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Next we gathered ice cubes and began grinding them according to package directions. Before too long the kids were enjoying yummy snow cones. I too tried a snow cone and found them to be delicious. I finished my first thimble sized snow cone and wanted another. Maybe it was the icey deliciousness, maybe it was the stress of the holidays but for some reason I could not get enough of these tiny snow cones. Later, after the kids went to bed I found myself up in the middle of the night grinding ice cubes like a fiend and eating snow cones by the vat. By morning the handle was broken off the snow cones maker, the syrup was gone and there I stood with a red mustache and all lying to the kids: . The shame of it all. But mommy's are magic. Voila- all better. We still eat too many icees, they are just so delicious. Only now its guilt free. Stir constantly until sugar is melted and water just begins to boil. Remove from heat and cool. Spoon or pour over shaved ice. 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Simple Minds - Someone Somewhere In Summertime (4: 3. Simple Minds - Colours Fly And Catherine Wheel (3: 5. Simple Minds - Promised You A Miracle (4: 2. Simple Minds - Big Sleep (5: 0. Simple Minds - Somebody Up There Likes You (5: 0. Simple Minds - New Gold Dream (8. Simple Minds - Glittering Prize (4: 3. Simple Minds - Hunter And The Hunted (5: 5. Simple Minds - King Is White And In The Crowd (7: 0. CD2 Extended Remixes: 0. Simple Minds - Promised You A Miracle (Extended) (4: 5. Simple Minds - Seeing Out The Angel (Instrumental Remix) (6: 3. Simple Minds - Promised You A Miracle (US Remix) (6: 0. Simple Minds - Promised You A Miracle (US Dub) (5: 2. Simple Minds - Promised You A Miracle (US Special Extended Remix) (6: 2. Simple Minds - Glittering Prize (Club Mix) (4: 5. Simple Minds - Glittering Prize (Extended Theme) (4: 5. Simple Minds - Someone Somewhere In Summertime (Extended) (6: 0. Simple Minds - New Gold Dream (8. German 1. 2' Mix) (6: 5. Simple Minds - King Is White And In The Crowd (Instrumental) (8: 4. Simple Minds - New Gold Dream (8. German 1. 2' Remix With Drums) (6: 5. Simple Minds - In Every Heaven (4: 2. CD3 Edits & B- Sides: 0. Simple Minds - Promised You A Miracle (Edit) (4: 0. Simple Minds - Theme For Great Cities (5: 5. Simple Minds - Glittering Prize (Edit) (4: 0. Simple Minds - Glittering Prize (Theme) (4: 0. Simple Minds - Someone Somewhere In Summertime (Edit) (4: 0. Simple Minds - Soundtrack For Every Heaven (4: 5. Simple Minds - New Gold Dream (8. Mix) (4: 4. 6)CD4 Radio Sessions: 0. Simple Minds - Promised You A Miracle (David Jensen Session 1. Simple Minds - In Trance As Mission (David Jensen Session 1. Simple Minds - King Is White And In The Crowd (David Jensen Session 1. Simple Minds - Promised You A Miracle (John Peel Session 1. Simple Minds - Love Song (John Peel Session 1. Simple Minds - Sons And Fascination (John Peel Session 1. Simple Minds - King Is White And In The Crowd (John Peel Session 1. Simple Minds - Someone Somewhere In Summertime (David Jensen Session 1. Simple Minds - Glittering Prize (David Jensen Session 1. Simple Minds - Hunter And The Hunted (David Jensen Session 1. CD5 Alternative Mixes, Roughs And Demos: 0. Simple Minds - Someone Somewhere In Summertime (full duration) (5: 4. Simple Minds - Colours Fly And Catherine Wheel (full duration instrumental) (4: 4. Simple Minds - Promised You A Miracle (remix for album - long) (4: 5. Simple Minds - Big Sleep (instrumental) (5: 1. Simple Minds - In Every Heaven (full duration) (5: 4. Simple Minds - Somebody Up There Likes You (full duration instrumental) (5: 1. Simple Minds - New Gold Dream (8. Simple Minds - Hunter And The Hunted (alternative take) (5: 1. Simple Minds - King Is White And In The Crowd (monitor mix) (7: 1. Simple Minds - In Every Heaven (early version) (4: 3. DOWNLOAD(Buy premium account for maximum speed and resuming ability). Simple Minds - New Gold Dream (8. Super Deluxe Edition) (2. Artist: Simple Minds. Title: New Gold Dream (8. Super Deluxe Edition)Year Of Release: 2. Label: Virgin. Genre: New Wave, Alternative, Pop Rock, Synth Pop. Quality: 3. 20 kbps . Someone Somewhere In Summertime. Colours Fly And Catherine Wheel. Promised You A Miracle. Somebody Up There Likes You. New Gold Dream (8. Hunter And The Hunted. King Is White And In The Crowd. CD 2: Extended Remixes. Promised You A Miracle - . Seeing Out The Angel - nstrumental Remix 1. Inch Single B1. 