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Sample records for a2 lp-pla2 activity

  1. Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, platelet-activating factor acetylhydrolase (PAF-AH) in leukocytes and body composition in healthy adults

    PubMed Central

    Detopoulou, Paraskevi; Nomikos, Tzortzis; Fragopoulou, Elizabeth; Panagiotakos, Demosthenis B; Pitsavos, Christos; Stefanadis, Christodoulos; Antonopoulou, Smaragdi

    2009-01-01

    Background Lipoprotein-associated phospholipase A2 (Lp-PLA2) also known as serum platelet activating factor acetylhydrolase (PAF-AH) activity constitutes a novel risk marker for cardiovascular disease. Leukocytes constitute one main cellular source of circulating Lp-PLA2. The aim of the present study was to evaluate the association of both serum and leukocyte PAF-AH activities with fat distribution and lean tissue. One hundred healthy volunteers without cardiovascular disease history participated in this study (n = 52 men, 44 ± 13 years and n = 48 women, 43 ± 13 years). Body composition was assessed with dual-energy X-ray absorptiometry, while anthropometrical indices were also measured. The activity of Lp-PLA2 and levels of lipid and glycemic parameters were determined in fasting samples. Results Mean Lp-PLA2 activity was 24.8 ± 4.5 and 19.6 ± 5.0 nmol/min/mL in men and women, respectively (P < 0.001). Mean activity of PAF-AH in leukocyte homogenates was 386 ± 127 pmol/min/mg and 292 ± 92 pmol/min/mg in men and women, correspondingly (P < 0.001). In multiple regression models upper and total adiposity measures were positively associated with Lp-PLA2 activity in men after adjusting for LDL-cholesterol, age, smoking, hs-CRP and physical activity, whereas no associations were found with PAF-AH leukocyte homogenates activity. Hierarchical analysis revealed that the variables with the highest explanatory ability of Lp-PLA2 activity in men, were DXA deriving L1–L4 region of interest and arms fat (increase in R2 = 0.136, P = 0.005 and increase in R2 = 0.118, P = 0.009, respectively), followed by trunk fat and total fat. In women, no association of body composition variables with Lp-PLA2 nor PAF-AH leukocyte homogenates activity was found. Conclusion Lp-PLA2 activity is differentiated across levels of adiposity and topology of adipose tissue, whereas no association was found regarding PAF-AH leukocyte homogenates activity. Our findings suggest that Lp-PLA2 may

  2. Discovery of Potent and Orally Active Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Inhibitors as a Potential Therapy for Diabetic Macular Edema.

    PubMed

    Chen, Xinde; Wang, Kai; Xu, Wenwei; Ma, Quanxin; Chen, Minli; Du, Lili; Mo, Mingguang; Wang, Yiping; Shen, Jianhua

    2016-03-24

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is considered to be a promising therapeutic target for several inflammation-associated diseases. Herein, we describe the discovery of a series of pyrimidone derivatives as Lp-PLA2 inhibitors. Systematic structural modifications led to the identification of several pyrimidone compounds with promising in vitro inhibitory potency and pharmacokinetic properties. Compound 14c, selected for in vivo evaluation, demonstrated decent pharmacokinetic profiles and robust inhibitory potency against Lp-PLA2 in Sprague-Dawley (SD) rats. Furthermore, 14c significantly inhibited retinal thickening in STZ-induced diabetic SD rats as a model of diabetic macular edema (DME) after oral dosing for 4 weeks. Taken together, these results suggested that 14c can serve as a valuable lead in the search for new Lp-PLA2 inhibitors for prevention and/or treatment of DME.

  3. Genome-Wide Association Study of Lp-PLA2 Activity and Mass in the Framingham Heart Study

    PubMed Central

    Suchindran, Sunil; Rivedal, David; Guyton, John R.; Milledge, Tom; Gao, Xiaoyi; Benjamin, Ashlee; Rowell, Jennifer; Ginsburg, Geoffrey S.; McCarthy, Jeanette J.

    2010-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an emerging risk factor and therapeutic target for cardiovascular disease. The activity and mass of this enzyme are heritable traits, but major genetic determinants have not been explored in a systematic, genome-wide fashion. We carried out a genome-wide association study of Lp-PLA2 activity and mass in 6,668 Caucasian subjects from the population-based Framingham Heart Study. Clinical data and genotypes from the Affymetrix 550K SNP array were obtained from the open-access Framingham SHARe project. Each polymorphism that passed quality control was tested for associations with Lp-PLA2 activity and mass using linear mixed models implemented in the R statistical package, accounting for familial correlations, and controlling for age, sex, smoking, lipid-lowering-medication use, and cohort. For Lp-PLA2 activity, polymorphisms at four independent loci reached genome-wide significance, including the APOE/APOC1 region on chromosome 19 (p = 6×10−24); CELSR2/PSRC1 on chromosome 1 (p = 3×10−15); SCARB1 on chromosome 12 (p = 1×10−8) and ZNF259/BUD13 in the APOA5/APOA1 gene region on chromosome 11 (p = 4×10−8). All of these remained significant after accounting for associations with LDL cholesterol, HDL cholesterol, or triglycerides. For Lp-PLA2 mass, 12 SNPs achieved genome-wide significance, all clustering in a region on chromosome 6p12.3 near the PLA2G7 gene. Our analyses demonstrate that genetic polymorphisms may contribute to inter-individual variation in Lp-PLA2 activity and mass. PMID:20442857

  4. Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes

    PubMed Central

    Canning, Paul; Kenny, Bridget-Ann; Prise, Vivien; Glenn, Josephine; Sarker, Mosharraf H.; Hudson, Natalie; Brandt, Martin; Lopez, Francisco J.; Gale, David; Luthert, Philip J.; Adamson, Peter; Turowski, Patric; Stitt, Alan W.

    2016-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA2, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA2 and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood–retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA2 inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA2 inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA2 may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents. PMID:27298369

  5. Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes.

    PubMed

    Canning, Paul; Kenny, Bridget-Ann; Prise, Vivien; Glenn, Josephine; Sarker, Mosharraf H; Hudson, Natalie; Brandt, Martin; Lopez, Francisco J; Gale, David; Luthert, Philip J; Adamson, Peter; Turowski, Patric; Stitt, Alan W

    2016-06-28

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA2, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA2 and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood-retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA2 inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA2 inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA2 may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents.

  6. Replacing with whole grains and legumes reduces Lp-PLA2 activities in plasma and PBMCs in patients with prediabetes or T2D.

    PubMed

    Kim, Minjoo; Jeung, Se Ri; Jeong, Tae-Sook; Lee, Sang-Hyun; Lee, Jong Ho

    2014-08-01

    To determine dietary effects on circulating lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and enzyme activity in peripheral blood mononuclear cells (PBMCs), 99 patients with impaired fasting glucose, impaired glucose tolerance, or newly-diagnosed T2D were randomly assigned to either a control group (usual diet with refined rice) or the whole grain and legume group. Substitution of whole grains and legumes for refined rice was associated with the replacement of 7% of energy from carbohydrates with energy from protein (about 4%) and fat. After 12 weeks, the whole grain and legume group showed a significant decrease in fasting glucose, insulin, homeostasis model assessment-insulin resistance, hemoglobin A1c, malondialdehyde, plasma Lp-PLA2 activity, and oxidized LDL (ox-LDL), and an increase in LDL particle size. The changes (Δs) in these variables in the whole grain and legume group were significantly different from those in controls after adjustment for the baseline levels. When all subjects were considered, Δ plasma Lp-PLA2 positively correlated with Δ glucose, Δ PBMC Lp-PLA2, Δ ox-LDL, and Δ urinary 8-epi-prostaglandin F2α after being adjusted for confounding factors. The Δ PBMC Lp-PLA2 correlated positively with Δ glucose and Δ ox-LDL, and negatively with Δ LDL particle size and baseline PBMC Lp-PLA2 The substitution of whole grains and legumes for refined rice resulted in a reduction in Lp-PLA2 activities in plasma and PBMCs partly through improved glycemic control, increased consumption of protein relative to carbohydrate, and reduced lipid peroxides.

  7. Prehypertension-Associated Elevation in Circulating Lysophosphatidlycholines, Lp-PLA2 Activity, and Oxidative Stress

    PubMed Central

    Kim, Minjoo; Jung, Saem; Kim, Su Yeon; Lee, Sang-Hyun; Lee, Jong Ho

    2014-01-01

    Prehypertension is a risk factor for atherosclerosis. We investigated alterations in plasma metabolites that are associated with prehypertension. A group of 53 individuals was identified who remained within the range of prehypertension during repeated measurements in a 3-year period. This group was compared with the control group of 53 normotensive subjects who were matched for age and gender. Metabolomic profiles were analyzed with UPLC-LTQ-Orbitrap mass spectrometry. The prehypertensive group showed higher levels of lysophosphatidylcholines (lysoPCs) containing C14:0, C16:1, C16:0, C18:2, C18:1, C18:0, C20:5, C20:4, C20:3, and C22:6, higher circulating Lp-PLA2 activity, oxidized LDL (ox-LDL), interleukin 6 (IL-6), urinary 8-epi-PGF2α, and higher brachial-ankle pulse wave velocity (ba-PWV), before and after adjusting for BMI, WHR, smoking, alcohol consumption, serum lipid profiles, glucose, and insulin. LysoPC (16:0) was the most important plasma metabolite for evaluating the difference between control and prehypertensive groups, with a variable important in the projection (VIP) value of 17.173, and it showed a positive and independent association with DBP and SBP. In the prehypertensive group, the levels of lysoPC (16:0) positively and significantly correlated with ox-LDL, Lp-PLA2 activity, 8-epi-PGF2α, ba-PWV, and IL-6 before and after adjusting for confounding variables. Prehypertension-associated elevations in lysoPCs, Lp-PLA2 activity, ox-LDL, urinary 8-epi-PGF2α, IL-6, and ba-PWV could indicate increased oxidative stress from Lp-PLA2-catalyzed PC hydrolysis during increased LDL oxidation, thereby enhancing proinflammation and arterial stiffness. PMID:24800806

  8. Exploitation of a Novel Binding Pocket in Human Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Discovered through X-ray Fragment Screening.

    PubMed

    Woolford, Alison J-A; Pero, Joseph E; Aravapalli, Sridhar; Berdini, Valerio; Coyle, Joseph E; Day, Philip J; Dodson, Andrew M; Grondin, Pascal; Holding, Finn P; Lee, Lydia Y W; Li, Peng; Manas, Eric S; Marino, Joseph; Martin, Agnes C L; McCleland, Brent W; McMenamin, Rachel L; Murray, Christopher W; Neipp, Christopher E; Page, Lee W; Patel, Vipulkumar K; Potvain, Florent; Rich, Sharna; Rivero, Ralph A; Smith, Kirsten; Somers, Donald O; Trottet, Lionel; Velagaleti, Ranganadh; Williams, Glyn; Xie, Ren

    2016-06-09

    Elevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA2) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA2 in order to identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by movement of a protein side chain (approximately 13 Å from the catalytic residue Ser273). Using structure guided design, we optimized a fragment that bound in this pocket to generate a novel low nanomolar chemotype, which did not interact with the catalytic residues.

  9. Antioxidant and inflammatory aspects of lipoprotein-associated phospholipase A2 (Lp-PLA2 ): a review

    PubMed Central

    2011-01-01

    The association of cardiovascular events with Lp-PLA2 has been studied continuously today. The enzyme has been strongly associated with several cardiovascular risk markers and events. Its discovery was directly related to the hydrolysis of the platelet-activating factor and oxidized phospholipids, which are considered protective functions. However, the hydrolysis of bioactive lipids generates lysophospholipids, compounds that have a pro-inflammatory function. Therefore, the evaluation of the distribution of Lp-PLA2 in the lipid fractions emphasized the dual role of the enzyme in the inflammatory process, since the HDL-Lp-PLA2 enzyme contributes to the reduction of atherosclerosis, while LDL-Lp-PLA2 stimulates this process. Recently, it has been verified that diet components and drugs can influence the enzyme activity and concentration. Thus, the effects of these treatments on Lp-PLA2 may represent a new kind of prevention of cardiovascular disease. Therefore, the association of the enzyme with the traditional assessment of cardiovascular risk may help to predict more accurately these diseases. PMID:21955667

  10. Fragment-Based Approach to the Development of an Orally Bioavailable Lactam Inhibitor of Lipoprotein-Associated Phospholipase A2 (Lp-PLA2).

    PubMed

    Woolford, Alison J-A; Day, Philip J; Bénéton, Véronique; Berdini, Valerio; Coyle, Joseph E; Dudit, Yann; Grondin, Pascal; Huet, Pascal; Lee, Lydia Y W; Manas, Eric S; McMenamin, Rachel L; Murray, Christopher W; Page, Lee W; Patel, Vipulkumar K; Potvain, Florent; Rich, Sharna J; Sang, Yingxia; Somers, Don O; Trottet, Lionel; Wan, Zehong; Zhang, Xiaomin

    2016-12-08

    Lp-PLA2 has been explored as a target for a number of inflammation associated diseases, including cardiovascular disease and dementia. This article describes the discovery of a new fragment derived chemotype that interacts with the active site of Lp-PLA2. The starting fragment hit was discovered through an X-ray fragment screen and showed no activity in the bioassay (IC50 > 1 mM). The fragment hit was optimized using a variety of structure-based drug design techniques, including virtual screening, fragment merging, and improvement of shape complementarity. A novel series of Lp-PLA2 inhibitors was generated with low lipophilicity and a promising pharmacokinetic profile.

  11. Genetic invalidation of Lp-PLA2 as a therapeutic target: Large-scale study of five functional Lp-PLA2-lowering alleles.

    PubMed

    Gregson, John M; Freitag, Daniel F; Surendran, Praveen; Stitziel, Nathan O; Chowdhury, Rajiv; Burgess, Stephen; Kaptoge, Stephen; Gao, Pei; Staley, James R; Willeit, Peter; Nielsen, Sune F; Caslake, Muriel; Trompet, Stella; Polfus, Linda M; Kuulasmaa, Kari; Kontto, Jukka; Perola, Markus; Blankenberg, Stefan; Veronesi, Giovanni; Gianfagna, Francesco; Männistö, Satu; Kimura, Akinori; Lin, Honghuang; Reilly, Dermot F; Gorski, Mathias; Mijatovic, Vladan; Munroe, Patricia B; Ehret, Georg B; Thompson, Alex; Uria-Nickelsen, Maria; Malarstig, Anders; Dehghan, Abbas; Vogt, Thomas F; Sasaoka, Taishi; Takeuchi, Fumihiko; Kato, Norihiro; Yamada, Yoshiji; Kee, Frank; Müller-Nurasyid, Martina; Ferrières, Jean; Arveiler, Dominique; Amouyel, Philippe; Salomaa, Veikko; Boerwinkle, Eric; Thompson, Simon G; Ford, Ian; Wouter Jukema, J; Sattar, Naveed; Packard, Chris J; Shafi Majumder, Abdulla Al; Alam, Dewan S; Deloukas, Panos; Schunkert, Heribert; Samani, Nilesh J; Kathiresan, Sekar; Nordestgaard, Børge G; Saleheen, Danish; Howson, Joanna Mm; Di Angelantonio, Emanuele; Butterworth, Adam S; Danesh, John

    2017-03-01

    Aims Darapladib, a potent inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2), has not reduced risk of cardiovascular disease outcomes in recent randomized trials. We aimed to test whether Lp-PLA2 enzyme activity is causally relevant to coronary heart disease. Methods In 72,657 patients with coronary heart disease and 110,218 controls in 23 epidemiological studies, we genotyped five functional variants: four rare loss-of-function mutations (c.109+2T > C (rs142974898), Arg82His (rs144983904), Val279Phe (rs76863441), Gln287Ter (rs140020965)) and one common modest-impact variant (Val379Ala (rs1051931)) in PLA2G7, the gene encoding Lp-PLA2. We supplemented de-novo genotyping with information on a further 45,823 coronary heart disease patients and 88,680 controls in publicly available databases and other previous studies. We conducted a systematic review of randomized trials to compare effects of darapladib treatment on soluble Lp-PLA2 activity, conventional cardiovascular risk factors, and coronary heart disease risk with corresponding effects of Lp-PLA2-lowering alleles. Results Lp-PLA2 activity was decreased by 64% ( p = 2.4 × 10(-25)) with carriage of any of the four loss-of-function variants, by 45% ( p < 10(-300)) for every allele inherited at Val279Phe, and by 2.7% ( p = 1.9 × 10(-12)) for every allele inherited at Val379Ala. Darapladib 160 mg once-daily reduced Lp-PLA2 activity by 65% ( p < 10(-300)). Causal risk ratios for coronary heart disease per 65% lower Lp-PLA2 activity were: 0.95 (0.88-1.03) with Val279Phe; 0.92 (0.74-1.16) with carriage of any loss-of-function variant; 1.01 (0.68-1.51) with Val379Ala; and 0.95 (0.89-1.02) with darapladib treatment. Conclusions In a large-scale human genetic study, none of a series of Lp-PLA2-lowering alleles was related to coronary heart disease risk, suggesting that Lp-PLA2 is unlikely to be a causal risk factor.

  12. Short-term fenofibrate treatment reduces elevated plasma Lp-PLA2 mass and sVCAM-1 levels in a subcohort of hypertriglyceridemic GOLDN participants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High levels of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) are associated with inflammation, atherosclerosis, and coronary heart disease events. In addition, Lp-PLA(2) has been linked to classical markers of endothelial activation, including soluble vascular cell adhesion molecule-1 (sVCAM...

  13. The elevation of apoB in hypercholesterolemic patients is primarily attributed to the relative increase of apoB/Lp-PLA2

    PubMed Central

    Tellis, Constantinos C.; Moutzouri, Eliza; Elisaf, Moses; Wolfert, Robert L.; Tselepis, Alexandros D.

    2013-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk factor of cardiovascular disease. Plasma Lp-PLA2 is mainly associated with apolipoprotein (apo)B-containing lipoproteins, primarily with low density lipoproteins (LDLs). Importantly, only a proportion of circulating lipoproteins contain Lp-PLA2. We determined the plasma levels of Lp-PLA2-bound apoB (apoB/Lp-PLA2) in patients with primary hypercholesterolemia. The effect of simvastatin therapy was also addressed. The plasma apoB/Lp-PLA2 concentration in 50 normolipidemic controls and 53 patients with primary hypercholesterolemia at baseline and at 3 months posttreatment with simvastatin (40 mg/day) was determined by an enzyme-linked immunosorbent assay. The concentration of the apoB-containing lipoproteins that do not bind Lp-PLA2 [apoB/Lp-PLA2(−)] was calculated by subtracting the apoB/Lp-PLA2 from total apoB. The apoB/Lp-PLA2 levels were 3.6-fold higher, while apoB/Lp-PLA2(−) were 1.3-fold higher in patients compared with controls. After 3 months of simvastatin treatment apoB/Lp-PLA2 and apoB/Lp-PLA2(−) levels were reduced by 52% and 33%, respectively. The elevation in apoB-containing lipoproteins in hypercholesterolemic patients is mainly attributed to the relative increase in the proatherogenic apoB/Lp-PLA2, while simvastatin reduces these particles to a higher extent compared with apoB/Lp-PLA2(−). Considering that Lp-PLA2 is proatherogenic, the predominance of apoB/Lp-PLA2 particles in hypercholesterolemic patients may contribute to their higher atherogenicity and incidence of cardiovascular disease. PMID:24092915

  14. Intensive Lifestyle Modification Reduces Lp-PLA2 in Dyslipidemic HIV/HAART Patients

    PubMed Central

    Wooten, Joshua S.; Nambi, Preethi; Gillard, Baiba K.; Pownall, Henry J.; Coraza, Ivonne; Scott, Lynne W.; Nambi, Vijay; Ballantyne, Christie M.; Balasubramanyam, Ashok

    2013-01-01

    Patients with dyslipidemia associated with HIV-1 infection and highly active antiretroviral therapy (HAART) have elevated levels of Lp-PLA2 and CCL5/RANTES, which may increase risk of cardiovascular disease. Purpose This study aimed to determine whether an intensive diet and exercise (D/E) program, independently or combined with fenofibrate or niacin, could reduce Lp-PLA2 or RANTES. Methods Hypertriglyceridemic HIV patients on stable HAART (n=107) were randomized to one of five interventions: 1) Usual Care (UC); 2) D/E with placebos; 3) D/E with fenofibrate and placebo; 4) D/E with niacin and placebo; or 5) D/E with fenofibrate and niacin for 24 weeks. Lp-PLA2 and RANTES concentrations were measured in fasting plasma samples at baseline and post-intervention. General linear models were used to compare Lp-PLA2 and RANTES levels between the five groups post-intervention, controlling for baseline levels, age, BMI, CD4+ T-cell count, viral load, duration of infection, and HAART. Results At baseline, fasting plasma Lp-PLA2 (388.5 ± 127.5 ng/mL) and RANTES (43.8 ± 25.5 ng/mL) levels were elevated when compared to healthy controls. Post-treatment Lp-PLA2 mass was lower in patients who received D/E only (323.0 ± 27.2 ng/mL), D/E plus fenofibrate (327.2 ± 25.9 ng/mL) and D/E plus niacin (311.1 ± 27.8 ng/mL) when compared to patients receiving UC (402.2 ± 25.3 ng/mL). RANTES concentrations were not significantly affected by any intervention. Conclusions Elevated plasma Lp-PLA2 mass can be reduced by an intensive diet and exercise program in patients with HIV/HAART-associated dyslipidemia. RANTES is elevated but is not reduced by lifestyle modification, fenofibrate or niacin. PMID:23299761

  15. Plasma levels of lipoprotein-associated phospholipase A2 are increased in patients with β-thalassemia

    PubMed Central

    Tselepis, Alexandros D.; Hahalis, George; Tellis, Constantinos C.; Papavasiliou, Eleni C.; Mylona, Panagiota T.; Kourakli, Alexandra; Alexopoulos, Dimitrios C.

    2010-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an independent cardiovascular risk factor. We investigated the plasma levels of Lp-PLA2 activity and mass as a function of plasma lipid levels, LDL subclass profile, and oxidative stress in patients with β-thalassemia. Thirty-five patients with β-thalassemia major (β-TM) and 25 patients with β-thalassemia intermedia (β-TI) participated in the study. Lp-PLA2 activity and mass were measured in total plasma, in apolipoprotein (apo)B-depleted plasma (HDL-Lp-PLA2), and in LDL subclasses. Lp-PLA2 activity produced and secreted from peripheral blood monocytes in culture was also determined. Patients with β-thalassemia are characterized by a predominance of small-dense LDL particles, increased oxidative stress, and very high plasma levels of Lp-PLA2 mass and activity, despite low LDL-cholesterol levels. A significant positive correlation between plasma Lp-PLA2 activity or mass and 8-isoprostane (8-epiPGF2a) and ferritin levels as well as intima-media thickness (IMT) values was observed. An increase in secreted and cell-associated Lp-PLA2 activity from monocytes in culture was observed in both patient groups. The HDL-Lp-PLA2 activity and mass as well as the ratio of HDL-Lp-PLA2/plasma Lp-PLA2 were significantly higher in both patient groups compared with the control group. In conclusion, patients with β-thalassemia exhibit high plasma Lp-PLA2 levels, attributed to increased enzyme secretion from monocytes/macrophages and to the predominance of sdLDL particles in plasma. Plasma Lp-PLA2 is correlated with carotid IMT, suggesting that this enzyme may be implicated in premature carotid atherosclerosis observed in β-thalassemia. PMID:20625038

  16. Structural and Thermodynamic Characterization of Protein-Ligand Interactions Formed between Lipoprotein-Associated Phospholipase A2 and Inhibitors.

    PubMed

    Liu, Qiufeng; Chen, Xinde; Chen, Wuyan; Yuan, Xiaojing; Su, Haixia; Shen, Jianhua; Xu, Yechun

    2016-05-26

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) represents a promising therapeutic target for atherosclerosis and Alzheimer's disease. Here we reported the first crystal structures of Lp-PLA2 bound with reversible inhibitors and the thermodynamic characterization of complexes. High rigidity of Lp-PLA2 structure and similar binding modes of inhibitors with completely different scaffolds are revealed. It not only provides the molecular basis for inhibitory activity but also sheds light on the essential features of Lp-PLA2 recognition with reversible inhibitors.

  17. Selective inhibitors and tailored activity probes for lipoprotein-associated phospholipase A2

    PubMed Central

    Nagano, Joseph M. G.; Hsu, Ku-Lung; Whitby, Landon R.; Niphakis, Micah J.; Speers, Anna E.; Brown, Steven J.; Spicer, Timothy; Fernandez-Vega, Virneliz; Ferguson, Jill; Hodder, Peter; Srinivasan, Prabhavathi; Gonzalez, Tara D.; Rosen, Hugh; Bahnson, Brian J.

    2013-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2 or PLA2G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA2 has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA2 have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA2 inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA2 and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA2 in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA2 function in biological systems. PMID:23260346

  18. Lipoprotein-associated phospholipase A2 and risk of incident peripheral arterial disease in a multi-ethnic cohort: The Multi-Ethnic Study of Atherosclerosis.

    PubMed

    Garg, Parveen K; Jorgensen, Neal W; McClelland, Robyn L; Jenny, Nancy S; Criqui, Michael H; Allison, Matthew A; Greenland, Philip; Rosenson, Robert S; Siscovick, David S; Cushman, Mary

    2017-02-01

    Prospective studies supporting a relationship between elevated lipoprotein-associated phospholipase A2 (Lp-PLA2) and incident peripheral arterial disease (PAD) are limited. We evaluated the association of Lp-PLA2 with incident PAD in a multi-ethnic cohort without clinical cardiovascular disease. A total of 4622 participants with measurement of Lp-PLA2 mass and Lp-PLA2 activity and an ankle-brachial index (ABI) between 0.9 and 1.4 were followed for the development of PAD (median follow-up = 9.3 years), defined as an ABI ⩽0.9 and decline from baseline ⩾0.15. There were 158 incident PAD events during follow-up. In adjusted logistic regression models, each higher standard deviation of both Lp-PLA2 activity and mass did not confer an increased risk of developing PAD [odds ratios, (95% confidence intervals)]: 0.92 (0.66-1.27) for Lp-PLA2 activity and 1.06 (0.85-1.34) for mass. Additionally, no significant interaction was found according to ethnicity: p=0.43 for Lp-PLA2 activity and p=0.55 for Lp-PLA2 mass. We found no evidence of an association between Lp-PLA2 and incident PAD.

  19. The role of lipoprotein-associated phospholipase A2 in a murine model of experimental autoimmune uveoretinitis.

    PubMed

    Crawford, G L; Boldison, J; Copland, D A; Adamson, P; Gale, D; Brandt, M; Nicholson, L B; Dick, A D

    2015-01-01

    Macrophage activation is, in part, regulated via hydrolysis of oxidised low density lipoproteins by Lipoprotein-Associated phospholipase A2 (Lp-PLA2), resulting in increased macrophage migration, pro-inflammatory cytokine release and chemokine expression. In uveitis, tissue damage is mediated as a result of macrophage activation; hence inhibition of Lp-PLA2 may limit macrophage activation and protect the tissue. Utilising Lp-PLA2 gene-deficient (KO) mice and a pharmacological inhibitor of Lp-PLA2 (SB-435495) we aimed to determine the effect of Lp-PLA2 suppression in mediating retinal protection in a model of autoimmune retinal inflammation, experimental autoimmune uveoretinitis (EAU). Following immunisation with RBP-3 (IRBP) 1-20 or 161-180 peptides, clinical disease was monitored and severity assessed, infiltrating leukocytes were enumerated by flow cytometry and tissue destruction quantified by histology. Despite ablation of Lp-PLA2 enzyme activity in Lp-PLA2 KO mice or wild-type mice treated with SB-435495, the number of infiltrating CD45+ cells in the retina was equivalent to control EAU animals, and there was no reduction in disease severity. Thus, despite the reported beneficial effects of therapeutic Lp-PLA2 depletion in a variety of vascular inflammatory conditions, we were unable to attenuate disease, show delayed disease onset or prevent progression of EAU in Lp-PLA2 KO mice. Although EAU exhibits inflammatory vasculopathy there is no overt defect in lipid metabolism and given the lack of effect following Lp-PLA2 suppression, these data support the hypothesis that sub-acute autoimmune inflammatory disease progresses independently of Lp-PLA2 activity.

  20. Nitro-oleic acid downregulates lipoprotein-associated phospholipase A2 expression via the p42/p44 MAPK and NFκB pathways

    PubMed Central

    Wang, Gangqi; Ji, Yuan; Li, Zhuang; Han, Xiaolei; Guo, Nannan; Song, Qi; Quan, Longquan; Wang, Tiedong; Han, Wenyu; Pang, Daxin; Ouyang, Hongsheng; Tang, Xiaochun

    2014-01-01

    Nitro-oleic acid (OA-NO2), acting as anti-inflammatory signaling mediators, are involved in multiple signaling pathways. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is well known as a cardiovascular risk biomarker. Our results showed that OA-NO2 downregulated the expression of Lp-PLA2 in a time- and dose-dependent manner, whereas native OA had no such effect. Furthermore, OA-NO2 could repress Lp-PLA2 expression in the peripheral blood mononuclear cells of apo CIII-transgenic (apo CIII TG) pigs, which exhibited higher Lp-PLA2 expression and activity than did wild-type (WT) pigs. OA-NO2 inhibited Lp-PLA2 expression in macrophages, independent of nitric oxide formation and PPARγ-activation. However, OA-NO2 downregulates Lp-PLA2 by inhibiting the p42/p44 mitogen-activated protein kinase (MAPK) and the nuclear factor κB (NFκB) pathways. When used to mediate anti-inflammatory signaling, the regulation of inflammatory cytokines and SOD by OA-NO2 might be associated with the reduction of Lp-PLA2. These results suggested that OA-NO2 might exert a vascular-protective effect partially via Lp-PLA2 inhibition. PMID:24809325

  1. Nitro-oleic acid downregulates lipoprotein-associated phospholipase A2 expression via the p42/p44 MAPK and NFκB pathways.

    PubMed

    Wang, Gangqi; Ji, Yuan; Li, Zhuang; Han, Xiaolei; Guo, Nannan; Song, Qi; Quan, Longquan; Wang, Tiedong; Han, Wenyu; Pang, Daxin; Ouyang, Hongsheng; Tang, Xiaochun

    2014-05-09

    Nitro-oleic acid (OA-NO2), acting as anti-inflammatory signaling mediators, are involved in multiple signaling pathways. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is well known as a cardiovascular risk biomarker. Our results showed that OA-NO2 downregulated the expression of Lp-PLA2 in a time- and dose-dependent manner, whereas native OA had no such effect. Furthermore, OA-NO2 could repress Lp-PLA2 expression in the peripheral blood mononuclear cells of apo CIII-transgenic (apo CIII TG) pigs, which exhibited higher Lp-PLA2 expression and activity than did wild-type (WT) pigs. OA-NO2 inhibited Lp-PLA2 expression in macrophages, independent of nitric oxide formation and PPARγ-activation. However, OA-NO2 downregulates Lp-PLA2 by inhibiting the p42/p44 mitogen-activated protein kinase (MAPK) and the nuclear factor κB (NFκB) pathways. When used to mediate anti-inflammatory signaling, the regulation of inflammatory cytokines and SOD by OA-NO2 might be associated with the reduction of Lp-PLA2. These results suggested that OA-NO2 might exert a vascular-protective effect partially via Lp-PLA2 inhibition.

  2. Evaluation of the safety, pharmacokinetics, pharmacodynamics, and drug-drug interaction potential of a selective Lp-PLA2 inhibitor (GSK2647544) in healthy volunteers

    PubMed Central

    Wu, Kai; Xu, Jianfeng; Fong, Regan; Yao, Xiaozhou; Xu, Yanmei; Guiney, William; Gray, Frank; Lockhart, Andrew

    2016-01-01

    Abstract. Objective: To evaluate in healthy volunteers the safety, pharmacokinetics (PK), pharmacodynamics (PD), and drug-drug interaction (DDI) potential of GSK2647544, (a selective lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitor). Methods: Study 1 was a single-blind, randomized, placebo-controlled, crossover study with healthy male volunteers randomized to receive single escalating oral doses (0.5 – 750 mg) of GSK2647544. Study 2 was a single-blind, randomized, placebo-controlled study with healthy volunteers randomized to receive repeat doses (80 mg) of GSK2647544. The drug-drug interaction of GSK2647544 with simvastatin was also evaluated in study 2. Results: Across both studies GSK2647544 doses were generally well tolerated with no GSK2647544-related clinically significant findings. GSK2647544 was readily absorbed and its plasma concentration declined bi-exponentially with a terminal half-life ranging from 8 to 16 hours. Plasma exposure of GSK2647544 increased approximately dose-proportionally. There was GSK2647544 dose-dependent inhibition of plasma Lp-PLA2 activity, with a trough inhibition (12 hours after dose) of 85.6% after 7-day twice daily dosing. The administration of simvastatin concomitantly with GSK2647544 increased the overall exposure (area under the plasma concentration-time curve and maximum plasma concentration) of simvastatin and simvastatin acid by 3.6- to 4.3-fold and 1.5- to 3.1-fold, respectively. Conclusions: GSK2647544 was generally well tolerated and had a reasonable PK-PD profile. The clinically significant drug-drug interaction led to an early termination of study 2. PMID:27719741

  3. Lipoprotein-associated phospholipase A2 prognostic role in atherosclerotic complications

    PubMed Central

    Maiolino, Giuseppe; Bisogni, Valeria; Rossitto, Giacomo; Rossi, Gian Paolo

    2015-01-01

    Atherosclerosis manifests itself clinically at advanced stages when plaques undergo hemorrhage and/or rupture with superimposed thrombosis, thus abruptly stopping blood supply. Identification of markers of plaque destabilization at a pre-clinical stage is, therefore, a major goal of cardiovascular research. Promising results along this line were provided by studies investigating the lipoprotein-associated phospholipase A2 (Lp-PLA2), a member of phospholipase A2 proteins family that plays a key role in the metabolism of pro-inflammatory phospholipids, as oxidized low-density lipoproteins, and in the generation of pro-atherogenic metabolites, including lysophosphatidylcholine and oxidized free fatty acids. We herein review the experimental and clinical studies supporting use of Lp-PLA2 activity for predicting cardiovascular events. To his end we considered not only Lp-PLA2 activity and mass, but also Lp-PLA2 gene variations and their association with incident coronary artery disease, stroke, and cardiovascular mortality. Based on these evidences the major scientific societies have included in their guidelines the measurement of Lp-PLA2 activity among the biomarkers that are useful in risk stratification of adult asymptomatic patients at intermediate cardiovascular risk. The results of two recently published major clinical trials with the Lp-PLA2 inhibitor darapladib, which seem to challenge the pathogenic role of Lp-PLA2, will also be discussed. PMID:26516415

  4. Lipoprotein-associated phospholipase A2 prognostic role in atherosclerotic complications.

    PubMed

    Maiolino, Giuseppe; Bisogni, Valeria; Rossitto, Giacomo; Rossi, Gian Paolo

    2015-10-26

    Atherosclerosis manifests itself clinically at advanced stages when plaques undergo hemorrhage and/or rupture with superimposed thrombosis, thus abruptly stopping blood supply. Identification of markers of plaque destabilization at a pre-clinical stage is, therefore, a major goal of cardiovascular research. Promising results along this line were provided by studies investigating the lipoprotein-associated phospholipase A2 (Lp-PLA2), a member of phospholipase A2 proteins family that plays a key role in the metabolism of pro-inflammatory phospholipids, as oxidized low-density lipoproteins, and in the generation of pro-atherogenic metabolites, including lysophosphatidylcholine and oxidized free fatty acids. We herein review the experimental and clinical studies supporting use of Lp-PLA2 activity for predicting cardiovascular events. To his end we considered not only Lp-PLA2 activity and mass, but also Lp-PLA2 gene variations and their association with incident coronary artery disease, stroke, and cardiovascular mortality. Based on these evidences the major scientific societies have included in their guidelines the measurement of Lp-PLA2 activity among the biomarkers that are useful in risk stratification of adult asymptomatic patients at intermediate cardiovascular risk. The results of two recently published major clinical trials with the Lp-PLA2 inhibitor darapladib, which seem to challenge the pathogenic role of Lp-PLA2, will also be discussed.

  5. Apolipoprotein CIII regulates lipoprotein-associated phospholipase A2 expression via the MAPK and NFκB pathways

    PubMed Central

    Han, Xiaolei; Wang, Tiedong; Zhang, Jifeng; Liu, Xingxing; Li, Zhuang; Wang, Gangqi; Song, Qi; Pang, Daxin; Ouyang, Hongsheng; Tang, Xiaochun

    2015-01-01

    Apolipoprotein CIII (apo CIII), a small glycoprotein that binds to the surfaces of certain lipoproteins, is associated with inflammatory and atherogenic responses in vascular cells. Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been proposed as an inflammatory biomarker and potential therapeutic target for cardiovascular disease (CVD). Here, we report that apo CIII increases Lp-PLA2 mRNA and protein levels in dose- and time- dependent manner in human monocytic THP-1 cells, and the increase can be abolished by MAPK and NFκB pathway inhibitors. Lp-PLA2 inhibitor, 1-linoleoyl glycerol attenuates the inflammation induced by apo CIII. In turn, exogenous Lp-PLA2 expression upregulates apo CIII and the upregulation can be inhibited by 1-linoleoyl glycerol in HepG2 cells. Moreover, plasma Lp-PLA2 level is correlated with apo CIII expression in pig liver. In vivo, Lp-PLA2 expression in monocytes and its activity in serum were significantly increased in human apo CIII transgenic porcine models compared with wild-type pigs. Our results suggest that Lp-PLA2 and apo CIII expression level is correlated with each other in vitro and in vivo. PMID:25836672

  6. Higher Levels of Lipoprotein Associated Phospholipase A2 is associated with Increased Prevalence of Cognitive Impairment: the APAC Study

    PubMed Central

    Jiang, Ruixuan; Chen, Shengyun; Shen, Yuan; Wu, Jianwei; Chen, Shuohua; Wang, Anxin; Wu, Shouling; Zhao, Xingquan

    2016-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a unique circulating phospholipase with inflammatory and oxidative activities and the limited data regarding the relationship between Lp-PLA2 and cognitive impairment are conflicted. We conducted a cross-sectional study including 1,374 Chinese adults recruited from 2010 to 2011, aiming to evaluate the relationship between Lp-PLA2 levels and the prevalence of cognitive impairment in a Chinese community-based population. Participants underwent standardized evaluation. Serum Lp-PLA2 mass was measured by ELISA. Cognition status was evaluated via the Mini-Mental Status Exam (MMSE) and cognitive impairment was identified as MMSE <24. Multivariable logistic regression models were used to assess the associations of Lp-PLA2 mass with cognitive impairment. Lp-PLA2 mass was significantly associated with the prevalence of cognitive impairment after adjusting for other potential confounding factors (compared with the first quartile, adjusted ORs of the second, third, and fourth quartile were 2.058 (95% CI, 0.876–4.835), 2.834 (95% CI, 1.255–6.398), and 4.882 (95% CI, 2.212–10.777), p < 0.0001). In conclusion, elevated level of Lp-PLA2 mass was independently associated with the prevalence of cognitive impairment in Chinese adults. PMID:27609335

  7. Apolipoprotein CIII regulates lipoprotein-associated phospholipase A2 expression via the MAPK and NFκB pathways.

    PubMed

    Han, Xiaolei; Wang, Tiedong; Zhang, Jifeng; Liu, Xingxing; Li, Zhuang; Wang, Gangqi; Song, Qi; Pang, Daxin; Ouyang, Hongsheng; Tang, Xiaochun

    2015-04-02

    Apolipoprotein CIII (apo CIII), a small glycoprotein that binds to the surfaces of certain lipoproteins, is associated with inflammatory and atherogenic responses in vascular cells. Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been proposed as an inflammatory biomarker and potential therapeutic target for cardiovascular disease (CVD). Here, we report that apo CIII increases Lp-PLA2 mRNA and protein levels in dose- and time- dependent manner in human monocytic THP-1 cells, and the increase can be abolished by MAPK and NFκB pathway inhibitors. Lp-PLA2 inhibitor, 1-linoleoyl glycerol attenuates the inflammation induced by apo CIII. In turn, exogenous Lp-PLA2 expression upregulates apo CIII and the upregulation can be inhibited by 1-linoleoyl glycerol in HepG2 cells. Moreover, plasma Lp-PLA2 level is correlated with apo CIII expression in pig liver. In vivo, Lp-PLA2 expression in monocytes and its activity in serum were significantly increased in human apo CIII transgenic porcine models compared with wild-type pigs. Our results suggest that Lp-PLA2 and apo CIII expression level is correlated with each other in vitro and in vivo.

  8. A previously unreported impact of a PLA2G7 gene polymorphism on the plasma levels of lipoprotein-associated phospholipase A2 activity and mass

    PubMed Central

    Qi, Yue; Zhao, Dong; Jia, Zhangrong; Wang, Wei; Wang, Miao; Sun, Jiayi; Liu, Jun; Li, Yan; Xie, Wuxiang; Liu, Jing

    2016-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) levels are associated with the development of atherosclerosis. We aimed to assess the genetic determinants of Lp-PLA2 activity and mass by genotyping multiple polymorphisms in PLA2G7, the gene encoding Lp-PLA2, among 1258 participants from the Chinese Multi-provincial Cohort Study-Beijing Project. The Sequenom MassARRAY system, Taqman assay and direct sequencing were adopted. For the first time, the rs13218408 polymorphism was found to be significantly associated with reduced Lp-PLA2 levels. We also confirmed the significant association of previously validated polymorphisms (rs1421378, rs1805018, rs16874954 and rs2216465), even after adjusting for traditional cardiovascular risk factors and for Bonferroni correction. Percentages of variance attributable to rs13218408 were 7.2% for activity and 13.3% for mass, and were secondary to those of rs16874954 (8.1% for activity and 16.9% for mass). A significant joint effect of rs13218408 and rs16874954 was observed on Lp-PLA2 activity (P = 0.058) and mass (P = 0.003), with their minor alleles together linking to the largest reduction in Lp-PLA2 levels (37.8% reduction in activity and 41.6% reduction in mass). Taken together, our findings show a significant association of a PLA2G7 polymorphism with Lp-PLA2 levels, which was previously unreported in any population. The functionality of this genetic variation deserves further investigations. PMID:27905470

  9. A previously unreported impact of a PLA2G7 gene polymorphism on the plasma levels of lipoprotein-associated phospholipase A2 activity and mass.

    PubMed

    Qi, Yue; Zhao, Dong; Jia, Zhangrong; Wang, Wei; Wang, Miao; Sun, Jiayi; Liu, Jun; Li, Yan; Xie, Wuxiang; Liu, Jing

    2016-12-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) levels are associated with the development of atherosclerosis. We aimed to assess the genetic determinants of Lp-PLA2 activity and mass by genotyping multiple polymorphisms in PLA2G7, the gene encoding Lp-PLA2, among 1258 participants from the Chinese Multi-provincial Cohort Study-Beijing Project. The Sequenom MassARRAY system, Taqman assay and direct sequencing were adopted. For the first time, the rs13218408 polymorphism was found to be significantly associated with reduced Lp-PLA2 levels. We also confirmed the significant association of previously validated polymorphisms (rs1421378, rs1805018, rs16874954 and rs2216465), even after adjusting for traditional cardiovascular risk factors and for Bonferroni correction. Percentages of variance attributable to rs13218408 were 7.2% for activity and 13.3% for mass, and were secondary to those of rs16874954 (8.1% for activity and 16.9% for mass). A significant joint effect of rs13218408 and rs16874954 was observed on Lp-PLA2 activity (P = 0.058) and mass (P = 0.003), with their minor alleles together linking to the largest reduction in Lp-PLA2 levels (37.8% reduction in activity and 41.6% reduction in mass). Taken together, our findings show a significant association of a PLA2G7 polymorphism with Lp-PLA2 levels, which was previously unreported in any population. The functionality of this genetic variation deserves further investigations.

  10. Admission Lipoprotein-Associated Phospholipase A2 Activity Is Not Associated with Long-Term Clinical Outcomes after ST-Segment Elevation Myocardial Infarction

    PubMed Central

    Woudstra, Pier; Damman, Peter; Kuijt, Wichert J.; Kikkert, Wouter J.; Grundeken, Maik J.; van Brussel, Peter M.; Stroobants, An K.; van Straalen, Jan P.; Fischer, Johan C.; Koch, Karel T.; Henriques, José P. S.; Piek, Jan J.; Tijssen, Jan G. P.; de Winter, Robbert J.

    2014-01-01

    Background Lipoprotein-associated phospholipase A2 (Lp-PLA2) activity is a biomarker predicting cardiovascular diseases in a real-world. However, the prognostic value in patients undergoing primary percutaneous coronary intervention (pPCI) for ST-segment elevation myocardial infarction (STEMI) on long-term clinical outcomes is unknown. Methods Lp-PLA2 activity was measured in samples obtained prior to pPCI from consecutive STEMI patients in a high-volume intervention center from 2005 until 2007. Five years all-cause mortality was estimated with the Kaplan-Meier method and compared among tertiles of Lp-PLA2 activity during complete follow-up and with a landmark at 30 days. In a subpopulation clinical endpoints were assessed at three years. The prognostic value of Lp-PLA2, in addition to the Thrombolysis In Myocardial Infarction or multimarker risk score, was assessed in multivariable Cox regression. Results The cohort (n = 987) was divided into tertiles (low <144, intermediate 144–179, and high >179 nmol/min/mL). Among the tertiles differences in baseline characteristics associated with long-term mortality were observed. However, no significant differences in five years mortality in association with Lp-PLA2 activity levels were found; intermediate versus low Lp-PLA2 (HR 0.97; CI 95% 0.68–1.40; p = 0.88) or high versus low Lp-PLA2 (HR 0.75; CI 95% 0.51–1.11; p = 0.15). Both in a landmark analysis and after adjustments for the established risk scores and selection of cases with biomarkers obtained, non-significant differences among the tertiles were observed. In the subpopulation no significant differences in clinical endpoints were observed among the tertiles. Conclusion Lp-PLA2 activity levels at admission prior to pPCI in STEMI patients are not associated with the incidence of short and/or long-term clinical endpoints. Lp-PLA2 as an independent and clinically useful biomarker in the risk stratification of STEMI patients still remains to be proven

  11. Effects of lipoprotein-associated phospholipase A2 on arginase/nitric oxide pathway in hemodialysis patients.

    PubMed

    Tektaş, Ayşegül Korkmaz; Uslu, Sema; Yalçin, Ahmet Uğur; Sahin, Garip; Temiz, Gökhan; Kara, Mehmet; Temel, Halide Edip; Demirkan, Emine Sütken; Colak, Ertuğrul; Colak, Omer

    2012-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) and arginase are recently described inflammatory biomarkers associated with cardiovascular disease. In this study, we aimed to investigate the possible effects of serum Lp-PLA2 mass levels on arginase/nitric oxide (NO) pathway as a cardiovascular risk marker in hemodialysis (HD) patients. Forty-three HD patients and 15 healthy subjects were included in this study. Lipid profile, high sensitivity C-reactive protein (hs-CRP), albumin, creatinine, body mass index (BMI), Lp-PLA2 and total nitrite levels, and arginase activity were determined in serum samples from patients and control subjects. Lp-PLA2 levels were found to be positively correlated with arginase, triglycerides, total cholesterol, low-density lipoprotein-cholesterol, and age and negatively correlated with high-density lipoprotein-cholesterol and total nitrite levels, while there was no correlation with BMI and hs-CRP, albumin, and creatinine levels in HD patients. We conclude that elevated Lp-PLA2 mass levels may contribute to impaired arginase/NO pathway in HD patients and that increased the arginase activity and Lp-PLA2 mass levels with decreased total nitrite levels seem to be useful biochemical markers in terms of reflecting endothelial dysfunction and associated cardiovascular risks in HD patients.

  12. Comparison of Lipoprotein-Associated Phospholipase A2 and High Sensitive C-Reactive Protein as Determinants of Metabolic Syndrome in Subjects without Coronary Heart Disease: In Search of the Best Predictor

    PubMed Central

    Acevedo, Mónica; Kramer, Verónica; Adasme, Marcela; Briones, Luisa

    2015-01-01

    High sensitivity C-reactive protein (hsCRP) is a marker of metabolic syndrome (MS) and cardiovascular (CV) disease. Lipoprotein-associated phospholipase A2 (Lp-PLA2) also predicts CV disease. There are no reports comparing these markers as predictors of MS. Methods. Cross-sectional study comparing Lp-PLA2 and hsCRP as predictors of MS in asymptomatic subjects was carried out; 152 subjects without known atherosclerosis participated. Data were collected on demographics, cardiovascular risk factors, anthropometric and biochemical measurements, and hsCRP and Lp-PLA2 activity levels. A logistic regression analysis was performed with each biomarker and receiver operating characteristic (ROC) curves were constructed for MS. Results. Mean age was 46 ± 11 years, and 38% of the subjects had MS. Mean Lp-PLA2 activity was 185 ± 48 nmol/mL/min, and mean hsCRP was 2.1 ± 2.2 mg/L. Subjects with MS had significantly higher levels of Lp-PLA2 (P = 0.03) and hsCRP (P < 0.0001) than those without MS. ROC curves showed that both markers predicted MS. Conclusion. Lp-PLA2 and hsCRP are elevated in subjects with MS. Both biomarkers were independent and significant predictors for MS, emphasizing the role of inflammation in MS. Further research is necessary to determine if inflammation predicts a higher risk for CV events in MS subjects. PMID:26089902

  13. Comparison of Lipoprotein-Associated Phospholipase A2 and High Sensitive C-Reactive Protein as Determinants of Metabolic Syndrome in Subjects without Coronary Heart Disease: In Search of the Best Predictor.

    PubMed

    Acevedo, Mónica; Varleta, Paola; Kramer, Verónica; Valentino, Giovanna; Quiroga, Teresa; Prieto, Carolina; Parada, Jacqueline; Adasme, Marcela; Briones, Luisa; Navarrete, Carlos

    2015-01-01

    High sensitivity C-reactive protein (hsCRP) is a marker of metabolic syndrome (MS) and cardiovascular (CV) disease. Lipoprotein-associated phospholipase A2 (Lp-PLA2) also predicts CV disease. There are no reports comparing these markers as predictors of MS. Methods. Cross-sectional study comparing Lp-PLA2 and hsCRP as predictors of MS in asymptomatic subjects was carried out; 152 subjects without known atherosclerosis participated. Data were collected on demographics, cardiovascular risk factors, anthropometric and biochemical measurements, and hsCRP and Lp-PLA2 activity levels. A logistic regression analysis was performed with each biomarker and receiver operating characteristic (ROC) curves were constructed for MS. Results. Mean age was 46 ± 11 years, and 38% of the subjects had MS. Mean Lp-PLA2 activity was 185 ± 48 nmol/mL/min, and mean hsCRP was 2.1 ± 2.2 mg/L. Subjects with MS had significantly higher levels of Lp-PLA2 (P = 0.03) and hsCRP (P < 0.0001) than those without MS. ROC curves showed that both markers predicted MS. Conclusion. Lp-PLA2 and hsCRP are elevated in subjects with MS. Both biomarkers were independent and significant predictors for MS, emphasizing the role of inflammation in MS. Further research is necessary to determine if inflammation predicts a higher risk for CV events in MS subjects.

  14. Plasma Lipoprotein-associated Phospholipase A2 in Patients with Metabolic Syndrome and Carotid Atherosclerosis

    PubMed Central

    2011-01-01

    Background Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a recently identified and potentially useful plasma biomarker for cardiovascular and atherosclerotic diseases. However, the correlation between the Lp-PLA2 activity and carotid atherosclerosis remains poorly investigated in patients with metabolic syndrome (MetS). The present study aimed to evaluate the potential role of Lp-PLA2 as a comprehensive marker of metabolic syndrome in individuals with and without carotid atherosclerosis. Methods We documented 118 consecutive patients with MetS and 70 age- and sex-matched healthy subjects served as controls. The patients were further divided into two groups: 39 with carotid plaques and 79 without carotid plaques to elucidate the influence of Lp-PLA2 on carotid atherosclerosis. The plasma Lp-PLA2 activity was measured by using ELISA method and carotid intimal-media thickness (IMT) was performed by ultrasound in all participants. Results Lp-PLA2 activity was significantly increased in MetS subgroups when compared with controls, and was higher in patients with carotid plaques than those without plaques (P < 0.05). Furthermore, we found that significant difference in Lp-PLA2 was obtained between patients with three and four disorders of metabolic syndrome (P < 0.01). Age (β = 0.183, P = 0.029), LDL-cholesterol (β = 0.401, P = 0.000) and waist-hip ratio (β = 0.410, P = 0.000) emerged as significant and independent determinants of Lp-PLA2 activity. Multiple stepwise regression analysis revealed that LDL-cholesterol (β = 0.309, P = 0.000), systolic blood pressure (β = 0.322, P = 0.002) and age (β = 0.235, P = 0.007) significantly correlated with max IMT, and Lp-PLA2 was not an independent predictor for carotid IMT. Conclusions Lp-PLA2 may be a modulating factor for carotid IMT via age and LDL-cholesterol, not independent predictor in the pathophysiological process of carotid atherosclerosis in patients with MetS. PMID:21247435

  15. Lipoprotein-associated phospholipase A2 decreases oxidized lipoprotein cellular association by human macrophages and hepatocytes.

    PubMed

    Yang, Ming; Chu, Eugene M; Caslake, Muriel J; Edelstein, Celina; Scanu, Angelo M; Hill, John S

    2010-02-01

    We investigated whether the presence of endogenous or exogenous lipoprotein-associated phospholipase A2 (Lp-PLA2) can modify the cellular association of oxidized low density lipoprotein (oxLDL) and oxidized lipoprotein(a) (oxLp(a)) by human monocyte-derived macrophages (MDM) and hepatocytes (HepG2). Purified recombinant Lp-PLA2 was used as a source of exogenous enzyme whereas Pefabloc (serine esterase inhibitor) was used to inhibit the endogenous Lp-PLA2 activity associated with isolated lipoproteins. Cellular association studies were performed with DiI-labeled oxLDL or oxLp(a) and human monocyte-derived macrophages and HepG2 cells. Active Lp-PLA2 decreased the cellular association of oxLDL and oxLp(a) in macrophages and HepG2 cells by approximately 30-40%, whereas the inactive enzyme did not significantly change oxidized lipoprotein cellular association by either cell type. OxLDL pretreated by Pefabloc increased oxLDL cellular association by MDM and HepG2 cells compared to untreated oxLDL. Therefore, unlike some lipases, Lp-PLA2 did not appear to have any catalytic independent function in oxLDL cellular association. To assess whether the reduced cellular association mediated by Lp-PLA2 was due to the hydrolysis of oxidized phosphatidylcholine (oxPC), we measured the concentration of lysophosphatidylcholine (lysoPC) in lipoprotein fractions after Lp-PLA2 treatment. LysoPC was increased by 20% (0.4 microM) and 87% (0.7 microM) by active Lp-PLA2 compared to inactive Lp-PLA2 for oxLDL and Lp(a), respectively. LysoPC at higher concentration dose-dependently increased the cellular association of oxLDL and oxLp(a) in MDM and HepG2 cells. We conclude that Lp-PLA2 mediates a decrease in oxidized lipoprotein cellular association in human macrophages and HepG2 cells by reducing the concentration of oxPC within these lipoproteins.

  16. Overexpression of porcine lipoprotein-associated phospholipase A2 in swine.

    PubMed

    Tang, Xiaochun; Wang, Gangqi; Liu, Xingxing; Han, Xiaolei; Li, Zhuang; Ran, Guangyao; Li, Zhanjun; Song, Qi; Ji, Yuan; Wang, Haijun; Wang, Yuhui; Ouyang, Hongsheng; Pang, Daxin

    2015-09-25

    Lipoprotein-associated phospholipase A 2 (Lp-PLA2) is associated with the risk of vascular disease. It circulates in human blood predominantly in association with low-density lipoprotein cholesterol (LDL-C) and hydrolyses oxidized phospholipids into pro-inflammatory products. However, in the mouse circulation, it predominantly binds to high-density lipoprotein cholesterol (HDL-C) and exhibits anti-inflammatory properties. To further investigate the effects of Lp-PLA2 in the circulation, we generated over-expressed Lp-PLA2 transgenic swine. The eukaryotic expression plasmid of porcine Lp-PLA2 which driven by EF1α promoter was constructed and generate transgenic swine via SCNT. The expression and activity of Lp-PLA2 in transgenic swine were evaluated, and the total cholesterol (TC), HDL-C, LDL-C and triglyceride (TG) levels in the fasting and fed states were also assessed. Compared with wild-type swine controls, the transgenic swine exhibited elevated Lp-PLA2 mRNA levels and activities, and the activity did not depend on the feeding state. The TC, HDL-C and LDL-C levels were not significantly increased. There was no change in the TG levels in the fasting state between transgenic and control pigs. However, in the fed state, the TG levels of transgenic swine were slightly increased compared with the control pigs and were significantly elevated compared with the fasting state. In addition, inflammatory gene (interleukin [IL]-6, monocyte chemotactic protein [MCP]-1 and tumor necrosis factor [TNF]-α) mRNA levels in peripheral blood mononuclear cells (PBMCs) were significantly increased. The results demonstrated that Lp-PLA2 is associated with triglycerides which may be helpful for understanding the relationship of this protein with cardiovascular disease.

  17. Investigation of a Bicyclo[1.1.1]pentane as a Phenyl Replacement within an LpPLA2 Inhibitor.

    PubMed

    Measom, Nicholas D; Down, Kenneth D; Hirst, David J; Jamieson, Craig; Manas, Eric S; Patel, Vipulkumar K; Somers, Don O

    2017-01-12

    We describe the incorporation of a bicyclo[1.1.1]pentane moiety within two known LpPLA2 inhibitors to act as bioisosteric phenyl replacements. An efficient synthesis to the target compounds was enabled with a dichlorocarbene insertion into a bicyclo[1.1.0]butane system being the key transformation. Potency, physicochemical, and X-ray crystallographic data were obtained to compare the known inhibitors to their bioisosteric counterparts, which showed the isostere was well tolerated and positively impacted on the physicochemical profile.

  18. The elevated lipoprotein-associated phospholipase A2 activity is associated with the occurrence and recurrence of acute cerebral infarction.

    PubMed

    Wei, Lingli; Ke, Zunyu; Zhao, Yu; Cai, Zhiyou

    2017-03-07

    There is a strong association between lipoprotein-associated phospholipase A2 (Lp-PLA2) levels and atherosclerosis-related diseases. The aim of this study was to investigate the role of Lp-PLA2 in the ischemic stroke and further offer clinical evidence that measuring Lp-PLA2 helps predict the risk of stroke occurrence and recurrence. A total of 328 hospitalized patients were recruited, including 179 cases of acute cerebral infarction (ACI) and 149 non-ACI controls. The serum level of Lp-PLA2 in ACI was significantly higher than non-ACI. The serum level of Lp-PLA2 in the recurrence of ACI was significantly higher than the nonrecurrence. The serum levels of Lp-PLA2 in large-artery atherosclerosis subtype were the highest among the subtypes of the Trial of Org 10172 in Acute Stroke Treatment and non-ACI controls. The level of Lp-PLA2 in large-artery atherosclerosis and the cardioembolism group was statistically significantly higher than that of the control cases. There was no statistically significant difference between the small-vessel occlusion group and the control cases. The present study confirmed that the elevated Lp-PLA2 level can be a risk factor for ischemic stroke in the Chinese population. The serum level of Lp-PLA2 may be a predictive factor for the recurrence of ACI.

  19. Effect of improving glycemic control in patients with type 2 diabetes mellitus on low-density lipoprotein size, electronegative low-density lipoprotein and lipoprotein-associated phospholipase A2 distribution.

    PubMed

    Sánchez-Quesada, José L; Vinagre, Irene; de Juan-Franco, Elena; Sánchez-Hernández, Juan; Blanco-Vaca, Francisco; Ordóñez-Llanos, Jordi; Pérez, Antonio

    2012-07-01

    The aim of this study was to determine the effect of intensified hypoglycemic therapy in patients with type 2 diabetes mellitus on the distribution of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity between high-density lipoprotein and low-density lipoprotein (LDL) and its relation with the lipid profile and other qualitative properties of LDL. Forty-two patients with type 2 diabetes on the basis of poor glycemic control and normal or near normal LDL cholesterol were recruited. Lifestyle counseling and pharmacologic hypoglycemic therapy were intensified to improve glycemic control, but lipid-lowering therapy was unchanged. At 4 ± 2 months, glycosylated hemoglobin had decreased by a mean of 2.1%, but the only effect on the lipid profile were statistically significant decreases in nonesterified fatty acids and apolipoprotein B concentration. LDL size increased and the proportion of electronegative LDL decreased significantly. In parallel, total Lp-PLA2 activity decreased significantly, promoting a redistribution of Lp-PLA2 activity toward a higher proportion in high-density lipoprotein. Improvements in glycemic control led to more marked changes in Lp-PLA2 activity and distribution in patients with diabetes who had not received previous lipid-lowering therapy. In conclusion, optimizing glycemic control in patients with type 2 diabetes promotes atheroprotective changes, including larger LDL size, decreased electronegative LDL, and a higher proportion of Lp-PLA2 activity in high-density lipoprotein.

  20. Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor.

    PubMed Central

    MacPhee, C H; Moores, K E; Boyd, H F; Dhanak, D; Ife, R J; Leach, C A; Leake, D S; Milliner, K J; Patterson, R A; Suckling, K E; Tew, D G; Hickey, D M

    1999-01-01

    A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine. PMID:10024526

  1. C-reactive protein and lipoprotein-associated phospholipase A2 in smokers and nonsmokers of the Ludwigshafen Risk and Cardiovascular Health study.

    PubMed

    Kleber, M E; Siekmeier, R; Delgado, G; Grammer, T B; Winkelmann, B R; Scharnagl, H; Boehm, B O; März, W

    2015-01-01

    Measurement of high sensitivity CRP (hsCRP) and lipoprotein-associated phospholipase A2 (LpPLA2) provides information on systemic inflammation and stability of atherosclerotic plaques. Data analyzing the effect of smoking on these parameters are sparse. The aim of our study was the analysis of these parameters in active smokers and never-smokers. The study included 777 smokers and 1,178 never-smokers, of whom 221 and 302 died during a follow-up, respectively. The values of LpPLA2 and hsCRP were significantly higher in smokers than in never-smokers. Mortality was highest in smokers and never-smokers with elevation of both biomarkers. Multivariate adjusted hazard ratios for patients in the highest tertile of both hsCRP and LpPLA2 compared with patients in the lowest tertile of both markers were 1.85 (1.04-3.28) in never-smokers and 1.94 (1.10-3.45) in smokers. Our data confirmed the predictive value of hsCRP and LpPLA2. However, there were a relevant number of patients with an increase of only one of these parameters. Therefore, beside other risk factors for cardiovascular disease, both parameters should be determined at least in high risk patients.

  2. Lipoprotein-associated phospholipase A2 as a predictive biomarker of sub-clinical inflammation in cardiovascular diseases

    PubMed Central

    Cojocaru, Manole; Cojocaru, Inimioara Mihaela; Silosi, Isabela

    2010-01-01

    ABSTRACT Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a predictor biomarker for incident atherosclerotic disease. Lp-PLA2 has been identified in atherosclerotic plaques, however, its role in atherosclerosis is still under investigation. Lp-PLA2 belongs to the superfamily of phospholipase A2 enzymes. It is produced by macrophages that appears to play a role in the atherosclerotic vessel wall. Emerging data seem to suggest that Lp-PLA2 may be proatherogenic, which is an effect thought to be mediated by lypophosphatidylcholine and oxidized nonesterified fatty acids, two mediators generated by Lp-PLA2. Phospholipase A2 plays an essential role in metabolism of membrane phospholipids, it is related to inflammatory reactions, secretion of amyloid precursor protein. Several studies have documented the strong association of Lp-PLA2 with coronary heart disease and stroke in the general population. Lp-PLA2 may be a stronger predictor of recurrent stroke risk. Inflammatory markers have been associated with ischemic stroke risk. Their relationship to prognosis after stroke is unsettled. The present review article focuses particularly on the characteristics of the Lp(a)-associated Lp-PLA2 and discusses the possible role of this enzyme in view of the new data. PMID:21977119

  3. Lipoprotein-associated phospholipase A2 as a predictive biomarker of sub-clinical inflammation in cardiovascular diseases.

    PubMed

    Cojocaru, Manole; Cojocaru, Inimioara Mihaela; Silosi, Isabela

    2010-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a predictor biomarker for incident atherosclerotic disease. Lp-PLA2 has been identified in atherosclerotic plaques, however, its role in atherosclerosis is still under investigation. Lp-PLA2 belongs to the superfamily of phospholipase A2 enzymes. It is produced by macrophages that appears to play a role in the atherosclerotic vessel wall. Emerging data seem to suggest that Lp-PLA2 may be proatherogenic, which is an effect thought to be mediated by lypophosphatidylcholine and oxidized nonesterified fatty acids, two mediators generated by Lp-PLA2. Phospholipase A2 plays an essential role in metabolism of membrane phospholipids, it is related to inflammatory reactions, secretion of amyloid precursor protein. Several studies have documented the strong association of Lp-PLA2 with coronary heart disease and stroke in the general population. Lp-PLA2 may be a stronger predictor of recurrent stroke risk. Inflammatory markers have been associated with ischemic stroke risk. Their relationship to prognosis after stroke is unsettled. The present review article focuses particularly on the characteristics of the Lp(a)-associated Lp-PLA2 and discusses the possible role of this enzyme in view of the new data.

  4. Discovery of a Novel Series of Imidazo[1,2-a]pyrimidine Derivatives as Potent and Orally Bioavailable Lipoprotein-Associated Phospholipase A2 Inhibitors.

    PubMed

    Chen, Xinde; Xu, Wenwei; Wang, Kai; Mo, Mingguang; Zhang, Wei; Du, Lili; Yuan, Xiaojing; Xu, Yechun; Wang, Yiping; Shen, Jianhua

    2015-11-12

    Inhibition of lipoprotein-associated phospholipase A2 (Lp-PLA2) has been suggested to be a promising therapeutic strategy for several inflammation-associated diseases, including atherosclerosis, Alzheimer's disease, and diabetic macular edema. Herein, we report the discovery of a novel series of Lp-PLA2 inhibitors constructed on an imidazo[1,2-a]pyrimidine scaffold through a conformational restriction strategy. Structure-activity relationship (SAR) analysis resulted in the identification of several compounds with high potency in vitro and good metabolic stability in liver S9 fractions. Compounds 7c and 14b selected for further exploration in vivo demonstrated excellent pharmacokinetic profiles and exhibited significant inhibitory efficacy in SD rats upon oral dosing.

  5. Platelet Aggregation Unchanged by Lipoprotein-Associated Phospholipase A2 Inhibition: Results from an In Vitro Study and Two Randomized Phase I Trials

    PubMed Central

    Shaddinger, Bonnie C.; Xu, Yanmei; Roger, James H.; Macphee, Colin H.; Handel, Malcolm; Baidoo, Charlotte A.; Magee, Mindy; Lepore, John J.; Sprecher, Dennis L.

    2014-01-01

    Background We explored the theorized upregulation of platelet-activating factor (PAF)– mediated biologic responses following lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibition using human platelet aggregation studies in an in vitro experiment and in 2 clinical trials. Methods and Results Full platelet aggregation concentration response curves were generated in vitro to several platelet agonists in human plasma samples pretreated with rilapladib (selective Lp-PLA2 inhibitor) or vehicle. This was followed by a randomized, double-blind crossover study in healthy adult men (n = 26) employing a single-agonist dose assay of platelet aggregation, after treatment of subjects with 250 mg oral rilapladib or placebo once daily for 14 days. This study was followed by a second randomized, double-blind parallel-group trial in healthy adult men (n = 58) also treated with 250 mg oral rilapladib or placebo once daily for 14 days using a full range of 10 collagen concentrations (0–10 µg/ml) for characterizing EC50 values for platelet aggregation for each subject. Both clinical studies were conducted at the GlaxoSmithKline Medicines Research Unit in the Prince of Wales Hospital, Sydney, Australia. EC50 values derived from multiple agonist concentrations were compared and no pro-aggregant signals were observed during exposure to rilapladib in any of these platelet studies, despite Lp-PLA2 inhibition exceeding 90%. An increase in collagen-mediated aggregation was observed 3 weeks post drug termination in the crossover study (15.4% vs baseline; 95% confidence interval [CI], 3.9–27.0), which was not observed during the treatment phase and was not observed in the parallel-group study employing a more robust EC50 examination. Conclusions Lp-PLA2 inhibition does not enhance platelet aggregation. Trial Registration 1) Study 1: ClinicalTrials.gov NCT01745458 2) Study 2: ClinicalTrials.gov NCT00387257 PMID:24475026

  6. Lipoprotein-associated phospholipase A2 levels are associated with erectile dysfunction in patients without known coronary artery disease.

