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Muscle microvascular permeability increase mediated via free radical generation
P. Thiagarajan, S.S. Yoharajan, P.A. Fraser* King’s College London, UK
[0643] The Arabidopsis thaliana GASA protein: possible roles in redox regulation and plant development L. Rubinovich*, D. Weiss The Hebrew University of Jerusalem, Israel Although the gibberellin (GA) signaling has been elucidated, very little is known about the steps linking first transcriptional activation to physiological responses. Among the few identified GA-induced genes are the plant-specific GAST1-like genes, which encode small proteins with a conserved cysteine-rich domain. The Arabidopsis GAST1-like gene family consists of 14 members, GASA1-14. Here we show that overexpression of GASA4 in Arabidopsis promoted typical GA responses. Suppression of several GASA genes using synthetic microRNA also promoted seed germination, probably due to the suppression of GASA5, which acts as a repressor of GA responses. Overexpression of GASA4 suppressed reactive oxygen species (ROS) accumulation and the transgenic seeds were partially resistant to the nitric oxide (NO) donor sodium nitroprusside (SNP). Moreover, Escherichia coli expressing GASA4 or GASA5 were resistant to H2O2 and SNP. A truncated version containing only the cysteine-rich domain of GASA4 was also resistant to SNP. Protein extracts from E. coli expressing GASA4 or GASA5 suppressed the oxidation of the fluorescent dye DCF-H2 by H2O2 and NO. In silico analysis of GAST-like genes suggested structural similarities to the cysteinerich domains of the ATP-Binding Cassette 1 (ABCE1) protein which bind iron-sulfur clusters [4Fe-4S]+2 via cysteine residues. These clusters are involved in electron transfer and redox regulation. Inductively coupled plasma mass spectrometry (ICP-MS) analysis indeed showed that recombinant GASA4 and GASA5 contain high levels of iron atoms. Mutated GASA4, in which conserved cysteines were replaced by alanines, lost its redox activity and the ability to promote GA responses, suggesting that the two functions are linked. We propose that GA induces some GAST1-like genes and suppresses others to regulate its own responses. We also suggest that the encoded proteins regulate the redox status of specific components to promote or suppress these responses. Keywords: redox regulation, plant gibberellin signaling, iron-sulfur clusters
development,
doi:10.1016/j.freeradbiomed.2012.08.431
S206
The cremaster microcirculation of freshly killed Wistar rats was perfused with a buffer containing FITC-albumin. Perfusion was stopped and the rate constant of fluorescence gradient decrease was used to calculate permeability. Bk application resulted in an increased permeability from 0.12 ± 0.02 × 10-6 cm.s-1 (mean ± sem, n = 139) to a dose-dependent logEC50 -5.3 ± 0.3 (maximal 2.7 ± 0.9 × 10-6 cm.s-1). Topical IL-1β (30 pM) 10 min. resulted Bk (1 μM) response from 0.18 ± 0.05 × 10-6 cm.s-1 (n = 9) to 1.1 ± 0.2 × 10-6 cm.s-1 (n = 17; p < 0.01 analysis of variance), which was completely blocked by either apocynin co-application with IL-1β (1 μM, 0.06 ± 0.02 × 10-6 cm.s-1; n = 9) or by scavenging ROS with a superoxide dismutase and catalase mixture during Bk application (0.1 ± 0.02 × 10-6cm.s-1; n=16). IL-1β activates sphingomyelinase to produce ceramide which in turn can activate PKC, and we tested whether ceramide application itself also rapidly potentiates the permeability response to Bk on the same preparation, but in the presence of the ACE inhibitor captopril (1mM) to increase the potency of Bk. Here the resting response to Bk gave a logEC50 -6.3±0.3, maximal 7.7 ± 1.9 x106 cm.s-1. Ceramide (1 mM) pretreatment moved the curve to the left (logEC50 -7.39 ± 0.3) with a maximal response 8.5 ± 1.4 x10-6cm.s-1 (n=30 vessels from 4 animals; p<0.01 analysis of co-variance). Thus ceramide is a possible mediator of the IL-1β rapid potentiation of the response to Bk. doi:10.1016/j.freeradbiomed.2012.08.432 Posters - Oxidation of Macromolecules [0046] Oxidative inhibition of erythrocyte Na+-K+ pump in diabetes: A functionally relevant circulating marker of oxidative stress C. Liu*1, N. Armstrong2, G. Figtree1 ,2, H. Rasmussen1 1 University of Sydney, Australia, 2Royal North Shore Hospital, Australia
[0672] Muscle microvascular permeability mediated via free radical generation
The burst in free radicals that disrupts the blood-brain barrier in ischemia-reperfusion is due to interaction of bradykinin (Bk) and IL-1β signaling pathways culminating in NOX2, cyclooxygenase and lipoxygenase generation of ROS (Woodfin; FRBM 2011, 50:518). We now show that this interaction also occurs in a muscle preparation.
