He buried his face in his hands, inconsolable before this room of teammates and reporters. "I could hear them crying. They were walking to their deaths."And he could not stop. Teammates put their hands on his shoulders. Reed began to speak again, but Burleson wept louder. He wasn't, for those moments, in the room with the rest of us so much as he was back against that wall. And he had taken the rest of us with him. A moment later, composed, he continued."These men, these athletes, they loaded them in the van, they took them to the helicopters, they release me, I turn and stand with the soldiers. Go to the Olympic Village see the helicopter fly off. From there you know what happens."Then he wept again, not the quite tears of sadness, but anguished sobs. "They were gong to die. These nine men. I hear them in my sleep. I hear them in my sleep."And he got up, and walked out of the room.No one had expected that. Burleson had told of that experience before. But maybe being with his teammates drew out some different emotions. It was a difficult thing to hear and see.It should have been. I have seen the film, "Munich." I've read about those events, heard many people talk about them. I never felt their impact until I saw and heard Burleson talk about it. We're generally not comfortable with such shows of emotions in this culture. But Burleson's were real and appropriate. The emotions he showed were ones any good man would feel.It's interesting, on a weekend designed to honor that team that had gold snatched away, not a single player failed to mention the bigger tragedy of the games, or to put his life into a larger context than those events. That, in the end, is how you win."I went there as a young 23-year-old kid who was concerned about the proper brand of clothing and whether I was wearing it or not," Davis said. "Then all of a sudden we see them take those Israeli kids out in those caskets. And I think all of us immediately went from whatever age we were as a youth to a pretty mature person. They were our age, ate the same food, lived in the same village, dreamed the same dreams. And they took them out in caskets. We all came back home and started families and had good careers and life has been good for us. We're still alive. It's been said what happened to us was unfortunate but what happened to them was a tragedy. And I believe that with all my heart."Davis said he doesn't think about his Olympic basketball experience so much anymore, only when people bring it up, which is every four years, like clockwork."We thought it would probably just go away like things do," he said. "But it seems like every four years when the Olympics begin again it refreshes itself in people's minds, and all of a sudden they want to hear the story again."Turns out, it's a story about winning, just like those players always said it was.Copyright 2012 WDRB News. All Rights Reserved.
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Definition of point of departure in English:
point of departureLine breaks: point of de¦part|ure
The starting point of a line of thought or course of action; an initial assumption: Keynes took these current events as his point of departure
More example sentences
- In the midst of all the clamor for redistribution, we should not forget our initial point of departure: that ordinary contracts produce gains from trade that are shared by all parties.
- Nonetheless, when numerous articles use the same assumption as a point of departure, that monotone can leave the false impression that the paper has chosen sides.
- The premature independence a young child acquires in a single-parent home marks a point of departure from the developmental course of the child from a never-divorced family.
Definition of point of departure in:
- The US English dictionary
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Feyereisen has been named
the Wisconsin Intercollegiate Athletic Conference Pitcher of the year
and six other Pointers earned conference year-end awards.
Feyereisen's selection marks the ninth year in a row, since 2005, the Pointers have had either a WIAC Position or Pitcher Player of the Year (sometimes both). Cam Seidl earned Pitcher of the Year honors for the Pointers in 2012.
Feyereisen led the WIAC with a 2.24 ERA and a boasted a 6-2 record. Feyereisen led the Pointers with 10 starts on the season and was second with 51 strikeouts in 64.1 innings pitched.
In addition to Feyereisen, four Pointers were named to the All-WIAC Team: shortstop Dan Douglas, first baseman Sean Gerber, starting pitcher Jacob Herbst, and catcher Ryan Schilter.
This marks the second time Douglas has been named to the All-WIAC Team, the first honor coming in 2011. Douglas led the WIAC with 76 hits and finished fourth in runs scored with 49.
Gerber has been named to the All-WIAC Team for the third consecutive season. Gerber led the WIAC in home runs (12) and RBIs (60). He also placed second in runs scored (60), second in doubles (17), and fourth in hits (70).
Herbst made 15 appearances (seven starts) for the Pointers while racking up a conference-high nine wins. Herbst led the Pointers with 66.1 innings pitched and 65 strikeouts. He finished the season with a 2.44 ERA.
