Journal articles on the topic 'Nuclear run-off analysis; Termination'

We have studied transcription elongation and termination in the human c-myc gene. Transcription of c-myc gene sequences with purified mammalian RNA polymerase II revealed several sites of transcription termination and pausing in the vicinity of the exon 1-intron 1 junction. This region previously has been shown to block transcription elongation in vivo by nuclear run-on analysis (D. Bentley and M. Groudine, Nature [London] 321:702-706, 1986). These sites were recognized by purified RNA polymerase II, and we therefore designated them intrinsic sites of termination and pausing. Two of these sites cause termination of RNA polymerase III transcription as well. RNA polymerase II terminated transcription in a cluster of seven consecutive T residues in the nontranscribed strand and paused during transcription at three additional sites in this region. The intrinsic sites of transcription termination and pausing described here correspond closely to the 3' ends of transcripts synthesized in Xenopus oocytes injected with plasmids containing the c-myc termination region (D. Bentley and M. Groudine, Cell 53:245-256, 1988). This correspondence suggests that the intrinsic recognition of these termination and pause sites by purified RNA polymerase II may play a role in the transcription elongation block observed in vivo.

We have studied transcription elongation and termination in the human c-myc gene. Transcription of c-myc gene sequences with purified mammalian RNA polymerase II revealed several sites of transcription termination and pausing in the vicinity of the exon 1-intron 1 junction. This region previously has been shown to block transcription elongation in vivo by nuclear run-on analysis (D. Bentley and M. Groudine, Nature [London] 321:702-706, 1986). These sites were recognized by purified RNA polymerase II, and we therefore designated them intrinsic sites of termination and pausing. Two of these sites cause termination of RNA polymerase III transcription as well. RNA polymerase II terminated transcription in a cluster of seven consecutive T residues in the nontranscribed strand and paused during transcription at three additional sites in this region. The intrinsic sites of transcription termination and pausing described here correspond closely to the 3' ends of transcripts synthesized in Xenopus oocytes injected with plasmids containing the c-myc termination region (D. Bentley and M. Groudine, Cell 53:245-256, 1988). This correspondence suggests that the intrinsic recognition of these termination and pause sites by purified RNA polymerase II may play a role in the transcription elongation block observed in vivo.

ABSTRACTHerpes simplex virus 1 (HSV-1) transcription is mediated by cellular RNA polymerase II (Pol II). Recent studies investigating how Pol II transcription of host genes is altered after HSV-1 are conflicting. Chromatin immunoprecipitation sequencing (ChIP-seq) studies suggest that Pol II is almost completely removed from host genes at 4 h postinfection (hpi), while 4-thiouridine (4SU) labeling experiments show that host transcription termination is extended at 7 hpi, implying that a significant amount of Pol II remains associated with host genes in infected cells. To address this discrepancy, we used precision nuclear run-on analysis (PRO-seq) to determine the location of Pol II to single-base-pair resolution in combination with quantitative reverse transcription-PCR (qRT-PCR) analysis at 3 hpi. HSV-1 decreased Pol II on approximately two-thirds of cellular genes but increased Pol II on others. For more than 85% of genes for which transcriptional termination could be statistically assessed, Pol II was displaced to positions downstream of the normal termination zone, suggesting extensive termination defects. Pol II amounts at the promoter, promoter-proximal pause site, and gene body were also modulated in a gene-specific manner. qRT-PCR of selected RNAs showed that HSV-1-induced extension of the termination zone strongly correlated with decreased RNA and mRNA accumulation. However, HSV-1-induced increases of Pol II occupancy on genes without termination zone extension correlated with increased cytoplasmic mRNA. Functional grouping of genes with increased Pol II occupancy suggested an upregulation of exosome secretion and downregulation of apoptosis, both of which are potentially beneficial to virus production.IMPORTANCEThis study provides a map of RNA polymerase II location on host genes after infection with HSV-1 with greater detail than previous ChIP-seq studies and rectifies discrepancies between ChIP-seq data and 4SU labeling experiments with HSV-1. The data show the effects that a given change in RNA Pol II location on host genes has on the abundance of different RNA types, including nuclear, polyadenylated mRNA and cytoplasmic, polyadenylated mRNA. It gives a clearer understanding of how HSV-1 augments host transcription of some genes to provide an environment favorable to HSV-1 replication.

A block to elongation of transcription has been shown to occur within the first exon of the human and murine c-myc genes. The extent of this block was found to vary with the physiological state of cells, indicating that modulation of the transcriptional block can serve to control the expression of this gene. To determine which sequences are required in cis for the transcriptional block, we generated a series of constructs containing various portions of murine c-myc 5'-flanking and exon 1 sequences. We established populations of HeLa and CV-1 cells stably transfected with these constructs. The transcription start sites were determined by S1 nuclease mapping analysis, and the extent of transcriptional block was measured by nuclear run-on transcription assays. Our results demonstrate that at least two cis-acting elements are necessary for the transcriptional block. A 3' element was found to be located in the region where transcription stopped and showed features reminiscent of some termination sites found in procaryotes. A 5' element was positioned between the P1 and P2 (C. Asselin, A. Nepveu, and K. B. Marcu, Oncogene 4:549-558, 1989). Removal of the more 3' binding site abolished the transcriptional block.

A block to elongation of transcription has been shown to occur within the first exon of the human and murine c-myc genes. The extent of this block was found to vary with the physiological state of cells, indicating that modulation of the transcriptional block can serve to control the expression of this gene. To determine which sequences are required in cis for the transcriptional block, we generated a series of constructs containing various portions of murine c-myc 5'-flanking and exon 1 sequences. We established populations of HeLa and CV-1 cells stably transfected with these constructs. The transcription start sites were determined by S1 nuclease mapping analysis, and the extent of transcriptional block was measured by nuclear run-on transcription assays. Our results demonstrate that at least two cis-acting elements are necessary for the transcriptional block. A 3' element was found to be located in the region where transcription stopped and showed features reminiscent of some termination sites found in procaryotes. A 5' element was positioned between the P1 and P2 (C. Asselin, A. Nepveu, and K. B. Marcu, Oncogene 4:549-558, 1989). Removal of the more 3' binding site abolished the transcriptional block.

Abstract Objectives To evaluate the influence of audio-guided self-hypnosis on claustrophobia in a high-risk cohort undergoing magnetic resonance (MR) imaging. Methods In this prospective observational 2-group study, 55 patients (69% female, mean age 53.6 ± 13.9) used self-hypnosis directly before imaging. Claustrophobia included premature termination, sedation, and coping actions. The claustrophobia questionnaire (CLQ) was completed before self-hypnosis and after MR imaging. Results were compared to a control cohort of 89 patients examined on the same open MR scanner using logistic regression for multivariate analysis. Furthermore, patients were asked about their preferences for future imaging. Results There was significantly fewer claustrophobia in the self-hypnosis group (16%; 9/55), compared with the control group (43%; 38/89; odds ratio .14; p = .001). Self-hypnosis patients also needed less sedation (2% vs 16%; 1/55 vs 14/89; odds ratio .1; p = .008) and non-sedation coping actions (13% vs 28%; 7/55 vs 25/89; odds ratio .3; p = .02). Self-hypnosis did not influence the CLQ results measured before and after MR imaging (p = .79). Self-hypnosis reduced the frequency of claustrophobia in the subgroup of patients above an established CLQ cut-off of .33 from 47% (37/78) to 18% (9/49; p = .002). In the subgroup below the CLQ cut-off of 0.33, there were no significant differences (0% vs 9%, 0/6 vs 1/11; p = 1.0). Most patients (67%; 35/52) preferred self-hypnosis for future MR examinations. Conclusions Self-hypnosis reduced claustrophobia in high-risk patients undergoing imaging in an open MR scanner and might reduce the need for sedation and non-sedation coping actions. Key Points • Forty percent of the patients at high risk for claustrophobia may also experience a claustrophobic event in an open MR scanner. • Self-hypnosis while listening to an audio in the waiting room before the examination may reduce claustrophobic events in over 50% of patients with high risk for claustrophobia. • Self-hypnosis may also reduce the need for sedation and other time-consuming non-sedation coping actions and is preferred by high-risk patients for future examinations.

ABSTRACT Haemophilus somnus undergoes antigenic and structural phase variation in its lipooligosaccharide (LOS). A gene (lob-1) containing repetitive 5′-CAAT-3′ sequences that may, in part, contribute to phase variation was cloned and sequenced (T. J. Inzana et al., Infect. Immun. 65:4675–4681, 1997). We have now identified another putative gene (lob-2A) immediately upstream from lob-1. Lob-2A contained homology to several LOS biosynthesis proteins of the family Pasteurellaceae and the LgtB and LgtE galactosyltransferases of Neisseria meningitidis and N. gonorrhoeae. Unlikelob-1, lob-2A contained 18 to 20 5′-GA-3′ repeats 141 bp upstream of the termination codon as determined by PCR amplification of DNA from individual colonies. Twenty repeats were most common, but when 19 5′-GA-3′ repeats were present a stop codon would occur 1 bp after the last 5′-GA-3′ repeat. A 630-bpSalI-BsgI fragment within lob-2Awas deleted, and a kanamycin resistance (Kmr) gene was inserted into this site to create pCAATΔlob2A. Following electroporation of pCAATΔlob2A into H. somnus 738, several allelic exchange mutants were isolated. The LOS electrophoretic profile of one mutant, strain 738-lob2A1::Km, was altered, and the phase variation rate was reduced but phase variation was not eliminated. A variant with 19 5′-GA-3′ repeats in lob-2Ahad an LOS profile similar to that of 738-lob2A1::Km, suggesting that lob-2A was turned off in this phase. Nanoelectrospray mass spectrometry (nES-MS) and nuclear magnetic resonance spectroscopy showed that 738-lob2A1::Km was deficient in the terminal βGal(1-3)βGlcNAc residue present in parent strain 738. Mutant 738-lob2A1::Km was significantly more sensitive to the bactericidal action of normal bovine serum and was less virulent in mice than was parent strain 738. When H. somnus 129Pt was electrotransformed with shuttle vector pLS88 containing lob-2A, its LOS electrophoretic profile was modified and additional N-acetylhexosamine residues were present, as determined by nES-MS analysis. These results indicated thatlob-2A may be an N-acetylglucosamine transferase involved in LOS biosynthesis and phase variation and that LOS structure is important to H. somnus virulence.

ABSTRACT Genome projects involving Leishmania and other trypanosomatids have revealed that most genes in these organisms are organized into large clusters of genes on the same DNA strand. We have previously shown that transcription of the entire Leishmania major Friedlin (LmjF) chromosome 1 (chr1) initiates bidirectionally between two divergent gene clusters. Here, we analyze transcription of LmjF chr3, which contains two convergent clusters of 67 and 30 genes, separated by a tRNA gene, with a single divergent protein-coding gene located close to the “left” telomere. Nuclear run-on analyses indicate that specific transcription of chr3 initiates bidirectionally between the single subtelomeric gene and the adjacent 67-gene cluster, close to the “right” telomere upstream of the 30-gene cluster, and upstream of the tRNA gene. Transcription on both strands terminates within the tRNA-gene region. Transient-transfection studies support the role of the tRNA-gene region as a transcription terminator for RNA polymerase II (Pol II) and Pol III, and also for Pol I.