    PubMed

    Otunctemur, A; Sahin, S; Ozbek, E; Cekmen, M; İnal, A; Tulubas, F; Dursun, M; Besiroglu, H; Koklu, I

    2015-08-01

    Endothelial dysfunction and microvascular damage play a crucial role in the pathogenesis of erectile dysfunction (ED). Lp-PLA2 is a calcium-independent member of the phospholipase A2 family and hydrolyses oxidised phospholipids on low-density lipoprotein (LDL) particles that plays a pivotal role in ox-LDL-induced endothelial dysfunction. The purpose of the current study was to determine the association between Lp-PLA2 levels and ED in patients without known coronary artery disease (CAD). All patients were evaluated for ED and divided into two groups: 88 patients suffering from ED for >1 year were enrolled as an experimental group and 88 patients without ED were enrolled as a control group in this study. Diagnosis of ED was based on the International Index of Erectile Function Score-5. Levels of Lp-PLA2 were measured in serum by colorimetric assay. The relationship between Lp-PLA2 levels and ED in patients was evaluated statistically. The mean age of patients with ED group was 59.4 ± 11.32 and 55.8 ± 9.67 in the control group. Plasma Lp-PLA2 levels were significantly higher in ED than in the control group (220.3 ± 66.90 and 174.8 ± 58.83 pg ml(-1) , respectively, P < 0.001). The Lp-PLA2 levels were negatively correlated with score of ED (r = -0.482, P < 0.05). In logistic regression analysis, enhanced plasma Lp-PLA2 levels result in approximately 1.2-fold increase in ED [1.22 (1.25-2.76)]. In this study, serum Lp-PLA2 levels were found to be associated with endothelial dysfunction predictive of ED. Serum Lp-PLA2 level appears to be a specific predictor of ED, and it may be used in early prediction of ED in the male population.

  7. Plasma Lipoprotein-Associated Phospholipase A2 Level Is an Independent Predictor of High Thrombus Burden in Patients With Acute ST-segment Elevation Myocardial Infarction.

    PubMed

    Wu, Xiangqi; Zhang, Yingqiang; Wu, Zhiming; You, Wei; Liang, Fengshuo; Ye, Fei; Chen, Shaoliang

    2016-12-02

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an independent risk factor for plaque rupture and atherothrombotic events. However, the associations between serum Lp-PLA2 level and thrombus burden in ST-segment elevation myocardial infarction (STEMI) patients remain unknown.We consecutively enrolled 351 STEMI patients who underwent primary percutaneous coronary intervention (pPCI). Patients were assigned to a high thrombus burden (HTB) group (n = 230) and a low thrombus burden (LTB) group (n = 121). Baseline data were recorded during hospital admission. Plasma Lp-PLA2 concentration, coronary angiography results, and in-hospital mortality were measured. Plasma Lp-PLA2 level had a high correlation with thrombus burden score (TBS) before pPCI and it was found to be a significant independent predictor of HTB in STEMI patients (P < 0.05). Moreover, TBS, corrected thrombolysis in myocardial infarction (TIMI) frame count (cTFC), and plasma Lp-PLA2 level after pPCI in patients with HTB were significantly higher than those in patients with LTB (P < 0.05). Meanwhile, TIMI flow grade (TFG) and TIMI myocardial perfusion grade (TMPG) of HTB patients were markedly lower than those of LTB patients (P < 0.05). Additionally, correlations of plasma Lp-PLA2 level before pPCI with TFG before pPCI and TBS, cTFC, and TMPG after pPCI were modest (P < 0.05). However, the associations of plasma Lp-PLA2 level after pPCI with TFG, TBS, cTFC and TMPG were low (P < 0.05).These results demonstrated that the plasma Lp-PLA2 level before pPCI is an independent predictor of HTB in STEMI patients, resulting in modestly predicting blood flow and myocardial perfusion of the culprit artery.

  8. [Meta-analysis for the relationship between lipoprotein-associated phospholipase A2 and ischemic stroke].

    PubMed

    Xia, Wencui; Hu, Zhongyang; Song, Zhi

    2017-02-28

    目的:用Meta分析的方法探讨脂蛋白相关磷脂酶A2(lipoprotein-associated phospholipase A2Lp-PLA2)与缺血性脑卒中发生的相关性。方法:在线检索PubMed,Web of Science,Embase和Cochrane Library以及中国生物医学文献数据库、中国期刊全文数据库、重庆维普和万方数字化期刊全文数据库中关于Lp-PLA2与缺血性脑卒中相关性的病例对照研究或队列研究,采用Stata 12.0软件对入选研究进行Meta分析。结果:共有8篇文献46 034例符合纳入标准。Meta分析结果显示:Lp-PLA2水平与缺血性脑卒中发生的合并效应量的危险比(95%可信区间)[RR(95% CI)]为1.04(0.98~1.11),提示Lp-PLA2水平与缺血性卒中发生无相关性。Lp-PLA2 活性与缺血性脑卒中发生的相关性合并效应量RR(95% CI)为1.03(0.96~1.10),提示Lp-PLA2活性与缺血性卒中发生无相关性。结论:目前不能认为Lp-PLA2水平及Lp-PLA2活性可以预测缺血性脑卒中的发生,高Lp-PLA2水平及高Lp-PLA2活性是缺血性脑卒中发病的危险因素的结论有待进一步论证。.

  9. Lipoprotein-associated phospholipase A2, myeloperoxidase and vascular endothelial growth factor - predictors of high vascular risk in respiratory bacterial infections.

    PubMed

    Seri, A; Marta, D S; Madalan, A; Popescu, M; Tiglea, A I; Moldoveanu, E

    2016-01-01

    Objective. Respiratory bacterial infections are associated with important coagulation disturbances that amplify the pulmonary lesions and determine a more severe course of the disease. The aim of our study was to investigate the correlation between the evolution of the general clinical parameters and the occurrence of thrombotic events on one side, and plasma levels of selected proteins involved in inflammation and coagulation on the other side, with the intent to establish and to validate a laboratory test panel for the assessment of the vascular risk in patients with bacterial respiratory infections. Methods. The study included 111 patients (divided into two groups, 61 without thrombosis and 50 with thrombosis) with bacterial respiratory infections and 30 healthy controls, age and gender-matched. The baseline evaluation of the patients included clinical, biological, and respiratory examination. LpPLA2 and MPO activities were measured by the spectrophotometric method. VEGF was quantified with an ELISA kit. Results. The collected data showed a correlation between the occurrence of superimposed thrombosis in respiratory infection patients, and the intensity of the inflammatory process, reflected by the increased MPO activity, and the dynamics of LpPLA2 and VEGF. Conclusion. Bacterial respiratory infections associate thrombotic vascular events of various degrees of severity, which correlate with the intensity of the inflammatory process, and the severity of endothelium dysfunction at the level of microcirculation. Starting from the recorded data, and based on the established severity scales in use, it is possible to compute a vascular risk score that takes into consideration the values of the three biomarkers under investigation. Abbreviations: COPD = chronic obstructive pulmonary disease,hsCRP = high sensitivity C reactive protein,EC = endothelial cells, ICAM-1 = intercellular adhesion molecule1, LpPLA2 = lipoprotein-associated phospholipase A2, MPO

  10. Lipoprotein-associated phospholipase A2, myeloperoxidase and vascular endothelial growth factor - predictors of high vascular risk in respiratory bacterial infections

    PubMed Central

    Seri, A; Marta, DS; Madalan, A; Popescu, M; Tiglea, AI; Moldoveanu, E

    2016-01-01

    Objective. Respiratory bacterial infections are associated with important coagulation disturbances that amplify the pulmonary lesions and determine a more severe course of the disease. The aim of our study was to investigate the correlation between the evolution of the general clinical parameters and the occurrence of thrombotic events on one side, and plasma levels of selected proteins involved in inflammation and coagulation on the other side, with the intent to establish and to validate a laboratory test panel for the assessment of the vascular risk in patients with bacterial respiratory infections. Methods. The study included 111 patients (divided into two groups, 61 without thrombosis and 50 with thrombosis) with bacterial respiratory infections and 30 healthy controls, age and gender-matched. The baseline evaluation of the patients included clinical, biological, and respiratory examination. LpPLA2 and MPO activities were measured by the spectrophotometric method. VEGF was quantified with an ELISA kit. Results. The collected data showed a correlation between the occurrence of superimposed thrombosis in respiratory infection patients, and the intensity of the inflammatory process, reflected by the increased MPO activity, and the dynamics of LpPLA2 and VEGF. Conclusion. Bacterial respiratory infections associate thrombotic vascular events of various degrees of severity, which correlate with the intensity of the inflammatory process, and the severity of endothelium dysfunction at the level of microcirculation. Starting from the recorded data, and based on the established severity scales in use, it is possible to compute a vascular risk score that takes into consideration the values of the three biomarkers under investigation. Abbreviations: COPD = chronic obstructive pulmonary disease,hsCRP = high sensitivity C reactive protein,EC = endothelial cells, ICAM-1 = intercellular adhesion molecule1, LpPLA2 = lipoprotein-associated phospholipase A2, MPO

  11. A phenome-wide association study of a lipoprotein-associated phospholipase A2 loss-of-function variant in 90 000 Chinese adults

    PubMed Central

    Millwood, Iona Y; Bennett, Derrick A; Walters, Robin G; Clarke, Robert; Waterworth, Dawn; Johnson, Toby; Chen, Yiping; Yang, Ling; Guo, Yu; Bian, Zheng; Hacker, Alex; Yeo, Astrid; Parish, Sarah; Hill, Michael R; Chissoe, Stephanie; Peto, Richard; Cardon, Lon; Collins, Rory; Li, Liming; Chen, Zhengming

    2016-01-01

    Background: Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been implicated in development of atherosclerosis; however, recent randomized trials of Lp-PLA2 inhibition reported no beneficial effects on vascular diseases. In East Asians, a loss-of-function variant in the PLA2G7 gene can be used to assess the effects of genetically determined lower Lp-PLA2. Methods: PLA2G7 V279F (rs76863441) was genotyped in 91 428 individuals randomly selected from the China Kadoorie Biobank of 0.5 M participants recruited in 2004–08 from 10 regions of China, with 7 years’ follow-up. Linear regression was used to assess effects of V279F on baseline traits. Logistic regression was conducted for a range of vascular and non-vascular diseases, including 41 ICD-10 coded disease categories. Results: PLA2G7 V279F frequency was 5% overall (range 3–7% by region), and 9691 (11%) participants had at least one loss-of-function variant. V279F was not associated with baseline blood pressure, adiposity, blood glucose or lung function. V279F was not associated with major vascular events [7141 events; odds ratio (OR) = 0.98 per F variant, 95% confidence interval (CI) 0.90-1.06] or other vascular outcomes, including major coronary events (922 events; 0.96, 0.79-1.18) and stroke (5967 events; 1.00, 0.92-1.09). Individuals with V279F had lower risks of diabetes (7031 events; 0.91, 0.84-0.98) and asthma (182 events; 0.53, 0.28-0.98), but there was no association after adjustment for multiple testing. Conclusions: Lifelong lower Lp-PLA2 activity was not associated with major risks of vascular or non-vascular diseases in Chinese adults. Using functional genetic variants in large-scale prospective studies with linkage to a range of health outcomes is a valuable approach to inform drug development and repositioning. PMID:27301456

  12. Is Lipoprotein-Associated Phospholipase A2 a Link between Inflammation and Subclinical Atherosclerosis in Rheumatoid Arthritis?

    PubMed Central

    Södergren, Anna; Karp, Kjell; Bengtsson, Christine; Möller, Bozena; Rantapää-Dahlqvist, Solbritt; Wållberg-Jonsson, Solveig

    2015-01-01

    Objective. Lipoprotein-associated phospholipase A2 (Lp-PLA2), a marker of vascular inflammation, is associated with cardiovascular disease. This prospective study of an inception cohort aimed to investigate whether the level of Lp-PLA2 is associated with subclinical atherosclerosis in patients with rheumatoid arthritis (RA). Methods. Patients from northern Sweden diagnosed with early RA were consecutively recruited into an ongoing prospective study. From these, all patients ≤60 years (n = 71) were included for measurements of subclinical atherosclerosis at inclusion (T0) and five years later (T5). Forty age- and sex-matched controls were included. The patients were clinically assessed, SCORE, Reynolds Risk Score, and Larsen score were calculated, and blood samples were drawn from all individuals at T0 and T5. Results. There was no significant difference in the level of Lp-PLA2 between patients with RA and controls (p > 0.05). In simple linear regression models among patients with RA, Lp-PLA2 at T0 was significantly associated with intima media thickness (IMT) at T0 and T5, flow mediated dilation (FMD) at T0 and T5, ever smoking, male sex, HDL-cholesterol (inversely), non-HDL-cholesterol, SCORE, Reynolds Risk Score, and Larsen score (p < 0.05). Conclusion. In this cohort of patients with early RA, the concentration of Lp-PLA2 was associated with both subclinical atherosclerosis and disease severity. PMID:26504820

  13. Lipoprotein-associated phospholipase A2 as a novel risk marker for cardiovascular disease: a systematic review of the literature.

    PubMed

    Madjid, Mohammad; Ali, Muzammil; Willerson, James T

    2010-01-01

    We sought to critically assess the role of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) in the prediction of cardiovascular events in primary and secondary prevention settings. The inclusion criteria for our study included population-based epidemiologic studies and the presence of clinical outcomes of interest, including atherosclerotic disease, coronary events, stroke, and cardiovascular death. Studies that lacked clinical outcomes or that involved animals were excluded. We included primary and secondary prevention studies of subjects in all ethnic groups and of either sex, with no age limitation. We searched MEDLINE, Google Scholar, and the Cochrane Library for studies with publication dates from January 1970 through July 2009, and we searched major cardiology meeting abstracts from 2000 through 2009. From each study, we used predictive ability-including relative risk, hazard ratio, odds ratio, and prevalence of high Lp-PLA(2) levels, with adjustment-along with baseline population characteristics.Of 33 studies that met our inclusion criteria, 30 showed a significant association between Lp-PLA(2) and cardiovascular events. Most of the studies had been adjusted for major Framingham risk factors and other variables that might influence the effect under question. After multivariate adjustments in cohort and nested case-control studies, increased levels of Lp-PLA(2) remained a significant predictor of cardiovascular events. The available body of evidence suggests that Lp-PLA(2) is a reliable marker of risk for cardiovascular events.

  14. [Role of secreted and lipoprotein-associated phospholipase A2 in cardiovascular risk].

    PubMed

    Ferri, Nicola; Corsini, Alberto

    2014-12-01

    Phospholipase A(2) (PLA(2)) are enzymes that hydrolyze the ester bond of glycerophospholipids releasing free fatty acids and lysophospholipids, including the arachidonic acid, the precursor of the eicosanoids and the inflammatory cascades. PLA(2) are present in the atherosclerotic plaques and their direct involvement in the proatherogenic inflammatory response is well documented. Epidemiological and genetic studies have demonstrated the correlation of the PLA(2) mass and enzymatic activity with the incidence of cardiovascular diseases. The potential pro-atherogenic role of PLA(2) led to the development of two small molecules, varespladib, a reversible sPLA(2) inhibitor, and darapladib, a selective Lp-PLA(2) inhibitor. Both molecules have demonstrated antiatherosclerotic properties in animal models, and positive effects on atherosclerotic plaque composition evaluated in phase 2 clinical trials. On these grounds, the results of three phase 3 studies have recently been published: the VISTA-16 study with varespladib in patients with acute coronary syndrome, and the STABILITY and SOLID-TIMI 52 studies with darapladib in patients with stable coronary heart disease and acute coronary syndrome, respectively. Unexpectedly, both studies did not demonstrate an additional protective action of PLA 2 inhibitors over the standard of care treatment with statins, antiplatelet drugs, and coronary revascularization. In the present article, the enzymatic properties and the involvement of sPLA(2) and Lp-PLA(2) in atherogenesis are reviewed, with a focus on the results of experimental studies and clinical studies with both varespladib and darapladib inhibitors.

  15. Correlation between plasma lipoprotein-associated phospholipase A2 and peripheral arterial disease.

    PubMed

    Li, Shuai-Bing; Yang, Fan; Jing, Li; Ma, Juan; Jia, Ya-Dan; Dong, Shao-Ying; Zheng, Wei-Feng; Zhao, Luo-Sha

    2013-05-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a recently identified and potentially useful plasma biomarker for cardiovascular diseases. However, its role in peripheral arterial disease (PAD) remains unclear. The objective of this study was to assess the independent association of Lp-PLA2 and other inflammatory markers with the reduced ankle-brachial blood pressure index (ABI), a marker of PAD. We performed a cross-sectional study in 982 individuals aged ≥40 years who were recruited from the First Affiliated Hospital of Zhengzhou University. PAD was defined as an ABI <0.9 in at least one leg. The individuals were further divided into two groups, 145 with PAD and 837 without PAD. Following adjustment for traditional cardiovascular risk factors, the odds ratios of PAD when comparing the highest to the lowest quartiles were 3.24 (95% CI, 1.68-3.94) for Lp-PLA2, 2.14 (95% CI, 1.07-3.11) for homocysteine, 1.93 (95% CI, 1.02-4.01) for fibrinogen, 2.26 (95% CI, 1.32-5.74) for apolipoprotein B and 1.3 (95% CI, 0.75-2.49) for high-sensitivity C-reactive protein (hsCRP). When Lp-PLA2 and inflammatory markers were simultaneously included in the full model, the corresponding odds ratios were 1.81 (95% CI, 1.14-3.68) for Lp-PLA2, 1.15 (95% CI, 0.49-2.69) for homocysteine, 1.21 (95% CI, 0.88-5.57) for fibrinogen, 0.98 (95% CI, 0.51-3.85) for apolipoprotein B and 1.23 (95% CI, 1.12-3.51) for hsCRP. Lp-PLA2 levels were significantly and independently associated with PAD following adjustment for other inflammatory markers. These findings reflect the potential role of circulating Lp-PLA2 as a marker of atherosclerosis.

  16. Lipoprotein-associated phospholipase A2 is associated with postpartum hypertension in women with history of preeclampsia.

    PubMed

    Zhou, Yuheng; Niu, Jianmin; Duan, Dongmei; Lei, Qiong; Wen, Jiying; Lin, Xiaohong; Lv, Lijuan; Chen, Longding

    2015-07-01

    Both hypertension and preeclampsia (PE) are considered as inflammatory diseases. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an inflammatory marker associated with lipid metabolism. We aimed to study the correlation and predictive value of Lp-PLA2 in postpartum hypertension after PE. A group of 160 PE patients (PE group) and a separate group of 160 normal pregnant women (control group) were recruited from January 2010 to October 2011. The average age in the PE group was 28.4 ± 4.5 years and the average gestational age was 34.7 ± 1.1 weeks. The average age in the control group was 27.8 ± 4.5 years and the average gestational age was 35.5 ± 1.2 weeks. General information (including age, gestational age, parity, history of metabolic disease, family history of high blood pressure, height, body weight before childbirth, and blood pressure) and blood samples were collected for measuring Lp-PLA2 and lipid parameters. From February to April in 2013, 153 cases in the PE group and 132 in the control group were re-called. We assessed their postpartum health, pregnancy, height, weight, and blood pressure. Serum mass of Lp-PLA2 in the PE group (210.67 ± 17.98 ng/mL) was significantly higher compared with that in the control group (174.72 ± 30.26 ng/mL) (P < 0.01). The pro-gestation BMI, systolic blood pressure (SBP), diastolic blood pressure, total cholesterol, triglyceride, and low-density lipoprotein-cholesterol (LDL-C) were also significantly higher. Correlation analysis showed that the level of Lp-PLA2 and SBP (r = 0.31), LDL-C (r = 0.37) were positively correlated. The incidence of postpartum hypertension in the PE group was higher than that in the normal control group. Logistic regression analysis showed that prenatal Lp-PLA2 mass was an independent risk factor for PE postpartum hypertension (OR 1.134,95 % CI 1.086-1.185). ROC curve analysis showed that the sensitivity of predicting postpartum hypertension was 73.2% and the specific degree was 86.6%, with

  17. Pathophysiological role and clinical significance of lipoprotein-associated phospholipase A₂ (Lp-PLA₂) bound to LDL and HDL.

    PubMed

    Tellis, Constantinos C; Tselepis, Alexandros D

    2014-01-01

    Lipoprotein-associated phospholipase A2 (Lp-PLA2), also named as platelet-activating factor (PAF)-acetylhydrolase, exhibits a Ca2+-independent phospholipase A2 activity and catalyzes the hydrolysis of the ester bond at the sn-2 position of PAF and oxidized phospholipids (oxPL). These phospholipids are formed under oxidative and inflammatory conditions, and may play important roles in atherogenesis. The vast majority of plasma Lp-PLA2 mass binds to low-density lipoprotein (LDL) while a smaller amount is associated with high-density lipoprotein (HDL). Lp-PLA2 is also bound to lipoprotein (a) [Lp(a)], very low-density lipoprotein (VLDL) and remnant lipoproteins. Several lines of evidence suggest that the role of plasma Lp-PLA2 in atherosclerosis may depend on the type of lipoprotein particle with which this enzyme is associated. Data from large Caucasian population studies have supported plasma Lp-PLA2 (primarily LDL-associated Lp-PLA2) as a cardiovascular risk marker independent of, and additive to, traditional risk factors. On the contrary, the HDL-associated Lp-PLA2 may express antiatherogenic activities and is also independently associated with lower risk for cardiac death. The present review presents data on the biochemical and enzymatic properties of Lp-PLA2 as well as its structural characteristics that determine the association with LDL and HDL. We also critically discuss the possible pathophysiological and clinical significance of the Lp- PLA2 distribution between LDL and HDL in human plasma, in view of the results of prospective epidemiologic studies on the association of Lp-PLA2 with future cardiovascular events as well the recent studies that evaluate the possible effectiveness of specific Lp-PLA2 inhibitors in reducing residual cardiovascular risk.

  18. Bioactive Lysophospholipids Generated by Hepatic Lipase Degradation of Lipoproteins Lead to Complement Activation via the Classical Pathway

    PubMed Central

    Ma, Wanchao; Paik, David C.; Barile, Gaetano R.

    2014-01-01

    Purpose. We determined bioactivity of lysophospholipids generated by degradation of the low-density (LDL), very low-density (VLDL), and high-density (HDL) lipoproteins with hepatic lipase (HL), cholesterol esterase (CE), and lipoprotein-associated phospholipase A2 (Lp-PLA2). Methods. The LDL, VLDL, and HDL were treated with HL, CE, and Lp-PLA2 after immobilization on plates, and complement activation studies were performed with diluted human serum. Complement component 3 (C3) fixation, a marker for complement activation, was determined with a monoclonal anti-human C3d antibody. Enzymatic properties of HL and CE were assayed with triglyceride and phosphatidylcholine substrates for triglyceride hydrolase and phospholipase A activities. The ARPE-19 cells were used for viability studies. Results. The HL degradation of human lipoproteins LDL, VLDL, or HDL results in the formation of modified lipoproteins that can activate the complement pathway. Complement activation is dose- and time-dependent upon HL and occurs via the classical pathway. Enzymatic studies suggest that the phospholipase A1 activity of HL generates complement-activating lysophospholipids. C-reactive protein (CRP), known to simultaneously interact with complement C1 and complement factor H (CFH), further enhances HL-induced complement activation. The lysophospholipids, 1-Palmitoyl-sn-glycero-3-phosphocholine and 1-Oleoyl-sn-glycero-3-phosphocholine, can be directly cytotoxic to ARPE-19 cells. Conclusions. The HL degradation of lipoproteins, known to accumulate in the outer retina and in drusen, can lead to the formation of bioactive lysophospholipids that can trigger complement activation and induce RPE cellular dysfunction. Given the known risk associations for age-related macular degeneration (AMD) with HL, CRP, and CFH, this study elucidates a possible damage pathway for age-related macular degeneration (AMD) in genetically predisposed individuals, that HL activity may lead to accumulation of

  19. Biosynthesis of oxidized lipid mediators via lipoprotein-associated phospholipase A2 hydrolysis of extracellular cardiolipin induces endothelial toxicity.

    PubMed

    Buland, Justin R; Wasserloos, Karla J; Tyurin, Vladimir A; Tyurina, Yulia Y; Amoscato, Andrew A; Mallampalli, Rama K; Chen, Bill B; Zhao, Jing; Zhao, Yutong; Ofori-Acquah, Solomon; Kagan, Valerian E; Pitt, Bruce R

    2016-08-01

    We (66) have previously described an NSAID-insensitive intramitochondrial biosynthetic pathway involving oxidation of the polyunsaturated mitochondrial phospholipid, cardiolipin (CL), followed by hydrolysis [by calcium-independent mitochondrial calcium-independent phospholipase A2-γ (iPLA2γ)] of oxidized CL (CLox), leading to the formation of lysoCL and oxygenated octadecadienoic metabolites. We now describe a model system utilizing oxidative lipidomics/mass spectrometry and bioassays on cultured bovine pulmonary artery endothelial cells (BPAECs) to assess the impact of CLox that we show, in vivo, can be released to the extracellular space and may be hydrolyzed by lipoprotein-associated PLA2 (Lp-PLA2). Chemically oxidized liposomes containing bovine heart CL produced multiple oxygenated species. Addition of Lp-PLA2 hydrolyzed CLox and produced (oxygenated) monolysoCL and dilysoCL and oxidized octadecadienoic metabolites including 9- and 13-hydroxyoctadecadienoic (HODE) acids. CLox caused BPAEC necrosis that was exacerbated by Lp-PLA2 Lower doses of nonlethal CLox increased permeability of BPAEC monolayers. This effect was exacerbated by Lp-PLA2 and partially mimicked by authentic monolysoCL or 9- or 13-HODE. Control mice plasma contained virtually no detectable CLox; in contrast, 4 h after Pseudomonas aeruginosa (P. aeruginosa) infection, 34 ± 8 mol% (n = 6; P < 0.02) of circulating CL was oxidized. In addition, molar percentage of monolysoCL increased twofold after P. aeruginosa in a subgroup analyzed for these changes. Collectively, these studies suggest an important role for 1) oxidation of CL in proinflammatory environments and 2) possible hydrolysis of CLox in extracellular spaces producing lysoCL and oxidized octadecadienoic acid metabolites that may lead to impairment of pulmonary endothelial barrier function and necrosis.

  20. Association between Ala379Val polymorphism of lipoprotein-associated phospholipase A2 and migraine without aura in Iranian population

    PubMed Central

    Haghdoost, Faraidoon; Gharzi, Mahsa; Faez, Farough; Hosseinzadeh, Elinaz; Tajaddini, Mohamadhasan; Rafiei, Laleh; Asgari, Fatemeh; Banihashemi, Mahboobeh; Masjedi, Samaneh Sadat; Zandifar, Alireza; Haghjooy-Javanmard, Shaghayegh

    2016-01-01

    Background: Migraine is a common neurovascular disorder with multifactorial and polygenic inheritance. The aim of this study was to investigate the association of a migraine without aura and Ala379Val polymorphism of lipoprotein-associated phospholipase A2 (Lp-PLA2) gene in the Iranian population. Methods: In this study, 103 migraine patients and 100 healthy controls were enrolled. DNA samples were extracted and the Ala379Val polymorphism of Lp-PLA2 gene was investigated. To assess severity of a headache, patients filled out the headache impact test (HIT-6) and migraine severity (MIGSEV) questionnaires. Results: Allele V had significantly lower frequency in the case group than control subjects [P = 0.001, odds ratio (OR) = 0.25, confidence interval (CI): 0.15-0.40]. The frequency of migraine patients that were a carrier of V allele (V/V and A/V) was statistically significant lower than the control group (P = 0.003, OR = 2.39, CI: 1.35-4.23). There was no significant difference of alleles frequency between three grades of MIGSEV (P = 0.316). Furthermore, total HIT-6 score was not significantly different between different genotypes (P = 0.466). Conclusion: Our results showed that Ala379Val gene polymorphism of LP-PLA2 is associated with lower risk of migraine but not with severity of headaches in an Iranian population. PMID:27326362

  1. Effects of overweight and the PLA2G7 V279F polymorphism on the association of age with systolic blood pressure

    PubMed Central

    Kim, Minjoo; Kim, Minkyung; Yoo, Hye Jin; Jang, Hye Young; Lee, Sang-Hyun

    2017-01-01

    This prospective study aimed to determine the effects of the persistence of overweight for three years and the PLA2G7 V279F polymorphism, as well as the interaction between these factors, on the association of age with blood pressure (BP). Healthy middle-aged subjects with normotensive BP were divided into the normal-weight and overweight groups. The PLA2G7 V279F genotype, BP, lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, and oxidized low-density lipoprotein (ox-LDL) were determined. Lp-PLA2 activity was lower in the F allele subjects (n = 111) than in those with the VV genotype (n = 389). The overweight individuals with the F allele had lower Lp-PLA2 activity and ox-LDL at both baseline and after three years and lower systolic and diastolic BP and LDL cholesterol after three years compared with those with the VV phenotype. After three years, the overweight subjects with the VV phenotype exhibited greater increases in Lp-PLA2 activity, systolic BP, and ox-LDL than those with the F allele and normal-weight subjects with the VV phenotype. A multivariate analysis revealed that the PLA2G7 V279F genotype, baseline BMI, changes in Lp-PLA2 activity and ox-LDL remained independently and positively associated with changes in systolic BP. The simultaneous presence of the PLA2G7 279VV genotype and persistence of overweight synergistically increases the risk for hypertension, whereas lower Lp-PLA2 activity in PLA2G7 279F allele carriers might offer certain protection against hypertension, even in individuals who have been overweight for over three years. PMID:28334001

  2. Oxidized phospholipids and lipoprotein-associated phospholipase A2 as important determinants of Lp(a) functionality and pathophysiological role.

    PubMed

    Tselepis, Alexandros D

    2016-04-02

    Lipoprotein(a) [Lp(a)] is composed of a low density lipoprotein (LDL)-like particle to which apolipoprotein (a) [apo(a)] is linked by a single disulfide bridge. Lp(a) is considered a causal risk factor for ischemic cardiovascular disease (CVD) and calcific aortic valve stenosis (CAVS). The evidence for a causal role of Lp(a) in CVD and CAVS is based on data from large epidemiological databases, mendelian randomization studies, and genomewide association studies. Despite the well-established role of Lp(a) as a causal risk factor for CVD and CAVS, the underlying mechanisms are not well understood. A key role in the Lp(a) functionality may be played by its oxidized phospholipids (OxPL) content. Importantly, most of circulating OxPL are associated with Lp(a); however, the underlying mechanisms leading to this preferential sequestration of OxPL on Lp(a) over the other lipoproteins, are mostly unknown. Several studies support the hypothesis that the risk of Lp(a) is primarily driven by its OxPL content. An important role in Lp(a) functionality may be played by the lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme that catalyzes the degradation of OxPL and is bound to plasma lipoproteins including Lp(a). The present review article discusses new data on the pathophysiological role of Lp(a) and particularly focuses on the functional role of OxPL and Lp-PLA2 associated with Lp(a).

  3. Antioxidant and antiatherogenic activity of cis-Hinokiresinol from Trapa pseudoincisa.

    PubMed

    Song, Myoung-Chong; Yang, Hye-Joung; Bang, Myun-Ho; Kim, Dae-Keun; Jeong, Tae-Sook; Kim, Jong-Pyung; Baek, Nam-In

    2007-11-01

    cis-Hinokiresinol, also known as (+)-nyasol, was isolated for the first time from an aquatic herbaceous plant, Trapa pseudoincisa NAKAI, via silica gel and octadecyl silica gel column chromatographies. The chemical structure was determined via analyses of the spectroscopic data, including NMR, MS and IR. cis-Hinokiresinol was also found to exhibit antioxidant and antiatherogenic activities. The ICso values for the scavenging activities of cis-hinokiresinol on ABTS cation and superoxide anion radicals were 45.6 and 40.5 microM, respectively. The IC50 values for the inhibitory effects on Lp-PLA2, hACAT1, hACAT2 and LDL-oxidation were 284.7, 280.6, 398.9 and 5.6 microM, respectively.

  4. Effect of 24 Weeks of Statin Therapy on Systemic and Vascular Inflammation in HIV-Infected Subjects Receiving Antiretroviral Therapy

    PubMed Central

    Eckard, Allison Ross; Jiang, Ying; Debanne, Sara M.; Funderburg, Nicholas T.; McComsey, Grace A.

    2014-01-01

    Background. Human immunodeficiency virus (HIV)–infected individuals are at increased risk of cardiovascular disease (CVD) due in part to inflammation. Statins decrease inflammation in the general population, but their effect during HIV infection is largely unknown. Methods. This is an ongoing randomized, double-blinded, placebo-controlled trial to evaluate the effect of statin therapy on inflammatory markers during HIV infection. Subjects received rosuvastatin 10 mg daily or placebo for 24 weeks. Subjects were receiving stable (>12 weeks) antiretroviral therapy and had a low-density lipoprotein (LDL) cholesterol level of ≤130 mg/dL and evidence of heightened immune activation or inflammation. This was a prespecified interim analysis. Results. A total of 147 subjects were enrolled (78% were male, 70% were black, and the median age was 47 years). By 24 weeks, LDL cholesterol levels had decreased in the statin group, compared with an increase in the placebo group (−28% vs +3.8%; P < .01). A 10% reduction in the lipoprotein-associated phospholipase A2 (Lp-PLA2) level was seen in the statin group, compared with a 2% reduction in the placebo group (P < .01). In multivariable regression, receipt of statin treatment and having a nadir CD4+ T-cell count of ≤100 cell/µL were the only statistically significant predictors of a decrease in Lp-PLA2 level. Markers of systemic inflammation did not change significantly between groups. Conclusions. Twenty-four weeks of rosuvastatin therapy significantly decreased the level of Lp-PLA2, a vascular-specific, inflammatory enzyme that predicts cardiovascular events in the general population. Statins may hold promise as a means of attenuating CVD risk in HIV-infected individuals by decreasing Lp-PLA2 levels. PMID:24415784

  5. Associations of MDR1, TBXA2R, PLA2G7, and PEAR1 genetic polymorphisms with the platelet activity in Chinese ischemic stroke patients receiving aspirin therapy

    PubMed Central

    Peng, Ling-ling; Zhao, Yuan-qi; Zhou, Zi-yi; Jin, Jing; Zhao, Min; Chen, Xin-meng; Chen, Ling-yan; Cai, Ye-feng; Li, Jia-li; Huang, Min

    2016-01-01

    Aim: Aspirin resistance has an incidence of 5%–65% in patients with ischemic stroke, who receive the standard dose of aspirin, but the platelet function is inadequately inhibited, thereby leading to thrombotic events. Numerous evidence shows that thromboxane A2 receptor (TXA2 receptor, encoded by TBXA2R), lipoprotein-associated phospholipase A2 (Lp-PLA2, encoded by PLA2G7) and platelet endothelial aggregation receptor-1 (PEAR1, encoded by PEAR1) are crucial in regulating platelet activation, and P-glycoprotein (P-gp, encoded by MDR1) influences the absorption of aspirin in the intestine. In this study we examined the correlation between MDR1, TBXA2R, PLA2G7, PEAR1 genetic polymorphisms and platelet activity in Chinese ischemic stroke patients receiving aspirin therapy. Methods: A total of 283 ischemic stroke patients receiving 100 mg aspirin for 7 d were genotyped for polymorphisms in MDR1 C3435T, TBXA2R (rs1131882), PLA2G7 (rs1051931, rs7756935), and PEAR1 (rs12566888, rs12041331). The platelet aggregation response was measured using an automatic platelet aggregation analyzer and a commercially available TXB2 ELISA kit. Results: Thirty-three patients (11.66%) were insensitive to aspirin treatment. MDR1 3435TT genotype carriers, whose arachidonic acid (AA) or adenosine diphosphate (ADP)-induced platelet aggregation was lower than that of CC+CT genotype carriers, were less likely to suffer from aspirin resistance (odds ratio=0.421, 95% CI: 0.233–0.759). The TBXA2R rs1131882 CC genotype, which was found more frequently in the aspirin-insensitive group (81.8% vs 62.4%) than in the sensitive group, was identified as a risk factor for aspirin resistance (odds ratio=2.712, 95% CI: 1.080–6.810) with a higher level of AA-induced platelet aggregation. Due to the combined effects of PLA2G7 rs1051931 and rs7756935, carriers of the AA-CC haplotype had a higher level of ADP-induced platelet aggregation, and were at considerably higher risk of aspirin resistance than

  6. Persistence of an atherogenic lipid profile after treatment of acute infection with brucella

    PubMed Central

    Apostolou, F.; Gazi, I. F.; Kostoula, A.; Tellis, C. C.; Tselepis, A. D.; Elisaf, M.; Liberopoulos, E. N.

    2009-01-01

    Serum lipid changes during infection may be associated with atherogenesis. No data are available on the effect of Brucellosis on lipids. Lipid parameters were determined in 28 patients with Brucellosis on admission and 4 months following treatment and were compared with 24 matched controls. Fasting levels of total cholesterol (TC), HDL-cholesterol (HDL-C), triglycerides, apolipoproteins (Apo) A, B, E CII, and CIII, and oxidized LDL (oxLDL) were measured. Activities of serum cholesterol ester transfer protein (CETP), paraoxonase 1 (PON1), and lipoprotein-associated phospholipase A2 (Lp-PLA2) and levels of cytokines [interleukins (IL)-1β, IL-6, and tumor necrosis factor (TNFa)] were also determined. On admission, patients compared with controls had 1) lower levels of TC, HDL-C, LDL-cholesterol (LDL-C), ApoB, ApoAI, and ApoCIII and higher LDL-C/HDL-C and ApoB/ApoAI ratios; 2) higher levels of IL-1b, IL-6, and TNFa; 3) similar ApoCII and oxLDL levels and Lp-PLA2 activity, lower PON1, and higher CETP activity; and 4) higher small dense LDL-C concentration. Four months later, increases in TC, HDL-C, LDL-C, ApoB, ApoAI, and ApoCIII levels, ApoB/ApoAI ratio, and PON1 activity were noticed compared with baseline, whereas CETP activity decreased. LDL-C/HDL-C ratio, ApoCII, and oxLDL levels, Lp-PLA2 activity, and small dense LDL-C concentration were not altered. Brucella infection is associated with an atherogenic lipid profile that is not fully restored 4 months following treatment. PMID:19535817

  7. Altered Monocyte and Endothelial Cell Adhesion Molecule Expression Is Linked to Vascular Inflammation in Human Immunodeficiency Virus Infection

    PubMed Central

    Kulkarni, Manjusha; Bowman, Emily; Gabriel, Janelle; Amburgy, Taylor; Mayne, Elizabeth; Zidar, David A.; Maierhofer, Courtney; Turner, Abigail Norris; Bazan, Jose A.; Koletar, Susan L.; Lederman, Michael M.; Sieg, Scott F.

    2016-01-01

    Background. Human immunodeficiency virus (HIV)-infected individuals have increased risk for vascular thrombosis, potentially driven by interactions between activated leukocytes and the endothelium. Methods. Monocyte subsets (CD14+CD16−, CD14+CD16+, CD14DimCD16+) from HIV negative (HIV−) and antiretroviral therapy-treated HIV positive (HIV+) participants (N = 19 and 49) were analyzed by flow cytometry for adhesion molecule expression (lymphocyte function-associated antigen 1 [LFA-1], macrophage-1 antigen [Mac-1], CD11c/CD18, very late antigen [VLA]-4) and the fractalkine receptor (CX3CR1); these receptors recognize ligands (intercellular adhesion molecules [ICAMs], vascular cell adhesion molecule [VCAM]-1, fractalkine) on activated endothelial cells (ECs) and promote vascular migration. Plasma markers of monocyte (soluble [s]CD14, sCD163) and EC (VCAM-1, ICAM-1,2, fractalkine) activation and systemic (tumor necrosis factor receptor [TNFR-I], TNFR-II) and vascular (lipoprotein-associated phospholipase A2 [Lp-PLA2]) inflammation were measured by enzyme-linked immunosorbent assay. Results. Proportions of CD16+ monocyte subsets were increased in HIV+ participants. Among all monocyte subsets, levels of LFA-1 were increased and CX3CR1 levels were decreased in HIV+ participants (P < .01). Levels of sCD163, sCD14, fractalkine, ICAM-1, VCAM-1, TNFR-II, and Lp-PLA2 were also increased in HIV+ participants (P < .05), and levels of sCD14, TNFR-I, and TNFR-II were directly related to ICAM-1 and VCAM-1 levels in HIV+ participants. Expression of CX3CR1 on monocyte subsets was inversely related to plasma Lp-PLA2 (P < .05 for all). Conclusions. Increased proportions of CD16+ monocytes, cells with altered adhesion molecule expression, combined with elevated levels of their ligands, may promote vascular inflammation in HIV infection. PMID:28066794

  8. LARGE PARTICLES IN ACTIVE ASTEROID P/2010 A2

    SciTech Connect

    Jewitt, David; Ishiguro, Masateru; Agarwal, Jessica

    2013-02-10

    The previously unknown asteroid P/2010 A2 rose to prominence in 2010 by forming a transient, comet-like tail consisting of ejected dust. The observed dust production was interpreted as the result of either a hypervelocity impact with a smaller body or a rotational disruption. We have re-observed this object, finding that large particles remain a full orbital period after the initial outburst. In the intervening years, particles smaller than {approx}3 mm in radius have been dispersed by radiation pressure, leaving only larger particles in the trail. Since the total mass is dominated by the largest particles, the radiation pressure filtering allows us to obtain a more reliable estimate of the debris mass than was previously possible. We find that the mass contained in the debris is {approx}5 Multiplication-Sign 10{sup 8} kg (assumed density 3000 kg m{sup -3}), the ratio of the total debris mass to the nucleus mass is {approx}0.1, and that events like P/2010 A2 contribute <3% to the Zodiacal dust production rate. Physical properties of the nucleus and debris are also determined.

  9. A2a and a2b adenosine receptors affect HIF-1α signaling in activated primary microglial cells.

    PubMed

    Merighi, Stefania; Borea, Pier Andrea; Stefanelli, Angela; Bencivenni, Serena; Castillo, Carlos Alberto; Varani, Katia; Gessi, Stefania

    2015-05-15

    Microglia are central nervous system (CNS)-resident immune cells, that play a crucial role in neuroinflammation. Hypoxia-inducible factor-1 (HIF-1), the main transcription factor of hypoxia-inducible genes, is also involved in the immune response, being regulated in normoxia by inflammatory mediators. Adenosine is an ubiquitous nucleoside that has an influence on many immune properties of microglia through interaction with four receptor subtypes. The aim of this study was to investigate whether adenosine may affect microglia functions by acting on HIF-1α modulation. Primary murine microglia were activated with lipopolysaccharide (LPS) with or without adenosine, adenosine receptor agonists and antagonists and HIF-1α accumulation and downstream genes regulation were determined. Adenosine increased LPS-induced HIF-1α accumulation leading to an increase in HIF-1α target genes involved in cell metabolism [glucose transporter-1 (GLUT-1)] and pathogens killing [inducible nitric-oxide synthase (iNOS)] but did not induce HIF-1α dependent genes related to angiogenesis [vascular endothelial growth factor (VEGF)] and inflammation [tumor necrosis factor-α (TNF-α)]. The stimulatory effect of adenosine on HIF-1α and its target genes was essentially exerted by activation of A2A through p44/42 and A2B subtypes via p38 mitogen-activated protein kinases (MAPKs) and Akt phosphorylation. Furthermore the nucleoside raised VEGF and decreased TNF-α levels, by activating A2B subtypes. In conclusion adenosine increases GLUT-1 and iNOS gene expression in a HIF-1α-dependent way, through A2A and A2B receptors, suggesting their role in the regulation of microglial cells function following injury. However, inhibition of TNF-α adds an important anti-inflammatory effect only for the A2B subtype. GLIA 2015.

  10. Periodontal microbiota and phospholipases: The Oral Infections and Vascular Disease Epidemiology Study (INVEST)

    PubMed Central

    Boillot, Adrien; Demmer, Ryan T.; Mallat, Ziad; Sacco, Ralph L.; Jacobs, David R.; Benessiano, Joelle; Tedgui, Alain; Rundek, Tatjana; Papapanou, Panos N.; Desvarieux, Moïse

    2016-01-01

    Objective Periodontal infections have been linked to cardiovascular disease, including atherosclerosis, and systemic inflammation has been proposed as a possible mediator. Secretory phospholipase A2 (s-PLA2) and Lipoprotein-associated PLA2 (Lp-PLA2) are inflammatory enzymes associated with athero-sclerosis. No data are available on the association between oral microbiota and PLA2s. We studied whether a relationship exists between periodontal microbiota and the activities of these enzymes. Methods The Oral Infection and Vascular Disease Epidemiology Study (INVEST) collected subgingival biofilms and serum samples from 593 dentate men and women (age 68.7 ± 8.6 years). 4561 biofilm samples were collected in the two most posterior teeth of each quadrant (average 7/participant) for quantitative assessment of 11 bacterial species using DNA–DNA checkerboard hybridization. Mean concentration of s-PLA2 and activities of s-PLA2 and Lp-PLA2 were regressed on tertiles of etiologic dominance (ED). ED is defined as the level of presumed periodontopathic species/combined level of all eleven species measured, and represents the relative abundance of periodontopathic organisms. Analyses were adjusted for age, sex, race/ethnicity, education, smoking, BMI, diabetes, LDL cholesterol and HDL cholesterol, and systolic blood pressure. Results Higher levels of s-PLA2 activity were observed across increasing tertiles of etiologic dominance (0.66 ± 0.04 nmol ml−1 min−1, 0.73 ± 0.04 nmol ml−1 min−1, 0.89 ± 0.04 nmol ml−1 min−1; p < 0.001), with also a trend of association between Lp-PLA2 activity and ED (p = 0.07), while s-PLA2 concentration was unrelated to ED. Conclusion Increasingly greater s-PLA2 activity at higher tertiles of etiologic dominance may provide a mechanistic explanatory link of the relationship between periodontal microbiota and vascular diseases. Additional studies investigating the role of s-PLA2 are needed. PMID:26282947

  11. HtrA2 suppresses autoimmune arthritis and regulates activation of STAT3

    PubMed Central

    Lee, Seung Hoon; Moon, Young-Mee; Seo, Hyeon-Beom; Kim, Se-Young; Kim, Eun-Kyung; Yi, Junyeong; Nam, Min-Kyung; Min, Jun-Ki; Park, Sung-Hwan; Rhim, Hyangshuk; Cho, Mi-La

    2016-01-01

    Rheumatoid arthritis (RA) is an autoimmune disease that is related to the induction of T helper (Th)17 cells, which secrete interleukin-17, and activation of the signal transducer and activator of transcription (STAT) 3. The expression of high-temperature requirement protein A (HtrA) 2, a serine protease involved in apoptosis, was decreased in RA patients nonresponsive to drug treatment of RA. The aim of this study was to determine whether overexpression of HtrA2 has a therapeutic effect on RA. Th17 differentiation, osteoclastogenesis, and lymphocyte activation are increased in motor neuron degeneration (mnd)2 mice, which lack HtrA2 activity because of a missense mutation (Ser276Cys) in the protease domain of HtrA2. The inhibitor of HtrA2 also increased Th17 differentiation. On the other hand, HtrA2 induced cleavage of STAT3 and overexpression of HtrA2 attenuated CIA in a mouse model. HtrA2 overexpression inhibited plaque development as well as the differentiation of Th17 in ApoE−/− mice after immunization with proteoglycans to induce a hyperlipidemia-based RA animal model. The therapeutic function of HtrA2 in inflammatory diseases is linked with Th17 development and the STAT3 pathway in splenocytes. These results suggest that HtrA2 participates in immunomodulatory activity where the upregulation of HtrA2 may shed light on therapeutic approaches to RA and hyperlipidemia. PMID:28008946

  12. Measurement of CYP1A2 activity: a focus on caffeine as a probe.

    PubMed

    Perera, Vidya; Gross, Annette S; McLachlan, Andrew J

    2012-06-01

    The drug metabolising enzyme CYP1A2 contributes to the metabolism of a number of medicines including clozapine, olanzapine and theophylline. These medicines display a high degree of inter-individual variability in pharmacokinetics and response. Measuring CYP1A2 activity in vivo can be an important tool to identify the factors that influence variability in drug pharmacokinetics and inform dose selection. Caffeine is the only currently accepted probe to conduct in vivo phenotyping of CYP1A2. Despite the number of proposed matrices (biological fluid containing the drug and/or metabolite/s of interest) and metrics (mathematical formula relating the drug and/or metabolite/s to enzyme activity) proposed to measure CYP1A2 activity using caffeine, many of these are compromised by factors related to the specific metabolic pathway studied or pharmacokinetic characteristics of caffeine and its metabolites. Furthermore, questions regarding the appropriate study design and methodology to conduct studies to evaluate CYP1A2 activity have often been overlooked. These issues include the potential influence of a methylxanthine abstinence period prior to caffeine CYP1A2 phenotyping and the impact of caffeine formulation on determining CYP1A2 activity. This review aims to discuss the various CYP1A2 matrices and metrics with a particular focus on unresolved methodological issues.

  13. A role of the SAM domain in EphA2 receptor activation

    PubMed Central

    Shi, Xiaojun; Hapiak, Vera; Zheng, Ji; Muller-Greven, Jeannine; Bowman, Deanna; Lingerak, Ryan; Buck, Matthias; Wang, Bing-Cheng; Smith, Adam W.

    2017-01-01

    Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization. PMID:28338017

  14. Tanshinone I increases CYP1A2 protein expression and enzyme activity in primary rat hepatocytes.

    PubMed

    Lee, Wayne Y W; Zhou, Xuelin; Or, Penelope M Y; Kwan, Yiu Wa; Yeung, John H K

    2012-01-15

    This study investigated the effects of Danshen and its active ingredients on the protein expression and enzymatic activity of CYP1A2 in primary rat hepatocytes. The ethanolic extract of Danshen roots (containing mainly tanshinones) inhibited CYP1A2-catalyzed phenacetin O-deethylation (IC(50)=24.6 μg/ml) in primary rat hepatocytes while the water extract containing mainly salvianolic acid B and danshenshu had no effect. Individual tanshinones such as cryptotanshinone, dihydrotanshinone, tanshinone IIA inhibited the CYP1A2-mediated metabolism with IC(50) values at 12.9, 17.4 and 31.9 μM, respectively. After 4-day treatment of the rat hepatocytes, the ethanolic extract of Danshen and tanshinone I increased rat CYP1A2 activity by 6.8- and 5.2-fold, respectively, with a concomitant up-regulation of CYP1A2 protein level by 13.5- and 6.5-fold, respectively. CYP1A2 induction correlated with the up-regulation of mRNA level of aryl hydrocarbon receptor (AhR), which suggested a positive feedback mechanism of tanshinone I-mediated CYP1A2 induction. A formulated Danshen pill (containing mainly danshensu and salvianolic acid B and the tanshinones) up-regulated CYP1A2 protein expression and enzyme activity, but danshensu and salvianolic acid B, when used individually, did not affect CYP1A2 activity. This study was the first report on the Janus action of the tanshinones on rat CYP1A2 activity.

  15. Annexin A2 Enhances Complement Activation by Inhibiting Factor H1

    PubMed Central

    Renner, Brandon; Tong, Hua Hua; Laskowski, Jennifer; Jonscher, Karen; Goetz, Lindsey; Woolaver, Rachel; Hannan, Jonathan; Li, Yong Xing; Hourcade, Dennis; Pickering, Matthew C.; Holers, V. Michael; Thurman, Joshua M.

    2015-01-01

    Factor H is a circulating protein that regulates activation of the alternative pathway (AP) of complement. Mutations and genetic variations of factor H are associated with several AP-mediated diseases, highlighting the critical role of factor H in AP regulation. AP-mediated inflammation is typically triggered by illness or tissue injury, however, and tissue injury can trigger AP activation in individuals with fully functional factor H. This suggests that factor H function is affected by local conditions within tissues. We hypothesized that inducible proteins impair the ability of factor H to locally control the AP, thereby increasing AP activation. We used purified murine factor H to immunoprecipitate binding partners from mouse kidneys. Using immunoaffinity liquid chromatography-mass spectrometry we then identified annexin A2 as a factor H binding partner. Further experiments showed that annexin A2 reduces the binding of factor H to cell surfaces. Recombinant annexin A2 impaired complement regulation by factor H, and increased complement activation on renal cell surfaces in vitro and in vivo. In a murine model of acute pneumococcal otitis media the administration of annexin A2 increased AP-mediated bacterial opsonization and clearance. In conclusion, the local production of annexin A2 within tissues suppresses regulation of the AP by factor H. Annexin A2 can contribute to AP-mediated tissue inflammation by locally impairing factor H function, but annexin A2 can also improve complement-mediated bacterial clearance. PMID:26729803

  16. Maternal and zygotic aldh1a2 activity is required for pancreas development in zebrafish.

    PubMed

    Alexa, Kristen; Choe, Seong-Kyu; Hirsch, Nicolas; Etheridge, Letitiah; Laver, Elizabeth; Sagerström, Charles G

    2009-12-11

    We have isolated and characterized a novel zebrafish pancreas mutant. Mutant embryos lack expression of isl1 and sst in the endocrine pancreas, but retain isl1 expression in the CNS. Non-endocrine endodermal gene expression is less affected in the mutant, with varying degrees of residual expression observed for pdx1, carbA, hhex, prox1, sid4, transferrin and ifabp. In addition, mutant embryos display a swollen pericardium and lack fin buds. Genetic mapping revealed a mutation resulting in a glycine to arginine change in the catalytic domain of the aldh1a2 gene, which is required for the production of retinoic acid from vitamin A. Comparison of our mutant (aldh1a2(um22)) to neckless (aldh1a2(i26)), a previously identified aldh1a2 mutant, revealed similarities in residual endodermal gene expression. In contrast, treatment with DEAB (diethylaminobenzaldehyde), a competitive reversible inhibitor of Aldh enzymes, produces a more severe phenotype with complete loss of endodermal gene expression, indicating that a source of Aldh activity persists in both mutants. We find that mRNA from the aldh1a2(um22) mutant allele is inactive, indicating that it represents a null allele. Instead, the residual Aldh activity is likely due to maternal aldh1a2, since we find that translation-blocking, but not splice-blocking, aldh1a2 morpholinos produce a phenotype similar to DEAB treatment. We conclude that Aldh1a2 is the primary Aldh acting during pancreas development and that maternal Aldh1a2 activity persists in aldh1a2(um22) and aldh1a2(i26) mutant embryos.

  17. Cytochrome P450 1A2 (CYP1A2) activity and risk factors for breast cancer: a cross-sectional study

    PubMed Central

    Hong, Chi-Chen; Tang, Bing-Kou; Hammond, Geoffrey L; Tritchler, David; Yaffe, Martin; Boyd, Norman F

    2004-01-01

    Introduction Breast cancer risk may be determined by various genetic, metabolic, and lifestyle factors that alter sex hormone metabolism. Cytochrome P450 1A2 (CYP1A2) is responsible for the metabolism of estrogens and many exogenous compounds, including caffeine. Methods In a cross-sectional study of 146 premenopausal and 149 postmenopausal women, we examined the relationships between CYP1A2 activity and known or suspected risk factors for breast cancer. Blood levels of sex hormones, lipids, and growth factors were measured. In vivo CYP1A2 activity was assessed by measuring caffeine metabolites in urine. Stepwise and maximum R regression analyses were used to identify covariates related to CYP1A2 activity after adjustment for ethnicity. Results In both menopausal groups CYP1A2 activity was positively related to smoking and levels of sex hormone binding globulin. In premenopausal women, CYP1A2 activity was also positively related to insulin levels, caffeine intake, age, and plasma triglyceride levels, and negatively related with total cholesterol levels and body mass index. In postmenopausal women CYP1A2 activity was positively associated with insulin-like growth factor-1, and negatively associated with plasma triglyceride, high-density lipoprotein cholesterol, and age at menarche. Conclusion These results suggest that CYP1A2 activity is correlated with hormones, blood lipids, and lifestyle factors associated with breast cancer risk, although some of the observed associations were contrary to hypothesized directions and suggest that increased CYP1A2 function may be associated with increased risk for breast cancer. PMID:15217502

  18. Cyclin A2 Mutagenesis Analysis: A New Insight into CDK Activation and Cellular Localization Requirements

    PubMed Central

    Bendris, Nawal; Lemmers, Bénédicte; Blanchard, Jean-Marie; Arsic, Nikola

    2011-01-01

    Cyclin A2 is essential at two critical points in the somatic cell cycle: during S phase, when it activates CDK2, and during the G2 to M transition when it activates CDK1. Based on the crystal structure of Cyclin A2 in association with CDKs, we generated a panel of mutants to characterize the specific amino acids required for partner binding, CDK activation and subcellular localization. We find that CDK1, CDK2, p21, p27 and p107 have overlapping but distinct requirements for association with this protein. Our data highlight the crucial importance of the N-terminal α helix, in conjunction with the α3 helix within the cyclin box, in activating CDK. Several Cyclin A2 mutants selectively bind to either CDK1 or CDK2. We demonstrate that association of Cyclin A2 to proteins such as CDK2 that was previously suggested as crucial is not a prerequisite for its nuclear localization, and we propose that the whole protein structure is involved. PMID:21829545

  19. Characterization of Serum Phospholipase A2 Activity in Three Diverse Species of West African Crocodiles

    PubMed Central

    Merchant, Mark; Juneau, Kate; Gemillion, Jared; Falconi, Rodolfo; Doucet, Aaron; Shirley, Matthew H.

    2011-01-01

    Secretory phospholipase A2, an enzyme that exhibits substantial immunological activity, was measured in the serum of three species of diverse West African crocodiles. Incubation of different volumes of crocodile serum with bacteria labeled with a fluorescent fatty acid in the sn-2 position of membrane lipids resulted in a volume-dependent liberation of fluorescent probe. Serum from the Nile crocodile (Crocodylus niloticus) exhibited slightly higher activity than that of the slender-snouted crocodile (Mecistops cataphractus) and the African dwarf crocodile (Osteolaemus tetraspis). Product formation was inhibited by BPB, a specific PLA2 inhibitor, confirming that the activity was a direct result of the presence of serum PLA2. Kinetic analysis showed that C. niloticus serum produced product more rapidly than M. cataphractus or O. tetraspis. Serum from all three species exhibited temperature-dependent PLA2 activities but with slightly different thermal profiles. All three crocodilian species showed high levels of activity against eight different species of bacteria. PMID:22110960

  20. [Diversity of culturable filamentous bacteria in the activated sludge from A2O wastewater treatment process].

    PubMed

    Gao, Sha; Jin, De-Cai; Zhao, Zhi-Rui; Qi, Rong; Peng, Xia-Wei; Bai, Zhi-Hui

    2013-07-01

    The anoxic-anaerobic-oxic (A2O) process is widely used in wastewater treatment plant, however, sludge bulking and foaming are the most frequent operational problems in this process. Activated sludge bulking is caused by the overgrowth of some types of filamentous bacteria, especially Microthrix parvicella. In the study, 17 strains of filamentous bacteria were isolated from the bulking sludge of A2O process using Gause's medium. The 16S rRNA genes of the 17 isolates were sequenced to analyze their diversity. The results showed all of the 17 isolates were Streptomyces. Further analysis of these strains by the repetitive sequence based on polymerase chain reaction (rep-PCR) technology showed that there was a high diversity in these isolated Streptomyces. The physiological properties of them were different from Microthrix parvicella. The settleability of activated sludge was improved when some of the isolates were inoculated.

  1. Triggering neurotrophic factor actions through adenosine A2A receptor activation: implications for neuroprotection

    PubMed Central

    Sebastião, Ana M; Ribeiro, Joaquim A

    2009-01-01

    G protein coupled receptors and tropomyosin-related kinase (Trk) receptors have distinct structure and transducing mechanisms; therefore, cross-talk among them was unexpected. Evidence has, however, accumulated showing that tonic adenosine A2A receptor activity is a required step to allow synaptic actions of neurotrophic factors, namely upon synaptic transmission at both pre- and post-synaptic level as well as upon synaptic plasticity. An enhancement of A2A receptor tonus upon ageing may partially compensate the loss of TrkB receptors, rescuing to certain degree the facilitatory action of brain derived neurotrophic factor in aged animals, which might prove particularly relevant in the prevention of neurodegeneration upon ageing. A2A receptors also trigger synaptic actions of other neurotrophic factors, such as glial derived neurotrophic factor at dopaminergic striatal nerve endings. The growing evidence that tonic adenosine A2A receptor activity is a crucial step to allow actions of neurotrophic factors in neurones will be reviewed and discussed in the light of therapeutic strategies for neurodegenerative diseases. PMID:19508402

  2. ATPase activity of Mycobacterium tuberculosis SecA1 and SecA2 proteins and its importance for SecA2 function in macrophages.

    PubMed

    Hou, Jie M; D'Lima, Nadia G; Rigel, Nathan W; Gibbons, Henry S; McCann, Jessica R; Braunstein, Miriam; Teschke, Carolyn M

    2008-07-01

    The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.

  3. Cytosolic phospholipase A2α regulates G1 progression through modulating FOXO1 activity

    PubMed Central

    Naini, Said Movahedi; Choukroun, Gabriel J.; Ryan, James R.; Hentschel, Dirk M.; Shah, Jagesh V.; Bonventre, Joseph V.

    2016-01-01

    Group IVA phospholipase A2 [cytosolic phospholipase A2α (cPLA2α)] is a key mediator of inflammation and tumorigenesis. In this study, by using a combination of chemical inhibition and genetic approaches in zebrafish and murine cells, we identify a mechanism by which cPLA2α promotes cell proliferation. We identified 2 cpla2α genes in zebrafish, cpla2αa and cpla2αb, with conserved phospholipase activity. In zebrafish, loss of cpla2α expression or inhibition of cpla2α activity diminished G1 progression through the cell cycle. This phenotype was also seen in both mouse embryonic fibroblasts and mesangial cells. G1 progression was rescued by the addition of arachidonic acid or prostaglandin E2 (PGE2), indicating a phospholipase-dependent mechanism. We further show that PGE2, through PI3K/AKT activation, promoted Forkhead box protein O1 (FOXO1) phosphorylation and FOXO1 nuclear export. This led to up-regulation of cyclin D1 and down-regulation of p27Kip1, thus promoting G1 progression. Finally, using pharmacologic inhibitors, we show that cPLA2α, rapidly accelerated fibrosarcoma (RAF)/MEK/ERK, and PI3K/AKT signaling pathways cooperatively regulate G1 progression in response to platelet-derived growth factor stimulation. In summary, these data indicate that cPLA2α, through its phospholipase activity, is a critical effector of G1 phase progression through the cell cycle and suggest that pharmacological targeting of this enzyme may have important therapeutic benefits in disease mechanisms that involve excessive cell proliferation, in particular, cancer and proliferative glomerulopathies.—Naini, S. M., Choukroun, G. J., Ryan, J. R., Hentschel, D. M., Shah, J. V., Bonventre, J. V. Cytosolic phospholipase A2α regulates G1 progression through modulating FOXO1 activity. PMID:26644349

  4. Ceramides increase the activity of the secretory phospholipase A2 and alter its fatty acid specificity.

    PubMed Central

    Koumanov, Kamen S; Momchilova, Albena B; Quinn, Peter J; Wolf, Claude

    2002-01-01

    Modulation of human recombinant secretory type II phospholipase A(2) activity by ceramide and cholesterol was investigated using model glycerophospholipid substrates composed of phosphatidylethanolamine and phosphatidylserine dispersed in aqueous medium. Enzyme activity was monitored by measurement of released fatty acids using capillary GC-MS. Fatty acids from the sn-2 position of the phospholipids were hydrolysed by the enzyme in proportion to the relative abundance of the phospholipid in the substrate. Addition of increasing amounts of ceramide to the substrate progressively enhanced phospholipase activity. The increased activity was accomplished largely by preferential hydrolysis of polyunsaturated fatty acids, particularly arachidonic acid, derived from phosphatidylethanolamine. The addition of sphingomyelin to the substrate glycerophospholipids inhibited phospholipase activity but its progressive substitution by ceramide, so as to mimic sphingomyelinase activity, counteracted the inhibition. The presence of cholesterol in dispersions of glycerophospholipid-substrate-containing ceramides suppressed activation of the enzyme resulting from the presence of ceramide. The molecular basis of enzyme modulation was investigated by analysis of the phase structure of the dispersed lipid substrate during temperature scans from 46 to 20 degrees C using small-angle synchrotron X-ray diffraction. These studies indicated that intermediate structures created after ceramide-dependent phase separation of hexagonal and lamellar phases represent the most susceptible form of the substrate for enzyme hydrolysis. PMID:11903045

  5. Phospholipase A2-activating protein is associated with a novel form of leukoencephalopathy.

    PubMed

    Falik Zaccai, Tzipora C; Savitzki, David; Zivony-Elboum, Yifat; Vilboux, Thierry; Fitts, Eric C; Shoval, Yishay; Kalfon, Limor; Samra, Nadra; Keren, Zohar; Gross, Bella; Chasnyk, Natalia; Straussberg, Rachel; Mullikin, James C; Teer, Jamie K; Geiger, Dan; Kornitzer, Daniel; Bitterman-Deutsch, Ora; Samson, Abraham O; Wakamiya, Maki; Peterson, Johnny W; Kirtley, Michelle L; Pinchuk, Iryna V; Baze, Wallace B; Gahl, William A; Kleta, Robert; Anikster, Yair; Chopra, Ashok K

    2017-02-01

    Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A2-activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E2 and cytosolic phospholipase A2 activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E2 levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein's ability to induce prostaglandin E2 and cytosolic phospholipase A2 synthesis in patients' fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E2 The non-functional phospholipase A2-activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance.

  6. Quantum dot-NBD-liposome luminescent probes for monitoring phospholipase A2 activity.

    PubMed

    Kethineedi, Venkata R; Crivat, Georgeta; Tarr, Matthew A; Rosenzweig, Zeev

    2013-12-01

    In this paper we describe the fabrication and characterization of new liposome encapsulated quantum dot-fluorescence resonance energy transfer (FRET)-based probes for monitoring the enzymatic activity of phospholipase A2. To fabricate the probes, luminescent CdSe/ZnS quantum dots capped with trioctylphosphine oxide (TOPO) ligands were incorporated into the lipid bilayer of unilamellar liposomes with an average diameter of approximately 100 nm. Incorporating TOPO capped quantum dots in liposomes enabled their use in aqueous solution while maintaining their hydrophobicity and excellent photophysical properties. The phospholipid bilayer was labeled with the fluorophore NBD C6-HPC (2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexa decanoyl-sn-glycero-3-phosphocholine). The luminescent quantum dots acted as FRET donors and the NBD dye molecules acted as FRET acceptors. The probe response was based on FRET interactions between the quantum dots and the NBD dye molecules. The NBD dye molecules were cleaved and released to the solution in the presence of the enzyme phospholipase A2. This led to an increase of the luminescence of the quantum dots and to a corresponding decrease in the fluorescence of the NBD molecules, because of a decrease in FRET efficiency between the quantum dots and the NBD dye molecules. Because the quantum dots were not attached covalently to the phospholipids, they did not hinder the enzyme activity as a result of steric effects. The probes were able to detect amounts of phospholipase A2 as low as 0.0075 U mL(-1) and to monitor enzyme activity in real time. The probes were also used to screen phospholipase A2 inhibitors. For example, we found that the inhibition efficiency of MJ33 (1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol) was higher than that of OBAA (3-(4-octadecyl)benzoylacrylic acid).

  7. Inosine attenuates spontaneous activity in the rat neurogenic bladder through an A2B pathway

    PubMed Central

    Doyle, Claire; Cristofaro, Vivian; Sack, Bryan S.; Lukianov, Stefan N.; Schäfer, Mattias; Chung, Yeun Goo; Sullivan, Maryrose P.; Adam, Rosalyn M.