Schilter makes the All-WIAC Team for the second consecutive season. His 21 stolen bases led the WIAC for the second consecutive season, and he also led the WIAC with 63 runs. Schilter's 73 hits are the second-most in the conference, and his nine home runs are good for fourth.
UWSP outfielder Jimmy Coady earned WIAC Honorable Mention. Coady finished fifth in the WIAC with 65 hits, and his .369 average is good for fourth on the team. Coady also finished third on the team in slugging percentage (.540), on-base percentage (.439) and total bases (95).
Senior Nick Douglas was named to the WIAC All-Sportsmanship team. Among Douglas's highlights with the Pointers is his very first at-bat at University Field in Stevens Point when he belted a home run.
After winning the 2013 WIAC Tournament championship the Pointers earned a No. 2 seed in the NCAA Midwest Regional. UWSP opens competition in Whitewater, Wis., against Benedictine at 3 p.m. on Wednesday, May 15.
2013 All-WIAC Baseball Team
Name, School, Year, Position, Hometown (High School)
Steve Bartlein, Whitewater, Senior, Shortstop, South Milwaukee, Wis.
Dan Douglas, Stevens Point, Senior, Shortstop, East Troy, Wis.
Joel Effertz, La Crosse, Junior, Righthanded Pitcher, Ladysmith, Wis.
TJ Evanson, Superior, Sophomore, Firstbase, Lakeville, Minn. (North)
JP Feyereisen, Stevens Point, Sophomore, Righthanded Pitcher, River Falls, Wis.
Jared Fon, Whitewater, Junior, Thirdbase, Germantown, Wis.
Sean Gerber, Stevens Point, Senior, Firstbase, Stoughton, Wis.
Jacob Herbst, Stevens Point, Senior, Righthanded Pitcher, Park Falls, Wis.
Marty Herum, Whitewater, Sophomore, Firstbase, River Falls, Wis.
Bobby Kachel, Oshkosh, Junior, Firstbase/Outfield, La Crosse, Wis. (Aquinas)
Tyler Kamps, Oshkosh, Senior, Secondbase/Firstbase, Appleton, Wis. (East)
Tom Kerndt, Whitewater, Senior, Righthanded Pitcher, Lansing, Iowa
Justin Lambert, Whitewater, Senior, Righthanded Pitcher, Mt. Horeb, Wis.
Joe Pavlovich, Oshkosh, Sophomore, Righthanded Pitcher, Denmark, Wis.
Mikole Pierce, Whitewater, Sophomore, Rightfield, De Pere, Wis.
Scott Plaza, Whitewater, Junior, Righthanded Pitcher, Arlington Heights, Ill.
Michael Polcyn, Oshkosh, Sophomore, Shortstop, Ripon, Wis.
Matt Roberts, Whitewater, Junior, Righthanded Pitcher, Manitowoc, Wis. (Roncalli)
Ryan Schilter, Stevens Point, Senior, Catcher, West Bend, Wis. (West)
Michael Swift, Oshkosh, Sophomore, Secondbase/Thirdbase, Kenosha, Wis. (Bradford)
Honorable Mention
Mike Boushley, Platteville, Sophomore, Righthanded Pitcher, Hortonville, Wis.
Brooks Braga, La Crosse, Senior, Outfield, Middleton, Wis.
David Cladis, Whitewater, Senior, Secondbase, Joliet, Ill. (Catholic)
Jimmy Coady, Stevens Point, Sophomore, Outfield, La Crosse, Wis. (Aquinas)
Adam Cordova, La Crosse, Senior, Designated Hitter, Merrill, Wis.
Dylan Friend, Whitewater, Junior, Centerfield, Burlington, Wis.
Dan Genrich, Superior, Senior, Lefthanded Pitcher, Wausau, Wis. (West)
Mitch LaVelle, Stout, Freshman, Shortstop, Chippewa Falls, Wis.
Santiago Morales, Stout, Senior, Centerfield, West St. Paul, Minn. (Henry Sibley)
Garrett Novinski, La Crosse, Senior, Shortstop, Middleton, Wis.
