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Bhandaru, Madhuri, Daniela S. Kempe, Anand Rotte, Rexhep Rexhepaj, Dietmar Kuhl, and Florian Lang. "Hyperaldosteronism, hypervolemia, and increased blood pressure in mice expressing defective APC." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 297, no. 3 (September 2009): R571—R575. http://dx.doi.org/10.1152/ajpregu.00070.2009.
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Adenomatous polyposis coli (APC) fosters degradation of β-catenin, a multifunctional protein upregulating the serum- and glucocorticoid-inducible kinase (SGK1). SGK1 regulates a wide variety of renal transport processes. The present study explored the possibility that APC influences renal function. To this end, metabolic cage experiments were performed in mice carrying a loss-of-function mutation in the APC gene ( apc Min/+), their wild-type littermates ( apc+/+), and apc Min/+ mice lacking functional SGK1 ( apc Min/+ /sgk1−/−). As a result, mean body weight, food intake, fluid intake, salt appetite, urinary flow, as well as plasma Na+ and K+ concentrations were similar in apc Min/+ mice, apc+/+ mice, and apc Min/+ /sgk1−/− mice. Glomerular filtration rate and absolute renal Na+ excretion were decreased, and fractional urinary K+ excretion was enhanced in apc Min/+ mice. The antinatriuresis, but not the hypofiltration and kaliuresis was partially reversed by additional lack of SGK1. Plasma corticosterone and aldosterone concentrations were significantly enhanced in apc Min/+ mice. While the plasma corticosterone concentration was similar in apc+/+ mice and apc Min/+ /sgk1−/− mice, plasma aldosterone was even higher in apc Min/+ /sgk1−/− mice than in apc Min/+ mice. The hyperaldosteronism of apc Min/+ mice was paralleled by significantly elevated plasma volume and blood pressure. The experiments reveal an influence of defective APC on adrenal hormone release and renal function, effects partially but not completely explained by APC dependence of SGK1 expression.
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Swietlicki, Elzbieta A., Shashi Bala, Jianyun Lu, Anisa Shaker, Gowri Kularatna, Marc S. Levin, and Deborah C. Rubin. "Epimorphin deletion inhibits polyposis in the Apcmin/+ mouse model of colon carcinogenesis via decreased myofibroblast HGF secretion." American Journal of Physiology-Gastrointestinal and Liver Physiology 305, no. 8 (October 15, 2013): G564—G572. http://dx.doi.org/10.1152/ajpgi.00486.2012.
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Interactions between the epithelium and surrounding mesenchyme/stroma play an important role in normal gut morphogenesis, the epithelial response to injury, and epithelial carcinogenesis. The tumor microenvironment, composed of stromal cells including myofibroblasts and immune cells, regulates tumor growth and the cancer stem cell niche. Deletion of epimorphin (Epim), a syntaxin family member expressed in myofibroblasts and macrophages, results in partial protection from colitis and from inflammation-induced colon cancer in mice. We sought to determine whether epimorphin deletion protects from polyposis in the Apc min/+ mouse model of intestinal carcinogenesis. Epim− /− mice were crossed to Apc min/+ mice; Apc min/+ and Apc min/+ /Epim− /− mice were killed at 3 mo of age. Polyp numbers and sizes were quantified in small intestine and colon, and gene expression analyses for pathways relevant to epithelial carcinogenesis were performed. Primary myofibroblast cultures were isolated, and expression and secretion of selected growth factors from Apc min/+ and Apc min/+ /Epim− /− myofibroblasts were examined by ELISA. Small bowel polyposis was significantly inhibited in Apc min/+ /Epim− /− compared with Apc min/+ mice. Apc min/+ /Epim− /− compared with Apc min/+ polyps and adjacent uninvolved intestinal mucosa had increased transforming growth factor-β (TGF-β) expression and signaling with increased P-Smad2/3 expression. Myofibroblasts isolated from Apc min/+ /Epim− /− vs. Apc min/+ mice had markedly decreased hepatocyte growth factor (HGF) expression and secretion. We concluded that Epim deletion inhibits polyposis in Apc min/+ mice, associated with increased mucosal TGF-β signaling and decreased myofibroblast HGF expression and secretion. Our data suggest that Epim deletion reduces tumorigenicity of the stromal microenvironment.
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Lin, Songbai, Sei-Jung Lee, Hyunsuk Shim, Jerold Chun, and C. Chris Yun. "The absence of LPA receptor 2 reduces the tumorigenesis by ApcMin mutation in the intestine." American Journal of Physiology-Gastrointestinal and Liver Physiology 299, no. 5 (November 2010): G1128—G1138. http://dx.doi.org/10.1152/ajpgi.00321.2010.
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Lysophosphatidic acid (LPA) is a lipid mediator that mediates several effects that promote cancer progress. The LPA receptor type 2 (LPA2) expression is often elevated in several types of cancers, including colorectal cancer (CRC). In this study, we investigated the role of LPA2 in the development of intestinal adenomas by comparing Apc Min/+ mice with Apc Min/+ /Lpar2 −/− mice. There were 50% fewer intestinal adenomas in Apc Min/+ /Lpar2 −/− mice than Apc Min/+ mice. Smaller-size adenomas (<1 mm) were found at higher frequencies in Apc Min/+ /Lpar2 −/− mice compared with Apc Min/+ mice at the two age groups examined. The expression level of LPA2 correlated with increased size of intestinal adenomas. Reduced tumor multiplicity and size in Apc Min/+ /Lpar2 −/− mice correlated with decreased proliferation of intestinal epithelial cells. Apc Min/+ /Lpar2 −/− mice showed an increased level of apoptosis, suggesting that LPA2-mediated signaling stimulates intestinal tumor development and progress by regulating both cell proliferation and survival. In addition, the expression levels of Krüpple-like factor 5 (KLF5), β-catenin, cyclin D1, c-Myc, and hypoxia-inducible factor-1α (HIF-1α) were significantly altered in Apc Min/+ /Lpar2 −/− mice compared with Apc Min/+ mice. In vitro studies using HCT116 cells showed that LPA induced cyclin D1, c-Myc, and HIF-1α expression, which was attenuated by knockdown of LPA2. In summary, intestinal tumor initiated by Apc mutations is altered by LPA2-mediated signaling, which regulates tumor growth and survival by altering multiple targets.
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Hutson, Anne M., Christina A. Sam, Yuko Mori-Akiyama, and Preethi H. Gunaratne. "MicroRNAs Dysregulation in APC(MIN/+) Mouse Adenomas." Gastroenterology 140, no. 5 (May 2011): S—191. http://dx.doi.org/10.1016/s0016-5085(11)60771-3.
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Kevin, Leo G., Peter Katz, Amadou K. S. Camara, Enis Novalija, Matthias L. Riess, and David F. Stowe. "Anesthetic Preconditioning." Anesthesiology 99, no. 2 (August 1, 2003): 385–91. http://dx.doi.org/10.1097/00000542-200308000-00020.
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Background Anesthetic preconditioning (APC) is protective for several aspects of cardiac function and structure, including left ventricular pressure, coronary flow, and infarction. APC may be protective, however, only if the duration of ischemia is within a certain, as yet undefined range. Brief ischemia causes minimal injury, and APC would be expected to provide little benefit. Conversely, very prolonged ischemia would ultimately cause serious injury with or without APC. Previous investigations used a constant ischemic time as the independent variable to assess ischemia-induced changes in dependent functional and structural variables. The purpose of the study was to define the critical limits of efficacy of APC by varying ischemic time. Methods Guinea pig hearts (Langendorff preparation; n = 96) underwent pretreatment with sevoflurane (APC) or no treatment (control), before global ischemia and 120 min reperfusion. Ischemia durations were 20, 25, 30, 35, 40, and 45 min. Results At 120 min reperfusion, developed (systolic-diastolic) left ventricular pressure was increased by APC compared with control for ischemia durations of 25-40 min. Infarction was decreased by APC for ischemia durations of 25-40 min, but not 20 or 45 min. APC improved coronary flow and vasodilator responses for all ischemia durations longer than 25 min, and decreased ventricular fibrillation on reperfusion for ischemia durations longer than 30 min. Conclusions Although APC protects against vascular dysfunction and dysrhythmias after prolonged ischemia, protection against contractile dysfunction and infarction in this model is restricted to a range of ischemia durations of 25-40 min. These results suggest that APC may be effective in a subset of patients who have cardiac ischemia of intermediate duration.
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McCully, James D., Yoshiya Toyoda, Masahisa Uematsu, Robert D. Stewart, and Sidney Levitsky. "Adenosine-enhanced ischemic preconditioning: adenosine receptor involvement during ischemia and reperfusion." American Journal of Physiology-Heart and Circulatory Physiology 280, no. 2 (February 1, 2001): H591—H602. http://dx.doi.org/10.1152/ajpheart.2001.280.2.h591.
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Adenosine-enhanced ischemic preconditioning (APC) extends the cardioprotection of ischemic preconditioning (IPC) by both significantly decreasing myocardial infarct size and significantly enhancing postischemic functional recovery. In this study, the role of adenosine receptors during ischemia-reperfusion was determined. Rabbit hearts ( n = 92) were used for Langendorff perfusion. Control hearts were perfused for 180 min, global ischemia hearts received 30-min ischemia and 120-min reperfusion, and IPC hearts received 5-min ischemia and 5-min reperfusion before ischemia. APC hearts received a bolus injection of adenosine coincident with IPC. Adenosine receptor (A1, A2, and A3) antagonists were used with APC before ischemia and/or during reperfusion. GR-69019X (A1/A3) and MRS-1191/MRS-1220 (A3) significantly increased infarct size in APC hearts when administered before ischemia and significantly decreased functional recovery when administered during both ischemia and reperfusion ( P < 0.05 vs. APC). DPCPX (A1) administered either before ischemia and/or during reperfusion had no effect on APC cardioprotection. APC-enhanced infarct size reduction is modulated by adenosine receptors primarily during ischemia, whereas APC-enhanced postischemic functional recovery is modulated by adenosine receptors during both ischemia and reperfusion.
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Novalija, Enis, Leo G. Kevin, Janis T. Eells, Michele M. Henry, and David F. Stowe. "Anesthetic Preconditioning Improves Adenosine Triphosphate Synthesis and Reduces Reactive Oxygen Species Formation in Mitochondria after Ischemia by a Redox Dependent Mechanism." Anesthesiology 98, no. 5 (May 1, 2003): 1155–63. http://dx.doi.org/10.1097/00000542-200305000-00018.
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Background Mitochondrial changes that characterize the heart after anesthetic preconditioning (APC) or the mechanisms by which mitochondrial triggering factors lead to protection are unknown. This study hypothesized that generation of reactive oxygen species (ROS) during APC is required to initiate the mitochondrial protective effects, and that APC leads to improved mitochondrial electron transport chain function and cardiac function during reperfusion. Methods Isolated guinea pig hearts were subject to 30 min ischemia and 120 min reperfusion. Prior to ischemia hearts were either untreated (I/R), or treated with sevoflurane (APC), in the presence or absence of the ROS scavenger tiron (TIR), or the superoxide dismutase mimetic MnTBAP (TBAP). Intracellular ROS were measured by spectrofluorometry using the fluorescent probe dihydroethidium (DHE). In another series of experiments, using the same protocol, hearts were reperfused for only 5 min and removed for measurement of adenosine triphosphate (ATP) synthesis by luciferin-luciferase luminometry and ROS generation by dichlorohydro-fluorescein (DCF) fluorescence in isolated mitochondria. Results The APC improved cardiac function and reduced infarction. Tiron or MnTBAP abrogated the protection afforded by APC. Mitochondrial ATP synthesis was decreased by 70 +/- 3% after IR alone, by only 7 +/- 3% after APC, by 69 +/- 2% after APC+TIR, and by 71 +/- 3% after APC + TBAP. Mitochondrial ROS formation (DCF) increased by 48 +/- 3% after IR alone, by 0 +/- 2% after APC, by 43 +/- 4% after APC + TIR, and by 46 +/- 3% after APC + TBAP. ROS generation (DHE) was increased in I/R group at 5 and 120 min reperfusion. This was attenuated by APC but this protective effect was abrogated in APC + TIR and APC + TBAP groups. Conclusions The results indicate that ROS are central both in triggering and mediating APC, and that the mitochondrion is the target for these changes.