    2017-01-01

    Neurogenic detrusor overactivity (NDO) is among the most challenging complications of spinal cord injury (SCI). A recent report by us demonstrated an improvement in NDO in SCI rats following chronic systemic treatment with the purine nucleoside inosine. The objective of this study was to investigate the mechanism of action of inosine underlying improvement of NDO. Male Sprague-Dawley rats underwent complete spinal cord transection at T8. Inosine (1 mM) delivered intravesically to SCI rats during conscious cystometry significantly decreased the frequency of spontaneous non-voiding contractions. In isolated tissue assays, inosine (1 mM) significantly decreased the amplitude of spontaneous activity (SA) in SCI bladder muscle strips. This effect was prevented by a pan-adenosine receptor antagonist CGS15943, but not by A1 or A3 receptor antagonists. The A2A antagonist ZM241385 and A2B antagonist PSB603 prevented the effect of inosine. The effect of inosine was mimicked by the adenosine receptor agonist NECA and the A2B receptor agonist BAY60-6583. The inhibition of SA by inosine was not observed in the presence of the BK antagonist, iberiotoxin, but persisted in the presence of KATP and SK antagonists. These findings demonstrate that inosine acts via an A2B receptor-mediated pathway that impinges on specific potassium channel effectors. PMID:28294142

  8. Amyloid-type fiber formation in control of enzyme action: interfacial activation of phospholipase A2.

    PubMed

    Code, Christian; Domanov, Yegor; Jutila, Arimatti; Kinnunen, Paavo K J

    2008-07-01

    The lag-burst behavior in the action of phospholipase A(2) (PLA(2)) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was investigated at temperatures slightly offset from the main phase transition temperature T(m) of this lipid, thus slowing down the kinetics of the activation process. Distinct stages leading to maximal activity were resolved using a combination of fluorescence parameters, including Förster resonance energy transfer between donor- and acceptor-labeled enzyme, fluorescence anisotropy, and lifetime, as well as thioflavin T fluorescence enhancement. We showed that the interfacial activation of PLA(2), evident after the preceding lag phase, coincides with the formation of oligomers staining with thioflavin T and subsequently with Congo red. Based on previous studies and our findings here, we propose a novel mechanism for the control of PLA(2), involving amyloid protofibrils with highly augmented enzymatic activity. Subsequently, these protofibrils form "mature" fibrils, devoid of activity. Accordingly, the process of amyloid formation is used as an on-off switch to obtain a transient burst in enzymatic catalysis.

  9. Amyloid-Type Fiber Formation in Control of Enzyme Action: Interfacial Activation of Phospholipase A2

    PubMed Central

    Code, Christian; Domanov, Yegor; Jutila, Arimatti; Kinnunen, Paavo K. J.

    2008-01-01

    The lag-burst behavior in the action of phospholipase A2 (PLA2) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was investigated at temperatures slightly offset from the main phase transition temperature Tm of this lipid, thus slowing down the kinetics of the activation process. Distinct stages leading to maximal activity were resolved using a combination of fluorescence parameters, including Förster resonance energy transfer between donor- and acceptor-labeled enzyme, fluorescence anisotropy, and lifetime, as well as thioflavin T fluorescence enhancement. We showed that the interfacial activation of PLA2, evident after the preceding lag phase, coincides with the formation of oligomers staining with thioflavin T and subsequently with Congo red. Based on previous studies and our findings here, we propose a novel mechanism for the control of PLA2, involving amyloid protofibrils with highly augmented enzymatic activity. Subsequently, these protofibrils form “mature” fibrils, devoid of activity. Accordingly, the process of amyloid formation is used as an on-off switch to obtain a transient burst in enzymatic catalysis. PMID:18339749

  10. Cognitive Stimulation Modulates Platelet Total Phospholipases A2 Activity in Subjects with Mild Cognitive Impairment

    PubMed Central

    Balietti, Marta; Giuli, Cinzia; Fattoretti, Patrizia; Fabbietti, Paolo; Postacchini, Demetrio; Conti, Fiorenzo

    2016-01-01

    We evaluated the effect of cognitive stimulation (CS) on platelet total phospholipases A2 activity (tPLA2A) in patients with mild cognitive impairment (MCI_P). At baseline, tPLA2A negatively correlated with Mini-Mental State Examination score (MMSE_s): patients with MMSE_s <26 (Subgroup 1) had significantly higher activity than those with MMSE_s ≥26 (Subgroup 2), who had values similar to the healthy elderly. Regarding CS effect, Subgroup 1 had a significant tPLA2A reduction, whereas Subgroup 2 did not significantly changes after training. Our results showed for the first time that tPLA2A correlates with the cognitive conditions of MCI_P, and that CS acts selectively on subjects with a dysregulated tPLA2A. PMID:26836161

  11. Cognitive Stimulation Modulates Platelet Total Phospholipases A2 Activity in Subjects with Mild Cognitive Impairment.

    PubMed

    Balietti, Marta; Giuli, Cinzia; Fattoretti, Patrizia; Fabbietti, Paolo; Postacchini, Demetrio; Conti, Fiorenzo

    2016-01-01

    We evaluated the effect of cognitive stimulation (CS) on platelet total phospholipases A2 activity (tPLA2A) in patients with mild cognitive impairment (MCI_P). At baseline, tPLA2A negatively correlated with Mini-Mental State Examination score (MMSE_s): patients with MMSE_s <26 (Subgroup 1) had significantly higher activity than those with MMSE_s ≥26 (Subgroup 2), who had values similar to the healthy elderly. Regarding CS effect, Subgroup 1 had a significant tPLA2A reduction, whereas Subgroup 2 did not significantly changes after training. Our results showed for the first time that tPLA2A correlates with the cognitive conditions of MCI_P, and that CS acts selectively on subjects with a dysregulated tPLA2A.

  12. Phospholipase A2 from bovine seminal plasma is a platelet-activating factor acetylhydrolase.

    PubMed Central

    Soubeyrand, S; Lazure, C; Manjunath, P

    1998-01-01

    The major phospholipase A2 activity from bovine seminal plasma was recently purified [Soubeyrand, Khadir, Brindle and Manjunath (1997) J. Biol. Chem. 272, 222-227]. We here show that the 60 kDa enzyme is in fact a platelet-activating factor acetylhydrolase (PAF-AH). Sequences of the N-terminus as well as of internal fragments showed 100% identity with the cDNA-deduced sequences of bovine plasma PAF-AH. The enzyme has kinetic properties similar to those of the human serum PAF-AH. Although capable of hydrolysing long-chained phosphatidylcholine, it displayed a highly preferential activity towards PAF. The enzyme activity towards phosphatidylcholine, but not PAF, was Ca2+-dependent. Biochemical characterization revealed that the enzyme is extensively N-glycosylated and that it exists predominantly as a dimer in solution. Western blot analysis revealed that the enzyme is highly heterogeneous in charge, with a maximal distribution at an isoelectric point of approx. 5.7. The enzyme was expressed exclusively in the seminal vesicles and the ampulla. No association of the enzyme with either epididymal or ejaculated spermatozoa could be detected. PMID:9405273

  13. Lithium activates brain phospholipase A2 and improves memory in rats: implications for Alzheimer's disease.

    PubMed

    Mury, Fábio B; da Silva, Weber C; Barbosa, Nádia R; Mendes, Camila T; Bonini, Juliana S; Sarkis, Jorge Eduardo Souza; Cammarota, Martin; Izquierdo, Ivan; Gattaz, Wagner F; Dias-Neto, Emmanuel

    2016-10-01

    Phospholipase A2 (Pla2) is required for memory retrieval, and its inhibition in the hippocampus has been reported to impair memory acquisition in rats. Moreover, cognitive decline and memory deficits showed to be reduced in animal models after lithium treatment, prompting us to evaluate possible links between Pla2, lithium and memory. Here, we evaluated the possible modulation of Pla2 activity by a long-term treatment of rats with low doses of lithium and its impact in memory. Wistar rats were trained for the inhibitory avoidance task, treated with lithium for 100 days and tested for perdurability of long-term memory. Hippocampal samples were used for quantifying the expression of 19 brain-expressed Pla2 genes and for evaluating the enzymatic activity of Pla2 using group-specific radio-enzymatic assays. Our data pointed to a significant perdurability of long-term memory, which correlated with increased transcriptional and enzymatic activities of certain members of the Pla2 family (iPla2 and sPla2) after the chronic lithium treatment. Our data suggest new possible targets of lithium, add more information on its pharmacological activity and reinforce the possible use of low doses of lithium for the treatment of neurodegenerative conditions such as the Alzheimer's disease.

  14. Monitoring Phospholipase A2 Activity with Gd-encapsulated Phospholipid Liposomes

    PubMed Central

    Cheng, Zhiliang; Tsourkas, Andrew

    2014-01-01

    To date, numerous analytical methods have been developed to monitor phospholipase A2 (PLA2) activity. However, many of these methods require the use of unnatural PLA2 substrates that may alter enzyme kinetics, and probes that cannot be extended to applications in more complex environments. It would be desirable to develop a versatile assay that monitors PLA2 activity based on interactions with natural phospholipids in complex biological samples. Here, we developed an activatable T1 magnetic resonance (MR) imaging contrast agent to monitor PLA2 activity. Specifically, the clinically approved gadolinium (Gd)-based MR contrast agent, gadoteridol, was encapsulated within nanometer-sized phospholipid liposomes. The encapsulated Gd exhibited a low T1-weighted signal, due to low membrane permeability. However, when the phospholipids within the liposomal membrane were hydrolyzed by PLA2, encapsulated Gd was released into bulk solution, resulting in a measureable change in the T1-relaxation time. These activatable MR contrast agents can potentially be used as nanosensors for monitoring of PLA2 activity in biological samples with minimal sample preparation. PMID:25376186

  15. A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities[S

    PubMed Central

    El Alaoui, Meddy; Soulère, Laurent; Noiriel, Alexandre; Popowycz, Florence; Khatib, Abdallah; Queneau, Yves; Abousalham, Abdelkarim

    2016-01-01

    A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase®, guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement. PMID:27194811

  16. Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A2 activities

    PubMed Central

    Manevich, Yefim; Shuvaeva, Tea; Dodia, Chandra; Kazi, Altaf; Feinstein, Sheldon I.; Fisher, Aron B.

    2010-01-01

    Peroxiredoxin 6 (Prdx6) differs from other mammalian peroxiredoxins both in its ability to reduce phospholipid hydroperoxides at neutral pH and in having phospholipase A2 (PLA2) activity that is maximal at acidic pH. We previously showed an active site C47 for peroxidase activity and a catalytic triad S32-H26-D140 necessary for binding of phospholipid and PLA2 activity. This study evaluated binding of reduced and oxidized phospholipid hydroperoxide to Prdx6 at cytosolic pH. Incubation of recombinant Prdx6 with 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PLPCOOH) resulted in peroxidase activity, cys47 oxidation as detected with Prdx6-SO2(3) antibody, and a marked shift in the Prdx6 melting temperature by circular dichroism analysis indicating that PLPCOOH is a specific substrate for Prdx6. Preferential Prdx6 binding to oxidized liposomes was detected by changes in DNS-PE or bis-Pyr fluorescence and by ultrafiltration. Site-specific mutation of S32 or H26 in Prdx6 abolished binding while D140 mutation had no effect. Treatment of A549 cells with peroxides led to lipid peroxidation and translocation of Prdx6 from the cytosol to the cell membrane. Thus, the pH specificity for the two enzymatic activities of Prdx6 can be explained by the differential binding kinetics of the protein; Prdx6 binds to reduced phospholipid at acidic pH but at cytosolic pH binds only phospholipid that is oxidized compatible with a role for Prdx6 in the repair of peroxidized cell membranes. PMID:19236840

  17. Regulation of human eosinophil degranulation and activation by endogenous phospholipase A2.

    PubMed Central

    White, S R; Strek, M E; Kulp, G V; Spaethe, S M; Burch, R A; Neeley, S P; Leff, A R

    1993-01-01

    The unique granular proteins of eosinophils may have a pathogenetic role in asthma and in the defense against parasitic infestations. However, the mechanisms regulating eosinophil degranulation are largely unknown. We examined the hypothesis that release of these proteins is regulated by endogenous activation of phospholipase A2. Human eosinophils (HE) were isolated from the peripheral blood of 42 subjects either by Percoll density separation or by negative-selection immunomagnetic fractionation. Eosinophil activation was initiated in vitro with 10(-6) M FMLP and 5 micrograms/ml cytochalasin B and was assessed by measurement of eosinophil peroxidase (EPO), leukotriene C4 (LTC4) and superoxide radical (.O2-) secretion. Treatment of HE with 100 microM mepacrine before activation blocked EPO release (2.0 +/- 0.2 vs 10.2 +/- 2.1% cell content for activated HE, P < 0.004, n = 9), .O2- generation (2.6 +/- 0.9 vs 44.2 +/- 10.8 nmol/ml per 10(6) HE, P < 0.002, n = 5), and LTC4 secretion (68.2 +/- 32.2 vs 1,125.2 +/- 526.8 pg/ml per 10(6) HE, P < 0.04, n = 8). Pretreatment of HE with 100 microM 4-bromophenacyl bromide before activation similarly blocked EPO release, .O2- generation and LTC4 secretion. Addition of AA to HE after treatment with 100 microM mepacrine and before subsequent activation reversed the inhibition of both EPO (10.4 +/- 2.2% with 1 microM AA vs 2.0 +/- 0.2% for mepacrine, n = 5, P < 0.02) and LTC4 secretion (695.1 +/- 412.9 with 10 microM AA vs 68.2 +/- 32.2 pg/ml per 10(6) HE for mepacrine, n = 8, P < 0.04), but did not reverse inhibition of .O2- generation by mepacrine. We demonstrate that secretion of preformed cytotoxic proteins and .O2- by eosinophils is regulated endogenously by phospholipase A2. PMID:8387540

  18. Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2.

    PubMed

    Cavazzini, Davide; Meschi, Francesca; Corsini, Romina; Bolchi, Angelo; Rossi, Gian Luigi; Einsle, Oliver; Ottonello, Simone

    2013-01-18

    Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A(2) (sPLA(2)s). TbSP1, the sPLA(2) primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A(2), whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA(2) overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation.

  19. Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2*

    PubMed Central

    Cavazzini, Davide; Meschi, Francesca; Corsini, Romina; Bolchi, Angelo; Rossi, Gian Luigi; Einsle, Oliver; Ottonello, Simone

    2013-01-01

    Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A2 (sPLA2s). TbSP1, the sPLA2 primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A2, whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA2 overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation. PMID:23192346

  20. Clinical correlates of change in inflammatory biomarkers: The Framingham Heart Study

    PubMed Central

    Fontes, Joao D.; Yamamoto, Jennifer F.; Larson, Martin G.; Wang, Na; Dallmeier, Dhayana; Rienstra, Michiel; Schnabel, Renate B.; Vasan, Ramachandran S.; Keaney, John F.; Benjamin, Emelia J.

    2013-01-01

    Objectives Traditional clinical risk factors are associated with inflammation cross-sectionally, but associations of longitudinal variation in inflammatory biomarkers with corresponding changes in clinical risk factors are incompletely described. We sought to analyze clinical factors associated with change in inflammation in the community. Methods We studied 3013 Framingham Offspring (n = 2735) and Omni Cohort (n = 278) participants (mean age 59 years, 55% women, 9% ethnic/racial minority) who attended two consecutive examination cycles (mean 6.7 years apart). We selected ten inflammatory biomarkers representing distinctive biological functions: C-reactive protein (CRP), intercellular adhesion molecule-1, interleukin-6, isoprostanes, lipoprotein-associated phospholipase-2 (Lp-PLA2) activity, Lp-PLA2-mass, monocyte chemoattractant protein-1, osteoprotegerin, P-selectin, and tumor necrosis factor receptor II (TNFRII). We constructed multivariable-adjusted regression models to assess the relations of baseline, follow-up and change in clinical risk factors with change in biomarker concentrations over time. Results Baseline, follow-up and change in clinical risk factors explain a moderate amount of the variation in biomarker concentrations across 2 consecutive examinations (ranging from r2 = 0.28 [TNFRII] up to 0.52 [Lp-PLA2-mass]). In multivariable models, increasing body-mass index, smoking initiation, worsening lipid profile, and increasing waist size were associated with increasing concentrations of several biomarkers. Conversely, hypercholesterolemia therapy and hormone replacement cessation were associated with decreasing concentrations of biomarkers such as CRP, Lp-PLA2-mass and activity. Conclusion Cardiovascular risk factors have different patterns of association with longitudinal change in inflammatory biomarkers and explain modest amounts of variability in biomarker concentrations. Nevertheless, a substantial proportion of longitudinal change in inflammatory

  1. Secretory phospholipase A2 activity in blood serum: the challenge to sense.

    PubMed

    Alekseeva, A S; Korotaeva, A A; Samoilova, E V; Volynsky, P E; Vodovozova, E L; Boldyrev, I A

    2014-11-07

    Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammatory diseases including vascular inflammation correlating with coronary events in coronary artery disease. Thus a method to monitor sPLA2 activity in blood serum is urgently needed. Such method is still a challenge since existing fluorescent probes do not allow to monitor sPLA2 activity directly in blood serum. Here we analyze and overcome barriers in sPLA2 sensing methodology and report a fluorescent probe and a kinetic model of its hydrolysis by sPLA2. New probe is designed with a fluorophore and a quencher not interfering binding to the enzyme. At the same time phospholipid matrix bearing the probe promotes efficient initial quenching of the fluorophore. Kinetic model of probe hydrolysis takes into account signal change due to the side processes. The probe and the kinetic model applied together prove the concept that the activity of sPLA can be measured directly in blood serum.

  2. WATER-ICE-DRIVEN ACTIVITY ON MAIN-BELT COMET P/2010 A2 (LINEAR)?

    SciTech Connect

    Moreno, F.; Ortiz, J. L.; Cabrera-Lavers, A.; Augusteijn, T.; Liimets, T.; Lindberg, J. E.; Pursimo, T.; RodrIguez-Gil, P.; Vaduvescu, O.

    2010-08-01

    The dust ejecta of Main-Belt Comet P/2010 A2 (LINEAR) have been observed with several telescopes at the Observatorio del Roque de los Muchachos on La Palma, Spain. Application of an inverse dust tail Monte Carlo method to the images of the dust ejecta from the object indicates that a sustained, likely water-ice-driven, activity over some eight months is the mechanism responsible for the formation of the observed tail. The total amount of the dust released is estimated to be 5 x 10{sup 7} kg, which represents about 0.3% of the nucleus mass. While the event could have been triggered by a collision, this cannot be determined from the currently available data.

  3. DYNAMICS OF LARGE FRAGMENTS IN THE TAIL OF ACTIVE ASTEROID P/2010 A2

    SciTech Connect

    Agarwal, Jessica; Jewitt, David; Weaver, Harold

    2013-05-20

    We examine the motions of large fragments at the head of the dust tail of the active asteroid P/2010 A2. In previous work, we showed that these fragments were ejected from the primary nucleus in early 2009, either following a hypervelocity impact or by rotationally induced breakup. Here, we follow their positions through a series of Hubble Space Telescope images taken during the first half of 2010. The orbital evolution of each fragment allows us to constrain its velocity relative to the main nucleus after leaving its sphere of gravitational influence. We find that the fragments constituting a prominent X-shaped tail feature were emitted in a direction opposite to the motion of the asteroid and toward the south of its orbital plane. Derived emission velocities of these primary fragments range between 0.02 and 0.3 m s{sup -1}, comparable to the {approx}0.08 m s{sup -1} gravitational escape speed from the nucleus. Their sizes are on the order of decimeters or larger. We obtain the best fits to our data with ejection velocity vectors lying in a plane that includes the nucleus. This may suggest that the cause of the disruption of P/2010 A2 is rotational breakup.

  4. Structural basis of the anionic interface preference and kcat* activation of pancreatic phospholipase A2.

    PubMed

    Yu, B Z; Poi, M J; Ramagopal, U A; Jain, R; Ramakumar, S; Berg, O G; Tsai, M D; Sekar, K; Jain, M K

    2000-10-10

    Pancreatic phospholipase A(2) (PLA2) shows a strong preference for the binding to the anionic interface and a consequent allosteric activation. In this paper, we show that virtually all the preference is mediated through 3 (Lys-53, -56, and -120) of the 12 cationic residues of bovine pancreatic PLA2. The lysine-to-methionine substitution enhances the binding of the enzyme to the zwitterionic interface, and for the K53,56,120M triple mutant at the zwitterionic interface is comparable to that for the wild type (WT) at the anionic interface. In the isomorphous crystal structure, the backbone folding of K53,56M K120,121A and WT are virtually identical, yet a significant change in the side chains of certain residues, away from the site of substitution, mostly at the putative contact site with the interface (i-face), is discernible. Such reciprocity, also supported by the spectroscopic results for the free and bound forms of the enzyme, is expected because a distal structural change that perturbs the interfacial binding could also affect the i-face. The results show that lysine-to-methionine substitution induces a structural change that promotes the binding of PLA2 to the interface as well as the substrate binding to the enzyme at the interface. The kinetic results are consistent with a model in which the interfacial Michaelis complex exists in two forms, and the complex that undergoes the chemical step is formed by the charge compensation of Lys-53 and -56. Analysis of the incremental changes in the kinetic parameters shows that the charge compensation of Lys-53 and -56 contributes to the activation and that of Lys-120 contributes only to the structural change that promotes the stability of the Michaelis complex at the interface. The charge compensation effects on these three residues also account for the differences in the anionic interface preference of the evolutionarily divergent secreted PLA2.

  5. Effects of smoke inhalation on surfactant phospholipids and phospholipase A2 activity in the mouse lung.

    PubMed Central

    Oulton, M.; Moores, H. K.; Scott, J. E.; Janigan, D. T.; Hajela, R.

    1991-01-01

    The effects of smoke inhalation on the pulmonary surfactant system were examined in mice exposed for 30 minutes to smoke generated from the burning of polyurethane foam. At 8 or 12 hours after exposure, surfactants were isolated separately from lung lavage (extracellular surfactant) and residual lung tissue (intracellular surfactant) for phospholipid analysis. Calcium-dependent phospholipase A2 (PLA2) was measured on a microsomal fraction prepared from the tissue homogenate. Smoke inhalation produced a twofold increase in extracellular surfactant total phospholipid. While there was no change in the total phospholipid or phosphatidylcholine (PC) content of the intracellular surfactant, smoke inhalation significantly decreased the disaturated species of PC (DSPC). The specific activity of PLA2 was reduced by more than 50% in both groups of exposed mice. Smoke inhalation appears to result in selective depletion of the DSPC of intracellular surfactant and PLA2 involved in its synthesis. This depletion may be compensated for by increased secretion or slower breakdown of the material present in the extracellular compartment. Images Figure 1 PMID:1987765

  6. Neuronal damage by secretory phospholipase A2: modulation by cytosolic phospholipase A2, platelet-activating factor, and cyclooxygenase-2 in neuronal cells in culture.

    PubMed

    Kolko, Miriam; Rodriguez de Turco, Elena B; Diemer, Nils H; Bazan, Nicolas G

    2003-02-27

    Activation of cytosolic phospholipase A(2) (cPLA(2)) is an early event in brain injury, which leads to the formation and accumulation of bioactive lipids: platelet-activating factor (PAF), free arachidonic acid, and eicosanoids. A cross-talk between secretory PLA(2) (sPLA(2)) and cPLA(2) in neural signal transduction has previously been suggested (J Biol Chem 271:32722; 1996). Here we show, using neuronal cell cultures, an up-regulation of cPLA(2) expression and an inhibition by the selective cPLA(2) inhibitor AACOCF3 after exposure to neurotoxic concentrations of sPLA(2)-OS2. Pretreatment of neuronal cultures with recombinant PAF acetylhydrolase (rPAF-AH) or the presynaptic PAF receptor antagonist, BN52021, partially blocked neuronal cell death induced by sPLA(2)-OS2. Furthermore, selective COX-2 inhibitors ameliorated sPLA(2)-OS2-induced neurotoxicity. We conclude that sPLA(2)-OS2 activates a neuronal signaling cascade that includes activation of cPLA(2), arachidonic acid release, PAF production, and induction of COX-2.

  7. pA2 values for antagonists of platelet activating factor on aggregation of rabbit platelets.

    PubMed Central

    O'Donnell, S. R.; Barnett, C. J.

    1988-01-01

    1. The relative potencies, and equilibrium dissociation constants, for nine antagonists of platelet activating factor (Paf) have been determined on rabbit platelets (in diluted platelet-rich plasma (PRP)) in experiments in which the aggregatory response to Paf was measured. 2. Log concentration-response (% maximum) curves to Paf were obtained in the absence (controls) and presence of different concentrations of each Paf antagonist drug. The antagonists shifted the Paf curves to a higher concentration range and the slopes of the Schild plots, constructed from these data, suggested that the drugs were competitive antagonists of Paf. The slopes of the Schild plots for CV-3988 and SRI 63-119 were greater than 1. 3. The pA2 values (pKB values in parentheses) were: WEB 2086 7.31 (7.63); SRI 63-119 6.95; L-652,731 6.71 (6.73); BN 52021 6.38 (6.47); SRI 63-072 6.36 (6.43); CV-3988 5.87; 48740 RP 4.97 (5.07); ketotifen 4.94 (4.95); thiazinamium 4.73 (4.76). 4. This study provides, for the first time, some functional response data for Paf antagonists (pKB values) which are in an appropriate form for use in classifying putative Paf receptors. The study also provides the comparative potencies of these Paf antagonists in inhibiting Paf-induced platelet aggregation. WEB 2086 was the most potent of the drugs examined. PMID:3293683

  8. Association between adjunctive metformin therapy in young type 1 diabetes patients with excess body fat and reduction of carotid intima-media thickness.

    PubMed

    Burchardt, Paweł; Zawada, Agnieszka; Kaczmarek, Jolanta; Marcinkaniec, Justyna; Wysocki, Henryk; Wierusz-Wysocka, Bogna; Grzymisławski, Marian; Rzeźniczak, Janusz; Zozulińska-Ziółkiewicz, Dorota; Naskręt, Dariusz

    2016-08-25

    INTRODUCTION    Lipoprotein-associated phospholipase A2 (Lp-PLA2) and cholesteryl ester lipase (CEL) may oxidize low-density lipoproteins (oxLDL). OBJECTIVES    The aim of the study was to determine the influence of metformin on the metabolism of atherogenic lipid fractions in relation to Lp-PLA2 and CEL levels, as well as assess consequent improvement in the intima-media thickness (IMT) of the common carotid artery in young type 1 diabetes patients with excess body fat. PATIENTS AND METHODS    It was an open-label randomized clinical trial that lasted 6 months. It included a total of 84 people with metabolic decompensation (glycated hemoglobin >7.5%, >58.5 mmol/mol) of diabetes. Adjunctive metformin therapy (in addition to insulin) was administered in 42 patients, and the remaining 42 patients received insulin alone. Glycated low-density lipoproteins (LDLs), oxLDL, Lp-PLA2, and CEL were assessed by commercially available enzyme-linked immunosorbent assay kits. Cartoid IMT was measured using the Carotid Analyser for Research tool. Biochemical analyses were performed using routine laboratory techniques. RESULTS    The reduction of mean carotid IMT was observed in young type 1 diabetic adults treated additionally with metformin (0.6 ±0.1 cm vs 0.53 ±0.1 cm; P = 0.002). This effect was probably due to weight reduction (90 ±16 kg vs 87 ±15 kg, P = 0.054) and the decrease in atherogenic glycated LDL levels (1.5 ±0.5 mg/dl vs 1.6 ±1.046 mg/dl, P = 0.006). No such correlations were observed in patients treated with insulin alone. Additionally, in patients receiving metformin, glycated LDL levels were inversely correlated with Lp-PLA2 levels (r = -0.31, P <0.05). CONCLUSIONS    Additional use of metformin in young type 1 diabetic patients with excess body fat leads to a significant reduction of mean IMT in the common carotid artery. Concentrations of CEL and Lp-PLA2 were significantly increased in both study arms despite improved glucose metabolism.

  9. Elevated Slit2 Activity Impairs VEGF-induced Angiogenesis and Tumor Neovascularization in EphA2-deficient Endothelium

    PubMed Central

    Youngblood, Victoria; Wang, Shan; Song, Wenqiang; Walter, Debra; Hwang, Yoonha; Chen, Jin; Brantley-Sieders, Dana M.

    2015-01-01

    Angiogenic remodeling during embryonic development and in adult tissue homeostasis is orchestrated by cooperative signaling between several distinct molecular pathways, which are often exploited by tumors. Indeed, tumors upregulate pro-angiogenic molecules while simultaneously suppressing angiostatic pathways in order to recruit blood vessels for growth, survival, and metastatic spread. Understanding how cancers exploit pro- and anti-angiogenic signals is a key step in developing new, molecularly targeted anti-angiogenic therapies. While EphA2, a receptor tyrosine kinase (RTK), is required for vascular endothelial growth factor (VEGF)-induced angiogenesis, the mechanism through which these pathways intersect remains unclear. Slit2 expression is elevated in EphA2-deficient endothelium, and here it is reported that inhibiting Slit activity rescues VEGF-induced angiogenesis in cell culture and in vivo, as well as VEGF-dependent tumor angiogenesis, in EphA2-deficient endothelial cells and animals. Moreover, blocking Slit activity or Slit2 expression in EphA2-deficient endothelial cells restores VEGF-induced activation of Src and Rac, both of which are required for VEGF-mediated angiogenesis. These data suggest that EphA2 suppression of Slit2 expression and Slit angiostatic activity enables VEGF-induced angiogenesis in vitro and in vivo, providing a plausible mechanism for impaired endothelial responses to VEGF in the absence of EphA2 function. PMID:25504371

  10. Influence of environmental and genetic factors on CYP1A2 activity in individuals of South Asian and European ancestry.

    PubMed

    Perera, V; Gross, A S; McLachlan, A J

    2012-10-01

    The drug-metabolizing enzyme CYP1A2 contributes to the metabolism of a number of commonly used medicines and displays wide interindividual variability. The aim of this study was to investigate CYP1A2 activity in a population of South Asian ancestry and compare it with a population of European ancestry. CYP1A2 activity was determined using the 4 h paraxanthine/caffeine saliva concentration ratio following a 100-mg oral dose of caffeine in healthy individuals of South Asian (n = 166) and European (n = 166) ancestry. Participants were surveyed for extrinsic ethnic factors and genotyped for polymorphisms in CYP1A2 and related genes. Significantly lower CYP1A2 activity was observed in South Asian participants (median: 0.42; range: 0.10-1.06) as compared with European participants (0.54; 0.12-1.64) (P < 0.01). Multiple linear regression indicated that 41% of the variability in CYP1A2 activity could be explained by the diet, lifestyle, and genetic factors studied.

  11. The first report on coagulation and phospholipase A2 activities of Persian Gulf lionfish, Pterois russelli, an Iranian venomous fish.

    PubMed

    Memar, Bahareh; Jamili, Shahla; Shahbazzadeh, Delavar; Bagheri, Kamran Pooshang

    2016-04-01

    Pterois russelli is a venomous fish belonging to scorpionidae family. Regarding to high significance value for tracing potential therapeutic molecules and special agents from venomous marine creatures, the present study was aimed to characterization of the Persian Gulf lionfish venom. Proteolytic, phospholipase, hemolytic, coagulation, edematogenic and dermonecrotic activities were determined for extracted venom. The LD50 of P. russelli venom was determined by intravenous injection in white Balb/c mice. Phospholipase A2 activity was recorded at 20 μg of total venom. Coagulation activity on human plasma was shown by Prothrombin Time (PT) and activated Partial Thromboplastin Time (APTT) assays and coagulation visualized after 7 and 14 s respectively for 60 μg of crude venom. LD50 was calculated as 10.5 mg/kg. SDS-PAGE revealed the presence of major and minor protein bands between 6 and 205 kDa. Different amounts of crude venom ranged from 1.87 to 30 μg showed proteolytic activity on casein. The highest edematic activity was detected at 20 μg. Our findings showed that the edematic activity was dose dependent and persisted for 48 h after injection. The crude venom did not induce dermonecrotic activity on rabbit skin and showed no hemolytic activity on human, mouse and rabbit erythrocytes. This is the first report for phospholipase A2 and coagulation activity in venomous fish and venomous marine animals respectively. Proteolytic activity of P. russelli venom is in accordance with the other genara of scorpionidae family. According to venom activity on intrinsic and extrinsic coagulation pathways, lionfish venom would be contained an interesting pharmaceutical agent. This study is pending to further characterization of phospholipase A2, coagulation, and protease activities and also in vivo activity on animal model of surface and internal bleeding.

  12. Expression of Calcineurin Activity after Lung Transplantation: A 2-Year Follow-Up

    PubMed Central

    Sanquer, Sylvia; Amrein, Catherine; Grenet, Dominique; Guillemain, Romain; Philippe, Bruno; Boussaud, Veronique; Herry, Laurence; Lena, Celine; Diouf, Alphonsine; Paunet, Michelle; Billaud, Eliane M.; Loriaux, Françoise; Jais, Jean-Philippe; Barouki, Robert; Stern, Marc

    2013-01-01

    The objective of this pharmacodynamic study was to longitudinally assess the activity of calcineurin during the first 2 years after lung transplantation. From March 2004 to October 2008, 107 patients were prospectively enrolled and their follow-up was performed until 2009. Calcineurin activity was measured in peripheral blood mononuclear cells. We report that calcineurin activity was linked to both acute and chronic rejection. An optimal activity for calcineurin with two thresholds was defined, and we found that the risk of rejection was higher when the enzyme activity was above the upper threshold of 102 pmol/mg/min or below the lower threshold of 12 pmol/mg/min. In addition, we report that the occurrence of malignancies and viral infections was significantly higher in patients displaying very low levels of calcineurin activity. Taken together, these findings suggest that the measurement of calcineurin activity may provide useful information for the management of the prevention therapy of patients receiving lung transplantation. PMID:23536885

  13. Formation of 8-iso-PGF(2alpha) and thromboxane A(2) by stimulation with several activators of phospholipase A(2) in the isolated human umbilical vein.

    PubMed

    Sametz, W; Hummer, K; Butter, M; Wintersteiger, R; Juan, H

    2000-09-01

    We investigated the effects of the phospholipase A(2) (PLA(2)) activators calcium ionophore A 23187, hydrogen peroxide (H(2)O(2)), bradykinin (BK), histamine and noradrenaline (NA) on the 8-iso-prostaglandin (PG)F(2alpha) formation in the isolated human umbilical vein and the isolated rabbit ear. For comparison, the influence of these substances on the thromboxane A(2) (TXA(2)) release was also investigated. The release of total (esterified as well as free) 8-iso-PGF(2alpha), free 8-iso-PGF(2alpha) and TXB(2), the stable metabolite of TXA(2), was determined by specific enzyme immunoassays. The results show that bolus injections of 5.4 mmol H(2)O(2), 30 nmol A 23187, 10 nmol BK, 50 nmol histamine and 20 nmol NA caused an increased release of total 8-iso-PGF(2alpha) in the umbilical vein and the rabbit ear. A perfusion with H(2)O(2) at a final concentration of 0.3 mM also increased the release of this isoprostane. Increased formation of free 8-iso-PGF(2alpha) was induced by A 23187 injection and by both modes of H(2)O(2) administration, but not by the other treatments. Bolus injections of A 23187, BK and histamine induced an increased release of TXB(2) in both organs. Both modes of H(2)O(2) administration and NA showed no releasing effects. In conclusion, our results show that the substances used are able to stimulate the formation of 8-iso-PGF(2alpha) concurrently with the release of PGs. This effect might be of pathophysiological relevance in inflammatory and cardiovascular diseases in which an enhanced release of free radicals, BK, histamine or NA play an important role.

  14. Characterization of phospholipase A(2) activity in serum of the American alligator (Alligator mississippiensis).

    PubMed

    Merchant, Mark; Heard, Raphiel; Monroe, Caleb

    2009-11-01

    PLA(2) is a diverse class of enzymes with a broad spectrum of physiological functions. Secretory PLA(2) isoforms have been reported to exhibit important innate immune function in higher vertebrates. This study was conducted to characterize PLA(2) activity in the serum of the American alligator (Alligator mississippiensis). We used a glycerophospholipid with a fatty acid in the sn-2 position labeled with a fluorescent probe (BODIPY) to detect and quantify alligator serum PLA(2) activity. Incubation of BODIPY-labeled bacteria with different concentrations of alligator serum resulted in a concentration-dependent detection of PLA(2) activity. Kinetic studies showed that product formation was rapid, with substantial activity within 5 min, and maximal activity at approximately 20 min. The alligator PLA(2) activity was temperature-dependent, with activity at lower temperatures (5-10 degrees C) approximately half of that observed at temperatures of 30-40 degrees C. In addition, the generation of fluorescent product was reduced by a specific inhibitor (p-bromophenacyl bromide) of secretory PLA(2) in a concentration-dependent manner, enforcing the idea that the observed activities are due to a secretory PLA(2) enzyme in alligator serum.

  15. IFN-γ prevents adenosine receptor (A2bR) upregulation to sustain the macrophage activation response

    PubMed Central

    Cohen, Heather B.; Ward, Amanda; Hamidzadeh, Kajal; Ravid, Katya; Mosser, David M.

    2015-01-01

    The priming of macrophages with IFN-γ prior to TLR stimulation results in enhanced and prolonged inflammatory cytokine production. Here, we demonstrate that following TLR stimulation, macrophages up regulate the adenosine 2b receptor (A2bR) to enhance their sensitivity to immunosuppressive extracellular adenosine. This up-regulation of A2bR leads to the induction of a macrophage with an immunoregulatory phenotype and the down regulation of inflammation. IFN-γ priming of macrophages, selectively prevents the induction of the A2bR in macrophages to mitigate sensitivity to adenosine and prevent this regulatory transition. IFN-γ-mediated A2bR blockade leads to a prolonged production of TNFα and IL-12 in response to TLR ligation. The pharmacological inhibition or the genetic deletion of the A2bR results in a hyper-inflammatory response to TLR ligation, similar to IFN-γ treatment of macrophages. Conversely, the overexpression of A2bR on macrophages blunts the IFN-γ effects and promotes the development of immunoregulatory macrophages. Thus, we propose a novel mechanism whereby IFN-γ contributes to host defense, by desensitizing macrophages to the immunoregulatory effects of adenosine. This mechanism overcomes the transient nature of TLR activation, and prolongs the anti-microbial state of the classically activated macrophage. This study may offer promising new targets to improve the clinical outcome of inflammatory diseases in which macrophage activation is dysregulated. PMID:26355158

  16. Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2.

    PubMed

    Vulfius, Catherine A; Kasheverov, Igor E; Starkov, Vladislav G; Osipov, Alexey V; Andreeva, Tatyana V; Filkin, Sergey Yu; Gorbacheva, Elena V; Astashev, Maxim E; Tsetlin, Victor I; Utkin, Yuri N

    2014-01-01

    Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

  17. Molecular modeling and snake venom phospholipase A2 inhibition by phenolic compounds: Structure-activity relationship.

    PubMed

    Alam, Md Iqbal; Alam, Mohammed A; Alam, Ozair; Nargotra, Amit; Taneja, Subhash Chandra; Koul, Surrinder

    2016-05-23

    In our earlier study, we have reported that a phenolic compound 2-hydroxy-4-methoxybenzaldehyde from Janakia arayalpatra root extract was active against Viper and Cobra envenomations. Based on the structure of this natural product, libraries of synthetic structurally variant phenolic compounds were studied through molecular docking on the venom protein. To validate the activity of eight selected compounds, we have tested them in in vivo and in vitro models. The compound 21 (2-hydroxy-3-methoxy benzaldehyde), 22 (2-hydroxy-4-methoxybenzaldehyde) and 35 (2-hydroxy-3-methoxybenzylalcohol) were found to be active against venom-induced pathophysiological changes. The compounds 20, 15 and 35 displayed maximum anti-hemorrhagic, anti-lethal and PLA2 inhibitory activity respectively. In terms of SAR, the presence of a formyl group in conjunction with a phenolic group was seen as a significant contributor towards increasing the antivenom activity. The above observations confirmed the anti-venom activity of the phenolic compounds which needs to be further investigated for the development of new anti-snake venom leads.

  18. Ultraslow Water-Mediated Transmembrane Interactions Regulate the Activation of A2A Adenosine Receptor

    NASA Astrophysics Data System (ADS)

    Lee, Yoonji; Kim, Songmi; Choi, Sun; Hyeon, Changbong

    2016-09-01

    Water molecules inside G-protein coupled receptor have recently been spotlighted in a series of crystal structures. To decipher the dynamics and functional roles of internal waters in GPCR activity, we studied A$_{\\text{2A}}$ adenosine receptor using $\\mu$sec-molecular dynamics simulations. Our study finds that the amount of water flux across the transmembrane (TM) domain varies depending on the receptor state, and that the water molecules of the TM channel in the active state flow three times slower than those in the inactive state. Depending on the location in solvent-protein interface as well as the receptor state, the average residence time of water in each residue varies from $\\sim\\mathcal{O}(10^2)$ psec to $\\sim\\mathcal{O}(10^2)$ nsec. Especially, water molecules, exhibiting ultraslow relaxation ($\\sim\\mathcal{O}(10^2)$ nsec) in the active state, are found around the microswitch residues that are considered activity hotspots for GPCR function. A continuous allosteric network spanning the TM domain, arising from water-mediated contacts, is unique in the active state, underscoring the importance of slow waters in the GPCR activation.

  19. Activation of nuclear receptor NR5A2 increases Glut4 expression and glucose metabolism in muscle cells

    SciTech Connect

    Bolado-Carrancio, A.; Riancho, J.A.; Sainz, J.; Rodríguez-Rey, J.C.

    2014-04-04

    Highlights: • NR5A2 expression in C2C12 is associated with myotube differentiation. • DLPC induces an increase in GLUT4 levels and glucose uptake in C2C12 myotubes. • In high glucose conditions the activation of NR5A2 inhibits fatty acids oxidation. - Abstract: NR5A2 is a nuclear receptor which regulates the expression of genes involved in cholesterol metabolism, pluripotency maintenance and cell differentiation. It has been recently shown that DLPC, a NR5A2 ligand, prevents liver steatosis and improves insulin sensitivity in mouse models of insulin resistance, an effect that has been associated with changes in glucose and fatty acids metabolism in liver. Because skeletal muscle is a major tissue in clearing glucose from blood, we studied the effect of the activation of NR5A2 on muscle metabolism by using cultures of C2C12, a mouse-derived cell line widely used as a model of skeletal muscle. Treatment of C2C12 with DLPC resulted in increased levels of expression of GLUT4 and also of several genes related to glycolysis and glycogen metabolism. These changes were accompanied by an increased glucose uptake. In addition, the activation of NR5A2 produced a reduction in the oxidation of fatty acids, an effect which disappeared in low-glucose conditions. Our results suggest that NR5A2, mostly by enhancing glucose uptake, switches muscle cells into a state of glucose preference. The increased use of glucose by muscle might constitute another mechanism by which NR5A2 improves blood glucose levels and restores insulin sensitivity.

  20. Electrochemical reduction of flavocytochromes 2B4 and 1A2 and their catalytic activity.

    PubMed

    Shumyantseva, V V; Bulko, T V; Bachmann, T T; Bilitewski, U; Schmid, R D; Archakov, A I

    2000-05-01

    The present study shows that cytochromes P450 2B4 and 1A2 with a covalently attached riboflavin (semisynthetic flavocytochromes RfP450 2B4 and RfP450 1A2) can be reduced electrochemically on rhodium-graphite electrodes at a potential of -500 mV (vs Ag/AgCl). In the presence of substrates such as aminopyrine, aniline, 7-ethoxyresorufin, and 7-pentoxyresorufin, N-demethylation, p-hydroxylation, and O-dealkylation reactions proceeded, as was confirmed by product analysis. Rates of electrocatalytically driven reactions are comparable to those obtained using NAD(P)H as the source of reducing equivalents. These results suggest the practicality of developing flavocytochrome P450s as catalysts for oxidation reactions with different classes of organic substrates.

  1. Predictive three-dimensional quantitative structure-activity relationship of cytochrome P450 1A2 inhibitors.

    PubMed

    Korhonen, Laura E; Rahnasto, Minna; Mähönen, Niina J; Wittekindt, Carsten; Poso, Antti; Juvonen, Risto O; Raunio, Hannu

    2005-06-02

    The purpose of this study was to determine the cytochrome P450 1A2 (CYP1A2) inhibition potencies of structurally diverse compounds to create a comprehensive three-dimensional quantitative structure-activity relationship (3D-QSAR) model of CYP1A2 inhibitors and to use this model to predict the inhibition potencies of an external set of compounds. Fifty-two compounds including naphthalene, lactone and quinoline derivatives were assayed in a 96-well plate format for CYP1A2 inhibition activity using 7-ethoxyresorufin O-dealkylation as the probe reaction. The IC50 values of the tested compounds varied from 2.3 microM to over 40,000 microM. On the basis of this data set, a comparative molecular field analysis (CoMFA) and GRID/GOLPE models were created that yielded novel structural information about the interaction between inhibitory molecules and the CYP1A2 active site. The created CoMFA model was able to accurately predict inhibitory potencies of several structurally unrelated compounds, including selective inhibitors of other cytochrome P450 forms.

  2. Effects of Eprosartan on Serum Metabolic Parameters in Patients with Essential Hypertension

    PubMed Central

    Rizos, Evangelos C; Spyrou, Athanasia; Liberopoulos, Evangelos N; Papavasiliou, Eleni C; Saougos, Vasilis; Tselepis, Alexandros D; Elisaf, Moses

    2007-01-01

    The effect of the anti-hypertensive drug eprosartan on metabolic parameters is currently not extensively documented. We evaluated the effect of eprosartan on parameters involved in atherogenesis, oxidative stress and clotting activity. This open-label unblinded intervention study included 40 adult patients with essential hypertension taking eprosartan. Eprosartan significantly reduced by 8% (p<0.001) the systolic and by 13% (p<.001) the diastolic blood pressure, and in-creased by 24% the time needed to produce oxidative by-products (p=0.001), a marker of oxidative stress. In contrast, ep-rosartan did not alter 8-isoprostane (8-epiPGF2a) levels, another marker of oxidative stress. Additionally, eprosartan re-duced by 14% aspartate aminotransferase and by 21% then alanine aminotransferase activity, while it had a neutral effect on the lipid profile and apolipoprotein levels and did not influence glucose homeostasis, creatinine and uric acid levels. Eprosartan did not affect the clotting/fibrinolytic status (estimated by plasminogen activator inhibitor 1, tissue plasmino-gen activator and a2 antiplasmin levels), or the enzymatic activity of the lipoprotein associated phospholipase A2 (Lp-PLA2) and paraoxonase 1 (PON1). In conclusion, eprosartan should be mainly considered as an anti-hypertensive agent with neutral effects on most of the metabolic parameters in hypertensive patients. PMID:18949087

  3. Evaluation of a 2-Year Physical Activity and Healthy Eating Intervention in Middle School Children

    ERIC Educational Resources Information Center

    Haerens, Leen; Deforche, Benedicte; Maes, Lea; Cardon, Greet; Stevens, Veerle; De Bourdeaudhuij, Ilse

    2006-01-01

    The aim of the present study was to evaluate the effects of a middle school physical activity and healthy eating intervention, including an environmental and computer-tailored component, and to investigate the effects of parental involvement. A random sample of 15 schools with seventh and eight graders was randomly assigned to one of three…

  4. A2A Adenosine Receptor Antagonism Enhances Synaptic and Motor Effects of Cocaine via CB1 Cannabinoid Receptor Activation

    PubMed Central

    Tozzi, Alessandro; de Iure, Antonio; Marsili, Valentina; Romano, Rosaria; Tantucci, Michela; Di Filippo, Massimiliano; Costa, Cinzia; Napolitano, Francesco; Mercuri, Nicola Biagio; Borsini, Franco; Giampà, Carmen; Fusco, Francesca Romana; Picconi, Barbara; Usiello, Alessandro; Calabresi, Paolo

    2012-01-01

    Background Cocaine increases the level of endogenous dopamine (DA) in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs) have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. Principal Findings Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. Conclusions The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine. PMID:22715379

  5. Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

    DOE PAGES

    Naranjo, Andrea N.; McNeely, Patrick M.; Katsaras, John; ...

    2016-05-27

    The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, ormore » DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. Furthermore, the studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.« less

  6. Deletion of striatal adenosine A(2A) receptor spares latent inhibition and prepulse inhibition but impairs active avoidance learning.

    PubMed

    Singer, Philipp; Wei, Catherine J; Chen, Jiang-Fan; Boison, Detlev; Yee, Benjamin K

    2013-04-01

    Following early clinical leads, the adenosine A(2A)R receptor (A(2A)R) has continued to attract attention as a potential novel target for treating schizophrenia, especially against the negative and cognitive symptoms of the disease because of A(2A)R's unique modulatory action over glutamatergic in addition to dopaminergic signaling. Through (i) the antagonistic interaction with the dopamine D(2) receptor, and (ii) the regulation of glutamate release and N-methyl-d-aspartate receptor function, striatal A(2A)R is ideally positioned to fine-tune the dopamine-glutamate balance, the disturbance of which is implicated in the pathophysiology of schizophrenia. However, the precise function of striatal A(2A)Rs in the regulation of schizophrenia-relevant behavior is poorly understood. Here, we tested the impact of conditional striatum-specific A(2A)R knockout (st-A(2A)R-KO) on latent inhibition (LI) and prepulse inhibition (PPI) - behavior that is tightly regulated by striatal dopamine and glutamate. These are two common cross-species translational tests for the assessment of selective attention and sensorimotor gating deficits reported in schizophrenia patients; and enhanced performance in these tests is associated with antipsychotic drug action. We found that neither LI nor PPI was significantly affected in st-A(2A)R-KO mice, although a deficit in active avoidance learning was identified in these animals. The latter phenotype, however, was not replicated in another form of aversive conditioning - namely, conditioned taste aversion. Hence, the present study shows that neither learned inattention (as measured by LI) nor sensory gating (as indexed by PPI) requires the integrity of striatal A(2A)Rs - a finding that may undermine the hypothesized importance of A(2A)R in the genesis and/or treatment of schizophrenia.

  7. Activation of A1, A2A, or A3 adenosine receptors attenuates lung ischemia-reperfusion injury

    PubMed Central

    Gazoni, Leo M.; Walters, Dustin M.; Unger, Eric B.; Linden, Joel; Kron, Irving L.; Laubach, Victor E.

    2010-01-01

    Objective Adenosine and the activation of specific adenosine receptors are implicated in the attenuation of inflammation and organ ischemia-reperfusion (IR) injury. We hypothesized that activation of A1, A2A, or A3 adenosine receptors would provide protection against lung IR injury. Methods Using an isolated, ventilated, blood-perfused rabbit lung model, lungs underwent 18 hours cold ischemia followed by 2 hours reperfusion. Lungs were administered either vehicle, adenosine, or selective A1, A2A, or A3 receptor agonists (CCPA, ATL-313, or IB-MECA, respectively) alone or with their respective antagonists (DPCPX, ZM241385, or MRS1191) during reperfusion. Results Compared to the vehicle-treated control group, treatment with A1, A2A, or A3 agonists significantly improved function (increased lung compliance and oxygenation and decreased pulmonary artery pressure), decreased neutrophil infiltration by myeloperoxidase activity, decreased edema, and reduced TNF-α production. Adenosine treatment was also protective but not to the level of the agonists. When each agonist was paired with its respective antagonist, all protective effects were blocked. The A2A agonist reduced pulmonary artery pressure and myeloperoxidase activity and increased oxygenation to a greater degree than the A1 or A3 agonists. Conclusions Selective activation of A1, A2A, or A3 adenosine receptors provides significant protection against lung IR injury. The decreased elaboration of the potent proinflammatory cytokine, TNF-α, and decreased neutrophil sequestration likely contribute to the overall improvement in pulmonary function. These results provide evidence for the therapeutic potential of specific adenosine receptor agonists in lung transplant recipients. PMID:20398911

  8. Central bombesin activates adrenal adrenaline- and noradrenaline-containing cells via brain thromboxane A2 in rats.

    PubMed

    Usui, Daisuke; Yamaguchi-Shima, Naoko; Okada, Shoshiro; Shimizu, Takahiro; Wakiguchi, Hiroshi; Yokotani, Kunihiko

    2009-05-11

    The sympathetic nervous system regulates peripheral organs via the adrenal chromaffin cells containing adrenaline (A-cells) or noradrenaline (NA-cells) and the sympathetic ganglia. We examined the effect of intracerebroventricularly administered bombesin on neuronal activities of adrenal A-cells and NA-cells and several kinds of sympathetic ganglia (superior cervical, stellate and celiac ganglia) using c-Fos (a marker for neuronal activation), with regard to brain prostanoid, in anesthetized rats. Bombesin induced c-Fos in both adrenal A-cells and NA-cells, but not in any of the sympathetic ganglia. Central pretreatment with either indomethacin (a cyclooxygenase inhibitor) or furegrelate (a thromboxane A(2) synthase inhibitor) abolished all bombesin-induced responses. These results suggest that bombesin centrally activates adrenal A-cells and NA-cells by brain thromboxane A(2)-mediated mechanisms in rats.

  9. A Role for Phospholipase A2 Activity in Membrane Tubule Formation and TGN Trafficking

    PubMed Central

    Schmidt, John A.; Kalkofen, Danielle N.; Donovan, Kirk W.; Brown, William J.

    2015-01-01

    We have investigated the role of phospholipase A2 (PLA2) enzymes in generating membrane tubules at the trans-Golgi network (TGN). Constitutive TGN membrane tubules and those induced by over-expressing kinase dead protein kinase D were inhibited by the PLA2 inhibitors ONO-RS-082 (ONO) and bromoenol lactone. These antagonists also inhibited secretory delivery of both soluble and transmembrane cargoes. Finally, use of the reversible antagonist ONO and time-lapse imaging revealed for the first time that PLA2 antagonists inhibit the initiation of membrane tubule formation at the TGN. Thus, PLA2 enzymes appear to have an important role in the earliest steps of membrane tubule formation at the TGN, which are utilized for membrane trafficking. PMID:20874826

  10. Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

    PubMed

    Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N

    2015-12-01

    Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

  11. Elevated adenosine signaling via adenosine A2B receptor induces normal and sickle erythrocyte sphingosine kinase 1 activity.

    PubMed

    Sun, Kaiqi; Zhang, Yujin; Bogdanov, Mikhail V; Wu, Hongyu; Song, Anren; Li, Jessica; Dowhan, William; Idowu, Modupe; Juneja, Harinder S; Molina, Jose G; Blackburn, Michael R; Kellems, Rodney E; Xia, Yang

    2015-03-05

    Erythrocyte possesses high sphingosine kinase 1 (SphK1) activity and is the major cell type supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is upregulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte SphK1 activity is regulated remains unknown. Here we report that adenosine induces SphK1 activity in human and mouse sickle and normal erythrocytes in vitro. Next, using 4 adenosine receptor-deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in human and mouse normal and sickle erythrocytes in vitro. Subsequently, we provide in vivo genetic evidence that adenosine deaminase (ADA) deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Finally, we revealed that protein kinase A-mediated extracellular signal-regulated kinase 1/2 activation functioning downstream of ADORA2B underlies adenosine-induced erythrocyte SphK1 activity. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms regulating SphK1 activity in normal and SCD.

  12. Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity

    SciTech Connect

    Lin, Y.W.; Robinson, H.; Yeung, N.; Gao, Y.-G.; Miner, K. D.; Lei, L.; Lu, Y.

    2010-07-28

    A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called Fe{sub B}Mb(-His)). A high resolution (1.65 {angstrom}) crystal structure of Cu(II)-CN?-Fe{sub B}Mb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe{sub B}Mb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-Fe{sub B}Mb(-His) and Fe(II)-Fe{sub B}Mb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N{sub 2}O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-Fe{sub B}Mb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-Fe{sub B}Mb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.

  13. Introducing a 2-His-1-Glu Nonheme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity

    SciTech Connect

    Y Lin; N Yeung; Y Gao; K Miner; L Lei; H Robinson; Y Lu

    2011-12-31

    A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called Fe{sub B}Mb(-His)). A high resolution (1.65 {angstrom}) crystal structure of Cu(II)-CN{sup -}-Fe{sub B}Mb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe{sub B}Mb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-Fe{sub B}Mb(-His) and Fe(II)-Fe{sub B}Mb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N{sub 2}O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-Fe{sub B}Mb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-Fe{sub B}Mb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.

  14. A 2D suspension of active agents: the role of fluid mediated interactions.

    PubMed

    Behmadi, Hojjat; Fazli, Zahra; Najafi, Ali

    2017-03-22

    Taking into account both the Vicsek short-range ordering and the far-field hydrodynamic interactions mediated by the ambient fluid, we investigate the role of long-range interactions in the ordering phenomena in a quasi 2-dimensional active suspension. By studying the number fluctuations, the velocity correlation functions and cluster size distribution function, we show that depending on the number density of swimmers and the strength of noise, the hydrodynamic interactions can have significant effects in a suspension. For a fixed value of noise, at larger density of particles, long-range interactions enhance the particle pairing and cluster formation in the system.

  15. A 2D suspension of active agents: the role of fluid mediated interactions

    NASA Astrophysics Data System (ADS)

    Behmadi, Hojjat; Fazli, Zahra; Najafi, Ali

    2017-03-01

    Taking into account both the Vicsek short-range ordering and the far-field hydrodynamic interactions mediated by the ambient fluid, we investigate the role of long-range interactions in the ordering phenomena in a quasi 2-dimensional active suspension. By studying the number fluctuations, the velocity correlation functions and cluster size distribution function, we show that depending on the number density of swimmers and the strength of noise, the hydrodynamic interactions can have significant effects in a suspension. For a fixed value of noise, at larger density of particles, long-range interactions enhance the particle pairing and cluster formation in the system.

  16. Antigen receptors on immature, but not mature, B and T cells are coupled to cytosolic phospholipase A2 activation: expression and activation of cytosolic phospholipase A2 correlate with lymphocyte maturation.

    PubMed

    Gilbert, J J; Stewart, A; Courtney, C A; Fleming, M C; Reid, P; Jackson, C G; Wise, A; Wakelam, M J; Harnett, M M

    1996-03-15

    The Ag receptors on mature B and T cells are not coupled to the activation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Moreover, phorbol esters such as PMA, which can activate cPLA2 via mitogen-activated protein (MAP) kinase in most cell types, also failed to induce the release of arachidonate from mature cells, suggesting that the cPLA2 pathway may not be functional in mature lymphocytes. Interestingly, Western blot analysis revealed that cPLA2, which had previously been thought to be expressed ubiquitously, is not expressed in mature B or T cells and that cytosolic phospholipase A2 expression could not be up-regulated in lymphocytes following culture with a range of cytokines most likely to be involved in an immune response such as IL-1 alpha, IL-3, or TNF-alpha. In contrast, cPLA2 was shown to be expressed and activated in thymocytes and immature B cells under conditions in which ligation of the Ag receptors led to growth arrest and/or apoptosis. Taken together, these data suggest that cPLA2 does not play a role in Ag receptor-mediated lymphocyte activation, but may be involved in the molecular mechanisms underlying lymphocyte maturation and/or self tolerance by clonal deletion.

  17. Enhancement of phosphorus removal in a low temperature A(2)/O process by anaerobic phosphorus release of activated sludge.

    PubMed

    Li, Jianzheng; Jin, Yu; Guo, Yaqiong; He, Junguo

    2013-01-01

    An anaerobic phosphorus release tank was introduced to an anaerobic-anoxic-aerobic (A(2)/O) process treating domestic sewage to enhance the phosphorus removal at low temperature. Phosphorus release of the activated sludge from the second sedimentation tank was evaluated at 14 °C by batch cultures, and the nutrient removal in the modified low temperature A(2)/O process was further investigated at the same temperature. The results showed that the feasible sludge retention time was 14 h for sequencing batch reaction and 12 h for continuous flow operation. The ratio of raw sewage to activated sludge from the second sedimentation tank was 1:1 in volume to meet the demand of carbon resource for the growth of phosphorus release microbes. The feasible chemical oxygen demand (COD) loading rate of the activated sludge in the phosphorus release tank was 0.015-0.02 g COD/g MLSS (mixed liquor suspended solids) and the nitrate concentration should be less than 5 mg/L. The phosphorus release was doubled when the sludge was blended intermittently and gently. The anaerobic phosphorus release of the activated sludge improved the phosphate removal remarkably, as well as the removal of NH4(+)-N and total nitrogen (TN) in the modified low temperature A(2)/O process. The effluent COD, NH4(+)-N, TN and total phosphorus could meet a stricter discharge standard.

  18. Effect of Darapladib Treatment on Endarterectomy Carotid Plaque Lipoprotein-Associated Phospholipase A2 Activity: A Randomized, Controlled Trial

    PubMed Central

    Johnson, Joel L.; Shi, Yi; Snipes, Rose; Janmohamed, Salim; Rolfe, Timothy E.; Davis, Bill; Postle, Anthony; Macphee, Colin H.

    2014-01-01

    Background The aim of this study was to assess the effects of darapladib, a selective oral investigational lipoprotein-associated phospholipase A2 inhibitor, on both plasma and plaque lipoprotein-associated phospholipase A2 activity. Methods Patients undergoing elective carotid endarterectomy were randomized to darapladib 40 mg (n = 34), 80 mg (n = 34), or placebo (n = 34) for 14 days, followed by carotid endarterectomy 24 hours after the last dose of study medication. Results Darapladib 40 mg and 80 mg reduced plasma lipoprotein-associated phospholipase A2 activity by 52% and 81%, respectively, versus placebo (both P<0.001). Significant reductions in plaque lipoprotein-associated phospholipase A2 activity were also observed compared with placebo (P<0.0001), which equated to a 52% and 80% decrease compared with placebo. No significant differences were observed between groups in plaque lysophosphatidylcholine content or other biomarkers, although a dose-dependent decrease in plaque matrix metalloproteinase-9 mRNA expression was observed with darapladib 80 mg (P = 0.053 vs placebo). In a post-hoc analysis, plaque caspase-3 (P<0.001) and caspase-8 (P<0.05) activity were found to be significantly lower in the darapladib 80-mg group versus placebo. No major safety concerns were identified in the study. Conclusions Short-term treatment (14±4 days) with darapladib produced a robust, dose-dependent reduction in plasma lipoprotein-associated phospholipase A2 activity. More importantly, darapladib demonstrated placebo-corrected reductions in carotid plaque lipoprotein-associated phospholipase A2 activity of similar magnitude. Darapladib was generally well tolerated and no safety concerns were identified. Additional studies of longer duration are needed to explore whether these pharmacodynamic effects are associated with improved clinical outcomes, as might be hypothesized. Trial Registration Information Name of Registry 1: ClinicalTrials.gov Registry Number

  19. An Asymptotic Analysis of a 2-D Model of Dynamically Active Compartments Coupled by Bulk Diffusion

    NASA Astrophysics Data System (ADS)

    Gou, J.; Ward, M. J.

    2016-08-01

    A class of coupled cell-bulk ODE-PDE models is formulated and analyzed in a two-dimensional domain, which is relevant to studying quorum-sensing behavior on thin substrates. In this model, spatially segregated dynamically active signaling cells of a common small radius ɛ ≪ 1 are coupled through a passive bulk diffusion field. For this coupled system, the method of matched asymptotic expansions is used to construct steady-state solutions and to formulate a spectral problem that characterizes the linear stability properties of the steady-state solutions, with the aim of predicting whether temporal oscillations can be triggered by the cell-bulk coupling. Phase diagrams in parameter space where such collective oscillations can occur, as obtained from our linear stability analysis, are illustrated for two specific choices of the intracellular kinetics. In the limit of very large bulk diffusion, it is shown that solutions to the ODE-PDE cell-bulk system can be approximated by a finite-dimensional dynamical system. This limiting system is studied both analytically, using a linear stability analysis and, globally, using numerical bifurcation software. For one illustrative example of the theory, it is shown that when the number of cells exceeds some critical number, i.e., when a quorum is attained, the passive bulk diffusion field can trigger oscillations through a Hopf bifurcation that would otherwise not occur without the coupling. Moreover, for two specific models for the intracellular dynamics, we show that there are rather wide regions in parameter space where these triggered oscillations are synchronous in nature. Unless the bulk diffusivity is asymptotically large, it is shown that a diffusion-sensing behavior is possible whereby more clustered spatial configurations of cells inside the domain lead to larger regions in parameter space where synchronous collective oscillations between the small cells can occur. Finally, the linear stability analysis for these cell

  20. Increased activity of group II phospholipase A2 in plasma in rat sodium deoxycholate induced acute pancreatitis

    PubMed Central

    Furue, S; Hori, Y; Kuwabara, K; Ikeuchi, J; Onoyama, H; Yamamoto, M; Tanaka, K

    1997-01-01

    Background—Two different types of secretory phospholipase A2 (PLA2), pancreatic group I (PLA2-I) and non-pancreatic group II (PLA2-II), have been identified and postulated to be associated with the pathogenesis of various diseases, such as acute pancreatitis, septic shock, and multiple organ failure. 
Aims—To investigate the type of secretory PLA2 responsible for its catalytic activity found in plasma and ascites of experimental acute pancreatitis. 
Methods—Acute pancreatitis of differing severity was induced by the injection of different concentrations (1% or 10%) of sodium deoxycholate (DCA) into the common biliopancreatic duct in rats, and catalytic PLA2 activity in plasma and ascites were differentiated by anti-PLA2-I antibody and specific inhibitor of PLA2-II. Survival rate and plasma amylase, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were also measured.
Results—In 1% and 10% DCA induced acute pancreatitis, plasma amylase values as well as PLA2 activity in ascites were greatly increased. PLA2 activity in plasma was also notably increased in 10% DCA induced acute pancreatitis, but not in 1% DCA induced acute pancreatitis. PLA2-I specific polyclonal antibody significantly inhibited PLA2 activity in ascites but not that in plasma. In contrast, plasma PLA2 activity was completely suppressed by PLA2-II specific inhibitor. In addition, a high mortality (93% at five hours) and a significant increase in plasma AST and ALT were noted in 10% DCA induced pancreatitis. 
Conclusion—Ascites PLA2 activity is mainly derived from PLA2-I, whereas plasma PLA2 activity is mostly derived from PLA2-II in severe acute pancreatitis, suggesting that increased plasma PLA2-II activity might be implicated in hepatic failure arising after severe acute pancreatitis. 

 Keywords: acute pancreatitis; phospholipase A2; sodium deoxycholate pancreatitis; hepatic failure PMID:9462218

  1. The MATROSHKA experiment: results and comparison from extravehicular activity (MTR-1) and intravehicular activity (MTR-2A/2B) exposure.

    PubMed

    Berger, Thomas; Bilski, Paweł; Hajek, Michael; Puchalska, Monika; Reitz, Günther

    2013-12-01

    Astronauts working and living in space are exposed to considerably higher doses and different qualities of ionizing radiation than people on Earth. The multilateral MATROSHKA (MTR) experiment, coordinated by the German Aerospace Center, represents the most comprehensive effort to date in radiation protection dosimetry in space using an anthropomorphic upper-torso phantom used for radiotherapy treatment planning. The anthropomorphic upper-torso phantom maps the radiation distribution as a simulated human body installed outside (MTR-1) and inside different compartments (MTR-2A: Pirs; MTR-2B: Zvezda) of the Russian Segment of the International Space Station. Thermoluminescence dosimeters arranged in a 2.54 cm orthogonal grid, at the site of vital organs and on the surface of the phantom allow for visualization of the absorbed dose distribution with superior spatial resolution. These results should help improve the estimation of radiation risks for long-term human space exploration and support benchmarking of radiation transport codes.

  2. Timosaponin AIII induces antiplatelet and antithrombotic activity via Gq-mediated signaling by the thromboxane A2 receptor

    PubMed Central

    Cong, Yue; Wang, Limei; Peng, Renjun; Zhao, Yang; Bai, Fan; Yang, Chao; Liu, Xiaolan; Wang, Daqian; Ma, Baiping; Cong, Yuwen

    2016-01-01

    The thromboxane (Tx) A2 pathway is a major contributor to the amplification of initial platelet activation and is therefore a key drug target. To identify potent small-molecule inhibitors of the thromboxane prostaglandin (TP) receptor, we screened a small steroidal saponin library using U46619-induced rat platelet aggregation assays. Timosaponin AIII (TAIII) was identified as a potent inhibitor of U46619-induced rat platelet aggregation and exhibited superior selectivity for the TP receptor versus other G protein-coupled receptors and a PKC activator. TAIII inhibited U46619-induced rat platelet aggregation independent of increases in cAMP and cGMP and the inhibition of TxA2 production. Both PKC and PLC activators restored TAIII-inhibited platelet aggregation, whereas TAIII did not inhibit platelet aggregation induced by co-activation of the G12/13 and Gz pathways. Furthermore, TAIII did not affect the platelet shape change or ROCK2 phosphorylation evoked by low-dose U46619. In vivo, TAIII prolonged tail bleeding time, reduced the mortality of animals with acute pulmonary thromboembolism and significantly reduced venous thrombus weight. Our study suggests that TAIII, by preferentially targeting Gq-mediated PLC/PKC signaling from the TP receptor, induces stronger in vitro antiplatelet activity and in vivo antithrombotic effects and may be an excellent candidate for the treatment of thrombotic disorders. PMID:27934923

  3. Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

    PubMed Central

    Hothersall, J. Daniel; Guo, Dong; Sarda, Sunil; Sheppard, Robert J.; Chen, Hongming; Keur, Wesley; Waring, Michael J.; IJzerman, Adriaan P.; Hill, Stephen J.; Dale, Ian L.