JT Schneider, Whitewater, Senior, Leftfield, Oak Creek, Wis.
Travis Sindles, Oshkosh, Junior, Lefthanded Pitcher, Twin Lakes, Wis. (Wilmot Union)
Robbie Veith, Superior, Senior, Outfield, Brainerd, Minn.
Tim Verthein, La Crosse, Senior, Righthanded Pitcher, La Crescent, Minn.
Adam Widder, Stout, Sophomore, Righthanded Pitcher, Wauwatosa, Wis. (Milwaukee Lutheran)
All-Sportsmanship Team
Steve Bartlein, Whitewater, Senior, Shortstop, South Milwaukee, Wis.
Adam Cordova, La Crosse, Senior, Designated Hitter, Merrill, Wis.
Casey Dittel, Superior, Senior, Secondbase, Woodbury, Minn.
Nick Douglas, Stevens Point, Senior, Outfield/Secondbase, East Troy, Wis.
Kevin Fessler, Platteville, Junior, Catcher, Wauconda, Ill.
Matt Guida, Stout, Senior, Catcher, Hawley, Minn.
Matt Wells, Oshkosh, Senior, Righthanded Pitcher, Little Chute, Wis.
Position Player of the Year: Marty Herum of Whitewater
Pitcher of the Year: JP Feyereisen of Stevens Point
Coach of the Year: John Vodenlich of Whitewater
Max Sparger Scholar-Athlete: Adam Cordova of La Crosse
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Q&A: Oxford's Mark McCarthy on Using Exome Arrays and Exome Sequencing to Study Diabetes
May 28, 2013
Q&A: Oxford's Mark McCarthy on Using Exome Arrays and Exome Sequencing to Study Diabetes
Justin Petrone
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Show Name: I Almost Got Away with It
Season 6 Episode 7
Episode Name: Got to Be Bonnie and Clyde
Genre: Crime
Aired: Mondays at 09:00 pm
Runtime: 60 Minutes
Season 6 Episode 7
Episode Name: Got to Be Bonnie and Clyde
Genre: Crime
Aired: Mondays at 09:00 pm
Runtime: 60 Minutes
For these perpetrators, their stories may be different, but their motives are always the same - to stay out of prison and live life on the lam. The people profiled in "I (ALMOST) GOT AWAY WITH IT" evaded justice for years, sometimes decades, by using their families, fake identification and even violence to avoid capture. Viewers learn how law enforcement uses all means necessary to capture these fugitives. Additionally, each episode features an interview from prison with these slick culprits, who spill where they found shelter, how they changed their identities and how they almost got away with it.
Might even be ahead of it's time, watch I Almost Got Away with It season 6 episode 7 live the time we get to spend in that half hour, it's truly relish! You have to push past this edge and be willing to step inside before discovering any depth. There is so much to recommend this show! I Almost Got Away with It has everything you'd expect from the TV show - sharply drawn characters, large-scale stories intercut with thrilling moments and a sense that you couldn't find something like it anywhere else on TV. Greatest show on earth right now at this moment. Considering most of what happened within a few days I Almost Got Away with It s06e07 is worth seeing. It can support the watching enjoyment with wonderful visuals and superb performances. This show is a near-perfect match with the established show, watch I Almost Got Away with It s06e07 online a caustic, sharp-edged mix of humor and drama that tiptoes along the edge of disaster but never drops into the looming abyss. It touches various subjects that many would be afraid to even think about.
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Track and Field opens 2007 outdoor season at FSU Relays on Thursday
March 28, 2007
JACKSONVILLE, Fla. - After two months of training, the JU track and field team will start their 2006 Atlantic Sun Conference Outdoor Track title defense on Thursday as they open the outdoor season at the FSU relays in Tallahassee, Fla.
"We are all anxious to get the outdoor season underway," said JU head coach Ron Grigg. "This first meet will be a good evaluation tool that will guide our training for the remainder of the season."
The last time the Dolphins were on the track they tallied 150 points to take their second straight A-Sun Indoor Track and Field Championship in Johnson City, Tenn. on Feb. 17.