increase
[0643] The Arabidopsis thaliana GASA protein: possible roles in redox regulation and plant development L. Rubinovich*, D. Weiss The Hebrew University of Jerusalem, Israel Although the gibberellin (GA) signaling has been elucidated, very little is known about the steps linking first transcriptional activation to physiological responses. Among the few identified GA-induced genes are the plant-specific GAST1-like genes, which encode small proteins with a conserved cysteine-rich domain. The Arabidopsis GAST1-like gene family consists of 14 members, GASA1-14. Here we show that overexpression of GASA4 in Arabidopsis promoted typical GA responses. Suppression of several GASA genes using synthetic microRNA also promoted seed germination, probably due to the suppression of GASA5, which acts as a repressor of GA responses. Overexpression of GASA4 suppressed reactive oxygen species (ROS) accumulation and the transgenic seeds were partially resistant to the nitric oxide (NO) donor sodium nitroprusside (SNP). Moreover, Escherichia coli expressing GASA4 or GASA5 were resistant to H2O2 and SNP. A truncated version containing only the cysteine-rich domain of GASA4 was also resistant to SNP. Protein extracts from E. coli expressing GASA4 or GASA5 suppressed the oxidation of the fluorescent dye DCF-H2 by H2O2 and NO. In silico analysis of GAST-like genes suggested structural similarities to the cysteinerich domains of the ATP-Binding Cassette 1 (ABCE1) protein which bind iron-sulfur clusters [4Fe-4S]+2 via cysteine residues. These clusters are involved in electron transfer and redox regulation. Inductively coupled plasma mass spectrometry (ICP-MS) analysis indeed showed that recombinant GASA4 and GASA5 contain high levels of iron atoms. Mutated GASA4, in which conserved cysteines were replaced by alanines, lost its redox activity and the ability to promote GA responses, suggesting that the two functions are linked. We propose that GA induces some GAST1-like genes and suppresses others to regulate its own responses. We also suggest that the encoded proteins regulate the redox status of specific components to promote or suppress these responses. Keywords: redox regulation, plant gibberellin signaling, iron-sulfur clusters
development,
doi:10.1016/j.freeradbiomed.2012.08.431
S206
The cremaster microcirculation of freshly killed Wistar rats was perfused with a buffer containing FITC-albumin. Perfusion was stopped and the rate constant of fluorescence gradient decrease was used to calculate permeability. Bk application resulted in an increased permeability from 0.12 ± 0.02 × 10-6 cm.s-1 (mean ± sem, n = 139) to a dose-dependent logEC50 -5.3 ± 0.3 (maximal 2.7 ± 0.9 × 10-6 cm.s-1). Topical IL-1β (30 pM) 10 min. resulted Bk (1 μM) response from 0.18 ± 0.05 × 10-6 cm.s-1 (n = 9) to 1.1 ± 0.2 × 10-6 cm.s-1 (n = 17; p < 0.01 analysis of variance), which was completely blocked by either apocynin co-application with IL-1β (1 μM, 0.06 ± 0.02 × 10-6 cm.s-1; n = 9) or by scavenging ROS with a superoxide dismutase and catalase mixture during Bk application (0.1 ± 0.02 × 10-6cm.s-1; n=16). IL-1β activates sphingomyelinase to produce ceramide which in turn can activate PKC, and we tested whether ceramide application itself also rapidly potentiates the permeability response to Bk on the same preparation, but in the presence of the ACE inhibitor captopril (1mM) to increase the potency of Bk. Here the resting response to Bk gave a logEC50 -6.3±0.3, maximal 7.7 ± 1.9 x106 cm.s-1. Ceramide (1 mM) pretreatment moved the curve to the left (logEC50 -7.39 ± 0.3) with a maximal response 8.5 ± 1.4 x10-6cm.s-1 (n=30 vessels from 4 animals; p<0.01 analysis of co-variance). Thus ceramide is a possible mediator of the IL-1β rapid potentiation of the response to Bk. doi:10.1016/j.freeradbiomed.2012.08.432 Posters - Oxidation of Macromolecules [0046] Oxidative inhibition of erythrocyte Na+-K+ pump in diabetes: A functionally relevant circulating marker of oxidative stress C. Liu*1, N. Armstrong2, G. Figtree1 ,2, H. Rasmussen1 1 University of Sydney, Australia, 2Royal North Shore Hospital, Australia
[0672] Muscle microvascular permeability mediated via free radical generation
The burst in free radicals that disrupts the blood-brain barrier in ischemia-reperfusion is due to interaction of bradykinin (Bk) and IL-1β signaling pathways culminating in NOX2, cyclooxygenase and lipoxygenase generation of ROS (Woodfin; FRBM 2011, 50:518). We now show that this interaction also occurs in a muscle preparation.
increase