    2017-01-01

    The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy. PMID:27803241

  4. Omeprazole does not enhance the metabolism of phenacetin, a marker of CYP1A2 activity, in healthy volunteers.

    PubMed

    Xiaodong, S; Gatti, G; Bartoli, A; Cipolla, G; Crema, F; Perucca, E

    1994-06-01

    Omeprazole has been reported to increase cytochrome P450IA2 (CYP1A2) activity in vitro, but whether this effect also occurs in vivo is controversial. To clarify this issue, the effect of omeprazole (20 mg/day for 8 days) on the kinetics and metabolism of phenacetin, an in vivo marker of CYP1A2 activity, was examined in 10 healthy volunteers. The pharmacokinetic parameters of phenacetin and metabolically derived paracetamol on the 8th day of omeprazole administration were very similar to those observed in a control session in the absence of omeprazole administration, the only significant difference being a higher peak plasma phenacetin concentration during omeprazole treatment. It is concluded that at the dosage used omeprazole does not increase the rate of oxidative and conjugative reactions involved in the metabolism of phenacetin and paracetamol respectively. These data are consistent with the hypothesis that omeprazole is generally devoid of inducing effects on CYP1A2 activity in vivo, at least in a Caucasian population with a low prevalence of the omeprazole-mephenytoin poor metabolizer phenotype.

  5. NcoA2-Dependent Inhibition of HIF-1α Activation Is Regulated via AhR.

    PubMed

    Tsai, Chi-Hao; Li, Ching-Hao; Liao, Po-Lin; Cheng, Yu-Wen; Lin, Cheng-Hui; Huang, Shih-Hsuan; Kang, Jaw-Jou

    2015-12-01

    High endogenous levels of aryl hydrocarbon receptor (AhR) contribute to hypoxia signaling pathway inhibition following exposure to the potent AhR ligand benzo[a]pyrene (B[a]P) and could alter cellular homeostasis and disease condition. Increasing evidence indicates that AhR might compete with AhR nuclear translocator (ARNT) for complex formation with hypoxia-inducible factor-1α (HIF-1α) for transactivation, which could alter several physiological variables. Nuclear receptor coactivator 2 (NcoA2) is a transcription coactivator that regulates transcription factor activation and inhibition of basic helix-loop-helix Per (Period)-ARNT-SIM (single-minded) (bHLH-PAS) family proteins, such as HIF-1α, ARNT, and AhR, through protein-protein interactions. In this study, we demonstrated that both hypoxia and hypoxia-mimic conditions decreased NcoA2 protein expression in HEK293T cells. Hypoxia response element (HRE) and xenobiotic-responsive element (XRE) transactivation also were downregulated with NcoA2 knockdown under hypoxic conditions. In addition, B[a]P significantly decreased NcoA2 protein expression be accompanied with AhR degradation. We next evaluated whether the absence of AhR could affect NcoA2 protein function under hypoxia-mimetic conditions. NcoA2 and HIF-1α nuclear localization decreased in both B[a]P-pretreated and AhR-knockdown HepG2 cells under hypoxia-mimic conditions. Interestingly, NcoA2 overexpression downregulated HRE transactivation by competing with HIF-1α and AhR to form protein complexes with ARNT. Both NcoA2 knockdown and overexpression inhibited endothelial cell tube formation in vitro. We also demonstrated using the in vivo plug assay that NcoA2-regulated vascularization decreased in mice. Taken together, these results revealed a biphasic role of NcoA2 between AhR and hypoxic conditions, thus providing a novel mechanism underlying the cross talk between AhR and hypoxia that affects disease development and progression.

  6. 2-Amino-N-pyrimidin-4-ylacetamides as A2A receptor antagonists: 2. Reduction of hERG activity, observed species selectivity, and structure-activity relationships.

    PubMed

    Slee, Deborah H; Moorjani, Manisha; Zhang, Xiaohu; Lin, Emily; Lanier, Marion C; Chen, Yongsheng; Rueter, Jaimie K; Lechner, Sandra M; Markison, Stacy; Malany, Siobhan; Joswig, Tanya; Santos, Mark; Gross, Raymond S; Williams, John P; Castro-Palomino, Julio C; Crespo, María I; Prat, Maria; Gual, Silvia; Díaz, José-Luis; Jalali, Kayvon; Sai, Yang; Zuo, Zhiyang; Yang, Chun; Wen, Jenny; O'Brien, Zhihong; Petroski, Robert; Saunders, John

    2008-03-27

    Previously we have described a series of novel A 2A receptor antagonists with excellent water solubility. As described in the accompanying paper, the antagonists were first optimized to remove an unsubstituted furyl moiety, with the aim of avoiding the potential metabolic liabilities that can arise from the presence of an unsubstituted furan. This effort identified a series of potent and selective methylfuryl derivatives. Herein, we describe the further optimization of this series to increase potency, maintain selectivity for the human A 2A vs the human A 1 receptor, and minimize activity against the hERG channel. In addition, the observed structure-activity relationships against both the human and the rat A 2A receptor are reported.

  7. Endogenous prostaglandin endoperoxides and prostacyclin modulate the thrombolytic activity of tissue plasminogen activator. Effects of simultaneous inhibition of thromboxane A2 synthase and blockade of thromboxane A2/prostaglandin H2 receptors in a canine model of coronary thrombosis.

    PubMed Central

    Golino, P; Rosolowsky, M; Yao, S K; McNatt, J; De Clerck, F; Buja, L M; Willerson, J T

    1990-01-01

    We tested the hypothesis that simultaneous inhibition of TxA2 synthase and blockade of TxA2/PHG2 receptors is more effective in enhancing thrombolysis and preventing reocclusion after discontinuation of tissue plasminogen activator (t-PA) than either intervention alone. Coronary thrombosis was induced in 35 dogs by placing a copper coil into the left anterior descending coronary artery. Coronary flow was measured with a Doppler flow probe. 30 min after thrombus formation, the animals received saline (controls, n = 10); SQ 29548 (0.4 mg/kg bolus + 0.4 mg/kg per h infusion), a TxA2/PGH2 receptor antagonist (n = 8); dazoxiben (5 mg/kg bolus + 5 mg/kg per h infusion), a TxA2 synthase inhibitor (n = 9); or R 68070 (5 mg/kg bolus + 5 mg/kg per h infusion), a drug that blocks TxA2/PGH2 receptors and inhibits TxA2 synthase (n = 8). Then, all dogs received heparin (200 U/kg) and a bolus of t-PA (80 micrograms/kg) followed by a continuous infusion (8 micrograms/kg per min) for up to 90 min or until reperfusion was achieved. The time to thrombolysis did not change significantly in SQ 29548-treated dogs as compared with controls (42 +/- 5 vs. 56 +/- 7 min, respectively, P = NS), but it was significantly shortened by R 68070 and dazoxiben (11 +/- 2 and 25 +/- 6 min, respectively, P less than 0.001 vs. controls and SQ 29548-treated dogs). R 68070 administration resulted in a lysis time significantly shorter than that observed in the dazoxiben-treated group (P less than 0.01). Reocclusion was observed in eight of eight control dogs, five of seven SQ 29548-treated dogs, seven of nine dazoxiben-treated dogs, and zero of eight R 68070-treated animals (P less than 0.001). TxB2 and 6-keto-PGF1 alpha, measured in blood samples obtained from the coronary artery distal to the thrombus, were significantly increased at reperfusion and at reocclusion in control animals and in dogs receiving SQ 29548. R 68070 and dazoxiben prevented the increase in plasma TxB2 levels, whereas 6-keto-PGF1

  8. PEDF Inhibits the Activation of NLRP3 Inflammasome in Hypoxia Cardiomyocytes through PEDF Receptor/Phospholipase A2

    PubMed Central

    Zhou, Zhongxin; Wang, Zhu; Guan, Qiuhua; Qiu, Fan; Li, Yufeng; Liu, Zhiwei; Zhang, Hao; Dong, Hongyan; Zhang, Zhongming

    2016-01-01

    The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome has been linked to sterile inflammation, which is involved in ischemic injury in myocardial cells. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein with many biological activities, such as anti-inflammatory, antioxidant and anti-angiogenic properties. However, it is not known whether and how PEDF acts to regulate the activation of the NLRP3 inflammasome in cardiomyocytes. In the present study, we used the neonatal cardiomyocytes models of ischemia-like conditions to evaluate the mitochondrial fission and the activation of the NLRP3 inflammasome. We also determined the mechanism by which PEDF inhibits hypoxia-induced activation of the NLRP3 inflammasome. We found that PEDF decreased the activation of the NLRP3 inflammasome in neonatal cardiomyocytes through pigment epithelial-derived factor receptor/calcium-independent phospholipase A2 (PEDFR/iPLA2). Meanwhile, PEDF reduced Drp1-induced mitochondrial fission and mitochondrial fission-induced mitochondrial DNA (mtDNA), as well as mitochondrial reactive oxygen species (mtROS) release into cytosol through PEDFR/iPLA2. We also found that PEDF inhibited mitochondrial fission-induced NLRP3 inflammasome activation. Furthermore, previous research has found that endogenous cytosolic mtDNA and mtROS can serve as activators of NLRP3 inflammasome activity. Therefore, we hypothesized that PEDF can protect against hypoxia-induced activation of the NLRP3 inflammasome by inhibiting mitochondrial fission though PEDFR/iPLA2. PMID:27973457

  9. PEDF Inhibits the Activation of NLRP3 Inflammasome in Hypoxia Cardiomyocytes through PEDF Receptor/Phospholipase A2.

    PubMed

    Zhou, Zhongxin; Wang, Zhu; Guan, Qiuhua; Qiu, Fan; Li, Yufeng; Liu, Zhiwei; Zhang, Hao; Dong, Hongyan; Zhang, Zhongming

    2016-12-12

    The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome has been linked to sterile inflammation, which is involved in ischemic injury in myocardial cells. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein with many biological activities, such as anti-inflammatory, antioxidant and anti-angiogenic properties. However, it is not known whether and how PEDF acts to regulate the activation of the NLRP3 inflammasome in cardiomyocytes. In the present study, we used the neonatal cardiomyocytes models of ischemia-like conditions to evaluate the mitochondrial fission and the activation of the NLRP3 inflammasome. We also determined the mechanism by which PEDF inhibits hypoxia-induced activation of the NLRP3 inflammasome. We found that PEDF decreased the activation of the NLRP3 inflammasome in neonatal cardiomyocytes through pigment epithelial-derived factor receptor/calcium-independent phospholipase A2 (PEDFR/iPLA2). Meanwhile, PEDF reduced Drp1-induced mitochondrial fission and mitochondrial fission-induced mitochondrial DNA (mtDNA), as well as mitochondrial reactive oxygen species (mtROS) release into cytosol through PEDFR/iPLA2. We also found that PEDF inhibited mitochondrial fission-induced NLRP3 inflammasome activation. Furthermore, previous research has found that endogenous cytosolic mtDNA and mtROS can serve as activators of NLRP3 inflammasome activity. Therefore, we hypothesized that PEDF can protect against hypoxia-induced activation of the NLRP3 inflammasome by inhibiting mitochondrial fission though PEDFR/iPLA2.

  10. Loss of HtrA2/Omi activity in non-neuronal tissues of adult mice causes premature aging.

    PubMed

    Kang, S; Louboutin, J-P; Datta, P; Landel, C P; Martinez, D; Zervos, A S; Strayer, D S; Fernandes-Alnemri, T; Alnemri, E S

    2013-02-01

    mnd2 mice die prematurely as a result of neurodegeneration 30-40 days after birth due to loss of the enzymatic activity of the mitochondrial quality control protease HtrA2/Omi. Here, we show that transgenic expression of human HtrA2/Omi in the central nervous system of mnd2 mice rescues them from neurodegeneration and prevents their premature death. Interestingly, adult transgenic mnd2 mice develop accelerated aging phenotypes, such as premature weight loss, hair loss, reduced fertility, curvature of the spine, heart enlargement, increased autophagy, and death by 12-17 months of age. These mice also have elevated levels of clonally expanded mitochondrial DNA (mtDNA) deletions in their tissues. Our results provide direct genetic evidence linking mitochondrial protein quality control to mtDNA deletions and aging in mammals.

  11. Pathophysiology of isoprostanes in the cardiovascular system: implications of isoprostane-mediated thromboxane A2 receptor activation.

    PubMed

    Bauer, Jochen; Ripperger, Anne; Frantz, Stefan; Ergün, Süleyman; Schwedhelm, Edzard; Benndorf, Ralf A

    2014-07-01

    Isoprostanes are free radical-catalysed PG-like products of unsaturated fatty acids, such as arachidonic acid, which are widely recognized as reliable markers of systemic lipid peroxidation and oxidative stress in vivo. Moreover, activation of enzymes, such as COX-2, may contribute to isoprostane formation. Indeed, formation of isoprostanes is considerably increased in various diseases which have been linked to oxidative stress, such as cardiovascular disease (CVD), and may predict the atherosclerotic burden and the risk of cardiovascular complications in the latter patients. In addition, several isoprostanes may directly contribute to the functional consequences of oxidant stress via activation of the TxA2 prostanoid receptor (TP), for example, by affecting endothelial cell function and regeneration, vascular tone, haemostasis and ischaemia/reperfusion injury. In this context, experimental and clinical data suggest that selected isoprostanes may represent important alternative activators of the TP receptor when endogenous TxA2 levels are low, for example, in aspirin-treated individuals with CVD. In this review, we will summarize the current understanding of isoprostane formation, biochemistry and (patho) physiology in the cardiovascular context.

  12. Structure-based activity prediction of CYP21A2 stability variants: A survey of available gene variations

    PubMed Central

    Bruque, Carlos D.; Delea, Marisol; Fernández, Cecilia S.; Orza, Juan V.; Taboas, Melisa; Buzzalino, Noemí; Espeche, Lucía D.; Solari, Andrea; Luccerini, Verónica; Alba, Liliana; Nadra, Alejandro D.; Dain, Liliana

    2016-01-01

    Congenital adrenal hyperplasia due to 21-hydroxylase deficiency accounts for 90–95% of CAH cases. In this work we performed an extensive survey of mutations and SNPs modifying the coding sequence of the CYP21A2 gene. Using bioinformatic tools and two plausible CYP21A2 structures as templates, we initially classified all known mutants (n = 343) according to their putative functional impacts, which were either reported in the literature or inferred from structural models. We then performed a detailed analysis on the subset of mutations believed to exclusively impact protein stability. For those mutants, the predicted stability was calculated and correlated with the variant’s expected activity. A high concordance was obtained when comparing our predictions with available in vitro residual activities and/or the patient’s phenotype. The predicted stability and derived activity of all reported mutations and SNPs lacking functional assays (n = 108) were assessed. As expected, most of the SNPs (52/76) showed no biological implications. Moreover, this approach was applied to evaluate the putative synergy that could emerge when two mutations occurred in cis. In addition, we propose a putative pathogenic effect of five novel mutations, p.L107Q, p.L122R, p.R132H, p.P335L and p.H466fs, found in 21-hydroxylase deficient patients of our cohort. PMID:27966633

  13. Structure-based activity prediction of CYP21A2 stability variants: A survey of available gene variations.

    PubMed

    Bruque, Carlos D; Delea, Marisol; Fernández, Cecilia S; Orza, Juan V; Taboas, Melisa; Buzzalino, Noemí; Espeche, Lucía D; Solari, Andrea; Luccerini, Verónica; Alba, Liliana; Nadra, Alejandro D; Dain, Liliana

    2016-12-14

    Congenital adrenal hyperplasia due to 21-hydroxylase deficiency accounts for 90-95% of CAH cases. In this work we performed an extensive survey of mutations and SNPs modifying the coding sequence of the CYP21A2 gene. Using bioinformatic tools and two plausible CYP21A2 structures as templates, we initially classified all known mutants (n = 343) according to their putative functional impacts, which were either reported in the literature or inferred from structural models. We then performed a detailed analysis on the subset of mutations believed to exclusively impact protein stability. For those mutants, the predicted stability was calculated and correlated with the variant's expected activity. A high concordance was obtained when comparing our predictions with available in vitro residual activities and/or the patient's phenotype. The predicted stability and derived activity of all reported mutations and SNPs lacking functional assays (n = 108) were assessed. As expected, most of the SNPs (52/76) showed no biological implications. Moreover, this approach was applied to evaluate the putative synergy that could emerge when two mutations occurred in cis. In addition, we propose a putative pathogenic effect of five novel mutations, p.L107Q, p.L122R, p.R132H, p.P335L and p.H466fs, found in 21-hydroxylase deficient patients of our cohort.

  14. Pulmonary toxicity of trichloroethylene: induction of changes in surfactant phospholipids and phospholipase A2 activity in the mouse lung.

    PubMed

    Scott, J E; Forkert, P G; Oulton, M; Rasmusson, M G; Temple, S; Fraser, M O; Whitefield, S

    1988-08-01

    Trichloroethylene (TCE) is a common organic solvent in use as a dry cleaning agent as well as an inhalant anesthetic. Nevertheless the effects of this material on the pulmonary surfactant which prevents alveolar collapse at maximal expiration is not known. Therefore, we have examined the effect of TCE on the intra- and extracellular surfactant pools and the activity of phospholipase A2, an enzyme which controls the remodeling of phosphatidylcholine to dipalmitoylphosphatidylcholine, the primary constituent of the pulmonary surfactant. Male CD-1 mice were treated ip with 2500 or 3000 mg/kg TCE. Twenty-four hours later mice were anesthetized and the lungs lavaged. Mice were then killed, the lungs perfused and excised, and subcellular fractions including lamellar bodies prepared. Some lungs were prepared for ultrastructural examination. Phospholipase A2 was assayed in all subcellular fractions. Phospholipid was assayed in the lavage (extracellular surfactant) and the lamellar bodies (intracellular surfactant). TCE (2500 mg/kg) caused selective exfoliation of Clara cells. However, only the dose of 3000 mg/kg TCE produced a significant decrease in the intracellular surfactant phospholipid. Minimal changes occurred in the phospholipid profiles. Phospholipase A2 specific activity was significantly decreased at both dosages within the lung microsomal fraction. In addition after treatment with 3000 mg/kg TCE the enzyme activity in the lamellar body fraction was significantly increased. These data suggest that inhalation of TCE may damage the enzymes which are responsible for synthesizing the pulmonary surfactant resulting in lower amounts of surfactant being stored and available for secretion into the alveolus.

  15. A1 and A2a receptors mediate inhibitory effects of adenosine on the motor activity of human colon.

    PubMed

    Fornai, M; Antonioli, L; Colucci, R; Ghisu, N; Buccianti, P; Marioni, A; Chiarugi, M; Tuccori, M; Blandizzi, C; Del Tacca, M

    2009-04-01

    Experimental evidence in animal models suggests that adenosine is involved in the regulation of digestive functions. This study examines the influence of adenosine on the contractile activity of human colon. Reverse transcription-polymerase chain reaction revealed A(1) and A(2a) receptor expression in colonic neuromuscular layers. Circular muscle preparations were connected to isotonic transducers to determine the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A(1) receptor antagonist), ZM 241385 (A(2a) receptor antagonist), CCPA (A(1) receptor agonist) and 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamide-adenosine (CGS 21680; A(2a) receptor agonist) on motor responses evoked by electrical stimulation or carbachol. Electrically evoked contractions were enhanced by DPCPX and ZM 241385, and reduced by CCPA and CGS 21680. Similar effects were observed when colonic preparations were incubated with guanethidine (noradrenergic blocker), L-732,138, GR-159897 and SB-218795 (NK receptor antagonists). However, in the presence of guanethidine, NK receptor antagonists and N(omega)-propyl-L-arginine (NPA; neuronal nitric oxide synthase inhibitor), the effects of DPCPX and CCPA were still evident, while those of ZM 241385 and CGS 21680 no longer occurred. Carbachol-induced contractions were unaffected by A(2a) receptor ligands, but they were enhanced or reduced by DPCPX and CCPA, respectively. When colonic preparations were incubated with guanethidine, NK antagonists and atropine, electrically induced relaxations were partly reduced by ZM 241385 or NPA, but unaffected by DPCPX. Dipyridamole or application of exogenous adenosine reduced electrically and carbachol-evoked contractions, whereas adenosine deaminase enhanced such motor responses. In conclusion, adenosine exerts an inhibitory control on human colonic motility. A(1) receptors mediate direct modulating actions on smooth muscle, whereas A(2a) receptors operate through inhibitory nitrergic nerve pathways.

  16. High plasma phospholipase A2 activity, inflammation markers, and LDL alterations in obesity with or without type 2 diabetes.

    PubMed

    Garces, Fatima; López, Flor; Niño, Cladimar; Fernandez, Anazita; Chacin, Luis; Hurt-Camejo, Eva; Camejo, Germán; Apitz-Castro, Rafael

    2010-10-01

    Plasma phospholipases A(2) (PLA(2)) hydrolyze phospholipids of circulating lipoproteins or deposited in arteries producing bioactive lipids believed to contribute to the atherosclerotic inflammatory response. PLA(2)(s) are elevated in obesity and type 2 diabetes (T2D) but it is not clear which of these conditions is the cause since they frequently coexist. This study attempts to evaluate if high plasma PLA(2)(s) activities and markers of their effects in lipoproteins are associated with obesity or T2D diabetes, or with both. Total PLA(2) and Ca(2+)-dependent and -independent activities, lipids, lipoproteins, apoAI, and apoB apolipoproteins and affinity of apoB-lipoproteins for arterial proteoglycans were measured, as well as Inflammation markers. These parameters were evaluated in plasma samples of four groups: (i) apparently healthy controls with normal BMI (nBMI), (ii) obese subjects with no T2D, (iii) patients with T2D but with nBMI, and (iv) obese patients with T2D. PLA(2) activities were measured in the presence and absence of Ca(2+) and in the presence of specific inhibitors. Obese subjects, with or without T2D, had high activities of total PLA(2) and of Ca(2+)-dependent and Ca(2+)-independent enzymes. The activities were correlated with inflammation markers in obese subjects with and without diabetes and with alterations of low-density lipoproteins (LDLs) that increased their affinity for arterial proteoglycans. Ca(2+)-dependent secretory (sPLA(2)) enzymes were the main responsible of the obesity-associated high activity. We speculate that augmented PLA(2)(s) activity that increases affinity of circulating LDL for arterial intima proteoglycans could be another atherogenic component of obesity.

  17. Genomics and the prospects of existing and emerging therapeutics for cardiovascular diseases.

    PubMed

    Zaiou, M; Benachour, H; Marteau, J B; Visvikis-Siest, S; Siest, G

    2009-01-01

    The growing knowledge about genetic influence on cardiovascular diseases (CVD) combined with the recently generated amounts of genomic data hold promise to the identification of new markers for atherosclerotic CVD. Cardiovascular pharmacogenomics and pharmacogenetics have now the potential for leading to identification of genetic contributors and therefore to the development of predictive genetic tests that could optimize drugs efficacy and minimize toxicity. Clinical studies have shown that genetic variations within cytochromes P450 (CYPs), 3-Hydroxyl-3-Methylglutaryl Coenzyme A Reductase (HMGCR) and apolipoprotein E (APOE) genes influence individual's response to lipid lowering statins. Furthermore, development of antagonists or inhibitors of molecules such as peroxisome proliferator-activated receptors (PPARs), lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), angiotensin-converting enzyme (ACE), angiotensin receptors and tumor necrosis factor (TNF)-alpha could be another alternative to prevent atherosclerosis. In addition, novel molecules under the name of biologics including family of peptides such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), urocortin, apelin and antimicrobial peptides (AMPs) could be considered as new targets for the prevention and treatment of CVD. In this article, we will focus mainly on recent genomic advances in the development of new markers and therapeutic agents for CVD. We present an array of molecules that could have pharmacological benefit for the treatment of heart disease. We also discuss in details new strategies including biologics, which are actually the focus of companies for clinical development of therapeutic drugs. All these efforts provide optimism and attractive promise to cure CVD.

  18. Activation of group IVC phospholipase A2 by polycyclic aromatic hydrocarbons induces apoptosis of human coronary artery endothelial cells

    PubMed Central

    Richards, Sean M.; Elgayyar, Mona A.; Menn, Fu-Minn; Vulava, Vijay M.; McKay, Larry; Sanseverino, John; Sayler, Gary; Tucker, Dawn E.; Leslie, Christina C.; Lu, Kim P.; Ramos, Kenneth S.

    2016-01-01

    Exposure to environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs) found in coal tar mixtures and tobacco sources, is considered a significant risk factor for the development of heart disease in humans. The goal of this study was to determine the influence of PAHs present at a Superfund site on human coronary artery endothelial cell (HCAEC) phospholipase A2 (PLA2) activity and apoptosis. Extremely high levels of 12 out of 15 EPA high-priority PAHs were present in both the streambed and floodplain sediments at a site where an urban creek and its adjacent floodplain were extensively contaminated by PAHs and other coal tar compounds. Nine of the 12 compounds and a coal tar mixture (SRM 1597A) activated group IVC PLA2 in HCAECs, and activation of this enzyme was associated with histone fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Genetic silencing of group IVC PLA2 inhibited both 3H-fatty acid release and histone fragmentation by PAHs and SRM 1597A, indicating that individual PAHs and a coal tar mixture induce apoptosis of HCAECs via a mechanism that involves group IVC PLA2. Western blot analysis of aortas isolated from feral mice (Peromyscus leucopus) inhabiting the Superfund site showed increased PARP and caspase-3 cleavage when compared to reference mice. These data suggest that PAHs induce apoptosis of HCAECs via activation of group IVC PLA2. PMID:21132278

  19. Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

    PubMed

    Furtado, Juliana L; Oliveira, George A; Pontes, Adriana S; Setúbal, Sulamita da S; Xavier, Caroline V; Lacouth-Silva, Fabianne; Lima, Beatriz F; Zaqueo, Kayena D; Kayano, Anderson M; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Zuliani, Juliana P

    2014-01-01

    In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

  20. Distribution of supplemental Escherichia coli AppA2 phytase activity in digesta of various gastrointestinal segments of young pigs.

    PubMed

    Pagano, A R; Roneker, K R; Lei, X G

    2007-06-01

    The objective of this study was to determine the functional location and disappearance of activity of a supplemental Escherichia coli AppA2 phytase and its impact on digesta P and Ca concentrations in the gastrointestinal tract of pigs. In Exp. 1, 18 pigs (8.3 +/- 0.2 kg of BW) were allotted to 3 groups (n = 6 each) and fed a low-P (0.4%) corn-soybean meal, basal diet (BD), BD + phytase [500 units (U)/kg of feed], or BD + inorganic P (iP, 0.1%) for 4 wk. In Exp. 2, 30 pigs (14.5 +/- 0.2 kg of BW) were allotted to 3 groups (n = 10 each) and fed BD, BD + 500 U of phytase/kg of feed, or BD + 2,000 U of phytase/kg of feed for 2 wk. Five or six pigs from each treatment group were killed at the end of both experiments to assay for digesta phytase activity and soluble P concentration in 6 segments of the digestive tract and digesta total P and Ca concentrations in stomach and colon. Compared with pigs fed BD, pigs fed BD + 500 U of phytase/kg of feed in Exp. 1 and BD + 2,000 U of phytase/kg of feed in Exp. 2 had greater (P < 0.05) phytase activities in the digesta of the stomach and upper jejunum (2 m aborally from the duodenum). No phytase activity was detected in the digesta of the lower jejunum (2.12 m cranial to the ileocecal junction) or ileum from any of the treatment groups in either trial. Concentrations of digesta-soluble P peaked in the upper jejunum of pigs fed BD in Exp. 1 and 2, but showed gradual decreases between the stomach and the upper jejunum of pigs fed BD + phytase or BD + iP. In both experiments, pigs fed only BD had greater (P < 0.05) colonic digesta phytase activity and soluble P concentrations than those fed phytase. In Exp. 2, total colonic digesta P or Ca concentrations, or both, of pigs displayed a phytase-dose-dependent reduction (P < 0.05). In conclusion, supplemental dietary AppA2 mainly functioned in the stomach and was associated with a reduced phytase activity in colonic digesta of weanling pigs.

  1. Inhibition of phospholipase A2 (PLA2) activity by nifedipine and nisoldipine is independent of their calcium-channel-blocking activity

    SciTech Connect

    Chang, J.; Blazek, E.; Carlson, R.P.

    1987-09-01

    The effects of several calcium antagonists on phospholipase A2 (PLA2) activity were examined. Nifedipine and nisoldipine inhibited a cell-free preparation of PLA2 in a dose-dependent manner with maximal inhibition of 71-77% observed at 100 microM. More potent or equipotent dihydropyridine calcium antagonists such as nitrendipine and felodipine did not inhibit PLA2 activity. In addition, nondihydropyridine calcium antagonists such as diltiazem, verapamil, and cinnarazine failed to reduce PLA2 activity markedly. Nifedipine and nisoldipine also reduced PLA2 activity in intact mouse peritoneal macrophages where PLA2 activity was monitored by free (/sup 14/C)arachidonic acid release from (/sup 14/C)arachidonic acid-prelabeled cells. When levels of PGE2 and LTC4 were measured by radioimmunoassay, it was found that the synthesis of these two metabolites was concomitantly inhibited by nifedipine and nisoldipine. In vivo, nifedipine and nisoldipine inhibited tetradecanoylphorbol acetate (TPA) induced ear edema. UV irradiation of nifedipine and nisoldipine (which destroys the slow calcium-channel-blocking activity of these compounds) did not result in a loss of PLA2 inhibitory activity. In fact, in both instances the UV-irradiated forms of nifedipine and nisoldipine were slightly more potent PLA2 inhibitors than the parent compound alone. We therefore conclude that the ability of nifedipine and nisoldipine to inhibit PLA2 was direct and unrelated to their actions on slow calcium channels.

  2. Activation of Presynaptic GABAB(1a,2) Receptors Inhibits Synaptic Transmission at Mammalian Inhibitory Cholinergic Olivocochlear–Hair Cell Synapses

    PubMed Central

    Wedemeyer, Carolina; Zorrilla de San Martín, Javier; Ballestero, Jimena; Gómez-Casati, María Eugenia; Torbidoni, Ana Vanesa; Fuchs, Paul A.; Bettler, Bernhard; Elgoyhen, Ana Belén

    2013-01-01

    The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca2+-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca2+-activated small-conductance type 2 (SK2)K+ channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABAB(1a,2) receptors [GABAB(1a,2)Rs] that downregulate the amount of ACh released at the OC–hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABABRs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABAB1–GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABAB1a isoform selectively inhibits release at efferent cholinergic synapses. PMID:24068816

  3. Activation of cytokine production by secreted phospholipase A2 in human lung macrophages expressing the M-type receptor.

    PubMed

    Granata, Francescopaolo; Petraroli, Angelica; Boilard, Eric; Bezzine, Sofiane; Bollinger, James; Del Vecchio, Luigi; Gelb, Michael H; Lambeau, Gerard; Marone, Gianni; Triggiani, Massimo

    2005-01-01

    Secreted phospholipases A(2) (sPLA(2)) are enzymes released in plasma and extracellular fluids during inflammatory diseases. Because human group IB and X sPLA(2)s are expressed in the lung, we examined their effects on primary human lung macrophages (HLM). Both sPLA(2)s induced TNF-alpha and IL-6 release in a concentration-dependent manner by increasing their mRNA expression. This effect was independent of their enzymatic activity because 1) the capacity of sPLA(2)s to mobilize arachidonic acid from HLM was unrelated to their ability to induce cytokine production; and 2) two catalytically inactive isoforms of group IB sPLA(2) (bromophenacyl bromide-inactivated human sPLA(2) and the H48Q mutant of the porcine sPLA(2)) were as effective as the catalytically active sPLA(2)s in inducing cytokine production. HLM expressed the M-type receptor for sPLA(2)s at both mRNA and protein levels, as determined by RT-PCR, immunoblotting, immunoprecipitation, and flow cytometry. Me-indoxam, which decreases sPLA(2) activity as well as binding to the M-type receptor, suppressed sPLA(2)-induced cytokine production. Incubation of HLM with the sPLA(2)s was associated with phosphorylation of ERK1/2, and a specific inhibitor of this pathway, PD98059, significantly reduced the production of IL-6 elicited by sPLA(2)s. In conclusion, two distinct sPLA(2)s produced in the human lung stimulate cytokine production by HLM via a mechanism that is independent of their enzymatic activity and involves activation of the ERK1/2 pathway. HLM express the M-type receptor, but its involvement in eliciting cytokine production deserves further investigation.

  4. Modulated mechanism of phosphatidylserine on the catalytic activity of Naja naja atra phospholipase A2 and Notechis scutatus scutatus notexin.

    PubMed

    Chiou, Yi-Ling; Lin, Shinne-Ren; Hu, Wan-Ping; Chang, Long-Sen

    2014-12-15

    Phosphatidylserine (PS) externalization is a hallmark for apoptotic death of cells. Previous studies showed that Naja naja atra phospholipase A2 (NnaPLA2) and Notechis scutatus scutatus notexin induced apoptosis of human cancer cells. However, NnaPLA2 and notexin did not markedly disrupt the integrity of cellular membrane as evidenced by membrane permeability of propidium iodide. These findings reflected that the ability of NnaPLA2 and notexin to hydrolyze membrane phospholipids may be affected by PS externalization. To address that question, this study investigated the membrane-interacted mode and catalytic activity of NnaPLA2 and notexin toward outer leaflet (phosphatidylcholine/sphingomyelin/cholesterol, PC/SM/Chol) and inner leaflet (phosphatidylserine/phosphatidylethanolamine/cholesterol, PS/PE/Chol) of plasma membrane-mimicking vesicles. PS incorporation promoted enzymatic activity of NnaPLA2 and notexin on PC and PC/SM vesicles, but suppressed NnaPLA2 and notexin activity on PC/SM/Chol and PE/Chol vesicles. PS incorporation increased the membrane fluidity of PC vesicles but reduced membrane fluidity of PC/SM, PC/SM/Chol and PE/Chol vesicles. PS increased the phospholipid order of all the tested vesicles. Moreover, PS incorporation did not greatly alter the binding affinity of notexin and NnaPLA2 with phospholipid vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that membrane-bound mode of notexin and NnaPLA2 varied with the targeted membrane compositions. The fine structure of catalytic site in NnaPLA2 and notexin in all the tested vesicles showed different changes. Collectively, the present data suggest that membrane-inserted PS modulates PLA2 interfacial activity via its effects on membrane structure and membrane-bound mode of NnaPLA2 and notexin, and membrane compositions determine the effect of PS on PLA2 activity.

  5. Activation of phospholipase A2 by temporin B: formation of antimicrobial peptide-enzyme amyloid-type cofibrils.

    PubMed

    Code, Christian; Domanov, Yegor A; Killian, J Antoinette; Kinnunen, Paavo K J

    2009-05-01

    Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate 1,2-dipalmitoyl-glycero-sn-3-phosphocholine (L-DPPC), exhibiting a lag-burst behaviour upon the initiation of the hydrolytic reaction by PLA2. Increasing concentrations of Cys-temporin B and its fluorescent Texas red derivative (TRC-temB) caused progressive shortening of the lag period. TRC-temB/PLA2D interaction was observed by Förster resonance energy transfer (FRET), with maximum efficiency coinciding with the burst in hydrolysis. Subsequently, supramolecular structures became visible by microscopy, revealing amyloid-like fibrils composed of both the activating peptide and PLA2. Reaction products, palmitic acid and 1-palmitoyl-2-lyso-glycero-sn-3-phosphocholine (lysoPC, both at >8 mol%) were required for FRET when using the non-hydrolysable substrate enantiomer 2,3-dipalmitoyl-glycero-sn-1-phosphocholine (D-DPPC). A novel mechanism of PLA2 activation by co-fibril formation and associated conformational changes is suggested.

  6. Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: The N-terminal region is more important than enzymatic activity

    PubMed Central

    Rouault, Morgane; Rash, Lachlan D.; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H.; Lambeau, Gérard

    2009-01-01

    Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, an homologous but non toxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1–22) of OS2, but not the central one (residues 58–89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102–119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity. PMID:16669624

  7. Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity.

    PubMed

    Rouault, Morgane; Rash, Lachlan D; Escoubas, Pierre; Boilard, Eric; Bollinger, James; Lomonte, Bruno; Maurin, Thomas; Guillaume, Carole; Canaan, Stéphane; Deregnaucourt, Christiane; Schrével, Joseph; Doglio, Alain; Gutiérrez, José María; Lazdunski, Michel; Gelb, Michael H; Lambeau, Gérard

    2006-05-09

    Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, a homologous but nontoxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1-22) of OS2, but not the central one (residues 58-89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102-119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera, which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity.

  8. Impact of Assay conditions on activity estimate and kinetics comparison of Aspergillus niger PhyA and Escherichia coli AppA2 phytases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was to compare three phytase activity assays and kinetics of Aspergillus niger PhyA and Escherichia coli AppA2 phytases expressed in Pichia pastoris at the observed stomach pH of 3.5. In Experiment 1, equivalent phytase activities in the crude preparations of PhyA and AppA2 were tested ...

  9. In-flight experiments on active TS-wave control on a 2D-laminar wing glove

    NASA Astrophysics Data System (ADS)

    Peltzer, Inken; Wicke, Kai; Pätzold, Andreas; Nitsche, Wolfgang

    In-flight measurements to delay laminar-turbulent transition by means of active Tollmien-Schlichting (TS) wave cancellation were carried out on a 2Dlaminar wing glove for a sailplane. The sensor-actuator system attached to the wing glove consisted of an array of surface hot-wire reference sensors to detect oncoming TS-waves upstream of a membrane actuator and surface hot-wire error sensors downstream of the actuator. The method applied was based on the dampening of naturally occurring instabilities through superimposition of a counter wave, which was calculated by a fast digital signal processor (DSP), using a closed loop feed-forward control algorithm. The flight experiments validated this system under varying atmospheric conditions successfully. Further attention was directed to the dampening of instabilities in the span-wise direction.

  10. Activation of J77A.1 Macrophages by Three Phospholipases A2 Isolated from Bothrops atrox Snake Venom

    PubMed Central

    Furtado, Juliana L.; Oliveira, George A.; Pontes, Adriana S.; Setúbal, Sulamita da S.; Xavier, Caroline V.; Lacouth-Silva, Fabianne; Lima, Beatriz F.; Zaqueo, Kayena D.; Kayano, Anderson M.; Calderon, Leonardo A.; Stábeli, Rodrigo G.; Soares, Andreimar M.; Zuliani, Juliana P.

    2014-01-01

    In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects. PMID:24592395

  11. Platelet-activating factor stimulates metabolism of phosphoinositides via phospholipase A2 in primary cultured rat hepatocytes

    SciTech Connect

    Okayasu, T.; Hasegawa, K.; Ishibashi, T.

    1987-07-01

    Addition of platelet-activating factor (PAF) to cells doubly labeled with (/sup 14/C)glycerol plus (/sup 3/H)arachidonic acid resulted in a transient decrease of (/sup 14/C)glycerol-labeled phosphatidylinositol (PI) and a transient increase of (/sup 14/C)glycerol-labeled lysophosphatidylinositol (LPI). (/sup 3/H)Arachidonate-labeled PI, on the other hand, decreased in a time-dependent manner. The radioactivity in phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and phosphatidylserine did not change significantly. The /sup 3/H//sup 14/C ratio decreased in PI in a time-dependent manner, suggesting the involvement of a phospholipase A2 activity. Although PAF also induced a gradual increase of diacylglycerol (DG), the increase of (/sup 14/C)glycerol-labeled DG paralleled the loss of triacyl (/sup 14/C)glycerol and the /sup 3/H//sup 14/C ratio of DG was 16 times smaller than that of PI. Thus, DG seemed not to be derived from PI. In myo- (/sup 3/H)inositol-prelabeled cells, PAF induced a transient decrease of (/sup 3/H)phosphatidylinositol-4,5-bis-phosphate (TPI) and (/sup 3/H)phosphatidylinositol-4-phosphate (DPI) at 1 min. PAF stimulation of cultured hepatocytes prelabeled with /sup 32/Pi induced a transient decrease of (/sup 32/P)polyphosphoinositides at 20 sec to 1 min. (/sup 32/P)LPI appeared within 10 sec after stimulation and paralleled the loss of (/sup 32/P)PI. (/sup 3/H)Inositol triphosphate, (/sup 3/H)inositol diphosphate, and (/sup 3/H)inositol phosphate, which increased in a time-dependent manner upon stimulation with adrenaline, did not accumulate with the stimulation due to PAF. These observations indicate that PAF causes degradation of inositol phospholipids via phospholipase A2 and induces a subsequent resynthesis of these phospholipids.

  12. Low Molecular Weight Hyaluronan Activates Cytosolic Phospholipase A2α and Eicosanoid Production in Monocytes and Macrophages* ♦

    PubMed Central

    Sokolowska, Milena; Chen, Li-Yuan; Eberlein, Michael; Martinez-Anton, Asuncion; Liu, Yueqin; Alsaaty, Sara; Qi, Hai-Yan; Logun, Carolea; Horton, Maureen; Shelhamer, James H.

    2014-01-01

    Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A2 group IVA (cPLA2α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA2α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA2α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E2 production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA2α inhibitor blocked HA-induced AA release and PGE2 production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA2α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE2 production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE2 production and COX2 expression were blocked in Tlr4−/− and Myd88−/− mice, but not in Cd44−/− mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA2α/COX2high and COX1/ALOX15/ALOX5/LTA4Hlow gene and PGE2/PGD2/15-HETEhigh and LXA4low eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism. PMID:24366870

  13. Phospholipase A2 activity-dependent and -independent fusogenic activity of Naja nigricollis CMS-9 on zwitterionic and anionic phospholipid vesicles.

    PubMed

    Chiou, Yi-Ling; Chen, Ying-Jung; Lin, Shinne-Ren; Chang, Long-Sen

    2011-11-01

    CMS-9, a phospholipase A(2) (PLA(2)) from Naja nigricollis venom, induced the death of human breast cancer MCF-7 cells accompanied with the formation of cell clumps without clear boundaries between cells. Annexin V-FITC staining indicated that abundant phosphatidylserine appeared on the outer membrane of MCF-7 cell clumps, implying the possibility that CMS-9 may promote membrane fusion via anionic phospholipids. To validate this proposition, fusogenic activity of CMS-9 on vesicles composed of zwitterionic phospholipid alone or a combination of zwitterionic and anionic phospholipids was examined. Although CMS-9-induced fusion of zwitterionic phospholipid vesicles depended on PLA(2) activity, CMS-9-induced fusion of vesicles containing anionic phospholipids could occur without the involvement of PLA(2) activity. Membrane-damaging activity of CMS-9 was associated with its fusogenicity. Moreover, CMS-9 induced differently membrane leakage and membrane fusion of vesicles with different compositions. Membrane fluidity and binding capability with phospholipid vesicles were not related to the fusogenicity of CMS-9. However, membrane-bound conformation and mode of CMS-9 depended on phospholipid compositions. Collectively, our data suggest that PLA(2) activity-dependent and -independent fusogenicity of CMS-9 are closely related to its membrane-bound modes and targeted membrane compositions.

  14. Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

    PubMed Central

    West, Ewan; Osborne, Craig; Nolan, William; Bate, Clive

    2015-01-01

    Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) and the loss of synapses. Aggregation of the cellular prion protein (PrPC) by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI) anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2) and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage. PMID:26043272

  15. Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA.

    PubMed

    Duchez, Anne-Claire; Boudreau, Luc H; Naika, Gajendra S; Bollinger, James; Belleannée, Clémence; Cloutier, Nathalie; Laffont, Benoit; Mendoza-Villarroel, Raifish E; Lévesque, Tania; Rollet-Labelle, Emmanuelle; Rousseau, Matthieu; Allaeys, Isabelle; Tremblay, Jacques J; Poubelle, Patrice E; Lambeau, Gérard; Pouliot, Marc; Provost, Patrick; Soulet, Denis; Gelb, Michael H; Boilard, Eric

    2015-07-07

    Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

  16. Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

    PubMed

    Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

    2015-10-07

    Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

  17. Neutralizing properties of Musa paradisiaca L. (Musaceae) juice on phospholipase A2, myotoxic, hemorrhagic and lethal activities of crotalidae venoms.

    PubMed

    Borges, M H; Alves, D L F; Raslan, D S; Piló-Veloso, D; Rodrigues, V M; Homsi-Brandeburgo, M I; de Lima, M E

    2005-04-08

    The use of plants as medicine has been referred to since ancient peoples, perhaps as early as Neanderthal man. Plants are a source of many biologically active products and nowadays they are of great interest to the pharmaceutical industry. The study of how people of different culture use plants in particular ways has led to the discovery of important new medicines. In this work, we verify the possible activity of Musa paradisiaca L. (Musaceae) against the toxicity of snake venoms. Musa paradisiaca, an important source of food in the world, has also been reported to be popularly used as an anti-venom. Interaction of Musa paradisiaca extract (MsE) with snake venom proteins has been examined in this study. Phospholipase A2 (PLA2), myotoxic and hemorrhagic activities, including lethality in mice, induced by crotalidae venoms were significantly inhibited when different amounts of MsE were mixed with these venoms before assays. On the other hand, mice that received MsE and venoms without previous mixture or by separated routes were not protected against venom toxicity. Partial chemical characterization of MsE showed the presence of polyphenols and tannins and they are known to non-specifically inactivate proteins. We suggest that these compounds can be responsible for the in vitro inhibition of the toxic effects of snake venoms. In conclusion, according to our results, using mice as experimental model, MsE does not show protection against the toxic effects of snake venoms in vivo, but if was very effective when the experiments were done in vitro.

  18. Pyrimidinoceptor-mediated activation of phospholipase C and phospholipase A2 in RAW 264.7 macrophages.

    PubMed Central

    Lin, W. W.; Lee, Y. T.

    1996-01-01

    1. As well as the presence of P2Z purinoceptors previously found in macrophages, we identified pyrimidinoceptors in RAW 264.7 cells, which activate phospholipase C (PLC) and phospholipase A2 (PLA2). 2. The relative potency of agonists to stimulate inositol phosphate (IP) formation and arachidonic acid (AA) release was UTP = UDP > > ATP, ATP gamma S, 2MeSATP. For both signalling pathways, the EC50 values for UTP and UDP (3 microM) were significantly lower than that for ATP and all other analogues tested (> 100 microM). 3. UTP and UDP displayed no additivity in terms of IP formation and AA release at maximally effective concentrations. 4. UTP-, but not ATP-, evoked AA release was 60% inhibited by pertussis toxin (PTX), while stimulation of IP formation by both agonists was unaffected. Short-term treatment with phorbol 12-myristate 13-acetate (PMA) led to a dose-dependent inhibition of IP responses to UTP and UDP, but failed to affect the AA responses. Removal of extracellular Ca2+ inhibited the PI response to UTP, but abolished its AA response. 5. ATP-induction of these two transmembrane signal pathways was decreased in high Mg(2+)-containing medium but potentiated by the removal of extracellular Mg2+. 6. Suramin and reactive blue displayed equal potency to inhibit the IP responses of UTP and ATP. 7. Both UTP and UDP (0.1-100 microM) induced a sustained increase in [Ca2+]i which lasted for more than 10 min. 8. Taken together, these results indicate that in mouse RAW 264.7 macrophages, pyrimidinoceptors with specificity for UTP and UDP mediate the activation of PLC and cytosolic (c) PLA2. The activation of PLC is via a PTX-insensitive G protein, whereas that of cPLA2 is via a PTX-sensitive G protein-dependent pathway. The sustained Ca2+ influx caused by UTP contributes to the activation of cPLA2. RAW 264.7 cells also possess P2z purinoceptors which mediate ATP(4-)-induced PLC and PLA2 activation. Images Figure 3 PMID:8886407

  19. RmpA2, an activator of capsule biosynthesis in Klebsiella pneumoniae CG43, regulates K2 cps gene expression at the transcriptional level.

    PubMed

    Lai, Yi-Chyi; Peng, Hwei-Ling; Chang, Hwan-You

    2003-02-01

    The rmpA2 gene, which encodes an activator for capsular polysaccharide (CPS) synthesis, was isolated from a 200-kb virulence plasmid of Klebsiella pneumoniae CG43. Based on the sequence homology with LuxR at the carboxyl-terminal DNA-binding motif, we hypothesized that RmpA2 exerts its effect by activating the expression of cps genes that are responsible for CPS biosynthesis. Two luxAB transcriptional fusions, each containing a putative promoter region of the K. pneumoniae K2 cps genes, were constructed and were found to be activated in the presence of multicopy rmpA2. The activation is likely due to direct binding of RmpA2 to the cps gene promoter through its C-terminal DNA binding motif. Moreover, the loss of colony mucoidy in a K. pneumoniae strain deficient in RcsB, a regulator for cps gene expression, could be recovered by complementing the strain with a multicopy plasmid carrying rmpA2. The CPS production in Lon protease-deficient K. pneumoniae significantly increased, and the effect was accompanied by an increase of RmpA2 stability. The expression of the rmpA2 gene was negatively autoregulated and could be activated when the organism was grown in M9 minimal medium. An IS3 element located upstream of the rmpA2 was required for the full activation of the rmpA2 promoter. In summary, our results suggest that the enhancement of K2 CPS synthesis in K. pneumoniae CG43 by RmpA2 can be attributed to its transcriptional activation of K2 cps genes, and the expression level of rmpA2 is autoregulated and under the control of Lon protease.

  20. The existence of phospholipase A(2) activity in plant mitochondria and its activation by hyperosmotic stress in durum wheat (Triticum durum Desf.).

    PubMed

    Trono, Daniela; Soccio, Mario; Laus, Maura N; Pastore, Donato

    2013-02-01

    The activity of mitochondrial phospholipase A(2) (PLA(2)) was shown for the first time in plants. It was observed in etiolated seedlings from durum wheat, barley, tomato, spelt and green seedlings of maize, but not in potato and topinambur tubers and lentil etiolated seedlings. This result was achieved by a novel spectrophotometric assay based on the coupled PLA(2)/lipoxygenase reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine as substrate; the mitochondrial localisation was assessed by checking recovery of marker enzymes. Durum wheat mitochondrial PLA(2) (DWM-PLA(2)) showed maximal activity at pH 9.0 and 1mM Ca(2+), hyperbolic kinetics (K(m)=90±6μM, V(max)=29±1nmolmin(-1)mg(-1) of protein) and inhibition by methyl arachidonyl fluorophosphonate, 5-(4-benzyloxyphenyl)-4S-(7-phenylheptanoylamino)pentanoic acid and palmityl trifluoromethyl ketone. Reactive oxygen species had no effect on DWM-PLA(2), that instead was activated by about 50% and 95%, respectively, under salt (0.21M NaCl) and osmotic (0.42M mannitol) stress imposed during germination. Contrarily, a secondary Ca(2+)-independent activity, having optimum at pH 7.0, was stress-insensitive. We propose that the activation of DWM-PLA(2) is responsible for the strong increase of free fatty acids recently measured in mitochondria under the same stress conditions [Laus, et al., J. Exp. Bot. 62 (2011) 141-154] that, in turn, activate potassium channel and uncoupling protein, able to counteract hyperosmotic stress.

  1. A pharmacometric approach to investigate the impact of methylxanthine abstinence and caffeine consumption on CYP1A2 activity.

    PubMed

    Perera, Vidya; Gross, Annette S; Forrest, Alan; Landersdorfer, Cornelia B; Xu, Hongmei; Ait-Oudhia, Sihem; McLachlan, Andrew J

    2013-11-01

    This study aimed to investigate the impact of methylxanthine abstinence (MA) periods on CYP1A2 activity in individuals with varying levels of caffeine consumption through development of a population pharmacokinetic model of caffeine and its major metabolite paraxanthine. This study developed and evaluated a mixed-effects pharmacokinetic model for caffeine and paraxanthine concentration-time data derived from a sequential single-dose cross-over study in healthy male volunteers (n = 30) who received oral 100 mg caffeine doses. Participants received caffeine with and without a MA period. Participants were classified as low (0-100 mg/d), medium (100-200 mg/d), or high (>200 mg/d) caffeine consumers (LCCs, MCCs, or HCCs, respectively). All caffeine and paraxanthine concentration-time data were simultaneously modeled. Caffeine pharmacokinetics was described by a two-compartment model with first-order absorption and two first-order elimination pathways. Paraxanthine was described by a one-compartment model with first-order absorption and elimination. Among LCCs (n = 16) and MCCs (n = 9), there was no difference in the mean (95% confidence interval) total apparent caffeine clearance (CL) between the MA period [LCCs: 6.88 (5.61-8.16 l/h); MCCs: 10.09 (7.57-12.60 l/h)] versus the no MA period [LCCs: 6.22 (4.97-7.46 l/h); MCCs: 9.68 (7.12-12.24 l/h)]. The mean CL among HCCs (n = 5) was considerably higher in the MA period [10.48 (5.62-15.33 l/h)] compared with the no MA period [6.30 (3.40-9.20 l/h)] (P < 0.05). The decrease in CL in the no MA period among HCC appears to be due to alternative caffeine elimination pathways, rather than CYP1A2.

  2. Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. III. Activation of phospholipase A2 in sperm homogenate by delta 9-tetrahydrocannabinol.

    PubMed

    Chang, M C; Berkery, D; Laychock, S G; Schuel, H

    1991-07-25

    Inhibition of the egg jelly induced acrosome reaction by delta 9-tetrahydrocannabinol (THC) is associated with the localized disruption of the nuclear envelope and the formation of lipid deposits in sea urchin sperm. This suggests that THC may activate phospholipase(s) within the sperm. We now report effects of THC on phospholipase A2 activity in homogenates of sea urchin sperm using 1-stearoyl-2-[1-14C]arachidonyl phosphatidylcholine as substrate. The release of radioactive arachidonic acid was measured after a 30-min incubation with the enzyme. In the absence of exogenous Ca2+, 100 microM THC produced a significant (P less than 0.001) increase in phospholipase A2 activity. THC activated phospholipase A2 in a concentration (1-100 microM) and time-dependent (0-30 min) manner. Exogenous calcium (10 mM) significantly augmented basal (P less than 0.001) and THC-stimulated (P less than 0.005) phospholipase A2 activity. Calcium chelators [ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)] inhibited the basal level of phospholipase A2 activity in the sperm homogenate, and prevented the activation of phospholipase A2 by THC. Submicromolar levels of free calcium ions were required for THC stimulation of phospholipase A2. Cannabinol which mimics the effects of THC on the acrosome reaction also activated phospholipase A2 in sperm homogenate. These results suggest that THC may alter lipid metabolism in sperm by activating calcium-dependent phospholipase A2. Putative metabolites derived from this process may inhibit the acrosome reaction and thereby reduce the fertilizing capacity of sea urchin sperm.

  3. Asp1 from Schizosaccharomyces pombe binds a [2Fe-2S](2+) cluster which inhibits inositol pyrophosphate 1-phosphatase activity.

    PubMed

    Wang, Huanchen; Nair, Vasudha S; Holland, Ashley A; Capolicchio, Samanta; Jessen, Henning J; Johnson, Michael K; Shears, Stephen B

    2015-10-27

    Iron-sulfur (Fe-S) clusters are widely distributed protein cofactors that are vital to cellular biochemistry and the maintenance of bioenergetic homeostasis, but to our knowledge, they have never been identified in any phosphatase. Here, we describe an iron-sulfur cluster in Asp1, a dual-function kinase/phosphatase that regulates cell morphogenesis in Schizosaccharomyces pombe. Full-length Asp1, and its phosphatase domain (Asp1(371-920)), were each heterologously expressed in Escherichia coli. The phosphatase activity is exquisitely specific: it hydrolyzes the 1-diphosphate from just two members of the inositol pyrophosphate (PP-InsP) signaling family, namely, 1-InsP7 and 1,5-InsP8. We demonstrate that Asp1 does not hydrolyze either InsP6, 2-InsP7, 3-InsP7, 4-InsP7, 5-InsP7, 6-InsP7, or 3,5-InsP8. We also recorded 1-phosphatase activity in a human homologue of Asp1, hPPIP5K1, which was heterologously expressed in Drosophila S3 cells with a biotinylated N-terminal tag, and then isolated from cell lysates with avidin beads. Purified, recombinant Asp1(371-920) contained iron and acid-labile sulfide, but the stoichiometry (0.8 atoms of each per protein molecule) indicates incomplete iron-sulfur cluster assembly. We reconstituted the Fe-S cluster in vitro under anaerobic conditions, which increased the stoichiometry to approximately 2 atoms of iron and acid-labile sulfide per Asp1 molecule. The presence of a [2Fe-2S](2+) cluster in Asp1(371-920) was demonstrated by UV-visible absorption, resonance Raman spectroscopy, and electron paramagnetic resonance spectroscopy. We determined that this [2Fe-2S](2+) cluster is unlikely to participate in redox chemistry, since it rapidly degraded upon reduction by dithionite. Biochemical and mutagenic studies demonstrated that the [2Fe-2S](2+) cluster substantially inhibits the phosphatase activity of Asp1, thereby increasing its net kinase activity.

  4. Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA

    PubMed Central

    Duchez, Anne-Claire; Boudreau, Luc H.; Naika, Gajendra S.; Bollinger, James; Belleannée, Clémence; Cloutier, Nathalie; Laffont, Benoit; Mendoza-Villarroel, Raifish E.; Lévesque, Tania; Rollet-Labelle, Emmanuelle; Rousseau, Matthieu; Allaeys, Isabelle; Tremblay, Jacques J.; Poubelle, Patrice E.; Lambeau, Gérard; Pouliot, Marc; Provost, Patrick; Soulet, Denis; Gelb, Michael H.; Boilard, Eric

    2015-01-01

    Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms. PMID:26106157

  5. Nanomolar vitamin E alpha-tocotrienol inhibits glutamate-induced activation of phospholipase A2 and causes neuroprotection.

    PubMed

    Khanna, Savita; Parinandi, Narasimham L; Kotha, Sainath R; Roy, Sashwati; Rink, Cameron; Bibus, Douglas; Sen, Chandan K

    2010-03-01

    Our previous works have elucidated that the 12-lipoxygenase pathway is directly implicated in glutamate-induced neural cell death, and that such that toxicity is prevented by nM concentrations of the natural vitamin E alpha-tocotrienol (TCT). In the current study we tested the hypothesis that phospholipase A(2) (PLA(2)) activity is sensitive to glutamate and mobilizes arachidonic acid (AA), a substrate for 12-lipoxygenase. Furthermore, we examined whether TCT regulates glutamate-inducible PLA(2) activity in neural cells. Glutamate challenge induced the release of [(3)H]AA from HT4 neural cells. Such response was attenuated by calcium chelators (EGTA and BAPTA), cytosolic PLA(2) (cPLA(2))-specific inhibitor (AACOCF(3)) as well as TCT at 250 nM. Glutamate also caused the elevation of free polyunsaturated fatty acid (AA and docosahexaenoic acid) levels and disappearance of phospholipid-esterified AA in neural cells. Furthermore, glutamate induced a time-dependent translocation and enhanced serine phosphorylation of cPLA(2) in the cells. These effects of glutamate on fatty acid levels and on cPLA(2) were significantly attenuated by nM TCT. The observations that AACOCF(3), transient knock-down of cPLA(2) as well as TCT significantly protected against the glutamate-induced death of neural cells implicate cPLA(2) as a TCT-sensitive mediator of glutamate induced neural cell death. This work presents first evidence recognizing glutamate-induced changes in cPLA(2) as a novel mechanism responsible for neuroprotection observed in response to nanomolar concentrations of TCT.

  6. Bee venom phospholipase A2 ameliorates motor dysfunction and modulates microglia activation in Parkinson's disease alpha-synuclein transgenic mice

    PubMed Central

    Ye, Minsook; Chung, Hwan-Suck; Lee, Chanju; Hyun Song, Joo; Shim, Insop; Kim, Youn-Sub; Bae, Hyunsu

    2016-01-01

    α-Synuclein (α-Syn) has a critical role in microglia-mediated neuroinflammation, which leads to the development of Parkinson's disease (PD). Recent studies have shown that bee venom (BV) has beneficial effects on PD symptoms in human patients or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin-induced PD mice. This study investigated whether treatment with BV-derived phospholipase A2 (bvPLA2) would improve the motor dysfunction and pathological features of PD in human A53T α-Syn mutant transgenic (A53T Tg) mice. The motor dysfunction of A53T Tg mice was assessed using the pole test. The levels of α-Syn, microglia and the M1/M2 phenotype in the spinal cord were evaluated by immunofluorescence. bvPLA2 treatment significantly ameliorated motor dysfunction in A53T Tg mice. In addition, bvPLA2 significantly reduced the expression of α-Syn, the activation and numbers of microglia, and the ratio of M1/M2 in A53T Tg mice. These results suggest that bvPLA2 could be a promising treatment option for PD. PMID:27388550

  7. Hyper- and Hypo- Induction of Cytochrome P450 activities with Aroclor 1254 and 3-Methylcholanthrene in Cyp1a2(−/−) mice

    PubMed Central

    Barker, Melissa L.; Hathaway, Laura B.; Arch, Dorinda D.; Westbroek, Mark L.; Kushner, James P.; Phillips, John D.; Franklin, Michael R.

    2009-01-01

    The response of hepatic mono-oxygenase activities to Aroclor 1254 or 3-methylcholanthrene was investigated in wild-type and Cyp1a2(−/−) mice. Cytochrome P450 concentrations were similar in naïve Cyp1a2(−/−) and wild-type mice. There was no difference between naïve wild-type and Cyp1a2(−/−) animals in 7-ethoxyresorufin and 7-ethoxy-4-trifluoromethylcoumarin dealkylase activities, nor was the induction response after 3-methylcholanthrene any different between the two genotypes. However, both activities were induced to a higher extent in Cyp1a2(−/−) mice after Aroclor 1254. In contrast, 7-pentoxyresorufin dealkylation activity was lower in Cyp1a2(−/−) mice and this differential was maintained during induction by both agents. 7-Methoxy- and 7-benzoxyresorufin dealkylation activities were also lower than wild-type in naïve Cyp1a2(−/−) animals and during 3-methylcholanthrene induction, but showed accelerated induction in Cyp1a2(−/−) mice with Aroclor 1254. Bufuralol 1′- and testosterone 6β-hydroxylation activities, and P450 characteristics were evaluated 48 hours after inducer administration. Bufuralol 1′-hydroxylation, a sexual dimorphic activity (female > male) showed no genotype differences in naïve animals. Activity changes varied across gender and genotype, with 3-methylcholanthrene and Aroclor 1254 inducing in male Cyp1a2(−/−), and Aroclor 1254 inducing in female wild-type. Testosterone 6β-hydroxylation activity was 16% higher in Cyp1a2(−/−) mice and neither 3-methylcholanthrene nor Aroclor 1254 elicited induction. After Aroclor 1254, a 24% increase in P450 concentration with a hypsochromic shift in the ferrous-CO maximum characteristic of CYP1A enzymes occurred in wild-type, compared to no change in either parameter in Cyp1a2(−/−) mice. Induction changes with 3-methylcholanthrene were greater in wild-type mice, a 60% increase in concentration and ~2 nm hypsochromic shift versus a 10% increase and ~1 nm hypsochromic

  8. Guanosine may increase absence epileptic activity by means of A2A adenosine receptors in Wistar Albino Glaxo Rijswijk rats.

    PubMed

    Lakatos, Renáta Krisztina; Dobolyi, Árpád; Todorov, Mihail Ivilinov; Kékesi, Katalin A; Juhász, Gábor; Aleksza, Magdolna; Kovács, Zsolt

    2016-06-01

    The non-adenosine nucleoside guanosine (Guo) was demonstrated to decrease quinolinic acid(QA)-induced seizures, spontaneously emerged absence epileptic seizures and lipopolysaccharide(LPS)-evoked induction of absence epileptic seizures suggesting its antiepileptic potential. It was also described previously that intraperitoneal (i.p.) injection of 20 and 50mg/kg Guo decreased the number of spike-wave discharges (SWDs) in a well investigated model of human absence epilepsy, the Wistar Albino Glaxo Rijswijk (WAG/Rij) rats during 4th (20mg/kg Guo) and 3rd as well as 4th (50mg/kg Guo) measuring hours. Guanosine can potentially decrease SWD number by means of its putative receptors but absence epileptic activity changing effects of Guo by means of increased extracellular adenosine (Ado) cannot be excluded. An increase in the dose of i.p. injected Guo is limited by its low solubility in saline, therefore, we addressed in the present study whether higher doses of Guo, diluted in sodium hydroxide (NaOH) solution, have more potent antiepileptic effect in WAG/Rij rats. We confirmed that i.p. 50mg/kg Guo decreased but, surprisingly, i.p. 100mg/kg Guo enhanced the number of SWDs in WAG/Rij rats. Combined i.p. injection of a non-selective Ado receptor antagonist theophylline (5mg/kg) or a selective Ado A2A receptor (A2AR) antagonist SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) (1mg/kg) and a cyclooxygenase 1 and 2/COX-1 and COX-2 inhibitor indomethacin (10mg/kg) with 100mg/kg Guo decreased the SWD number compared to i.p. 100mg/kg Guo alone. The results suggest that i.p. 100mg/kg Guo can increase SWD number by means of the adenosinergic system.

  9. The phzA2-G2 Transcript Exhibits Direct RsmA-Mediated Activation in Pseudomonas aeruginosa M18

    PubMed Central

    Ren, Bin; Shen, Huifeng; Lu, Zhi John; Liu, Haiming; Xu, Yuquan

    2014-01-01

    In bacteria, RNA-binding proteins of the RsmA/CsrA family act as post-transcriptional regulators that modulate translation initiation at target transcripts. The Pseudomonas aeruginosa genome contains two phenazine biosynthetic (phz) gene clusters, phzA1-G1 (phz1) and phzA2-G2 (phz2), each of which is responsible for phenazine-1-carboxylic acid (PCA) biosynthesis. In the present study, we show that RsmA exhibits differential gene regulation on two phz clusters in P. aeruginosa M18 at the post-transcriptional level. Based on the sequence analysis, four GGA motifs, the potential RsmA binding sites, are found on the 5′-untranslated region (UTR) of the phz2 transcript. Studies with a series of lacZ reporter fusions, and gel mobility shift assays suggest that the third GGA motif (S3), located 21 nucleotides upstream of the Shine-Dalgarno (SD) sequence, is involved in direct RsmA-mediated activation of phz2 expression. We therefore propose a novel model in which the binding of RsmA to the target S3 results in the destabilization of the stem-loop structure and the enhancement of ribosome access. This model could be fully supported by RNA structure prediction, free energy calculations, and nucleotide replacement studies. In contrast, various RsmA-mediated translation repression mechanisms have been identified in which RsmA binds near the SD sequence of target transcripts, thereby blocking ribosome access. Similarly, RsmA is shown to negatively regulate phz1 expression. Our new findings suggest that the differential regulation exerted by RsmA on the two phz clusters may confer an advantage to P. aeruginosa over other pseudomonads containing only a single phz cluster in their genomes. PMID:24586939

  10. An Anti-Parkinson’s Disease Drug via Targeting Adenosine A2A Receptor Enhances Amyloid-β Generation and γ-Secretase Activity

    PubMed Central

    Li, Xiaohang; Wang, Xin; Zhou, Yue; Yang, Wenjuan; Chen, Ming; Zhao, Jian; Pei, Gang

    2016-01-01

    γ-secretase mediates the intramembranous proteolysis of amyloid precursor protein (APP) and determines the generation of Aβ which is associated with Alzheimer’s disease (AD). Here we identified that an anti-Parkinson’s disease drug, Istradefylline, could enhance Aβ generation in various cell lines and primary neuronal cells of APP/PS1 mouse. Moreover, the increased generation of Aβ42 was detected in the cortex of APP/PS1 mouse after chronic treatment with Istradefylline. Istradefylline promoted the activity of γ-secretase which could lead to increased Aβ production. These effects of Istradefylline were reduced by the knockdown of A2AR but independent of A2AR-mediated G protein- or β-arrestin-dependent signal pathway. We further observed that A2AR colocalized with γ-secretase in endosomes and physically interacted with the catalytic subunit presenilin-1 (PS1). Interestingly, Istradefylline attenuated the interaction in time- and dosage-dependent manners. Moreover the knockdown of A2AR which in theory would release PS1 potentiated both Aβ generation and γ-secretase activity. Thus, our study implies that the association of A2AR could modulate γ-secretase activity. Istradefylline enhance Aβ generation and γ-secretase activity possibly via modulating the interaction between A2AR and γ-secretase, which may bring some undesired effects in the central nervous system (CNS). PMID:27835671

  11. Structure-activity relationships and mechanism of action of Eph-ephrin antagonists: interaction of cholanic acid with the EphA2 receptor

    PubMed Central

    Tognolini, Massimiliano; Incerti, Matteo; Mohamed, Iftiin Hassan; Giorgio, Carmine; Russo, Simonetta; Bruni, Renato; Lelli, Barbara; Bracci, Luisa; Noberini, Roberta; Pasquale, Elena B.; Barocelli, Elisabetta; Vicini, Paola; Mor, Marco

    2012-01-01

    The Eph–ephrin system, including the EphA2 receptor and the ephrin-A1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. We previously identified (3α,5β)-3-hydroxycholan-24-oic acid (lithocholic acid) as an Eph-ephrin antagonist able to inhibit EphA2 receptor activation and therefore potentially useful as a novel EphA2 receptor targeting agent. Here, we explore the structure-activity relationships of a focused set of lithocholic acid derivatives, based on molecular modelling investigation and displacement binding assays. Our exploration shows that while the 3-α-hydroxyl group of lithocholic acid has a negligible role in the recognition of the EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrin-A1 to the EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic acid (cholanic acid) as a novel compound that competitively inhibits EphA2-ephrin-A1 interaction with higher potency than lithocholic acid. Surface plasmon resonance analysis indicates that cholanic acid binds specifically and reversibly to the ligand-binding domain of EphA2, with a steady-state dissociation constant (KD) in the low micromolar range. Furthermore, cholanic acid blocks the phosphorylation of EphA2 and cell retraction and rounding in PC3 prostate cancer cells, two effects that depend on EphA2 activation by the ephrin-A1 ligand. These findings suggest that cholanic acid can be used as a template structure to design effective EphA2 antagonists, with potential impact in the elucidation of the role played by this receptor in pathological conditions. PMID:22529030

  12. Clonal Evolution and Blast Crisis Correlate with Enhanced Proteolytic Activity of Separase in BCR-ABL b3a2 Fusion Type CML under Imatinib Therapy.