Senior Janita Ocana was named "Most Outstanding Field Performer" after winning the long jump with championship record jump of 19'06.75", while Alyssa Deal was the second Dolphin in two conference indoor meets to earn "Most Outstanding Freshman Performer". Grigg was named "Coach of the Year" for the second consecutive indoor season to round out the JU individual honors.
Sophomore Natasha Harvey preps for her second outdoor track campaign after becoming the third Dolphin track athlete in school history to earn All-America accolades by finishing fourth in the long jump with a mark of 20'10.75" (6.37m) at the NCAA Indoor Track and Field Championships on March 9th
The meet is set to begin 11 a.m. with the heptathlon for the women.


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Wedding Blackmore and Vellacott 35
12:21pm Friday 19th August 2011 in Weddings By Contributed report
Photo: A and B Photography, Minehead. ANDREW Blackmore from Withypool and Alison Vellacott from Dulverton were married at St Andrew's Church, Withypool. Kathryn Vellacott, Emily Marlow and Zara Blackmore were bridesmaids and Dominic Blackmore and Thomas Crossman were pageboys. The reception was held at Liscombe Lodge.


text:
A Wausau high school is going green. Newman Catholic expects to save several hundred dollars a year, all thanks to a new solar-electric system. But officials say it's not just about money.
10 solar-electric panels now sit on the roof of Newman Catholic High School in Wausau.
The panels are part of a system being installed to help produce electricity at the school.
According to Wisconsin Public Service, the system will power at least three classrooms in just seconds.
"Light strikes the solar panels and that converts it into electricity," said Steve Knudsen.
Newman Catholic High School science teacher Steve Knudsen is excited.
"They chose us this year to get a system, we're lucky to get one," said Knudsen.
Knudsen says the system has several benefits.
"We also have the opportunity to monitor and study the energy coming in through these panels so that we can incorporate that information into our science curriculum for our students," said Knudsen.
He says it gives learning a new meaning.
"This is going to give students direct hands-on ability to study a large solar-energy system that they really didn't have before," said Knudsen.
The system was donated to the school by the WPS Community Foundation through a program that teaches students about energy.
"The "SolarWise for Schools" system gives the panels and it's wired up so that the teacher and the students can see all the data," said Project Manager, Michael Moore.
That, teachers say is a valuable hands-on tool.
"This is going to be a really big step up for teaching students about how to go green because they'll see how practical and how useful a system like this is," said Knudsen.
WPS officials say the installation should be finished by tomorrow.
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Essential Oil Stories – PastTense Oil Blend
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PastTense Oil Blend – A Healing Story by Amanda
One fall day I met with my son's elementary school teacher for a Parent-Teacher Conference while my kids played on the school playground. After a few minutes my kids came running into the classroom telling me that my five year old daughter had been stung by a bee or maybe a wasp.
I wasn't sure what to do since I wasn't at home with my usual first aid supplies. I had a bottle of PastTense in my purse and decided to roll that on the red and swollen bite. A minute after rolling on the Past Tense, the pain and itching was gone. After a few more applications the redness and swelling was gone too.
By the end of that day my five year old and I had both forgotten that she had been stung by a bee earlier that day. There was no sign of it! Now I know that PastTense is not only good for headaches and muscle pains, it's great for bee stings too.


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A collection of news and information related to John Irving published by this site and its partners.
Top John Irving Articles
Displaying items 34-44
A rainy National Book Festival whets readers' appetites
Paperback Writers: Slaughter and rubble
Writer David Foster Wallace found dead
Annual Hay Festival is one for the books
Cultural capitals of EuropeStory | Jan 14, 2009 | 4:29 PMThe newly anointed Cultural Capitals of Europe for 2009 are Linz, Austria, and Vilnius, Lithuania, both a bit off the continent's prime tourist paths. Separated by the Czech Republic and Poland, these central and northern European cities are near...
Weiner Talks 'Mad Men'Blog | Oct 23, 2008 | 4:30 PMThis morning's Variety has what I believe to be one of the more exhaustive discussions of "Mad Men" I've seen, and the guy doing the discussing is none other than creator Matthew Weiner. I could send you to Variety,......