The photosynthetic non-sulphur bacterium Rhodospirillum rubrum contains a cluster of five genes encoding the subunits of F1-ATPase [Falk, Hampe & Walker (1985) Biochem. J. 228, 391-407]. Transcription of these genes has been studied by two methods, transcriptional mapping with S1 nuclease and primer extension analysis. Thereby a 5'-end in RNA derived from this region has been demonstrated at a guanine residue 236 bases before the initiation codon of the gene for the delta-subunit, the first in this cluster. DNA sequences on the 5' side of this nucleotide show some similarity to promoters in Escherichia coli, but are not apparently related to sequences upstream of the Rhodopseudomonas blastica atp operon. A 3'-end in RNA derived from this gene cluster has been demonstrated by S1-nuclease mapping. This is found before a run of thymidylate residues in the DNA, on the 3' side of a region of dyad symmetry. In E. coli these features are characteristic of rho-independent transcriptional termination signals. It appears from these studies and from the organization of the genes that the five genes in the atp cluster may be co-transcribed from this promoter and that transcripts terminate at the region of dyad symmetry.

The inducible form of nitric oxide synthase (iNOS) is expressed during inflammation of the intestine and may contribute to tissue injury. We have examined iNOS transcriptional regulation in DLD-1 cells, a human intestinal epithelial line that produces large amounts of nitric oxide and iNOS mRNA in response to a combination of the proinflammatory cytokines interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma). Levels of iNOS mRNA are extremely low in unstimulated DLD-1 cells but increase dramatically after cytokine treatment. Nuclear run-on analyses demonstrated that transcriptional activation, which accounts for a portion of this increase, is dependent on both IL-1 beta and IFN-gamma and requires de novo protein synthesis. Transfection of DLD-1 cells with reporter constructs containing deletions of the iNOS promoter showed that sequences located between 8.7 and 10.7 kb upstream of the transcription initiation site are necessary for cytokine responsiveness. This region contains potential binding sites for several cytokine-induced transcription factors and was shown to function in either orientation when placed upstream of a basal iNOS promoter segment terminating at-1.1 kb. The extremely distal location of the cytokine-responsive region contrasts with the reported positions of elements involved in the regulation of iNOS transcription in other cell types. Our data also suggest that posttranscriptional events could play a significant role in regulating iNOS gene expression in human intestinal epithelia.

Abstract Using the technique of differential cDNA library screening, we have molecularly cloned a gene that is highly expressed in an undifferentiated myeloid multipotent and growth factor-dependent stem cell line (FDCP-Mix) and that downregulates as these cells are induced to differentiate along monocytic, granulocytic, and erythroid cell lineages. Sequence analysis of this gene has shown homology with a previously cloned gene, cytotoxic cell protease 1 (CCP1 or Granzyme ‘B’), that has been shown to be expressed only in thymocytes, activated T cells, a mast cell line, and peritoneal exudate leukocytes. In situ hybridization, Northern blot analysis, and nuclear run-off assay has confirmed that expression of CCP1 is restricted to the phenotypically primitive multipotent undifferentiated. FDCP-Mix cells that are undergoing self-renewal in the presence of growth factors such as interleukin-3.

Using the technique of differential cDNA library screening, we have molecularly cloned a gene that is highly expressed in an undifferentiated myeloid multipotent and growth factor-dependent stem cell line (FDCP-Mix) and that downregulates as these cells are induced to differentiate along monocytic, granulocytic, and erythroid cell lineages. Sequence analysis of this gene has shown homology with a previously cloned gene, cytotoxic cell protease 1 (CCP1 or Granzyme ‘B’), that has been shown to be expressed only in thymocytes, activated T cells, a mast cell line, and peritoneal exudate leukocytes. In situ hybridization, Northern blot analysis, and nuclear run-off assay has confirmed that expression of CCP1 is restricted to the phenotypically primitive multipotent undifferentiated. FDCP-Mix cells that are undergoing self-renewal in the presence of growth factors such as interleukin-3.

As more human retinas affected with genetic or immune-based diseases become available for morphological analysis, it is important to identify immunocytochemical markers for specific subtypes of retinal neurons. In this study, we have focused on bipolar cell markers in central retina. We have done single and double labeling using several antisera previously utilized in macaque monkey or human retinal studies and two new antisera (1) to correlate combinations of antisera labeling with morphological types of bipolar cells in human retina, and (2) to compare human labeling patterns with those in monkey retina. Human bipolar cells showed a wide range of labeling patterns with at least ten different bipolar cell types identified from their anatomy and marker content. Many bipolar cell bodies in the outer part of the inner nuclear layer contained combinations of protein kinase C alpha (PKCα), Islet-1, glycine, and Goα. Bipolar cells labeled with these markers had axons terminating in the inner half of the inner plexiform layer (IPL), consistent with ON bipolar cells. Bipolar cell bodies adjacent to the amacrine cells and with axons in the outer half of the IPL contained combinations of recoverin, glutamate transporter-1, and PKCβ, or CD15 and calbindin. Bipolar cells labeled with these markers were presumed OFF bipolar cells. Calcium-binding protein 5 (CaB5) labeled both putative ON and OFF bipolar cells. Using this cell labeling as a criteria, most cell bodies close to the horizontal cells were ON bipolar cells and almost all bipolar cells adjacent to the amacrine cells were OFF with a band in the middle 2–3 cell bodies thick containing intermixed ON and OFF bipolar cells. Differences were found between human and monkey bipolar cell types labeled by calbindin, CaB5, and CD15. Two new types were identified. One was morphologically similar to the DB3, but labeled for CD15 and CaB5. The other had a calbindin-labeled cell body adjacent to the horizontal cell bodies, but did not contain any accepted ON markers. These results support the use of macaque monkey retina as a model for human, but caution against the assumption that all labeling patterns are identical in the two primates.

The expression of endothelin-1 (ET-1) and endothelial constitutive nitric oxide synthase (ecNOS) was assessed in two independent in vitro models: asynchronously differentially proliferating cultures and wounded endothelial cell monolayers. Northern blot analysis of RNA isolated from preconfluent, confluent, and postconfluent cells revealed a fourfold rise in ET-1 mRNA transcripts, whereas levels of ecNOS mRNA transcripts were reduced twofold in proliferating cells. Nuclear run-off analysis demonstrated that increased steady-state ET-1 mRNA content in proliferating cells was mediated, in part, by increased gene transcription. In contrast, ecNOS transcription rates in proliferating cells were not decreased compared with quiescent nonproliferating cells, indicating that mRNA destabilization mediated the decreased ecNOS mRNA levels in proliferating endothelium. Concordant changes in protein expression were documented for both ET-1 and ecNOS. In injured endothelial cell monolayers, in situ cRNA hybridization demonstrated increased mRNA transcript levels for ET-1 in growth fronts of injured endothelial monolayers. These data are taken to indicate that expression of ET-1 and ecNOS is reciprocally regulated in two different models of endothelial cell proliferation.

Nerve growth factor (NGF) leads to neuronal differentiation of PC12 cells and promotes their survival in serum-free medium. Past studies have shown that purine analogues block some of the effects of NGF but not others and thus that they can be used to dissect the mechanistic pathways of its action. In the present work we used 2-aminopurine (2-AP) and 6-thioguanine (6-TG) to examine whether NGF causes activation of primary response genes through a single signaling pathway or via multiple pathways. Northern blot analysis and nuclear run-off transcription assays were used to assess the activation of c-fos, c-jun, TIS1, TIS8, and TIS11 after exposure of PC12 cells to NGF in the presence or absence of 2-AP and 6-TG. Our findings indicate that NGF appears to employ at least three distinct pathways to induce early genes in PC12 cells. This suggests that the NGF signaling mechanism diverges at an early point after interaction of NGF with its receptor.

This study examined the regulation of murine beta 3-receptor mRNA and determined whether the recently described mRNA splice variants are differentially regulated by agents that alter total beta 3-receptor mRNA levels. In vivo treatment of mice with the beta 3-receptor agonist BRL-26830 reduced total beta 3-transcripts by 64% in white adipose tissue but did not alter the mRNA splicing pattern. Further analysis in cultured 3T3-F442A adipocytes showed that isoproterenol, dexamethasone, or phorbol 12-myristate 13-acetate also greatly reduced beta 3-receptor mRNA levels without selectively altering poly-U-containing transcripts. Blockade of transcription with actinomycin D produced a rapid loss of beta 3-receptor mRNA, which was prevented by blockade of mRNA translation with cycloheximide. However, neither actinomycin D nor cycloheximide altered the splicing pattern of beta 3-receptor mRNA. Analysis of transcription rate by nuclear run-off assay indicated that 8-bromoadenosine 3',5'-cyclic monophosphate and phorbol 12-myristate 13-acetate reduce beta 3-receptor gene transcription and that suppression of transcription is sufficient to account for the reduction in beta 3-receptor mRNA levels by these agents.

Abstract. A 2600 yr long composite palaeoflood record is reconstructed from high-resolution delta plain sediments of the Hasli–Aare floodplain on the northern slope of the Swiss Alps. Natural proxies compiled from sedimentary, geochemical and geomorphological data were calibrated by textual and factual sources and instrumental data. No fewer than 12 of the 14 historically recorded extreme events between 1480 and the termination of the Hasli–Aare Correction in 1875 were also identified by coarse-grained flood layers, log(Zr/Ti) peaks and Factor 1 anomalies. Geomorphological, historical and instrumental data provide evidence for flood damage intensities and discharge estimations of severe and catastrophic historical floods. Spectral analysis of the geochemical and documentary flood series and several climate proxies (TSI, δ18O, tree-rings, NAO, SNAO) identify similar periodicities of around 60, 80, 100, 120 and 200 years during the last millennia, indicating the influence of the North Atlantic circulation and solar forcing on alpine flood dynamics. The composite floodplain record illustrates that periods of organic soil formation and deposition of phyllosilicates (from the medium high catchment area) match those of Total Solar Irradiance maxima, suggesting reduced flood activity during warmer climate pulses. Aggradation of clusters of flood layers with increased contribution of siliciclasts from the highest catchment area (plutonic bedrock) (e.g., 1300–1350, 1420–1480, 1550–1620, 1650–1720 and 1811–1851 cal yr AD) occurred predominantly during periods with reduced solar irradiance, lower δ18O anomalies, cooler summer temperatures and phases of drier spring climate in the Alps. Increased water storage by glaciers, snow cover and snow patches susceptible to melting processes associated with rainfall episodes and abrupt rises in temperature substantially increased surface run-off on slopes and discharges of alpine rivers. This interpretation is in agreement with the findings that the severe and catastrophic historical floods in the Aare since 1670 occurred mostly during positive SNAO pulses after years or even decades dominated by negative SNAO and cooler annual temperatures.