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Riess, Matthias L., Amadou K. S. Camara, Qun Chen, Enis Novalija, Samhita S. Rhodes, and David F. Stowe. "Altered NADH and improved function by anesthetic and ischemic preconditioning in guinea pig intact hearts." American Journal of Physiology-Heart and Circulatory Physiology 283, no. 1 (July 1, 2002): H53—H60. http://dx.doi.org/10.1152/ajpheart.01057.2001.
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NADH increases during ischemia because O2 shortage limits NADH oxidation at the electron transport chain. Ischemic (IPC) and anesthetic preconditioning (APC) attenuate cardiac reperfusion injury. We examined whether IPC and APC similarly alter NADH, i.e., mitochondrial metabolism. NADH fluorescence was measured at the left ventricular wall of 40 Langendorff-prepared guinea pig hearts. IPC was achieved by two 5-min periods of ischemia and APC by exposure to 0.5 or 1.3 mM sevoflurane for 15 min, each ending 30 min before 30 min of global ischemia. During ischemia, NADH initially increased in nonpreconditioned control hearts and then gradually declined below baseline levels. This increase in NADH was lower after APC but not after IPC. The subsequent decline was slower after IPC and APC. On reperfusion, NADH was less decreased after IPC or APC, mechanical and metabolic functions were improved, and infarct size was lower compared with controls. Our results indicate that IPC and APC cause distinctive changes in mitochondrial metabolism during ischemia and thus lead to improved function and tissue viability on reperfusion.
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Riess, Matthias L., Enis Novalija, Amadou K. S. Camara, Janis T. Eells, Qun Chen, and David F. Stowe. "Preconditioning with Sevoflurane Reduces Changes in Nicotinamide Adenine Dinucleotide during Ischemia–Reperfusion in Isolated Hearts." Anesthesiology 98, no. 2 (February 1, 2003): 387–95. http://dx.doi.org/10.1097/00000542-200302000-00019.
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Background Ischemia causes an imbalance in mitochondrial metabolism and accumulation of nicotinamide adenine dinucleotide (NADH). We showed that anesthetic preconditioning (APC), like ischemic preconditioning, improved mitochondrial NADH energy balance during ischemia and improved function and reduced infarct size on reperfusion. Opening adenosine triphosphate-sensitive potassium (K(atp)) channels may be involved in triggering APC. The authors tested if effects of APC on NADH concentrations before, during, and after ischemia are reversible by 5-hydroxydecanoate (5-HD), a putative mitochondrial K channel blocker. Methods Nicotinamide adenine dinucleotide fluorescence was measured in 60 guinea pig Langendorff-prepared hearts assigned into five groups: (1) no treatment before ischemia; (2) APC by exposure to 1.3 mm sevoflurane for 15 min; (3) 200 microm 5-HD from 5 min before to 15 min after sevoflurane exposure; (4) 35 min 5-HD alone; and (5) no treatment and no ischemia. Sevoflurane was washed out for 30 min, and 5-HD for 15 min, before 30-min ischemia and 120-min reperfusion. Results Nicotinamide adenine dinucleotide was reversibly increased during sevoflurane exposure before ischemia, and the increase and rate of decline in NADH during ischemia were reduced after APC. 5-HD abolished these changes in NADH. On reperfusion, function was improved and infarct size reduced after APC compared with other groups. Conclusion Anesthetic preconditioning was evidenced by improved mitochondrial bioenergetics as assessed from NADH concentrations during ischemia and by attenuated reperfusion injury. Reversal of APC by bracketing sevoflurane exposure with 5-HD suggests that APC is triggered by mitochondrial K channel opening or, alternatively, by attenuated mitochondrial respiration without direct involvement of mitochondrial K channel opening.
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Kohno, Masataka, Michiko Momoi, Myat Lin Oo, Ji-Hye Paik, Yong-Moon Lee, Krishnan Venkataraman, Youxi Ai, et al. "Intracellular Role for Sphingosine Kinase 1 in Intestinal Adenoma Cell Proliferation." Molecular and Cellular Biology 26, no. 19 (October 1, 2006): 7211–23. http://dx.doi.org/10.1128/mcb.02341-05.
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ABSTRACT Sphingosine kinase (Sphk) enzymes are important in intracellular sphingolipid metabolism as well as in the biosynthesis of sphingosine 1-phosphate (S1P), an extracellular lipid mediator. Here, we show that Sphk1 is expressed and is required for small intestinal tumor cell proliferation in Apc Min/+ mice. Adenoma size but not incidence was dramatically reduced in Apc Min/+ Sphk −/ − mice. Concomitantly, epithelial cell proliferation in the polyps was significantly attenuated, suggesting that Sphk1 regulates adenoma progression. Although the S1P receptors (S1P1R, S1P2R, and S1P3R) are expressed, polyp incidence or size was unaltered in Apc Min/+ S1p2r −/ −, Apc Min/+ S1p3r −/ −, and Apc Min/+ S1p1r +/ − bigenic mice. These data suggest that extracellular S1P signaling via its receptors is not involved in adenoma cell proliferation. Interestingly, tissue sphingosine content was elevated in the adenomas of Apc Min/ + Sphk1 −/ − mice, whereas S1P levels were not significantly altered. Concomitantly, epithelial cell proliferation and the expression of the G1/S cell cycle regulator CDK4 and c-myc were diminished in the polyps of Apc Min/ + Sphk1 −/ − mice. In rat intestinal epithelial (RIE) cells in vitro, Sphk1 overexpression enhanced cell cycle traverse at the G1/S boundary. In addition, RIE cells treated with sphingosine but not C6-ceramide exhibited reduced cell proliferation, reduced retinoblastoma protein phosphorylation, and cyclin-dependent kinase 4 (Cdk4) expression. Our findings suggest that Sphk1 plays a critical role in intestinal tumor cell proliferation and that inhibitors of Sphk1 may be useful in the control of intestinal cancer.
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Novalija, Enis, Srinivasan G. Varadarajan, Amadou K. S. Camara, Jianzhong An, Qun Chen, Matthias L. Riess, Neil Hogg, and David F. Stowe. "Anesthetic preconditioning: triggering role of reactive oxygen and nitrogen species in isolated hearts." American Journal of Physiology-Heart and Circulatory Physiology 283, no. 1 (July 1, 2002): H44—H52. http://dx.doi.org/10.1152/ajpheart.01056.2001.
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We postulated that anesthetic preconditioning (APC) is triggered by reactive oxygen/nitrogen species (ROS/RNS). We used the isolated guinea pig heart perfused withl-tyrosine, which reacts with ROS and RNS to form strong oxidants, principally peroxynitrite (ONOO−), and then forms fluorescent dityrosine. ROS scavengers superoxide dismutase, catalase, and glutathione (SCG) and NO· synthesis inhibitor N G-nitro-l-arginine methyl ester (l-NAME) were given 5 min before and after sevoflurane preconditioning stimuli. Drugs were washed out before 30 min of ischemia and 120 min of reperfusion. Groups were control (nontreated ischemia control), APC (two, 2-min periods of perfusion with 0.32 ± 0.02 mM of sevoflurane; separated by a 6-min period of perfusion without sevoflurane), SCG, APC + SCG,l-NAME, and APC + l-NAME. Effluent dityrosine at 1 min reperfusion was 56 ± 6 (SE)‡, 15 ± 5, 40 ± 5‡, 39 ± 4‡, 35 ± 4‡, and 33 ± 5‡ units (‡ P < 0.05 vs. APC), respectively; left ventricular pressure (%baseline) at 60 min of reperfusion was 30 ± 5‡, 60 ± 4, 35 ± 5‡, 37 ± 5‡, 44 ± 4, and 47 ± 4; and infarct size (%total heart weight) was 50 ± 5‡, 19 ± 2, 48 ± 3‡, 46 ± 4‡, 42 ± 4‡, and 45 ± 2‡. Thus APC is initiated by ROS as shown by improved function, reduced infarct size, and reduced dityrosine on reperfusion; protective and ROS/RNS-reducing effect of APC were attenuated when bracketed by ROS scavengers or NO· inhibition.
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Wei, Huijun, Jin Shang, CarolAnn Keohane, Min Wang, Qiu Li, Weihua Ni, Kim O’Neill, and Madhu Chintala. "A novel approach to assess the spontaneous gastrointestinal bleeding risk of antithrombotic agents using Apc min/+ mice." Thrombosis and Haemostasis 111, no. 06 (2014): 1121–32. http://dx.doi.org/10.1160/th13-11-0926.
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SummaryAssessment of the bleeding risk of antithrombotic agents is usually performed in healthy animals with some form of vascular injury to peripheral organs to induce bleeding. However, bleeding observed in patients with currently marketed antithrombotic drugs is typically spontaneous in nature such as intracranial haemorrhage (ICH) and gastrointestinal (GI) bleeding, which happens most frequently on top of preexisting pathologies such as GI ulcerations and polyps. Apc min/+ mice are reported to develop multiple adenomas through the entire intestinal tract and display progressive anaemia. In this study, we evaluated the potential utility of Apc min/+ mice as a model for assessing spontaneous GI bleeding with antithrombotic agents. Apc min/+ mice exhibited progressive blood loss starting at the age of nine weeks. Despite the increase in bleeding, Apc min/+ mice were in a hypercoagulable state and displayed an age-dependent increase in thrombin generation and circulating fibrinogen as well as a significant decrease in clotting times. We evaluated the effect of warfarin, dabigatran etexilate, apixaban and clopidogrel in this model by administering them in diet or in the drinking water to mice for 1–4 weeks. All of these marketed drugs significantly increased GI bleeding in Apc min/+ mice, but not in wild-type mice. Although different exposure profiles of these antithrombotic agents make it challenging to compare the bleeding risk of compounds, our results indicate that the Apc min/+ mouse may be a sensitive preclinical model for assessing the spontaneous GI bleeding risk of novel antithrombotic agents.
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Baltgalvis, Kristen A., Franklin G. Berger, Maria Marjorette O. Peña, J. Mark Davis, and James A. Carson. "Effect of exercise on biological pathways in ApcMin/+ mouse intestinal polyps." Journal of Applied Physiology 104, no. 4 (April 2008): 1137–43. http://dx.doi.org/10.1152/japplphysiol.00955.2007.
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Many epidemiological studies have demonstrated that level of exercise is associated with reduced colorectal cancer risk. Treadmill training can decrease Apc Min/+ mouse intestinal polyp number and size, but the mechanisms remain unclear. Understanding the molecular changes in the tumor following exercise training may provide insight on the mechanism by which exercise decreases Apc Min/+ mouse polyp formation and growth. The purpose of this study was to determine if exercise can modulate Apc Min/+ mouse intestinal polyp cellular signaling related to tumor formation and growth. Male Apc Min/+ mice were randomly assigned to control ( n = 20) or exercise ( n = 20) treatment groups. Exercised mice ran on a treadmill at a moderate intensity (18 m/min, 60 min, 6 days/wk, 5% grade) for 9 wk. Polyps from Apc Min/+ mice were used to quantify markers of polyp inflammation, apoptosis, and β-catenin signaling. Exercise decreased the number of macrophages in polyps by 35%. Related to apoptosis, exercise decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells by 73% in all polyps. Bax protein expression in polyps was decreased 43% by exercise. β-Catenin phosphorylation was elevated 3.3-fold in polyps from exercised mice. Moderate-intensity exercise training alters cellular pathways in Apc Min/+ mouse polyps, and these changes may be related to the exercise-induced reduction in polyp formation and growth.