John Irving, poolside in 1998Story | Oct 25, 2009 | 4:33 PMWhen "The World According to Garp" was published more than 30 years ago, John Irving found himself on bestseller lists for the first time. Since then he's won a shelf of awards, including an Oscar for the adaptation of his......
Thursday's TV Talk Shows: 'Charlie Rose' studies the brainStory | Oct 29, 2009 | 4:34 AMClick here to download TV listings for the week of Oct. 25 - 31 in PDF format This week's TV Movies The Early Show Harry Smith appears on 'A Prairie Home Companion'; artist Stephen Wiltshire. (N) 7 a.m. KCBS Today......
Tuesday's TV Talk Shows: Gov. Arnold Schwarzenegger visits 'The Jay Leno Show'Story | Nov 2, 2009 | 9:34 PMThe Early Show The cast of 'Murphy Brown' reunites; Vanilla Ice performs. (N) 7 a.m. KCBS Today Author John Grisham ('Ford County'). (N) 7 a.m. KNBC KTLA Morning News (N) 7 a.m. KTLA Good Morning America Carrie Underwood performs; author......
Door in the Floor director to do Paranormal Activity 2Story | Mar 29, 2010 | 4:39 PMTod "Kip" Williams didn't make box office waves his The Door in the Floor, an inventive and beautifully acted John Irving adaptation starring Jeff Bridges. Perhaps he'll have a hit on his hands with the Paranormal Activity sequel. Perhaps not....
Summer reading: Marcy Dermansky on F. Scott FitzgeraldStory | Jun 7, 2010 | 4:34 PMAs summer gets underway, we've created the L.A. Times list of 60 books for 92 days. All of these are new titles, being released in time for summer 2010. At Jacket Copy, we're asking writers and other bookish types about......


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Ether Nudes
Olivier De Rycke Photography
High qualitty erotic art nudes pictures for the price of a fashion magazine only !
Set of 6 pictures.Model: [Désirée] , last updated 2014-08-12 13:27:20.
About Ether Nudes
It is all and only the work of the French photographer Olivier De Rycke. Beauty throught a photographer eye with a trendy touch, catching with a keen eye the very essence of sensuality. You will not find those pictures anywhere else than here.
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Olivier De Rycke is a professional photographer based between Riga (Latvia) and Paris (France) for Portraits - Beauty - Fashion. But he has produced an extensive portfolio of nudes as well, that is published here on this site. Also a first book 'My body My Soul' was published in 2008 by the Editing House Ragage (Paris France).
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Lot of choices but in any case if you have a request, do not hesitate to contact me.


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- Game Info
- Articles
- Review
- Videos
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- Downloads
- Guide
- Cheats
The next generation in online FPS action. UT 2007 is built on the Unreal Engine 3.
VideoGamer.com Score:8
Our Verdict: Unreal Tournament 3 for the Xbox 360 is a stunning looking, slick and polished FPS that every competitive 360 gamer should add to their collection.
Latest Articles for Unreal Tournament 3
Epic Games launches official online store
Purchase specially designed merchandise and apparel based on the firm's brands.
Epic Games president Mike Capps retires to start family
Transitioning into an advisory role and will remain on the Epic board of directors.
Cliff Bleszinski leaves Epic Games after 20 years
The man formerly known as CliffyB leaves behind a legacy of triple-A blockbuster titles.
Epic isn't working on a new Unreal game
Remains a 'huge influence' on the studio now most famous for Gears of War and the Unreal Engine.
Rein: Unreal Engine 4 is 'a long way off'
Next generation engine built for massively multi-core processors.
Steve Polge on Epic's future plans
News on potential single-player Unreal, Gears of War 2 PC and great looking UE3 games.
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Epic Games launches official online store in Article Comments
Purchase specially designed merchandise and apparel based on the firm's brands.
Cliff Bleszinski leaves Epic Games after 20 years in Article Comments
Gutting news very talanted but I hope he's not too long on his break and starts something new very soon.
VideoGamer.com Rumour mill in Article Comments
A round-up of the juiciest rumours being talked about across the internet.