✓ The role of N-myc, c-src, and major histocompatibility complex (MHC, H-2 in the mouse) class I antigen gene expressions in dimethyl sulfoxide (DMSO)-induced differentiation and intracerebral tumorigenicity was examined using a mouse MNB85 neuroblastoma cell line. A fluorescence-activated cell sorter disclosed cell-surface MHC enhancement by DMSO, causing an increase in cytotoxic T-lymphocyte sensitivity. Southern blot analysis verified a single copy of the proto-oncogenes and MHC deoxyribonucleic acids in both untreated and DMSO-treated MNB85 cells. Northern blot analysis indicated that DMSO treatment induced a decrease in N-myc and an increase in c-src and MHC messenger ribonucleic acids. Nuclear run-off transcription assay revealed down-regulation of N-myc at a posttranscriptional level, contrasted with primary up-regulation of c-src at a transcriptional level. Immunoprecipitation after treatment with enzyme endo-beta-N-acetyl-glycoseamidase H proved that the terminal glycosylation of MHC heavy-chain gene products normally occurs in the Golgi apparatus of MNB85 cells. Intracerebral tumorigenicity assay showed that cells highly MHC-expressed by DMSO were less tumorigenic than untreated cells in association with DMSO-augmented cytotoxic T-lymphocyte susceptibility. These results suggest that proto-oncogenes may be linked to cellular differentiation, while cell-surface MHC gene expression influences intracerebral immunosurveillance.

We have used the Tpr-Met oncogene as a model to examine signaling pathways of growth factors and tyrosine kinase oncogenes that can increase parathyroid hormone-related peptide (PTHRP) production. PTHRP production in Tpr-Met transfected cells, when assessed by Northern blot analysis and radioimmunoassay, was increased four- to eightfold. Treatment of these cells with the transcriptional inhibitor actinomycin D and nuclear run-off assays showed that the major cause of increased PTHRP mRNA was enhanced gene transcription. To analyze the intracellular signaling molecules involved in PTHRP production, stable cell lines expressing a Tyr489 Phe mutant of the Tpr-Met oncoprotein were examined. The mutant fails to activate phosphatidylinositol (PI)-3 kinase or associate with the Grb-2 adaptor protein and caused a significant reduction in PTHRP production. Treatment of wild-type Tpr-Met transfected cells with wortmannin, a PI-3 kinase inhibitor, had no effect on PTHRP production; however, treatment of these cells with lovastatin, an inhibitor of p21ran isoprenylation, significantly reduced PTHRP expression. These results show that PTHRP is a downsteam target of the Tpr-Met oncogene and indicate that the PTHRP stimulating activity is mediated via the Ras signaling pathway.

Platelet-derived growth factor (PDGF) has been implicated in the process of mesangial cell (MC) proliferation in vitro and in vivo. To investigate early changes in gene expression that couple biochemical events with changes in phenotype in PDGF-stimulated cultured MC, we studied expression of the early growth response gene 1 (Egr-1), a member of the family of immediate early genes. Our findings show that protein tyrosine phosphorylation is required for induction of Egr-1 mRNA and proliferation by PDGF in MC. Nuclear run-off assays show that Egr-1 induction occurs at the transcriptional level. An 11.3-fold increase in Egr-1 transcription rate was observed as early as 5 min after PDGF stimulation of MC. Promoter deletion analysis revealed that the region critical for Egr-1 inducibility by PDGF contains serum response element (SRE) consensus sequences. Sequential deletion of the Egr-1 SREs led to a stepwise drop in promoter activity, suggesting that PDGF induces Egr-1 transcription through SREs in the Egr-1 promoter region. Interestingly, electrophoretic mobility shift assays, with an Egr-1 SRE as probe, demonstrate that protein-SRE complexes of differing size undergo modest quantitative changes following PDGF stimulation. These data in MC suggest that the upstream SREs mediate the transcriptional induction of Egr-1 by PDGF.

Off-pump coronary artery bypass (OPCAB) requires heart manipulation during exposure of the lateral and posterior walls of the heart, which may cause hemodynamic instability, mainly through right ventricular dysfunction. A coaxial atrial cannula connected to a minicentrifugal pump was developed to bypass the right heart. This study was designed to test the hemocompatibility of this pump ongoing for 6 h. In five calves (bodyweight, 70.3± 4.2 kg), the pump was inserted and set to its maximal motor speed of 7000 rpm. Blood samples were taken for blood gas analyses, hematology and chemistry on an hourly basis. ANOVA was used for statistical analysis. During the 6-h run, hematocrit and red blood cell count were stable ( p= 0.77 and 0.87, respectively). Platelet count was not significantly altered ( p= 0.55). LDH was stable ( p= 0.61) and plasma free hemoglobin remained below 100 mg/l throughout the experiment. Adequate tissue perfusion was maintained as reflected by the stable mixed venous oxygen saturation (baseline, 72.5± 2%, and 6 h, 65.6± 3.4%) and no defect of any pump system was detected during this 6-h testing. This right heart minipump appears to have a minimal impact on red cells and platelets when set at its maximal speed for 6 h, underlining the hematological safety of the system.

Abstract The mechanisms that regulate the mRNA levels of interleukin-5 (IL-5) were compared with those regulating the mRNA levels of two other coordinately expressed lymphokines in the murine T lymphoma EL4.23. Our results indicate that IL-5 mRNA levels are independently regulated from those of IL-2 and granulocyte-macrophage colony-stimulating factor (GM- CSF) mRNAs. The induction of IL-5 mRNA by phorbol 12-myristate 13- acetate (PMA) stimulation was found to be cyclosporin A-resistant, in contrast to the induction of IL-2 and GM-CSF mRNAs. Although the three lymphokine mRNAs were not detected in unstimulated cells by Northern blot analysis, the GM-CSF gene was found by nuclear run-off analysis to be constitutively transcribed. However, the IL-2 and IL-5 genes were transcriptionally inactive in the absence of PMA stimulation. The induction of IL-5 mRNA by PMA stimulation primarily involved increased transcriptional activity. In contrast, GM-CSF mRNA induction predominantly involved enhanced mRNA stability. Both transcriptional and mRNA stabilization mechanisms appeared to regulate IL-2 mRNA induction. The activation of IL-2 and IL-5 gene transcription was dependent on de novo protein synthesis. Cellular treatment with cycloheximide enhanced IL-2 gene transcription once activation was initiated, implicating the involvement of a labile repressor(s). Furthermore, IL-5 mRNA was more stable than IL-2 and GM-CSF mRNAs. These latter two species were stabilized by cycloheximide, suggesting that a labile mechanism may regulate their degradation.

The mechanisms that regulate the mRNA levels of interleukin-5 (IL-5) were compared with those regulating the mRNA levels of two other coordinately expressed lymphokines in the murine T lymphoma EL4.23. Our results indicate that IL-5 mRNA levels are independently regulated from those of IL-2 and granulocyte-macrophage colony-stimulating factor (GM- CSF) mRNAs. The induction of IL-5 mRNA by phorbol 12-myristate 13- acetate (PMA) stimulation was found to be cyclosporin A-resistant, in contrast to the induction of IL-2 and GM-CSF mRNAs. Although the three lymphokine mRNAs were not detected in unstimulated cells by Northern blot analysis, the GM-CSF gene was found by nuclear run-off analysis to be constitutively transcribed. However, the IL-2 and IL-5 genes were transcriptionally inactive in the absence of PMA stimulation. The induction of IL-5 mRNA by PMA stimulation primarily involved increased transcriptional activity. In contrast, GM-CSF mRNA induction predominantly involved enhanced mRNA stability. Both transcriptional and mRNA stabilization mechanisms appeared to regulate IL-2 mRNA induction. The activation of IL-2 and IL-5 gene transcription was dependent on de novo protein synthesis. Cellular treatment with cycloheximide enhanced IL-2 gene transcription once activation was initiated, implicating the involvement of a labile repressor(s). Furthermore, IL-5 mRNA was more stable than IL-2 and GM-CSF mRNAs. These latter two species were stabilized by cycloheximide, suggesting that a labile mechanism may regulate their degradation.

Abstract In this study we have examined the expression of a heat-shock protein (HSP) 70 gene in normal human peripheral blood leukocytes. Northern blot analysis showed that appreciable levels of hsp70 mRNA are present in monocytes and granulocytes, whereas transcript levels were barely detectable or absent in lymphocytes. Monocytes functionally activated by bacterial lipopolysaccharide (LPS) showed an early (15 minutes) increase of hsp70 transcripts that was shown, by actinomycin D blocking and nuclear run-off experiments, to be dependent on transcriptional activation of the gene. LPS did not appreciably affect the hsp70 mRNA half-life. Monocytes exposed to inactivated streptococci, phorbol-12- myristate-13-acetate, and tumor necrosis factor showed augmented levels of hsp70 transcripts, whereas interferon-gamma and monocyte, granulocyte, and granulocyte-monocyte colony-stimulating factors had no effect. Adherence to plastic augmented hsp70 expression in monocytes. S1 protection analysis indicated that the gene expressed in monocytes is indeed a heat-inducible member of the hsp70 gene family rather than a constitutively expressed heat-shock cognate gene. Western blot analysis showed that a heat-inducible HSP72 was present in monocytes and, at augmented levels, in LPS-treated monocytes. LPS-activated monocytes were more resistant to heat shock than unstimulated cells. These data indicate that a heat-inducible hsp70 gene can be efficiently expressed in myelomonocytic cells at physiologic temperatures. Expression of hsp70 genes in monocytes suggests a possible role of heat- inducible genes in the differentiation and/or functional activation of terminally differentiated nonproliferating elements of the myelomonocytic lineage.

In this study we have examined the expression of a heat-shock protein (HSP) 70 gene in normal human peripheral blood leukocytes. Northern blot analysis showed that appreciable levels of hsp70 mRNA are present in monocytes and granulocytes, whereas transcript levels were barely detectable or absent in lymphocytes. Monocytes functionally activated by bacterial lipopolysaccharide (LPS) showed an early (15 minutes) increase of hsp70 transcripts that was shown, by actinomycin D blocking and nuclear run-off experiments, to be dependent on transcriptional activation of the gene. LPS did not appreciably affect the hsp70 mRNA half-life. Monocytes exposed to inactivated streptococci, phorbol-12- myristate-13-acetate, and tumor necrosis factor showed augmented levels of hsp70 transcripts, whereas interferon-gamma and monocyte, granulocyte, and granulocyte-monocyte colony-stimulating factors had no effect. Adherence to plastic augmented hsp70 expression in monocytes. S1 protection analysis indicated that the gene expressed in monocytes is indeed a heat-inducible member of the hsp70 gene family rather than a constitutively expressed heat-shock cognate gene. Western blot analysis showed that a heat-inducible HSP72 was present in monocytes and, at augmented levels, in LPS-treated monocytes. LPS-activated monocytes were more resistant to heat shock than unstimulated cells. These data indicate that a heat-inducible hsp70 gene can be efficiently expressed in myelomonocytic cells at physiologic temperatures. Expression of hsp70 genes in monocytes suggests a possible role of heat- inducible genes in the differentiation and/or functional activation of terminally differentiated nonproliferating elements of the myelomonocytic lineage.