    PubMed

    Haaß, Wiltrud; Kleiner, Helga; Weiß, Christel; Haferlach, Claudia; Schlegelberger, Brigitte; Müller, Martin C; Hehlmann, Rüdiger; Hofmann, Wolf-Karsten; Fabarius, Alice; Seifarth, Wolfgang

    2015-01-01

    Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors

  13. Clonal Evolution and Blast Crisis Correlate with Enhanced Proteolytic Activity of Separase in BCR-ABL b3a2 Fusion Type CML under Imatinib Therapy

    PubMed Central

    Haaß, Wiltrud; Kleiner, Helga; Weiß, Christel; Haferlach, Claudia; Schlegelberger, Brigitte; Müller, Martin C.; Hehlmann, Rüdiger; Hofmann, Wolf-Karsten; Fabarius, Alice; Seifarth, Wolfgang

    2015-01-01

    Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors

  14. Echinacea purpurea up-regulates CYP1A2, CYP3A4 and MDR1 gene expression by activation of pregnane X receptor pathway

    PubMed Central

    Awortwe, Charles; Manda, Vamshi K.; Avonto, Cristina; Khan, Shabana I.; Khan, Ikhlas A.; Walker, Larry A.; Bouic, Patrick J.; Rosenkranz, Bernd

    2015-01-01

    This study investigated the mechanism underlying Echinacea-mediated induction of CYP1A2, CYP3A4 and MDR1 in terms of human pregnane X receptor (PXR) activation. Crude extracts and fractions of Echinacea purpurea were tested for PXR activation in HepG2 cells by a reporter gene assay. Quantitative real-time PCR was carried out to determine their effects on CYP1A2 and CYP3A4 mRNA expressions. Capsules and fractions were risk ranked as high, intermediate and remote risk of drug-metabolizing enzymes induction based on EC50 values determined for respective CYPs. Fractions F1, F2 and capsule (2660) strongly activated PXR with 5-, 4- and 3.5-fold increase in activity, respectively. Echinacea preparations potentiated up-regulation of CYP1A2, CYP3A4 and MDR1 via PXR activation. Thus E. purpurea preparations cause herb–drug interaction by up-regulating CYP1A2, CYP3A4 and P-gp via PXR activation. PMID:25377539

  15. Fun with Foodella: A Pilot Study for Determining the Efficacy of a 2nd Grade Nutrition and Physical Activity Curriculum

    ERIC Educational Resources Information Center

    Winter, Elizabeth M.; Stluka, Suzanne; Wells, Karlys; Wey, Howard; Kemmer, Teresa M.

    2012-01-01

    Fun with Foodella is a nutrition and physical activity workbook designed for elementary-aged youth. The objective was to determine if the Fun with Foodella program increased participant preference for fruit, vegetables, low-fat dairy products, and physical activity. Four intervention (53 students) and four control (68 students) schools…

  16. Impact of assay conditions on activity estimate and kinetics comparison of Aspergillus niger PhyA and Escherichia coli AppA2 phytases.

    PubMed

    Weaver, Jeremy D; Ullah, Abul H J; Sethumadhavan, Kandan; Mullaney, Edward J; Lei, Xin Gen

    2009-06-24

    Aspergillus niger PhyA and Escherichia coli AppA2 are increasingly used in animal feed for phosphorus nutrition and environmental protection. The objective of this study was to determine the impacts of assay conditions on activity estimates of these two phytases and to compare their biochemical characteristics at a pH similar to the stomach environment. The activities of the unpurified AppA2 were more variable than those of PhyA with three commonly used phytase activity assays. The variations associated with AppA2 were accounted for by buffer, pH, and the inclusion of Triton X-100 and BSA by approximately one-third each. At the commonly observed stomach pH of 3.5, the purified AppA2 had a lower affinity to phytate (a higher K(m)), but greater V(max), k(cat), and k(cat)/K(m) than those of PhyA. In summary, differences between AppA2 and PhyA in responses to activity assay conditions and in inherent kinetic properties should be considered in interpreting their feeding efficacy.

  17. The first metagenome of activated sludge from full-scale anaerobic/anoxic/oxic (A2O) nitrogen and phosphorus removal reactor using Illumina sequencing.

    PubMed

    Tian, Mei; Zhao, Fangqing; Shen, Xin; Chu, Kahou; Wang, Jinfeng; Chen, Shuai; Guo, Yan; Liu, Hanhu

    2015-09-01

    The anaerobic/anoxic/oxic (A2O) process is globally one of the widely used biological sewage treatment processes. This is the first report of a metagenomic analysis using Illumina sequencing of full-scale A2O sludge from a municipal sewage treatment plant. With more than 530,000 clean reads from different taxa and metabolic categories, the metagenome results allow us to gain insight into the functioning of the biological community of the A2O sludge. There are 51 phyla and nearly 900 genera identified from the A2O activated sludge ecosystem. Proteobacteria, Bacteroidetes, Nitrospirae and Chloroflexi are predominant phyla in the activated sludge, suggesting that these organisms play key roles in the biodegradation processes in the A2O sewage treatment system. Nitrospira, Thauera, Dechloromonas and Ignavibacterium, which have abilities to metabolize nitrogen and aromatic compounds, are most prevalent genera. The percent of nitrogen and phosphorus metabolism in the A2O sludge is 2.72% and 1.48%, respectively. In the current A2O sludge, the proportion of Candidatus Accumulibacter is 1.37%, which is several times more than that reported in a recent study of A2O sludge. Among the four processes of nitrogen metabolism, denitrification related genes had the highest number of sequences (76.74%), followed by ammonification (15.77%), nitrogen fixation (3.88%) and nitrification (3.61%). In phylum Planctomycetes, four genera (Planctomyces, Pirellula, Gemmata and Singulisphaera) are included in the top 30 abundant genera, suggesting the key role of ANAMMOX in nitrogen metabolism in the A2O sludge.

  18. The transcription factors MS188 and AMS form a complex to activate the expression of CYP703A2 for sporopollenin biosynthesis in Arabidopsis thaliana.

    PubMed

    Xiong, Shuang-Xi; Lu, Jie-Yang; Lou, Yue; Teng, Xiao-Dong; Gu, Jing-Nan; Zhang, Cheng; Shi, Qiang-Sheng; Yang, Zhong-Nan; Zhu, Jun

    2016-12-01

    The sexine layer of pollen grain is mainly composed of sporopollenins. The sporophytic secretory tapetum is required for the biosynthesis of sporopollenin. Although several enzymes involved in sporopollenin biosynthesis have been reported, the regulatory mechanism of these enzymes in tapetal layer remains elusive. ABORTED MICROSPORES (AMS) and MALE STERILE 188/MYB103/MYB80 (MS188/MYB103/MYB80) are two tapetal cell-specific transcription factors required for pollen wall formation. AMS functions upstream of MS188. Here we report that AMS and MS188 target the CYP703A2 gene, which is involved in sporopollenin biosynthesis. We found that AMS and MS188 were localized in tapetum while CYP703A2 was localized in both tapetum and locule. Chromatin immunoprecipitation (ChIP) showed that MS188 directly bound to the promoter of CYP703A2 and luciferase-inducible assay showed that MS188 activated the expression of CYP703A2. Yeast two-hybrid and electrophoretic mobility shift assays (EMSAs) further demonstrated that MS188 complexed with AMS. The expression of CYP703A2 could be partially restored by the elevated levels of MS188 in the ams mutant. Therefore, our data reveal that MS188 coordinates with AMS to activate CYP703A2 in sporopollenin biosynthesis of plant tapetum.

  19. On the anti-inflammatory and anti-phospholipase A(2) activity of extracts from lanostane-rich species.

    PubMed

    Giner-Larza, E M; Máñez, S; Giner-Pons, R M; Carmen Recio, M; Ríos, J L

    2000-11-01

    We have studied extracts from three species rich in lanostane triterpenes for their activity against different in vivo models of inflammation induced by TPA, EPP and PLA(2). The inhibitory effect against PLA(2) in vitro was also studied. When the Poria cocos extract was tested against PLA(2)-induced mouse paw edema, it was active by the oral and parenteral routes. Its effect was greater in both magnitude and duration than that of Pistacia terebinthus and Ganoderma lucidum extracts. P. terebinthus was effective against chronic and acute inflammation, and according to a preliminary chromatographic analysis, its seems to be a good source of lanostane anti-inflammatory agents. G. lucidum was the least effective of the three species studied and, unlike the other two, failed to inhibit the activity of PLA(2) in vitro.

  20. Involvement of cAMP-PKA pathway in adenosine A1 and A2A receptor-mediated regulation of acetaldehyde-induced activation of HSCs.

    PubMed

    Yang, Yaru; Wang, He; Lv, Xiongwen; Wang, Qi; Zhao, Han; Yang, Feng; Yang, Yan; Li, Jun

    2015-08-01

    The present study was undertaken to investigate the mechanism by which adenosine receptors (ARs)-mediated the cAMP/PKA/CREB signal pathway regulates the activation of acetaldehyde-induced hepatic stellate cells (HSCs). Primary HSCs were isolated from SD rats, cultured in vitro, and activated with different concentrations of acetaldehyde at different time points. Quantitative real-time PCR and Western blotting were used to quantify both protein and mRNA levels of the four AR (A1R, A2AR, A2BR, and A3R) in rat HSCs. Selective inhibitors of PDEs and the Gi/o protein pathway, general AR agonists, and AR subtype specific agents were used to study the AR signaling. The level of cAMP was measured by radio-immunoassay, and the expression of α-SMA, collagen type I and III, PKA and p-CREB were also detected by Western blotting. Acetaldehyde could significantly promote HSC proliferation, with a maximum stimulatory effect observed at 48 h after exposure to 200 μM acetaldehyde. All four AR subtypes could be present in rat HSCs, and the mRNA and protein expression levels for A2AR and A1R in much greater abundance than those for A2BR and A3R. The expression of A2AR and A1R was significantly increased in acetaldehyde-induced HSCs as compared with that of control group, whereas the expression of A2BR and A3R remained unaffected by the addition of acetaldehyde. Curiously, there is coupling of A2AR to the Gs-AC signaling, as well as coupling of A1R to the Gi/o-AC signaling pathway in acetaldehyde-induced HSCs. Both the A2AR and A1R antagonists could suppress the activation of HSC, although they have opposing effects on cAMP signal transduction. These results suggested that a combination of cAMP/PKA/CREB signals via A2AR and A1R likely mediate the activation of acetaldehyde-induced HSCs, and A1R coupled to the Gi/o-AC signaling pathway may be masked by the more predominant A2AR that coupled to the Gs-AC signaling pathway.

  1. Limited Effects of a 2-Year School-Based Physical Activity Intervention on Body Composition and Cardiorespiratory Fitness in 7-Year-Old Children

    ERIC Educational Resources Information Center

    Magnusson, Kristjan Thor; Hrafnkelsson, Hannes; Sigurgeirsson, Ingvar; Johannsson, Erlingur; Sveinsson, Thorarinn

    2012-01-01

    The aim of this study was to assess the effects of a 2-year cluster-randomized physical activity and dietary intervention program among 7-year-old (at baseline) elementary school participants on body composition and objectively measured cardiorespiratory fitness. Three pairs of schools were selected and matched, then randomly selected as either an…

  2. HPLC Determination of Caffeine and Paraxanthine in Urine: An Assay for Cytochrome P450 1A2 Activity

    ERIC Educational Resources Information Center

    Furge, Laura Lowe; Fletke, Kyle J.

    2007-01-01

    Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. However, the activity of the different cytochrome P450 enzymes varies among…

  3. Three-dimensional solution structure of bottromycin A2: a potent antibiotic active against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci.

    PubMed

    Gouda, Hiroaki; Kobayashi, Yutaka; Yamada, Takeshi; Ideguchi, Tetsuya; Sugawara, Akihiro; Hirose, Tomoyasu; Omura, Satoshi; Sunazuka, Toshiaki; Hirono, Shuichi

    2012-01-01

    The three-dimensional (3D) structure of bottromycin A(2), a natural anti-methicillin-resistant Staphylococcus aureus (MRSA) and anti-vancomycin-resistant Enterococci (VRE) agent consisting of seven amino acids, has been investigated through NMR spectroscopy. On the basis of 57 experimental constraints, a total of 34 converged structures were obtained. The average pairwise atomic root mean square difference is 0.74±0.59 Å for all heavy atoms. The resulting structure indicates an interesting feature in that the three C-terminal residues of bottromycin A(2) fold back on the 12-membered cyclic skeleton made by the four N-terminal residues. Thus, MePro(2) and Thia-β-Ala-OMe(7), modification of which significantly affects the antibacterial activities of bottromycin A(2), are located on one side of its 3D structure. These distinct structural features might be important for the binding of bottromycin A(2) with the bacterial ribosome.

  4. Effects of tissue plasminogen activator and annexin A2 combination therapy on long-term neurological outcomes of rat focal embolic stroke

    PubMed Central

    Wang, Xiaoshu; Fan, Xiang; Yu, Zhanyang; Liao, Zhengbu; Zhao, Jianhua; Mandeville, Emiri; Guo, Shuzhen; Lo, Eng H.; Wang, Xiaoying

    2014-01-01

    Background and Purpose Tissue type plasminogen activator (tPA) in combination with recombinant annexin A2 (rA2) is known to reduce acute brain damage after focal ischemia. Here, we ask whether tPA plus rA2 combination therapy can lead to sustained long term neurological improvements as well. Methods We compared the effects of intravenous high-dose tPA alone (10mg/kg) versus a combination of low-dose tPA (5mg/kg) plus 10 mg/kg rA2 in a model of focal embolic cerebral ischemia in rats. All rats were treated at 3 hours after embolization. Brain tissue and neurological outcomes were assessed at 1 month. Surrogate biomarkers for endogenous neurovascular remodeling in peri-infarct area were analyzed by immunohistochemistry. Results Compared to high-dose tPA alone, low-dose tPA plus rA2 significantly decreased infarction and improved neurological function at 1 month post-stroke. In peri-infarct areas, tPA-plus-rA2 combination therapy also significantly augmented microvessel density, VEGF and synaptophysin expression. Conclusions Compared to conventional high-dose tPA alone, combination low-dose tPA plus rA2 therapy may provide a safe and effective way to improve long term neurological outcomes after stroke. PMID:24368559

  5. Synthesis, crystal structures and antitumor activities of copper(II) complexes with a 2-acetylpyrazine isonicotinoyl hydrazone ligand

    NASA Astrophysics Data System (ADS)

    Xu, Jun; Zhou, Tao; Xu, Zhou-Qing; Gu, Xin-Nan; Wu, Wei-Na; Chen, Hong; Wang, Yuan; Jia, Lei; Zhu, Tao-Feng; Chen, Ru-Hua

    2017-01-01

    Five complexes, [Cu(L)2]·4.5H2O (1), [Cu(HL)2](NO3)2·CH3OH (2) {[Cu2(L)2(NO3)(H2O)2]·(NO3)}n (3), [Cu2(HL)2(SO4)2]·2CH3OH (4) and [Cu4(L)4Cl4]·5H2O (5) based on HL (where HL = 2-acetylpyrazine isonicotinoyl hydrazone) have been synthesized and characterized by X-ray diffraction analyses. The counter anion and organic base during the synthesis procedure influence the structures of the complexes efficiently, which generate five complexes as mono-, bi-, tetra-nuclear and one-dimensional structures. The antitumor activities of the complexes 1-5 (except for complex 3 with the poor solubility) against the Patu8988 human pancreatic cancer, ECA109 human esophagus cancer and SGC7901 human gastric cancer cell lines are screened by MTT assay. The results indicate that the chelation of Cu(II) with the ligand is responsible for the observed high cytotoxicity of the copper(II) complexes and the 1:2 copper species 1 and 2 demonstrate lower antitumor activities than that of the 1:1 copper species 4 and 5. In addition, the in vitro apoptosis inducing activity of the copper(II) complex 5 against SGC7901 cell line is determined. And the results show that the complex can bring about apoptosis of the cancerous cells in vitro.

  6. Histamine H3 Receptor Activation Counteracts Adenosine A2A Receptor-Mediated Enhancement of Depolarization-Evoked [3H]-GABA Release from Rat Globus Pallidus Synaptosomes

    PubMed Central

    2014-01-01

    High levels of histamine H3 receptors (H3Rs) are found in the globus pallidus (GP), a neuronal nucleus in the basal ganglia involved in the control of motor behavior. By using rat GP isolated nerve terminals (synaptosomes), we studied whether H3R activation modified the previously reported enhancing action of adenosine A2A receptor (A2AR) stimulation on depolarization-evoked [3H]-GABA release. At 3 and 10 nM, the A2AR agonist CGS-21680 enhanced [3H]-GABA release induced by high K+ (20 mM) and the effect of 3 nM CGS-21680 was prevented by the A2AR antagonist ZM-241385 (100 nM). The presence of presynaptic H3Rs was confirmed by the specific binding of N-α-[methyl-3H]-histamine to membranes from GP synaptosomes (maximum binding, Bmax, 1327 ± 79 fmol/mg protein; dissociation constant, Kd, 0.74 nM), which was inhibited by the H3R ligands immepip, clobenpropit, and A-331440 (inhibition constants, Ki, 0.28, 8.53, and 316 nM, respectively). Perfusion of synaptosomes with the H3R agonist immepip (100 nM) had no effect on K+-evoked [3H]-GABA release, but inhibited the stimulatory action of A2AR activation. In turn, the effect of immepip was blocked by the H3R antagonist clobenpropit, which had no significant effect of its own on K+-induced [3H]-GABA release. These data indicate that H3R activation selectively counteracts the facilitatory action of A2AR stimulation on GABA release from striato-pallidal projections. PMID:24884070

  7. Mechanistic characterization of a 2-thioxanthine myeloperoxidase inhibitor and selectivity assessment utilizing click chemistry--activity-based protein profiling.

    PubMed

    Ward, Jessica; Spath, Samantha N; Pabst, Brandon; Carpino, Philip A; Ruggeri, Roger B; Xing, Gang; Speers, Anna E; Cravatt, Benjamin F; Ahn, Kay

    2013-12-23

    Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Despite a high level of interest in MPO as a therapeutic target, there have been limited reports about MPO inhibitors that are suitable for evaluating MPO in pharmacological studies. 2-Thioxanthine, 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (A), has recently been reported to inhibit MPO by covalently modifying the heme prosthetic group. Here we report a detailed mechanistic characterization demonstrating that A possesses all the distinguishing features of a mechanism-based inactivator. A is a time-dependent MPO inhibitor and displays saturable inactivation kinetics consistent with a two-step mechanism of inactivation and a potency (k(inact)/K(I) ratio) of 8450 ± 780 M⁻¹ s⁻¹. MPO inactivation by A is dependent on MPO catalysis and is protected by substrate. A reduces MPO compound I to compound II with a second-order rate constant of (0.801 ± 0.056) × 10⁶ M⁻¹ s⁻¹, and its irreversible inactivation of MPO occurs prior to release of the activated inhibitory species. Despite its relatively high selectivity against a broad panel of more than 100 individual targets, including enzymes, receptors, transporters, and ion channels, we demonstrate that A labels multiple other protein targets in the presence of MPO. By synthesizing an alkyne analogue of A and utilizing click chemistry-activity-based protein profiling, we present that the MPO-activated inhibitory species can diffuse away to covalently modify other proteins, as reflected by the relatively high partition ratio of A, which we determined to be 15.6. This study highlights critical methods that can guide the discovery and development of next-generation MPO inhibitors.

  8. Sequential injection system for phospholipase A2 activity evaluation: studies on liposomes using an environment-sensitive fluorescent probe.

    PubMed

    Araujo, André R T S; Gaspar, Diana; Lúcio, Marlene; Reis, Salette; Saraiva, M Lúcia M F S; Lima, José L F C

    2009-09-15

    This work reports the development of an automatic methodology based on the use of 1-anilinonaphthalene-8-sulfonate (ANS) as an interfacial fluorescent probe for detecting the hydrophobic environment shift around the probe, caused by the hydrolytic action of PLA(2) on the liposomes. The implementation of this reaction in a sequential injection analysis (SIA) system along with the use of the mixing chambers permitted the evaluation of PLA(2) activity and assessment of the inhibitory effect of the non-steroidal anti-inflammatory drugs (NSAIDs) on PLA(2) activity. Several studies were performed with the aim of establishing the appropriate flow system configuration: the liposome substrate; PLA(2) and ANS optimum concentrations and incubation times before and after the enzyme addition. Based on these studies, the optimum reaction conditions were selected. It was shown that PLA(2) is effectively inhibited by the NSAIDs tested (meloxicam, tolmetin and ibuprofen) and by the alpha-lipoic acid, used as a positive control. Results obtained from the flow system are in agreement with those provided by the comparison batch procedures. The proposed methodology is in fact more efficient and rapid than the comparison batch experiments, enabling the exact timing of fluidic manipulations and precise control of the reaction conditions.

  9. Hyperthermia-induced seizures alter adenosine A1 and A2A receptors and 5'-nucleotidase activity in rat cerebral cortex.

    PubMed

    León-Navarro, David Agustín; Albasanz, José L; Martín, Mairena

    2015-08-01

    Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia-induced seizures on adenosine A1 and A2A receptors and 5'-nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real-time PCR, and 5'-nucleotidase activity assays. Hyperthermic seizures were induced in 13-day-old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A1 receptor density was observed using [(3) H]DPCPX as radioligand, and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density was detected, using [(3) H]ZM241385 as radioligand, 48 h after hyperthermia-evoked convulsions. These short-term changes in A1 and A2A receptors were also accompanied by a loss of 5'-nucleotidase activity. No significant variations either in A1 or A2A receptor density or 5'-nucleotidase were observed 5 and 20 days after hyperthermic seizures. Taken together, both regulation of A1 and A2A receptors and loss of 5'-nucleotidase in the cerebral cortex suggest the existence of a neuroprotective mechanism against seizures. Febrile seizure is one of the most common convulsive disorders in children. The consequences of hyperthermia-induced seizures (animal model of febrile seizures) on adenosine A1 and A2A receptors and 5'-nucleotidase activity have been studied at different periods in cerebral cortical area. A significant increase in A1 receptor density and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density and 5'-nucleotidase activity was detected 48 h after convulsions evoked by hyperthermia

  10. Activation of phospholipase A2 by 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine in vitro.

    PubMed

    Code, Christian; Mahalka, Ajay K; Bry, Kristian; Kinnunen, Paavo K J

    2010-08-01

    Oxidative stress leads to drastic modifications of both the biophysical properties of biomembranes and their associated chemistry imparted upon the formation of oxidatively modified lipids. To this end, oxidized phospholipid derivatives bearing an aldehyde function, such as 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) can covalently react with proteins that come into direct contact. Intriguingly, we observed PoxnoPC in a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) matrix to shorten and abolish the lag time in the action of phospholipase A2 (PLA2) on this composite substrate, with concomitant augmented decrement in pH, indicating more extensive hydrolysis, which was in keeping with enhanced 90 degrees light scattering. The latter was abolished by the aldehyde scavenger methoxyamine, thus suggesting the involvement of Schiff base. Enhanced hydrolysis of a fluorescent phospholipid analogue was seen for PLA2 preincubated with PoxnoPC. Mixing PLA2 with submicellar (22 microM) PoxnoPC caused a pronounced increase in Thioflavin T fluorescence, in keeping with the formation of amyloid-type fibers, which were seen also by electron microscopy.

  11. Activation of the A2B adenosine receptor in B16 melanomas induces CXCL12 expression in FAP-positive tumor stromal cells, enhancing tumor progression

    PubMed Central

    Sorrentino, Claudia; Miele, Lucio; Porta, Amalia; Pinto, Aldo; Morello, Silvana

    2016-01-01

    The A2B receptor (A2BR) can mediate adenosine-induced tumor proliferation, immunosuppression and angiogenesis. Targeting the A2BR has proved to be therapeutically effective in some murine tumor models, but the mechanisms of these effects are still incompletely understood. Here, we report that pharmacologic inhibition of A2BR with PSB1115, which inhibits tumor growth, decreased the number of fibroblast activation protein (FAP)-expressing cells in tumors in a mouse model of melanoma. This effect was associated with reduced expression of fibroblast growth factor (FGF)-2. Treatment of melanoma-associated fibroblasts with the A2BR agonist Bay60-6583 enhanced CXCL12 and FGF2 expression. This effect was abrogated by PSB1115. The A2AR agonist CGS21680 did not induce CXCL12 or FGF2 expression in tumor associated fibroblasts. Similar results were obtained under hypoxic conditions in skin-derived fibroblasts, which responded to Bay60-6583 in an A2BR-dependent manner, by stimulating pERK1/2. FGF2 produced by Bay60-6583-treated fibroblasts directly enhanced the proliferation of melanoma cells. This effect could be reversed by PSB1115 or an anti-FGF2 antibody. Interestingly, melanoma growth in mice receiving Bay60-6583 was attenuated by inhibition of the CXCL12/CXCR4 pathway with AMD3100. CXCL12 and its receptor CXCR4 are involved in angiogenesis and immune-suppression. Treatment of mice with AMD3100 reduced the number of CD31+ cells induced by Bay60-6583. Conversely, CXCR4 blockade did not affect the accumulation of tumor-infiltrating MDSCs or Tregs. Together, our data reveal an important role for A2BR in stimulating FGF2 and CXCL12 expression in melanoma-associated fibroblasts. These factors contribute to create a tumor-promoting microenvironment. Our findings support the therapeutic potential of PSB1115 for melanoma. PMID:27590504

  12. The Human UDP-glucuronosyltransferase UGT2A1 and UGT2A2 enzymes are highly active in bile acid glucuronidation.

    PubMed

    Perreault, Martin; Gauthier-Landry, Louis; Trottier, Jocelyn; Verreault, Mélanie; Caron, Patrick; Finel, Moshe; Barbier, Olivier

    2013-09-01

    Bile acids (BA) are essential modulators of lipid, glucose, and cholesterol homeostasis, but they exert cytotoxic effects in the cholestatic liver. Glucuronidation, catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes is a pharmacologically relevant BA detoxification process. The present study characterized the BA-conjugating activity of the little-studied human UGTs of subfamily 2A: UGT2A1, 2A2, and 2A3. Recombinant UGT2As, expressed in baculovirus-infected insect cells, were assayed for the glucuronidation of six major bile acids: chenodeoxycholic acid (CDCA), cholic acid (CA), lithocholic acid (LCA), deoxycholic acid (DCA), hyocholic acid (HCA) and hyodeoxycholic acid (HDCA). UGT2A3 exhibited detectable but very low activity with all the tested BA substrates. UGT2A1 was highly efficient in forming LCA-3 and LCA-24G, CDCA-24, DCA-24, HCA-24, and HDCA-24G, whereas UGT2A2 was the most active enzyme for CA-24G and CDCA-24G formation and also was able to generate HDCA-6G, HDCA-24G, LCA-24G, and HCA-24G. The Km values of UGT2A1 varied between 102.2 ± 14.3 µM and 2.4 ± 1.2 mM. With the exception of CA-24G, a low affinity substrate for UGT2A2, all the Km values for UGT2A2 were in the 100 to 400 µM range. We demonstrate the high reactivity of the human UGT2A1 and UGT2A2 for bile acid glucuronidation. The physiologic importance of these reactions to BA disposition remains, however, to be clarified in vivo.

  13. The human UDP-glucuronosyltransferase UGT2A1 and UGT2A2 enzymes are highly active in bile acid glucuronidation

    PubMed Central

    Perreault, Martin; Gauthier-Landry, Louis; Trottier, Jocelyn; Verreault, Mélanie; Caron, Patrick; Finel, Moshe; Barbier, Olivier

    2013-01-01

    Bile acids (BA) are essential modulators of lipid, glucose and cholesterol homeostasis, but exert cytotoxic effects in the cholestatic liver. Glucuronidation, catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes is a pharmacologically-relevant BA detoxification process. The present study aimed at characterizing the BA-conjugating activity of the little-studied human UGTs of subfamily 2A, UGT2A1, 2A2 and 2A3. Recombinant UGT2As, expressed in baculovirus-infected insect cells, were assayed for the glucuronidation of 6 major bile acids, chenodeoxycholic (CDCA), cholic (CA), lithocholic (LCA), deoxycholic (DCA), hyocholic (HCA) and hyodeoxycholic (HDCA) acids. UGT2A3 exhibited detectable, but very low, activity with all the tested BAs substrates. UGT2A1 was highly efficient in forming LCA-3 and -24G, CDCA-24, DCA-24, HCA-24 and HDCA-24G, while UGT2A2 was the most active enzyme for CA-24G and CDCA-24G formation, and was also able to generate HDCA-6G, HDCA-24G, LCA-24G and HCA-24G. The Km values of UGT2A1 varied between 102.2 ± 14.3 μM and 2.4 ± 1.2 mM. With the exception of CA-24G, a low affinity substrate for UGT2A2, all the Km values for UGT2A2 were in the 100 to 400 μM range. In conclusion, the present study demonstrates the high reactivity of the human UGT2A1 and UGT2A2 for bile acid glucuronidation. The physiological importance of these reactions to BA disposition remains, however, to be clarified in vivo. PMID:23756265

  14. Ceria-Zirconia Particles Wrapped in a 2D Carbon Envelope: Improved Low-Temperature Oxygen Transfer and Oxidation Activity.

    PubMed

    Aneggi, Eleonora; Rico-Perez, Veronica; de Leitenburg, Carla; Maschio, Stefano; Soler, Lluís; Llorca, Jordi; Trovarelli, Alessandro

    2015-11-16

    Engineering the interface between different components of heterogeneous catalysts at nanometer level can radically alter their performances. This is particularly true for ceria-based catalysts where the interactions are critical for obtaining materials with enhanced properties. Here we show that mechanical contact achieved by high-energy milling of CeO2-ZrO2 powders and carbon soot results in the formation of a core of oxide particles wrapped in a thin carbon envelope. This 2D nanoscale carbon arrangement greatly increases the number and quality of contact points between the oxide and carbon. Consequently, the temperatures of activation and transfer of the oxygen in ceria are shifted to exceptionally low temperatures and the soot combustion rate is boosted. The study confirms the importance of the redox behavior of ceria-zirconia particles in the mechanism of soot oxidation and shows that the organization of contact points at the nanoscale can significantly modify the reactivity resulting in unexpected properties and functionalities.

  15. Inhibition of secretory phospholipase A2 activity attenuates acute cardiogenic pulmonary edema induced by isoproterenol infusion in mice after myocardial infarction.

    PubMed

    Kawabata, Kenichi; Fujioka, Daisuke; Kobayashi, Tsuyoshi; Saito, Yukio; Obata, Jun-Ei; Nakamura, Takamitsu; Yano, Toshiaki; Watanabe, Kazuhiro; Watanabe, Yosuke; Mishina, Hideto; Kugiyama, Kiyotaka

    2010-10-01

    Several types of secretory phospholipase A2 (sPLA2) are expressed in lung tissue, yielding various eicosanoids that might cause pulmonary edema. This study examined whether inhibition of sPLA2 activity attenuates acute cardiogenic pulmonary edema in mice. Acute cardiogenic pulmonary edema was induced in C57BL/6J male mice by an increase in heart rate with continuous intravenous infusion of isoproterenol (ISP) (10 mg/kg/h) at 2 weeks after the creation of myocardial infarction by left coronary artery ligation. Just before ISP infusion, a single intraperitoneal injection of 100 mg/kg LY374388, a prodrug of LY329722 that inhibits sPLA2 activity, or vehicle was administered. The ISP infusion after myocardial infarction induced interstitial and alveolar edema on lung histology. Furthermore, it increased the lung-to-body weight ratio, pulmonary vascular permeability evaluated by the Evans blue extravasation method, lung activity of sPLA2, and lung content of thromboxane A2 and leukotriene B4. These changes were significantly attenuated by LY374388 treatment. In Kaplan-Meier analysis, the survival rate during the ISP infusion after myocardial infarction was significantly higher in LY374388- than in vehicle-treated mice. Similar results were obtained with another inhibitor of sPLA2 activity, para-bromophenacyl bromide. In conclusion, inhibition of sPLA2 activity suppressed acute cardiogenic pulmonary edema.

  16. Predicting caries by measuring its activity using quantitative light-induced fluorescence in vivo: a 2-year caries increment analysis.

    PubMed

    Meller, C; Santamaria, R M; Connert, T; Splieth, C

    2012-01-01

    The aim of this study was to analyse the predictive power of several clinical baseline parameters and the de-/remineralisation properties of in vivo etched sites measured with quantitative light-induced fluorescence (QLF) for subsequent 2-year caries increment. At baseline, in 44 children (8.23 ± 1.5 years) two areas (diameter 2 mm) of the buccal surface of a primary posterior tooth were etched with 36% phosphoric acid gel for 1 and 4 min, respectively. The etched sites were analysed immediately after etching (ΔQ1) and 24 h (ΔQ2) later by QLF. Additionally, caries status (deft/DMFT and initial caries), approximal plaque, bleeding on probing, and the patient's current use of fluorides were recorded. In the 2-year follow-up, 29 children were re-assessed. After clinical examination, the caries increment was calculated (ΔDMFT) and correlated with the baseline clinical variables and the QLF readings. Results showed a significant positive correlation between ΔQ(1 min) and the ΔDMFT (r = 0.44, p = 0.02). The ΔDMFT was significantly correlated with the baseline deft (r = 0.56, p = 0.002), cavitated active caries lesions (r = 0.52, p = 0.003), and filled teeth (r = 0.53, p = 0.003). In a regression analysis the use of fluoridated salt (SC = -0.10) and fluoride gel (SC = -0.14) were negatively associated with ΔDMFT. In conclusion, these findings suggest that the demineralisation properties of the etched sites and the outcome of the 24-hour measurements with QLF are significantly associated with caries increment. Previous caries experience strongly correlated with caries increment in this group of children.

  17. Activities of cytochrome P450 1A2, N-acetyltransferase 2, xanthine oxidase, and cytochrome P450 2D6 are unaltered in children with cystic fibrosis.

    PubMed

    Kennedy, Mary Jayne; Scripture, Charity D; Kashuba, Angela D M; Scott, Christy S; Gaedigk, Andrea; Kearns, Gregory L

    2004-03-01

    The activities of hepatic cytochrome P450 (CYP) 1A2, N-acetyltransferase 2 (NAT-2), xanthine oxidase (XO), and CYP2D6 were evaluated in 12 young children (aged 3-8 years) with mild cystic fibrosis (CF) and 12 age-matched healthy control subjects by use of standard caffeine and dextromethorphan phenotyping methods. Subjects were given 4 oz of Coca-Cola (approximately 35 mg caffeine) (The Coca-Cola Company, Atlanta, Ga) and a single 0.5-mg/kg dose of dextromethorphan. Urine was collected for 8 hours after biomarker administration, and enzyme activity was assessed by use of previously validated caffeine and dextromethorphan molar ratios. CYP2D6 genotyping was also performed in 10 of 12 subjects with CF and 11 of 12 control subjects. There were no significant differences in the urinary molar ratios for any of the enzyme systems evaluated. These data suggest that CF does not alter the activities of CYP1A2, NAT-2, XO, and CYP2D6. Altered biotransformation of drugs in this patient population is likely enzyme- and isoform-specific and thus is apparent for only selected compounds that are substrates for enzymes other than CYP1A2, NAT-2, XO, and CYP2D6.

  18. Changes in CYP1A2 activity in humans after 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) administration using caffeine as a probe drug.

    PubMed

    Yubero-Lahoz, Samanta; Pardo, Ricardo; Farre, Magí; Mathuna, Brian Ó; Torrens, Marta; Mustata, Cristina; Perez-Mañá, Clara; Langohr, Klaus; Carbó, Marcel Lí; de la Torre, Rafael

    2012-01-01

    3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) is a ring-substituted amphetamine widely used for recreational purposes. MDMA is predominantly O-demethylenated in humans by cytochrome P450 (CYP) 2D6, and is also a potent mechanism-based inhibitor of the enzyme. After assessing the inhibition and recovery of CYP2D6 in a previous study, the aim of this work was to study in humans the activity of CYP1A2 in vivo after CYP2D6 had been inhibited by MDMA, using caffeine as a probe drug. Twelve male and nine female recreational MDMA users were included. In session 1, 100 mg of caffeine was given at 0 h. In session 2, a 1.5 mg/kg MDMA dose (range 75-100 mg) was given at 0 h followed by a 100 mg dose of caffeine 4 h later. Aliquots of plasma were assayed for caffeine (137X) and paraxanthine (17X) and statistically significant differences were assessed with a one-way ANOVA. There were significant gender differences at basal condition, which persisted after MDMA administration. CYP1A2 activity was higher in both genders after drug administration, with an increase in 40% in females and 20% in males. Results show an increase in CYP1A2 activity when CYP2D6 is inhibited by MDMA in both genders, being more pronounced in females.

  19. MicroRNA-410 acts as oncogene in NSCLC through downregulating SLC34A2 via activating Wnt/β-catenin pathway

    PubMed Central

    Pu, Qiang; Yuan, Yue; Yang, Weihan; Luo, Xinmei; Jiang, Qianqian; Hu, Xueting; Gong, Yi; Tang, Kui; Su, Xiaolan; Liu, Lunxu; Zhu, Wen; Wei, Yuquan

    2016-01-01

    SLC34A2 had been reported to be down-regulated in human NSCLC cells and patient tissues, and played a significant role in lung cancer. However, the mechanism of its unusual expressionin NSCLC has not been fully elucidated. In present study, we identified SLC34A2 was a direct target of miR-410 and could be inhibited by miR-410 transcriptionally and post-transcriptionally. MiR-410 promoted the growth, invasion and migration of NSCLC cells in vitro. An orthotopic xenograft nude mouse model further affirmed that miR-410 promoted NSCLC cell growth and metastasis in vivo. Moreover, restoring SLC34A2 expression effectively reversed the miR-410-mediated promotion of cell growth, invasion and migration in NSCLC cells. In addition, miR-410high /SLC34A2low expression signature frequently existed in NSCLC cells and tumor tissues. MiR-410 significantly increased the expression of DVL2 and β-catenin protein while decreased that of Gsk3β protein of Wnt/β-catenin signaling pathway, while SLC34A2 partly blocked the effects of miR-410 on those protein expressions. Hence, our data for the first time delineated that unusual expression of SLC34A2 was modulated by miR-410, and miR-410 might positivelycontribute to the tumorigenesis and development of NSCLC by down-regulating SLC34A2 and activating Wnt/β-catenin signaling pathway. MiR-410 might be a new potential therapeutic target for NSCLC. PMID:26910912

  20. A predominate role of CYP1A2 for the metabolism of nabumetone to the active metabolite, 6-methoxy-2-naphthylacetic acid, in human liver microsomes.

    PubMed

    Turpeinen, Miia; Hofmann, Ute; Klein, Kathrin; Mürdter, Thomas; Schwab, Matthias; Zanger, Ulrich M

    2009-05-01

    Nabumetone, a widely used nonsteroidal anti-inflammatory drug, requires biotransformation into 6-methoxy-2-naphthylacetic acid (6-MNA), a close structural analog to naproxen, to achieve its analgesic and anti-inflammatory effects. Despite its wide use, the enzymes involved in metabolism have not been identified. In the present study, several in vitro approaches were used to identify the cytochrome P450 (P450) enzyme(s) responsible for 6-MNA formation. In human liver microsomes (HLMs) 6-MNA formation displayed monophasic Michaelis-Menten kinetics with apparent K(m) and V(max) values (mean +/- S.D.) of 75.1 +/- 15.3 microM and 1304 +/- 226 pmol/min/mg protein, respectively, and formation rate of 6-MNA varied approximately 5.5-fold (179-983 pmol/min/mg protein). 6-MNA activity correlated strongly with both CYP1A2-mediated phenacetin O-deethylation activity and CYP1A2 protein content (r = 0.85 and 0.74, respectively; p < 0.0001 for both). Additional correlations were found with model activities of CYP2C19 and CYP3A4. Of 11 cDNA-expressed recombinant P450s used, recombinant CYP1A2 was the major form catalyzing the 6-MNA formation with an apparent K(m) of 45 microM and V(max) of 8.7 pmol/min/pmol P450. Minor fractions were catalyzed by recombinant P450s CYP1A1, CYP2B6, CYP2C19, CYP2D6, and CYP2E1. Experiments with P450-selective chemical inhibitors and monoclonal anti-P450 antibodies showed that furafylline, a mechanism-based inhibitor CYP1A2, and anti-CYP1A2 antibody markedly inhibited 6-MNA formation, whereas inhibitors for other P450s did not show significant inhibitory effects. Taken together, these studies indicate that the formation of the active metabolite of nabumetone, 6-MNA, is predominantly catalyzed by CYP1A2 in HLMs with only minor contribution of other P450s.

  1. Activation of the thromboxane A2 receptor by 8-isoprostane inhibits the pro-angiogenic effect of vascular endothelial growth factor in scleroderma

    PubMed Central

    Tsou, Pei-Suen; Amin, M. Asif; Campbell, Phil; Zakhem, George; Balogh, Beatrix; Edhayan, Gautam; Ohara, Ray A.; Schiopu, Elena; Khanna, Dinesh; Koch, Alisa E.; Fox, David A.

    2015-01-01

    The pathogenesis of scleroderma (SSc) includes components of autoimmunity, vascular dysfunction, and accumulation of extracellular matrix. 8-isoprostane, an oxidized lipid created by oxidative stress, activates the thromboxane A2 receptor (TXAR) and ROCK pathway. In this study we determined whether the TXAR was activated by 8-isoprostane in SSc endothelial cells (ECs), and whether this pathway inhibited VEGF-induced angiogenesis. Elevated 8-isoprostane was observed in plasma and conditioned media from SSc patients. SSc conditioned media inhibited EC tube formation, while addition of vitamin E, by reducing 8-isoprostane, increased tube formation. VEGF did not induce angiogenesis in SSc ECs, but vitamin E or TXAR inhibition restored its effect. The expression of TXAR, RhoA, and ROCK1/2 were elevated in SSc ECs. ROCK activity and 8-isoprostane-induced ROCK activation were significantly higher in SSc ECs while VEGF had no effect. The hyper-activation of the TXAR leads to inhibition of VEGF-induced angiogenesis, as inhibition of the TXAR pathway results in blockade of 8-isoprostane induced ROCK activation and restoration of VEGF activity. These results suggest that the TXAR pathway plays a crucial role in angiogenesis and that 8-isoprostane is not just a by-product of oxidative stress, but also plays a significant role in the impaired angiogenesis that characterizes SSc. PMID:26288351

  2. Thromboxane A2 receptor antagonist SQ29548 reduces ischemic stroke-induced microglia/macrophages activation and enrichment, and ameliorates brain injury

    PubMed Central

    Yan, Aijuan; Zhang, Tingting; Yang, Xiao; Shao, Jiaxiang; Fu, Ningzhen; Shen, Fanxia; Fu, Yi; Xia, Weiliang

    2016-01-01

    Thromboxane A2 receptor (TXA2R) activation is thought to be involved in thrombosis/hemostasis and inflammation responses. We have previously shown that TXA2R antagonist SQ29548 attenuates BV2 microglia activation by suppression of ERK pathway, but its effect is not tested in vivo. The present study aims to explore the role of TXA2R on microglia/macrophages activation after ischemia/reperfusion brain injury in mice. Adult male ICR mice underwent 90-min transient middle cerebral artery occlusion (tMCAO). Immediately and 24 h after reperfusion, SQ29548 was administered twice to the ipsilateral ventricle (10 μl, 2.6 μmol/ml, per dose). Cerebral infarction volume, inflammatory cytokines release and microglia/macrophages activation were measured using the cresyl violet method, quantitative polymerase chain reaction (qPCR), and immunofluorescence double staining, respectively. Expression of TXA2R was significantly increased in the ipsilateral brain tissue after ischemia/reperfusion, which was also found to co-localize with activated microglia/macrophages in the infarct area. Administration of SQ29548 inhibited microglia/macrophages activation and enrichment, including both M1 and M2 phenotypes, and attenuated ischemia-induced IL-1ß, IL-6, and TNF-α up-regulation and iNOS release. TXA2R antagonist SQ29548 inhibited ischemia-induced inflammatory response and furthermore reduced microglia/macrophages activation and ischemic/reperfusion brain injury. PMID:27775054

  3. Coffee and tea consumption, genotype-based CYP1A2 and NAT2 activity and colorectal cancer risk-results from the EPIC cohort study.

    PubMed

    Dik, Vincent K; Bueno-de-Mesquita, H B As; Van Oijen, Martijn G H; Siersema, Peter D; Uiterwaal, Cuno S P M; Van Gils, Carla H; Van Duijnhoven, Fränzel J B; Cauchi, Stéphane; Yengo, Loic; Froguel, Philippe; Overvad, Kim; Bech, Bodil H; Tjønneland, Anne; Olsen, Anja; Boutron-Ruault, Marie-Christine; Racine, Antoine; Fagherazzi, Guy; Kühn, Tilman; Campa, Daniele; Boeing, Heiner; Aleksandrova, Krasimira; Trichopoulou, Antonia; Peppa, Eleni; Oikonomou, Eleni; Palli, Domenico; Grioni, Sara; Vineis, Paolo; Tumino, Rosaria; Panico, Salvatore; Peeters, Petra H M; Weiderpass, Elisabete; Engeset, Dagrun; Braaten, Tonje; Dorronsoro, Miren; Chirlaque, María-Dolores; Sánchez, María-José; Barricarte, Aurelio; Zamora-Ros, Raul; Argüelles, Marcial; Jirström, Karin; Wallström, Peter; Nilsson, Lena M; Ljuslinder, Ingrid; Travis, Ruth C; Khaw, Kay-Tee; Wareham, Nick; Freisling, Heinz; Licaj, Idlir; Jenab, Mazda; Gunter, Marc J; Murphy, Neil; Romaguera-Bosch, Dora; Riboli, Elio

    2014-07-15

    Coffee and tea contain numerous antimutagenic and antioxidant components and high levels of caffeine that may protect against colorectal cancer (CRC). We investigated the association between coffee and tea consumption and CRC risk and studied potential effect modification by CYP1A2 and NAT2 genotypes, enzymes involved in the metabolization of caffeine. Data from 477,071 participants (70.2% female) of the European Investigation into Cancer and Nutrition (EPIC) cohort study were analyzed. At baseline (1992-2000) habitual (total, caffeinated and decaffeinated) coffee and tea consumption was assessed with dietary questionnaires. Cox proportional hazards models were used to estimate adjusted hazard ratio's (HR) and 95% confidence intervals (95% CI). Potential effect modification by genotype-based CYP1A2 and NAT2 activity was studied in a nested case-control set of 1,252 cases and 2,175 controls. After a median follow-up of 11.6 years, 4,234 participants developed CRC (mean age 64.7 ± 8.3 years). Total coffee consumption (high vs. non/low) was not associated with CRC risk (HR 1.06, 95% CI 0.95-1.18) or subsite cancers, and no significant associations were found for caffeinated (HR 1.10, 95% CI 0.97-1.26) and decaffeinated coffee (HR 0.96, 95% CI 0.84-1.11) and tea (HR 0.97, 95% CI 0.86-1.09). High coffee and tea consuming subjects with slow CYP1A2 or NAT2 activity had a similar CRC risk compared to non/low coffee and tea consuming subjects with a fast CYP1A2 or NAT2 activity, which suggests that caffeine metabolism does not affect the link between coffee and tea consumption and CRC risk. This study shows that coffee and tea consumption is not likely to be associated with overall CRC.

  4. Kaempferol enhances endothelium-dependent relaxation in the porcine coronary artery through activation of large-conductance a2+-activated K+ channels

    PubMed Central

    Xu, Y C; Leung, S W S; Leung, G P H; Man, R Y K

    2015-01-01

    Background and Purpose Kaempferol, a plant flavonoid present in normal human diet, can modulate vasomotor tone. The present study aimed to elucidate the signalling pathway through which this flavonoid enhanced relaxation of vascular smooth muscle. Experimental Approach The effect of kaempferol on the relaxation of porcine coronary arteries to endothelium-dependent (bradykinin) and -independent (sodium nitroprusside) relaxing agents was studied in an in vitro organ chamber setup. The whole-cell patch-clamp technique was used to determine the effect of kaempferol on potassium channels in porcine coronary artery smooth muscle cells (PCASMCs). Key Results At a concentration without direct effect on vascular tone, kaempferol (3 × 10−6 M) enhanced relaxations produced by bradykinin and sodium nitroprusside. The potentiation by kaempferol of the bradykinin-induced relaxation was not affected by Nω-nitro-L-arginine methyl ester, an inhibitor of NO synthase (10−4 M) or TRAM-34 plus UCL 1684, inhibitors of intermediate- and small-conductance calcium-activated potassium channels, respectively (10−6 M each), but was abolished by tetraethylammonium chloride, a non-selective inhibitor of calcium-activated potassium channels (10−3 M), and iberiotoxin, a selective inhibitor of large-conductance calcium-activated potassium channel (KCa1.1; 10−7 M). Iberiotoxin also inhibited the potentiation by kaempferol of sodium nitroprusside-induced relaxations. Kaempferol stimulated an outward-rectifying current in PCASMCs, which was abolished by iberiotoxin. Conclusions and Implications The present results suggest that, in smooth muscle cells of the porcine coronary artery, kaempferol enhanced relaxations caused by endothelium-derived and exogenous NO as well as those due to endothelium-dependent hyperpolarization. This vascular effect of kaempferol involved the activation of KCa1.1 channels. PMID:25652142

  5. Potentiation by tonic A2a-adenosine receptor activation of CGRP-facilitated [3H]-ACh release from rat motor nerve endings.

    PubMed Central

    Correia-de-Sá, P.; Ribeiro, J. A.

    1994-01-01

    1. The effect of calcitonin gene-related peptide (CGRP) on [3H]-acetylcholine ([3H]-ACh) release from motor nerve endings and its interaction with presynaptic facilitatory A2a-adenosine and nicotinic acetylcholine receptors was studied on rat phrenic nerve-hemidiaphragm preparations loaded with [3H]-choline. 2. CGRP (100-400 nM) increased electrically evoked [3H]-ACh release from phrenic nerve endings in a concentration-dependent manner. 3. The magnitude of CGRP excitation increased with the increase of the stimulation pulse duration from 40 microseconds to 1 ms, keeping the frequency, the amplitude and the train length constants. With 1 ms pulses, the evoked [3H]-ACh release was more intense than with 40 microseconds pulse duration. 4. Both the nicotinic acetylcholine receptor agonist, 1,1-dimethyl-4-phenylpiperazinium, and the A2a adenosine receptor agonist, CGS 21680C, increased evoked [3H]-ACh release, but only CGS 21680C potentiated the facilitatory effect of CGRP. This potentiation was prevented by the A2a adenosine receptor antagonist, PD 115,199. 5. Adenosine deaminase prevented the excitatory effect of CGRP (400 nM) on [3H]-ACh release. This effect was reversed by the non-hydrolysable A2a-adenosine receptor agonist, CGS 21680C. 6. The nicotinic antagonist, tubocurarine, did not significantly change, whereas the A2-adenosine receptor antagonist, PD 115,199, blocked the CGRP facilitation. The A1-adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, potentiated the CGRP excitatory effect. 7. The results suggest that the facilitatory effect of CGRP on evoked [3H]-ACh release from rat phrenic motor nerve endings depends on the presence of endogenous adenosine which tonically activates A2a-adenosine receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8004402

  6. Comparison of CYP106A1 and CYP106A2 from Bacillus megaterium - identification of a novel 11-oxidase activity.

    PubMed

    Kiss, Flora Marta; Schmitz, Daniela; Zapp, Josef; Dier, Tobias K F; Volmer, Dietrich A; Bernhardt, Rita

    2015-10-01

    The CYP106A subfamily hydroxylates steroids, diterpenes, and triterpenes in a regioselective and stereoselective manner, which is a challenging task for synthetic chemistry. The well-studied CYP106A2 enzyme, from the Bacillus megaterium strain ATCC 13368, is a highly promising candidate for the pharmaceutical industry. It shares 63 % amino acid sequence identity with CYP106A1 from B. megaterium DSM319, which was recently characterized. A focused steroid library was screened with both CYP106A1 and CYP106A2. Out of the 23 tested steroids, 19 were successfully converted by both enzymes during in vitro and in vivo reactions. Thirteen new substrates were identified for CYP106A1, while the substrate spectrum of CYP106A2 was extended by seven new members. Finally, six chosen steroids were further studied on a preparative scale employing a recombinant B. megaterium MS941 whole-cell system, yielding sufficient amounts of product for structure characterization by nuclear magnetic resonance. The hydroxylase activity was confirmed at positons 6β, 7β, 9α, and 15β. In addition, the CYP106A subfamily showed unprecedented 11-oxidase activity, converting 11β-hydroxysteroids to their 11-keto derivatives. This novel reaction and the diverse hydroxylation positions on pharmaceutically relevant compounds underline the role of the CYP106A subfamily in drug development and production.

  7. Adenosine A2 receptor activation ameliorates mitochondrial oxidative stress upon reperfusion through the posttranslational modification of NDUFV2 subunit of complex I in the heart.

    PubMed

    Xu, Jingman; Bian, Xiyun; Liu, Yuan; Hong, Lan; Teng, Tianming; Sun, Yuemin; Xu, Zhelong

    2017-05-01

    While it is well known that adenosine receptor activation protects the heart from ischemia/reperfusion injury, the precise mitochondrial mechanism responsible for the action remains unknown. This study probed the mitochondrial events associated with the cardioprotective effect of 5'-(N-ethylcarboxamido) adenosine (NECA), an adenosine A2 receptor agonist. Isolated rat hearts were subjected to 30min ischemia followed by 10min of reperfusion, whereas H9c2 cells experienced 20min ischemia and 10min reperfusion. NECA prevented mitochondrial structural damage, decreases in respiratory control ratio (RCR), and collapse of mitochondrial membrane potential (ΔΨm). Both the adenosine A2A receptor antagonist SCH58261 and A2B receptor antagonist MRS1706 inhibited the action of NECA. NECA reduced mitochondrial proteins carbonylation, H2O2, and superoxide generation at reperfusion, but did not change superoxide dismutase (SOD) activity. In support, the protective effects of NECA and Peg-SOD on ΔΨm upon reperfusion were additive, implying that NECA's protection is attributable to the reduced superoxide generation but not to the enhancement of the superoxide-scavenging capacity. NECA increased the mitochondrial Src tyrosine kinase activity and suppressed complex I activity at reperfusion in a Src-dependent manner. NECA also reduced mitochondrial superoxide through Src tyrosine kinase. Studies with liquid chromatography-mass spectrometer (LC-MS) identified Tyr118 of the NDUFV2 subunit of complex 1 as a likely site of the tyrosine phosphorylation. Furthermore, the complex I activity of cells transfected with the Y118F mutant was increased, suggesting that this site might be a negative regulator of complex I activity. In support, NECA failed to suppress complex I activity at reperfusion in cells transfected with the Y118F mutant of NDUFV2. In conclusion, NECA prevents mitochondrial oxidative stress by decreasing mitochondrial superoxide generation through inhibition of complex I

  8. Genetic modification of potato against microbial diseases: in vitro and in planta activity of a dermaseptin B1 derivative, MsrA2.

    PubMed

    Osusky, Milan; Osuska, Lubica; Kay, William; Misra, Santosh

    2005-08-01

    Dermaseptin B1 is a potent cationic antimicrobial peptide found in skin secretions of the arboreal frog Phyllomedusa bicolor. A synthetic derivative of dermaseptin B1, MsrA2 (N-Met-dermaseptin B1), elicited strong antimicrobial activities against various phytopathogenic fungi and bacteria in vitro. To assess its potential for plant protection, MsrA2 was expressed at low levels (1-5 microg/g of fresh tissue) in the transgenic potato (Solanum tuberosum L.) cv. Desiree. Stringent challenges of these transgenic potato plants with a variety of highly virulent fungal phytopathogens--Alternaria, Cercospora, Fusarium, Phytophthora, Pythium, Rhizoctonia and Verticillium species--and with the bacterial pathogen Erwinia carotovora demonstrated that the plants had an unusually broad-spectrum and powerful resistance to infection. MsrA2 profoundly protected both plants and tubers from diseases such as late blight, dry rot and pink rot and markedly extended the storage life of tubers. Due to these properties in planta, MsrA2 is proposed as an ideal antimicrobial peptide candidate to significantly increase resistance to phytopathogens and improve quality in a variety of crops worldwide with the potential to obviate fungicides and facilitate storage under difficult conditions.

  9. Basal adenosine modulates the functional properties of AMPA receptors in mouse hippocampal neurons through the activation of A1R A2AR and A3R

    PubMed Central

    Di Angelantonio, Silvia; Bertollini, Cristina; Piccinin, Sonia; Rosito, Maria; Trettel, Flavia; Pagani, Francesca; Limatola, Cristina; Ragozzino, Davide

    2015-01-01

    Adenosine is a widespread neuromodulator within the CNS and its extracellular level is increased during hypoxia or intense synaptic activity, modulating pre- and postsynaptic sites. We studied the neuromodulatory action of adenosine on glutamatergic currents in the hippocampus, showing that activation of multiple adenosine receptors (ARs) by basal adenosine impacts postsynaptic site. Specifically, the stimulation of both A1R and A3R reduces AMPA currents, while A2AR has an opposite potentiating effect. The effect of ARs stimulation on glutamatergic currents in hippocampal cultures was investigated using pharmacological and genetic approaches. A3R inhibition by MRS1523 increased GluR1-Ser845 phosphorylation and potentiated AMPA current amplitude, increasing the apparent affinity for the agonist. A similar effect was observed blocking A1R with DPCPX or by genetic deletion of either A3R or A1R. Conversely, impairment of A2AR reduced AMPA currents, and decreased agonist sensitivity. Consistently, in hippocampal slices, ARs activation by AR agonist NECA modulated glutamatergic current amplitude evoked by AMPA application or afferent fiber stimulation. Opposite effects of AR subtypes stimulation are likely associated to changes in GluR1 phosphorylation and represent a novel mechanism of physiological modulation of glutamatergic transmission by adenosine, likely acting in normal conditions in the brain, depending on the level of extracellular adenosine and the distribution of AR subtypes. PMID:26528137

  10. Activity of 8F4, a T-cell receptor-like anti-PR1/HLA-A2 antibody, against primary human AML in vivo.

    PubMed

    Sergeeva, A; He, H; Ruisaard, K; St John, L; Alatrash, G; Clise-Dwyer, K; Li, D; Patenia, R; Hong, R; Sukhumalchandra, P; You, M J; Gagea, M; Ma, Q; Molldrem, J J

    2016-07-01

    The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a high-affinity T-cell receptor-like murine monoclonal antibody, 8F4, that binds to the PR1/HLA-A2 complex, mediates lysis of AML and inhibits leukemia colony formation. Here, we explored whether 8F4 was active in vivo against chemotherapy-resistant AML, including secondary AML. In a screening model, coincubation of AML with 8F4 ex vivo prevented engraftment of all tested AML subtypes in immunodeficient NSG (NOD scid IL-2 receptor γ-chain knockout) mice. In a treatment model of established human AML, administration of 8F4 significantly reduced or eliminated AML xenografts and extended survival compared with isotype antibody-treated mice. Moreover, in secondary transfer experiments, mice inoculated with bone marrow from 8F4-treated mice showed no evidence of AML engraftment, supporting the possible activity of 8F4 against the subset of AML with self-renewing potential. Our data provide evidence that 8F4 antibody is highly active in AML, including chemotherapy-resistant disease, supporting its potential use as a therapeutic agent in patients with AML.

  11. Hindbrain A2 noradrenergic neuron adenosine 5'-monophosphate-activated protein kinase activation, upstream kinase/phosphorylase protein expression, and receptivity to hormone and fuel reporters of short-term food deprivation are regulated by estradiol.

    PubMed

    Briski, Karen P; Alenazi, Fahaad S H; Shakya, Manita; Sylvester, Paul W

    2016-09-12

    Estradiol (E) mitigates acute and postacute adverse effects of 12 hr-food deprivation (FD) on energy balance. Hindbrain 5'-monophosphate-activated protein kinase (AMPK) regulates hyperphagic and hypothalamic metabolic neuropeptide and norepinephrine responses to FD in an E-dependent manner. Energy-state information from AMPK-expressing hindbrain A2 noradrenergic neurons shapes neural responses to metabolic imbalance. Here we investigate the hypothesis that FD causes divergent changes in A2 AMPK activity in E- vs. oil (O)-implanted ovariectomized female rats, alongside dissimilar adjustments in circulating metabolic fuel (glucose, free fatty acids [FFA]) and energy deficit-sensitive hormone (corticosterone, glucagon, leptin) levels. FD decreased blood glucose in oil (O)- but not E-implanted ovariectomized female rats and elevated and reduced glucagon levels in O and E, respectively. FD decreased circulating leptin in O and E, but increased corticosterone and FFA concentrations in E only. Western blot analysis of laser-microdissected A2 neurons showed that glucocorticoid receptor type II and very-long-chain acyl-CoA synthetase 3 protein profiles were amplified in FD/E vs. FD/O. A2 total AMPK protein was elevated without change in activity in FD/O, whereas FD/E exhibited increased AMPK activation along with decreased upstream phosphatase expression. The catecholamine biosynthetic enzyme dopamine-β-hydroxylase (DβH) was increased in FD/O but not FD/E A2 cells. The data show discordance between A2 AMPK activation and glycemic responses to FD; sensor activity was refractory to glucose decrements in FD/O but augmented in FD/E despite stabilized glucose and elevated FFA levels. E-dependent amplification of AMPK activity may reflect adaptive conversion to fatty acid oxidation and/or glucocorticoid stimulation. FD augmentation of A2 DβH protein profiles in FD/O but not FD/E animals suggests that FD may correspondingly regulate NE synthesis vs. metabolism/release in the

  12. Construction of a 2D Graphene-Like MoS2/C3N4 Heterojunction with Enhanced Visible-Light Photocatalytic Activity and Photoelectrochemical Activity.

    PubMed

    Yan, Jia; Chen, Zhigang; Ji, Haiyan; Liu, Zheng; Wang, Xin; Xu, Yuanguo; She, Xiaojie; Huang, Liying; Xu, Li; Xu, Hui; Li, Huaming

    2016-03-24

    A novel graphene-like MoS2 /C3N4 (GL-MoS2/C3N4) composite photocatalyst has been synthesized by a facile ethylene glycol (EG)-assisted solvothermal method. The structure and morphology of this GL-MoS2/C3N4 photocatalyst have been investigated by a wide range of characterization methods. The results showed that GL-MoS2 was uniformly distributed on the surface of GL-C3N4 forming a heterostructure. The obtained composite exhibited strong absorbing ability in the ultraviolet (UV) and visible regions. When irradiated with visible light, the composite photocatalyst showed high activity superior to those of the respective individual components GL-MoS2 and GL-C3N4 in the degradation of methyl orange. The enhanced photocatalytic activity of the composite may be attributed to the efficient separation of electron-hole pairs as a result of the matching band potentials between GL-MoS2 and GL-C3N4. Furthermore, a photocatalytic mechanism for the composite material has been proposed, and the photocatalytic reaction kinetics has been measured. Moreover, GL-MoS2/C3N4 could serve as a novel sensor for trace amounts of Cu(2+) since it exhibited good selectivity for Cu(2+) detection in water.

  13. Expression, purification and characterization of a human serine-dependent phospholipase A2 with high specificity for oxidized phospholipids and platelet activating factor.

    PubMed Central

    Rice, S Q; Southan, C; Boyd, H F; Terrett, J A; MacPhee, C H; Moores, K; Gloger, I S; Tew, D G

    1998-01-01

    Using expressed sequence tag (EST) homology screening, a new human serine dependent phospholipase A2 (HSD-PLA2) was identified that has 40% amino acid identity with human low density lipoprotein-associated phospholipase A2 (LDL-PLA2). HSD-PLA2 has very recently been purified and cloned from brain tissue but named PAF-AH II. However, because the homology with LDL-PLA2 suggested a broader substrate specificity than simply platelet activating factor (PAF), we have further characterized this enzyme using baculovirus-expressed protein. The recombinant enzyme, which was purified 21-fold to homogeneity, had a molecular mass of 44kDa and possessed a specific activity of 35 micromol min-1 mg-1 when assayed against PAF. Activity could also be measured using 1-decanoyl-2-(4-nitrophenylglutaryl) phosphate (DNGP) as substrate. Like LDL-PLA2, HSD-PLA2 was able to hydrolyse oxidatively modified phosphatidylcholines when supplemented to human LDL prior to copper-stimulated oxidation. A GXSXG motif evident from sequence information and inhibition of its activity by 3,4, dichloroisocoumarin, diisopropyl fluorophosphate (DFP) and diethyl p-nitrophenyl phosphate (DENP) confirm that the enzyme is serine dependent. Moreover, sequence comparison indicates the HSD-PLA2 probable active site triad positions are shared with LDL-PLA2 and a C. elegans homologue, suggesting that these sequences comprise members of a new enzyme family. Although clearly structurally related with similar substrate specificities further work reported here shows HSD-PLA2 and LDL-PLA2 to be different with respect to chromosomal localization and tissue distribution. PMID:9494101

  14. 1alpha,25(OH)2D3 causes a rapid increase in phosphatidylinositol-specific PLC-beta activity via phospholipase A2-dependent production of lysophospholipid.

    PubMed

    Schwartz, Z; Shaked, D; Hardin, R R; Gruwell, S; Dean, D D; Sylvia, V L; Boyan, B D

    2003-05-01

    1alpha,25(OH)(2)D(3) activates protein kinase C (PKC) in rat growth plate chondrocytes via mechanisms involving phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A(2) (PLA(2)). The purpose of this study was to determine if 1alpha,25(OH)(2)D(3) activates PI-PLC directly or through a PLA(2)-dependent mechanism. We determined which PLC isoforms are present in the growth plate chondrocytes, and determined which isoform(s) of PLC is(are) regulated by 1alpha,25(OH)(2)D(3). Inhibitors and activators of PLA(2) were used to assess the inter-relationship between these two phospholipid-signaling pathways. PI-PLC activity in lysates of prehypertrophic and upper hypertrophic zone (growth zone) cells that were incubated with 1alpha,25(OH)(2)D(3), was increased within 30s with peak activity at 1-3 min. PI-PLC activity in resting zone cells was unaffected by 1alpha,25(OH)(2)D(3). 1beta,25(OH)(2)D(3), 24R,25(OH)(2)D(3), actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, 1alpha,25(OH)(2)D(3) regulation of PI-PLC enzyme activity is stereospecific, cell maturation-dependent, and nongenomic. PLA(2)-activation (mastoparan or melittin) increased PI-PLC activity to the same extent as 1alpha,25(OH)(2)D(3); PLA(2)-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF(3)) reduced the effect of 1alpha,25(OH)(2)D(3). Neither arachidonic acid (AA) nor its metabolites affected PI-PLC. In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) activated PI-PLC (LPE>LPC). 1alpha,25(OH)(2)D(3) stimulated PI-PLC and PKC activities via Gq; GDPbetaS inhibited activity, but pertussis toxin did not. RT-PCR showed that the cells express PLC-beta1a, PLC-beta1b, PLC-beta3 and PLC-gamma1 mRNA. Antibodies to PLC-beta1 and PLC-beta3 blocked the 1alpha,25(OH)(2)D(3) effect; antibodies to PLC-delta and PLC-gamma did not. Thus, 1alpha,25(OH)(2)D(3) regulates PLC-beta through PLA(2)-dependent production of

  15. Simultaneous activation of p38 and JNK by arachidonic acid stimulates the cytosolic phospholipase A2-dependent synthesis of lipid droplets in human monocytes

    PubMed Central

    Guijas, Carlos; Pérez-Chacón, Gema; Astudillo, Alma M.; Rubio, Julio M.; Gil-de-Gómez, Luis; Balboa, María A.; Balsinde, Jesús

    2012-01-01

    Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LDs are formed by two different routes: (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling, leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. Both routes can be dissociated by the arachidonyl-CoA synthetase inhibitor triacsin C, which prevents the former but not the latter. LD formation by AA-induced signaling predominates, accounting for 60–70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A2α (cPLA2α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA2α activation by phosphorylation. Collectively, these results suggest that concomitant activation of p38 and JNK by AA cooperate to activate cPLA2α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis. PMID:22949356

  16. Effect of Chlorogenic Acid (5-Caffeoylquinic Acid) Isolated from Baccharis oxyodonta on the Structure and Pharmacological Activities of Secretory Phospholipase A2 from Crotalus durissus terrificus

    PubMed Central

    Toyama, Daniela O.; Ferreira, Marcelo J. P.; Romoff, Paulete; Fávero, Oriana A.; Gaeta, Henrique H.; Toyama, Marcos H.