03. Promised You A Miracle - S Remix 1. Inch Single A1. 04. Promised You A Miracle - S Dub 1. Inch Single B1 FIRST TIME ON CD0. Promised You A Miracle - S Special Extended Remix 1. Inch Promo Single A1 FIRST TIME ON CD0. Glittering Prize - . Glittering Prize - . Someone Somewhere In Summertime - . King Is White And In The Crowd - nstr (Unedited & Restored Mix Originally Released With Touch Tape Mag) FIRST TIME ON CD1. In Every Heaven - Outtake. CD 3: Edits & B- Sides. Promised You A Miracle - . Theme From Great Cities - 7. Inch Single B1. 03. Glittering Prize - . Glittering Prize - (Theme) 7. Inch Single B1 FIRST TIME ON CD0. Someone Somewhere (In Summertime) - 7. Inch Promo Single A1. Soundtrack For Every Heaven - 1. Inch Single B1. 07. Recorded 1. 1th February 1. Promised You A Miracle - BC Session. In Trance As Mission - BC Session. John Peel Session. Recorded 1. 5th February 1. King Is White And In The Crowd - BC Session FIRST TIME ON CD0. Promised You A Miracle - BC Session FIRST TIME ON CD0. Love Song - BC Session FIRST TIME ON CD0. Sons And Fascination - BC Session FIRST TIME ON CD0. King Is White And In The Crowd - BC Session. Kid Jensen Session. Recorded 1. 3th August 1. Someone Somewhere In Summertime - BC Session. Glittering Prize - BC Session. Hunter And The Hunted - BC Session. CD 5: Alternative Mixes, Roughs And Demos. Someone Somewhere In Summertime - . Colours Fly And Catherine Wheel - . Promised You A Miracle - . Big Sleep - nstrumental PREVIOUSLY UNRELEASED0. Somebody Up There Likes You - . New Gold Dream (8. Hunter And The Hunted - . King Is White And In The Crowd - . Disc one features a 2. Andrew Walter at Abbey Road) approved by Charlie Burchill and the second disc gathers 1. CD debut. Various edits and B- sides can be found on the third CD in the set while disc four features previously unreleased BBC John Peel and Kid Jensen radio sessions, recorded in February and August 1. All ten tracks on disc five are previously unreleased; made up of alternative mixes and demos and the icing on the cake is the sixth and final disc which is a DVD, featuring Charlie Burchill and Ronald Prent's 5. DVD- Audio in 2. 00. This mix of the album is a unique 'full duration' mix which is different to the standard version. DVD also includes promo videos and a few Top of the Pops performances. Note, this is a DVD- V unlike the DVD- A/V disc from previous Simple Minds box sets. 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Pivotal role of JNK- dependent FOXO1 activation in downregulation of kallistatin expression by oxidative stress. Abstract. Oxidative stress has been shown to suppress endothelial nitric oxide synthase expression through activation of the transcription factor forkhead box O 1 (FOXO1) in cultured endothelial cells. We previously reported that circulating kallistatin levels are markedly reduced in rats with chronic oxidative organ damage. In this study, we investigated the potential role of oxidative stress in suppression of kallistatin expression via FOXO1 activation. In Dahl salt- sensitive (DSS) rats, we found that high salt intake induced a time- dependent correlation of increased thiobarbituric acid reactive substances (TBARS, an indicator of lipid peroxidation) with reduced serum kallistatin levels. Moreover, salt loading provoked an elevation of in situ aortic superoxide formation in association with reduced kallistatin levels. Expression of kallistatin was identified in cultured endothelial cells by immunocytochemistry and flow cytometry; however, H2. O2 dose- dependently lowered kallistatin m. RNA and protein levels as determined by real- time PCR and Western blot, respectively. Downregulation of kallistatin synthesis by oxidative stress was restored by knockdown of FOXO1 expression with small- interfering RNA. H2. O2 rapidly induced FOXO1 nuclear translocation, but the effect was blocked by c- Jun NH2- terminal kinase (JNK) inhibitor. Physiology, aging, and calorie restriction. NAD-dependent ADP-ribosyltransferase activity. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Export citation (RIS) Email citation. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase Brunet, A.; Sweeney, L. Renal Physiology Published 1 August. Inhibition of JNK by pharmacological inhibitor or small- interfering RNA reversed H2. O2's effect on kallistatin expression in endothelial cells. This study demonstrates that an inverse relationship exists between oxidative stress and kallistatin levels in the circulation and blood vessels and that kallistatin expression is negatively regulated by oxidative stress via JNK- dependent FOXO1 activation in cultured endothelial cells. Forkhead box O 1oxidative stressc- Jun NH2- terminal kinaseblood vesselkallistatin, a plasma protein that belongs to the serine protease inhibitor (serpin) family, is widely expressed in organs such as liver, kidney, and blood vessel (9, 1. Previous studies have demonstrated kallistatin to be a potent anti- inflammatory agent. For example, kallistatin reduced lipopolysaccharide- induced inflammation and lethality in kallistatin transgenic mice (1. 16 FOXO Transcription Factors as Potential Therapeutic Targets in Neuroblastoma Michael J. Ausserlechner 1,3, Judith Hagenbuchner 2,3, Stefan Fuchs 3, Kathrin Geiger 3 and Petra Obexer 2,3 Medical University Innsbruck. A biweekly scientific journal publishing high-quality research in molecular biology and genetics, cancer biology, biochemistry, and related fields. JNK-dependent FOXO1 activation in downregulation of kallistatin. Physiology - Heart and Circulatory Physiology Published 1. FoxO transcription factors in the maintenance of cellular homeostasis during aging Dervis AM Salih and Anne Brunet. Of AMPK in worms increases stress resistance and extends longevity in a FOXO/DAF-16-dependent. This is a PDF file of an unedited manuscript that has been accepted for.Kallistatin administration via gene delivery significantly decreased neutrophil accumulation and joint swelling in a rat model of arthritis (3. Moreover, kallistatin improved cardiac function and diminished oxidative stress, cardiomyocyte apoptosis, and inflammatory cell accumulation after acute myocardial ischemia- reperfusion (1. Kallistatin also reduced myocardial infarct size, inflammation, and ventricular remodeling after myocardial infarction and attenuated salt- induced renal damage, inflammation, and fibrosis in association with reduced oxidative stress and increased nitric oxide (NO) levels (1. Kallistatin is a negative acute phase protein, since its levels are rapidly decreased after endotoxin shock and experimental inflammation (1. Moreover, circulating kallistatin levels are reduced in NO- deficient and spontaneously hypertensive rats as well as in normotensive rats with ischemic cerebral injury and gentamicin- induced kidney damage (7, 8, 1. Increasing evidence indicates that reactive oxygen species (ROS) are the common factors underlying the pathogenesis of hypertension, inflammation, and cardiovascular and renal diseases (1, 3. These combined findings suggest that oxidative stress may play an important role in the suppression of kallistatin synthesis. However, the molecular mechanism underlying negative regulation of kallistatin expression has not been explored. Forkhead box O (FOXO) transcription factors promote a variety of cellular responses, including cell differentiation and stress response by modulating a series of specific target gene expression (1. Accumulating evidence has shown that growth factor- activated protein kinase B (Akt) and stress- activated c- Jun N- terminal kinase (JNK) have opposing effects on FOXO: Akt prevents FOXO nuclear localization and inhibits its activity, whereas JNK increases FOXO activity by promoting its import in the nucleus (4, 1. Thus the survival pathway and the stress pathway appear to be in a tight balance to regulate FOXO activation. FOXO1, the most abundant FOXO isoform in endothelial cells, has been shown to bind to the consensus sequences in endothelial NO synthase (e. NOS) promoter and suppress e. NOS expression in response to oxidative stress (3. Exposing endothelial cells to hypoxia resulted in significant reduction in e. NOS expression (2. Because both kallistatin and e. NOS levels are reduced in rats with salt- induced hypertension (3. FOXO1 may also be involved in the negative regulation of kallistatin expression under oxidative stress. Our present study was designed to determine the role and mechanism of oxidative stress in suppressing kallistatin expression in cultured endothelial cells. MATERIALS AND METHODSAnimal treatments. All procedures complied with the standards for care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Resources, National Academy of Sciences, Bethesda, MD). The protocol for our animal study was approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina. Four- week- old, male Dahl salt- sensitive (DSS) rats (Sprague- Dawley Harlan, Madison, WI) were fed either with