Introduction Heparin-bonded expanded polytetrafluoroethylene grafts (Propaten, WL Gore, Flagstaff, AZ, USA) have been shown to have superior patency compared to standard prosthetic grafts in leg bypass. This study analyzed the outcomes of Propaten grafts with distal anastomotic patch versus autogenous saphenous vein grafts in tibial artery bypass. Methods A retrospective analysis of prospective collected data was performed during a recent 15-year period. Sixty-two Propaten bypass grafts with distal anastomotic patch (Propaten group) were compared with 46 saphenous vein graft (vein group). Pertinent clinical variables including graft patency and limb salvage were analyzed. Results Both groups had similar clinical risk factors, bypass indications, and target vessel for tibial artery anastomoses. Decreased trends of operative time (196 ± 34 min vs. 287 ± 65 min, p = 0.07) and length of hospital stay (5.2 ± 2.3 days vs. 7.5 ± 3.6, p = 0.08) were noted in the Propaten group compared to the vein group. Similar primary patency rates were noted at four years between the Propaten and vein groups (85%, 71%, 64%, and 57%, vs. 87%, 78%, 67%, and 61% respectively; p = 0.97). Both groups had comparable secondary patency rates yearly in four years (the Propaten group: 84%, 76%, 74%, and 67%, respectively; the vein group: 88%, 79%, 76%, and 72%, respectively; p = 0.94). The limb salvage rates were equivalent between the Propaten and vein group at four years (84% vs. 92%, p = 0.89). Multivariate analysis showed active tobacco usage and poor run-off score as predictors for graft occlusion. Conclusions Propaten grafts with distal anastomotic patch have similar clinical outcomes compared to the saphenous vein graft in tibial artery bypass. Our data support the use of Propaten graft with distal anastomotic patch as a viable conduit of choice in patients undergoing tibial artery bypass.

Pyrazole, cobalt and phenobarbital increase the activity of coumarin 7-hydroxylase (COH) in mouse liver. To study the mechanism of this increase, we measured the expression of the cytochrome P-450 2a-4/5 (Cyp2a-4/5) complex, which mediates testosterone 15 alpha-hydroxylase and COH activities, as a function of dose and time after the treatment of C57BL/6 (B6) and DBA/2 (D2) male mice with the inducers. COH activity and Cyp2a-4/5 steady-state mRNA levels were increased in both strains in response to the inducers. No marked effect occurred with testosterone 15 alpha-hydroxylase or activities associated with Cyp1a-1 or Cyp2e-1. A 2-7-fold increase in response to the inducers was seen in the amount of P-450Coh (cytochrome P-450 isoenzyme catalysing coumarin 7-hydroxylation) protein in Western immunoblots. PCR amplification of a 1 kb region in Cyp2a-4/5-mRNA-derived cDNA, followed by cutting at the diagnostic PstI site, showed that most of the steady-state mRNA consisted of Cyp2a-5, which is also the form most affected by pyrazole. Nuclear run-off analysis revealed no increase in the transcription rate of Cyp2a-4/5 after pyrazole or cobalt treatment, whereas a 2-3-fold increase occurred after phenobarbital pretreatment in B6 mice. Together with previous reports [Aida & Negishi (1991) Biochemistry 30, 8041-8045], the current data suggest that both pyrazole and cobalt increase COH catalytic activity by affecting Cyp2a-5 by post-transcriptional mechanisms in mice.

The role of inorganic metals and metalloporphyrins in the induction of mRNAs for haem oxygenase and heat-shock protein 70 (hsp70), the two heat-shock proteins, was examined in human HepG2 and Hep3B hepatoma cells. SnCl2, but not Sn-protoporphyrin, was found to be a potent inducer of both haem oxygenase and hsp70 mRNAs. In contrast, CoCl2, ZnCl2 and FeCl2 caused little induction of haem oxygenase and hsp70 mRNAs, whereas the porphyrin complexes of these metals strongly induced haem oxygenase mRNA, without influencing the level of hsp70 mRNA. The induction process was largely transcriptional, as judged by the inhibition of induction by actinomycin D, but not by cycloheximide, and by increased transcription demonstrated by nuclear run-off analysis. Since CoCl2 is a potent inducer of haem oxygenase in vivo in animals, the possibility of the biosynthesis of Co-protoporphyrin was examined in human hepatoma cells by incubating them with CoCl2 and protoporphyrin, or delta-aminolaevulinate (ALA), the precursor of protoporphyrin. Both types of treatment led to a potent induction of haem oxygenase mRNA. Co-protoporphyrin formation was also spectrally demonstrated in cells incubated with the metal and ALA. The results of this study indicate that certain metals, e.g. SnCl2, may directly induce haem oxygenase mRNA, whereas with other elements, incorporation of the metal into the porphyrin macrocycle is necessary for induction. Therefore CoCl2, like haemin, may activate the haem oxygenase gene via a haem-responsive transcription factor, whereas SnCl2 may exert its effect via a metal-responsive transcription factor.

We have characterized tissue type plasminogen activator (tPA) promoter elements and nuclear factors required for follicle-stimulating hormone (FSH)-induced transcription of the rat tPA gene in granulosa cells and constitutive expression of the gene in the rat neuroblastoma cell line B103. Run-on transcription analysis of isolated nuclei revealed that B103 cells transcribe the tPA gene at a high and constitutive level, while FSH was found to induce tPA gene transcription in a rapid and transient manner in granulosa cells. The maximal FSH-induced transcription rate was obtained after 20 min and was similar in the absence or presence of the protein synthesis inhibitor cycloheximide. However, in the presence of cycloheximide, tPA transcription was not turned off but continued at a high rate for several hours. This phenomenon may at least partly explain the earlier finding that tPA mRNA is superinduced by FSH in the presence of cycloheximide. DNase I footprinting analysis of the first 621 bp of the tPA promoter revealed a total of six regions that interact with nuclear factors from B103 and granulosa cells. Deletion of the promoter region from positions -269 to -621, a region that includes the two most-upstream footprints, had no effect on constitutive or FSH-induced transcription in transient expression experiments. Nuclear extracts from both granulosa cells and B103 cells showed strong binding to a consensus cyclic AMP-responsive element (CRE) at positions -178 to -185 and a neighboring binding site for nuclear factor 1 (NF1) at positions -145 to -158. The factors binding to these two regions were identified as members of the CRE-binding protein and NF1 families of transcription factors, respectively. Footprints were also obtained over two GC boxes at positions -64 to -71 and -41 to -49. These footprints were more pronounced with nuclear extracts from B103 cells than with extracts from untreated or FSH-treated granulosa cells, but gel shift assays indicate that similar amounts of two distinct factors bind to the two GC boxes in both cell types. Transfection experiments using promoter constructs with inactivated promoter elements indicate that both the CRE and NF1 sites contribute to the FSH responsiveness of the rat tPA gene in granulosa cells, while only the NF1 site is important for constitutive expression in B103 cells. The two GC boxes were found to be necessary both for constitutive expression in B103 cells and for FSH-induced expression in granulosa cells, and inactivation of both GC boxes essentially eliminated the tPA promoter activity in both cell types.

We have characterized tissue type plasminogen activator (tPA) promoter elements and nuclear factors required for follicle-stimulating hormone (FSH)-induced transcription of the rat tPA gene in granulosa cells and constitutive expression of the gene in the rat neuroblastoma cell line B103. Run-on transcription analysis of isolated nuclei revealed that B103 cells transcribe the tPA gene at a high and constitutive level, while FSH was found to induce tPA gene transcription in a rapid and transient manner in granulosa cells. The maximal FSH-induced transcription rate was obtained after 20 min and was similar in the absence or presence of the protein synthesis inhibitor cycloheximide. However, in the presence of cycloheximide, tPA transcription was not turned off but continued at a high rate for several hours. This phenomenon may at least partly explain the earlier finding that tPA mRNA is superinduced by FSH in the presence of cycloheximide. DNase I footprinting analysis of the first 621 bp of the tPA promoter revealed a total of six regions that interact with nuclear factors from B103 and granulosa cells. Deletion of the promoter region from positions -269 to -621, a region that includes the two most-upstream footprints, had no effect on constitutive or FSH-induced transcription in transient expression experiments. Nuclear extracts from both granulosa cells and B103 cells showed strong binding to a consensus cyclic AMP-responsive element (CRE) at positions -178 to -185 and a neighboring binding site for nuclear factor 1 (NF1) at positions -145 to -158. The factors binding to these two regions were identified as members of the CRE-binding protein and NF1 families of transcription factors, respectively. Footprints were also obtained over two GC boxes at positions -64 to -71 and -41 to -49. These footprints were more pronounced with nuclear extracts from B103 cells than with extracts from untreated or FSH-treated granulosa cells, but gel shift assays indicate that similar amounts of two distinct factors bind to the two GC boxes in both cell types. Transfection experiments using promoter constructs with inactivated promoter elements indicate that both the CRE and NF1 sites contribute to the FSH responsiveness of the rat tPA gene in granulosa cells, while only the NF1 site is important for constitutive expression in B103 cells. The two GC boxes were found to be necessary both for constitutive expression in B103 cells and for FSH-induced expression in granulosa cells, and inactivation of both GC boxes essentially eliminated the tPA promoter activity in both cell types.

Abstract 2D-Nuclear magnetic resonance (NMR) spectra are used in the (structural) analysis of small molecules. In contrast to 1D-NMR spectra, 2D-NMR spectra correlate the chemical shifts of 1H and 13C at the same time. A spectrum consists of several peaks in a two--dimensional space. The most important information of a peak is the location of its center, which captures the bonding relationships of hydrogen and carbon atoms. A spectrum contains much information about the chemical structure of a product, but in most cases the structure cannot be read off in a simple and straightforward manner. Structure elucidation involves a considerable amount (manual) efforts. Using high-field NMR spectrometers, many 2D-NMR spectra can be recorded in short time. So the common situation is that a lab or company has a repository of 2D-NMR spectra, partially annotated with the structural information. For the remaining spectra the structure in unknown. In case two research labs are collaborating, the repositories will be merged and annotations shared. We reduce that problem to the task of finding duplicates in a given set of 2D-NMR spectra. Therefore, we propose a simple but robust definition of 2D-NMR duplicates, which allows for small measurement errors. We give a quadratic algorithm for the problem, which can be implemented in SQL. Further, we analyze a more abstract class of heuristics, which are based on selecting particular peaks. Such a heuristic works as a filter step on the pairs of possible duplicates and allows false positives. We compare all methods with respect to their run time. Finally we discuss the effectiveness of the duplicate definition on real data.

Abstract We have examined the signal transduction pathways leading to the expression of the interleukin-1 beta (IL-1 beta) gene in human myeloid leukemia cells lines. Two cell lines representing different stages of differentiation were used (HL-60, promyelocytic, and THP-1, mature monocytic). In accordance with previous studies, it was observed that a protein kinase C (PKC) activator, phorbol myristate acetate (PMA), was a sufficient stimulus for induction of the IL-1 beta messenger RNA (mRNA) expression and IL-1 beta protein production in both of these cell lines. A structural analog of cyclic adenosine monophosphate (dbcAMP) or agents elevating the endogenous cAMP levels (prostaglandin E2, forskolin) were not alone able to induce IL-1 beta expression, but they strongly enhanced the PMA-induced IL-1 beta production and IL-1 beta mRNA accumulation. Nuclear run off analysis showed that this elevation in IL-1 beta mRNA levels was due to an increased rate of transcription. If dbcAMP was added 6 hours before PMA to the cultures, no enhancement in the IL-1 beta production was seen, implying that for this enhancing effect both of these signals must be present simultaneously. PKC inhibitor, H7, also blocked effectively the PMA plus dbcAMP induced IL-1 beta production, while the protein kinase A (PKA) inhibitor, HA1004, had no effect, suggesting that PKA activation is not involved in the mechanism of action of cAMP in this case. Collectively, the present findings show that cAMP-dependent signals can have a positive regulatory effect on the PKC-dependent activation of the IL-1 beta gene in cells derived from different stages of myeloid differentiation.