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Bienengraeber, Martin, Molly Pellitteri-Hahn, Naoyuki Hirata, Tesfaye M. Baye, Zeljko J. Bosnjak, and Michael Olivier. "Quantitative characterization of changes in the cardiac mitochondrial proteome during anesthetic preconditioning and ischemia." Physiological Genomics 45, no. 5 (March 1, 2013): 163–70. http://dx.doi.org/10.1152/physiolgenomics.00117.2012.
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Changes in mitochondrial bioenergetics have been proposed to be critical for triggering and effecting anesthetic-induced preconditioning (APC) against cardiac ischemia and reperfusion injury. The objective of this study was to analyze changes in mitochondrial protein levels and link those changes to potential functional changes. A 18O-labeling method was applied for relative comparison of cardiac mitochondrial samples from control and isoflurane exposed rats before and after ischemia and reperfusion. Wistar rats were exposed to isoflurane for 30 min (APC) or did not receive the anesthetic (control). Rats were subjected to 30 min coronary occlusion and 15 min reperfusion without (ischemia) or after APC (ischemia + APC). The following comparisons were made: control vs. APC, control vs. ischemia, and APC vs. ischemia + APC. Proteins were analyzed by liquid chromatography-mass spectrometry. A total of 98 proteins currently annotated as mitochondrial proteins in the UniProt database were positively identified from three replicate experiments. Most of the changes during APC and ischemia occur in complexes of the electron transport chain. Overall, fewer changes in ETC complexes were found when comparing APC with APC + ischemia than when comparing control and ischemia. This corresponds to the preservation of bioenergetics due to APC after ischemia and reperfusion as indicated by preserved ATP level and generation. APC itself induced changes in complex I, but those changes were not correlated with activity changes in mitochondria after APC. Thus, a proteomic mass spectral approach does not only assess quantitative changes without prior knowledge of proteins, but also allows insight into the mechanisms of ischemia and reperfusion injury and APC.
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Meledeo, Michael A., Melanie V. Valenciana, Armando C. Rodriguez, and Andrew P. Cap. "Synergistic Anticoagulant Effect of Activated Protein C and Tissue Factor Pathway Inhibitor As a Mechanism for Acute Traumatic Coagulopathy." Blood 124, no. 21 (December 6, 2014): 1487. http://dx.doi.org/10.1182/blood.v124.21.1487.1487.
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Abstract Introduction Severe trauma with tissue damage and shock can rapidly (<30 min) result in abnormal coagulation function which is independent of consumption or dilution effects; this acute traumatic coagulopathy (ATC) has been linked to increased transfusion requirements, morbidity and mortality. It has been suggested that ATC is caused by an increase in activated protein C (aPC; from 40 pM in normal to 175 pM) which has been identified in trauma patients [Cohen MJ, Ann Surg 255:379 (2012)]. aPC inactivates factor Va (FVa) as part of normal hemostasis, but our previous results indicate that aPC at trauma levels is not sufficient to prevent coagulation, particularly when platelets are present, as platelet fVa is resistant to aPC [Campbell JE, PLoS ONE 9:e99181 (2014); Camire RM, Blood 91:2818 (1998)]. Tissue Factor Pathway Inhibitor (TFPI) is an endothelial-bound protein which inhibits coagulation through effects on factor Xa and the factor VIIa-tissue factor complex. It is released in the plasma (normal level 2.5nM, pathophysiologic states including trauma up to 10nM) and is typically cleared by the liver or kidneys. TFPIα accounts for 20% of circulating TFPI and binds FV and FVa [Ndonwi M, J Thromb Haemost 10:1944 (2012)]. Recently, a truncated FV (termed FV-short) produced by an autosomal dominant mutation was found to form complexes with TFPIα, resulting in retention of TFPIα in a 10-fold increase over normal levels in affected individuals [Vincent LM, J Clin Invest 123:3777 (2013)]. FV-short has significantly reduced thrombogenic potential and, by concomitantly raising TFPIα, causes a bleeding disorder. We hypothesize that the activation of PC in acute trauma may result in the production of FV-degradation products which could stabilize TFPIα similarly to the effect of FV-short. The combination of reduced FVa and increased TFPIα may contribute to ATC. Methods Whole blood from healthy volunteers was drawn by phlebotomy into ACD-containing tubes. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were collected by centrifugation (200g for 10 min and 1000g for 15 min, respectively). Calibrated automated thrombogram (CAT) assays and thromboelastography (TEG) were conducted using PRP and PPP with exogenously delivered aPC (0, 100pM, or 1nM) and TFPI (0, 2.5nM, or 10nM). An immunodepleted FV-deficient (<1% normal) plasma (FVdp) was used as a reference. Results CAT assays verified that aPC and TFPI each delay and suppress thrombin generation in PPP in a dose-dependent manner, but the combination of aPC and TFPI had a synergistic effect at the highest doses tested. The endogenous thrombin potential (ETP) was eliminated in PPP (control: 1663 nM-min; 1nM aPC + 10nM TFPI: 0 nM-min; P<0.001); lag time was similarly affected (control: 2.5 min; 1nM aPC + 10nM TFPI: >60 min; P<0.001). FVdp also featured a delayed lag time (11.17 min, P<0.001 versus control), but the delay induced by the addition of 10nM TFPI was not nearly as severe in FVdp (19.67 min) as in the PPP + 10nM aPC sample (>60 min, P<0.001). For PRP, there was no statistical difference between control and the highest doses of aPC and TFPI in ETP (1636 nM-min versus 1388 nM-min) or lag time (8.72 min versus 9.13 min). Conclusions In vitro studies suggest that PC activation is not the sole cause of ATC; the concentrations of aPC measured in trauma patient blood have little effect on the coagulation potential of plasma with or without platelets when aPC is delivered exogenously. The results here demonstrate that there is a synergistic effect between aPC and TFPI. When TFPI is added to FV deficient plasma, the thrombin generation is delayed and suppressed; however, in normal plasma digested with aPC (containing fragmented FVa), the addition of TFPI is sufficient to suppress the generation of thrombin beyond the window of measurement (>60 min). The addition of platelets to the milieu eliminated the effects of aPC, TFPI, and the combination on thrombin generation, highlighting the central role of platelets in maintaining hemostatic function. Disclosures No relevant conflicts of interest to declare.
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Zhong, Caiyun, Yamei Zhou, and Hong Liu. "Nuclear Factor κB and Anesthetic Preconditioning during Myocardial Ischemia-Reperfusion." Anesthesiology 100, no. 3 (March 1, 2004): 540–46. http://dx.doi.org/10.1097/00000542-200403000-00012.
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Background Volatile anesthetic preconditioning (APC) protects against myocardial ischemia-reperfusion (IR) injury, but the precise mechanisms underlying this phenomenon remain undefined. To investigate the molecular mechanism of APC in myocardial protection, the activation of nuclear factor (NF) kappaB and its regulated inflammatory mediators expression were examined in the current study. Methods Hearts from male rats were isolated, Langendorff perfused, and randomly assigned to one of three groups: (1) the control group: hearts were continuously perfused for 130 min; (2) the IR group: 30 min of equilibration, 15 min of baseline, 25 min of ischemia, 60 min of reperfusion; and (3) the APC + IR group: 30 min of equilibration, 10 min of sevoflurane exposure and a 5-min washout, 25 min of global ischemia, 60 min of reperfusion. Tissue samples were acquired at the end of reperfusion. NF-kappaB activity was determined by electrophoretic mobility shift assay. The NF-kappaB inhibitor, IkappaB-alpha, was determined by Western blot analysis. Myocardial inflammatory mediators, including tumor necrosis factor alpha, interleukin 1, intercellular adhesion molecule 1, and inducible nitric oxide synthase, were also assessed by Western blot analysis. Results Nuclear factor kappaB-DNA binding activity was significantly increased at the end of reperfusion in rat myocardium, and cytosolic IkappaB-alpha was decreased. Supershift assay revealed the involvement of NF-kappaB p65 and p50 subunits. APC with sevoflurane attenuated NF-kappaB activation and reduced the expression of tumor necrosis factor alpha, interleukin 1, intercellular adhesion molecule 1, and inducible nitric oxide synthase. APC also reduced infarct size and creatine kinase release and improved myocardial left ventricular developed pressure during IR. Conclusions The results of this study indicate that attenuation of NF-kappaB activation and subsequent down-regulation of NF-kappaB-dependent inflammatory gene expression plays an important role in the protective mechanism of APC against acute myocardial IR injury.
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Madka, Venkateshwar, Srikanth Chiliveru, Yuting Zhang, Nicole Stratton, Anh Bao, Nandini Kumar, and Chinthalapally V. Rao. "Abstract A011: Effect of IL-23 knockdown on obesity driven CRC in high-fat diet fed Apcmin/+ mice." Cancer Prevention Research 15, no. 12_Supplement_2 (December 1, 2022): A011. http://dx.doi.org/10.1158/1940-6215.tacpad22-a011.
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Abstract Colorectal cancer (CRC) is the 3rd common cancer in the United States with an estimated 151,030 new cases and 52,580 deaths in 2022. Several studies suggest that western-style diet-induced obesity contributes to CRC promotion via systemic inflammatory mediators. Recently, we have reported significant elevation of circulating IL-23 levels in obese human subjects (BMI>30). Also, TCGA gene expression data indicated IL-23A over expression in colonic tumors (CT) that correlated with tumor stage, patient obesity, nodal metastasis, and poor survival rates. Importantly, we have shown that IL-23 knockdown in Apc min/+ mice led to significant suppression of the intestinal tumors strongly supporting our hypothesis that pro-inflammatory cytokine IL-23 could be a possible link between obesity and CRC. Here we explored the contribution of IL-23 in obesity induce CRC in vivo using high-fat diet (HFD) fed Apc min/+ mouse model. For this study, Apc min/+ and IL-23 KO (IL-23A p19-/- mice; MMMRC) were cross-bred to generate Apc min/+ mice with IL-23 normal (IL-23+/+) or KO (IL-23-/-) genotypes. Six-week old Apc min/+ mice (N≥15/gender) were grouped by IL-23 genotypes and fed HFD (60 Kcal) until termination. Mice were euthanized at 20 weeks age, intestines were harvested and evaluated for tumors incidence and multiplicity. In Apc min/+ mice, IL-23 deletion had significant suppressive effect on HFD driven CT and small intestinal tumors (SIT). IL-23-/- Apc min/+ male mice had 50% less CT (0.81+0.25 vs 1.61±0.27; p<0.05) while female mice had 40% less CT (0.40±0.16 vs 0.94±0.21; p=0.05) compared to the IL-23+/+ Apc min/+ control mice. Similarly, CT incidence was also suppressed by 36% and 48% in male and female IL-23-/- Apc min/+ mice respectively compared to control mice. SIT multiplicity was also inhibited by 48% in male (15.00±1.13 vs 28.54±1.11; p<0.0001) and by 41% in female (14.83±1.35 vs 25.18±1.71; p<0.0005) IL23-/- Apc min/+ compared to control mice. Tumor data was complemented by significant reduction in markers of proliferation, immune evasion, circulating proinflammatory cytokine and chemokines (IL-1, IL- 17, IL-23, IL-10, CCL-3, CCL-2, CCL-5, IFNγ, TNFα, etc.) in IL-23 KO compared to control mice. Our data clearly indicates that IL-23 is a promising target for CRC prevention in high-risk obese individuals and warrants further investigation. (Supported in part by P30CA225520 and Kerley-Cade Endowed Chair). Citation Format: Venkateshwar Madka, Srikanth Chiliveru, Yuting Zhang, Nicole Stratton, Anh Bao, Nandini Kumar, Chinthalapally V. Rao. Effect of IL-23 knockdown on obesity driven CRC in high-fat diet fed Apcmin/+ mice [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A011.