    2014-01-01

    The aim of this paper was to investigate the effect of chlorogenic acid (5-caffeoylquinic acid, 5CQA), isolated from Baccharis oxyodonta, on the structure and pharmacological effect of secretory phospholipase A2 (sPLA2) from Crotalus durissus terrificus. All in vitro and in vivo experiments were conducted using a purified sPLA2 compared under the same experimental conditions with sPLA2 : 5CQA. 5CQA induced several discrete modifications in the secondary structure and the hydrophobic characteristics of native sPLA2 that induced slight changes in the α-helical content, increase in the random coil structure, and decrease of fluorescence of native sPLA2. Moreover, 5CQA significantly decreased the enzymatic activity and the oedema and myonecrosis induced by native sPLA2. As the catalytic activity of sPLA2 plays an important role in several of its biological and pharmacological properties, antibacterial activity was used to confirm the decrease in its enzymatic activity by 5CQA, which induced massive bacterial cell destruction. We found that 5CQA specifically abolished the enzymatic activity of sPLA2 and induced discrete protein unfolding that mainly involved the pharmacological site of sPLA2. These results showed the potential application of 5CQA in the snake poisoning treatment and modulation of the pathological effect of inflammation induced by secretory PLA2. PMID:25258715

  17. Effect of GABAergic ligands on the anxiolytic-like activity of DOI (a 5-HT(2A/2C) agonist) in the four-plate test in mice.

    PubMed

    Massé, Fabienne; Hascoët, Martine; Bourin, Michel

    2007-01-01

    5-HTergic and GABAergic systems are involved in neurobiology of anxiety. Precedent studies have demonstrated that SSRIs possessed an anxiolytic-like effect in the four-plate test (FPT) at doses that did not modify spontaneous locomotor activity. This effect seems to be mediated through the activation of 5-HT(2A) postsynaptic receptors. The purpose of the present study was to examine the implication of GABA system in the anxiolytic-like activity of DOI in the FPT. To achieve this, the co-administration of DOI (5-HT(2A/2C) receptor agonists) with GABA(A) and GABA(B) receptor ligands was evaluated in the FPT. Alprazolam, diazepam and muscimol (for higher dose) potentiated the anxiolytic-like effect of DOI. Bicuculline, picrotoxin and baclofen inhibited the anxiolytic-like effect of DOI. Flumazenil and CGP 35348 had no effect on the anxiolytic-like activity of DOI. These results suggest that the GABA system seems to be strongly implicated in the anxiolytic-like activity of DOI in the FPT.

  18. Constitutive androstane receptor transcriptionally activates human CYP1A1 and CYP1A2 genes through a common regulatory element in the 5'-flanking region.

    PubMed

    Yoshinari, Kouichi; Yoda, Noriaki; Toriyabe, Takayoshi; Yamazoe, Yasushi

    2010-01-15

    Phenobarbital has long been known to increase cellular levels of CYP1A1 and CYP1A2 possibly through a pathway(s) independent of aryl hydrocarbon receptor. We have investigated the role of constitutive androstane receptor (CAR), a xenobiotic-responsive nuclear receptor, in the transactivation of human CYP1A1 and CYP1A2. These genes are located in a head-to-head orientation, sharing a 5'-flanking region. Reporter assays were thus performed with dual-reporter constructs, containing the whole or partially deleted human CYP1A promoter between two different reporter genes. In this system, human CAR (hCAR) enhanced the transcription of both genes through common promoter regions from -461 to -554 and from -18089 to -21975 of CYP1A1. With reporter assays using additional deleted and mutated constructs, electrophoresis mobility shift assays and chromatin immunoprecipitation assays, an ER8 motif (everted repeat separated by eight nucleotides), located at around -520 of CYP1A1, was identified as an hCAR-responsive element and a binding motif of hCAR/human retinoid X receptor alpha heterodimer. hCAR enhanced the transcription of both genes also in the presence of an aryl hydrocarbon receptor ligand. Finally, hCAR activation increased CYP1A1 and CYP1A2 mRNA levels in cultured human hepatocytes. Our results indicate that CAR transactivates human CYP1A1 and CYP1A2 in human hepatocytes through the common cis-element ER8. Interestingly, the ER8 motif is highly conserved in the CYP1A1 proximal promoter sequences of various species, suggesting a fundamental role of CAR in the xenobiotic-induced expression of CYP1A1 and CYP1A2 independent of aryl hydrocarbon receptor.

  19. A2A adenosine-receptor-mediated facilitation of noradrenaline release in rat tail artery involves protein kinase C activation and betagamma subunits formed after alpha2-adrenoceptor activation.

    PubMed

    Fresco, Paula; Oliveira, Jorge M A; Kunc, Filip; Soares, Ana Sofia; Rocha-Pereira, Carolina; Gonçalves, Jorge; Diniz, Carmen

    2007-07-01

    This work aimed to investigate the molecular mechanisms involved in the interaction of alpha2-adrenoceptors and adenosine A2A-receptor-mediated facilitation of noradrenaline release in rat tail artery, namely the type of G-protein involved in this effect and the step or steps where the signalling cascades triggered by alpha2-adrenoceptors and A2A-receptors interact. The selective adenosine A2A-receptor agonist 2-p-(2-carboxy ethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 100 nM) enhanced tritium overflow evoked by trains of 100 pulses at 5 Hz. This effect was abolished by the selective adenosine A2A-receptor antagonist 5-amino-7-(2-phenyl ethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine (SCH 58261; 20 nM) and by yohimbine (1 microM). CGS 21680-mediated effects were also abolished by drugs that disrupted G(i/o)-protein coupling with receptors, PTX (2 microg/ml) or NEM (40 microM), by the anti-G(salpha) peptide (2 microg/ml) anti-G(betagamma) peptide (10 microg/ml) indicating coupling of A2A-receptors to G(salpha) and suggesting a crucial role for G(betagamma) subunits in the A(2A)-receptor-mediated enhancement of tritium overflow. Furthermore, phorbol 12-myristate 13-acetate (PMA; 1 microM) or forskolin (1 microM), direct activators of protein kinase C and of adenylyl cyclase, respectively, also enhanced tritium overflow. In addition, PMA-mediated effects were not observed in the presence of either yohimbine or PTX. Results indicate that facilitatory adenosine A2A-receptors couple to G(salpha) subunits which is essential, but not sufficient, for the release facilitation to occur, requiring the involvement of G(i/o)-protein coupling (it disappears after disruption of G(i/o)-protein coupling, PTX or NEM) and/or G(betagamma) subunits (anti-G(betagamma)). We propose a mechanism for the interaction in study suggesting group 2 AC isoforms as a plausible candidate for the interaction site, as these isoforms can integrate inputs from G

  20. Daboxin P, a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity

    PubMed Central

    Iyer, Janaki Krishnamurthy; Shih, Norrapat; Majumder, Munmi; Mattaparthi, Venkata Satish Kumar; Mukhopadhyay, Rupak; Doley, Robin

    2016-01-01

    In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity. PMID:27089306

  1. Phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in MCII mast cells mediated by high-affinity Fc receptor for IgE.

    PubMed Central

    Currie, S; Roberts, E F; Spaethe, S M; Roehm, N W; Kramer, R M

    1994-01-01

    In the present study we examined the activation of Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) after aggregation of cell-surface high-affinity Fc receptors for IgE (Fc epsilon RI) on mast cells. MCII mast cells (a factor-dependent bone-marrow-derived murine mast cell line) produce significant amounts of leukotriene C4 (LTC4) (70 ng/10(6) cells) on cross-linking of Fc epsilon RI. Using enzymic and immunochemical analysis we found that cPLA2 is the predominant form of this enzyme in MCII mast cells (0.2 micrograms/mg of total protein) and other forms (i.e. secretory PLA2 or Ca2+ independent cytosolic PLA2) could not be detected. Therefore MCII mast cells represent an excellent cellular model for the study of the biochemical mechanism(s) responsible for Fc epsilon RI-induced activation of cPLA2 and the involvement of cPLA2 in Fc epsilon RI-mediated production of LTC4. After activation of Fc epsilon RI by cross-linking, cPLA2 in MCII mast cells exhibited a decreased electrophoretic mobility and its enzyme activity was increased 3-fold. Treatment with phosphatase reversed both the altered electrophoretic mobility and the enhanced enzyme activity demonstrating that they were the result of Fc epsilon RI-induced phosphorylation. On cross-linking of Fc epsilon RI, cPLA2 was phosphorylated within 30 s and appeared to be an early substrate for Fc epsilon RI-activated protein kinases in MCII mast cells. Tyrosine phosphorylation may be a critical component in this process, as genistein, an inhibitor of protein tyrosine kinases, blocked the activation of cPLA2. Using anti-phosphotyrosine antibodies we observed that the activating phosphorylation was not on tyrosine residues of cPLA2, indicating that tyrosine kinases participate upstream in the signalling cascade that couples Fc epsilon RI to cPLA2. We conclude that in MCII mast cells cPLA2 is activated by kinase-dependent mechanisms and may be responsible for Fc epsilon RI-induced mobilization of arachidonic acid for the

  2. A novel protein from the serum of Python sebae, structurally homologous with type-γ phospholipase A(2) inhibitor, displays antitumour activity.

    PubMed

    Donnini, Sandra; Finetti, Federica; Francese, Simona; Boscaro, Francesca; Dani, Francesca R; Maset, Fabio; Frasson, Roberta; Palmieri, Michele; Pazzagli, Mario; De Filippis, Vincenzo; Garaci, Enrico; Ziche, Marina

    2011-12-01

    Cytotoxic and antitumour factors have been documented in the venom of snakes, although little information is available on the identification of cytotoxic products in snake serum. In the present study, we purified and characterized a new cytotoxic factor from serum of the non-venomous African rock python (Python sebae), endowed with antitumour activity. PSS (P. sebae serum) exerted a cytotoxic activity and reduced dose-dependently the viability of several different tumour cell lines. In a model of human squamous cell carcinoma xenograft (A431), subcutaneous injection of PSS in proximity of the tumour mass reduced the tumour volume by 20%. Fractionation of PSS by ion-exchange chromatography yielded an active protein fraction, F5, which significantly reduced tumour cell viability in vitro and, strikingly, tumour growth in vivo. F5 is composed of P1 (peak 1) and P2 subunits interacting in a 1:1 stoichiometric ratio to form a heterotetramer in equilibrium with a hexameric form, which retained biological activity only when assembled. The two peptides share sequence similarity with PIP {PLI-γ [type-γ PLA(2) (phospholipase A(2)) inhibitor] from Python reticulatus}, existing as a homohexamer. More importantly, although PIP inhibits the hydrolytic activity of PLA(2), the anti-PLA(2) function of F5 is negligible. Using high-resolution MS, we covered 87 and 97% of the sequences of P1 and P2 respectively. In conclusion, in the present study we have identified and thoroughly characterized a novel protein displaying high sequence similarity to PLI-γ and possessing remarkable cytotoxic and antitumour effects that can be exploited for potential pharmacological applications.

  3. Activation of mitochondrial calcium-independent phospholipase A2γ (iPLA2γ) by divalent cations mediating arachidonate release and production of downstream eicosanoids.

    PubMed

    Moon, Sung Ho; Jenkins, Christopher M; Liu, Xinping; Guan, Shaoping; Mancuso, David J; Gross, Richard W

    2012-04-27

    Calcium-independent phospholipase A(2)γ (iPLA(2)γ) (PNPLA8) is the predominant phospholipase activity in mammalian mitochondria. However, the chemical mechanisms that regulate its activity are unknown. Here, we utilize iPLA(2)γ gain of function and loss of function genetic models to demonstrate the robust activation of iPLA(2)γ in murine myocardial mitochondria by Ca(2+) or Mg(2+) ions. Calcium ion stimulated the production of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) from 1-palmitoyl-2-[(14)C]arachidonoyl-sn-glycero-3-phosphocholine during incubations with wild-type heart mitochondrial homogenates. Furthermore, incubation of mitochondrial homogenates from transgenic myocardium expressing iPLA(2)γ resulted in 13- and 25-fold increases in the initial rate of radiolabeled 2-AA-LPC and arachidonic acid (AA) production, respectively, in the presence of calcium ion. Mass spectrometric analysis of the products of calcium-activated hydrolysis of endogenous mitochondrial phospholipids in transgenic iPLA(2)γ mitochondria revealed the robust production of AA, 2-AA-LPC, and 2-docosahexaenoyl-LPC that was over 10-fold greater than wild-type mitochondria. The mechanism-based inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (iPLA(2)γ selective), but not its enantiomer, (S)-BEL (iPLA(2)β selective) or pyrrolidine (cytosolic PLA(2)α selective), markedly attenuated Ca(2+)-dependent fatty acid release and polyunsaturated LPC production. Moreover, Ca(2+)-induced iPLA(2)γ activation was accompanied by the production of downstream eicosanoid metabolites that were nearly completely ablated by (R)-BEL or by genetic ablation of iPLA(2)γ. Intriguingly, Ca(2+)-induced iPLA(2)γ activation was completely inhibited by long-chain acyl-CoA (IC(50) ∼20 μm) as well as by a nonhydrolyzable acyl-CoA thioether analog. Collectively, these results demonstrate that mitochondrial iPLA(2)γ is activated by divalent cations and inhibited by acyl

  4. Creatine, similarly to ketamine, affords antidepressant-like effects in the tail suspension test via adenosine A₁ and A2A receptor activation.

    PubMed

    Cunha, Mauricio P; Pazini, Francis L; Rosa, Julia M; Ramos-Hryb, Ana B; Oliveira, Ágatha; Kaster, Manuella P; Rodrigues, Ana Lúcia S

    2015-06-01

    The benefits of creatine supplementation have been reported in a broad range of central nervous systems diseases, including depression. A previous study from our group demonstrated that creatine produces an antidepressant-like effect in the tail suspension test (TST), a predictive model of antidepressant activity. Since depression is associated with a dysfunction of the adenosinergic system, we investigated the involvement of adenosine A1 and A2A receptors in the antidepressant-like effect of creatine in the TST. The anti-immobility effect of creatine (1 mg/kg, po) or ketamine (a fast-acting antidepressant, 1 mg/kg, ip) in the TST was prevented by pretreatment of mice with caffeine (3 mg/kg, ip, nonselective adenosine receptor antagonist), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (2 mg/kg, ip, selective adenosine A1 receptor antagonist), and 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo-{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)-phenol (ZM241385) (1 mg/kg, ip, selective adenosine A2A receptor antagonist). In addition, the combined administration of subeffective doses of creatine and adenosine (0.1 mg/kg, ip, nonselective adenosine receptor agonist) or inosine (0.1 mg/kg, ip, nucleoside formed by the breakdown of adenosine) reduced immobility time in the TST. Moreover, the administration of subeffective doses of creatine or ketamine combined with N-6-cyclohexyladenosine (CHA) (0.05 mg/kg, ip, selective adenosine A1 receptor agonist), N-6-[2-(3,5-dimethoxyphenyl)-2-(methylphenyl)ethyl]adenosine (DPMA) (0.1 mg/kg, ip, selective adenosine A2A receptor agonist), or dipyridamole (0.1 μg/mouse, icv, adenosine transporter inhibitor) produced a synergistic antidepressant-like effect in the TST. These results indicate that creatine, similarly to ketamine, exhibits antidepressant-like effect in the TST probably mediated by the activation of both adenosine A1 and A2A receptors, further reinforcing the potential of targeting the purinergic system to the management of mood disorders.

  5. Neuroinflammatory pathways in binge alcohol-induced neuronal degeneration: oxidative stress cascade involving aquaporin, brain edema, and phospholipase A2 activation.

    PubMed

    Collins, Michael A; Neafsey, Edward J

    2012-01-01

    Chronic binge alcohol exposure in adult rat models causes neuronal degeneration in the cortex and hippocampus that is not reduced by excitotoxic receptor antagonists, but is prevented by antioxidants. Neuroinflammatory (glial-neuronal) signaling pathways are believed to underlie the oxidative stress and brain damage. Based on our experimental results as well as increased knowledge about the pro-neuroinflammatory potential of glial water channels, we propose that induction of aquaporin-4 can be a critical initiating factor in alcohol's neurotoxic effects, through the instigation of cellular edema-based neuroinflammatory cascades involving increased phospholipase A2 activities, polyunsaturated fatty acid release/membrane depletion, decreased prosurvival signaling, and oxidative stress. A testable scheme for this pathway is presented that incorporates recent findings in the alcohol-brain literature indicating a role for neuroimmune activation (upregulation of NF-kappaB, proinflammatory cytokines, and toll-like receptors). We present the argument that such neuroimmune activation could be associated with or even dependent on increased aquaporin-4 and glial swelling as well.

  6. Activation of Th1 and Tc1 cell adenosine A2A receptors directly inhibits IL-2 secretion in vitro and IL-2-driven expansion in vivo.

    PubMed

    Erdmann, Andreas A; Gao, Zhan-Guo; Jung, Unsu; Foley, Jason; Borenstein, Todd; Jacobson, Kenneth A; Fowler, Daniel H

    2005-06-15

    To evaluate the direct effect of adenosine on cytokine-polarized effector T cells, murine type 1 helper T cells (Th1) and type 1 cytotoxic T lymphocytes (Tc1) and Th2/Tc2 cells were generated using an antigen-presenting cell (APC)-free method. Tc1 and Tc2 cells had similar adenosine signaling, as measured by intracellular cyclic AMP (cAMP) increase upon adenosine A(2A) receptor agonism by CGS21680 (CGS). CGS greatly reduced Tc1 and Tc2 cell interleukin 2 (IL-2) and tumor necrosis factor alpha (TNF-alpha) secretion, with nominal effect on interferon gamma (IFN-gamma) secretion. Tc2 cell IL-4 and IL-5 secretion was not reduced by CGS, and IL-10 secretion was moderately reduced. Agonist-mediated inhibition of IL-2 and TNF-alpha secretion occurred via A(2A) receptors, with no involvement of A(1), A(2B), or A(3) receptors. Adenosine agonist concentrations that abrogated cytokine secretion did not inhibit Tc1 or Tc2 cell cytolytic function. Adenosine modulated effector T cells in vivo, as CGS administration reduced CD4(+)Th1 and CD8(+)Tc1 cell expansion to alloantigen and, in a separate model, reduced antigen-specific CD4(+) Th1 cell numbers. Remarkably, agonist-mediated T-cell inhibition was abrogated by in vivo IL-2 therapy. Adenosine receptor activation therefore preferentially inhibits type I cytokine secretion, most notably IL-2. Modulation of adenosine receptors may thus represent a suitable target primarily for inflammatory conditions mediated by Th1 and Tc1 cells.

  7. Inhibition of small HA fragment activity and stimulation of A2A adenosine receptor pathway limit apoptosis and reduce cartilage damage in experimental arthritis.

    PubMed

    Campo, Giuseppe M; Micali, Antonio; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Pisani, Antonina; Bruschetta, Antongiulio; Calatroni, Alberto; Puzzolo, Domenico; Campo, Salvatore

    2015-05-01

    Recent studies have found that the inactivation of small hyaluronan (HA) fragments originating from native HA during inflammation reduced the inflammatory response in models of experimental arthritis. The stimulation of adenosine receptors A2A reduced inflammation by inhibiting NF-kB activation. The combination of both treatments was significantly more effective than either of the individual treatments. The aim of this study was to further investigate the effects of a combined treatment using the HA inhibitor Pep-1 and a selective A2AR agonist (CV-1808) on the structure and ultrastructure of the articular cartilage and on apoptosis in a model of collagen-induced arthritis (CIA) in mice. Arthritic mice were treated with Pep-1 and/or CV-1808 intraperitoneally daily for 20 days. At day 35, the hind limbs were processed for light microscopy (hematoxylin/eosin and Safranin-O-Fast Green) and for transmission and scanning electron microscopy. CIA increased IL-6, caspase-3 and caspase-7 mRNA expression and the related protein levels in arthritic articular cartilage, and significantly increased concentrations of Bcl-2-associated X protein (Bax), while B cell-lymphoma-2 protein (Bcl-2) was markedly reduced. The combined Pep-1/CV-1808 treatment significantly reduced CIA injury, particularly at the highest doses, demonstrated by the presence of Safranin-O-positive cartilage, with a smooth surface and normal chondrocytes in the superficial, intermediate and deep zones. Morphological data and histological scoring were strongly supported by the reduction in inflammation and apoptotic markers. The results further support the role of HA degradation and A2A receptors in arthritis.

  8. Novel chromium doped perovskites A2ZnTiO6 (A = Pr, Gd): Synthesis, crystal structure and photocatalytic activity under simulated solar light irradiation

    NASA Astrophysics Data System (ADS)

    Zhu, Hekai; Fang, Minghao; Huang, Zhaohui; Liu, Yan'gai; Chen, Kai; Guan, Ming; Tang, Chao; Zhang, Lina; Wang, Meng

    2017-01-01

    Double perovskite related oxides A2ZnTiO6 (A = Pr, Gd) have been successfully synthesized by solid state reaction and investigated as photocatalysts for the first time. The two layered titanates mainly demonstrate absorbances under UV irradiation, except for several sharp absorption bands above 400 nm for Pr2ZnTiO6. Therefore, a series of photocatalysts by doping A2ZnTiO6 (A = Pr, Gd) with Cr have been developed in the hope to improve their absorption in the visible light region. The successful incorporation of Cr was detected by XRD and XPS, and the prepared samples have also been characteriazed by SEM, UV-vis DRS and PL. The characterization results suggested that Cr was present mainly in the form of Cr3+, with only a small amount of Cr6+ species. It served as an efficient dopant for the extension of visible light absorbance and improved photocatalytic activities under solar light irradiation. For both Pr2ZnTiO6 and Gd2ZnTiO6, the valence band (VB) was composed of hybridized states of the Zn 3d, O 2p and the conduction band (CB) has major contribution from Zn 4s, Ti 3d orbitals. For Cr doped samples, the newly formed spin-polarized valence band in the middle of the band gap that primarily arises from Cr 3d orbitals was responsible for the improved optical and photocatalytic properties.

  9. Ectopically Expressed Pro-group X Secretory Phospholipase A2 Is Proteolytically Activated in Mouse Adrenal Cells by Furin-like Proprotein Convertases

    PubMed Central

    Layne, Joseph D.; Shridas, Preetha; Webb, Nancy R.

    2015-01-01

    Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA2 is produced as a pro-enzyme (pro-GX sPLA2) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA2 activation have not been identified. We previously reported that GX sPLA2 negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA2 expression construct (FLAG-pro-GX sPLA2), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA2 processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA2 processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA2 processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA2 to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA2 processing and sPLA2 activity in Y1 cells, and it significantly attenuated GX sPLA2-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA2 is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells. PMID:25623068

  10. Oxidations of p-alkoxyacylanilides catalyzed by human cytochrome P450 1A2: structure-activity relationships and simulation of rate constants of individual steps in catalysis.

    PubMed

    Yun, C H; Miller, G P; Guengerich, F P

    2001-04-10

    Human cytochrome P450 (P450) 1A2 is involved in the oxidation of many important drugs and carcinogens. The prototype substrate phenacetin is oxidized to an acetol as well as the O-dealkylation product [Yun, C.-H., Miller, G. P., and Guengerich, F. P. (2000) Biochemistry 39, 11319-11329]. In an effort to improve rates of catalysis of P450 1A2 enzymes, we considered a set of p-alkoxyacylanilide analogues of phenacetin and found that variations in the O-alkyl and N-acyl substituents altered the rates of the two oxidation reactions and the ratio of acetol/phenol products. Moving one methylene group of phenacetin from the O-alkyl group to the N-acyl moiety increased rates of both oxidations approximately 5-fold and improved the coupling efficiency (oxidation products formed/NADPH consumed) from 6% to 38%. Noncompetitive kinetic deuterium isotope effects of 2-3 were measured for all O-dealkylation reactions examined with wild-type P450 1A2 and the E225I mutant, which has 6-fold higher activity. A trend of decreasing kinetic deuterium isotope effect for E225I > wild-type > mutant D320A was observed for O-demethylation of p-methoxyacetanilide, which follows the trend for k(cat). The set of O-dealkylation and acetol formation results for wild-type P450 1A2 and the E225I mutant with several of the protiated and deuterated substrates were fit to a model developed for the basic catalytic cycle and a set of microscopic rate constants in which the only variable was the rate of product formation (substrate oxygenation, including hydrogen abstraction). In this model, k(cat) is considerably less than any of the microscopic rate constants and is affected by several individual rate constants, including the rate of formation of the oxygenating species, the rate of substrate oxidation by the oxygenating species, and the rates of generation of reduced oxygen species (H(2)O(2), H(2)O). This analysis of the effects of the individual rate constants provides a framework for consideration of

  11. Expression, purification and refolding of active durum wheat (Triticum durum Desf.) secretory phospholipase A2 from inclusion bodies of Escherichia coli.

    PubMed

    Verlotta, Angelo; Trono, Daniela

    2014-09-01

    Recently, a durum wheat (Triticum durum Desf.) secretory phospholipase A2 (TdsPLA2III) was identified in leaves as potentially involved in plant responses to conditions of limiting water supply. Therefore, to allow future functional studies on TdsPLA2III and shed further light on the involvement of sPLA2 isoforms in specific plant functions, here we report a protocol for the overexpression of TdsPLA2III in Escherichia coli in the form of inclusion bodies, and for its purification and refolding. The use of the Gateway system (Invitrogen) allows the expression of a large quantity of the mature form (without the signal peptide) of TdsPLA2III with an N-terminal 6×His-tag, for purification using Ni-affinity chromatography. The purified recombinant 6×His-TdsPLA2III fusion protein is then refolded using a step-wise dialysis approach. About 40mg purified and active protein was obtained from 1L of cell culture. This recombinant 6×His-TdsPLA2III protein shows PLA2 activity, as it can hydrolyze linoleate from the sn-2 position of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine. Moreover, it has some features that are typical of other known plant sPLA2s: Ca(2+)-dependence, inhibition by the disulfide bond reducing agent dithiothreitol, and resistance to high temperature.

  12. Disulfide-Trapping Identifies a New, Effective Chemical Probe for Activating the Nuclear Receptor Human LRH-1 (NR5A2)

    PubMed Central

    de Jesus Cortez, Felipe; Suzawa, Miyuki; Irvy, Sam; Bruning, John M.; Sablin, Elena; Jacobson, Matthew P.; Fletterick, Robert J.; Ingraham, Holly A.

    2016-01-01

    Conventional efforts relying on high-throughput physical and virtual screening of large compound libraries have failed to yield high-efficiency chemical probes for many of the 48 human nuclear receptors. Here, we investigated whether disulfide-trapping, an approach new to nuclear receptors, would provide effective lead compounds targeting human liver receptor homolog 1 (hLRH-1, NR5A2). Despite the fact that hLRH-1 contains a large ligand binding pocket and binds phospholipids with high affinity, existing synthetic hLRH-1 ligands are of limited utility due to poor solubility, low efficacy or significant off-target effects. Using disulfide-trapping, we identified a lead compound that conjugates with remarkably high-efficiency to a native cysteine residue (Cys346) lining the hydrophobic cavity in the ligand binding domain of hLRH-1. Guided by computational modeling and cellular assays, the lead compound was elaborated into ligands PME8 and PME9 that bind hLRH-1 reversibly (no cysteine reactivity) and increase hLRH-1 activity in cells. When compared with the existing hLRH-1 synthetic agonist RJW100, both PME8 and PME9 showed comparable induction of the LRH-1 dependent target gene CYP24A1 in human HepG2 cells, beginning as early as 3 h after drug treatment. The induction is specific as siRNA-mediated knock-down of hLRH-1 renders both PME8 and PME9 ineffective. These data show that PME8 and PME9 are potent activators of hLRH-1 and suggest that with further development this lead series may yield useful chemical probes for manipulating LRH-1 activity in vivo. PMID:27467220

  13. Inhibition of serine-peptidase activity enhances the generation of a survivin-derived HLA-A2-presented CTL epitope in colon-carcinoma cells.

    PubMed

    Preta, G; Marescotti, D; Fortini, C; Carcoforo, P; Castelli, C; Masucci, M; Gavioli, R

    2008-12-01

    Cytotoxic T lymphocytes eliminate tumor cells expressing antigenic peptides in the context of MHC-I molecules. Peptides are generated during protein degradation by the proteasome and resulting products, surviving cytosolic amino-peptidases activity, may be presented by MHC-I molecules. The MHC-I processing pathway is altered in a large number of malignancies and modulation of antigen generation is one strategy employed by cells to evade immune control. In this study we analyzed the generation and presentation of a survivin-derived CTL epitope in HLA-A2-positive colon-carcinoma cells. Although all cell lines expressed the anti-apoptotic protein survivin, some tumors were poorly recognized by ELTLGEFLKL (ELT)-specific CTL cultures. The expression of MHC-I or TAP molecules was similar in all cell lines suggesting that tumors not recognized by CTLs may present defects in the generation of the ELT-epitope which could be due either to lack of generation or to subsequent degradation of the epitope. The cells were analyzed for the expression and the activity of extra-proteasomal peptidases. A significant overexpression and higher activity of TPPII was observed in colon-carcinoma cells which are not killed by ELT-specific CTLs, suggesting a possible role of TPPII in the degradation of the ELT-epitope. To confirm the role of TPPII in the degradation of the ELT-peptide, we showed that treatment of colon-carcinoma cells with a TPPII inhibitor resulted in a dose-dependent increased sensitivity to ELT-specific CTLs. These results suggest that TPPII is involved in degradation of the ELT-peptide, and its overexpression may contribute to the immune escape of colon-carcinoma cells.

  14. ASB14780, an Orally Active Inhibitor of Group IVA Phospholipase A2, Is a Pharmacotherapeutic Candidate for Nonalcoholic Fatty Liver Disease.

    PubMed

    Kanai, Shiho; Ishihara, Keiichi; Kawashita, Eri; Tomoo, Toshiyuki; Nagahira, Kazuhiro; Hayashi, Yasuhiro; Akiba, Satoshi

    2016-03-01

    We have previously shown that high-fat cholesterol diet (HFCD)-induced fatty liver and carbon tetrachloride (CCl4)-induced hepatic fibrosis are reduced in mice deficient in group IVA phospholipase A2 (IVA-PLA2), which plays a role in inflammation. We herein demonstrate the beneficial effects of ASB14780 (3-[1-(4-phenoxyphenyl)-3-(2-phenylethyl)-1H-indol-5-yl]propanoic acid 2-amino-2-(hydroxymethyl)propane-1,3-diol salt), an orally active IVA-PLA2 inhibitor, on the development of fatty liver and hepatic fibrosis in mice. The daily coadministration of ASB14780 markedly ameliorated liver injury and hepatic fibrosis following 6 weeks of treatment with CCl4. ASB14780 markedly attenuated the CCl4-induced expression of smooth muscle α-actin (α-SMA) protein and the mRNA expression of collagen 1a2, α-SMA, and transforming growth factor-β1 in the liver, and inhibited the expression of monocyte/macrophage markers, CD11b and monocyte chemotactic protein-1, while preventing the recruitment of monocytes/macrophages to the liver. Importantly, ASB14780 also reduced the development of fibrosis even in matured hepatic fibrosis. Additionally, ASB14780 also reduced HFCD-induced lipid deposition not only in the liver, but also in already established fatty liver. Furthermore, treatment with ASB14780 suppressed the HFCD-induced expression of lipogenic mRNAs. The present findings suggest that an IVA-PLA2 inhibitor, such as ASB14780, could be useful for the treatment of nonalcoholic fatty liver diseases, including fatty liver and hepatic fibrosis.

  15. Phospholipase A(2) activation by poultry particulate matter is mediated through extracellular signal-regulated kinase in lung epithelial cells: regulation of interleukin-8 release.

    PubMed

    Kotha, Sainath R; Piper, Melissa G; Patel, Rishi B; Sliman, Sean; Malireddy, Smitha; Zhao, Lingying; Baran, Christopher P; Nana-Sinkam, Patrick S; Wewers, Mark D; Romberger, Debra; Marsh, Clay B; Parinandi, Narasimham L

    2013-11-01

    The mechanisms of poultry particulate matter (PM)-induced agricultural respiratory disorders are not thoroughly understood. Hence, it is hypothesized in this article that poultry PM induces the release of interleukin-8 (IL-8) by lung epithelial cells that is regulated upstream by the concerted action of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK). To test this hypothesis, the widely used cultured human lung epithelial cells (A549) were chosen as the model system. Poultry PM caused a significant activation of PLA2 in A549 cells, which was attenuated by AACOCF3 (cPLA2 inhibitor) and PD98059 (ERK-1/2 upstream inhibitor). Poultry PM induced upstream ERK-1/2 phosphorylation and downstream cPLA2 serine phosphorylation, in a concerted fashion, in cells with enhanced association of ERK-1/2 and cPLA2. The poultry PM-induced cPLA2 serine phosphorylation and IL-8 release were attenuated by AACOCF3, PD98059, and by transfection with dominant-negative ERK-1/2 DNA in cells. The poultry PM-induced IL-8 release by the bone marrow-derived macrophages of cPLA2 knockout mice was significantly lower. For the first time, this study demonstrated that the poultry PM-induced IL-8 secretion by human lung epithelial cells was regulated by cPLA2 activation through ERK-mediated serine phosphorylation, suggesting a mechanism of airway inflammation among poultry farm workers.

  16. Synthesis, structural study and biological activity of new derivatives of chrysin containing a 2-mercaptopyridyl or 5-(trifluoromethyl)-2-mercaptopyridyl fragments

    NASA Astrophysics Data System (ADS)

    Valdez-Calderón, Alejandro; González-Montiel, Simplicio; Martínez-Otero, Diego; Martínez-Torres, Ataulfo; Vásquez-Pérez, José Manuel; Molina-Vera, Carlos; Torres-Valencia, J. Martín; Alvarado-Rodríguez, José G.; Cruz-Borbolla, Julian

    2016-04-01

    New derivatives of chrysin containing 2-mercaptopyridine (2a-2e) or 5-(trifluoromethyl)-2-mercaptopyridine (3a-3e) moieties were prepared from the reaction between bromides (1a-1e) and 2-mercaptopyridine or 5-(trifluoromethyl)-2-mercaptopyridine, respectively. Their structures were elucidated by NMR, IR and elemental analysis. The molecular structure of compounds 1a, 1c-1e, 2b-2e and 3a was determined by single-crystal X-ray diffraction studies. All rings in these structures are nearly coplanar and they showed an intramolecular hydrogen bond between the phenolic hydroxyl H atom and the carbonyl O atom that forms a six membered ring. The crystal packing also showed a wide variety of intermolecular contacts such as C-H⋯A, π-π, C-H⋯π and lone pair⋯π interactions which were supported by quantum theory of atoms in molecules (QTAIM), Hirshfeld surface, and fingerprint plot analyses. Biological activity of all compounds was tested in growth assays of the nematode Caenorhabiditis elegans. Compounds 2e, 3b and 3c inhibited larval development.

  17. Assembly of an Intact Golgi Complex Requires Phospholipase A2 (PLA2) Activity, Membrane Tubules, and Dynein-Mediated Microtubule Transport

    PubMed Central

    Judson, Bret L.; Brown, William J.

    2009-01-01

    Previous studies have shown that treatment of mammalian cells with phospholipase A2 (PLA2) antagonists cause the normally interconnected Golgi ribbon to break up into large fragments of stacked Golgi cisternae (“mini-stacks”) that remain located in the juxtanuclear region. Using the reversible PLA2 antagonist, ONO-RS-082 (ONO) and live-cell, time-lapse microscopy to image the Golgi reassembly process, we found that Golgi mini-stacks underwent a burst of membrane tubule formation following washout of ONO: before washout only 4.3 ± 3.8 tubules/cell/10 min were formed, whereas after washout 29.9 ± 11.9 tubules/cell/10 min formed. These membranes tubules formed bridges between physically separate mini-stacks, thus mediating their coalescence into intact Golgi ribbons. Formation of inter-stack tubules and an intact Golgi ribbon was also facilitated by microtubules because treatment with nocodazole significantly inhibited both processes. This microtubule-dependent process was also dependent on dynein because the dynein inhibitor nordihydroguaiaretic acid (NDGA) inhibited reassembly. These studies show that a late stage of Golgi assembly occurs via membrane tubules, whose formation is dependent on PLA2 activity and microtubules. Considering these results together, we concluded that the maintenance and assembly of normal Golgi architecture is dependent on the PLA2-mediated, dynamic formation of inter-Golgi membrane tubules. PMID:19747452

  18. Mechanism of inhibition of protein kinase C by 14-3-3 isoforms. 14-3-3 isoforms do not have phospholipase A2 activity.

    PubMed Central

    Robinson, K; Jones, D; Patel, Y; Martin, H; Madrazo, J; Martin, S; Howell, S; Elmore, M; Finnen, M J; Aitken, A

    1994-01-01

    The ability of individual members of the 14-3-3 protein family to inhibit protein kinase C (PKC) has been studied by using a synthetic peptide based on the specific 80 kDa substrate for PKC (MARCKS protein) in two different assay systems. Recombinant 14-3-3 and isoforms renatured by a novel method after separation by reverse-phase h.p.l.c. were studied. The detailed effects of diacylglycerol and the phorbol ester phorbol 12-myristate 13-acetate on the inhibition were also investigated. This suggests that one of the sites of interaction of 14-3-3 may be the cysteine-rich (C1) domain in PKC. Since a region in secreted phospholipase A2 (PLA2) shares similarity with this domain, the ability of 14-3-3 to interact with mammalian PLA2 was studied. Cytosolic PLA2 has some similarity to the C2 region of PKC, and the effect of 14-3-3 on this class of PLA2 was also analysed. In contrast with a previous report, no PLA2 activity was found in brain 14-3-3, nor in any of the recombinant proteins tested. These include zeta 14-3-3 isoform, on which the original observation was made. Images Figure 2 PMID:8192676

  19. TNF-α induces cytosolic phospholipase A2 expression via Jak2/PDGFR-dependent Elk-1/p300 activation in human lung epithelial cells.

    PubMed

    Yang, Chuen-Mao; Lee, I-Ta; Chi, Pei-Ling; Cheng, Shin-Ei; Hsiao, Li-Der; Hsu, Chih-Kai

    2014-03-15

    Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA2 expression in human lung epithelial cells (HPAEpiCs) were not completely understood. Here, we demonstrated that TNF-α induced cPLA2 mRNA and protein expression, promoter activity, and PGE2 secretion in HPAEpiCs. These responses induced by TNF-α were inhibited by pretreatment with the inhibitor of Jak2 (AG490), platelet-derived growth factor receptor (PDGFR) (AG1296), phosphoinositide 3 kinase (PI3K) (LY294002), or MEK1/2 (PD98059) and transfection with siRNA of Jak2, PDGFR, Akt, or p42. We showed that TNF-α markedly stimulated Jak2, PDGFR, Akt, and p42/p44 MAPK phosphorylation, which were attenuated by their respective inhibitors. Moreover, TNF-α stimulated Akt activation via a Jak2/PDGFR pathway in HPAEpiCs. In addition, TNF-α-induced p42/p44 MAPK phosphorylation was reduced by AG1296 or LY294002. On the other hand, TNF-α could induce Akt and p42/p44 MAPK translocation from the cytosol into the nucleus, which was inhibited by AG490, AG1296, or LY294002. Finally, we showed that TNF-α stimulated Elk-1 phosphorylation, which was reduced by LY294002 or PD98059. We also observed that TNF-α time dependently induced p300/Elk-1 and p300/Akt complex formation in HPAEpiCs, which was reduced by AG490, AG1296, or LY294002. The activity of cPLA2 protein upregulated by TNF-α was reflected on the PGE2 release, which was reduced by AG490, AG1296, LY294002, or PD98059. Taken together, these results demonstrated that TNF-α-induced cPLA2 expression and PGE2 release were mediated through a Jak2/PDGFR/PI3K/Akt/p42/p44 MAPK/Elk-1 pathway in HPAEpiCs.

  20. 26 CFR 1.367(a)-2T - Exception for transfers of property for use in the active conduct of a trade or business...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... for the purpose of combining the research activities of X and Y. Z contracts all of its operational and research activities to Y for an arm's-length fee. Z's activities do not constitute the active... business activities, future plant replacement, or future business contingencies. (c) Property...

  1. Prediction of cross-recognition of peptide-HLA A2 by Melan-A-specific cytotoxic T lymphocytes using three-dimensional quantitative structure-activity relationships.

    PubMed

    Fagerberg, Theres; Zoete, Vincent; Viatte, Sebastien; Baumgaertner, Petra; Alves, Pedro M; Romero, Pedro; Speiser, Daniel E; Michielin, Olivier

    2013-01-01

    The cross-recognition of peptides by cytotoxic T lymphocytes is a key element in immunology and in particular in peptide based immunotherapy. Here we develop three-dimensional (3D) quantitative structure-activity relationships (QSARs) to predict cross-recognition by Melan-A-specific cytotoxic T lymphocytes of peptides bound to HLA A*0201 (hereafter referred to as HLA A2). First, we predict the structure of a set of self- and pathogen-derived peptides bound to HLA A2 using a previously developed ab initio structure prediction approach [Fagerberg et al., J. Mol. Biol., 521-46 (2006)]. Second, shape and electrostatic energy calculations are performed on a 3D grid to produce similarity matrices which are combined with a genetic neural network method [So et al., J. Med. Chem., 4347-59 (1997)] to generate 3D-QSAR models. The models are extensively validated using several different approaches. During the model generation, the leave-one-out cross-validated correlation coefficient (q (2)) is used as the fitness criterion and all obtained models are evaluated based on their q (2) values. Moreover, the best model obtained for a partitioned data set is evaluated by its correlation coefficient (r = 0.92 for the external test set). The physical relevance of all models is tested using a functional dependence analysis and the robustness of the models obtained for the entire data set is confirmed using y-randomization. Finally, the validated models are tested for their utility in the setting of rational peptide design: their ability to discriminate between peptides that only contain side chain substitutions in a single secondary anchor position is evaluated. In addition, the predicted cross-recognition of the mono-substituted peptides is confirmed experimentally in chromium-release assays. These results underline the utility of 3D-QSARs in peptide mimetic design and suggest that the properties of the unbound epitope are sufficient to capture most of the information to determine the

  2. Ca2+ signalling in K562 human erythroleukaemia cells: effect of dimethyl sulphoxide and role of G-proteins in thrombin- and thromboxane A2-activated pathways.

    PubMed Central

    Thomas, C P; Dunn, M J; Mattera, R

    1995-01-01

    The human leukaemic cell line K562 is a pluripotent stem cell with the potential to mature along a megakaryocytic or erythroid line. In these cells, thrombin and U46619 (9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F2 alpha), a thromboxane A2 analogue, increased intracellular Ca2+ in a rapid and concentration-dependent manner. The peak transient observed with both thrombin and U46619 was preserved upon stimulation in the absence of extracellular calcium and blunted with phorbol myristate acetate, suggestive of activation of phospholipase C. Short-term treatment with leupeptin abolished the calcium response to thrombin, but did not alter that to U46619. Both pertussis toxin (PT) and DMSO pretreatment inhibited thrombin- but not U46619-stimulated intracellular calcium elevation, indicating that these agonists signal through different G-proteins. Western blot analysis of crude membranes from K562 cells revealed the presence of G12 alpha and G13 alpha; the other known PT-substrates, Gi1 alpha and G0 alpha, were not detected. Consistent with this observation, ADP-ribosylation experiments revealed the presence of two PT substrates which co-migrated with human erythrocyte G12 alpha and G13 alpha. An antibody raised against Gq/11 alpha, a subfamily of G-protein alpha subunits unmodified by PT, specifically recognized 42 kDa protein(s) in K562 cells. PCR amplification of reverse-transcribed K562 RNA followed by DNA sequencing showed that these cells express messages for both Gq alpha and G11 alpha. Treatment of K562 cells with DMSO reduced the levels of thrombin receptor mRNA, without simultaneous changes in the expression of G12 alpha and G13 alpha. We have thus identified Ca(2+)-mobilizing agonists and related G-proteins in K562 cells, together with changes induced by DMSO in this signalling pathway. Images Figure 5 Figure 6 Figure 7 Figure 9 PMID:7492305

  3. HsfA2 Controls the Activity of Developmentally and Stress-Regulated Heat Stress Protection Mechanisms in Tomato Male Reproductive Tissues1[OPEN

    PubMed Central

    Simm, Stefan; Paupière, Marine Josephine; Theres, Klaus; Bovy, Arnaud; Schleiff, Enrico; Scharf, Klaus-Dieter

    2016-01-01

    Male reproductive tissues are more sensitive to heat stress (HS) compared to vegetative tissues, but the basis of this phenomenon is poorly understood. Heat stress transcription factors (Hsfs) regulate the transcriptional changes required for protection from HS. In tomato (Solanum lycopersicum), HsfA2 acts as coactivator of HsfA1a and is one of the major Hsfs accumulating in response to elevated temperatures. The contribution of HsfA2 in heat stress response (HSR) and thermotolerance was investigated in different tissues of transgenic tomato plants with suppressed HsfA2 levels (A2AS). Global transcriptome analysis and immunodetection of two major Hsps in vegetative and reproductive tissues showed that HsfA2 regulates subsets of HS-induced genes in a tissue-specific manner. Accumulation of HsfA2 by a moderate HS treatment enhances the capacity of seedlings to cope with a subsequent severe HS, suggesting an important role for HsfA2 in regulating acquired thermotolerance. In pollen, HsfA2 is an important coactivator of HsfA1a during HSR. HsfA2 suppression reduces the viability and germination rate of pollen that received the stress during the stages of meiosis and microspore formation but had no effect on more advanced stages. In general, pollen meiocytes and microspores are characterized by increased susceptibility to HS due to their lower capacity to induce a strong HSR. This sensitivity is partially mitigated by the developmentally regulated expression of HsfA2 and several HS-responsive genes mediated by HsfA1a under nonstress conditions. Thereby, HsfA2 is an important factor for the priming process that sustains pollen thermotolerance during microsporogenesis. PMID:26917685

  4. Structure-activity relationship studies on 1-heteroaryl-3-phenoxypropan-2-ones acting as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: replacement of the activated ketone group by other serine traps.

    PubMed

    Sundermann, Tom; Hanekamp, Walburga; Lehr, Matthias

    2016-08-01

    Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are serine hydrolases. cPLA2α is involved in the generation of pro-inflammatory lipid mediators, FAAH terminates the anti-inflammatory effects of endocannabinoids. Therefore, inhibitors of these enzymes may represent new drug candidates for the treatment of inflammation. We have reported that certain 1-heteroarylpropan-2-ones are potent inhibitors of cPLA2α and FAAH. The serine reactive ketone group of these compounds, which is crucial for enzyme inhibition, is readily metabolized resulting in inactive alcohol derivatives. In order to obtain metabolically more stable inhibitors, we replaced this moiety by α-ketoheterocyle, cyanamide and nitrile serine traps. Investigations on activity and metabolic stability of these substances revealed that in all cases an increased metabolic stability was accompanied by a loss of inhibitory potency against cPLA2α and FAAH, respectively.

  5. Effect of polyunsaturated fatty acids ω-3 on the induction of activity and expression of CYP1A1 and CYP1A2 genes in the liver of rats under the influence of indole-3-carbinol.

    PubMed

    Kravchenko, L V; Tutel'yan, V A; Trusov, N V; Guseva, G V; Aksenov, I V

    2014-01-01

    Supplementation of the ration with eicosapentaenoic and docosahexaenoic ω-3 polyunsaturated fatty acids (PUFA) in doses of 0.3 and 1 g/kg body weight for 4 weeks had no effect on ethoxyresorufin O-dealkylase (EROD) activity and expression of the CYP1A1 gene in male Wistar rats, but caused a dose-dependent increase in methoxyresorufin O-dealkylase (MROD) activity of CYP1A2 (by 28 and 73%, respectively) without significant changes in CYP1A2 mRNA expression. ω-3 PUFA had no effect on the indole-3-carbinol-induced (20 mg/kg body weight over the last 7 days of the experiment) EROD activity and expression of CYP1A1 mRNA. The indole-3-carbinol-induced MROD activity was shown to increase by 6.2 times in rats not receiving ω-3 PUFA and only by 3.9 and 2.7 times in animals receiving ω-3 PUFA. The indole-3-carbinol-induced expression of CYP1A2 mRNA slightly increased in animals receiving ω-3 PUFA. Our results suggest that the effect of ω-3 PUFA on the induced and basal activity of CYP1A2 is not related to modulation of CYP1A2 gene expression.

  6. Overexpression of SLC34A2 is an independent prognostic indicator in bladder cancer and its depletion suppresses tumor growth via decreasing c-Myc expression and transcriptional activity.

    PubMed

    Ye, Wen; Chen, Cui; Gao, Ying; Zheng, Zou-Shan; Xu, Yi; Yun, Miao; Weng, Hui-Wen; Xie, Dan; Ye, Sheng; Zhang, Jia-Xing

    2017-02-02

    Solute carrier family 34 member 2 (SLC34A2), a pH-sensitive sodium-dependent phosphate transporter, is associated with several human cancers. In this study, we investigate the clinical significance of SLC34A2 and its function in human bladder cancer (BC). The expression dynamics of SLC34A2 were examined in two independent cohorts of BC samples by quantitative PCR, western blotting and immunohistochemical staining. In the training cohort (156 cases), we applied the X-tile program software to assess the optimal cutoff points for biomarkers in order to accurately classify patients according to clinical outcome. In the validation cohort (130 cases), the cutoff score derived from X-title analysis was investigated to determine the association of SLC34A2 expression with survival outcome. A series of in vitro and in vivo assays were then performed to elucidate the function of SLC34A2 in BC and its underlying mechanisms. Results showed that SLC34A2 was significantly upregulated in BC cell lines and clinical samples. In both two cohorts of BC samples, high expression of SLC34A2 was associated with large tumor size, advanced T status and poor patients' survival. The depletion of SLC34A2 in BC suppressed cellular viability, colony formation and anchorage-independent growth in vitro, and inhibited xenograft tumor growth in vivo, whereas overexpression of SLC34A2 had the converse effect. Simultaneously, downregulation of SLC34A2 decreased the transcriptional activity and protein expression level of c-Myc in BC cells, whereas restoration of c-Myc expression could compromise the anti-proliferation effect of SLC34A2 depletion. Furthermore, miR-214 was proved as a negative regulator of SLC34A2. Our present study illustrated that SLC34A2 has an important role in promoting proliferation and tumorigenicity of BC, and may represent a novel therapeutic target for this disease.

  7. 26 CFR 1.367(a)-2T - Exception for transfers of property for use in the active conduct of a trade or business...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... could constitute) an independent economic enterprise carried on for profit. For example, the activities... officers and employees of the corporation carry out substantial managerial and operational activities. A... determining whether the officers and employees of the corporation carry out substantial managerial...

  8. Extraction and Inhibition of Enzymatic Activity of Botulinum Neurotoxins/A1, /A2, and /A3 by a Panel of Monoclonal Anti-BoNT/A Antibodies

    DTIC Science & Technology

    2009-04-01

    a disease that is contracted by ingestion of food containing the toxin [1,2], colonization of the bacteria in the gastrointestinal tract of infants...In addition, commer- cially purified BoNT/A1 (strain Hall) and BoNT/A2 (strain FRI- honey ) complex toxins from Metabiologics (Madison, WI) were used

  9. Effects of the adenosine A2A receptor antagonist SCH 58621 on cyclooxygenase-2 expression, glial activation, and brain-derived neurotrophic factor availability in a rat model of striatal neurodegeneration.

    PubMed

    Minghetti, Luisa; Greco, Anita; Potenza, Rosa Luisa; Pezzola, Antonella; Blum, David; Bantubungi, Kadiombo; Popoli, Patrizia

    2007-05-01

    Inhibition of adenosine A2A receptors (A2ARs) is neuroprotective in several experimental models of striatal diseases. However, the mechanisms elicited by A2AR blockade are only partially known, and critical aspects about the potential beneficial effects of A2AR antagonism in models of neurodegeneration still await elucidation. In the present study, we analyzed the influence of the selective A2AR antagonist SCH 58261 in a rat model of striatal excitotoxicity obtained by unilateral intrastriatal injection of quinolinic acid (QA). We found that SCH 58261 differently affected the expression of cyclooxygenase-2 (COX-2) induced by QA in cortex and striatum. The antagonist enhanced COX-2 expression in cortical neurons and prevented it in striatal microglia-like cells. Similarly, SCH 58261 differently regulated astrogliosis and microglial activation in the 2 brain regions. In addition, the A2AR antagonist prevented the QA-induced increase in striatal brain-derived neurotrophic factor levels. Because COX-2 activity has been linked to excitotoxic processes and because brain-derived neurotrophic factor depletion has been observed in mouse models as well as in patients with Huntington disease, we suggest that the final outcome of A2AR blockade (namely neuroprotection vs neurodegeneration) is likely to depend on the balance among its various and region-specific effects.

  10. Chronic and acute adenosine A2A receptor blockade prevents long-term episodic memory disruption caused by acute cannabinoid CB1 receptor activation.

    PubMed

    Mouro, Francisco M; Batalha, Vânia L; Ferreira, Diana G; Coelho, Joana E; Baqi, Younis; Müller, Christa E; Lopes, Luísa V; Ribeiro, Joaquim A; Sebastião, Ana M

    2017-05-01

    Cannabinoid-mediated memory impairment is a concern in cannabinoid-based therapies. Caffeine exacerbates cannabinoid CB1 receptor (CB1R)-induced memory deficits through an adenosine A1 receptor-mediated mechanism. We now evaluated how chronic or acute blockade of adenosine A2A receptors (A2ARs) affects long-term episodic memory deficits induced by a single injection of a selective CB1R agonist. Long-term episodic memory was assessed by the novel object recognition (NOR) test. Mice received an intraperitoneal (i.p.) injection of the CB1/CB2 receptor agonist WIN 55,212-2 (1 mg/kg) immediately after the NOR training, being tested for novelty recognition 24 h later. Anxiety levels were assessed by the Elevated Plus Maze test, immediately after the NOR. Mice were also tested for exploratory behaviour at the Open Field. For chronic A2AR blockade, KW-6002 (istradefylline) (3 mg/kg/day) was administered orally for 30 days; acute blockade of A2ARs was assessed by i.p. injection of SCH 58261 (1 mg/kg) administered either together with WIN 55,212-2 or only 30 min before the NOR test phase. The involvement of CB1Rs was assessed by using the CB1R antagonist, AM251 (3 mg/kg, i.p.). WIN 55,212-2 caused a disruption in NOR, an action absent in mice also receiving AM251, KW-6002 or SCH 58261 during the encoding/consolidation phase; SCH 58251 was ineffective if present during retrieval only. No effects were detected in the Elevated Plus maze or Open Field Test. The finding that CB1R-mediated memory disruption is prevented by antagonism of adenosine A2ARs, highlights a possibility to prevent cognitive side effects when therapeutic application of CB1R drugs is desired.

  11. Crystal structure of phospholipase A2 complex with the hydrolysis products of platelet activating factor: equilibrium binding of fatty acid and lysophospholipid-ether at the active site may be mutually exclusive.

    PubMed

    Pan, Ying H; Yu, Bao-Zhu; Berg, Otto G; Jain, Mahendra K; Bahnson, Brian J

    2002-12-17

    We have solved the 1.55 A crystal structure of the anion-assisted dimer of porcine pancreatic group IB phospholipase A2 (PLA2), complexed with the products of hydrolysis of the substrate platelet activating factor. The dimer contains five coplanar phosphate anions bound at the contact surface between the two PLA2 subunits. This structure parallels a previously reported anion-assisted dimer that mimics the tetrahedral intermediate of PLA2 bound to a substrate interface [Pan, Y. H., et al. (2001) Biochemistry 40, 609-617]. The dimer structure has a molecule of the product acetate bound in subunit A and the other product 1-octadecyl-sn-glycero-3-phosphocholine (LPC-ether) to subunit B. Therefore, this structure is of the two individual product binary complexes and not of a ternary complex with both products in one active site of PLA2. Protein crystals with bound products were only obtained by cocrystallization starting from the initial substrate. In contrast, an alternate crystal form was obtained when PLA2 was cocrystallized with LPC-ether and succinate, and this crystal form did not contain bound products. The product bound structure has acetate positioned in the catalytic site of subunit A such that one of its oxygen atoms is located 3.5 A from the catalytic calcium. Likewise, a longer than typical Ca-to-Gly(32) carbonyl distance of 3.4 A results in a final Ca coordination that is four-coordinate and has distorted geometry. The other oxygen of acetate makes hydrogen bonds with N(delta)(1)-His(48), O(delta)(1)-Asp(49), and the catalytic assisting water (w7). In contrast, the glycerophosphocholine headgroup of LPC-ether in subunit B makes no contacts with calcium or with the catalytic residues His(48) or Asp(49). The tail of the LPC-ether is located near the active site pocket with the last nine carbons of the sn-1- acyl chain refined in two alternate conformations. The remaining atoms of the LPC-ether product have been modeled into the solvent channel but have their

  12. Modulation of Ca2+-currents by sequential and simultaneous activation of adenosine A1 and A 2A receptors in striatal projection neurons.

    PubMed

    Hernández-González, O; Hernández-Flores, T; Prieto, G A; Pérez-Burgos, A; Arias-García, M A; Galarraga, E; Bargas, J

    2014-01-01

    D(1)- and D(2)-types of dopamine receptors are located separately in direct and indirect pathway striatal projection neurons (dSPNs and iSPNs). In comparison, adenosine A(1)-type receptors are located in both neuron classes, and adenosine A(2A)-type receptors show a preferential expression in iSPNs. Due to their importance for neuronal excitability, Ca(2+)-currents have been used as final effectors to see the function of signaling cascades associated with different G protein-coupled receptors. For example, among many other actions, D(1)-type receptors increase, while D(2)-type receptors decrease neuronal excitability by either enhancing or reducing, respectively, CaV1 Ca(2+)-currents. These actions occur separately in dSPNs and iSPNs. In the case of purinergic signaling, the actions of A(1)- and A(2A)-receptors have not been compared observing their actions on Ca(2+)-channels of SPNs as final effectors. Our hypotheses are that modulation of Ca(2+)-currents by A(1)-receptors occurs in both dSPNs and iSPNs. In contrast, iSPNs would exhibit modulation by both A(1)- and A2A-receptors. We demonstrate that A(1)-type receptors reduced Ca(2+)-currents in all SPNs tested. However, A(2A)-type receptors enhanced Ca(2+)-currents only in half tested neurons. Intriguingly, to observe the actions of A(2A)-type receptors, occupation of A(1)-type receptors had to occur first. However, A(1)-receptors decreased Ca(V)2 Ca(2+)-currents, while A(2A)-type receptors enhanced current through Ca(V)1 channels. Because these channels have opposing actions on cell discharge, these differences explain in part why iSPNs may be more excitable than dSPNs. It is demonstrated that intrinsic voltage-gated currents expressed in SPNs are effectors of purinergic signaling that therefore play a role in excitability.

  13. Isolation, properties and amino acid sequences of a phospholipase A2 and its homologue without activity from the venom of a sea snake, Laticauda colubrina, from the Solomon Islands.

    PubMed

    Takasaki, C; Kimura, S; Kokubun, Y; Tamiya, N

    1988-08-01

    A phospholipase A2, Laticauda colubrina phospholipase A2 II (LcPLA-II), and a phospholipase A2 homologue, Laticauda colubrina phospholipase A2 homologue I (LcPLH-I), were isolated from the venom of the yellow-lipped sea snake, Laticauda colubrina, from the Solomon Islands. LcPLA-II showed phospholipase A2 activity towards egg-yolk phosphatidylcholine (24 mumol/min per mg at optimal conditions at 37 degrees C) and lethal potency (LD50 45 micrograms/kg body wt. intravenously in mice). Both of the activities were lost by treatment with p-bromophenacyl bromide. LcPLH-I showed neither phospholipase A2 activity nor lethal potency at a dose of 4.5 mg/kg body wt. in mice. It was not modified by the treatment with p-bromophenacyl bromide. LcPLA-II and LcPLH-I bound Ca2+ at a 1:1 molar ratio with KCa values of 105 microM and 44 microM at pH 8.0 respectively. Elucidation of the amino acid sequences of these two proteins showed that each protein consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. The two sequences are different from each other at 22 residues and highly homologous to those from other sources. The essential histidine residue for the phospholipase A2 activity at position 48 is replaced by an asparagine residue in the homologue LcPLH-I. Details of the separation of the peptides obtained by proteinase digestions of LcPLA-II and LcPLA-I and the determination of their amino acid sequences are given in Supplementary Publication SUP 50145 (14 pages), which has been deposited at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.

  14. 2-Amino-N-pyrimidin-4-ylacetamides as A2A receptor antagonists: 1. Structure-activity relationships and optimization of heterocyclic substituents.

    PubMed

    Slee, Deborah H; Chen, Yongsheng; Zhang, Xiaohu; Moorjani, Manisha; Lanier, Marion C; Lin, Emily; Rueter, Jaimie K; Williams, John P; Lechner, Sandra M; Markison, Stacy; Malany, Siobhan; Santos, Mark; Gross, Raymond S; Jalali, Kayvon; Sai, Yang; Zuo, Zhiyang; Yang, Chun; Castro-Palomino, Julio C; Crespo, María I; Prat, Maria; Gual, Silvia; Díaz, José-Luis; Saunders, John

    2008-03-27

    Previously we have described a novel series of potent and selective A 2A receptor antagonists (e.g., 1) with excellent aqueous solubility. While these compounds are efficacious A 2A antagonists in vivo, the presence of an unsubstituted furyl moiety was a cause of some concern. In order to avoid the potential metabolic liabilities that could arise from an unsubstituted furyl moiety, an optimization effort was undertaken with the aim of replacing the unsubstituted furan with a more metabolically stable group while maintaining potency and selectivity. Herein, we describe the synthesis and SAR of a range of novel heterocyclic systems and the successful identification of a replacement for the unsubstituted furan moiety with a methylfuran or thiazole moiety while maintaining potency and selectivity.

  15. Pharmacophore generation, atom-based 3D-QSAR, HQSAR and activity cliff analyses of benzothiazine and deazaxanthine derivatives as dual A2A antagonists/MAO‑B inhibitors.

    PubMed

    Bhayye, S S; Roy, K; Saha, A

    2016-02-12

    Dual inhibition of A2A and MAO-B is an emerging strategy in neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). In this study, atom-based three-dimensional quantitative structure-activity relationship (3D-QSAR) and hologram quantitative structure-activity relationship (HQSAR) models were generated with benzothiazine and deazaxanthine derivatives. Based on activity against A2A and MAO-B, two statistically significant 3D-QSAR models (r(2) = 0.96, q(2) = 0.76 and r(2) = 0.91, q(2) = 0.63) and HQSAR models (r(2) = 0.93, q(2) = 0.68 and r(2) = 0.97, q(2) = 0.58) were developed. In an activity cliff analysis, structural outliers were identified by calculating the Mahalanobis distance for a pair of compounds with A2A and MAO-B inhibitory activities. The generated 3D-QSAR and HQSAR models, activity cliff analysis, molecular docking and dynamic studies for dual target protein inhibitors provide key structural scaffolds that serve as building blocks in designing drug-like molecules for neurodegenerative diseases.

  16. Regulation of activator protein-1 by 8-iso-prostaglandin E2 in a thromboxane A2 receptor-dependent and -independent manner

    SciTech Connect

    Weber, Thomas J.; Markillie, Lye MENG.

    2003-05-01

    The thromboxane (TX) A{sub 2} receptor (TP) encompasses two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP activity may be modulated by novel ligands, termed the isoprostanes, that paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes 8-iso-prostaglandin (PG)F{sub 2{alpha}} and 8-iso-PGE{sub 2} regulate the activity of TP isoforms expressed in Chinese hamster ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist [9,11-dideoxy-9{alpha},11{alpha}-methanoepoxy PGF{sub 2{alpha}} (U46619)] in CHO cells transfected with the human TP-P and TP-E receptors, and this response was fully inhibited by TP antagonists [1S-[1{alpha},2{beta}(Z),3{alpha},5{alpha}

  17. Pyranoflavones: A Group of Small-molecule Probes for Exploring the Active Site Cavities of Cytochrome P450 Enzymes 1A1, 1A2, and 1B1

    PubMed Central

    Liu, Jiawang; Taylor, Shannon F.; Dupart, Patrick S.; Arnold, Corey L.; Sridhar, Jayalakshmi; Jiang, Quan; Wang, Yuji; Skripnikova, Elena V.; Zhao, Ming; Foroozesh, Maryam

    2013-01-01

    Selective inhibition of P450 enzymes is the key to block the conversion of environmental procarcinogens to their carcinogenic metabolites in both animals and humans. To discover highly potent and selective inhibitors of P450s 1A1, 1A2, and 1B1, as well as to investigate active site cavities of these enzymes, 14 novel flavone derivatives were prepared as chemical probes. Fluorimetric enzyme inhibition assays were used to determine the inhibitory activities of these probes towards P450s 1A1, 1A2, 1B1, 2A6, and 2B1. A highly selective P450 1B1 inhibitor, 5-hydroxy-4′-propargyloxyflavone (5H4′FPE) was discovered. Some tested compounds also showed selectivity between P450s 1A1 and 1A2. Alpha-naphthoflavone-like and 5-hydroxyflavone derivatives preferentially inhibited P450 1A2, while beta-naphthoflavone-like flavone derivatives showed selective inhibition of P450 1A1. On the basis of structural analysis, the active site cavity models of P450 enzymes 1A1 and 1A2 were generated, demonstrating a planar long strip cavity and a planar triangular cavity, respectively. PMID:23600958

  18. Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1

    PubMed Central

    Milichovský, Jan; Bárta, František; Schmeiser, Heinz H.; Arlt, Volker M.; Frei, Eva; Stiborová, Marie; Martínek, Václav

    2016-01-01

    Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are NAD(P)H:quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using 32P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast

  19. Young patients with cystic fibrosis: attitude toward physical activity and influence on physical fitness and spirometric values of a 2-week training course.

    PubMed

    Stanghelle, J K; Winnem, M; Roaldsen, K; de Wit, S; Notgewitch, J H; Nilsen, B R

    1988-02-01

    The purpose of this investigation was to study the attitude toward physical activity in young patients with cystic fibrosis (CF) and the influence of 2 weeks of training on physical fitness and spirometric values. Two groups of CF patients with mean ages of 11 and 15 years, respectively (total of 13 patients, range 9-21 years), participated in the study. Two healthy control groups with mean ages of 11 and 14 years, respectively, performed the same tests as the CF patients. The CF patients answered questionnaires regarding their attitude toward the activities both during the camp and at home and accomplished spirometric and exercise tests. The CF childrens' parents were interviewed concerning their children's attitude toward physical activity. The attitude toward physical activity of the CF children was very positive both during the camps and at home. The CF patients had at least as good a motivation for exercise as the healthy controls. The physical fitness of the CF patients was reduced, but improved during the training period. The spirometric tests were unchanged during the first course, but increased significantly during the other camp, even if lung physiotherapy was stopped during the training period. The study indicates that CF patients can be motivated for physical exercise, even for training for endurance and at a high enough level to replace lung physiotherapy, at least for shorter periods.

  20. Activation of apoptosis, but not necrosis, during Mycobacterium tuberculosis infection correlated with decreased bacterial growth: role of TNF-alpha, IL-10, caspases and phospholipase A2.

    PubMed

    Arcila, Mary Luz; Sánchez, María Dulfary; Ortiz, Blair; Barrera, Luis Fernando; García, Luis F; Rojas, Mauricio

    2007-10-01

    Monocyte/macrophage cell death is an important event during mycobacterial infection. To get insights about the influence of mononuclear phagocyte maturation in this event we compared the response to Mycobacterium tuberculosis (Mtb) infection of fresh isolated monocytes and monocyte-derived macrophages (MDM) from healthy tuberculin positive individuals. Both monocytes and MDM underwent apoptosis, however, there was a higher numbers of apoptotic macrophages with active Caspases 8 and 9. We also compared Mtb-induced cell death in U937 pro-monocytes and PMA-differentiated cells (U937D). In response to Mtb infection, U937D cells underwent apoptosis and promonocytes both apoptosis and necrosis. There were high number of U937D cells producing TNF-alpha and high number of IL-10+ promonocytes. These evidences suggest that U937 could be a valid model to study the mechanisms that rule Mtb-induced cell death. Experiments with the cell line and fresh isolated mononuclear cells with pharmacological inhibitors showed that induction of necrosis involved calcium and cAMP signals resulting in IL-10 production. Necrosis also correlated with Caspase 3, PLA2 activity and bacterial growth. In U937D cells and monocytes from healthy donors there was activation of calcium, TNF-alpha and Caspase 8 activation and decreased bacterial load. Understanding the mechanisms that control the dichotomy events between apoptosis and necrosis/oncosis associated with cell maturity might open new strategies to better control the course of mycobacterial infections.