We have examined the signal transduction pathways leading to the expression of the interleukin-1 beta (IL-1 beta) gene in human myeloid leukemia cells lines. Two cell lines representing different stages of differentiation were used (HL-60, promyelocytic, and THP-1, mature monocytic). In accordance with previous studies, it was observed that a protein kinase C (PKC) activator, phorbol myristate acetate (PMA), was a sufficient stimulus for induction of the IL-1 beta messenger RNA (mRNA) expression and IL-1 beta protein production in both of these cell lines. A structural analog of cyclic adenosine monophosphate (dbcAMP) or agents elevating the endogenous cAMP levels (prostaglandin E2, forskolin) were not alone able to induce IL-1 beta expression, but they strongly enhanced the PMA-induced IL-1 beta production and IL-1 beta mRNA accumulation. Nuclear run off analysis showed that this elevation in IL-1 beta mRNA levels was due to an increased rate of transcription. If dbcAMP was added 6 hours before PMA to the cultures, no enhancement in the IL-1 beta production was seen, implying that for this enhancing effect both of these signals must be present simultaneously. PKC inhibitor, H7, also blocked effectively the PMA plus dbcAMP induced IL-1 beta production, while the protein kinase A (PKA) inhibitor, HA1004, had no effect, suggesting that PKA activation is not involved in the mechanism of action of cAMP in this case. Collectively, the present findings show that cAMP-dependent signals can have a positive regulatory effect on the PKC-dependent activation of the IL-1 beta gene in cells derived from different stages of myeloid differentiation.

Abstract Recent studies indicate that a purified protein, activin A, belongs to the transforming growth factor beta (TGF-beta) superfamily. Similar to TGF-beta, activin A can have different biologic activities, depending on the target tissues. We used recombinant activin A to demonstrate a possible regulatory role of this protein in modulating human erythroid differentiation in the human erythroid cell line, K562. Using genomic probes containing the second exon of alpha, beta, gamma, and epsilon globins, relative abundance of various types of globin transcripts in untreated and activin-treated K562 cells was examined with S1 nuclease analysis. Despite considerable homology amongst various globin sequences, these globin probes were highly specific for their unique mRNA species in the analyses. It was shown that the abundance of specific globin probe fragments for gamma and epsilon globins (209 nucleotides) as well as alpha (180 nucleotides), which were protected from S1 digestion, increased many fold in K562 cells treated with activin A. In contrast, there were no specific transcripts of beta globin detected in either the control or activin-treated cells. The increases in the level of fetal and embryonic beta-like and alpha globin transcripts also confirmed earlier studies of Northern and slot- blot analyses using globin cDNA as probes. In addition, nuclear run-off transcription assay using isolated nuclei indicated that most of the increase in the globin transcripts after activin treatment could be attributed to the stimulation of transcription rate for globin genes. Transient transfection assays also provide evidence that activin A significantly stimulated transcriptional activity of an epsilon globin promoter in K562, but not in the nonerythroid Chinese hamster ovary cells. Therefore, it was concluded that activin A exerts its effects on globin gene expression at the level of transcription in erythroid cells.

Recent studies indicate that a purified protein, activin A, belongs to the transforming growth factor beta (TGF-beta) superfamily. Similar to TGF-beta, activin A can have different biologic activities, depending on the target tissues. We used recombinant activin A to demonstrate a possible regulatory role of this protein in modulating human erythroid differentiation in the human erythroid cell line, K562. Using genomic probes containing the second exon of alpha, beta, gamma, and epsilon globins, relative abundance of various types of globin transcripts in untreated and activin-treated K562 cells was examined with S1 nuclease analysis. Despite considerable homology amongst various globin sequences, these globin probes were highly specific for their unique mRNA species in the analyses. It was shown that the abundance of specific globin probe fragments for gamma and epsilon globins (209 nucleotides) as well as alpha (180 nucleotides), which were protected from S1 digestion, increased many fold in K562 cells treated with activin A. In contrast, there were no specific transcripts of beta globin detected in either the control or activin-treated cells. The increases in the level of fetal and embryonic beta-like and alpha globin transcripts also confirmed earlier studies of Northern and slot- blot analyses using globin cDNA as probes. In addition, nuclear run-off transcription assay using isolated nuclei indicated that most of the increase in the globin transcripts after activin treatment could be attributed to the stimulation of transcription rate for globin genes. Transient transfection assays also provide evidence that activin A significantly stimulated transcriptional activity of an epsilon globin promoter in K562, but not in the nonerythroid Chinese hamster ovary cells. Therefore, it was concluded that activin A exerts its effects on globin gene expression at the level of transcription in erythroid cells.

Short-term (less than 2 h) glucose stimulation of isolated pancreatic islets specifically increases the biosynthesis of proinsulin and its converting enzymes PC2 and PC3 at the translation level. To determine whether gene expression of PC2 and PC3 was also regulated by longer-term (more than 6 h) glucose stimulation along with that of preproinsulin, studies were performed with the βTC3 insulin-producing cell line. By Northern blot analysis, glucose maintained PC2 and PC3 mRNA levels in parallel with those of preproinsulin. After 48 h, mRNA levels of preproinsulin, PC2 and PC3 were, respectively, 2.9 (P < 0.05), 3.0 (P < 0.005) and 5.3 (P < 0.001) times greater in the presence of glucose than in βTC3 cells cultured in the absence of glucose. Glucose-regulated PC2 and PC3 gene expression, like that of preproinsulin, was maximal at glucose concentrations above 5.5 mM. Studies of mRNA stability showed that the half-lives of PC2 (9 h) and PC3 (5 h) mRNA were much shorter than that of preproinsulin mRNA (over 24 h), but little effect of glucose on stability of these mRNAs was observed. Nuclear run-off analysis indicated that transcription of preproinsulin, PC2 and PC3 was modestly induced after 1 h exposure to 16.7 mM glucose. Therefore preproinsulin, PC2 and PC3 mRNA levels in βTC3 cells were most probably maintained at the level of gene transcription. In contrast, elevation of cyclic AMP by forskolin had no effect on mRNA levels or gene transcription of preproinsulin, PC2 and PC3, despite a cyclic-AMP-induced phosphorylation of the cyclic AMP response element binding protein that correlated with a marked increase in cJun and cFos gene transcription in the same β-cells. These results suggest that preproinsulin, PC2 and PC3 gene transcription can be specifically glucose-regulated in a mechanism that is unlikely to involve a key role for cyclic AMP. The co-ordinate increase in PC2 and PC3 mRNA levels with that of preproinsulin mRNA in response to chronic glucose represents a long-term means of catering for an increased demand on proinsulin conversion.

Abstract Bromodomain and extra-terminal (BET) inhibitors may be efficacious for treatment of acute myeloid leukemia because they attenuate the expression of critical oncogenes including MYC and BCL2. These BET inhibitors (BETi) disrupt the transcriptional elongation process by displacing BET family members BRD2,3, and 4 off of chromatin, and causing RNA polymerase promoter-proximal pausing. We used precision nuclear run-on transcription sequencing (PROseq) to directly measure the effects of INCB054329, a potent BETi, on RNA polymerase II pausing and elongation. We found dramatic reductions on the elongation of key oncogenes such as MYC and BCL2 within 15 min of adding the drug. These effects became more significant over time, eventually affecting nearly two thousand genes. By four hours after drug addition, we found a loss of ribosomal gene expression and a loss of mitochondrial gene expression that is characteristic of genes regulated by MYC, suggesting that these were secondary to turning off MYC expression. When we examined the potential of the BETi INCB054329 for therapeutic efficacy in AML using Alamar Blue assays, which measure cellular redox potential, we noted marked growth inhibition of AML cell lines. However, growth assays and measurements of apoptosis using Annexin V staining found that BETi induced minimal apoptosis and cells were largely cytostatic. BrdU incorporation assays showed that INCB054329 caused the cells to accumulate in the G0/G1 phase of the cell cycle. Metabolic studies indicated that INCB054329 treatment for 48 hours caused disruption of mitochondrial respiration rate and severely reduced glycolytic capacity. Taken together, the growth inhibition, cell cycle arrest and reduced metabolic rate suggests that INCB054329 promoted quiescence in AML cells, but that this is reversible, consistent with the clinical experience of single-agent treatment of hematologic malignancies with BETi. MLL fusion proteins enhance transcription by stimulating RNA polymerase elongation, suggesting INCB054329may provide a therapeutic option to reverse this effect. However, the cell cycle arrest suggested that a second compound may be needed to trigger cell death. We first performed in vivo studies with INCB054329 using a systemic AML xenograft model of MV4-11 cells that express MLL-AF4. Engrafted NSGS mice received INCB054329 in 3 different doses (vehicle vs 10, 30 and 75mg/kg q.d) daily. During treatment, the kinetics of MV-4-11 expansion was monitored via flow cytometry for the detection of human AML in the blood. At approximately 4 weeks after transplant, the vehicle mice became moribund, and all experimental groups were sacrificed for analysis of chimerism. Significant decreases in leukemic expansion were evident in the bone marrow (vehicle vs75mg/kg, p<.001) and spleen (vehicle vs. 75mg/kg, p <.001) of treated mice. As BETi decreases expression of BCL2, we posited that BH3 directed therapy with the BCL-2 inhibitor venetoclax (VEN) could be enhanced by INCB054329. In vitro, we found that the combination of INCB054329 and VEN resulted in significant growth inhibition and apoptosis of treated AML cells. This finding prompted us to test the combination of INCB054329 with VEN in vivo. Mice engrafted with human AML cells received INCB054329 (50mg/kg q.d), VEN (25mg/kg q.d) or the combination. Four weeks after transplant, analyses by flow cytometric measurement of human CD45 of combination treated mice revealed significant decreases of AML cells in the bone marrow (vehicle vs. BRDi/VEN p = 0.004) and spleen (vehicle vs.BRDi/VEN, p = 0.001). Further studies are underway to test this combination in both VEN sensitive and resistant AML primary xenograftmodels. These preliminary data suggest that INCB054329 may serve as a non-cytotoxic priming agent for BH3 directed therapy, and the combination of INCB054329 +VEN may provide a potent therapy in a variety of genetically distinct subtypes of AML. Disclosures Stubbs: Incyte: Employment. Liu:Incyte: Employment. Rathmell:Calithera: Research Funding. Hiebert:Incyte: Research Funding. Savona:Boehringer Ingelheim: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Background The usefulness of exercise Doppler echocardiography (EDE) is uncertain in identifying patients with pulmonary hypertension (PH). Recently the cut off value for the definition of PH was changed and was set at the level of mean pulmonary arterial pressure (PAP) &gt;20 mmHg, measured by right heart catheterization (RHC). Purpose We examined whether EDE can unmask the presence of PH in patients with systemic sclerosis (SSc) whose baseline echocardiographic assessment for PH is non-diagnostic. Methods Forty-one patients with SSc (2 men and 39 women; mean age 61.2 ± 10 years) underwent treadmill symptom-limited EDE using a modified Bruce protocol. Tricuspid regurgitation velocity (TRV) was recorded within 60 seconds after the termination of the test. Inclusion criteria comprised: preserved left (ejection fraction &gt;50%), and right ventricular function (tricuspid annulus plane systolic excursion &gt;15 mm), lack of left side moderate or severe valvulopathy, presence of mild or moderate TR, satisfactory exercise tolerance and baseline TRV in the range of 2.6-3.0 m/s. All patients had RHC within 48 hours after EDE. Results In 5 cases the quality of post-exercise TRV was poor and further analysis was confined to 36 patients. RHC confirmed the presence of PH (mean PAP &gt;20 mmHg) in 14 cases (38.9%). All patients had pulmonary capillary wedge pressure &lt;15 mmHg. The increase in TRV from baseline to post-exercise was 1.25 ± 0.6 m/s. Ten patients developed post-exercise TRV ≥3.7 m/s of whom in 8 RHC validated PH. Post-exercise TRV was positively correlated with mean PAP obtained by RHC (r = 0,652, p &lt; 0.001). A cut-off value of post-exercise TRV ≥3.7 m/s had a sensitivity of 57.1%, a specificity of 90% and a diagnostic accuracy of 77.8% in detecting PH validated by RHC. Conclusions EDE has a moderate diagnostic accuracy for the identification of PH in patients with SSc whose baseline echocardiographic measurements for PH lie in the gray zone.