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Sniecinski, Roman, and Hong Liu. "Reduced Efficacy of Volatile Anesthetic Preconditioning with Advanced Age in Isolated Rat Myocardium." Anesthesiology 100, no. 3 (March 1, 2004): 589–97. http://dx.doi.org/10.1097/00000542-200403000-00019.
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Background Ischemic preconditioning and anesthetic preconditioning (APC) are reported to decrease myocardial infarct size during ischemia-reperfusion injury. However, the beneficial effects of ischemic preconditioning have been shown to decrease with advancing age. Although the mechanisms of ischemic preconditioning and APC are thought to be similar, it is not known whether the beneficial effects of APC are also reduced in the aged myocardium. Methods Male Fischer 344 rats of three age groups (2-4, 10-12, and 20-24 months) were used. Hearts were Langendorff perfused. Six hearts in each age group were pretreated with 10 min of sevoflurane and a 5-min washout before 25 min of ischemia and 60 min of reperfusion. Six control hearts in each age group received no treatment before ischemia. Nuclear magnetic resonance was used to measure intracellular Na, intracellular Ca, and intracellular pH, respectively. Left ventricular developed pressure, creatine kinase, and infarct size were measured. Results Ischemia decreases intracellular pH and increases intracellular Na and intracellular Ca in all age groups. APC blunts the pH decreases in young adult and middle-aged rats, but not in aged rats. APC decreased intracellular Na and intracellular Ca accumulation during ischemia in young adult and middle-aged hearts. APC improved adenosine triphosphate recovery in young rats but not in aged rats. Creatine kinase and infarct sizes were significantly reduced and left ventricular developed pressure was improved with APC in the young adult and middle-aged groups but not the aged group. Conclusions The benefits of APC are significantly reduced with advanced age in an isolated rat heart model.
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Ousingsawat, Jiraporn, Melanie Spitzner, Rainer Schreiber, and Karl Kunzelmann. "Upregulation of colonic ion channels in APC Min/+ mice." Pflügers Archiv - European Journal of Physiology 456, no. 5 (February 5, 2008): 847–55. http://dx.doi.org/10.1007/s00424-008-0451-3.
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Xu, Haibo, Gary H. Posner, Michael Stevenson, and Frederick C. Campbell. "Apc MIN modulation of vitamin D secosteroid growth control." Carcinogenesis 31, no. 8 (May 20, 2010): 1434–41. http://dx.doi.org/10.1093/carcin/bgq098.
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Nagamine, Claude M., Jane J. Sohn, Barry H. Rickman, Arlin B. Rogers, James G. Fox, and David B. Schauer. "Helicobacter hepaticus Infection Promotes Colon Tumorigenesis in the BALB/c-Rag2−/−ApcMin/+ Mouse." Infection and Immunity 76, no. 6 (April 14, 2008): 2758–66. http://dx.doi.org/10.1128/iai.01604-07.
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ABSTRACT Adenomatous polyposis coli (APC) mutations are linked to human and mouse colorectal cancers. The Apc multiple intestinal neoplasia (Min) mouse mutation causes adenomas to develop throughout the small and large intestines. The BALB-Min (C.B6-Apc Min/+) congenic strain was generated by backcrossing into BALB/c the Apc Min allele from C57BL/6J-Apc Min/+ mice. BALB-Min mice have a low tumor multiplicity (27.4 small intestine tumors/mouse) and a relatively long life span (>1 year) that makes them amenable to long-term studies. To investigate the interplay of the adaptive immune system and intestinal tumorigenesis, the immunodeficient compound mutant strain BALB-RagMin (C.Cg-Rag2 −/− Apc Min/+) was generated. BALB-RagMin mice had a significant increase in tumors in the small, but not large, intestine relative to their BALB-Min counterparts (43.0 versus 24.0 tumors/mouse, respectively). The results suggest that the adaptive immune system plays a role in either the elimination or the equilibrium phase of cancer immunoediting in the small intestine in this model. We investigated the effect of the enterohepatic bacterial pathogen Helicobacter hepaticus on liver and intestine tumorigenesis in BALB-RagMin mice. H. hepaticus-infected BALB-RagMin mice developed moderate hepatitis, moderate typhlitis, and mild colitis. There were no differences in small intestine and cecal tumor multiplicity, regionality, or size relative to that in uninfected mice. However, H. hepaticus-infected BALB-RagMin mice had a significant increase in colon tumor incidence relative to uninfected BALB-RagMin mice (23.5% versus 1.7%, respectively). The data suggest that H. hepaticus, which is present in many research colonies, promotes colon tumorigenesis in the BALB-RagMin mouse and that it has the potential to confound colon tumorigenesis studies.
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An, Jianzhong, Samhita S. Rhodes, Ming Tao Jiang, Zeljko J. Bosnjak, Ming Tian, and David F. Stowe. "Anesthetic Preconditioning Enhances Ca2+Handling and Mechanical and Metabolic Function Elicited by Na+–Ca2+Exchange Inhibition in Isolated Hearts." Anesthesiology 105, no. 3 (September 1, 2006): 541–49. http://dx.doi.org/10.1097/00000542-200609000-00018.
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Background Anesthetic preconditioning (APC) is well known to protect against myocardial ischemia-reperfusion injury. Studies also show the benefit of Na+-Ca2+ exchange inhibition on ischemia-reperfusion injury. The authors tested whether APC plus Na+-Ca2+ exchange inhibitors given just on reperfusion affords additive protection in intact hearts. Methods Cytosolic [Ca2+] was measured by fluorescence at the left ventricular wall of guinea pig isolated hearts using indo-1 dye. Sarcoplasmic reticular Ca2+-cycling proteins, i.e., Ca2+ release channel (ryanodine receptor [RyR2]), sarcoplasmic reticular Ca2+-pump adenosine triphosphatase (SERCA2a), and phospholamban were measured by Western blots. Hearts were assigned to seven groups (n = 8 each): (1) time control; (2) ischemia; (3, 4) 10 microM Na+-Ca2+ exchange inhibitor KB-R7943 (KBR) or 1 microM SEA0400 (SEA), given during the first 10 min of reperfusion; (5) APC initiated by sevoflurane (2.2%, 0.41 +/- 0.03 mm) given for 15 min and washed out for 15 min before ischemia-reperfusion; (6, 7) APC plus KBR or SEA. Results The authors found that APC reduced the increase in systolic [Ca2+], whereas KBR and SEA both reduced the increase in diastolic [Ca2+] on reperfusion. Each intervention improved recovery of left ventricular function. Moreover, APC plus KBR or SEA afforded better functional recovery than APC, KBR, or SEA alone (P < 0.05). Ischemia-reperfusion-induced degradation of major sarcoplasmic reticular Ca2+-cycling proteins was attenuated by APC, but not by KBR or SEA. Conclusions APC plus Na+-Ca2+ exchange inhibition exerts additive protection in part by reducing systolic and diastolic Ca2+ overload, respectively, during ischemia-reperfusion. Less degradation of sarcoplasmic reticular Ca2+-cycling proteins may also contribute to cardiac protection.
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Wang, Huan, Lingnan Guan, Jing Li, Maode Lai, and Xiaodong Wen. "The Effects of Berberine on the Gut Microbiota in Apc min/+ Mice Fed with a High Fat Diet." Molecules 23, no. 9 (September 8, 2018): 2298. http://dx.doi.org/10.3390/molecules23092298.
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Background: Berberine (BBR) has been extensively reported to inhibit colorectal cancer (CRC) development, though its bioavailability is poor. Nowadays, an increasing number of studies have shown that BBR significantly accumulates in the intestines and could regulate gut microbiota in obesity. The purpose of this study was to further explore the effects of BBR on gut microbiota in Apc min/+ mice receiving a high fat diet (HFD). Methods: Apc min/+ mice received either HFD alone or HFD and BBR for 12 weeks. The intestinal tissues were collected to evaluate the efficiency of BBR on neoplasm development by hematoxylin and eosin staining. Meanwhile, immunohistochemistry was conducted to investigate the effects of BBR on cyclin D1 and β-catenin in colon tissues. Fecal samples were subjected to 16S rRNA sequencing. Results: BBR significantly reduced intestinal tumor development and altered the structure of gut microbiota in Apc min/+ mice fed with an HFD. At the phylum level, it was able to significantly inhibit the increase in Verrucomicrobia. At the genus level, it was able to suppress Akkermansia and elevate some short chain fat acid (SCFA)-producing bacteria. Conclusions: BBR significantly alleviated the development of CRC in Apc min/+ mice fed with HFD and restored the enteric microbiome community.
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Adam, Jacqueline, Klara Eriksson, Benno Schnyder, Stefano Fontana, Werner Pichler, and Daniel Yerly. "Non-covalent interaction of Abacavir with HLA-B*5701 generates an antigenic complex recognized by specific T cells (100.13)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 100.13. http://dx.doi.org/10.4049/jimmunol.186.supp.100.13.
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Abstract Rationale: Abacavir (ABC) hypersensitivity is mediated by specific CD8+ T cells that interact with ABC in HLA-B*5701+ individuals. To understand how ABC is presented and recognized by T cells, we generated ABC specific T cell clones (ABC-TCC) and analysed the kinetic of ABC recognition. Methods: ABC-TCC from ABC naïve HLA-B*5701+ subjects were generated by limiting dilution. TCC activation by ABC was measured by means of Ca2+ influx kinetic analyses and Annexin V staining. Results: ABC-TCC in presence of autologous antigen-presenting cells (APC) reacted within a short time (3-25 min) to ABC in solution. Interestingly, APC that internalized the drug were able to present it already after 30 min on the cell surface leading to an immediate TCC activation (60 sec). Furthermore, no mRNA for alcoholdehydrogenases (ADH), the enzymes involved in ABC metabolism, was detected in the immune cells of peripheral blood. Conclusions: The rapid kinetic of TCC reactivity to ABC presented by HLA-B*5701 and the lack of ADH activity in peripheral blood mononuclear cells is best explained by a direct, metabolism and processing independent interaction of ABC with the restricting HLA-B*5701 molecule. This interaction either occurs intracellular or on the cell surface, leading to a modification of the HLA-complex able to induce a full T cell response. Our data underline that the immune stimulatory capacity of drugs is not uniquely restricted to the ability to form covalent bonds to proteins.
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Von Drygalski, Annette, Andrew J. Gale, Laurent Burnier, Thomas J. Cramer, John H. Griffin, and Laurent O. Mosnier. "An Engineered Factor Fva Prevents Bleeding Induced By Anticoagulant Wild Type Activated Protein C." Blood 122, no. 21 (November 15, 2013): 203. http://dx.doi.org/10.1182/blood.v122.21.203.203.