  1. Roles of the AMPA receptor subunit GluA1 but not GluA2 in synaptic potentiation and activation of ERK in the anterior cingulate cortex.

    PubMed

    Toyoda, Hiroki; Zhao, Ming-Gao; Ulzhöfer, Bettina; Wu, Long-Jun; Xu, Hui; Seeburg, Peter H; Sprengel, Rolf; Kuner, Rohini; Zhuo, Min

    2009-08-10

    Cortical areas including the anterior cingulate cortex (ACC) are important for pain and pleasure. Recent studies using genetic and physiological approaches have demonstrated that the investigation of basic mechanism for long-term potentiation (LTP) in the ACC may reveal key cellular and molecular mechanisms for chronic pain in the cortex. Glutamate N-methyl D-aspartate (NMDA) receptors in the ACC are critical for the induction of LTP, including both NR2A and NR2B subunits. However, cellular and molecular mechanisms for the expression of ACC LTP have been less investigated. Here, we report that the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit, GluA1 but not GluA2 contributes to LTP in the ACC using genetic manipulated mice lacking GluA1 or GluA2 gene. Furthermore, GluA1 knockout mice showed decreased extracellular signal-regulated kinase (ERK) phosphorylation in the ACC in inflammatory pain models in vivo. Our results demonstrate that AMPA receptor subunit GluA1 is a key mechanism for the expression of ACC LTP and inflammation-induced long-term plastic changes in the ACC.

  2. Regulatory xenobiotic responsive elements in the distal 5'-flanking region of the mouse Cyp1a2 gene required for transcriptional activation by 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    PubMed

    Kawasaki, Yuki; Sakuma, Tsutomu; Goto, Yuma; Nemoto, Nobuo

    2010-10-01

    We examined the xenobiotic responsive element (XRE) responsible for induction of the mouse Cyp1a2 gene by 3-methylcholanthrene (3MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) using a reporter gene assay in mouse hepatocytes in primary culture. Although, the 5'-flanking region up to -9.5 kilobase pairs did not show a significant increase in transcriptional activity after treatment with 3MC or TCDD, a further distal 5'-flanking region from -13,958 to -12,520 containing 12 putative XREs (5'-GCGTG-3') demonstrated distinctive transcriptional activity after treatment with 3MC or TCDD. When a mutation was introduced into XRE14 at -12,972, the activation was decreased, and concurrent mutations in XRE14, XRE13, and XRE15 completely abolished it. However, mutations in XRE13, XRE15, XRE16, or XRE17 did not affect the inducible transcriptional activation of the mouse Cyp1a2 gene. These results suggest that XRE14 is important and that XRE13 at -12,897 and/or XRE15 at -13,061 are cooperative to the inducible transcriptional activation of the mouse Cyp1a2 gene by ligands of the aryl hydrocarbon receptor.

  3. The infarct-sparing effect of IB-MECA against myocardial ischemia/reperfusion injury in mice is mediated by sequential activation of adenosine A3 and A 2A receptors.

    PubMed

    Tian, Yikui; Marshall, Melissa; French, Brent A; Linden, Joel; Yang, Zequan

    2015-03-01

    Conflicting results exist regarding the role of A3 adenosine receptors (A3ARs) in mediating cardioprotection during reperfusion following myocardial infarction. We hypothesized that the effects of the A3AR agonist IB-MECA to produce cardioprotection might involve activation of other adenosine receptor subtypes. C57Bl/6 (B6), A3AR KO, A2AAR KO, and A2AAR KO/WT bone marrow chimeric mice were assigned to 12 groups undergoing either hemodynamic studies or 45 min of LAD occlusion and 60 min of reperfusion. IB-MECA (100 μg/kg) or vehicle was administered by iv bolus 5 min before reperfusion. Radioligand binding assays showed that IB-MECA has high affinity for the mouse A3AR (K i = 0.17 ± 0.05 nM), but also can bind with lower affinity to the A1AR (9.0 ± 2.4 nM) or the A2AAR (56.5 ± 10.2 nM). IB-MECA caused bi-phasic hemodynamic changes, which were completely absent in A3AR KO mice and were modified by A2AAR blockade or deletion. IB-MECA stimulated histamine release, increased heart rate, and significantly reduced IF size in B6 mice from 61.5 ± 1.4 to 48.6 ± 2.4% of risk region (RR; 21% reduction, p < 0.05) but not in A3AR KO mice. Compared to B6, A3AR KO mice had significantly reduced IF size (p < 0.05). In B6/B6 bone marrow chimeras, IB-MECA caused a 47% reduction of IF size (from 47.3 ± 3.9 to 24.7 ± 4.5, p < 0.05). However, no significant cardioprotective effect of IB-MECA was observed in A2AARKO/B6 mice, which lacked A2AARs only on their bone marrow-derived cells. Activation of A3ARs induces a bi-phasic hemodynamic response, which is partially mediated by activation of A2AARs. The cardioprotective effect of IB-MECA is due to the initial activation of A3AR followed by activation of A2AARs in bone marrow-derived cells.

  4. A 2,5-Dihydroxybenzoic Acid-Gelatin Conjugate: The Synthesis, Antiviral Activity and Mechanism of Antiviral Action Against Two Alphaherpesviruses.

    PubMed

    Lisov, Alexander; Vrublevskaya, Veronika; Lisova, Zoy; Leontievsky, Alexey; Morenkov, Oleg

    2015-10-15

    Various natural and synthetic polyanionic polymers with different chemical structures are known to exhibit potent antiviral activity in vitro toward a variety of enveloped viruses and may be considered as promising therapeutic agents. A water-soluble conjugate of 2,5-dihydroxybezoic acid (2,5-DHBA) with gelatin was synthesized by laccase-catalyzed oxidation of 2,5-DHBA in the presence of gelatin, and its antiviral activity against pseudorabies virus (PRV) and bovine herpesvirus type 1 (BoHV-1), two members of the Alphaherpesvirinae subfamily, was studied. The conjugate produced no direct cytotoxic effect on cells, and did not inhibit cell growth at concentrations up to 1000 µg/mL. It exhibited potent antiviral activity against PRV (IC50, 1.5-15 µg/mL for different virus strains) and BoHV-1 (IC50, 0.5-0.7 µg/mL). When present during virus adsorption, the conjugate strongly inhibited the attachment of PRV and BoHV-1 to cells. The 2,5-DHBA-gelatin conjugate had no direct virucidal effect on the viruses and did not influence their penetration into cells, cell-to-cell spread, production of infectious virus particles in cells, and expression of PRV glycoproteins E and B. The results indicated that the 2,5-DHBA-gelatin conjugate strongly inhibits the adsorption of alphaherpesviruses to cells and can be a promising synthetic polymer for the development of antiviral formulations against alphaherpesvirus infections.

  5. A 2,5-Dihydroxybenzoic Acid–Gelatin Conjugate: The Synthesis, Antiviral Activity and Mechanism of Antiviral Action Against Two Alphaherpesviruses

    PubMed Central

    Lisov, Alexander; Vrublevskaya, Veronika; Lisova, Zoy; Leontievsky, Alexey; Morenkov, Oleg

    2015-01-01

    Various natural and synthetic polyanionic polymers with different chemical structures are known to exhibit potent antiviral activity in vitro toward a variety of enveloped viruses and may be considered as promising therapeutic agents. A water-soluble conjugate of 2,5-dihydroxybezoic acid (2,5-DHBA) with gelatin was synthesized by laccase-catalyzed oxidation of 2,5-DHBA in the presence of gelatin, and its antiviral activity against pseudorabies virus (PRV) and bovine herpesvirus type 1 (BoHV-1), two members of the Alphaherpesvirinae subfamily, was studied. The conjugate produced no direct cytotoxic effect on cells, and did not inhibit cell growth at concentrations up to 1000 µg/mL. It exhibited potent antiviral activity against PRV (IC50, 1.5–15 µg/mL for different virus strains) and BoHV-1 (IC50, 0.5–0.7 µg/mL). When present during virus adsorption, the conjugate strongly inhibited the attachment of PRV and BoHV-1 to cells. The 2,5-DHBA–gelatin conjugate had no direct virucidal effect on the viruses and did not influence their penetration into cells, cell-to-cell spread, production of infectious virus particles in cells, and expression of PRV glycoproteins E and B. The results indicated that the 2,5-DHBA–gelatin conjugate strongly inhibits the adsorption of alphaherpesviruses to cells and can be a promising synthetic polymer for the development of antiviral formulations against alphaherpesvirus infections. PMID:26501311

  6. Effect of polysaccharides extract of rhizoma atractylodis macrocephalae on thymus, spleen and cardiac indexes, caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein and mRNA expression levels in aged rats.

    PubMed

    Guo, Ling; Sun, Yong Le; Wang, Ai Hong; Xu, Chong En; Zhang, Meng Yuan

    2012-10-01

    This study was designed to determine the possible protective effect of polysaccharides extract of rhizoma atractylodis macrocephalae on heart function in aged rats. Polysaccharides extract of rhizoma atractylodis macrocephalae was administered to aged rats. Results showed that thymus, spleen and cardiac indexs were significantly increased, whereas caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein expression, Smac/DIABLO and HtrA2/Omi mRNA expression levels were markedly reduced. It can be concluded that polysaccharides extract of rhizoma atractylodis macrocephalae may enhance immunity and improve heart function in aged rats.

  7. Simultaneous imaging of the topography and electrochemical activity of a 2D carbon nanotube network using a dual functional L-shaped nanoprobe.

    PubMed

    Lee, Eunjoo; Sung, Jungwoo; An, Taechang; Shin, Heungjoo; Nam, Hong Gil; Lim, Geunbae

    2015-05-07

    The application of nanomaterials for biosensors and fuel cells is becoming more common, but it requires an understanding of the relationship between the structure and electrochemical characteristics of the materials at the nanoscale. Herein, we report the development of scanning electrochemical microscopy-atomic force microscopy (SECM-AFM) nanoprobes for collecting spatially resolved data regarding the electrochemical activity of nanomaterials such as carbon nanotube (CNT) networks. The fabrication of the nanoprobe begins with the integration of a CNT-bundle wire into a conventional AFM probe followed by the deposition of an insulating layer and cutting of the probe end. In addition, a protrusive insulating tip is integrated at the end of the insulated CNT-bundle wire to maintain a constant distance between the nanoelectrode and the substrate; this yields an L-shaped nanoprobe. The resulting nanoprobes produced well-fitted maps of faradaic current data with less than 300 nm spatial resolution and topographical images of CNT networks owing to the small effective distance (of the order of tens of nanometers) between the electrode and the substrate. Electrochemical imaging using the L-shaped nanoprobe revealed that the electrochemical activity of the CNT network is not homogeneous and provided further understanding of the relationship between the topography and electrochemical characteristics of CNT networks.

  8. Limitations of short range Mexican hat connection for driving target selection in a 2D neural field: activity suppression and deviation from input stimuli

    PubMed Central

    Mégardon, Geoffrey; Tandonnet, Christophe; Sumner, Petroc; Guillaume, Alain

    2015-01-01

    Dynamic Neural Field models (DNF) often use a kernel of connection with short range excitation and long range inhibition. This organization has been suggested as a model for brain structures or for artificial systems involved in winner-take-all processes such as saliency localization, perceptual decision or target/action selection. A good example of such a DNF is the superior colliculus (SC), a key structure for eye movements. Recent results suggest that the superficial layers of the SC (SCs) exhibit relatively short range inhibition with a longer time constant than excitation. The aim of the present study was to further examine the properties of a DNF with such an inhibition pattern in the context of target selection. First we tested the effects of stimulus size and shape on when and where self-maintained clusters of firing neurons appeared, using three variants of the model. In each model variant, small stimuli led to rapid formation of a spiking cluster, a range of medium sizes led to the suppression of any activity on the network and hence to no target selection, while larger sizes led to delayed selection of multiple loci. Second, we tested the model with two stimuli separated by a varying distance. Again single, none, or multiple spiking clusters could occur, depending on distance and relative stimulus strength. For short distances, activity attracted toward the strongest stimulus, reminiscent of well-known behavioral data for saccadic eye movements, while for larger distances repulsion away from the second stimulus occurred. All these properties predicted by the model suggest that the SCs, or any other neural structure thought to implement a short range MH, is an imperfect winner-take-all system. Although, those properties call for systematic testing, the discussion gathers neurophysiological and behavioral data suggesting that such properties are indeed present in target selection for saccadic eye movements. PMID:26539103

  9. Results of a 2-year randomized, controlled obesity prevention trial: Effects on diet, activity and sleep behaviors in an at-risk young adult population.

    PubMed

    Laska, Melissa N; Lytle, Leslie A; Nanney, Marilyn S; Moe, Stacey G; Linde, Jennifer A; Hannan, Peter J

    2016-08-01

    Excess weight gain tends to occur in young adulthood. However, research examining effective weight-related interventions for this age group has been limited. As one of seven trials in the EARLY Trials consortium (Early Adult Reduction of weight through LifestYle intervention), the CHOICES Study (Choosing Healthy Options in College Environments and Settings) tested effects of a technology-integrated, young adult weight gain prevention intervention. It was a randomized controlled trial with assessments at baseline (2011) and 4-, 12- and 24-months post-intervention initiation and included 441 participants (ages 18-35) who were students at three Minnesota community colleges. The 24-month intervention included a 1-credit academic course and social networking and support online intervention. This analysis examined effects on 12 secondary behavioral outcomes across three domains: diet (fast food, sugary beverages, breakfast, at-home meal preparation), physical activity/screen time (minutes and energy expenditure in leisure time physical activity, television viewing, leisure time computer use) and sleep (hours of sleep, time required to fall asleep, days not getting enough rest, difficulty staying awake). The intervention resulted in significant reductions in fast food (p=0.007) but increases in difficulty staying awake (p=0.015). There was limited evidence of other behavior changes at 4months (0.05

  10. Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2*

    PubMed Central

    Pallan, Pradeep S.; Nagy, Leslie D.; Lei, Li; Gonzalez, Eric; Kramlinger, Valerie M.; Azumaya, Caleigh M.; Wawrzak, Zdzislaw; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

    2015-01-01

    Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116–119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity. PMID:25533464

  11. Integrating porphyrin nanoparticles into a 2D graphene matrix for free-standing nanohybrid films with enhanced visible-light photocatalytic activity.

    PubMed

    Chen, Yingzhi; Huang, Zheng-Hong; Yue, Mengbin; Kang, Feiyu

    2014-01-21

    Organic nanostructures in terms of porphyrin building blocks have shown great potential in visible-light photocatalytic applications because of their optical, electrical, and catalytic properties. Graphenes are known to provide a high-quality two-dimensional (2D) support for inorganic semiconductor nanostructures to increase the adsorption capability of the photocatalysts and an electron-transfer medium with attractive potential to enhance photogenerated charge separation. A combination of porphyrin nanostructures with graphene sheets, particularly in the form of free-standing films, is highly desirable due to its photocatalysing feasibility and convenience. Toward this aim, we demonstrate a facile method to integrate porphyrin (meso-tetra(p-hydroxyphenyl)porphyrin, p-THPP) nanoparticles (NPs) into macroscopic graphene (reduced graphene oxide, rGO) films through vacuum filtration of the co-colloids of graphene oxide (GO) and p-THPP nanoparticles (NPs) followed by gaseous reduction. The obtained p-THPP/rGO nanohybrid film exhibits enhanced visible-light photocatalytic activity compared to each moiety of the hybrid, and this photocatalyst can be easily separated and recycled for successive use with excellent stability. The results show that this facile fabrication of the p-THPP/rGO nanohybrid film makes it available for high-performance optoelectronic applications, as well as for device integration.

  12. Role of the non-opioid dynorphin peptide des-Tyr-dynorphin (DYN-A(2-17)) in food intake and physical activity, and its interaction with orexin-A.

    PubMed

    Gac, L; Butterick, T A; Duffy, C M; Teske, J A; Perez-Leighton, C E

    2016-02-01

    Food intake and physical activity are regulated by multiple neuropeptides, including orexin and dynorphin (DYN). Orexin-A (OXA) is one of two orexin peptides with robust roles in regulation of food intake and spontaneous physical activity (SPA). DYN collectively refers to several peptides, some of which act through opioid receptors (opioid DYN) and some whose biological effects are not mediated by opioid receptors (non-opioid DYN). While opioid DYN is known to increase food intake, the effects of non-opioid DYN peptides on food intake and SPA are unknown. Neurons that co-express and release OXA and DYN are located within the lateral hypothalamus. Limited evidence suggests that OXA and opioid DYN peptides can interact to modulate some aspects of behaviors classically related to orexin peptide function. The paraventricular hypothalamic nucleus (PVN) is a brain area where OXA and DYN peptides might interact to modulate food intake and SPA. We demonstrate that injection of des-Tyr-dynorphin (DYN-A(2-17), a non opioid DYN peptide) into the PVN increases food intake and SPA in adult mice. Co-injection of DYN-A(2-17) and OXA in the PVN further increases food intake compared to DYN-A(2-17) or OXA alone. This is the first report describing the effects of non-opioid DYN-A(2-17) on food intake and SPA, and suggests that DYN-A(2-17) interacts with OXA in the PVN to modulate food intake. Our data suggest a novel function for non-opioid DYN-A(2-17) on food intake, supporting the concept that some behavioral effects of the orexin neurons result from combined actions of the orexin and DYN peptides.

  13. Serum levels of osteoprotegerin and receptor activator of nuclear factor -κB ligand in children with early juvenile idiopathic arthritis: a 2-year prospective controlled study

    PubMed Central

    2010-01-01

    Background The clinical relevance of observations of serum levels of osteoprotegerin (OPG) and receptor activator of nuclear factor -κB ligand (RANKL) in juvenile idiopathic arthritis (JIA) is not clear. To elucidate the potential role of OPG and RANKL in JIA we determined serum levels of OPG and RANKL in patients with early JIA compared to healthy children, and prospectively explored changes in relation to radiographic score, bone and lean mass, severity of the disease, and treatment. Methods Ninety children with early oligoarticular or polyarticular JIA (ages 6-18 years; mean disease duration 19.4 months) and 90 healthy children individually matched for age, sex, race, and county of residence, were examined at baseline and 2-year follow-up. OPG and RANKL were quantified by enzyme-immunoassay. Data were analyzed with the use of t-tests, ANOVA, and multiple regression analyses. Results Serum OPG was significantly lower in patients than controls at baseline, and there was a trend towards higher RANKL and a lower OPG/RANKL ratio. Patients with polyarthritis had significantly higher increments in RANKL from baseline to follow-up, compared to patients with oligoarthritis. RANKL was a significant negative predictor for increments in total body lean mass. Patients who were receiving corticosteroids (CS) or disease-modifying antirheumatic drugs (DMARDs) at follow-up had higher OPG/RANKL ratio compared with patients who did not receive this medication. Conclusions The data supports that levels of OPG are lower in patients with JIA compared to healthy children, and higher levels of RANKL is associated with more serious disease. RANKL was a significant negative predictor of lean mass in patients with JIA. The OPG/RANKL ratio was higher in patients on DMARDs or CS treatment. PMID:21134287

  14. Incorporation of marine lipids into mitochondrial membranes increases susceptibility to damage by calcium and reactive oxygen species: evidence for enhanced activation of phospholipase A2 in mitochondria enriched with n-3 fatty acids.

    PubMed Central

    Malis, C D; Weber, P C; Leaf, A; Bonventre, J V

    1990-01-01

    Experiments were designed to evaluate the susceptibility of mitochondrial membranes enriched with n-3 fatty acids to damage by Ca2+ and reactive oxygen species. Fatty acid content and respiratory function were assessed in renal cortical mitochondria isolated from fish-oil- and beef-tallow-fed rats. Dietary fish oils were readily incorporated into mitochondrial membranes. After exposure to Ca2+ and reactive oxygen species, mitochondria enriched in n-3 fatty acids, and using pyruvate and malate as substrates, had significantly greater changes in state 3 and uncoupled respirations, when compared with mitochondria from rats fed beef tallow. Mitochondrial site 1 (NADH coenzyme Q reductase) activity was reduced to 45 and 85% of control values in fish-oil- and beef-tallow-fed groups, respectively. Exposure to Ca2+ and reactive oxygen species enhance the release of polyunsaturated fatty acids enriched at the sn-2 position of phospholipids from mitochondria of fish-oil-fed rats when compared with similarly treated mitochondria of beef-tallow-fed rats. This release of fatty acids was partially inhibited by dibucaine, the phospholipase A2 inhibitor, which we have previously shown to protect mitochondria against damage associated with Ca2+ and reactive oxygen species. The results indicate that phospholipase A2 is activated in mitochondria exposed to Ca2+ and reactive oxygen species and is responsible, at least in part, for the impairment of respiratory function. Phospholipase A2 activity and mitochondrial damage are enhanced when mitochondrial membranes are enriched with n-3 fatty acids. PMID:2123344

  15. The neuromuscular activity of Bothriopsis bilineata smaragdina (forest viper) venom and its toxin Bbil-TX (Asp49 phospholipase A2) on isolated mouse nerve-muscle preparations.

    PubMed

    Floriano, Rafael Stuani; Rocha, Thalita; Carregari, Victor Corasolla; Marangoni, Sergio; da Cruz-Höfling, Maria Alice; Hyslop, Stephen; Rodrigues-Simioni, Léa; Rowan, Edward G

    2015-03-01

    ; Bbil-TX did not change the calcium fluorescence in TSn-m preparations. Immunohistochemical analysis of toxin-treated PND preparations revealed labeling of junctional ACh receptors but a loss of the presynaptic proteins synaptophysin and SNAP25. Together, these data confirm the presynaptic action of Bbil-TX and show that it involves modulation of K(+) channel activity and presynaptic protein expression.

  16. TSU-16, (Z)-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone, is a potent activator of aryl hydrocarbon receptor and increases CYP1A1 and CYP1A2 expression in human hepatocytes.

    PubMed

    Matsuoka-Kawano, Kazuaki; Yoshinari, Kouichi; Nagayama, Sekio; Yamazoe, Yasushi

    2010-04-15

    (Z)-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (TSU-16), is a potent anti-angiogenic agent that inhibits the tyrosine kinase of vascular endothelial growth factor receptor-2. In clinical trials with daily or twice weekly intravenous administration of TSU-16, its increased clearance was observed. To understand the mechanism underlying this observation, we have investigated the TSU-16-mediated regulation of cytochrome P450 expression. In human hepatocytes, TSU-16 increased mRNA levels of CYP1A1 and CYP1A2, but not CYP2B6 and CYP3A4. The extent of increase and profiles of the time-dependent changes in CYP1A1 and CYP1A2 mRNA levels after TSU-16 treatment were similar to those after treatment with 3-methylcholanthrene (3MC), a well-known activator of the aryl hydrocarbon receptor (AhR). In reporter assays using a plasmid construct that contained the human CYP1A1 5'-flanking region including the region crucial for the AhR-dependent transcription of both human CYP1A1 and CYP1A2, TSU-16 treatment increased reporter activities to an extent similar to that obtained with 3MC. Treatment of HepG2 cells and human hepatocytes with AhR-targeting siRNA suppressed the increase in both mRNA levels and CYP1A activities after treatment with TSU-16 as well as after that with omeprazole or 3MC. TSU-16 also time-dependently reduced cellular AhR protein levels in HepG2 cells to a similar extent with 3MC treatment. Furthermore, we demonstrated that unlabeled TSU-16 and 3MC but not omeprazole completely inhibited the specific binding of [(3)H]-3MC to mouse Hepa1c1c7 cytosol, suggesting TSU-16 as an AhR ligand. In conclusion, our present results suggest that TSU-16 binds to and activates AhR to enhance the expression of both human CYP1A1 and CYP1A2. Because TSU-16 is metabolized mainly by CYP1A2, its increased clearance after repeated dosing may be attributed to the enhanced expression of hepatic CYP1A2.

  17. Anticoagulant mechanism and platelet deaggregation property of a non-cytotoxic, acidic phospholipase A2 purified from Indian cobra (Naja naja) venom: inhibition of anticoagulant activity by low molecular weight heparin.

    PubMed

    Dutta, Sumita; Gogoi, Debananda; Mukherjee, Ashis K

    2015-03-01

    In the present study, anticoagulant and platelet modulating activities of an acidic phospholipase A2 (NnPLA2-I) purified from Indian cobra Naja naja venom was investigated. The NnPLA2-I displayed a mass of 15.2 kDa and 14,186.0 Da when analyzed by SDS-PAGE and MALDI-TOF-MS, respectively. Peptide mass fingerprinting analysis of the NnPLA2-I showed its significant similarity with phospholipase A2 enzymes purified from cobra venom. BLAST analysis of one tryptic peptide sequence of NnPLA2-I demonstrated putative conserved domains of the PLA2-like superfamily. The Km and Vmax values of NnPLA2-I toward hydrolysis of its most preferred substrate-phosphotidylcholine (PC)-were determined to be 0.72 mM and 29.3 μmol min(-1) mg(-1), respectively. The anticoagulant activity of NnPLA2-I was found to be higher than the anticoagulant activity of heparin/AT-III or warfarin. The histidine modifying reagent, monovalent and polyvalent antivenom differentially inhibited the catalytic and anticoagulant activities of NnPLA2-I. Low molecular weight heparin did not inhibit the catalytic and platelet deaggregation activity of NnPLA2-I, albeit its anticoagulant activity was significantly reduced. The NnPLA2-I showed a non-enzymatic, mixed inhibition of thrombin with a Ki value of 9.3 nM. Heparin significantly decreased, with an IC50 value of 15.23 mIU, the thrombin inhibitory activity of NnPLA2-I. The NnPLA2-I uniquely increased the amidolytic activity of FXa without influencing its prothrombin activating property. NnPLA2-I showed dose-dependent deaggregation of platelet rich plasma (PRP) and inhibited the collagen and thrombin-induced aggregation of PRP. However, deaggregation of washed platelets by NnPLA2-I demonstrated in presence of PC or platelet poor plasma. Alkylation of histidine residue of NnPLA2-I resulted in 95% and 21% reduction of its platelet deaggregation and platelet binding properties, respectively. NnPLA2-I did not show cytotoxicity against human glioblastoma U87MG cells

  18. Inhibition of secreted phospholipase A2. 4-glycerol derivatives of 4,5-dihydro-3-(4-tetradecyloxybenzyl)-1,2,4-4H-oxadiazol-5-one with broad activities.

    PubMed

    Touaibia, Mohamed; Djimdé, Atimé; Cao, Fei; Boilard, Eric; Bezzine, Sofiane; Lambeau, Gérard; Redeuilh, Catherine; Lamouri, Aazdine; Massicot, France; Chau, François; Dong, Chang-Zhi; Heymans, Françoise

    2007-04-05

    Secreted phospholipases A2 (sPLA2s) have been reported to play an important role in various inflammatory conditions and thus represent an attractive therapeutic target. Previous SAR studies from our laboratory have revealed certain important features of our recently discovered specific hGIIA sPLA2 inhibitors, and we report here the synthesis and biological activities of glycerol-containing derivatives of our lead compound III (Figure 1). Efficient and selective synthesis methods have been developed to make glycerol trisubstituted by different groups on desired positions. In terms of biological activities, the best compounds (A3, A6, and A15) are more active than III (Figure 1), as potent as Me-Indoxam, an sPLA2s inhibitor of reference, against hGIIA, hGV, and hGX sPLA2s and at least 10 times less active toward the GIB enzymes in two in vitro assay systems. By synthesis of enantiopure (S)-A6, we demonstrated that no important improvement of the inhibitory potency could be achieved by this approach. Furthermore, the results show that the global lipophilicity is likely responsible for the anti-PLA2 activity and two oxadiazolone moieties seem too big to be accommodated by the active site of the hGIIA enzyme.

  19. Hax1 lacks BH modules and is peripherally associated to heavy membranes: implications for Omi/HtrA2 and PARL activity in the regulation of mitochondrial stress and apoptosis.

    PubMed

    Jeyaraju, D V; Cisbani, G; De Brito, O M; Koonin, E V; Pellegrini, L

    2009-12-01

    Hax1 has an important role in immunodeficiency syndromes and apoptosis. A recent report (Chao et al., Nature, 2008) proposed that the Bcl-2-family-related protein, Hax1, suppresses apoptosis in lymphocytes and neurons through a mechanism that involves its association to the inner mitochondrial membrane rhomboid protease PARL, to proteolytically activate the serine protease Omi/HtrA2 and eliminate active Bax. This model implies that the control of cell-type sensitivity to pro-apoptotic stimuli is governed by the PARL/Hax1 complex in the mitochondria intermembrane space and, more generally, that Bcl-2-family-related proteins can control mitochondrial outer-membrane permeabilization from inside the mitochondrion. Further, it defines a novel, anti-apoptotic Opa1-independent pathway for PARL. In this study, we present evidence that, in vivo, the activity of Hax1 cannot be mechanistically coupled to PARL because the two proteins are confined in distinct cellular compartments and their interaction in vitro is an artifact. We also show by sequence analysis and secondary structure prediction that Hax1 is extremely unlikely to be a Bcl-2-family-related protein because it lacks Bcl-2 homology modules. These results indicate a different function and mechanism of Hax1 in apoptosis and re-opens the question of whether mammalian PARL, in addition to apoptosis, regulates mitochondrial stress response through Omi/HtrA2 processing.

  20. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis.

    PubMed

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-02-26

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.

  1. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis*

    PubMed Central

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-01-01

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy. PMID:26724804

  2. Nicotine and ethanol activate protein kinase A synergistically via G(i) betagamma subunits in nucleus accumbens/ventral tegmental cocultures: the role of dopamine D(1)/D(2) and adenosine A(2A) receptors.

    PubMed

    Inoue, Yuichiro; Yao, Lina; Hopf, F Woodward; Fan, Peidong; Jiang, Zhan; Bonci, Antonello; Diamond, Ivan

    2007-07-01

    Tobacco and alcohol are the most commonly used drugs of abuse and show the most serious comorbidity. The mesolimbic dopamine system contributes significantly to nicotine and ethanol reinforcement, but the underlying cellular signaling mechanisms are poorly understood. Nicotinic acetylcholine (nACh) receptors are highly expressed on ventral tegmental area (VTA) dopamine neurons, with relatively low expression in nucleus accumbens (NAcb) neurons. Because dopamine receptors D(1) and D(2) are highly expressed on NAcb neurons, nicotine could influence NAcb neurons indirectly by activating VTA neurons to release dopamine in the NAcb. To investigate this possibility in vitro, we established primary cultures containing neurons from VTA or NAcb separately or in cocultures. Nicotine increased cAMP response element-mediated gene expression only in cocultures; this increase was blocked by nACh or dopamine D(1) or D(2) receptor antagonists. Furthermore, subthreshold concentrations of nicotine with ethanol increased gene expression in cocultures, and this increase was blocked by nACh, D(2) or adenosine A(2A) receptor antagonists, Gbetagamma or protein kinase A (PKA) inhibitors, and adenosine deaminase. These results suggest that nicotine activated VTA neurons, causing the release of dopamine, which in turn stimulated both D(1) and D(2) receptors on NAcb neurons. In addition, subthreshold concentrations of nicotine and ethanol in combination also activated NAcb neurons through synergy between D(2) and A(2A) receptors. These data provide a novel cellular mechanism, involving Gbetagamma subunits, A(2A) receptors, and PKA, whereby combined use of tobacco and alcohol could enhance the reinforcing effect in humans as well as facilitate long-term neuroadaptations, increasing the risk for developing coaddiction.

  3. N9-benzyl-substituted 1,3-dimethyl- and 1,3-dipropyl-pyrimido[2,1-f]purinediones: synthesis and structure-activity relationships at adenosine A1 and A2A receptors.

    PubMed

    Drabczyńska, Anna; Müller, Christa E; Karolak-Wojciechowska, Janina; Schumacher, Britta; Schiedel, Anke; Yuzlenko, Olga; Kieć-Kononowicz, Katarzyna

    2007-07-15

    Synthesis and physicochemical properties of N-benzyl pyrimido[2,1-f]purinediones are described. These derivatives were synthesized by the cyclization of 7-chloropropylo-8-bromo-1,3-dimethyl- or 1,3-dipropyl xanthine derivatives with corresponding (un)substituted benzylamines. Dipropyl derivatives were obtained under microwave irradiation conditions either. The obtained compounds (1-20) were evaluated for their affinity to adenosine A1 and A2A receptors, selected compounds were additionally investigated for affinity to the A3 receptor subtype. The results of the radioligand binding assays to A1 and A2A adenosine receptors showed that most of the 1,3-dimethyl-9-benzylpyrimidopurinediones exhibited selective affinity to A2A receptors at micromolar or submicromolar concentrations (for example, derivative 9 with o-methoxy substituent displayed a Ki value of 0.699 microM at rat A2A receptor with more than 36-fold selectivity). Contrary to previously described arylpyrimido[2,1-f]purinediones dipropyl derivatives (compounds 15-20) showed affinity to both kinds of receptors increased, however A1 affinity increased to a larger extent, with the result that A2A selectivity was abolished. The best adenosine A1 receptor ligand was m-chlorobenzyl derivative 18 (Ki=0.089 microM and 5-fold A1 selectivity). Structure-activity relationships were discussed with the analysis of lipophilic and spatial properties of the investigated compounds. Pharmacophore model of adenosine A1 receptor antagonist was adopted for this purpose.

  4. Maternal protein restriction during lactation modulated the expression and activity of rat offspring hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2, and CYP2E1 during development.

    PubMed

    Da Costa, N Meireles; Visoni, S B C; Dos Santos, I L; Barja-Fidalgo, T C; Ribeiro-Pinto, L F

    2016-01-01

    Early nutrition plays a long-term role in the predisposition to chronic diseases and influences the metabolism of several drugs. This may happen through cytochromes P450 (CYPs) regulation, which are the main enzymes responsible for the metabolism of xenobiotics. Here, we analyzed the effects of maternal protein restriction (MPR) on the expression and activity of hepatic offspring's CYPs during 90 days after birth, using Wistar rats as a mammal model. Hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2 and CYP2E1 mRNA and protein expression, and associated catalytic activities (ECOD, EROD, MROD, BROD, PROD and PNPH) were evaluated in 15-, 30-, 60-, and 90-day-old offspring from dams fed with either a 0% protein (MPR groups) or a standard diet (C groups) during the 10 first days of lactation. Results showed that most CYP genes were induced in 60- and 90-day-old MPR offspring. The inductions detected in MPR60 and MPR90 were of 5.0- and 2.0-fold (CYP1A2), 3.7- and 2.0-fold (CYP2B2) and 9.8- and 5.8- fold (CYP2E1), respectively, and a 3.8-fold increase of CYP2B1 in MPR90. No major alterations were detected in CYP protein expression. The most relevant CYP catalytic activities' alterations were observed in EROD, BROD and PNPH. Nevertheless, they did not follow the same pattern observed for mRNA expression, except for an induction of EROD in MPR90 (3.5-fold) and of PNPH in MPR60 (2.2-fold). Together, these results suggest that MPR during lactation was capable of altering the expression and activity of the hepatic CYP enzymes evaluated in the offspring along development.

  5. Metabolic activation of o-phenylphenol to a major cytotoxic metabolite, phenylhydroquinone: role of human CYP1A2 and rat CYP2C11/CYP2E1.

    PubMed

    Ozawa, S; Ohta, K; Miyajima, A; Kurebayashi, H; Sunouchi, M; Shimizu, M; Murayama, N; Matsumoto, Y; Fukuoka, M; Ohno, Y

    2000-10-01

    1. The in vitro metabolic activation of o-phenylphenol has been evaluated as yielding a toxic metabolite, 2,5-dihydroxybiphenyl (phenylhydroquinone), by p-hydroxylation in liver microsomes of rat and human. The involvement of rat CYP2C11, CYP2E1 and human CYP1A2 in the p-hydroxylation of o-phenylphenol is suggested. 2. 2,3- and phenylhydroquinone, which induced DNA single-strand scission in the presence of 1 microM CuCl2, were the most cytotoxic chemicals examined to cultured mammalian cell lines among o-phenylphenol, m-phenylphenol, p-phenylphenol, 2,2'-, 4,4'-, 2,3- and phenylhydroquinone. 3. Rat and human liver microsomes catalysed the formation of phenylhydroquinone, but not 2,3-dihydroxybiphenyl, using o-phenylphenol as a substrate. A higher rate of metabolic activation of o-phenylphenol was observed with livers of the male than the female rats by 5.6- and 2.6-fold respectively. 4. Inhibitory antibodies against the male-specific CYP2C11 inhibited hepatic o-phenylphenol p-hydroxylation in the male F344 and Sprague-Dawley rat by > 70%. Liver microsomes from the isoniazid-treated rats produced 1.8- and 3-fold induction of o-phenylphenol p-hydroxylation and chlorzoxazone 6-hydroxylation (a CYP2E1-dependent activity) respectively. 5. Human CYP1A2, expressed by baculovirus-mediated cDNA expression systems, exhibited a remarkably higher capacity for o-phenylphenol p-hydroxylation at concentrations of 5 (> 5-fold), 50 (> 2-fold) and 500 microM (> 2-fold) than CYP2A, CYP2B, CYP2Cs, CYP2D6, CYP2E1 and CYP3A4 on the basis of pmol P450. 6. Among various CYP inhibitors tested here, 7,8-benzoflavone and furafylline, typical human CYP1A2 inhibitors, inhibited the microsomal p-hydroxylation of o-phenylphenol in human livers most potently by 70 and 50% respectively. 7. The results thus indicate the involvement of rat CYP2C11/CYP2E1 and human CYP1A2 in the hepatic p-hydroxylation of o-phenylphenol.

  6. In neurons, activity-dependent association of dendritically transported mRNA transcripts with the transacting factor CBF-A is mediated by A2RE/RTS elements.

    PubMed

    Raju, Chandrasekhar S; Fukuda, Nanaho; López-Iglesias, Carmen; Göritz, Christian; Visa, Neus; Percipalle, Piergiorgio

    2011-06-01

    In neurons certain mRNA transcripts are transported to synapses through mechanisms that are not fully understood. Here we report that the heterogeneous nuclear ribonucleoprotein CBF-A (CArG Box binding Factor A) facilitates dendritic transport and localization of activity-regulated cytoskeleton-associated protein (Arc), brain-derived neurotrophic factor (BDNF), and calmodulin-dependent protein kinase II (CaMKIIα) mRNAs. We discovered that, in the adult mouse brain, CBF-A has a broad distribution. In the nucleus, CBF-A was found at active transcription sites and interchromosomal spaces and close to nuclear pores. In the cytoplasm, CBF-A localized to dendrites as well as pre- and postsynaptic sites. CBF-A was found in synaptosomal fractions, associated with Arc, BDNF, and CaMKIIα mRNAs. Electrophoretic mobility shift assays demonstrated a direct interaction mediated via their hnRNP A2 response element (A2RE)/RNA trafficking sequence (RTS) elements located in the 3' untranslated regions. In situ hybridization and microscopy on live hippocampal neurons showed that CBF-A is in dynamic granules containing Arc, BDNF, and CaMKIIα mRNAs. N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) postsynaptic receptor stimulation led to CBF-A accumulation in dendrites; increased Arc, BDNF, and CaMKIIα mRNA levels; and increased amounts of transcripts coprecipitating with CBF-A. Finally, CBF-A gene knockdown led to decreased mRNA levels. We propose that CBF-A cotranscriptionally binds RTSs in Arc, BDNF, and CaMKIIα mRNAs and follows the transcripts from genes to dendrites, promoting activity-dependent nuclear sorting of transport-competent mRNAs.

  7. Maternal protein restriction during lactation modulated the expression and activity of rat offspring hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2, and CYP2E1 during development

    PubMed Central

    Da Costa, N. Meireles; Visoni, S.B.C.; Dos Santos, I.L.; Barja-Fidalgo, T.C.; Ribeiro-Pinto, L.F.

    2016-01-01

    Early nutrition plays a long-term role in the predisposition to chronic diseases and influences the metabolism of several drugs. This may happen through cytochromes P450 (CYPs) regulation, which are the main enzymes responsible for the metabolism of xenobiotics. Here, we analyzed the effects of maternal protein restriction (MPR) on the expression and activity of hepatic offspring’s CYPs during 90 days after birth, using Wistar rats as a mammal model. Hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2 and CYP2E1 mRNA and protein expression, and associated catalytic activities (ECOD, EROD, MROD, BROD, PROD and PNPH) were evaluated in 15-, 30-, 60-, and 90-day-old offspring from dams fed with either a 0% protein (MPR groups) or a standard diet (C groups) during the 10 first days of lactation. Results showed that most CYP genes were induced in 60- and 90-day-old MPR offspring. The inductions detected in MPR60 and MPR90 were of 5.0- and 2.0-fold (CYP1A2), 3.7- and 2.0-fold (CYP2B2) and 9.8- and 5.8– fold (CYP2E1), respectively, and a 3.8-fold increase of CYP2B1 in MPR90. No major alterations were detected in CYP protein expression. The most relevant CYP catalytic activities’ alterations were observed in EROD, BROD and PNPH. Nevertheless, they did not follow the same pattern observed for mRNA expression, except for an induction of EROD in MPR90 (3.5-fold) and of PNPH in MPR60 (2.2-fold). Together, these results suggest that MPR during lactation was capable of altering the expression and activity of the hepatic CYP enzymes evaluated in the offspring along development. PMID:27828666

  8. Comparative Photoluminescence Properties and Judd-Ofelt Analysis of Eu3+ Ion-Activated Metal Molybdate Phosphors A2MoO6:Eu3+ (A = La, Y, Gd and Bi)

    NASA Astrophysics Data System (ADS)

    Han, Bing; Liu, Bingkun; Zhang, Jie; Li, Pengju; Shi, Hengzhen

    2017-03-01

    A class of red-emitting Eu3+ ion-activated metal molybdate A2MoO6:Eu3+ (A = La, Y, Gd and Bi) phosphors were synthesized by a conventional high-temperature solid-state reaction method. The x-ray diffraction patterns, scanning electron microscope images, Fourier transform infrared spectra, ultraviolet-visible diffuse reflection spectra as well as photoluminescence properties were measured to characterize the as-prepared samples. The photoluminescence properties including excitation/emission spectra, decay curves, Commission Internationale de L'Eclairage chromaticity coordinates and quantum efficiency were comparatively investigated in detail. The Judd-Ofelt theory was also applied to understand the radiative properties of f-f transitions of Eu3+ ions in this system for the first time. The as-prepared phosphors can be effectively excited with near-ultraviolet and/or blue light, and exhibit red emission belonging to the prevailing 5D0 → 7F2 transitions of Eu3+ with short decay time (millisecond level). The results demonstrated that A2MoO6:Eu3+ (A = La, Y, Gd and Bi) phosphors could have potential application as red-emitting phosphors in white light-emitting diodes based on near-ultraviolet and/or blue light-emitting diode chips.

  9. Ectopically expressed pro-group X secretory phospholipase A2 is proteolytically activated in mouse adrenal cells by furin-like proprotein convertases: implications for the regulation of adrenal steroidogenesis.

    PubMed

    Layne, Joseph D; Shridas, Preetha; Webb, Nancy R

    2015-03-20

    Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA2 is produced as a pro-enzyme (pro-GX sPLA2) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA2 activation have not been identified. We previously reported that GX sPLA2 negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA2 expression construct (FLAG-pro-GX sPLA2), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA2 processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA2 processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA2 processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA2 to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA2 processing and sPLA2 activity in Y1 cells, and it significantly attenuated GX sPLA2-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA2 is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.

  10. Loss of Interdependent Binding by the FoxO1 and FoxA1/A2 Forkhead Transcription Factors Culminates in Perturbation of Active Chromatin Marks and Binding of Transcriptional Regulators at Insulin-sensitive Genes.

    PubMed

    Yalley, Akua; Schill, Daniel; Hatta, Mitsutoki; Johnson, Nicole; Cirillo, Lisa Ann

    2016-04-15

    FoxO1 binds to insulin response elements located in the promoters of insulin-like growth factor-binding protein 1 (IGFBP1) and glucose-6-phosphatase (G6Pase), activating their expression. Insulin-mediated phosphorylation of FoxO1 promotes cytoplasmic translocation, inhibiting FoxO1-mediated transactivation. We have previously demonstrated that FoxO1 opens and remodels chromatin assembled from the IGFBP1 promoter via a highly conserved winged helix motif. This finding, which established FoxO1 as a "pioneer" factor, suggested a model whereby FoxO1 chromatin remodeling at regulatory targets facilitates binding and recruitment of additional regulatory factors. However, the impact of FoxO1 phosphorylation on its ability to bind chromatin and the effect of FoxO1 loss on recruitment of neighboring transcription factors at its regulatory targets in liver chromatin is unknown. In this study, we demonstrate that an amino acid substitution that mimics insulin-mediated phosphorylation of a serine in the winged helix DNA binding motif curtails FoxO1 nucleosome binding. We also demonstrate that shRNA-mediated loss of FoxO1 binding to the IGFBP1 and G6Pase promoters in HepG2 cells significantly reduces binding of RNA polymerase II and the pioneer factors FoxA1/A2. Knockdown of FoxA1 similarly reduced binding of RNA polymerase II and FoxO1. Reduction in acetylation of histone H3 Lys-27 accompanies loss of FoxO1 and FoxA1/A2 binding. Interdependent binding of FoxO1 and FoxA1/A2 possibly entails cooperative binding because FoxO1 and FoxA1/A2 facilitate one another's binding to IGFPB1 promoter DNA. These results illustrate how transcription factors can nucleate transcriptional events in chromatin in response to signaling events and suggest a model for regulation of hepatic glucose metabolism through interdependent FoxO/FoxA binding.

  11. The prevalent deep intronic c. 639+919 G>A GLA mutation causes pseudoexon activation and Fabry disease by abolishing the binding of hnRNPA1 and hnRNP A2/B1 to a splicing silencer.

    PubMed

    Palhais, Bruno; Dembic, Maja; Sabaratnam, Rugivan; Nielsen, Kira S; Doktor, Thomas Koed; Bruun, Gitte Hoffmann; Andresen, Brage Storstein

    2016-11-01

    Fabry disease is an X-linked recessive inborn disorder of the glycosphingolipid metabolism, caused by total or partial deficiency of the lysosomal α-galactosidase A enzyme due to mutations in the GLA gene. The prevalent c.639+919 G>A mutation in GLA leads to pathogenic insertion of a 57bp pseudoexon sequence from intron 4, which is responsible for the cardiac variant phenotype. In this study we investigate the splicing regulatory mechanism leading to GLA pseudoexon activation. Splicing analysis of GLA minigenes revealed that pseudoexon activation is influenced by cell-type. We demonstrate that the wild-type sequence harbors an hnRNP A1 and hnRNP A2/B1-binding exonic splicing silencer (ESS) overlapping the 5'splice site (5'ss) that prevents pseudoexon inclusion. The c.639+919 G>A mutation disrupts this ESS allowing U1 snRNP recognition of the 5'ss. We show that the wild-type GLA 5'ss motif with the ESS is also able to inhibit inclusion of an unrelated pseudoexon in the FGB gene, and that also in the FGB context inactivation of the ESS by the c.639+919 G>A mutation causes pseudoexon activation, underscoring the universal nature of the ESS. Finally, we demonstrate that splice switching oligonucleotide (SSO) mediated blocking of the pseudoexon 3'ss and 5'ss effectively restores normal GLA splicing. This indicates that SSO based splicing correction may be a therapeutic alternative in the treatment of Fabry disease.

  12. Effects of central activation of serotonin 5-HT2A/2C or dopamine D2/3 receptors on the acute and repeated effects of clozapine in the conditioned avoidance response test

    PubMed Central

    Feng, Min; Gao, Jun; Sui, Nan; Li, Ming

    2014-01-01

    Rationale: Acute administration of clozapine (a gold standard of atypical antipsychotics) disrupts avoidance response in rodents, while repeated administration often causes a tolerance effect. Objective: The present study investigated the neuroanatomical basis and receptor mechanisms of acute and repeated effects of clozapine treatment in the conditioned avoidance response test in male Sprague-Dawley rats. Methods: DOI (2,5-dimethoxy-4-iodo-amphetamine, a preferential 5-HT2A/2C agonist) or quinpirole (a preferential dopamine D2/3 agonist) was microinjected into the medial prefrontal cortex (mPFC) or nucleus accumbens shell (NAs), and their effects on the acute and long-term avoidance-disruptive effect of clozapine were tested. Results: Intra-mPFC microinjection of quinpirole enhanced the acute avoidance disruptive effect of clozapine (10 mg/kg, sc), while DOI microinjections reduced it marginally. Repeated administration of clozapine (10 mg/kg, sc) daily for 5 days caused a progressive decrease in its inhibition of avoidance responding, indicating tolerance development. Intra-mPFC microinjection of DOI at 25.0 (but not 5.0) μg/side during this period completely abolished the expression of clozapine tolerance. This was indicated by the finding that clozapine-treated rats centrally infused with 25.0 μg/side DOI did not show higher levels of avoidance responses than the vehicle-treated rats in the clozapine challenge test. Microinjection of DOI into the mPFC immediately before the challenge test also decreased the expression of clozapine tolerance. Conclusions: Acute behavioral effect of clozapine can be enhanced by activation of the D2/3 receptors in the mPFC. Clozapine tolerance expression relies on the neuroplasticity initiated by its antagonist action against 5-HT2A/2C receptors in the mPFC. PMID:25288514

  13. A comparison of vanadate to a 2′–5′ linkage at the active site of a small ribozyme suggests a role for water in transition-state stabilization

    PubMed Central

    Torelli, Andrew T.; Krucinska, Jolanta; Wedekind, Joseph E.

    2007-01-01

    The potential for water to participate in RNA catalyzed reactions has been the topic of several recent studies. Here, we report crystals of a minimal, hinged hairpin ribozyme in complex with the transition-state analog vanadate at 2.05 Å resolution. Waters are present in the active site and are discussed in light of existing views of catalytic strategies employed by the hairpin ribozyme. A second structure harboring a 2′,5′-phosphodiester linkage at the site of cleavage was also solved at 2.35 Å resolution and corroborates the assignment of active site waters in the structure containing vanadate. A comparison of the two structures reveals that the 2′,5′ structure adopts a conformation that resembles the reaction intermediate in terms of (1) the positioning of its nonbridging oxygens and (2) the covalent attachment of the 2′-O nucleophile with the scissile G+1 phosphorus. The 2′,5′-linked structure was then overlaid with scissile bonds of other small ribozymes including the glmS metabolite-sensing riboswitch and the hammerhead ribozyme, and suggests the potential of the 2′,5′ linkage to elicit a reaction-intermediate conformation without the need to form metalloenzyme complexes. The hairpin ribozyme structures presented here also suggest how water molecules bound at each of the nonbridging oxygens of G+1 may electrostatically stabilize the transition state in a manner that supplements nucleobase functional groups. Such coordination has not been reported for small ribozymes, but is consistent with the structures of protein enzymes. Overall, this work establishes significant parallels between the RNA and protein enzyme worlds. PMID:17488874

  14. Monitoring disappearance of monomers and generation of resistance to proteolysis during the formation of the activation domain of human procarboxypeptidase A2 (ADA2h) amyloid fibrils by matrix-assisted laser-desorption ionization-time-of-flight-MS.

    PubMed Central

    Villanueva, Josep; Villegas, Virtudes; Querol, Enrique; Avilés, Francesc X; Serrano, Luis

    2003-01-01

    The term 'amyloidosis' is used to represent a group of protein misfolding diseases characterized by the polymerization of normally innocuous and soluble proteins or peptides into insoluble proteinaceous deposits. One of the several questions that remain unclear regarding the process of amyloid fibril formation is related to the status of the protein when such a process begins. Protein engineering is one of the selected approaches to study amyloidosis. Characterization of many variants of a protein can give information about why a soluble protein aggregates to form fibrils. In the present study, we report information on the conformational changes that precede the formation of fibrils, monitored by the complementary use of exoproteolysis and matrix-assisted laser-desorption ionization-time-of-flight-MS. This is a novel application of an easy and fast approach. In addition, we used it to evaluate the ability of the model protein ADA2h (activation domain of human procarboxypeptidase A2) and their mutants to generate amyloid fibrils. It could be a useful test to screen protein variants and to study to what extent some physicochemical parameters affect fibrillogenesis. PMID:12765547

  15. Inhibition of cAMP-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes

    PubMed Central

    Ji, X.; Maxwell, J. T.; Mignery, G. A.; Samarel, A. M.; Lipsius, S. L.

    2016-01-01

    We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates β2-adrenergic receptor (β2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates β2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 μM; zint-β2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 μM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 μM GW5074), phospholipase C (PLC; 0.5 μM edelfosine), PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 μM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in–PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in–PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In–PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in–PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the

  16. Activity.

    ERIC Educational Resources Information Center

    Clearing: Nature and Learning in the Pacific Northwest, 1984

    1984-01-01

    Presents three activities: (1) investigating succession in a schoolground; (2) investigating oak galls; and (3) making sun prints (photographs made without camera or darkroom). Each activity includes a list of materials needed and procedures used. (JN)

  17. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities

    PubMed Central

    Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

    2015-01-01

    Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG’ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG’ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG’ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells

  18. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

    PubMed

    Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-Ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

    2015-01-01

    Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and

  19. Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells.

    PubMed

    Caporarello, Nunzia; Salmeri, Mario; Scalia, Marina; Motta, Carla; Parrino, Cristina; Frittitta, Lucia; Olivieri, Melania; Cristaldi, Martina; Avola, Roberto; Bramanti, Vincenzo; Toscano, Maria Antonietta; Anfuso, Carmelina Daniela; Lupo, Gabriella

    2016-01-01

    It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion.

  20. Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells

    PubMed Central

    Scalia, Marina; Motta, Carla; Parrino, Cristina; Frittitta, Lucia; Olivieri, Melania; Cristaldi, Martina; Avola, Roberto; Bramanti, Vincenzo; Toscano, Maria Antonietta; Anfuso, Carmelina Daniela; Lupo, Gabriella

    2016-01-01

    It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion. PMID:27631977

  1. Activities.

    ERIC Educational Resources Information Center

    Bippert, Judy

    1993-01-01

    Presents activities designed to give students an opportunity to solve concrete problems involving spatial relationships and logical thinking utilizing hands-on manipulatives. Provides teacher instructions and four reproducible worksheets. (MDH)

  2. Activities.

    ERIC Educational Resources Information Center

    Kincaid, Charlene; And Others

    1993-01-01

    Presents an activity in which students collect and organize data from a real-world simulation of the scientific concept of half life. Students collect data using a marble sifter, analyze the data using a graphing calculator, and determine an appropriate mathematical model. Includes reproducible worksheets. (MDH)

  3. A2A Adenosine Receptor (A2AAR) as a Therapeutic Target in Diabetic Retinopathy

    PubMed Central

    Ibrahim, Ahmed S.; El-shishtawy, Mamdouh M.; Zhang, Wenbo; Caldwell, Ruth B.; Liou, Gregory I.

    2011-01-01

    In diabetic retinopathy (DR), abnormalities in vascular and neuronal function are closely related to the local production of inflammatory mediators whose potential source is microglia. A2A adenosine receptor (A2AAR) has been shown to possess anti-inflammatory properties that have not been studied in DR. Here, we evaluate the role of A2AAR and its underlying signaling in retinal complications associated with diabetes. Initial studies in wild-type mice revealed that the treatment with the A2AAR agonist resulted in marked decreases in hyperglycemia-induced retinal cell death and tumor necrosis factor (TNF)-α release. To further assess the role of A2AAR in DR, we studied the effects of A2AAR ablation on diabetes-induced retinal abnormalities. Diabetic A2AAR−/− mice had significantly more terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells, TNF-α release, and intercellular adhesion molecule-1 expression compared with diabetic wild-type mice. To explore a potential mechanism by which A2AAR signaling regulates inflammation in DR, we performed additional studies using microglial cells treated with Amadori-glycated albumin, a risk factor in diabetic disorders. The results showed that activation of A2AAR attenuated Amadori-glycated albumin-induced TNF-α release in a cAMP/exchange protein directly activated by cAMP-dependent mechanism and significantly repressed the inflammatory cascade, C-Raf/extracellular signal-regulated kinase (ERK), in activated microglia. Collectively, this work provides pharmacological and genetic evidence for A2AAR signaling as a control point of cell death in DR and suggests that the retinal protective effect of A2AAR is mediated by abrogating the inflammatory response that occurs in microglia via interaction with C-Raf/ERK pathway. PMID:21514428

  4. Impact of chronic subthalamic high-frequency stimulation on metabolic basal ganglia activity: a 2-deoxyglucose uptake and cytochrome oxidase mRNA study in a macaque model of Parkinson's disease.

    PubMed

    Meissner, Wassilios; Guigoni, Celine; Cirilli, Laetitia; Garret, Maurice; Bioulac, Bernard H; Gross, Christian E; Bezard, Erwan; Benazzouz, Abdelhamid

    2007-03-01

    The mechanisms of action of high-frequency stimulation (HFS) of the subthalamic nucleus (STN) remain only partially understood. Hitherto, experimental studies have suggested that STN-HFS reduces the activity of STN neurons. However, some recent reports have challenged this view, showing that STN-HFS might also increase the activity of globus pallidus internalis (GPi) neurons that are under strong excitatory drive of the STN. In addition, most results emanate from studies applying acute STN-HFS, while parkinsonian patients receive chronic stimulation. Thus, the present study was designed to assess the effect of chronic (10 days) STN-HFS in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primate. For this purpose, 2-deoxyglucose (2-DG) uptake, a measure of global synaptic activity, was assessed in the basal ganglia and the motor thalamus after chronic unilateral STN-HFS. Cytochrome oxidase subunit 1 (COI) mRNA expression, a marker of efferent metabolic activity, was additionally assessed in the globus pallidus. Chronic STN-HFS (i) reversed abnormally decreased 2-DG uptake in the STN of parkinsonian nonhuman primates, (ii) reversed abnormally increased 2-DG accumulation in the GPi while COI mRNA expression was increased, suggesting global activation of GPi neurons, and (iii) reversed abnormally increased 2-DG uptake in the ventrolateral motor thalamus nucleus. The simultaneous decrease in 2-DG uptake and increase in COI mRNA expression are difficult to reconcile with the current model of basal ganglia function and suggest that the mechanisms by which STN-HFS exerts its clinical benefits are more complex than a simple reversal of abnormal activity in the STN and its targets.

  5. Role of hepatitis B virus X repression of C/EBPbeta activity in the down-regulation of glutathione S-transferase A2 gene: implications in other phase II detoxifying enzyme expression.

    PubMed

    Cho, I J; Ki, S H; Brooks, C; Kim, S G

    2009-02-01

    1. A genome-wide in silico screening rendered the genes of phase II enzymes in the rat genome whose promoters contain the putative DNA elements interacting with CCAAT/enhancer binding protein (C/EBP) and NF-E2-related factor (Nrf2). The hepatitis B virus X (HBx) protein strongly modulates the transactivation and/or the repression of genes regulated by some bZIP transcription factors. 2. This study investigated the effects of HBx on the induction of phase II enzymes with the aim of elucidating the role of HBx interaction with C/EBPbeta or Nrf2 bZIP transcription factors in hepatocyte-derived cells. 3. Immunoblot and reporter gene analyses revealed that transfection of HBx interfered with the constitutive and inducible GSTA2 transactivation promoted by oltipraz (C/EBPbeta activator), but not that by tert-butylhydroquinone (t-BHQ, Nrf2 activator). Moreover, HBx transfection completely inhibited GSTA2 reporter gene activity induced by C/EBPbeta, but failed to inhibit that by Nrf2. 4. Gel shift assays identified that HBx inhibited the increase in C/EBPbeta-DNA complex formation by oltipraz, but not the increase in Nrf2-DNA complex by t-BHQ. Immunoprecipitation and immunoblot assays verified the direct interaction between HBx and C/EBPbeta. Moreover, chromatin immunoprecipitation assays confirmed HBx inhibition of C/EBPbeta binding to its binding site in the GSTA2 gene promoter. HBx repressed the induction of other phase II enzymes including GSTP, UDP-glucuronyltransferase 1A, microsomal epoxide hydrolase, GSTM1, GSTM2, and gamma-glutamylcysteine synthase. 5. These results demonstrate that HBx inhibits the induction of phase II detoxifying enzymes, which is mediated by its interaction with C/EBPbeta, but not Nrf2, substantiating the specific role of HBx in phase II detoxifying capacity.

  6. Activation of TLR4 is required for the synergistic induction of dual oxidase 2 and dual oxidase A2 by IFN-γ and lipopolysaccharide in human pancreatic cancer cell lines.

    PubMed

    Wu, Yongzhong; Lu, Jiamo; Antony, Smitha; Juhasz, Agnes; Liu, Han; Jiang, Guojian; Meitzler, Jennifer L; Hollingshead, Melinda; Haines, Diana C; Butcher, Donna; Roy, Krishnendu; Doroshow, James H

    2013-02-15

    Pancreatitis is associated with release of proinflammatory cytokines and reactive oxygen species and plays an important role in the development of pancreatic cancer. We recently demonstrated that dual oxidase (Duox)2, an NADPH oxidase essential for reactive oxygen species-related, gastrointestinal host defense, is regulated by IFN-γ-mediated Stat1 binding to the Duox2 promoter in pancreatic tumor lines. Because LPS enhances the development and invasiveness of pancreatic cancer in vivo following TLR4-related activation of NF-κB, we examined whether LPS, alone or combined with IFN-γ, regulated Duox2. We found that upregulation of TLR4 by IFN-γ in BxPC-3 and CFPAC-1 pancreatic cancer cells was augmented by LPS, resulting in activation of NF-κB, accumulation of NF-κB (p65) in the nucleus, and increased binding of p65 to the Duox2 promoter. TLR4 silencing with small interfering RNAs, as well as two independent NF-κB inhibitors, attenuated LPS- and IFN-γ-mediated Duox2 upregulation in BxPC-3 cells. Induction of Duox2 expression by IFN-γ and LPS may result from IFN-γ-related activation of Stat1 acting in concert with NF-κB-related upregulation of Duox2. Sustained extracellular accumulation of H(2)O(2) generated by exposure to both LPS and IFN-γ was responsible for an ∼50% decrease in BxPC-3 cell proliferation associated with a G(1) cell cycle block, apoptosis, and DNA damage. We also demonstrated upregulation of Duox expression in vivo in pancreatic cancer xenografts and in patients with chronic pancreatitis. These results suggest that inflammatory cytokines can interact to produce a Duox-dependent pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation and pancreatic cancer cells.

  7. The A2 Experiment Program at MAMI

    NASA Astrophysics Data System (ADS)

    Briscoe, William; A2 Collaboration

    2014-09-01

    The Mainz Microtron MAMI is an accelerator for electron beams run by the Institut für Kernphysik of the Johannes Gutenberg-Universität Mainz used for hadron physics experiments. Of it's three active experimental halls, the A2 facility, which features the presence of the SLAC Crystal Ball detector, has produced a plethora of experimental results, which has contributed to the understanding of the structure of the nucleon. An overview and update of the current A2 program will be presented. The Mainz Microtron MAMI is an accelerator for electron beams run by the Institut für Kernphysik of the Johannes Gutenberg-Universität Mainz used for hadron physics experiments. Of it's three active experimental halls, the A2 facility, which features the presence of the SLAC Crystal Ball detector, has produced a plethora of experimental results, which has contributed to the understanding of the structure of the nucleon. An overview and update of the current A2 program will be presented. Funded in part by SFB 1044. US collaborators funded by USDOE and USNSF.

  8. DNA binding and antiproliferative activity toward human carcinoma cells of copper(II) and zinc(II) complexes of a 2,5-diphenyl[1,3,4]oxadiazole derivative.

    PubMed

    Terenzi, Alessio; Fanelli, Mirco; Ambrosi, Gianluca; Amatori, Stefano; Fusi, Vieri; Giorgi, Luca; Turco Liveri, Vincenzo; Barone, Giampaolo

    2012-04-21

    The interaction of calf thymus DNA with [CuL(ClO(4))]ClO(4)·H(2)O (1) and [ZnLBr]Br·H(2)O (2) (L = 9,12,15,18,27,28-hexaaza-29-oxatetracyclo[24.2.1.0(2,7).0(20,25)]enneicosa-2,4,6,20,22,24,26,28(1)-octaene) dicationic complexes in aqueous solution at neutral pH, was investigated by variable-temperature UV-vis absorption, circular dichroism and fluorescence spectroscopy. The values of the DNA-binding constants of these complexes, determined by competitive binding spectrofluorimetric titrations of ethidium bromide (EB)-DNA solutions, are (6.7 ± 0.5) × 10(6) M(-1) for CuL(2+) and (4.7 ± 0.5) × 10(5) M(-1) for ZnL(2+). These data together with a through analysis of the spectroscopic behaviour consistently suggest that both compounds are effective DNA binders. Interestingly, the DNA-binding strength of these complexes has been found to be correlated to their in vitro cytotoxic activity toward human breast carcinoma cells, although the complex with lower DNA-binding affinity is more active. In fact, biological studies showed that when the compounds are delivered through the cell membrane by a lipidic carrier, the cell survival is sensibly reduced, up to 58% with 1 and to 31% with 2.

  9. A renal metanephric adenoma showing both a 2p16e24 deletion and BRAF V600E mutation: a synergistic role for a tumor suppressor gene on chromosome 2p and BRAF activation?

    PubMed

    Dadone, Bérengère; Ambrosetti, Damien; Carpentier, Xavier; Duranton-Tanneur, Valérie; Burel-Vandenbos, Fanny; Amiel, Jean; Pedeutour, Florence

    2013-01-01

    Metanephric adenomas (MAs) are rare benign tumors that may be difficult to recognize. Specific genetic anomalies might aid in diagnosis, but genomic data are limited and conflicting. Consistent mutations of the BRAF gene have been recently reported in MAs and could become useful as a discriminative marker among renal tumors. We report here a case of MA, showing both a BRAF V600E mutation and a segmental loss within bands 2p16 and 2p24 as the sole quantitative genomic anomaly. We compared the borders and size of the deleted region in our case to those of five cases of MAs previously reported. We identified a common minimal region containing 87 genes, among which several tumor suppressor genes could be candidate actors in the pathogenesis of MA. We ruled out MSH2 and MSH6 as target gene candidates, both located in the deleted region, on the basis of preserved expression and microsatellite sequence stability. Our study confirms the recurrence of a BRAF mutation and of 2p alterations in MAs. This first case showing simultaneous presence of a BRAF mutation and a 2p deletion raises the question of a synergistic role for these two anomalies in the pathogenesis of MAs.

  10. IL-29 and IFN-α regulate the expression of MxA, 2',5'-OAS and PKR genes in association with the activation of Raf-MEK-ERK and PI3K-AKT signal pathways in HepG2.2.15 cells.

    PubMed

    Chai, Yu; Huang, Hai-Liang; Hu, Dao-Jun; Luo, Xin; Tao, Qian-Shan; Zhang, Xiao-Ling; Zhang, Sheng-Quan

    2011-01-01

    Interferons (IFNs) can activate the PI3K-AKT and Raf-MEK-ERK signal pathways and induce antiviral proteins (MxA, 2',5'-OAS and PKR) expression in specific cell lines. However, the relationship between those antiviral proteins expression and signal pathways remains unknown at present. Thus our experiments were designed to determine the exact relationship in HepG2.2.15 cell line. The results demonstrated that IFN-α and IL-29 were both able to activate PI3K-AKT and Raf-MEK-ERK signal pathways, and IFN-α up-regulated the expression of MxA, 2',5'-OAS and PKR whereas IL-29 increased mRNA expression of MxA and 2',5'-OAS and had no influence on PKR. Furthermore, MxA, 2',5'-OAS and PKR expression were down-regulated while PI3K-AKT signal pathway was blocked by LY294002. And MxA was up-regulated after Raf-MEK-ERK signal pathway being blocked by PD98059. These findings indicate that the expression of MxA, 2',5'-OAS and PKR are up-regulate by PI3K-AKT signal pathway, and Raf-MEK-ERK signal pathway has a negative regulatory effect on the expression of MxA and no significant effect on 2',5'-OAS and PKR.