Abstract Aims Incidental detection of left atrial appendage (LAA) filling defects is a common finding on multi-detector computed tomography in aortic stenosis patients under evaluation for transcatheter aortic valve implantation (TAVI). We aimed to investigate the incidence of LAA filling defects before TAVI and its impact on clinical outcomes. Methods and results In a prospective registry, LAA filling defects were retrospectively evaluated and categorized into one of four sub-types: thrombus-like, heterogeneous, horizontal, and Hounsfield Unit (HU)-run-off. The primary endpoint was the composite of cardiovascular death or disabling stroke up to 1-year follow-up. Among 1621 patients undergoing TAVI between August 2007 and June 2018, LAA filling defects were present in 177 patients (11%), and categorized as thrombus-like in 22 (1.4%), heterogeneous in 37 (2.3%), horizontal in 80 (4.9%), and HU-run-off in 38 (2.4%). Compared to patients with normal LAA filling, patients with LAA filling defects had greater prevalence of atrial fibrillation (84.7% vs. 26.4%, P &lt; 0.001) and history of cerebrovascular events (16.4% vs. 10.9%, P = 0.045). The primary endpoint occurred in 131 patients (9.2%) with normal LAA filling and in 36 patients (21.2%) with LAA filling defects (P &lt; 0.001). Subgroup analysis suggested that the risk of disabling stroke was greatest in the thrombus-like pattern (23.0%), followed by the HU-run-off (8.0%), the heterogeneous (6.2%), and the horizontal pattern (1.2%). Conclusion LAA filling defects were observed in 11% of aortic stenosis patients undergoing TAVI and associated with an increased risk of cardiovascular death and disabling stroke up to 1 year following TAVI. Trial Registration https://www.clinicaltrials.gov. NCT01368250.

Abstract FASERν is a newly proposed detector whose main mission is to detect the neutrino flux from the collision of the proton beams at the ATLAS Interaction Point (IP) during the run III of the LHC in 2022–2024. We show that this detector can also test certain beyond standard model scenarios, especially the ones in which the neutrino interaction with matter fields can produce new unstable particles decaying back into charged leptons. Models of this kind are motivated by the MiniBooNE anomaly. We show that, if the new physics involves multi-muon production by neutrinos scattering off matter fields, including the neutrino flux interactions in the rock before the detector in the analysis (i.e., accounting for the through-going muon pairs) can significantly increase the effective detector mass and its sensitivity to new physics. We propose a concrete model that can give rise to such a multi-muon signal.

Exhaust gas recirculation (EGR) is extensively employed in diesel combustion engines to achieve nitrogen oxides emission targets. The EGR is often cooled in order to increase the effectiveness of the strategy, even though this leads to a further undesired impact on particulate matter and hydrocarbons. Experimental tests were carried out on a diesel engine at a dynamometer rig under steady-state speed and load working conditions that were considered relevant for the New European Driving Cycle. Two different shell and tube-type EGR coolers were compared, in terms of the pressure and temperature of the exhaust and intake lines, to evaluate thermal effectiveness and induced pumping losses. All the relevant engine parameters were acquired along EGR trade-off curves, in order to perform a detailed comparison of the two coolers. The effect of intake throttling operation on increasing the EGR ratio was also investigated. A purposely designed aging procedure was run in order to characterize the deterioration of the thermal effectiveness and verify whether clogging of the EGR cooler occurred. The EGR mass flow-rate dependence on the pressure and temperature upstream of the turbine as well as the pressure downstream of the EGR control valve was modeled by means of the expression for convergent nozzles. The restricted flow-area at the valve-seat passage and the discharge coefficient were accurately determined as functions of the valve lift.

Introduction: The aim of this study was to identify factors associated with primary graft patency 1 year following open lower limb revascularisation (LLR) at a tertiary referral vascular service. Methods: A retrospective analysis of patients undergoing infra-inguinal bypass surgery between January 2016 and May 2017 at a tertiary vascular centre (St Mary’s Hospital, London) was performed. Data regarding patient demographics, comorbidities, type of operation and post-operative anti-thrombotic strategy were collected. Quality of run-off score was assessed from pre-operative imaging. Results: Seventy-seven cases were included in the analysis. Overall, the primary patency rate at 1-year was 63.6% ( n = 49/77) and the secondary patency rate was 67.5% ( n = 52/77). Independent variables with statistically significant inferior patency rates at 1-year were (1) bypasses with below knee targets (p = 0.0096), (2) chronic limb threatening ischaemia indication (p = 0.038), (3) previous ipsilateral revascularisation (p < 0.001) and (4) absence of hypertension history (p = 0.041). There was also a trend towards significance for American Society of Anesthesiologists (ASA) grade (p = 0.06). Independent variables with log-rank test p values of <0.1 were included in a Cox proportional hazards model. The only variable with a statistically significant impact on primary patency rates was previous open or endovascular ipsilateral revascularisation (HR 2.44 (1.04–5.7), p = 0.04). Conclusion: At 1-year follow-up, previous ipsilateral revascularisation was the most significant factor in affecting patency rates. Patients in this subgroup should therefore be deemed high-risk, which should be reflected in the informed consent and peri-operative management.

Objective The aim of the study was to evaluate the outcome of first line hybrid treatment in patients with chronic limb threatening ischemia (CLTI) and to evaluate possible predictors of primary patency (PP) loss and limb clinical improvement (LCI). Methods This was a retrospective non-randomized study. All patients underwent one-stage hybrid common femoral artery (CFA) endarterectomy combined with an inflow and/or outflow endovascular revascularization procedure. Demographic, clinical, and lesion characteristics for each patient were reported. Primary patency analysis was performed using Kaplan–Meier life tables, and univariate and multivariate analysis was used to assess possible predictors of PP loss and clinical improvement. Results Complete data were obtained from 132 patients. Patients were divided into two groups according to their Rutherford’s category (RC), group 1 (Rutherford 4) and group 2 (Rutherford 5 and 6). Technical success was 98%. The overall surgical peri-operative complication rate was 8%. At a mean follow-up of 32 ± 23 months, the rate of major adverse limb events (MALE) was 30%; only the rate of major amputation between two groups was significant statistically different ( p = .006). Group 1 had significantly lower amputation rate at 36 months ( p = .01). The presence of high iliac peripheral artery calcium scoring system (PACCS) grade (HR 9.43, 95% CI 2.40–36.9, p = .001), the poor run-off of leg vessels (HR 0.15, 95% CI 0.02–0.92, p = .04), and undergoing CFA endarterectomy combined with outflow endovascular revascularization procedure (HR 4.25, 95% CI 1.07–16.89, p = .04) were independent predictors of PP loss, while severe iliac artery stenosis (OR 0.09, 95% CI 0.02–0.32, p = <.001) and the presence of pre-operative patent leg vessels (OR 8.03, 95% CI 2015–29.95, p = .002) were the significant independent predictors of LCI. Conclusion The use of hybrid first line approach in patients with CLTI is a safe and feasible technique. From the analysis of the current study, it is clear that any effort should be made to achieve as many patency leg vessels as possible in order to obtain better and longer lasting clinical outcomes.

AbstractThis paper describes an aerodynamic design optimization of a highly loaded compressor stator blade using parameterized free-form deformation (FFD). The optimization methodology presented utilizes a B-spline-based FFD control volume to map the blade from the object space to the parametric space via transformation operations in order to perturb the blade surface. Coupled with a multi-objective genetic algorithm (MOGA) and a Gaussian process-based response surface method (RSM), a fully automated iterative loop was used to run the optimization on a fitted correlation function. A weighted average reduction of 6.1% and 36.9% in total pressure loss and exit whirl angle was achieved, showing a better compromise of objective functions with smoother blade shape than other results obtained in the open literature. Data mining of the Pareto set of optimums revealed four groups of data interactions of which some design variables were found to have skewed scatter relationship with objective functions and can be redefined for further improvement of performance. Analysis of the flow field showed that the thinning of the blade at midspan and reduction in camber distribution were responsible for the elimination of the focal-type separation vortex by redirecting the secondary flow in an axially forward direction toward the midspan and near the hub endwall downstream. Furthermore, the reduction in exit whirl angle especially at the shroud was due to the mild bow shape which generated radial forces on the flow field thereby reducing the flow diffusion rate at the suction surface corner. This effect substantially delayed or eliminated the formation of corner separation at design and off-design operating conditions. Parameterized FFD was found to have superior benefits of smooth surface generation with low number of design variables while maintaining a good compromise between objective functions when coupled with a genetic algorithm.