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5 Deuce Hoover Gangster Crips
From Rap Dictionary
The 5 Deuce Hoover Crips are the only Hoover set still using the Crip identity at the end of their name, the rest of the Hoover Gangs refer to themselves as Hoover Criminals not Crips even though they're still under the Crip Alliance. New term description goes here. Quote demonstrating new term use -- MC Artist (Name Of The Song) [1]
Categories: Terms | Nouns


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Here is all the information you need to make sure you catch all the live footy on the box so that you don't miss a minute of action. Pre-season club tournaments will begin winding down at the beginning of this month, with the International Champions Cub Final airing on Tuesday 5th. The FA Community Shield will pair Arsenal and Man City on Sunday 10th at Wembley in the traditional season curtain-raiser. Finally, the long-awaited start to the Premier League season will happen on Saturday 16th August, with Man United taking on Swansea City at Old Trafford on BT Sport.
This TV guide contains all the games live on all British TV channels, including Sky Sports, BT Sport, BBC, ITV, ESPN, British Eurosport, MUTV and Chelsea TV, as well as Irish channels Setanta Ireland and RTE. So whether you live in England, Scotland, Wales, Northern Ireland or The Republic of Ireland, you have no excuses to miss any upcoming live football. So make sure you make a note of all the games that you think will be unmissable viewing, and settle down in front of the TV or get yourself to the local pub. Whether you follow British, European, American or even Japanese football, there are plenty of games to look forward to in August.
Sat 2nd Aug
Sun 3rd Aug
Tue 5th Aug
Wed 6th Aug
Thu 7th Aug
Fri 8th Aug
Sat 9th Aug
Sun 10th Aug
Mon 11th Aug
Tue 12th Aug
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Thu 14th Aug
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Executive Director & Founder
Nicole Baran, MSW, is the Executive Director and Founder of the Center for Relationship Abuse Awareness. She has been dedicated to the domestic violence movement since 2000, working both as an advocate with survivors of domestic violence and at the policy level for systemic change. Nicole has conducted national trainings and contributed to publications to improve the institutional response to domestic violence since 2001. She has delivered over 300 customized workshops, locally and nationwide, at organizations such as Kaiser Permanente, the San Francisco Bar Association, and California's State Domestic Violence Coalition. Nicole has been teaching in the Feminist Studies Program at Stanford University since 2007 after developing Violence Against Women: Theory, Issues and Prevention. In 2006, she successfully partnered with Stanford University to institutionalize a comprehensive response to gender-based violence on campus. She managed a five-year grant from the USDOJ Office on Violence Against Women and was instrumental in implementing Stanford's first Office on Sexual Assault and Relationship Abuse. Nicole received her BA and MA in English from Stanford University and a Masters in Social Work from Washington University in St. Louis, with a specialization in management and a focus on women's rights. Nicole Baran is also the Executive Director of the Peggy and Jack Baskin Foundation which funds innovative strategies for eliminating obstacles facing marginalized populations, with an emphasis on empowering women and promoting educational equality. She established the infrastructure and direction of the Foundation and became the first Executive Director in 2009.
Read Nicole's interviews with Seneca International and Maestro Market or listen her interview on Stanford Radio KZSU's "Culture Lens" to learn more about her work.
Program Director
Shannon Moskitis is the Program Director for the Center for Relationship Abuse Awareness. She received her BA from Stanford University in Urban Studies with a minor in Feminist Studies, concentrating in Gender Violence and Human Rights. Her honors thesis, "'It's a Catch 22′: The Intersection of Homelessness, Domestic Violence, and the Shelter System's Response", based on in-depth interviews with women utilizing homeless shelter systems in the Bay Area, was awarded the 2011 Firestone Medal in Urban Studies. Shannon served as an intern with the Center for four years, as a peer educator with a focus on relationship abuse in the LGBT/Queer community, before joining the Center's staff. She plans to pursue an MSW and continued work against gender violence. She also currently serves as a rape crisis counselor and advocate in Alameda County through Bay Area Women Against Rape.
"The Center has given me the courage to speak out, with the facts, with compassion, with the knowledge that the movement to end violence against women is a movement that affects all of us. In no small way, my life has been changed by this internship." – Intern


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We found 3 Marquees which matched your search.