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Abstract Increased risk of bleeding is observed in patients receiving therapy with a variety of anticoagulant drugs, and there is an unmet need for prohemostatic agents that reduce bleeding risk. In some clinical trials, wild type (wt) APC therapy was associated with an increased risk of serious bleeding. In most animal models of inflammatory injury and disease where APC was beneficial, APC’s cytoprotective effects were responsible for the protective effects of APC therapy whereas its anticoagulant effects were neither required nor contributing. Thus, wt APC’s anticoagulant activities and associated risk of bleeding may be a limiting factor for potential novel wt APC therapies. The availability of a wt APC-anticoagulant specific antidote or reversal agent that does not affect wt APC’s cytoprotective activities would be highly desirable. We hypothesized that superFVa, an engineered FVa-variant that potently normalizes hemostasis in hemophilia, fits the criteria for a prohemostatic biologic that could reduce wt APC-anticoagulant-induced bleeding, and here we test this hypothesis. SuperFVa has enhanced specific activity compared to wt FVa due to an engineered disulfide bond (Cys609-Cys1691) between the A2 and A3 domains (FV(A2-SS-A3)) and, its biological activity is augmented by mutations of the APC cleavage sites (Arg506/306/679Gln). As a result of these modifications, superFVa was found to be resistant to wt APC and normalized hemostatic properties and prevented bleeding in a hemophilic mouse model much more efficiently than wt FVa. In aPTT clotting assays both superFVa and wt FVa dose-dependently normalized clotting times when wt-APC was used to prolong clotting (aPTT > 100 sec). However, superFVa normalized the aPTT at 100-fold lower concentrations compared to wt FVa. In thrombin generation assays using either human or murine plasma, superFVa fully restored thrombin generation at concentrations where wt FVa did not show effects. In an ex vivo whole blood aPTT assay, intravenous (iv) injection of murine recombinant wt APC (0.5 mg/kg) in Balb/c mice doubled clotting times from 30 sec to ∼ 60 sec (n=40). Addition of superFVa (1 nM) to whole blood significantly normalized aPTT clotting times whereas wt FVa failed to show a significant effect. In a tail clip-bleeding model in Balb/c mice, injection (iv) of human recombinant or plasma-derived wt APC induced significant bleeding at 1.25 mg/kg and mean blood loss increased from 3.4 µL/g with saline to 27 µL/g with wt APC treatment. SuperFVa injected (iv 3.5 mg/kg) 2 min prior to wt APC administration reduced bleeding significantly to 9.2 µL/g (n∼10 per group). In another bleeding model, liver laceration was used because it provides important information concerning microvessel-mediated bleeding after acute organ injury. After adaptation and technical modifications of the model used in rats and rabbits for mouse anatomy and validation in hemophilia versus control mice, this model provided a reliable assessment of bleeding with a ∼ 4-fold reduced inter-individual range of blood loss compared to the tail clip-bleeding model. Injection (iv) of human wt APC increased blood loss from 29 µL/g to 49 µL/g, and the excessive bleeding was associated with a ∼40% mortality rate. SuperFVa reduced wt APC-induced bleeding after 20 min significantly to 29 µL/g and abolished bleeding-induced mortality. Remarkably, bleeding patterns in the tail clip and liver laceration models were different when blood loss was determined separately for the 1st and 2nd 10 min after injury. In the tail clip-model wt-APC-induced bleeding during both 1st and 2nd 10 min after tail clip and superFVa decreased blood loss during both phases. In the liver laceration model, most blood loss occurred immediately after injury and bleeding during the 2nd 10 min was less pronounced. In this model, however, superFVa corrected blood loss entirely during the 1st 10 min phase and fully prevented bleeding during the 2nd 10 min phase. Our results provide proof of principle that superFVa is effective in the prevention and reversal of wt APC-induced bleeding. Thus, in addition to improving hemostasis in hemophilia, superFVa protects against bleeding in 2 different mouse models where bleeding was induced by wt APC. Hence, superFVa deserves future consideration for development as a prohemostatic agent in situations where bleeding is a serious risk. Disclosures: No relevant conflicts of interest to declare.
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Sergeev, Pavel, Rafaela da Silva, Eliana Lucchinetti, Kathrin Zaugg, Thomas Pasch, Marcus C. Schaub, and Michael Zaugg. "Trigger-dependent Gene Expression Profiles in Cardiac Preconditioning." Anesthesiology 100, no. 3 (March 1, 2004): 474–88. http://dx.doi.org/10.1097/00000542-200403000-00005.
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Background DNA chips facilitate genomic-wide exploration of gene expression. The authors hypothesized that ischemic (IPC) and anesthetic preconditioning (APC) would differentially modulate gene expression in hearts. Methods Affymetrix rat U34A gene chips were used to explore the transcriptional response to IPC and APC, sustained ischemia (110 min) without reperfusion, and time-matched perfusion in isolated rat hearts. IPC was induced by three cycles of 5 min of ischemia, and APC was induced by 1.5 minimum alveolar concentration isoflurane (110 min). For each heart, a separate chip was used for hybridization. Data were analyzed for significant > or = 2.0-fold changes in gene expression. Microarray results were confirmed by quantitative real-time reverse-transcription polymerase chain reaction. Results Of the 8,799 genes represented on U34A, 217 transcripts in the APC group, 234 in the IPC group, and 29 in the ischemia group displayed significant > or = 2.0-fold up-regulation in messenger RNA levels, and 185 transcripts in the APC group, 55 in the IPC group, and 49 in the ischemia group displayed significant > or = 2.0-fold down-regulation. Many of these transcripts were unknown genes. A high number of commonly regulated genes were found in IPC and APC (39 up-regulated, 17 down-regulated). Genes commonly regulated included those associated with cell defense (heat shock protein 10, aldose reductase, Bcl-xS). Conversely, a pool of protective and antiprotective genes was differentially regulated in APC versus IPC (heat shock protein 27/70, programmed cell death 8), suggesting trigger-dependent transcriptome variability. Conclusions The novel microarray technology provides evidence for distinct cardioprotective phenotypes in IPC and APC. The observed transcriptional changes raise the possibility of a second window of protection by volatile anesthetics. The authors' molecular portraits are the first global genomic comparison between IPC and APC.
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van der Meer, Felix J. M., Nico H. van Tilburg, Aat van Wijngaarden, Irma K. van der Linden, Ernest Briët, and Rogier M. Bertina. "A Second Plasma Inhibitor of Activated Protein C: α1-Antitrypsin." Thrombosis and Haemostasis 62, no. 02 (1989): 756–62. http://dx.doi.org/10.1055/s-0038-1646897.
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SummaryInactivation of activated protein C (APC) in normal human plasma was studied in the absence and presence of heparin. In the absence of heparin APC inactivation followed pseudo-first order kinetics. In the presence of heparin the neutralization of APC was found to be biphasic. Up to 500 nM APC could be readily inactivated in normal plasma, indicating that the concentration of the APC inhibitor must be higher than previously assumed. Plasma deficient in the protein C inhibitor (PCI-I, as described by Suzuki and coworkers) and deficient in p2-glycoprotein I still possessed APC neutralizing capacity, presumably through the formation of complexes of APC with another plasma protein as was demonstrated by immunoblotting with anti-protein C antibodies. Together these data made us to conclude that a second inhibitor of APC (PCI-II) must be present in normal human plasma. This second inhibitor should be heparin independent, have a relatively high plasma concentration and form complexes with APC.Subsequently, we purified this PCI-II by isolating APC-PCI-II complexes from plasma deficient of vitamin K dependent proteins, PCI-I and β2-glycoprotein-I, to which purified human APC had been added. Purified PCI-II has a molecular weight of 50,000 daltons and aminoacid analysis revealed that PCI-II is identical with α1-antitrypsin (α1-AT). The second order rate constant for the reaction between purified α1-AT and APC was found to be 269 M−1 min−1 in the absence of calcium and 602 M−1 min−1 in the presence of calcium.Finally, the analysis of the kinetics of APC inactivation in the plasma of a patient congenitally deficient in α1-AT (3% α1-AT antigen) revealed that there probably is a third inhibitor of APC in plasma, different from PCI-I and from α1-AT.
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Riess, Matthias L., Leo G. Kevin, Amadou K. S. Camara, James S. Heisner, and David F. Stowe. "Dual Exposure to Sevoflurane Improves Anesthetic Preconditioning in Intact Hearts." Anesthesiology 100, no. 3 (March 1, 2004): 569–74. http://dx.doi.org/10.1097/00000542-200403000-00016.
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Background Anesthetic preconditioning (APC) with sevoflurane reduces myocardial ischemia-reperfusion injury. The authors tested whether two brief exposures to sevoflurane would lead to a better preconditioning state than would a single longer exposure and whether dual exposure to a lower (L) concentration of sevoflurane would achieve an outcome similar to that associated with a single exposure to a higher (H) concentration. Methods Langendorff-prepared guinea pig hearts were exposed to 0.4 mM sevoflurane once for 15 min (H1-15; n = 8) or 0.4 mM (H2-5; n = 8) or 0.2 mM sevoflurane (L2-5; n = 8) twice for 5 min, with a 5-min washout period interspersed. Sevoflurane was then washed out for 20 min before 30 min of global no-flow ischemia and 120 min of reperfusion. Control hearts (n = 8) were not subjected to APC. Left ventricular pressure was measured isovolumetrically. Ventricular infarct size was determined by tetrazolium staining and cumulative planimetry. Values are expressed as mean +/- SD. Results The authors found a better functional return and a lesser percentage of infarction on reperfusion in H2-5 (28 +/- 9%) than in H1-15 (36 +/- 8%; P < 0.05), L2-5 (43 +/- 6%; P < 0.05), or control hearts (52 +/- 7%; P < 0.05). Conclusion These results suggest that APC depends not only on the concentration but also on the protocol used for preconditioning. Similarly to ischemic preconditioning, repeated application of the volatile anesthetic seems to be more important than the duration of exposure in initiating the signaling sequence that elicits APC at clinically relevant concentrations. Therefore, repeated cycles of anesthetic exposure followed by volatile anesthetic-free periods may be beneficial for APC in the clinical setting.
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Novalija, Enis, Leo G. Kevin, Amadou K. S. Camara, Zeljko J. Bosnjak, John P. Kampine, and David F. Stowe. "Reactive Oxygen Species Precede the ε Isoform of Protein Kinase C in the Anesthetic Preconditioning Signaling Cascade." Anesthesiology 99, no. 2 (August 1, 2003): 421–28. http://dx.doi.org/10.1097/00000542-200308000-00024.
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Background Protein kinase C (PKC) and reactive oxygen species (ROS) are known to have a role in anesthetic preconditioning (APC). Cardiac preconditioning by triggers other than volatile anesthetics, such as opioids or brief ischemia, is known to be isoform selective, but the isoform required for APC is not known. The authors aimed to identify the PKC isoform that is involved in APC and to elucidate the relative positions of PKC activation and ROS formation in the APC signaling cascade. Methods Isolated guinea pig hearts were subjected to 30 min of ischemia and 120 min of reperfusion. Before ischemia, hearts were either untreated or treated with sevoflurane (APC) in the absence or presence of the nonspecific PKC inhibitor chelerythrine, the PKC-delta inhibitor PP101, or the PKC-epsilon inhibitor PP149. Spectrofluorometry and the fluorescent probes dihydroethidium were used to measure intracellular ROS, and effluent dityrosine as used to measure extracellular ROS release. Results Previous sevoflurane exposure protected the heart against ischemia-reperfusion injury, as previously described. Chelerythrine or PP149 abolished protection, but PP101 did not. ROS formation was observed during sevoflurane exposure and was not altered by any of the PKC inhibitors. Conclusions APC is mediated by PKC-epsilon but not by PKC-delta. Furthermore, PKC activation probably occurs downstream of ROS generation in the APC signaling cascade.
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Lian, Hailing, and Molly Wang. "Prof. Min Li: three heads are better than one." Annals of Pancreatic Cancer 1 (September 2018): 27. http://dx.doi.org/10.21037/apc.2018.08.04.
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Souris, Jeffrey S., Hannah J. Zhang, Urszula Dougherty, Nai-Tzu Chen, Joseph V. Waller, Leu-Wei Lo, John Hart, Chin-Tu Chen, and Marc Bissonnette. "A novel mouse model of sporadic colon cancer induced by combination of conditional Apc genes and chemical carcinogen in the absence of Cre recombinase." Carcinogenesis 40, no. 11 (March 12, 2019): 1376–86. http://dx.doi.org/10.1093/carcin/bgz050.