a normal salt (0. Na. Cl) or a high- salt (4% Na. Cl) diet for 6 wk. Blood was collected each week for thiobarbituric acid reactive substances (TBARS) and rat kallistatin measurements. On the day of death, serum was collected by cardiac puncture, and kidneys and aortas were removed for histological and biochemical analyses. Cell culture. Human umbilical vein endothelial cells (HUVECs) were acquired from Cambrex Bioscience and cultured in endothelial cell basal medium- 2 supplemented with EGM- 2 single. Quots kit (Lonza, Allendale, NJ). Analyses of kallistatin expression. Kallistatin levels in rat serum and in kidney extracts were determined using an enzyme- linked immunosorbent assay specific for rat kallistatin as previously described (2. Kallistatin expression in rat aorta was detected by immunohistochemistry. Briefly, aortic sections were incubated at 4. Immunohistochemistry was performed using the Vectastain Universal Elite ABC Kit (Vector Laboratories, Burlingame, CA), following the supplied instructions. Expression of human kallistatin in HUVECs was detected by immunocytochemistry using a specific monoclonal antibody (1. For flow cytometric analysis of human kallistatin expression in HUVECs, cells were incubated with 3 . After washing and centrifugation, the cells were resuspended in 0. PBS for immediate acquisition by flow cytometry. Assays for TBARS and superoxide formation. Serum lipid peroxidation, an indicator of oxidative stress, was determined by measuring circulating TBARS levels at 5. Superoxide production was measured in kidney extracts by ferricytochrome c reduction assay using a modified protocol (5). In situ detection of superoxide in rat aorta. Aortic ring segments were immediately placed in optimum- cutting temperature embedding medium at the time of death and stored at . Aortic ring segments were cut into 3. Slides were incubated in a light- protected humidified chamber at 3. Images were obtained with a laser- scanning confocal microscope equipped with a krypton/argon laser. Laser settings were identical for acquisition of images for all slides. Fluorescence was detected with a 5. RNA extraction and quantitative PCR. Total RNA was isolated from cultured cells with Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. Total RNA was reverse- transcribed using the High Capacity c. DNA Reverse Transcription Kit. Real- time quantitative RT- PCR was performed with the Taq. Man Gene Expression Assay and was normalized against 1. S RNA using an ABI 7. PCR System (Applied Biosystems, Foster City, CA). Kallistatin expression was analyzed using SYBR Green Supermix with custom primers: 5. FOXO1 cellular localization was detected by immunofluorescence labeling as previously described (4, 1. HUVECs were fixed with 4% formaldehyde, permeabilized with 0. Triton X- 1. 00, and stained with an anti- FOXO1 antibody (Cell Signaling, Danvers, MA) and Cy. Ig. G secondary antibody. Cells were counterstained with 1 . Louis, MO) to visualize the nucleus. A matched control Ig. G was used as a negative control to demonstrate specificity. Images were acquired using a fluorescence microscope. Fluorescence intensity was measured separately within the nucleus and cytoplasm. To calculate relative nuclear fluorescence, nuclear fluorescence was divided by the total amount of cellular fluorescence. All quantitative values represent averages of at least 3. Small- interfering RNA transfection. Expression of FOXO1 and JNK in HUVECs was inhibited by small- interfering RNA (si. RNA) oligonucleotides. Knockdown was performed by transfection of si. RNA into cells using the Dharma. FECT1 transfection reagent according to the manufacturer's instructions. As a control, cells were also transfected with scrambled si. RNA. Knockdown efficiency was verified by real- time PCR and Western blot. HUVECs with or without si. RNA transfection were treated with H2. O2 for 2. 4 h, and kallistatin expression was determined by real- time PCR and Western blot. Western blot analysis. Total cell lysates (5. Nonspecific binding sites were blocked with a blocking solution containing 5% nonfat dry milk in Tris- buffered saline (p. H 7. 6) with 0. 1% Tween 2. TBST) at room temperature for 1 h. The membrane was then incubated overnight at 4. After three washes, the membrane was then incubated at room temperature for 1 h with horseradish peroxidase- conjugated anti- mouse Ig. G (1: 3,0. 00 dilution; Jackson Immuno. |
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