Unconventional energy sources have become increasingly important to the global energy mix. These include coal seam gas, shale gas and shale oil. The unconventional gas industry was pioneered in the United States and embraced following the first oil shock in 1973 (Rogers). As has been the case with many global resources (Hiscock), many of the same companies that worked in the USA carried their experience in this industry to early Australian explorations. Recently the USA has secured significant energy security with the development of unconventional energy deposits such as the Marcellus shale gas and the Bakken shale oil (Dobb; McGraw). But this has not come without environmental impact, including contamination to underground water supply (Osborn, Vengosh, Warner, Jackson) and potential greenhouse gas contributions (Howarth, Santoro, Ingraffea; McKenna). The environmental impact of unconventional gas extraction has raised serious public concern about the introduction and growth of the industry in Australia. In coal rich Australia coal seam gas is currently the major source of unconventional gas. Large gas deposits have been found in prime agricultural land along eastern Australia, such as the Liverpool Plains in New South Wales and the Darling Downs in Queensland. Competing land-uses and a series of environmental incidents from the coal seam gas industry have warranted major protest from a coalition of environmentalists and farmers (Berry; McLeish). Conflict between energy companies wanting development and environmentalists warning precaution is an easy script to cast for frontline media coverage. But historical perspectives are often missing in these contemporary debates. While coal mining and natural gas have often received “boosting” historical coverage (Diamond; Wilkinson), and although historical themes of “development” and “rushes” remain predominant when observing the span of the industry (AGA; Blainey), the history of unconventional gas, particularly the history of its environmental impact, has been little studied. Few people are aware, for example, that the first shale gas exploratory well was completed in late 2010 in the Cooper Basin in Central Australia (Molan) and is considered as a “new” frontier in Australian unconventional gas. Moreover many people are unaware that the first coal seam gas wells were completed in 1976 in Queensland. The first four wells offer an important moment for reflection in light of the industry’s recent move into Central Australia. By locating and analysing the first four coal seam gas wells, this essay identifies the roots of the unconventional gas industry in Australia and explores the early environmental impact of these wells. By analysing exploration reports that have been placed online by the Queensland Department of Natural Resources and Mines through the lens of environmental history, the dominant developmental narrative of this industry can also be scrutinised. These narratives often place more significance on economic and national benefits while displacing the environmental and social impacts of the industry (Connor, Higginbotham, Freeman, Albrecht; Duus; McEachern; Trigger). This essay therefore seeks to bring an environmental insight into early unconventional gas mining in Australia. As the author, I am concerned that nearly four decades on and it seems that no one has heeded the warning gleaned from these early wells and early exploration reports, as gas exploration in Australia continues under little scrutiny. Arrival The first four unconventional gas wells in Australia appear at the beginning of the industry world-wide (Schraufnagel, McBane, and Kuuskraa; McClanahan). The wells were explored by Houston Oils and Minerals—a company that entered the Australian mining scene by sharing a mining prospect with International Australian Energy Company (Wiltshire). The International Australian Energy Company was owned by Black Giant Oil Company in the US, which in turn was owned by International Royalty and Oil Company also based in the US. The Texan oilman Robert Kanton held a sixteen percent share in the latter. Kanton had an idea that the Mimosa Syncline in the south-eastern Bowen Basin was a gas trap waiting to be exploited. To test the theory he needed capital. Kanton presented the idea to Houston Oil and Minerals which had the financial backing to take the risk. Shotover No. 1 was drilled by Houston Oil and Minerals thirty miles south-east of the coal mining town of Blackwater. By late August 1975 it was drilled to 2,717 metres, discovered to have little gas, spudded, and, after a spend of $610,000, abandoned. The data from the Shotover well showed that the porosity of the rocks in the area was not a trap, and the Mimosa Syncline was therefore downgraded as a possible hydrocarbon location. There was, however, a small amount of gas found in the coal seams (Benbow 16). The well had passed through the huge coal seams of both the Bowen and Surat basins—important basins for the future of both the coal and gas industries. Mining Concepts In 1975, while Houston Oil and Minerals was drilling the Shotover well, US Steel and the US Bureau of Mines used hydraulic fracture, a technique already used in the petroleum industry, to drill vertical surface wells to drain gas from a coal seam (Methane Drainage Taskforce 102). They were able to remove gas from the coal seam before it was mined and sold enough to make a profit. With the well data from the Shotover well in Australia compiled, Houston returned to the US to research the possibility of harvesting methane in Australia. As the company saw it, methane drainage was “a novel exploitation concept” and the methane in the Bowen Basin was an “enormous hydrocarbon resource” (Wiltshire 7). The Shotover well passed through a section of the German Creek Coal measures and this became their next target. In September 1976 the Shotover well was re-opened and plugged at 1499 meters to become Australia’s first exploratory unconventional gas well. By the end of the month the rig was released and gas production tested. At one point an employee on the drilling operation observed a gas flame “the size of a 44 gal drum” (HOMA, “Shotover # 1” 9). But apart from the brief show, no gas flowed. And yet, Houston Oil and Minerals was not deterred, as they had already taken out other leases for further prospecting (Wiltshire 4). Only a week after the Shotover well had failed, Houston moved the methane search south-east to an area five miles north of the Moura township. Houston Oil and Minerals had researched the coal exploration seismic surveys of the area that were conducted in 1969, 1972, and 1973 to choose the location. Over the next two months in late 1976, two new wells—Kinma No.1 and Carra No.1—were drilled within a mile from each other and completed as gas wells. Houston Oil and Minerals also purchased the old oil exploration well Moura No. 1 from the Queensland Government and completed it as a suspended gas well. The company must have mined the Department of Mines archive to find Moura No.1, as the previous exploration report from 1969 noted methane given off from the coal seams (Sell). By December 1976 Houston Oil and Minerals had three gas wells in the vicinity of each other and by early 1977 testing had occurred. The results were disappointing with minimal gas flow at Kinma and Carra, but Moura showed a little more promise. Here, the drillers were able to convert their Fairbanks-Morse engine driving the pump from an engine run on LPG to one run on methane produced from the well (Porter, “Moura # 1”). Drink This? Although there was not much gas to find in the test production phase, there was a lot of water. The exploration reports produced by the company are incomplete (indeed no report was available for the Shotover well), but the information available shows that a large amount of water was extracted before gas started to flow (Porter, “Carra # 1”; Porter, “Moura # 1”; Porter, “Kinma # 1”). As Porter’s reports outline, prior to gas flowing, the water produced at Carra, Kinma and Moura totalled 37,600 litres, 11,900 and 2,900 respectively. It should be noted that the method used to test the amount of water was not continuous and these amounts were not the full amount of water produced; also, upon gas coming to the surface some of the wells continued to produce water. In short, before any gas flowed at the first unconventional gas wells in Australia at least 50,000 litres of water were taken from underground. Results show that the water was not ready to drink (Mathers, “Moura # 1”; Mathers, “Appendix 1”; HOMA, “Miscellaneous Pages” 21-24). The water had total dissolved solids (minerals) well over the average set by the authorities (WHO; Apps Laboratories; NHMRC; QDAFF). The well at Kinma recorded the highest levels, almost two and a half times the unacceptable standard. On average the water from the Moura well was of reasonable standard, possibly because some water was extracted from the well when it was originally sunk in 1969; but the water from Kinma and Carra was very poor quality, not good enough for crops, stock or to be let run into creeks. The biggest issue was the sodium concentration; all wells had very high salt levels. Kinma and Carra were four and two times the maximum standard respectively. In short, there was a substantial amount of poor quality water produced from drilling and testing the three wells. Fracking Australia Hydraulic fracturing is an artificial process that can encourage more gas to flow to the surface (McGraw; Fischetti; Senate). Prior to the testing phase at the Moura field, well data was sent to the Chemical Research and Development Department at Halliburton in Oklahoma, to examine the ability to fracture the coal and shale in the Australian wells. Halliburton was the founding father of hydraulic fracture. In Oklahoma on 17 March 1949, operating under an exclusive license from Standard Oil, this company conducted the first ever hydraulic fracture of an oil well (Montgomery and Smith). To come up with a program of hydraulic fracturing for the Australian field, Halliburton went back to the laboratory. They bonded together small slabs of coal and shale similar to Australian samples, drilled one-inch holes into the sample, then pressurised the holes and completed a “hydro-frac” in miniature. “These samples were difficult to prepare,” they wrote in their report to Houston Oil and Minerals (HOMA, “Miscellaneous Pages” 10). Their program for fracturing was informed by a field of science that had been evolving since the first hydraulic fracture but had rapidly progressed since the first oil shock. Halliburton’s laboratory test had confirmed that the model of Perkins and Kern developed for widths of hydraulic fracture—in an article that defined the field—should also apply to Australian coals (Perkins and Kern). By late January 1977 Halliburton had issued Houston Oil and Minerals with a program of hydraulic fracture to use on the central Queensland wells. On the final page of their report they warned: “There are many unknowns in a vertical fracture design procedure” (HOMA, “Miscellaneous Pages” 17). In July 1977, Moura No. 1 became the first coal seam gas well hydraulically fractured in Australia. The exploration report states: “During July 1977 the well was killed with 1% KCL solution and the tubing and packer were pulled from the well … and pumping commenced” (Porter 2-3). The use of the word “kill” is interesting—potassium chloride (KCl) is the third and final drug administered in the lethal injection of humans on death row in the USA. Potassium chloride was used to minimise the effect on parts of the coal seam that were water-sensitive and was the recommended solution prior to adding other chemicals (Montgomery and Smith 28); but a word such as “kill” also implies that the well and the larger environment were alive before fracking commenced (Giblett; Trigger). Pumping recommenced after the fracturing fluid was unloaded. Initially gas supply was very good. It increased from an average estimate of 7,000 cubic feet per day to 30,000, but this only lasted two days before coal and sand started flowing back up to the surface. In effect, the cleats were propped open but the coal did not close and hold onto them which meant coal particles and sand flowed back up the pipe with diminishing amounts of gas (Walters 12). Although there were some interesting results, the program was considered a failure. In April 1978, Houston Oil and Minerals finally abandoned the methane concept. Following the failure, they reflected on the possibilities for a coal seam gas industry given the gas prices in Queensland: “Methane drainage wells appear to offer no economic potential” (Wooldridge 2). At the wells they let the tubing drop into the hole, put a fifteen foot cement plug at the top of the hole, covered it with a steel plate and by their own description restored the area to its “original state” (Wiltshire 8). Houston Oil and Minerals now turned to “conventional targets” which included coal exploration (Wiltshire 7). A Thousand Memories The first four wells show some of the critical environmental issues that were present from the outset of the industry in Australia. The process of hydraulic fracture was not just a failure, but conducted on a science that had never been tested in Australia, was ponderous at best, and by Halliburton’s own admission had “many unknowns”. There was also the role of large multinationals providing “experience” (Briody; Hiscock) and conducting these tests while having limited knowledge of the Australian landscape. Before any gas came to the surface, a large amount of water was produced that was loaded with a mixture of salt and other heavy minerals. The source of water for both the mud drilling of Carra and Kinma, as well as the hydraulic fracture job on Moura, was extracted from Kianga Creek three miles from the site (HOMA, “Carra # 1” 5; HOMA, “Kinma # 1” 5; Porter, “Moura # 1”). No location was listed for the disposal of the water from the wells, including the hydraulic fracture liquid. Considering the poor quality of water, if the water was disposed on site or let drain into a creek, this would have had significant environmental impact. Nobody has yet answered the question of where all this water went. The environmental issues of water extraction, saline water and hydraulic fracture were present at the first four wells. At the first four wells environmental concern was not a priority. The complexity of inter-company relations, as witnessed at the Shotover well, shows there was little time. The re-use of old wells, such as the Moura well, also shows that economic priorities were more important. Even if environmental information was considered important at the time, no one would have had access to it because, as handwritten notes on some of the reports show, many of the reports were “confidential” (Sell). Even though coal mines commenced filing Environmental Impact Statements in the early 1970s, there is no such documentation for gas exploration conducted by Houston Oil and Minerals. A lack of broader awareness for the surrounding environment, from floral and faunal health to the impact on habitat quality, can be gleaned when reading across all the exploration reports. Nearly four decades on and we now have thousands of wells throughout the world. Yet, the challenges of unconventional gas still persist. The implications of the environmental history of the first four wells in Australia for contemporary unconventional gas exploration and development in this country and beyond are significant. Many environmental issues were present from the beginning of the coal seam gas industry in Australia. Owning up to this history would place policy makers and regulators in a position to strengthen current regulation. The industry continues to face the same challenges today as it did at the start of development—including water extraction, hydraulic fracturing and problems associated with drilling through underground aquifers. Looking more broadly at the unconventional gas industry, shale gas has appeared as the next target for energy resources in Australia. Reflecting on the first exploratory shale gas wells drilled in Central Australia, the chief executive of the company responsible for the shale gas wells noted their deliberate decision to locate their activities in semi-desert country away from “an area of prime agricultural land” and conflict with environmentalists (quoted in Molan). Moreover, the journalist Paul Cleary recently complained about the coal seam gas industry polluting Australia’s food-bowl but concluded that the “next frontier” should be in “remote” Central Australia with shale gas (Cleary 195). It appears that preference is to move the industry to the arid centre of Australia, to the ecologically and culturally unique Lake Eyre Basin region (Robin and Smith). 