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AbstractAlthough valuable insights into colon cancer biology have been garnered from human colon cancer cell lines and primary colonic tissues, and animal studies using human colon cancer xenografts, immunocompetent mouse models of spontaneous or chemically induced colon cancer better phenocopy human disease. As most sporadic human colon tumors present adenomatous polyposis coli (APC) gene mutations, considerable effort has gone into developing mice that express mutant Apc alleles that mimic human colon cancer pathogenesis. A serious limitation of many of these Apc-mutant murine models, however, is that these mice develop numerous tumors in the small intestine but few, if any, in the colon. In this work, we examined three spontaneous mouse models of colon tumorigenesis based upon the widely used multiple intestinal neoplasia (Min) mouse: mice with either constitutive or conditional Apc mutations alone or in combination with caudal-related homeobox transcription factor CDX2P-Cre transgene — either with or without exposure to the potent colon carcinogen azoxymethane. Using the CDX2 promoter to drive Cre recombinase transgene expression effectively inactivated Apc in colonocytes, creating a model with earlier tumor onset and increased tumor incidence/burden, but without the Min mouse model’s small intestine tumorigenesis and susceptibility to intestinal perforation/ulceration/hemorrhage. Most significantly, azoxymethane-treated mice with conditional Apc expression, but absent the Cre recombinase gene, demonstrated nearly 50% tumor incidence with two or more large colon tumors per mouse of human-like histology, but no small intestine tumors — unlike the azoxymethane-resistant C57BL/6J-background Min mouse model. As such this model provides a robust platform for chemoprevention studies.
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Omata, Ryo, Yusuke Hattori, Tetsuo Sasaki, Tomoaki Sakamoto, and Makoto Otsuka. "Elucidation of the Molecular Mechanism of Wet Granulation for Pharmaceutical Standard Formulations in a High-Speed Shear Mixer Using Near-Infrared Spectroscopy." Pharmaceuticals 13, no. 9 (August 31, 2020): 226. http://dx.doi.org/10.3390/ph13090226.
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The granulation process of pharmaceutical standard formulation in a high-speed shear wet granulation (HSWG) was measured by in-line near-infrared spectroscopy (NIRS) and agitation power consumption (APC) methods. The F-1, F-2, and F-3 formulations (500 g) contained 96% w/w α-lactose monohydrate (LA), potato starch (PS), and a LA:PS = 7:3 mixture, respectively, and all the formulations contained 4% w/w hydroxypropyl cellulose. While adding purified water at 10 mL/min, the sample powder was mixed. The calibration models to measure the amount of binding water (Wa) and APC of the HSWG formulations were established based on NIRS of the samples measured for 60 min by partial least-squares regression analysis (PLS). Molecular interaction related to APC between the particle surface and binding liquor was analyzed based on NIRS. The predicted values of Wa and APC for all formulations were superimposed with the measured values on a straight line, respectively. The regression vector (RV) of the calibration model for Wa indicated the chemical information of all the water in the samples. In contrast, the RV for APC suggested that APC changes in the processes are related to powder aggregation because of surface tension of binding water between particles.
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Matsuda, Akitoshi, Naohisa Kuriyama, Hiroyuki Kato, Akihiro Tanemura, Yasuhiro Murata, Yoshinori Azumi, Masashi Kishiwada, et al. "Comparative Study on the Cytoprotective Effects of Activated Protein C Treatment in Nonsteatotic and Steatotic Livers under Ischemia-Reperfusion Injury." BioMed Research International 2015 (2015): 1–13. http://dx.doi.org/10.1155/2015/635041.
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Activated protein C (APC) has cytoprotective effects on liver ischemia-reperfusion injury (IRI). However, it is unclear whether APC is beneficial in steatotic liver IRI. We compared the cytoprotective effects of APC in nonsteatotic and steatotic liver IRI.Methods.Mice fed either normal diets (ND mice) or high fat diets (HF mice), were treated with APC or saline (control) and were performed 60 min partial IRI. Moreover, primary steatotic hepatocytes were either untreated or treated with APC and then incubated with H2O2.Results.APC significantly reduced serum transaminase levels and the inflammatory cells infiltration compared with control at 4 h in ND mice and at 24 h in HF mice. APC inhibited sinusoidal endothelial injury in ND mice, but not in HF mice. In contrast, APC activated adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in HF mice, but not in ND mice. In the in vitro study, APC significantly increased AMPK phosphorylation, ATP concentration, and survival rates of hepatocytes compared with control.Conclusion.During IRI in normal liver, APC attenuated initial damage by inhibiting inflammatory cell infiltration and sinusoidal endothelial injury, but not in steatotic liver. However, in steatotic liver, APC might attenuate late damage via activation of AMPK.
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Moran, Amy E., Adelaide M. Carothers, Michael J. Weyant, Mark Redston, and Monica M. Bertagnolli. "Carnosol Inhibits β-Catenin Tyrosine Phosphorylation and Prevents Adenoma Formation in the C57BL/6J/Min/+ (Min/+) Mouse." Cancer Research 65, no. 3 (February 1, 2005): 1097–104. http://dx.doi.org/10.1158/0008-5472.1097.65.3.
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Abstract Carnosol, a constituent of the herb, rosemary, has shown beneficial medicinal and antitumor effects. Using the C57BL/6J/Min/+ (Min/+) mouse, a model of colonic tumorigenesis, we found that dietary administration of 0.1% carnosol decreased intestinal tumor multiplicity by 46%. Previous studies showed that tumor formation in the Min/+ mouse was associated with alterations in the adherens junctions, including an increased expression of tyrosine-phosphorylated β-catenin, dissociation of β-catenin from E-cadherin, and strongly reduced amounts of E-cadherin located at lateral plasma membranes of histologically normal enterocytes. Here, we confirm these findings and show that treatment of Min/+ intestinal tissue with carnosol restored both E-cadherin and β-catenin to these enterocyte membranes, yielding a phenotype similar to that of the Apc+/+ wild-type (WT) littermate. Moreover, treatment of WT intestine with the phosphatase inhibitor, pervanadate, removed E-cadherin and β-catenin from the lateral membranes of enterocytes, mimicking the appearance of the Min/+ tissue. Pretreatment of WT tissue with carnosol inhibited the pervanadate-inducible expression of tyrosine-phosphorylated β-catenin. Thus, the ApcMin allele produces adhesion defects that involve up-regulated expression of tyrosine-phosphorylated proteins, including β-catenin. Moreover, these data suggest that carnosol prevents Apc-associated intestinal tumorigenesis, potentially via its ability to enhance E-cadherin-mediated adhesion and suppress β-catenin tyrosine phosphorylation.
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Linden, M. D., M. Schneider, and W. N. Erber. "Factor VLEIDEN and cardiopulmonary bypass: investigation of haemostatic parameters and the effect of aprotinin using an ex vivo model." Perfusion 16, no. 6 (December 2001): 476–84. http://dx.doi.org/10.1177/026765910101600607.
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It has been suggested that aprotinin results in significantly increased risk for perioperative thrombotic complications in patients with Factor VLEIDEN (F5L) due to its ability to competitively inhibit activated protein C (APC) function in vitro. No clinical studies have been performed to assess the effect of aprotinin on APC function of F5L in vivo. We developed an ex vivo model to mimic the effects of cardiopulmonary bypass with the exclusion of the patient in order to assess APC function. Blood from normal ( n = 2) and F5L heterozygous donors ( n = 2) was treated with aprotinin or placebo (saline). The blood was heparinized, added to the prime and circulated at 2 l/min through a modified cardiopulmonary bypass circuit. After 60 min of circulation, the heparin was neutralized with protamine sulfate. Blood samples, drawn at specific time points, were analysed for APC ratio. Results showed a decrease in APC ratio for both F5L and normal bloods with the addition of aprotinin (18% and 40%, respectively). APC ratios also decreased with the commencement of extracorporeal circulation for all bloods, resulting in an APC ratio of 1.35 in normal placebo-treated blood and 0.67 in F5L placebo-treated blood. The combined effect of aprotinin and extracorporeal circulation resulted in APC ratios of 0.90 for normal blood and 0.63 for F5L blood, corresponding to a severe dysfunction of APC intraoperatively (reference range 1.9-4.0). The data from this model predict an increased risk of perioperative thrombosis due to inhibition of APC function in cardiac surgical patients heterozygous for the F5L mutation. Aprotinin further compounds the severity of APC dysfunction, though the effect is more severe in normal blood. The ex vivo model employed was an effective tool for the investigation of the haemostatic effect of aprotinin. This model may be exploited for other applications such as the investigation of novel or emerging haemostatic agents prior to clinical trial.
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Deane, Rashid, Barbra LaRue, Abhay P. Sagare, Francis J. Castellino, Zhihui Zhong, and Berislav V. Zlokovic. "Endothelial Protein C Receptor-Assisted Transport of Activated Protein C across the Mouse Blood—Brain Barrier." Journal of Cerebral Blood Flow & Metabolism 29, no. 1 (October 8, 2008): 25–33. http://dx.doi.org/10.1038/jcbfm.2008.117.
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Activated protein C (APC), a serine-protease with anticoagulant, anti-inflammatory, and cytoprotective activities, is neuroprotective and holds potential to treat different neurologic disorders. It is unknown whether APC crosses the blood—brain barrier (BBB) to reach its therapeutic targets in the brain. By using a brain vascular perfusion technique, we show that 125I-labeled plasma-derived mouse APC enters the brain from cerebrovascular circulation by a concentration-dependent mechanism. The permeability surface area product of 125I-APC (0.1 nmol/L) in different forebrain regions ranged from 3.11 to 4.13 μL/min/g brain. This was approximately 80- to 110-fold greater than for 14C-inulin, a simultaneously infused reference tracer. The Km value for APC BBB cortical transport was 1.6±0.2 nmol/L. Recombinant APC variants with reduced anticoagulant activity, 5A-APC and 3K3A-APC, but not protein C, exhibited high affinity for the APC BBB transport system. Blockade of APC-binding site on endothelial protein C receptor (EPCR), but not blockade of its protease-activated receptor-1 (PAR1) catalytic site, inhibited by > 85% APC entry into the brain. APC brain uptake was reduced by 64% in severely deficient EPCR mice, but not in PAR1 null mice. These data suggest that APC and its variants with reduced anticoagulant activity cross the BBB via EPCR-mediated saturable transport.
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Uecker, Marina, Rafaela da Silva, Thomas Grampp, Thomas Pasch, Marcus C. Schaub, and Michael Zaugg. "Translocation of Protein Kinase C Isoforms to Subcellular Targets in Ischemic and Anesthetic Preconditioning." Anesthesiology 99, no. 1 (July 1, 2003): 138–47. http://dx.doi.org/10.1097/00000542-200307000-00023.