  11. Group XV phospholipase A2, a lysosomal phospholipase A2

    PubMed Central

    Shayman, James A.; Kelly, Robert; Kollmeyer, Jessica; He, Yongqun; Abe, Akira

    2010-01-01

    A phospholipase A2 was identified from MDCK cell homogenates with broad specificity toward glycerophospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. The phospholipase has the unique ability to transacylate short chain ceramides. This phospholipase is calcium-independent, localized to lysosomes, and has an acidic pH optimum. The enzyme was purified from bovine brain and found to be a water-soluble glycoprotein consisting of a single peptide chain with a molecular weight of 45 kDa. The primary structure deduced from the DNA sequences is highly conserved between chordates. The enzyme was named lysosomal phospholipase A2 (LPLA2) and subsequently designated group XV phospholipase A2. LPLA2 has 49 percent of amino acid sequence identity to lecithin cholesterol acyltransferase and is a member of the αβ-hydrolase superfamily. LPLA2 is highly expressed in alveolar macrophages. A marked accumulation of glycerophospholipids and extensive lamellar inclusion bodies, a hallmark of cellular phospholipidosis, is observed in alveolar macrophages in LPLA2−/− mice. This defect can also be reproduced in macrophages that are exposed to cationic amphiphilic drugs such as amiodarone. In addition, older LPLA2−/− mice develop a phenotype similar to human autoimmune disease. These observations indicate that LPLA2 may play a primary role in phospholipid homeostasis, drug toxicity, and host defense. PMID:21074554

  12. Adenosine A2A Receptors and A2A Receptor Heteromers as Key Players in Striatal Function

    PubMed Central

    Ferré, Sergi; Quiroz, César; Orru, Marco; Guitart, Xavier; Navarro, Gemma; Cortés, Antonio; Casadó, Vicent; Canela, Enric I.; Lluis, Carme; Franco, Rafael

    2011-01-01

    A very significant density of adenosine A2A receptors (A2ARs) is present in the striatum, where they are preferentially localized postsynaptically in striatopallidal medium spiny neurons (MSNs). In this localization A2ARs establish reciprocal antagonistic interactions with dopamine D2 receptors (D2Rs). In one type of interaction, A2AR and D2R are forming heteromers and, by means of an allosteric interaction, A2AR counteracts D2R-mediated inhibitory modulation of the effects of NMDA receptor stimulation in the striatopallidal neuron. This interaction is probably mostly responsible for the locomotor depressant and activating effects of A2AR agonist and antagonists, respectively. The second type of interaction involves A2AR and D2R that do not form heteromers and takes place at the level of adenylyl cyclase (AC). Due to a strong tonic effect of endogenous dopamine on striatal D2R, this interaction keeps A2AR from signaling through AC. However, under conditions of dopamine depletion or with blockade of D2R, A2AR-mediated AC activation is unleashed with an increased gene expression and activity of the striatopallidal neuron and with a consequent motor depression. This interaction is probably the main mechanism responsible for the locomotor depression induced by D2R antagonists. Finally, striatal A2ARs are also localized presynaptically, in cortico-striatal glutamatergic terminals that contact the striato-nigral MSN. These presynaptic A2ARs heteromerize with A1 receptors (A1Rs) and their activation facilitates glutamate release. These three different types of A2ARs can be pharmacologically dissected by their ability to bind ligands with different affinity and can therefore provide selective targets for drug development in different basal ganglia disorders. PMID:21731559

  13. Dermatan sulfate in tunicate phylogeny: Order-specific sulfation pattern and the effect of [→4IdoA(2-Sulfate)β-1→3GalNAc(4-Sulfate)β-1→] motifs in dermatan sulfate on heparin cofactor II activity

    PubMed Central

    2011-01-01

    Background Previously, we have reported the presence of highly sulfated dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra) and Stolidobranchia (Halocynthia pyriformis and Styela plicata). Despite the identical disaccharide backbone, consisting of [→4IdoA(2S)β-1→3GalNAcβ-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi) and Phlebobranchia (Ciona intestinalis), aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units. Results Disaccharide analyses indicated a high content of disulfated disaccharide units in the dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfate)β-1→3GalNAcβ-1→] modulate heparin

  14. EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

    PubMed Central

    Subbarayal, Prema; Karunakaran, Karthika; Winkler, Ann-Cathrin; Rother, Marion; Gonzalez, Erik; Meyer, Thomas F.; Rudel, Thomas

    2015-01-01

    The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and

  15. Metabolic Syndrome and Selective Inflammatory Markers in Psoriatic Patients

    PubMed Central

    Vachatova, Simona; Andrys, Ctirad; Salavec, Miloslav; Ettler, Karel; Rehacek, Vit; Cermakova, Eva; Malkova, Andrea

    2016-01-01

    The presented article studies the role of selected inflammatory and anti-inflammatory serum markers of psoriatic patients in the pathogenesis of metabolic syndrome (MS) and psoriasis. The study is based on the comparison between the group of psoriatic patients (74) and the control group (65). We found significantly higher BMI (p < 0.05) and diastolic blood pressure (p < 0.05) in the psoriatic patients. The values of waist circumference and BMI were significantly higher (p < 0.05) in the male patients compared to the men in the control group. The analysis revealed significantly higher CRP (p < 0.001), Lp-PLA2 (p < 0.001), leptin (p < 0.01), and resistin (p < 0.01) levels in the psoriatic patients. Significantly higher levels of CRP (p < 0.01), Lp-PLA2 (p < 0.001), leptin (p < 0.01), and resistin (p < 0.05) were found in the patients with MS compared to the controls with MS. The level of adiponectin was significantly lower (p < 0.01) in the patients with MS. Finally, we found significantly higher level of Lp-PLA2 (p < 0.001) in the group of patients without MS compared to the controls without MS. In conclusion, observed inflammatory and anti-inflammatory markers (CRP, adiponectin, leptin, resistin, and Lp-PLA2) are involved in both pathogenesis of MS and pathogenesis of psoriasis. The level of Lp-PLA2 indicates the presence of subclinical atherosclerosis (cardiovascular risk) in psoriatic patients. PMID:28097156

  16. Chromomycin A2 induces autophagy in melanoma cells.

    PubMed

    Guimarães, Larissa Alves; Jimenez, Paula Christine; Sousa, Thiciana da Silva; Freitas, Hozana Patrícia S; Rocha, Danilo Damasceno; Wilke, Diego Veras; Martín, Jesús; Reyes, Fernando; Deusdênia Loiola Pessoa, Otília; Costa-Lotufo, Letícia Veras

    2014-12-04

    The present study highlights the biological effects of chromomycin A2 toward metastatic melanoma cells in culture. Besides chromomycin A2, chromomycin A3 and demethylchromomycin A2 were also identified from the extract derived from Streptomyces sp., recovered from Paracuru Beach, located in the northeast region of Brazil. The cytotoxic activity of chromomycin A2 was evaluated across a panel of human tumor cell lines, which found IC50 values in the nM-range for exposures of 48 and 72 h. MALME-3M, a metastatic melanoma cell line, showed the highest sensitivity to chromomycin A2 after 48h incubation, and was chosen as a model to investigate this potent cytotoxic effect. Treatment with chromomycin A2 at 30 nM reduced cell proliferation, but had no significant effect upon cell viability. Additionally, chromomycin A2 induced accumulation of cells in G0/G1 phase of the cell cycle, with consequent reduction of S and G2/M and unbalanced expression of cyclins. Chromomycin A2 treated cells depicted several cellular fragments resembling autophagosomes and increased expression of proteins LC3-A and LC3-B. Moreover, exposure to chromomycin A2 also induced the appearance of acidic vacuolar organelles in treated cells. These features combined are suggestive of the induction of autophagy promoted by chromomycin A2, a feature not previously described for chromomycins.

  17. Chromomycin A2 Induces Autophagy in Melanoma Cells

    PubMed Central

    Guimarães, Larissa Alves; Jimenez, Paula Christine; Sousa, Thiciana da Silva; Freitas, Hozana Patrícia S.; Rocha, Danilo Damasceno; Wilke, Diego Veras; Martín, Jesús; Reyes, Fernando; Pessoa, Otília Deusdênia Loiola; Costa-Lotufo, Letícia Veras

    2014-01-01

    The present study highlights the biological effects of chromomycin A2 toward metastatic melanoma cells in culture. Besides chromomycin A2, chromomycin A3 and demethylchromomycin A2 were also identified from the extract derived from Streptomyces sp., recovered from Paracuru Beach, located in the northeast region of Brazil. The cytotoxic activity of chromomycin A2 was evaluated across a panel of human tumor cell lines, which found IC50 values in the nM-range for exposures of 48 and 72 h. MALME-3M, a metastatic melanoma cell line, showed the highest sensitivity to chromomycin A2 after 48h incubation, and was chosen as a model to investigate this potent cytotoxic effect. Treatment with chromomycin A2 at 30 nM reduced cell proliferation, but had no significant effect upon cell viability. Additionally, chromomycin A2 induced accumulation of cells in G0/G1 phase of the cell cycle, with consequent reduction of S and G2/M and unbalanced expression of cyclins. Chromomycin A2 treated cells depicted several cellular fragments resembling autophagosomes and increased expression of proteins LC3-A and LC3-B. Moreover, exposure to chromomycin A2 also induced the appearance of acidic vacuolar organelles in treated cells. These features combined are suggestive of the induction of autophagy promoted by chromomycin A2, a feature not previously described for chromomycins. PMID:25486109

  18. Annexin A2 Regulates TRPA1-Dependent Nociception

    PubMed Central

    Avenali, Luca; Narayanan, Pratibha; Rouwette, Tom; Cervellini, Ilaria; Sereda, Michael

    2014-01-01

    The transient receptor potential A1 (TRPA1) channel is essential for vertebrate pain. Even though TRPA1 activation by ligands has been studied extensively, the molecular machinery regulating TRPA1 is only poorly understood. Using an unbiased proteomics-based approach we uncovered the physical association of Annexin A2 (AnxA2) with native TRPA1 in mouse sensory neurons. AnxA2 is enriched in a subpopulation of sensory neurons and coexpressed with TRPA1. Furthermore, we observe an increase of TRPA1 membrane levels in cultured sensory neurons from AnxA2-deficient mice. This is reflected by our calcium imaging experiments revealing higher responsiveness upon TRPA1 activation in AnxA2-deficient neurons. In vivo these findings are associated with enhanced nocifensive behaviors specifically in TRPA1-dependent paradigms of acute and inflammatory pain, while heat and mechanical sensitivity as well as TRPV1-mediated pain are preserved in AnxA2-deficient mice. Our results support a model whereby AnxA2 limits the availability of TRPA1 channels to regulate nociceptive signaling in vertebrates. PMID:25355205

  19. Annexin A2 regulates TRPA1-dependent nociception.

    PubMed

    Avenali, Luca; Narayanan, Pratibha; Rouwette, Tom; Cervellini, Ilaria; Sereda, Michael; Gomez-Varela, David; Schmidt, Manuela

    2014-10-29

    The transient receptor potential A1 (TRPA1) channel is essential for vertebrate pain. Even though TRPA1 activation by ligands has been studied extensively, the molecular machinery regulating TRPA1 is only poorly understood. Using an unbiased proteomics-based approach we uncovered the physical association of Annexin A2 (AnxA2) with native TRPA1 in mouse sensory neurons. AnxA2 is enriched in a subpopulation of sensory neurons and coexpressed with TRPA1. Furthermore, we observe an increase of TRPA1 membrane levels in cultured sensory neurons from AnxA2-deficient mice. This is reflected by our calcium imaging experiments revealing higher responsiveness upon TRPA1 activation in AnxA2-deficient neurons. In vivo these findings are associated with enhanced nocifensive behaviors specifically in TRPA1-dependent paradigms of acute and inflammatory pain, while heat and mechanical sensitivity as well as TRPV1-mediated pain are preserved in AnxA2-deficient mice. Our results support a model whereby AnxA2 limits the availability of TRPA1 channels to regulate nociceptive signaling in vertebrates.

  20. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  1. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  2. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  3. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  4. 42 CFR 5a.2 - Applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Applicability. 5a.2 Section 5a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS RURAL PHYSICIAN... Public Health Service Act....

  5. 18 CFR 3a.2 - Authority.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Authority. 3a.2 Section 3a.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION General § 3a.2 Authority. Official information...

  6. 18 CFR 3a.2 - Authority.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Authority. 3a.2 Section 3a.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION General § 3a.2 Authority. Official information...

  7. 18 CFR 3a.2 - Authority.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Authority. 3a.2 Section 3a.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION General § 3a.2 Authority. Official information...

  8. 18 CFR 3a.2 - Authority.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Authority. 3a.2 Section 3a.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION General § 3a.2 Authority. Official information...

  9. 18 CFR 3a.2 - Authority.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Authority. 3a.2 Section 3a.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES NATIONAL SECURITY INFORMATION General § 3a.2 Authority. Official information...

  10. 42 CFR 51a.2 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Definitions. 51a.2 Section 51a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS PROJECT GRANTS FOR MATERNAL AND CHILD HEALTH § 51a.2 Definitions. Act means the Social Security Act, as amended. Genetic diseases...

  11. 45 CFR 12a.2 - Applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Applicability. 12a.2 Section 12a.2 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION USE OF FEDERAL REAL PROPERTY TO ASSIST THE HOMELESS § 12a.2 Applicability. (a) This part applies to Federal real property which has been designated...

  12. 45 CFR 12a.2 - Applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 45 Public Welfare 1 2012-10-01 2012-10-01 false Applicability. 12a.2 Section 12a.2 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION USE OF FEDERAL REAL PROPERTY TO ASSIST THE HOMELESS § 12a.2 Applicability. (a) This part applies to Federal real property which has been designated...

  13. 45 CFR 12a.2 - Applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 45 Public Welfare 1 2013-10-01 2013-10-01 false Applicability. 12a.2 Section 12a.2 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION USE OF FEDERAL REAL PROPERTY TO ASSIST THE HOMELESS § 12a.2 Applicability. (a) This part applies to Federal real property which has been designated...

  14. 45 CFR 12a.2 - Applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 45 Public Welfare 1 2014-10-01 2014-10-01 false Applicability. 12a.2 Section 12a.2 Public Welfare Department of Health and Human Services GENERAL ADMINISTRATION USE OF FEDERAL REAL PROPERTY TO ASSIST THE HOMELESS § 12a.2 Applicability. (a) This part applies to Federal real property which has been designated...

  15. 45 CFR 12a.2 - Applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 1 2011-10-01 2011-10-01 false Applicability. 12a.2 Section 12a.2 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION USE OF FEDERAL REAL PROPERTY TO ASSIST THE HOMELESS § 12a.2 Applicability. (a) This part applies to Federal real property which has been designated...

  16. 32 CFR 168a.2 - Applicability.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 1 2010-07-01 2010-07-01 false Applicability. 168a.2 Section 168a.2 National Defense Department of Defense OFFICE OF THE SECRETARY OF DEFENSE DEFENSE CONTRACTING NATIONAL DEFENSE SCIENCE AND ENGINEERING GRADUATE FELLOWSHIPS § 168a.2 Applicability. This part applies to the Office...

  17. 32 CFR 168a.2 - Applicability.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 1 2011-07-01 2011-07-01 false Applicability. 168a.2 Section 168a.2 National Defense Department of Defense OFFICE OF THE SECRETARY OF DEFENSE DEFENSE CONTRACTING NATIONAL DEFENSE SCIENCE AND ENGINEERING GRADUATE FELLOWSHIPS § 168a.2 Applicability. This part applies to the Office...

  18. 29 CFR 1912a.2 - Membership.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 7 2014-07-01 2014-07-01 false Membership. 1912a.2 Section 1912a.2 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) NATIONAL ADVISORY COMMITTEE ON OCCUPATIONAL SAFETY AND HEALTH § 1912a.2 Membership. The Committee is...

  19. 29 CFR 1912a.2 - Membership.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 7 2012-07-01 2012-07-01 false Membership. 1912a.2 Section 1912a.2 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) NATIONAL ADVISORY COMMITTEE ON OCCUPATIONAL SAFETY AND HEALTH § 1912a.2 Membership. The Committee is...

  20. 29 CFR 1912a.2 - Membership.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 7 2010-07-01 2010-07-01 false Membership. 1912a.2 Section 1912a.2 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) NATIONAL ADVISORY COMMITTEE ON OCCUPATIONAL SAFETY AND HEALTH § 1912a.2 Membership. The Committee is...

  1. 29 CFR 1912a.2 - Membership.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 7 2013-07-01 2013-07-01 false Membership. 1912a.2 Section 1912a.2 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) NATIONAL ADVISORY COMMITTEE ON OCCUPATIONAL SAFETY AND HEALTH § 1912a.2 Membership. The Committee is...

  2. 29 CFR 1912a.2 - Membership.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 7 2011-07-01 2011-07-01 false Membership. 1912a.2 Section 1912a.2 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) NATIONAL ADVISORY COMMITTEE ON OCCUPATIONAL SAFETY AND HEALTH § 1912a.2 Membership. The Committee is...

  3. 32 CFR 352a.2 - Applicability.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 2 2010-07-01 2010-07-01 false Applicability. 352a.2 Section 352a.2 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) ORGANIZATIONAL CHARTERS DEFENSE FINANCE AND ACCOUNTING SERVICE (DFAS) § 352a.2 Applicability. This part applies to...

  4. 42 CFR 63a.2 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Definitions. 63a.2 Section 63a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES FELLOWSHIPS, INTERNSHIPS, TRAINING NATIONAL INSTITUTES OF HEALTH TRAINING GRANTS § 63a.2 Definitions. As used in this part: Act means...

  5. 42 CFR 63a.2 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Definitions. 63a.2 Section 63a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES FELLOWSHIPS, INTERNSHIPS, TRAINING NATIONAL INSTITUTES OF HEALTH TRAINING GRANTS § 63a.2 Definitions. As used in this part: Act means...

  6. 42 CFR 63a.2 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Definitions. 63a.2 Section 63a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES FELLOWSHIPS, INTERNSHIPS, TRAINING NATIONAL INSTITUTES OF HEALTH TRAINING GRANTS § 63a.2 Definitions. As used in this part: Act means...

  7. 42 CFR 63a.2 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Definitions. 63a.2 Section 63a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES FELLOWSHIPS, INTERNSHIPS, TRAINING NATIONAL INSTITUTES OF HEALTH TRAINING GRANTS § 63a.2 Definitions. As used in this part: Act means...

  8. 42 CFR 63a.2 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Definitions. 63a.2 Section 63a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES FELLOWSHIPS, INTERNSHIPS, TRAINING NATIONAL INSTITUTES OF HEALTH TRAINING GRANTS § 63a.2 Definitions. As used in this part: Act means...

  9. 22 CFR 9a.2 - General policy.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false General policy. 9a.2 Section 9a.2 Foreign... ENERGY PROGRAMS; RELATED MATERIAL § 9a.2 General policy. (a) The United States has entered into the... the IEA. Confidentiality is essential to assure the free and open discussion necessary to...

  10. Potential therapeutic relevance of adenosine A2B and A2A receptors in the central nervous system.

    PubMed

    Popoli, Patrizia; Pepponi, Rita

    2012-09-01

    Adenosine A2B and, much more importantly, adenosine A2A receptors modulate many physiological and pathological processes in the brain. In this review, the most recent evidence concerning the role of such receptors and their potential therapeutic relevance is discussed. The low affinity of A2B receptors for adenosine implies that they might represent a good therapeutic target, since they are activated only under pathological conditions (when adenosine levels raise up to micromolar concentrations). The availability of selective ligands for A2B receptors would allow exploration of such an hypothesis. Since adenosine A2A receptors mediate both potentially neuroprotective and potentially neurotoxic effects, their role in neurodegenerative diseases is highly controversial. Nevertheless, A2A receptor antagonists have shown clear antiparkinsonian effects, and a great interest exists on the role of A2A receptors in Alzheimer's disease, brain ischaemia, spinal cord injury, drug addiction and other conditions. In order to establish whether such receptors represent a target for CNS diseases, at least two conditions are needed: the full comprehension of A2A-dependent mechanisms and the availability of ligands capable of discriminating among the different receptor populations.

  11. COL4A2 mutations impair COL4A1 and COL4A2 secretion and cause hemorrhagic stroke.

    PubMed

    Jeanne, Marion; Labelle-Dumais, Cassandre; Jorgensen, Jeff; Kauffman, W Berkeley; Mancini, Grazia M; Favor, Jack; Valant, Valerie; Greenberg, Steven M; Rosand, Jonathan; Gould, Douglas B

    2012-01-13

    Collagen, type IV, alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers and are abundant components of basement membranes, including those of the cerebral vasculature. COL4A1 mutations are an increasingly recognized cause of multisystem disorders, including highly penetrant cerebrovascular disease and intracerebral hemorrhage (ICH). Because COL4A1 and COL4A2 are structurally and functionally associated, we hypothesized that variants in COL4A2 would also cause ICH. We sequence COL4A2 in 96 patients with ICH and identify three rare, nonsynonymous coding variants in four patients that are not present in a cohort of 144 ICH-free individuals. All three variants change evolutionarily conserved amino acids. Using a cellular assay, we show that these putative mutations cause intracellular accumulation of COL4A1 and COL4A2 at the expense of their secretion, which supports their pathogenecity. Furthermore, we show that Col4a2 mutant mice also have completely penetrant ICH and that mutations in mouse and human lead to retention of COL4A1 and COL4A2 within the endoplasmic reticulum (ER). Importantly, two of the three putative mutations found in patients trigger ER stress and activate the unfolded protein response. The identification of putative COL4A2 mutations that might contribute to ICH in human patients provides insight into the pathogenic mechanisms of this disease. Our data suggest that COL4A2 mutations impair COL4A1 and COL4A2 secretion and can also result in cytotoxicity. Finally, our findings suggest that, collectively, mutations in COL4A1 and COL4A2 contribute to sporadic cases of ICH.

  12. 42 CFR 59a.2 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Definitions. 59a.2 Section 59a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL LIBRARY OF MEDICINE GRANTS... have the same meaning as provided in the Act. As used in this subpart: Act means the Public...

  13. 42 CFR 59a.2 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Definitions. 59a.2 Section 59a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL LIBRARY OF MEDICINE GRANTS... have the same meaning as provided in the Act. As used in this subpart: Act means the Public...

  14. 42 CFR 59a.2 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Definitions. 59a.2 Section 59a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL LIBRARY OF MEDICINE GRANTS... have the same meaning as provided in the Act. As used in this subpart: Act means the Public...

  15. 42 CFR 59a.2 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Definitions. 59a.2 Section 59a.2 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL LIBRARY OF MEDICINE GRANTS... have the same meaning as provided in the Act. As used in this subpart: Act means the Public...

  16. Dnmt3a2: a hub for enhancing cognitive functions.

    PubMed

    Oliveira, A M M; Hemstedt, T J; Freitag, H E; Bading, H

    2016-08-01

    The mechanisms responsible for fear memory formation and extinction are far from being understood. Uncovering the molecules and mechanisms regulating these processes is vital for identifying molecular targets for the development of novel therapeutic strategies for anxiety and fear disorders. Cognitive abilities require the activation of gene expression necessary to the consolidation of lasting changes in neuronal function. In this study we established a key role for an epigenetic factor, the de novo DNA methyltransferase, Dnmt3a2, in memory formation and extinction. We found that Dnmt3a2 overexpression in the hippocampus of young adult mice induced memory enhancements in a variety of situations; it converted a weak learning experience into long-term memory, enhanced fear memory formation and facilitated fear memory extinction. Dnmt3a2 overexpression was also associated with the increased expression of plasticity-related genes. Furthermore, the knockdown of Dnmt3a2 expression impaired the animals' ability to extinguish memories, identifying Dnmt3a2 as a key player in extinction. Thus, Dnmt3a2 is at the core of memory processes and represents a novel target for cognition-enhancing therapies to ameliorate anxiety and fear disorders and boost memory consolidation.

  17. Enzymatic Comparisons of Aspergillus niger PhyA and Escherichia coli AppA2 Phytases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was to compare three phytase activity assays and kinetics of Aspergillus niger PhyA and Escherichia coli AppA2 phytases expressed in Pichia pastoris at the observed stomach pH of 3.5. In Experiment 1, equivalent phytase activities in the crude preparations of PhyA and AppA2 were tested ...

  18. Ruthenium-cluster-mediated activation of all bonds of a methyl group of 6,6'-dimethyl-2,2'-bipyridine and 2,9-dimethyl-1,10-phenanthroline: transformation of the latter into a 2-alkenyl-9-methyl-1,10-phenanthroline ligand.

    PubMed

    Cabeza, Javier A; del Río, Ignacio; Martínez-Méndez, Lorena; Miguel, Daniel

    2006-02-01

    The treatment of [Ru3(CO)12] with 6,6'-dimethyl-2,2'-bipyridine (Me2bipy) or 2,9-dimethyl-1,10-phenanthroline (Me2phen) in THF at reflux temperature gives the trinuclear dihydride complexes [Ru3(mu-H)2(mu3-L1)(CO)8] (L1 = HCbipyMe 1 a, HCphenMe 1 b), which result from the activation of two C-H bonds of a methyl group. The hexa-, hepta-, and pentanuclear derivatives [Ru6(mu3-H)(mu5-L2)(mu-CO)3(CO)13] (L2 = CbipyMe 2 a, CphenMe 2 b), [Ru7(mu3-H)(mu5-L2)(mu-CO)2(CO)16] (L2 = CbipyMe 3 a, CphenMe 3 b), and [Ru5(mu-H)(mu5-C)(mu-L3)(CO)13] (L3 = bipyMe 4 a, phenMe 4 b) can also be obtained by treating 1 a and 1 b with [Ru3(CO)12]. Compounds 2 a and 2 b have a basal edge-bridged square-pyramidal metallic skeleton with a carbyne-type C atom capping the four Ru atoms of the pyramid base. The structures of 3 a and 3 b are similar to those of 2 a and 2 b, respectively, but an additional Ru atom now caps a triangular face of the square-pyramidal fragment of the metallic skeleton. The most interesting feature of 2 a, 2 b, 3 a, and 3 b is that their carbyne-type C atoms were originally bound to three hydrogen atoms in Me2bipy or Me2phen and, therefore, they arise from the unprecedented activation of all three C-H bonds of C-bound methyl groups. The pentanuclear compounds 4 a and 4 b contain a carbide ligand surrounded by five Ru atoms in a distorted trigonal-bipyramidal environment. They are the products of a series of processes that includes the activation of all bonds (three C-H and one C-C) of organic methyl groups, and are the first examples of complexes having carbide ligands that arise from C-bonded methyl groups. The alkenyl derivatives [Ru5(mu5-C)(mu-p-MeC6H4CHCHphenMe)(CO)13] (5 b), [Ru5(mu-H)(mu5-C)(mu-p-MeC6H4CHCHphenMe)(p-tolC2)(CO)12] (6 b), and [Ru5(mu-H)(mu5-C)(mu-PhCHCHphenMe)(PhC2)(CO)12] (7 b) have been obtained by treating 4 b with p-tolyl- and phenylacetylene, respectively. Their heterocyclic ligands contain an alkenyl fragment in the position that was

  19. METSAT: Advanced Microwave Sounding Unit-A2 (AMSU-A2) structural mathematical model

    NASA Technical Reports Server (NTRS)

    Ely, Wayne

    1995-01-01

    This plan describes the Structural Mathematical Model of the METSAT AMSU-A2 instrument. The model is used to verify the structural adequacy of the AMSU-A2 instrument for the specified loading environments.

  20. 42 CFR 52a.2 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ..., psychological, prevention and treatment research and related activities on breast cancer; (d) For purposes of..., prevention and treatment methods for cancer; (c) For purposes of grants authorized by section 417 of the Act..., and related activities on prostate cancer; (e) For purposes of grants authorized by section 422 of...

  1. 42 CFR 52a.2 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ..., psychological, prevention and treatment research and related activities on breast cancer; (d) For purposes of..., prevention and treatment methods for cancer; (c) For purposes of grants authorized by section 417 of the Act..., and related activities on prostate cancer; (e) For purposes of grants authorized by section 422 of...

  2. 42 CFR 52a.2 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ..., psychological, prevention and treatment research and related activities on breast cancer; (d) For purposes of..., prevention and treatment methods for cancer; (c) For purposes of grants authorized by section 417 of the Act..., and related activities on prostate cancer; (e) For purposes of grants authorized by section 422 of...

  3. 42 CFR 2a.2 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS PROTECTION OF IDENTITY-RESEARCH SUBJECTS § 2a.2 Definitions. (a) Secretary means the Secretary of Health and Human Services and any other officer or employee of the Department of Health and Human Services to whom the...

  4. 42 CFR 2a.2 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS PROTECTION OF IDENTITY-RESEARCH SUBJECTS § 2a.2 Definitions. (a) Secretary means the Secretary of Health and Human Services and any other officer or employee of the Department of Health and Human Services to whom the...

  5. 42 CFR 2a.2 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS PROTECTION OF IDENTITY-RESEARCH SUBJECTS § 2a.2 Definitions. (a) Secretary means the Secretary of Health and Human Services and any other officer or employee of the Department of Health and Human Services to whom the...

  6. 42 CFR 2a.2 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS PROTECTION OF IDENTITY-RESEARCH SUBJECTS § 2a.2 Definitions. (a) Secretary means the Secretary of Health and Human Services and any other officer or employee of the Department of Health and Human Services to whom the...

  7. 29 CFR 4041A.2 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... plan year, available resources as described in section 4245(b)(3) of ERISA. Benefits subject to... Relating to Labor (Continued) PENSION BENEFIT GUARANTY CORPORATION PLAN TERMINATIONS TERMINATION OF MULTIEMPLOYER PLANS General Provisions § 4041A.2 Definitions. The following terms are defined in § 4001.1...

  8. 42 CFR 51a.2 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CHILD HEALTH § 51a.2 Definitions. Act means the Social Security Act, as amended. Genetic diseases means... factor. Institution of higher learning means any college or university accredited by a regionalized body... has higher learning among its purposes and functions and which has a formal affiliation with...

  9. 42 CFR 51a.2 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CHILD HEALTH § 51a.2 Definitions. Act means the Social Security Act, as amended. Genetic diseases means inherited disorders caused by the transmission of certain aberrant genes from one generation to another. Hemophilia means a genetically transmitted bleeding disorder resulting from a deficiency of a plasma...

  10. 42 CFR 51a.2 - Definitions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CHILD HEALTH § 51a.2 Definitions. Act means the Social Security Act, as amended. Genetic diseases means inherited disorders caused by the transmission of certain aberrant genes from one generation to another. Hemophilia means a genetically transmitted bleeding disorder resulting from a deficiency of a plasma...

  11. 42 CFR 51a.2 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CHILD HEALTH § 51a.2 Definitions. Act means the Social Security Act, as amended. Genetic diseases means inherited disorders caused by the transmission of certain aberrant genes from one generation to another. Hemophilia means a genetically transmitted bleeding disorder resulting from a deficiency of a plasma...

  12. 32 CFR 383a.2 - Applicability.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Defense Agencies; and the DoD Field Activities (hereafter referred to collectively as “DoD Components”). The term “Military Services,” as used herein, refers to the Army, Navy, Air Force, and Marine Corps....

  13. 32 CFR 383a.2 - Applicability.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...; the Unified and Specified Commands; the Inspector General of the Department of Defense (IG, DoD); the Defense Agencies; and the DoD Field Activities (hereafter referred to collectively as “DoD...

  14. 32 CFR 383a.2 - Applicability.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...; the Unified and Specified Commands; the Inspector General of the Department of Defense (IG, DoD); the Defense Agencies; and the DoD Field Activities (hereafter referred to collectively as “DoD...

  15. 32 CFR 383a.2 - Applicability.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...; the Unified and Specified Commands; the Inspector General of the Department of Defense (IG, DoD); the Defense Agencies; and the DoD Field Activities (hereafter referred to collectively as “DoD...

  16. 32 CFR 383a.2 - Applicability.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...; the Unified and Specified Commands; the Inspector General of the Department of Defense (IG, DoD); the Defense Agencies; and the DoD Field Activities (hereafter referred to collectively as “DoD...

  17. 42 CFR 85a.2 - Definitions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH... means research projects, experiments, demonstrations, studies, and similar activities of NIOSH which are..., Public Health Service, Department of Health and Human Services. (g) NIOSH authorized representative...

  18. 42 CFR 85a.2 - Definitions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH... means research projects, experiments, demonstrations, studies, and similar activities of NIOSH which are..., Public Health Service, Department of Health and Human Services. (g) NIOSH authorized representative...

  19. 42 CFR 85a.2 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH... means research projects, experiments, demonstrations, studies, and similar activities of NIOSH which are..., Public Health Service, Department of Health and Human Services. (g) NIOSH authorized representative...

  20. 42 CFR 85a.2 - Definitions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH... means research projects, experiments, demonstrations, studies, and similar activities of NIOSH which are..., Public Health Service, Department of Health and Human Services. (g) NIOSH authorized representative...

  1. Role of adenosine A2b receptor overexpression in tumor progression.

    PubMed

    Sepúlveda, Cesar; Palomo, Iván; Fuentes, Eduardo

    2016-12-01

    The adenosine A2b receptor is a G-protein coupled receptor. Its activation occurs with high extracellular adenosine concentration, for example in inflammation or hypoxia. These conditions are generated in the tumor environment. Studies show that A2b receptor is overexpressed in various tumor lines and biopsies from patients with different cancers. This suggests that A2b receptor can be used by tumor cells to promote progression. Thus A2b participates in different events, such as angiogenesis and metastasis, besides exerting immunomodulatory effects that protect tumor cells. Therefore, adenosine A2b receptor appears as an interesting therapeutic target for cancer treatment.

  2. A2 noradrenergic neurons regulate forced swim test immobility.

    PubMed

    Nam, Hyungwoo; Kerman, Ilan A

    2016-10-15

    The Wistar-Kyoto (WKY) rat is a widely used animal model of depression, which is characterized by dysregulation of noradrenergic signaling. We previously demonstrated that WKY rats show a unique behavioral profile on the forced swim test (FST), characterized by high levels of immobility upon initial exposure and a greater learning-like response by further increasing immobility upon re-exposure than the genetically related Wistar rats. In the current study we aimed to determine whether altered activation of brainstem noradrenergic cell groups contributes to this behavioral profile. We exposed WKY and Wistar rats, to either 5min of forced swim or to the standard two-day FST (i.e. 15min forced swim on Day 1, followed by 5min on Day 2). We then stained their brains for FOS/tyrosine hydroxylase double-immunocytochemistry to determine potential differences in the activation of the brainstem noradrenergic cell groups. We detected a relative hyperactivation in the locus coeruleus of WKY rats when compared to Wistars in response to both one- and two-day forced swim. In contrast, within the A2 noradrenergic cell group, WKY rats exhibited diminished levels of FOS across both days of the FST, suggesting their lesser activation. We followed up these observations by selectively lesioning the A2 neurons, using anti-dopamine-β-hydroxylase-conjugated saporin, in Wistar rats, which resulted in increased FST immobility on both days of the test. Together these data indicate that the A2 noradrenergic cell group regulates FST behavior, and that its hypoactivation may contribute to the unique behavioral phenotype of WKY rats.

  3. Importance of UDP-glucuronosyltransferases 2A2 and 2A3 in tobacco carcinogen metabolism.

    PubMed

    Bushey, Ryan T; Dluzen, Douglas F; Lazarus, Philip

    2013-01-01

    UDP-glucuronosyltransferase A1 (UGT2A1) is expressed in the lung and exhibits activity against polycyclic aromatic hydrocarbons (PAHs), suggesting UGT2A1 involvement in the local metabolism of PAH tobacco carcinogens. The goal of the present study was to investigate the importance of two additional UGT2A enzymes, UGT2A2 and UGT2A3, in tobacco carcinogen metabolism. Real-time polymerase chain reaction suggested that wild-type UGT2A2 had the highest expression in the breast, followed by trachea > larynx > kidney. A novel splice variant of UGT2A2 lacking exon 3 (termed UGT2A2Δexon3) was investigated, with UGT2A2Δexon3 expression determined to be 25-50% that of wild-type UGT2A2 in all tissues examined. UGT2A3 was determined to be well expressed in the liver and colon, followed by pancreas > kidney > lung > tonsil > trachea > larynx. Cell homogenates prepared from human embryonic kidney (HEK)293 cells overexpressing wild-type UGT2A2 (termed UGT2A2_i1) exhibited glucuronidation activity, as observed by reverse-phase ultra-pressure liquid chromatography, against 1-hydroxy-(OH)-pyrene, 1-naphthol, and hydroxylated benzo(a)pyrene metabolites, whereas homogenates prepared from HEK293 cells overexpressing UGT2A3 only showed activity against simple PAHs like 1-OH-pyrene and 1-naphthol. Activity assays showed the UGT2A2Δexon3 protein (termed UGT2A2_i2) exhibited no detectable glucuronidation activity against all substrates examined; however, coexpression studies suggested that UGT2A2_i2 negatively modulates UGT2A2_i1 activity. Both UGT2A2 and UGT2A3 exhibited no detectable activity against complex PAH proximate carcinogens, tobacco-specific nitrosamines, or heterocyclic amines. These data suggest that, although UGT2A1 is the only UGT2A enzyme active against PAH proximate carcinogens (including PAH diols), both UGTs 2A1 and 2A2 play an important role in the local detoxification of procarcinogenic monohydroxylated PAH metabolites.

  4. Foci of cyclin A2 interact with actin and RhoA in mitosis

    PubMed Central

    Loukil, Abdelhalim; Izard, Fanny; Georgieva, Mariya; Mashayekhan, Shaereh; Blanchard, Jean-Marie; Parmeggiani, Andrea; Peter, Marion

    2016-01-01

    Cyclin A2 is a key player in the regulation of the cell cycle. Its degradation in mid-mitosis depends primarily on the ubiquitin-proteasome system (UPS), while autophagy also contributes. However, a fraction of cyclin A2 persists beyond metaphase. In this work, we focus on cyclin A2-rich foci detected in mitosis by high resolution imaging and analyse their movements. We demonstrate that cyclin A2 interacts with actin and RhoA during mitosis, and that cyclin A2 depletion induces a dramatic decrease in active RhoA in mitosis. Our data suggest cyclin A2 participation in RhoA activation in late mitosis. PMID:27279564

  5. Protein kinase A can block EphA2 receptor–mediated cell repulsion by increasing EphA2 S897 phosphorylation

    PubMed Central

    Barquilla, Antonio; Lamberto, Ilaria; Noberini, Roberta; Heynen-Genel, Susanne; Brill, Laurence M.; Pasquale, Elena B.

    2016-01-01

    The EphA2 receptor tyrosine kinase plays key roles in tissue homeostasis and disease processes such as cancer, pathological angiogenesis, and inflammation through two distinct signaling mechanisms. EphA2 “canonical” signaling involves ephrin-A ligand binding, tyrosine autophosphorylation, and kinase activity; EphA2 “noncanonical” signaling involves phosphorylation of serine 897 (S897) by AKT and RSK kinases. To identify small molecules counteracting EphA2 canonical signaling, we developed a high-content screening platform measuring inhibition of ephrin-A1–induced PC3 prostate cancer cell retraction. Surprisingly, most hits from a screened collection of pharmacologically active compounds are agents that elevate intracellular cAMP by activating G protein–coupled receptors such as the β2-adrenoceptor. We found that cAMP promotes phosphorylation of S897 by protein kinase A (PKA) as well as increases the phosphorylation of several nearby serine/threonine residues, which constitute a phosphorylation hotspot. Whereas EphA2 canonical and noncanonical signaling have been viewed as mutually exclusive, we show that S897 phosphorylation by PKA can coexist with EphA2 tyrosine phosphorylation and block cell retraction induced by EphA2 kinase activity. Our findings reveal a novel paradigm in EphA2 function involving the interplay of canonical and noncanonical signaling and highlight the ability of the β2-adrenoceptor/cAMP/PKA axis to rewire EphA2 signaling in a subset of cancer cells. PMID:27385333

  6. Preclinical evaluation of human secretoglobin 3A2 in mouse models of lung development and fibrosis

    PubMed Central

    Cai, Yan; Winn, Melissa E.; Zehmer, John K.; Gillette, William K.; Lubkowski, Jacek T.; Pilon, Aprile L.

    2013-01-01

    Secretoglobin (SCGB) 3A2 is a member of the SCGB gene superfamily of small secreted proteins, predominantly expressed in lung airways. We hypothesize that human SCGB3A2 may exhibit anti-inflammatory, growth factor, and antifibrotic activities and be of clinical utility. Recombinant human SCGB3A2 was expressed, purified, and biochemically characterized as a first step to its development as a therapeutic agent in clinical settings. Human SCGB3A2, as well as mouse SCGB3A2, readily formed a dimer in solution and exhibited novel phospholipase A2 inhibitory activity. This is the first demonstration of any quantitative biochemical measurement for the evaluation of SCGB3A2 protein. In the mouse as an experimental animal, human SCGB3A2 exhibited growth factor activity by promoting embryonic lung development in both ex vivo and in vivo systems and antifibrotic activity in the bleomycin-induced lung fibrosis model. The results suggested that human SCGB3A2 can function as a growth factor and an antifibrotic agent in humans. When SCGB3A2 was administered to pregnant female mice through the tail vein, the protein was detected in the dam's serum and lung, as well as the placenta, amniotic fluids, and embryonic lungs at 10 min postadministration, suggesting that SCGB3A2 readily crosses the placenta. The results warrant further development of recombinant SCGB3A2 as a therapeutic agent in treating patients suffering from lung diseases or preterm infants with respiratory distress. PMID:24213919

  7. Proteolysis of EphA2 converts it from a tumor suppressor to an oncoprotein

    PubMed Central

    KOSHIKAWA, Naohiko; HOSHINO, Daisuke; TANIGUCHI, Hiroaki; MINEGISHI, Tomoko; TOMARI, Taizo; NAM, Sung-Ouk; AOKI, Mikiko; SUETA, Takayuki; NAKAGAWA, Takashi; MIYAMOTO, Shingo; NABESHIMA, Kazuki; WEAVER, Alissa M.; SEIKI, Motoharu

    2015-01-01

    Eph receptor tyrosine kinases are considered candidate therapeutic targets in cancer, but they can exert opposing effects on cell growth. In presence of its ligands, Eph receptor EphA2 suppresses signaling by other growth factor receptors, including ErbB, whereas ligand-independent activation of EphA2 augments ErbB signaling. To deploy EphA2-targeting drugs effectively in tumors, the anti-oncogenic ligand-dependent activation state of EphA2 must be discriminated from its oncogenic ligand-independent state. Since the molecular basis for the latter is little understood, we investigated how the activation state of EphA2 can be switched in tumor tissue. We found that ligand-binding domain of EphA2 is cleaved frequently by the membrane metalloproteinase MT1-MMP, a powerful modulator of the pericellular environment in tumor cells. EphA2 immunostaining revealed a significant loss of the N-terminal portion of EphA2 in areas of tumor tissue that expressed MT1-MMP. Moreover, EphA2 phosphorylation patterns that signify ligand-independent activation were observed specifically in these areas of tumor tissue. Mechanistic experiments revealed that processing of EphA2 by MT1-MMP promoted ErbB signaling, anchorage-independent growth, and cell migration. Conversely, expression of a proteolysis-resistant mutant of EphA2 prevented tumorigenesis and metastasis of human tumor xenografts in mice. Overall, our results showed how the proteolytic state of EphA2 in tumors determines its effector function and influences its status as a candidate biomarker for targeted therapy. PMID:26130649

  8. INHIBITION OF HUMAN AND RAT CYP1A2 BY TCDD AND DIOXIN-LIKE CHEMICALS

    EPA Science Inventory

    Dioxins have been shown to bind and induce rodent CYP1A2, producing a dose-dependent hepatic sequestration in vivo. The induction of CYP1A2 activity has been used as a noninvasive biomarker for human exposure to dioxins; while there is a consistent relationship between exposure ...

  9. Ostrich pancreatic phospholipase A(2): purification and biochemical characterization.

    PubMed

    Ben Bacha, Abir; Gargouri, Youssef; Bezzine, Sofiane; Mosbah, Habib; Mejdoub, Hafedh

    2007-09-15

    Ostrich pancreatic phospholipase A(2) (OPLA(2)) was purified from delipidated pancreases. Pure protein was obtained after heat treatment (70 degrees C), precipitation by ammonium sulphate and ethanol, respectively followed by sequential column chromatography on MonoQ Sepharose and size exclusion HPLC column. Purified OPLA(2), which is not a glycosylated protein, was found to be monomeric protein with a molecular mass of 13773.93 Da. A specific activity of 840U/mg for purified OPLA(2) was measured at optimal conditions (pH 8.2 and 37 degrees C) in the presence of 4 mM NaTDC and 10 mM CaCl(2) using PC as substrate. This enzyme was also found to be able to hydrolyze, at low surface pressure, 1,2-dilauroyl-sn-glycero-3 phosphocholine (di C(12)-PC) monolayers. Maximal activity was measured at 5-8 mNm(-1). The sequence of the first 22 amino-acid residues at the N-terminal extremity of purified bird PLA(2) was determined by automatic Edman degradation and showed a high sequence homology with known mammal pancreatic secreted phospholipases A(2).

  10. X-15A-2 with dummy ramjet

    NASA Technical Reports Server (NTRS)

    1967-01-01

    This photo shows the X-15A-2 (56-6671) on a research flight with a dummy ramjet engine attached to the bottom of its wedge-shaped vertical tail. One of the experiments planned for the X-15A-2 involved tests of a functional ramjet at speeds above Mach 5. This photo was taken with a dummy ramjet. On this research flight, the X-15A-2 did not carry the two drop tanks used on its Mach 6.7 flight. It also had not yet been covered with an ablative coating. The X-15A-2 made several flights with the dummy ramjet, leading to the record Mach 6.7 flight on October 3, 1967. Delays in producing the operational ramjet, aerodynamic heating damage to the aircraft during the record flight (despite the ablative coating), and the end of the X-15 program in 1968 resulted in no flights with the actual ramjet. The X-15 was a rocket-powered aircraft. The original three aircraft were about 50 ft long with a wingspan of 22 ft. The modified #2 aircraft (X-15A-2 was longer.) They were a missile-shaped vehicles with unusual wedge-shaped vertical tails, thin stubby wings, and unique side fairings that extended along the side of the fuselage. The X-15 weighed about 14,000 lb empty and approximately 34,000 lb at launch. The XLR-99 rocket engine, manufactured by Thiokol Chemical Corp., was pilot controlled and was rated at 57,000 lb of thrust, although there are indications that it actually achieved up to 60,000 lb. North American Aviation built three X-15 aircraft for the program. The X-15 research aircraft was developed to provide in-flight information and data on aerodynamics, structures, flight controls, and the physiological aspects of high-speed, high-altitude flight. A follow-on program used the aircraft as testbeds to carry various scientific experiments beyond the Earth's atmosphere on a repeated basis. For flight in the dense air of the usable atmosphere, the X-15 used conventional aerodynamic controls such as rudder surfaces on the vertical stabilizers to control yaw and movable

  11. High salt diet modulates vascular response in A2AAR (+/+) and A 2AAR (-/-) mice: role of sEH, PPARγ, and K ATP channels.

    PubMed

    Pradhan, Isha; Ledent, Catherine; Mustafa, S Jamal; Morisseau, Christophe; Nayeem, Mohammed A

    2015-06-01

    This study aims to investigate the signaling mechanism involved in HS-induced modulation of adenosine-mediated vascular tone in the presence or absence of adenosine A2A receptor (A2AAR). We hypothesized that HS-induced enhanced vascular relaxation through A2AAR and epoxyeicosatrienoic acid (EETs) is dependent on peroxisome proliferator-activated receptor gamma (PPARγ) and ATP-sensitive potassium channels (KATP channels) in A2AAR(+/+) mice, while HS-induced vascular contraction to adenosine is dependent on soluble epoxide hydrolase (sEH) that degrades EETs in A2AAR(-/-) mice. Organ bath and Western blot techniques were conducted in HS (4 % NaCl) and normal salt (NS, 0.45 % NaCl)-fed A2AAR(+/+) and A2AAR(-/-) mouse aorta. We found that enhanced vasodilation to A2AAR agonist, CGS 21680, in HS-fed A2AAR(+/+) mice was blocked by PPARγ antagonist (T0070907) and KATP channel blocker (Glibenclamide). Also, sEH inhibitor (AUDA)-dependent vascular relaxation was mitigated by PPARγ antagonist. PPARγ agonist (Rosiglitazone)-induced relaxation in HS-A2AAR(+/+) mice was attenuated by KATP channel blocker. Conversely, HS-induced contraction in A2AAR(-/-) mice was attenuated by sEH inhibitor. Overall, findings from this study that implicates the contribution of EETs, PPARγ and KATP channels downstream of A2AAR to mediate enhanced vascular relaxation in response to HS diet while, role of sEH in mediating vascular contraction in HS-fed A2AAR(-/-) mice.

  12. EphA2 signaling following endocytosis: role of Tiam1.

    PubMed

    Boissier, Pomme; Chen, Jin; Huynh-Do, Uyen

    2013-12-01

    Eph receptors and their membrane-bound ligands, the ephrins, represent a complex subfamily of receptor tyrosine kinases (RTKs). Eph/ephrin binding can lead to various and opposite cellular behaviors such as adhesion versus repulsion, or cell migration versus cell-adhesion. Recently, Eph endocytosis has been identified as one of the critical steps responsible for such diversity. Eph receptors, as many RTKs, are rapidly endocytosed following ligand-mediated activation and traffic through endocytic compartments prior to degradation. However, it is becoming obvious that endocytosis controls signaling in many different manners. Here we showed that activated EphA2 are degraded in the lysosomes and that about 35% of internalized receptors are recycled back to the plasma membrane. Our study is also the first to demonstrate that EphA2 retains the capacity to signal in endosomes. In particular, activated EphA2 interacted with the Rho family GEF Tiam1 in endosomes. This association led to Tiam1 activation, which in turn increased Rac1 activity and facilitated Eph/ephrin endocytosis. Disrupting Tiam1 function with RNA interference impaired both ephrinA1-dependent Rac1 activation and ephrinA1-induced EphA2 endocytosis. In summary, our findings shed new light on the regulation of EphA2 endocytosis, intracellular trafficking and signal termination and establish Tiam1 as an important modulator of EphA2 signaling.

  13. Secreted phospholipase A2 and mast cells.

    PubMed

    Murakami, Makoto; Taketomi, Yoshitaka

    2015-01-01

    Phospholipase A2s (PLA2s) are a group of enzymes that hydrolyze the sn-2 position of phospholipids to release (typically unsaturated) fatty acids and lysophospholipids, which serve as precursors for a variety of bioactive lipid mediators. Among the PLA2 superfamily, secreted PLA2 (sPLA2) enzymes comprise the largest subfamily that includes 11 isoforms with a conserved His-Asp catalytic dyad. Individual sPLA2 enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. Recent studies using transgenic and knockout mice for individual sPLA2 isofoms have revealed their involvement in various pathophysiological events. Here, we overview the current state of knowledge about sPLA2s, specifically their roles in mast cells (MCs) in the context of allergology. In particular, we highlight group III sPLA2 (PLA2G3) as an "anaphylactic sPLA2" that promotes MC maturation and thereby anaphylaxis through a previously unrecognized lipid-orchestrated circuit.

  14. Casein Kinase 2 Is a Novel Regulator of the Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) Trafficking.

    PubMed

    Chan, Ting; Cheung, Florence Shin Gee; Zheng, Jian; Lu, Xiaoxi; Zhu, Ling; Grewal, Thomas; Murray, Michael; Zhou, Fanfan

    2016-01-04

    Human organic anion transporting polypeptides (OATPs) mediate the influx of many important drugs into cells. Casein kinase 2 (CK2) is a critical protein kinase that phosphorylates >300 protein substrates and is dysregulated in a number of disease states. Among the CK2 substrates are several transporters, although whether this includes human OATPs has not been evaluated. The current study was undertaken to evaluate the regulation of human OATP1A2 by CK2. HEK-239T cells in which OATP1A2 was overexpressed were treated with CK2 specific inhibitors or transfected with CK2 specific siRNA, and the activity, expression, and subcellular trafficking of OATP1A2 was evaluated. CK2 inhibition decreased the uptake of the prototypic OATP1A2 substrate estrone-3-sulfate (E3S). Kinetic studies revealed that this was due to a decrease in the maximum velocity (Vmax) of E3S uptake, while the Michaelis constant was unchanged. The cell surface expression, but not the total cellular expression of OATP1A2, was impaired by CK2 inhibition and knockdown of the catalytic α-subunits of CK2. CK2 inhibition decreased the internalization of OATP1A2 via a clathrin-dependent pathway, decreased OATP1A2 recycling, and likely impaired OATP1A2 targeting to the cell surface. Consistent with these findings, CK2 inhibition also disrupted the colocalization of OATP1A2 and Rab GTPase (Rab)4-, Rab8-, and Rab9-positive endosomal and secretory vesicles. Taken together, CK2 has emerged as a novel regulator of the subcellular trafficking and stability of OATP1A2. Because OATP1A2 transports many molecules of physiological and pharmacological importance, the present data may inform drug selection in patients with diseases in which CK2 and OATP1A2 are dysregulated.

  15. Role of Vibrio cholerae exochitinase ChiA2 in horizontal gene transfer.

    PubMed

    Mondal, Moumita; Chatterjee, Nabendu Sekhar

    2016-03-01

    Vibrio cholerae exochitinase ChiA2 plays a key role in acquisition of nutrients by chitin hydrolysis in the natural environment as well as in pathogenesis in the intestinal milieu. In this study we demonstrate the importance of ChiA2 in horizontal gene transfer in the natural environment. We found that the expression of ChiA2 and TfoX, the central regulator of V. cholerae horizontal gene transfer, varied with changes in environmental conditions. The activity of ChiA2 was also dependent on these conditions. In 3 different environmental conditions tested here, we observed that the supporting environmental condition for maximum expression and activity of ChiA2 was 20 °C, pH 5.5, and 100 mmol/L salinity in the presence of chitin. The same condition also induced TfoX expression and was favorable for horizontal gene transfer in V. cholerae. High-performance liquid chromatography analysis showed that ChiA2 released a significant amount of (GlcNAc)2 from chitin hydrolysis under the favorable condition. We hypothesized that under the favorable environmental condition, ChiA2 was upregulated and maximally active to produce a significant amount of (GlcNAc)2 from chitin. The same environmental condition also induced tfoX expression, followed by its translational activation by the (GlcNAc)2 produced, leading to efficient horizontal gene transfer.

  16. A lipid switch unlocks Parkinson’s disease-associated ATP13A2

    PubMed Central

    Holemans, Tine; Sørensen, Danny Mollerup; van Veen, Sarah; Martin, Shaun; Hermans, Diane; Kemmer, Gerdi Christine; Van den Haute, Chris; Baekelandt, Veerle; Günther Pomorski, Thomas; Agostinis, Patrizia; Wuytack, Frank; Palmgren, Michael; Eggermont, Jan; Vangheluwe, Peter

    2015-01-01

    ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor–Rakeb syndrome and Parkinson’s disease (PD), providing protection against α-synuclein, Mn2+, and Zn2+ toxicity in various model systems. So far, the molecular function and regulation of ATP13A2 remains undetermined. Here, we demonstrate that ATP13A2 contains a unique N-terminal hydrophobic extension that lies on the cytosolic membrane surface of the lysosome, where it interacts with the lysosomal signaling lipids phosphatidic acid (PA) and phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2]. We further demonstrate that ATP13A2 accumulates in an inactive autophosphorylated state and that PA and PI(3,5)P2 stimulate the autophosphorylation of ATP13A2. In a cellular model of PD, only catalytically active ATP13A2 offers cellular protection against rotenone-induced mitochondrial stress, which relies on the availability of PA and PI(3,5)P2. Thus, the N-terminal binding of PA and PI(3,5)P2 emerges as a key to unlock the activity of ATP13A2, which may offer a therapeutic strategy to activate ATP13A2 and thereby reduce α-synuclein toxicity or mitochondrial stress in PD or related disorders. PMID:26134396

  17. Estimation of crop gross primary production (GPP): fAPAR_chl versus MOD15A2 FPAR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Within leaf chloroplasts chlorophylls absorb photosynthetically active radiation (PAR) for photosynthesis (PSN). The MOD15A2 FPAR (fraction of PAR absorbed by canopy, i.e., fAPARcanopy) product has been widely used to compute absorbed PAR for PSN (APARPSN). The MOD17A2 algorithm uses MOD15A2 FPAR i...

  18. Dosimetric characterization of a 2D polycrystalline CVD diamond detector

    NASA Astrophysics Data System (ADS)

    Bartoli, A.; Cupparo, I.; Baldi, A.; Scaringella, M.; Pasquini, A.; Pallotta, S.; Talamonti, C.; Bruzzi, M.

    2017-03-01

    A bidimensional pixelated dosimeter composed of two polycrystalline Chemical Vapour Deposited diamond films, 2.5 × 2.5 cm2 each placed aside, has been manufactured so as to obtain a detector with a 2 mm pitch over a total active area of 5.0 × 2.5 cm2. We performed the dosimetric characterization of the detector with an Elekta Synergy linear accelerator using a 6 MV photon beam. Uniformity maps, rise and fall times, signal repeatability, dependence on dose rate, linearity with dose and sensitivity show that the device is suitable for dosimetric evaluations in Intensity Modulated Radiation Therapy and Volumetric Modulated Arc Therapy (VMAT) treatments. Then, a first quantitative evaluation of the dose distribution in a lung VMAT treatment plan has been carried out, by comparing data from our device with Treatment Planning Sistem values by means of a Γ test, with promising results.

  19. Adenosine receptors and diabetes: Focus on the A(2B) adenosine receptor subtype.

    PubMed

    Merighi, Stefania; Borea, Pier Andrea; Gessi, Stefania

    2015-09-01

    Over the last two decades, diabetes mellitus has become one of the most challenging health problems worldwide. Diabetes mellitus, classified as type I and II, is a pathology concerning blood glucose level in the body. The nucleoside adenosine has long been known to affect insulin secretion, glucose homeostasis and lipid metabolism, through activation of four G protein coupled adenosine receptors (ARs), named A1, A2A, A2B and A3. Currently, the novel promising subtype to develop new drugs for diabetes treatment is the A2BAR subtype. The use of selective agonists and antagonists for A2BAR subtype in various diabetic animal models allowed us to identify several effects of A2BAR signaling in cell metabolism. In particular, the focus of this review is to summarize the studies on purinergic signaling associated with diabetes through A2BARs modulation.

  20. Stabilization of EphA2 dimers as a novel anti-cancer strategy

    NASA Astrophysics Data System (ADS)

    Singh, Deo; Ahmed, Fozia; Salloto, Matt; Hristova, Kalina

    We have recently shown that EphA2 receptors exist in a monomer-dimer equilibrium in the absence of ligand. The monomers promote tumorigenic activity and thus a therapeutic strategy that minimizes the monomer population may be beneficial in the clinic. The YSA peptide is an EphA2-targeting peptide that effectively delivers anticancer agents to cancer tumors. The quantitative measurements of the dimerization of EphA2 receptors in the presence of these peptides using quantitative spectral Forster resonance transfer (QS-FRET) methodology in conjunction with two-photon microscopy that has been developed recently in our lab suggests that this peptide stabilizes the EphA2 dimers. Thus, such peptides that stabilize the EphA2 dimers may be used for the treatment of some cancers that overexpress EphA2.

  1. Involvement of free radicals followed by the activation of phospholipase A2 in the mechanism that underlies the combined effects of methamphetamine and morphine on subacute toxicity or lethality in mice: comparison of the therapeutic potential of fullerene, mepacrine, and cooling.

    PubMed

    Mori, Tomohisa; Ito, Shinobu; Namiki, Mizuho; Suzuki, Tadashi; Kobayashi, Shizuko; Matsubayashi, Kenji; Sawaguchi, Toshiko

    2007-07-17

    An increase in polydrug abuse is a major problem worldwide. The coadministration of methamphetamine and morphine increased subacute toxicity or lethality in rodents. However, the underlying mechanisms by which lethality is increased by the coadministration of methamphetamine and morphine are not yet fully understood. Coadministered methamphetamine and morphine induced lethality by more than 80% in BALB/c mice, accompanied by the rupture of cells in the kidney and liver, and an increase in poly (ADP-ribose) polymerase (PARP)-immunoreactive cells in the heart, kidney and liver. The lethal effect and the increase in the incidence of rupture or PARP-immunoreactive cells induced by the coadministration of methamphetamine and morphine was significantly attenuated by pretreatment with mepacrine (phospholipase A(2) inhibitor) or fullerene (a radical scavenger), or by cooling from 30 to 90 min after drug administration. Furthermore, based on the results of the electron spin resonance spin-trapping technique, hydroxyl radicals were increased by the administration of methamphetamine and morphine, and these increased hydroxyl radicals were potently attenuated by fullerene and cooling. These results suggest that hydroxyl radicals plays an important role in the increased lethality induced by the coadministration of methamphetamine plus morphine. The potency of cooling or drugs for decreasing the subacute toxicity or lethality induced by the coadministration of methamphetamine and morphine was in the order fullerene=cooling>mepacrine. These results indicate that fullerene and cooling are beneficial for preventing death that is induced by the coadministration of methamphetamine and morphine.

  2. Functions, dysfunctions and possible therapeutic relevance of adenosine A2A receptors in Huntington's disease.

    PubMed

    Popoli, Patrizia; Blum, David; Martire, Alberto; Ledent, Catherine; Ceruti, Stefania; Abbracchio, Maria P

    2007-04-01

    The aim of this review is to summarize and critically discuss the complex role played by adenosine A(2A) receptors (A(2A)Rs) in Huntington's disease (HD). Since A(2A)Rs are mainly localized on the neurons, which degenerate early in HD, and given their ability to stimulate glutamate outflow and inflammatory gliosis, it was hypothesized that they could be involved in the pathogenesis of HD, and that A(2A)R antagonists could be neuroprotective. This was further sustained by the demonstration that A(2A)Rs and underlying signaling systems undergo profound changes in cellular and animal models of HD. More recently, however, the equation A(2A) receptor blockade=neuroprotection has appeared too simplistic. First, it is now definitely clear that, besides mediating 'bad' responses (for example, stimulation of glutamate outflow and excessive glial activation), A(2A)Rs also promote 'good' responses (such as trophic and antinflammatory effects). This implies that A(2A)R blockade results either in pro-toxic or neuroprotective effects according to the mechanisms involved in a given experimental model. Second, since HD is a chronically progressive disease, the multiple mechanisms involving A(2A)Rs may play different relative roles along the degenerative process. Such different mechanisms can be influenced by A(2A)R activation or blockade in different ways, even leading to opposite outcomes depending on the time of agonist/antagonist administration. The number, and the complexity, of the possible scenarios is further increased by the influence of mutant Huntingtin on both the expression and functions of A(2A)Rs, and by the strikingly different effects mediated by A(2A)Rs expressed by different cell populations within the brain.

  3. DNA Methyl Transferase 1 Reduces Expression of SRD5A2 in the Aging Adult Prostate

    PubMed Central

    Ge, Rongbin; Wang, Zongwei; Bechis, Seth K.; Otsetov, Alexander G.; Hua, Shengyu; Wu, Shulin; Wu, Chin-Lee; Tabatabaei, Shahin; Olumi, Aria F.

    2016-01-01

    5-α Reductase type 2 (SRD5A2) is a critical enzyme for prostatic development and growth. Inhibition of SRD5A2 by finasteride is used commonly for the management of urinary obstruction caused by benign prostatic hyperplasia. Contrary to common belief, we have found that expression of SRD5A2 is variable and absent in one third of benign adult prostates. In human samples, absent SRD5A2 expression is associated with hypermethylation of the SRD5A2 promoter, and in vitro SRD5A2 promoter activity is suppressed by methylation. We show that methylation of SRD5A2 is regulated by DNA methyltransferase 1, and inflammatory mediators such as tumor necrosis factor α, NF-κB, and IL-6 regulate DNA methyltransferase 1 expression and thereby affect SRD5A2 promoter methylation and gene expression. Furthermore, we show that increasing age in mice and humans is associated with increased methylation of the SRD5A2 promoter and concomitantly decreased protein expression. Artificial induction of inflammation in prostate primary epithelial cells leads to hypermethylation of the SRD5A2 promoter and silencing of SRD5A2, whereas inhibition with tumor necrosis factor α inhibitor reactivates SRD5A2 expression. Therefore, expression of SRD5A2 is not static and ubiquitous in benign adult prostate tissues. Methylation and expression of SRD5A2 may be used as a gene signature to tailor therapies for more effective treatment of prostatic diseases. PMID:25700986

  4. Ligand targeting of EphA2 enhances keratinocyte adhesion and differentiation via desmoglein 1.

    PubMed

    Lin, Samantha; Gordon, Kristin; Kaplan, Nihal; Getsios, Spiro

    2010-11-15

    EphA2 is a receptor tyrosine kinase that is engaged and activated by membrane-linked ephrin-A ligands residing on adjacent cell surfaces. Ligand targeting of EphA2 has been implicated in epithelial growth regulation by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2)-mitogen activated protein kinase (MAPK) pathway. Although contact-dependent EphA2 activation was required for dampening Erk1/2-MAPK signaling after a calcium switch in primary human epidermal keratinocytes, the loss of this receptor did not prevent exit from the cell cycle. Incubating keratinocytes with a soluble ephrin-A1-Fc peptide mimetic to target EphA2 further increased receptor activation leading to its down-regulation. Moreover, soluble ligand targeting of EphA2 restricted the lateral expansion of epidermal cell colonies without limiting proliferation in these primary cultures. Rather, ephrin-A1-Fc peptide treatment promoted epidermal cell colony compaction and stratification in a manner that was associated with increased keratinocyte differentiation. The ligand-dependent increase in keratinocyte adhesion and differentiation relied largely upon the up-regulation of desmoglein 1, a desmosomal cadherin that maintains the integrity and differentiated state of suprabasal keratinocytes in the epidermis. These data suggest that keratinocytes expressing EphA2 in the basal layer may respond to ephrin-A1-based cues from their neighbors to facilitate entry into a terminal differentiation pathway.

  5. Effects of glycosaminoglycans and glycosphingolipids on cytosolic phospholipases A2 from bovine brain.

    PubMed Central

    Yang, H C; Farooqui, A A; Horrocks, L A

    1994-01-01

    Two forms of Ca(2+)-independent cytosolic phospholipase A2 activity (110 kDa and 39 kDa) were found in bovine brain. They were separated by Sephadex G-75 column chromatography. The 110 kDa phospholipase A2 was much more active with phosphatidylethanolamine and was not affected by glycosaminoglycans, whereas the 39 kDa phospholipase A2 was much more active with ethanolamine plasmalogen and was markedly inhibited by glycosaminoglycans. Heparan sulphate was the most potent inhibitor, followed by chondroitin sulphate, hyaluronic acid and heparin. Gangliosides, especially the GM3 ganglioside, but not other glycosphingolipids, inhibited the activity of the 39 kDa phospholipase A2 in a dose-dependent manner. The heat-inactivation profiles of the 110 kDa and 39 kDa phospholipases A2 provide further evidence for the differences between these cytosolic enzymes. Interactions between glycosaminoglycans, gangliosides and phospholipases A2 may be involved in the maintenance of membrane function. PMID:8166664

  6. GPR88 in A2AR Neurons Enhances Anxiety-Like Behaviors

    PubMed Central

    Meirsman, Aura Carole; Robé, Anne

    2016-01-01

    Abstract GPR88 is an orphan G-protein-coupled receptor highly expressed in striatal dopamine D1 (receptor) R- and D2R-expressing medium spiny neurons. This receptor is involved in activity and motor responses, and we previously showed that this receptor also regulates anxiety-like behaviors. To determine whether GPR88 in D2R-expressing neurons contributes to this emotional phenotype, we generated conditional Gpr88 knock-out mice using adenosine A2AR (A2AR)-Cre-driven recombination, and compared anxiety-related responses in both total and A2AR-Gpr88 KO mice. A2AR-Gpr88 KO mice showed a selective reduction of Gpr88 mRNA in D2R-expressing, but not D1R-expressing, neurons. These mutant mice showed increased locomotor activity and decreased anxiety-like behaviors in light/dark and elevated plus maze tests. These phenotypes were superimposable on those observed in total Gpr88 KO mice, demonstrating that the previously reported anxiogenic activity of GPR88 operates at the level of A2AR-expressing neurons. Further, A2AR-Gpr88 KO mice showed no change in novelty preference and novelty-suppressed feeding, while these responses were increased and decreased, respectively, in the total Gpr88 KO mice. Also, A2AR-Gpr88 KO mice showed intact fear conditioning, while the fear responses were decreased in total Gpr88 KO. We therefore also show for the first time that GPR88 activity regulates approach behaviors and conditional fear; however, these behaviors do not seem mediated by receptors in A2AR neurons. We conclude that Gpr88 expressed in A2AR neurons enhances ethological anxiety-like behaviors without affecting conflict anxiety and fear responses. PMID:27570825

  7. Compression Induces Ephrin-A2 in PDL Fibroblasts via c-fos

    PubMed Central

    Sen, S.; Diercke, K.; Zingler, S.; Lux, C. J.

    2015-01-01

    Ephrin-A2–EphA2 and ephrin-B2–EphB4 int