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If one morning I walked on top of the water across the Potomac River, the headline that afternoon would read: "President Can't Swim". —Lyndon B. Johnson Introduction The term “spin” implies manipulating the truth, and this concept, along with “spin doctoring,” is now common in media and public discourse. The prevalence of “spin zones” in American politics is undeniable; media outlets themselves, such as Bill O’Reilly’s “No Spin Zone” on Fox News, now run segments on the topic. Despite this apparent media certainty about what constitutes “spin” there is a lack of conceptual clarity regarding the term among those who study media and politics. This article will draw on previous literature to identify two competing yet overlapping spin zones in American politics: the media’s spin zone and the President’s spin zone. Highlighting examples from the two most recent American presidential election campaigns, the article will evaluate the interplay of these zones and the consequences for future campaigns. Spin Zones In the United States, the press and the President are engaged in a struggle over providing information. Ever since the Watergate Scandal, the media is increasingly expected to be a “watchdog” that informs citizens and keeps the Executive accountable (Coronel 13) The President, conversely, may attempt to use the power of his position to set the discursive agenda or frame the political debate in his favor. Furthermore, with the rise of multi-media access and information provision, the lines between the spin doctoring of the Executive and the media have become even more blurred. Because of the complexities of these overlapping spin zones, many scholars disagree on how to define and/or precisely measure these effects. The following section briefly describes the ‘spin zone’ tools of agenda setting, framing, and priming, and then considers the example of a candidate who failed to prime his negative evaluation and a President who primes his image and successfully counterattacks his negative evaluation. The literature recognises two separate, yet interrelated zones that are integral to understanding these media/presidential relations: what we term the presidential spin zone and the media spin zone. The interplay between these zones comes together around three key concepts—agenda setting, framing, and priming. A key difficulty for scholars is that the President, his electoral challengers, and the press are engaged in agenda setting, framing and priming, sometimes simultaneously. Agenda setting is a broad concept and refers to focusing on certain issues to the exclusion of others. Framing is defined as the decision by the news media to “emphasise certain elements to define the ‘public’s belief’ about social and political issues” (Van Gorp 488). Other scholars describe priming as “a disproportionate amount of public comments with the hope . . . of causing voters to base their selection among the candidates on [that] issue” (Druckman et al. 1181; see also Druckman “Framing Effects”; Nelson, Clawson and Oxley; Van Gorp). Candidates may also undertake “image priming,” which is proposed by James Druckman et al., as a tool that can be used to counteract negative candidate evaluations (1182–1183). The definition of the media spin zone is, in most instances, synonymous with priming. Defining the presidential spin zone is more complex. Clearly the presidential spin zone involves both the previously-discussed “issue framing abilities of the president” and how he “set[s] the agenda” (Miller and Krosnick 301; see also, Gamson and Modigliano, Baumgardner and Jones; Druckman, “Framing Effects”). Mark Rozell, for instance, found that the Ford and Carter administrations had difficulty controlling the public agenda since many issues were either beyond their control, or because the president and his advisors lacked the strategy or skill to affect media coverage. The Reagan White House however was able to use his “image” to control the media (85–86). Similarly, George W. Bush’s administration was able to implement policies concerning the invasion of Iraq after the 9-11 through “issue framing” scare tactics, which were constantly reinforced by media outlets (Kellner 643). However, the President can also be engaged in priming at any given time. In other words, the President (or candidate) may attempt to prime what the media has already spun about him/her. A problem, of course, is that the President or candidate, in attempting to prime an issue that has already been spun in a sense tacitly admits they have lost the opportunity to set the agenda in the first place. However, this is when he can seize the aforementioned opportunity to use “image priming” to counterattack the media. In the examples that follow we examine whether the President or candidate can use priming to effectively counterattack the media spin zone, with a focus on two political tools that have been historically reserved for the President or candidates, namely, holding the base and wedge issues. Holding the Base and the Media Spin Zone Holding the base has been defined as a way in which candidates or Presidents can use the media to strengthen support among voters who already identify with their political party (Iyengar and McGrady 246). A classic example of this is the 1984 Reagan/Bush re-election campaign, the “The Bear.” This featured a bear in the woods that “some” could “see” and others didn’t “see at all” which was an implicit threat regarding Soviet communism and a reminder that Reagan was tough on foreign policy (“The Bear”). However, the evidence indicates that the media has increasingly begun “holding the base” on its own to facilitate its partisan framing and priming of candidates or Presidents. The Swift Boat Veterans for Truth attack advertisements on 2004 Democratic presidential candidate John Kerry is a key example of a media attempt to “hold the base.” In these advertisements, former “Swift Boat Veterans attack[ed] his [Kerry’s] military record” (Muravchik A17). While this initiative began as a means to collect Republican donations, Shanto Iyengar and Jennifer McGrady maintain that the amount was “trivial” and that the real impact came with “the torrent of news reports across the country” (150). Indeed, Kathleen Jamieson and Joseph Capella found that by August 2004, “viewers of Fox News were more likely than other network viewers to say that candidate John Kerry did not earn his Vietnam medals” (279). Their evaluation of this data demonstrated the power of the media spin zone: “He (Limbaugh) employs intense language, disparaging information and negative framing to distance perceptions of the Democratic candidate from those of the anointed Republican candidate” (Jamieson and Capella 228). The coverage of disputes surrounding Kerry’s military record was augmented by the media’s simultaneous coverage of the threat of terrorism. This priming “in the media continued, reaching a high peak of 55 threat messages in August 2004, a month later 25% of the public was very concerned about another major terrorist attack in the US—two months before the presidential election” (Nacos, Bloch-Elkon and Shapiro 120). Both President Bush and Candidate Kerry acknowledged that their respective win/loss could be attributed in some measure to the press coverage of the “war on terror” (Nacos, Bloch-Elkon and Shapiro 124). While questions loomed about his military experience against the backdrop of the war on terror, Senator Kerry won the first two Presidential debates by significant margins. Alec Gallup and Frank Newport suggested that the Kerry camp had “won the spin contest … to characterize their own candidate as the winner” (406). So, what happened to Kerry? The media spin zone stopped him. The presidential debate wins were 30 September 2004 and 8 October 2004, respectively. Iyengar and McGrady demonstrate that before the debates even began the number of Swift Boat veteran stories primed in the national and international press went from under 100 to over 500 (151). According to Kim Fridkin et al. the media’s spin was a significant factor in the third debate. They found that media coverage concerning Senator Kerry’s response to one question on whether homosexuality was a choice affected citizens’ evaluations of the candidate. In the post debate coverage, the tone “in newspapers, on the Internet, and on television was uniformly negative in its assessment of Senator Kerry’s comments” (Fridkin et al. 30). The impact of this negative framing was sufficiently strong to override positive evaluations of Kerry held by those who watched the debate. In sum, the “perfect storm of media coverage lessened the bounce that Senator Kerry received from the actual debate and led people to develop negative impressions of Kerry a mere three weeks before Election Day” (Fridkin 43). Despite these liabilities, Kerry should have counterattacked the media spin zone. He should have “counterpunched,” as noted by Drew Westen, priming the media that he was “a different kind of Democrat”—“one who knows when it’s time to take off the gloves” (337). Westen’s advice is echoed in Druckman’s call for further research in this area as well as by his own research findings. The media’s framing and priming led to negative evaluations of Kerry, which afforded him the opportunity to prime his “image” in a counterattack, as Druckman suggests (1183). Overcoming the Wedge Issues of the Media Spin Zone President Obama, however, orchestrates a different outcome in dealing with the media spin zone attack against him which centered on a “wedge” or “us verses them” issue. Iyengar and McGrady note that “wedge issues are designed to pit groups against each other, to appeal to voters’ sense of group identity” (145). However, they define wedge issues within the context of presidential spin zones; thus, the candidate or the president would be framing the “us versus them” topic. In this instance, the media framed a wedge issue, the status of President Obama’s citizenship, against him. In this case the birther movement, oft-promoted by conservative radio host Rush Limbaugh, argued that President Obama was not a US citizen. This issue became so prominent that it was soon adopted by the media spin zone. The media framing demanded proof in addition to the short form birth certificate that the President had already released (Wilson 109). For his part, President Obama handled the media spin zone’s wedge issue with great aplomb, responding in a brief statement to the public on 27 April 2011: “We do not have time for this kind of silliness” (Shear). Moreover, he did not alienate the media for framing the birther movement, but he placed the blame implicitly on Donald Trump who had taken up the birther gauntlet thrown down by Rush Limbaugh. It was “clearly Trump” he was priming when he indicated that he did not want to be “distracted by sideshows and carnival barkers” (Shear). Moreover, his strategic focus on “silliness” is an illustration of “image priming”. He did not allow himself to be drawn into the race-baiting or religious controversy that was a component of some of the media talk show discussions. The Washington Post reported after Obama’s speech that the percentage of Americans who questioned his legitimacy to serve as President dropped from 20% to 10%—thus legitimating his choice to address the nation. This result meant that the President responded to an attack from the media spin zone with a counterattack of his own; he effectively counterattacked to prime his image. Interestingly, Stephen Ansolobehare and Iyengar have indirectly demonstrated the efficacy of counterattacks in presidential spin zone situations by evaluating situations where one candidate attacks another and the “victim” of the attack either, does not respond, responds with a positive message or responds with a counterattack (143). They found overwhelming evidence that voters prefer their party’s candidate to counterattack rather than be victimised. Conclusion In this paper we have furthered the call for conceptual clarity in the field by joining Druckman et al. in emphasising the need for more research on “image priming” on the part of candidates and Presidents in the interplay between the press and the presidency. If used properly, image priming seems a viable way for the presidency to counterattack against media framing and priming, but squandered opportunities may irreparably harm candidates. President Obama faced a difficult wedge issue that had undercurrents of both racial and religious tensions, but he deftly avoided those issues and found a way to “use Trump as a foil and present the president as a more serious leader” (Shear). His counterattack against the wedge used by the media spin zone was successful. Senator Kerry, on the other hand, failed to counterattack the media spin zone’s rallying of the base. His silence allowed the media to generate both issue and image frames and priming against him. 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