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Background Translocation of protein kinase C (PKC) to subcellular targets is a pivotal signaling step in ischemic preconditioning (IPC). However, to date, it is unknown whether PKC isoforms translocate in anesthetic preconditioning (APC). Methods The PKC blockers chelerythrine and rottlerin and the adenosine triphosphate-dependent potassium (K(ATP)) channel blockers HMR-1098 and 5-hydroxydecanoate were used to assess the role of PKC and K(ATP) channels in isolated perfused rat hearts subjected to IPC or APC (1.5 minimum alveolar concentration isoflurane) followed by 40 min of ischemia and 30 min of reperfusion. Immunohistochemical techniques were used to visualize PKC translocation after preconditioning. In addition, the phosphorylation status of PKC isoforms was assessed. Results Chelerythrine, rottlerin, and 5-hydroxydecanoate blocked IPC and APC with respect to functional recovery, albeit IPC at higher concentrations. HMR-1098 did not affect IPC or APC. PKCdelta and PKCepsilon translocated to nuclei in both IPC and APC, which was inhibited by chelerythrine and rottlerin. PKCdelta translocated to mitochondria but not to the sarcolemma, and PKCepsilon translocated to the sarcolemma and intercalated disks but not to mitochondria. Interestingly, PKCepsilon was accumulated at the intercalated disks in control and preconditioned hearts. Phosphorylation of PKCdelta on serine643 was increased in IPC and APC and blocked by chelerythrine and rottlerin, whereas phosphorylation of PKCdelta on threonine505 was increased only in IPC and not blocked by chelerythrine or rottlerin. PKCepsilon on serine729 did not change its phosphorylation status. Conclusions This study indicates that translocation of PKCdelta plays a pivotal role in IPC and APC and suggests that phosphorylation of PKCdelta on serine643 may be of particular relevance in transferring the APC stimulus to mitochondrial K(ATP) channels.
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Ishigamori, Rikako, Masami Komiya, Shinji Takasu, Michihiro Mutoh, Toshio Imai, and Mami Takahashi. "Osteopontin Deficiency Suppresses Intestinal Tumor Development in Apc-Deficient Min Mice." International Journal of Molecular Sciences 18, no. 5 (May 14, 2017): 1058. http://dx.doi.org/10.3390/ijms18051058.
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Jacobasch, Gisela, Gerhard Dongowski, Simone Florian, Katrin Müller-Schmehl, Barbara Raab, and Detlef Schmiedl. "Pectin Does Not Inhibit Intestinal Carcinogenesis in APC-Deficient Min/+ Mice." Journal of Agricultural and Food Chemistry 56, no. 4 (February 2008): 1501–10. http://dx.doi.org/10.1021/jf070872l.
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McCart, A., J. Haines, N. Suraweera, I. Tomlinson, and A. Silver. "Characterisation of an intestinal neoplasm modifier locus in Apc Min mice." European Journal of Cancer Supplements 6, no. 9 (July 2008): 38. http://dx.doi.org/10.1016/s1359-6349(08)71321-1.
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Tao, Yun, Jeannette S. Messer, Kathleen H. Goss, John Hart, Marc Bissonnette, and Eugene B. Chang. "Hsp70 exerts oncogenic activity in the Apc mutant Min mouse model." Carcinogenesis 37, no. 7 (May 4, 2016): 731–39. http://dx.doi.org/10.1093/carcin/bgw056.
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Rühl, Heiko, Janine Rossa, Christina Berens, Anna Winterhagen, Johannes Oldenburg, Jens Müller, and Bernd Pötzsch. "Evidence of Thrombin-Independent Generation of Activated Protein C." Blood 128, no. 22 (December 2, 2016): 2568. http://dx.doi.org/10.1182/blood.v128.22.2568.2568.
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Abstract Introduction: In a recent study, we performed autologous serum infusions to evaluate the elimination kinetics of hemostasis-related biomarkers in healthy human subjects. In order to monitor a serum-induced activation of coagulation, we measured free thrombin in the infused serum and in plasma samples taken during and after infusion, but did not detect any de novo thrombin formation [PLoS One. 2015; 10(12): e0145012]. To study if the low levels of free thrombin in the infused serum induce generation of activated protein C (APC) we additionally measured APC in samples drawn after autologous serum infusion and in vitro in a purified system. Methods: Autologous serum was infused (50 mL/30 min) into 19 healthy volunteers. Four of them were simultaneously receiving infusions of the thrombin inhibitor argatroban (1 µg/kg/min), initiated 1 h before and ceased 1 h after starting the infusion of serum. Thrombin and APC were measured in serum and in plasma samples drawn before and in 15-min intervals during the infusion of serum, using a highly-sensitive oligonucleotide-based enzyme capture assay (OECA) platform. In in vitro experiments, APC formation was induced by addition of purified thrombin or serum to buffer containing protein C and thrombomodulin in excess, and CaCl2 at physiological concentrations. The formation of APC was subsequently measured by OECA. Results: In the autologous serum median (interquartile range) concentrations of thrombin and APC were 6.68 (4.63 - 8.73) ng/mL and 9.17 (7.63 - 13.91) ng/mL, thus doses of 0.12 (0.07 - 0.15) ng/mL of thrombin and 0.16 (0.14 - 0.22) ng/mL of APC were infused per mL of the subjects' plasma volume. In the plasma of probands, that did not receive argatroban, peak thrombin levels of 0.04 (0.00 - 0.08) ng/mL were measured, indicating a rapid inactivation of thrombin by endogenous inhibitors present in the plasma. However, with 1.41 (0.76 - 2.97) ng/mL peak APC levels exceeded the infused APC doses by a multiple. This was also true for the plasma samples from the probands that received argatroban, in which peak levels of APC of 0.94 (0.79 - 1.22) ng/mL were measured despite thrombin inhibition indicated by prolongation of the aPTT of 42.9 (40.1 - 44.4) s and thrombin time of 78.3 (69.3 - 87.2) s. In the in vitro experiments addition of argatroban at the concentrations achieved in the probands completely abolished APC generation up to a thrombin concentration of 5 ng/ml. Addition of human serum as a source for thrombin in the same purified system consistently induced generation of greater amounts of APC than expected on the basis of the amount of thrombin present in the serum samples. Conclusions: The data obtained provide evidence for a thrombin-independent mechanism of APC formation. Further in vitro studies with endothelial cells are required to identify the components that are involved in this alternative way of APC generation. Disclosures Rühl: CSL Behring: Research Funding; Bayer: Consultancy, Honoraria. Müller:Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.
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Molinari, J. F., W. R. Moore, J. Clark, R. Tanaka, J. H. Butterfield, and W. M. Abraham. "Role of tryptase in immediate cutaneous responses in allergic sheep." Journal of Applied Physiology 79, no. 6 (December 1, 1995): 1966–70. http://dx.doi.org/10.1152/jappl.1995.79.6.1966.
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In this study, we used a specific tryptase inhibitor, APC-366 [N-(1-hydroxy-2-napthoyl)-L-arginyl-L- prolinamide hydrochloride] to investigate the effect of intradermally administered tryptase and tryptase released by antigen challenge on the immediate cutaneous reaction (ICR) in allergic sheep. The surface areas of cutaneous wheals produced by intradermal injections (0.05 ml) of 1 and 10 ng tryptase alone, tryptase combined with 3 U heparin (tryptase-heparin), or Ascaris suum antigen (10(-5) dilution) with or without pretreatment with APC-366 (1 mg/ml) were measured at 20 and 60 min after challenge. Intradermal injections of 1 and 10 ng tryptase alone (n = 7) produced an ICR of < or = 20% of that obtained after injection of histamine (5% wt/vol). Intradermal injection of tryptase-heparin (n = 7), however, resulted in 50 (1 ng) and 82% (10 ng) of the ICR to histamine (both, P < 0.05 vs. tryptase alone). APC-366 inhibited (P < 0.05) the ICR to 1 and 10 ng tryptase-heparin by > or = 70% at all times (n = 8) but had no effect on the histamine-induced ICR (n = 3). A combination of the histamine H1 antagonist chlorpheniramine (2 mg/kg iv) and the H2 antagonist metiamide (3 mg/kg iv) given 40 min before challenge (n = 8) inhibited the response to 1 and 10 ng tryptase-heparin by 42 and 62% at 20 min and by 96 and 86% at 60 min, respectively (all, P < 0.05). APC-366 also blocked the ICR to A. suum antigen by 68% (P < 0.05) in nine sheep. These results indicate that intradermal injection of tryptase-heparin can induce an ICR. This ICR can be inhibited by APC-366 or a combination of the histamine H1 and H2 antagonists, suggesting that the tryptase response is mediated by histamine. APC-366 also blocks the mast cell-mediated ICR to intradermally injected A. suum antigen. Collectively, these results suggest that tryptase may modulate mast cell histamine release.
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Gruber, Andras, Jose A. Fernandez, Ulla M. Marzec, Leslie Bush, John H. Griffin, Stephen R. Hanson, and Enrico Di Cera. "Sustained Pharmacological Activation of Protein C (PC) in Baboons." Blood 104, no. 11 (November 16, 2004): 3499. http://dx.doi.org/10.1182/blood.v104.11.3499.3499.
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Abstract The introduction of two mutations at the specificity sites S3-S4 of thrombin (W215A and E217A) results in a mutant thrombin (WE) with more than 1000-fold reduced activity towards fibrinogen and PAR1. WE retains its antithrombotic activity via selective activation of the circulating PC pool and elevation of activated PC (APC) levels when administered as a bolus to primates (Gruber et al. Blood 2002). We investigated whether increased APC levels can be sustained and/or the PC pool exhausted by a 4 to 6 hour long infusion of WE in 15 baboons. In order to assess whether intravascular thrombomodulin availability is rate limiting in pharmacological PC activation, the effect of high dose WE administration on circulating APC (enzyme capture assay) and PC activity (Protac-activatable PC) levels were tested with or without co-administration of equimolar doses of recombinant human soluble thrombomodulin (rhsTM). Infusion of a very high dose of WE alone (110 ug/kg bolus followed by 110 ug/kg/h) gave a peak APC concentration of 201 ng/mL by 10 min. The endogenous APC concentration then sharply decreased with an apparent initial half life (T1/2) of 25 min - similar to that of exogenous human APC in baboons and almost returned to baseline (8 ng/mL) by the end of the treatment despite continued infusion of high dose WE. Thrombin-antithrombin (TAT) complex levels gradually increased reaching an apparent equilibrium at 104 ng/mL by the 4th hour. D-Dimer levels increased throughout the treatment and reached a level that was 20-fold of the baseline value by the end of the treatment. Co-administration of rhsTM (180 ug/kg bolus + 180 ug/kg/h infusion) with the high dose WE also resulted in rapid PC activation and an early APC peak (240 ng/ml at 10 min). The enzyme peak was followed by a decline in APC concentration with an apparent initial T1/2 of 51 min to an average equilibrium concentration of 40 ng/mL by the end of the treatment. TAT levels sharply increased from the 3.5 ng/mL baseline level, reaching greater than 240 ng/ml by the 10th min of the treatment, and remained outside the range of the test. There was a nonsignificant increase in D-Dimer levels towards the end of the WE+rhsTM treatment. Approximately 40% and 80% of the Protac-activatable circulating PC pool was consumed by administration of WE and WE+rhsTM, respectively. Based on the findings we propose that: 1) there is sufficient PC substrate for a sustained pharmacological increase in endogenous APC levels; 2) either complex formation of WE with antithrombin is significantly accelerated by rhsTM or clearance of TAT is inhibited by rhsTM; 3) the existing endogenous soluble and/or endothelial thrombomodulin pool appears to be transiently depleted upon WE infusion; and 4) the circulating PC pool might not be homogeneous such that part of the PC pool is not activatable in vivo by infused WE+rhsTM. In summary, significant and sustained levels of endogenous circulating APC can be generated in baboons by infusion of rhsTM with the highly selective WE thrombin mutant.
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Schilder, Louise, S. Azam Nurmohamed, Pieter M. ter Wee, Nanne J. Paauw, Armand R. J. Girbes, Albertus Beishuizen, Robert H. J. Beelen, and A. B. Johan Groeneveld. "Coagulation, Fibrinolysis and Inhibitors in Failing Filters during Continuous Venovenous Hemofiltration in Critically Ill Patients with Acute Kidney Injury: Effect of Anticoagulation Modalities." Blood Purification 39, no. 4 (2015): 297–305. http://dx.doi.org/10.1159/000380904.