Worcestershire-based Fews Marquees has over 14 years' expertise in providing high-quality structures for hire, and counts JCB and Red Bull among its loyal client base. The award-winning company… more »
A popular supplier of alternative event structures, Ten By Fifteen offer stretch-fabric structures for corporate and private events that can be adapted to fit just about any space. The fabric… more »
This sister company to Ten by Fifteen Structures provides Nordic tipi-style tents for events such as weddings, festivals and corporate parties. The eye-catching structures are also well suited… more »


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[Source Description: 6 January 1964: Letter from (Sender Unknown) to President William Friday. - From the Records of the Office of the President - William C. Friday Files (#40009), University Archives, Wilson Library, University of North Carolina at Chapel Hill.]
Document Description
Calling attention to the case of Rosemary Ezra (mistakenly referred to as "Rosemary Eliza"), the letter writer complained to President Friday that "[s]uch students reflect credit on what they may have learned from a given institution."


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DISTRESSSun, Oct 12, 1|12c
SEASON 2, EPISODE 216 - "The BAU investigates the murders of three people who have been killed in the last 48 hours at various construction sites in Houstons Fifth Ward. What they discover is that the killings are not at the hands of a serial killer but a war veteran suffering from severe Post Traumatic Stress Syndrome. He thinks hes in the middle of a war zone and is trying to stay alive until hes rescued. The BAU stage an extraction to rescue this veteran before


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the one approach Is Lucy! TOWIE's leave out Mecklenburgh voted best bikini body of …
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cleaning soap star Michelle Keegan and Victoria's Secret model Rosie Huntington-Whiteley took a good seventh situation, with each garnering four per cent of the votes. Contender: In close second location came Rihanna, with one in 5 girls mentioning she.
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Last Updated: Monday, March 18, 2013, 17:33
Demanding pay parity with UGC grades, hundreds of teachers affiliated to Madhya Pradesh Medical Teachers` Association Monday went on mass casual leave affecting health services in government run hospitals in the city.
Last Updated: Wednesday, October 10, 2012, 14:33
The proposed mass leave by about 70,000 policemen in Bihar on Wednesday was deferred till November 30.
Last Updated: Monday, October 08, 2012, 16:26
Over 70,000 policemen in Bihar have decided to go on a mass leave for five days from October 10 to press for hike in salaries and perks among other demands.
Last Updated: Thursday, August 18, 2011, 22:17
Teachers of Lucknow University (LU)
today went on mass casual leave in support of Anna Hazare`s
agitation on Lokpal Bill issue.
Last Updated: Tuesday, July 12, 2011, 10:23
Government doctors in Rajasthan, who had announced to go on a two-day mass leave, withdrew their plan early on Tuesday after a meeting with a sub-committee of ministers.
Last Updated: Monday, November 30, 2009, 17:43
Teaching work was disrupted in
state universities and colleges across Uttar Pradesh as
18,000 teachers went on mass casual leave demanding payment of
Last Updated: Tuesday, August 25, 2009, 00:45
Over 200 professors from IIT-Bombay
on Monday went on mass leave demanding pay parity with government scientists.
more videos >>


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Superfund Reauthorization
Committee members heard testimony from administration officials and others concerning reauthorization and modification through S.1285 of… read more
Committee members heard testimony from administration officials and others concerning reauthorization and modification through S.1285 of the Superfund program, through which corporations convicted of violating environmental regulations pay for cleaning up the sites. The legislation would attempt to speed up the clean up of hazardous waste sites in several ways. This was the first in a series of two hearings. close
People in this video
- Browner, Carol M. Administrator Environmental Protection Agency
- Florini, Karen Attorney Environmental Defense Fund
- Goodman, Sherri Wasserman Undersecretary Department of Defense->Environment
- Grumbly, Thomas Undersecretary Department of Energy
- Hall, Douglas Assistant Secretary Department of Commerce->Oceans and Atmosphere
- Laws, Elliott Assistant Administrator Environmental Protection Agency->Office of Solid Waste
- Schiffer, Lois Assistant Attorney General Department of Justice->Environment and Natural Resources
- Spisak, John Representative Superfund Reform '95
- Wilson, Lawrence Representative Chemical Manufacturers Association
Hosting Organization
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