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Introduction: The mechanisms of early filter failure and clotting with different anticoagulation modalities during continuous venovenous hemofiltration (CVVH) are largely unknown. Methods: Citrate, heparin and no anticoagulation were compared. Blood was drawn pre- and post filter up to 720 min. Concentrations of the thrombin-antithrombin (TAT), activated protein C-protein C inhibitor (APC-PCI), and type I plasminogen activator inhibitor (PAI-1) were determined. Results: In case of early filter failure (<24 h), inlet concentrations of TAT and APC-PCI were higher over time, irrespective of anticoagulation. There was more production of APC-PCI and platelet-derived PAI-1 in the filter after 10 min in the heparin group than in other groups. In clotting filters, production of APC-PCI and PAI was also higher with heparin than citrate. Conclusion: Coagulation activation in plasma and inhibition of anticoagulation in plasma and filter may partly determine early CVVH filter failure due to clotting, particularly when heparin is used. Regional anticoagulation by citrate circumvents the inhibition of anticoagulation and fibrinolysis by platelet activation following heparin.
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Inaba, K., and R. M. Steinman. "Accessory cell-T lymphocyte interactions. Antigen-dependent and -independent clustering." Journal of Experimental Medicine 163, no. 2 (February 1, 1986): 247–61. http://dx.doi.org/10.1084/jem.163.2.247.
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Previous work documented the capacity of dendritic cells (DC) to stimulate primary immune responses and to physically cluster with the responding lymphocytes. Rapid cell-cell aggregation assays were used here to study the interaction of DC and other types of APC with T lymphocytes. Graded doses of APC were sedimented with T cells that had been primed to alloantigens, soluble proteins, or lectin, and then labeled with carboxyfluorescein diacetate. The number of clustered T cells was measured after 10 min at 4 or 37 degrees C. At 4 degrees, binding was antigen-dependent and included greater than 50% of the added T cells. Clustering was mediated by all types of APC tested, including DC, macrophages, B lymphocytes, and fresh Langerhans cells, although DC were the most effective. Specificity was evident in the findings that alloreactive T lymphoblasts bound to allogeneic but not syngeneic APC; KLH- and OVA-reactive T cells bound to syngeneic APC in the presence of specific protein: and Con A blasts needed lectin to cluster. A 30 min pretreatment with chloroquine, a drug known to inhibit APC activity, markedly blocked the specific binding of alloreactive and protein-specific T blasts at 4 degrees C. Since Lyt-2- alloreactive blasts should specifically recognize Ia, presentation of Ia seems to be altered by chloroquine. Binding assays at 37 degrees C gave similar results to those performed at 4 degrees C, with one exception. When DC were used as APC, striking antigen-independent clustering occurred. DC could efficiently cluster primed T cells in the absence of alloantigen, soluble protein, or lectin. We suggest that antigen-independent binding contributes to the distinctive capacity of DC to prime T cells in the afferent limb of the immune response, whereas antigen-dependent binding between other APC and sensitized lymphocytes is critical in the efferent limb.
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Mehl, Kristen A., J. Mark Davis, Julie M. Clements, Franklin G. Berger, Maria M. Pena, and James A. Carson. "Decreased intestinal polyp multiplicity is related to exercise mode and gender in ApcMin/+ mice." Journal of Applied Physiology 98, no. 6 (June 2005): 2219–25. http://dx.doi.org/10.1152/japplphysiol.00975.2004.
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Moderate-intensity treadmill running can alter male Apc Min/+ mouse polyp formation. This purpose of this study was to examine whether exercise mode differentially affects Apc Min/+ mouse intestinal polyp development in male and female mice. Male and female Apc Min/+ mice were randomly assigned to control, treadmill (18 m/min; 60 min/day; 6 days/wk), or voluntary wheel running (24-h access) groups. Nine weeks of training decreased total intestinal polyps by 29% in male treadmill runners (66 ± 9; P = 0.038) compared with male controls (93 ± 7). The number of large polyps (≥1-mm diameter) were also reduced by 38% in male treadmill runners (49 ± 6; P = 0.005) compared with male controls (79 ± 6). Treadmill running in female Apc Min/+ mice and wheel running in both genders did not affect polyp number or size. Spleen weight decreased in male treadmill runners (91 ± 9 mg; P = 0.011) and wheel runners (75 ± 6 mg; P = 0.004) compared with controls (141 ± 13 mg). Plasma IL-6 was reduced by 96% in male treadmill runners (1.2 ± 0.6 pg/ml) and 78% in male wheel runners (6.6 ± 3.3 pg/ml) compared with control mice (27.9 ± 2.8 pg/ml; P < 0.05). Female mice responded similarly with an 86% decrease in plasma IL-6 with treadmill running (3.2 ± 1.2 pg/ml) and 90% decrease with wheel running (2.9 ± 2.0 pg/ml) compared with control mice (21.1 ± 5.3 pg/ml; P < 0.05). The crypt depth-to-villus height ratio in the intestine, an indirect marker of intestinal inflammation, decreased by 21 ( P = 0.024) and 24% ( P = 0.029), respectively, in male and female treadmill runners but not wheel runners. Physical activity-induced attenuation of intestinal polyp number and size is dependent on exercise mode and differs between genders. The modulation of systemic and intestinal inflammation may also depend on exercise mode.
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Amour, Julien, Anna K. Brzezinska, Dorothee Weihrauch, Amie R. Billstrom, Jacek Zielonka, John G. Krolikowski, Martin W. Bienengraeber, David C. Warltier, Philip F. Pratt, and Judy R. Kersten. "Role of Heat Shock Protein 90 and Endothelial Nitric Oxide Synthase during Early Anesthetic and Ischemic Preconditioning." Anesthesiology 110, no. 2 (February 1, 2009): 317–25. http://dx.doi.org/10.1097/aln.0b013e3181942cb4.
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Background Nitric oxide is known to be essential for early anesthetic preconditioning (APC) and ischemic preconditioning (IPC) of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, the authors tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC. Methods Myocardial infarct size was measured in rabbits after coronary occlusion and reperfusion in the absence or presence of preconditioning within 30 min of isoflurane (APC) or 5 min of coronary artery occlusion (IPC), and with or without pretreatment with geldanamycin or radicicol, two chemically distinct Hsp90 inhibitors, or N-nitro-L-arginine methyl ester, a nonspecific nitric oxide synthase NOS inhibitor. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells or mouse cardiomyocytes, in the absence or presence of Hsp90 inhibitors or N-nitro-L-arginine methyl ester. Interactions between Hsp90 and eNOS, and eNOS activation, were assessed with immunoprecipitation, immunoblotting, and confocal microscopy. Results APC and IPC decreased infarct size (by 50% and 59%, respectively), and this action was abolished by Hsp90 inhibitors. N-nitro-L-arginine methyl ester blocked APC but not IPC. Isoflurane increased nitric oxide production in human coronary artery endothelial cells concomitantly with an increase in Hsp90-eNOS interaction (immunoprecipitation, immunoblotting, and immunohistochemistry). Pretreatment with Hsp90 inhibitors abolished isoflurane-dependent nitric oxide production and decreased Hsp90-eNOS interactions. Isoflurane did not increase nitric oxide production in mouse cardiomyocytes, and eNOS was below the level of detection. Conclusion The results indicate that Hsp90 plays a critical role in mediating APC and IPC through protein-protein interactions, and suggest that endothelial cells are important contributors to nitric oxide-mediated signaling during APC.
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Orthner, Carolyn L., Billy Kolen, and William N. Drohan. "A Sensitive and Facile Assay for the Measurement of Activated Protein C Activity Levels in Vivo." Thrombosis and Haemostasis 69, no. 05 (1993): 441–47. http://dx.doi.org/10.1055/s-0038-1651630.
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SummaryActivated protein C (APC) is a serine protease which plays an important role as a naturally occurring antithrombotic enzyme. APC, which is formed by thrombin-catalyzed limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic inactivation of the coagulation cofactors VIIIa and Va. APC is inhibited by several members of the serpin family as well a by α2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment of thrombosis. We have developed an assay to quantify circulating levels of enzymatically active APC during its administration to patients, in healthy individuals, and in various disease states. This assay utilizes an EDTA-dependent anti-protein C monoclonal antibody (Mab) 7D7B10 to capture both APC and protein C from plasma, prepared from blood collected in an anticoagulant supplemented with the reversible inhibitor p-aminobenzamidine. Mab 7D7B10-derivatized agarose beads are added to the wells of a 96-well filtration plate, equilibrated with Tris-buffered saline, and incubated for 10 min with 200 μl of plasma. After washing, APC and protein C are eluted from the immunosorbent beads with a calcium-containing buffer into the wells of a 96-well microtiter plate containing antithrombin III (ATIII) and heparin. The amidolytic activity of APC is then measured on a kinetic plate reader following the addition of L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate.The rate of substrate hydrolysis was proportional to APC concentration over a 200-fold concentration range (5.0 to 1,000 ng/ml) when measured continuously over a 15 to 30 min time period. The coefficient of variation was 5.9% at 35 ng/ml and 8.8% at 350 ng/ml APC. The sensitivity of the assay could be increased by measuring the amount of color produced after longer incubation times in the endpoint mode. The measured APC activity levels were little affected by varying protein C or prothrombin over the extremes of 0 to 150% of normal plasma concentrations. By constructing the standard curve in protein C-deficient plasma, the concentration of APC activity in normal pooled plasma was determined to be 2.8 ng/ml (45 pM), which represents 0.08% of the protein C concentration. The assay was approximately 50-fold more sensitive than the identical assay, but using Mab-coated microtiter wells rather than immunosorbent beads as the capture step.
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Yoshida, Michihiro, Luqing Zhao, Gevorg Grigoryan, Hyunsuk Shim, Peijian He, and C. Chris Yun. "Deletion of Na+/H+ exchanger regulatory factor 2 represses colon cancer progress by suppression of Stat3 and CD24." American Journal of Physiology-Gastrointestinal and Liver Physiology 310, no. 8 (April 15, 2016): G586—G598. http://dx.doi.org/10.1152/ajpgi.00419.2015.
Full textAbstract:
The Na+/H+ exchanger regulatory factor (NHERF) family of proteins is scaffolds that orchestrate interaction of receptors and cellular proteins. Previous studies have shown that NHERF1 functions as a tumor suppressor. The goal of this study is to determine whether the loss of NHERF2 alters colorectal cancer (CRC) progress. We found that NHERF2 expression is elevated in advanced-stage CRC. Knockdown of NHERF2 decreased cancer cell proliferation in vitro and in a mouse xenograft tumor model. In addition, deletion of NHERF2 in Apc Min/+ mice resulted in decreased tumor growth in Apc Min/+ mice and increased lifespan. Blocking NHERF2 interaction with a small peptide designed to bind the second PDZ domain of NHERF2 attenuated cancer cell proliferation. Although NHERF2 is known to facilitate the effects of lysophosphatidic acid receptor 2 (LPA2), transcriptome analysis of xenograft tumors revealed that NHERF2-dependent genes largely differ from LPA2-regulated genes. Activation of β-catenin and ERK1/2 was mitigated in Apc Min/+; Nherf2− /− adenomas. Moreover, Stat3 phosphorylation and CD24 expression levels were suppressed in Apc Min/+; Nherf2− /− adenomas. Consistently, NHERF2 knockdown attenuated Stat3 activation and CD24 expression in colon cancer cells. Interestingly, CD24 was important in the maintenance of Stat3 phosphorylation, whereas NHERF2-dependent increase in CD24 expression was blocked by inhibition of Stat3, suggesting that NHERF2 regulates Stat3 phosphorylation through a positive feedback mechanism between Stat3 and CD24. In summary, this study identifies NHERF2 as a novel oncogenic protein and a potential target for cancer treatment. NHERF2 potentiates the oncogenic effects in part by regulation of Stat3 and CD24.