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The role of nitric oxide in the fission yeast Schizosaccharomyces pombe under oxidative stress conditions
Abstracts/Nitric Oxide 42 (2014) 99–153
incorporated the recombinant oxygenase subunit of the enzyme into miniature lipid membranes called nanodiscs which are 12.9 nm in diameter. These nanodiscs based on membrane scaffold proteins provide a unique system that mimics the enzyme’s native microenvironment, yet the prepared enzyme/nanodisc assemblies can be conveniently studied in solution like any soluble enzyme preparation. Homogenous eNOS/nanodisc samples are purified using size exclusion chromatography. The average size of nanodisc diameter was confirmed by particle analysis based on dynamic light scattering. Griess assay is used to measure activity of free and nanodisc-bound enzymes. As compared to the free enzyme, the specific activity of nanodiscbound eNOS oxygenase appears to be much lower. These data suggest that the membrane environment affects the catalytic properties of eNOS oxygenase. Keywords: Cardiovascular disease; eNOS; Nanodiscs; Membrane scaffold protein.
P102. The role of nitric oxide in the fission yeast Schizosaccharomyces pombe under oxidative stress conditions http://dx.doi.org/10.1016/j.niox.2014.09.050 Rika Indri Astuti, Hiroshi Takagi Nara Institute of Science and Technology
Nitric oxide (NO) confers oxidative stress tolerance by enhancing cellular antioxidative activity in mammals and plants. In the unicellular eukaryote yeast cells, NO may be involved in stress response pathways, but the synthetic mechanism and the physiological role of NO is poorly understood due to the lack of mammalian and bacterial NO synthase (NOS) orthologues in the genome. Our lab recently revealed the novel antioxidative mechanism mediated by NO in the budding yeast Saccharomyces cerevisiae, indicating that increased conversion of l-proline into l-arginine led to NO production in response to elevated temperature that induces intracellular reactive oxygen species generation. Here, we found that the fission yeast Schizosaccharomyces pombe cells possess both high mitochondrial respiratory activity with the nitrosyl thiolate compound accumulation and low activity which consequently induces the expression of a NO detoxification enzyme, S-nitrosoglutathione reductase (GSNOR). In addition to NOS-like activity, the mitochondrial activity, in particular cytochrome-c oxidase, is partially involved in NO synthesis. KCN and l-NAME treatments indeed abolish NO signaling, as confirmed by the loss of GSNOR expression. It should be noteworthy that NO is regulated by the constitutive expression of NO deoxygenase (NOD) through growth phases, presumably for NO homeostasis. Interestingly, l-arginine treatment enhances NO generation, most likely via NOS-like activity, simultaneously in response to H2O2-induced oxidative stress, which confers tolerance on yeast cells. Treatment with the exogenous NO donor (DetaNONOate) increased cell viability under oxidative stress conditions, while NO scavenger (c-PTIO) resulted in the contrary phenotype. Furthermore, NO-mediated stress tolerant mechanism is likely to occur via the pathway of the transcription factor Pap1 that has a role in the S. pombe stress response. Taken together, the fission yeast cells endogenously produce NO potentially involved in oxidative stress tolerance. Keywords: Fission yeast; Schizosaccharomyces pombe; NO; GSNOR; NOS; Oxidative stress.
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P103. Restoring perivascular nitric oxide gradients normalizes breast cancer vasculature http://dx.doi.org/10.1016/j.niox.2014.09.051 Suboj Babykutty, Takahiro Heishi, Kosuke Tsukada, Yuhui Huang, Sergey Kozin, David Conner, Qingcong Lin, Raju Kucherlapati, Rakesh K. Jain, Dai Fukumura Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
Objective: Impaired blood supply hinders the delivery and efficacy of therapeutics in solid tumors. Normalization of aberrant tumor vasculature can overcome these physiological barriers. We have reported that nitric oxide (NO) produced by vascular endothelial cells is crucial for angiogenesis and vessel maturation, while NO from non-vascular sources such as neuronal NO synthase in glioma cells interferes with these processes. Breast cancers often overexpress inducible nitric oxide synthase (iNOS). We hypothesized that heterogeneous NO gradients in breast cancer tissues contribute to the formation of abnormal tumor vasculature and resultant microenvironment. Here, we determined the role of iNOS in murine breast cancers on their vasculature and response to radiation therapy. Methods: We grew murine breast cancers (MCaIV and E0771) orthotopically in the mammary fat pad in syngeneic C3H and C57BL/6 mice, respectively. To determine vessel maturation, we visualized perivascular cells using fluorescent protein reporter mice (a-smooth muscle actin promoter driven green fluorescent protein or DsRed). We then determined tissue oxygenation using a redox marker pimonidazole. To block iNOS in these tumors, we silenced iNOS expression in tumor cells by shRNA and also used an iNOS selective inhibitor 1400 W (10 mg/ kg/day). Fractionated radiation therapy (5 Gy/day × 3 days) was performed when mammary fat pad tumors reached 100 mm3. Results: Murine breast cancers express high levels of iNOS. Tumor cells were the major source of iNOS in the tumor microenvironment. We found that genetic and pharmacological inhibition of iNOS resulted in improved morphology and function of tumor vasculature such as an increase in functional blood vessels and their maturation, and a decrease in the abnormal vessel dilation and vascular hyperpermeability. Finally, iNOS silencing in the tumor alleviated tumor hypoxia and that was translated into improved tumor response to radiation therapy. Conclusion: We found that restoring perivascular NO gradients could normalize tumor vasculature both morphologically and functionally in murine breast cancers, followed by the improvements in tissue oxygenation and efficacy of radiation therapy. These data suggest that targeting non-vascular sources of NO is a potential mean to potentiate anti-tumor therapies. (This work was supported by NIH grant CA096915.) Keywords: Nitric oxide; Tumor microenvironment; Vessel normalization. P104. A systemically-injected targeted nitric oxide-delivery vehicle durably inhibits neointimal hyperplasia after arterial injury http://dx.doi.org/10.1016/j.niox.2014.09.052 Edward Bahnson, Tyson Moyer, Hussein Kassam, Janet Vercammen, Samuel Stupp, Melina Kibbe Northwestern University
Introduction: Vascular interventions continue to fail from restenosis secondary to neointimal hyperplasia. We developed a novel, systemically delivered, targeted therapy that will prevent restenosis following all cardiovascular interventions, using a highly customizable peptide amphiphile (PA). We previously showed that a PA targeted to a unique collagen-binding sequence (since collagen is exposed to the circulation only after endothelial
incorporated the recombinant oxygenase subunit of the enzyme into miniature lipid membranes called nanodiscs which are 12.9 nm in diameter. These nanodiscs based on membrane scaffold proteins provide a unique system that mimics the enzyme’s native microenvironment, yet the prepared enzyme/nanodisc assemblies can be conveniently studied in solution like any soluble enzyme preparation. Homogenous eNOS/nanodisc samples are purified using size exclusion chromatography. The average size of nanodisc diameter was confirmed by particle analysis based on dynamic light scattering. Griess assay is used to measure activity of free and nanodisc-bound enzymes. As compared to the free enzyme, the specific activity of nanodiscbound eNOS oxygenase appears to be much lower. These data suggest that the membrane environment affects the catalytic properties of eNOS oxygenase. Keywords: Cardiovascular disease; eNOS; Nanodiscs; Membrane scaffold protein.
P102. The role of nitric oxide in the fission yeast Schizosaccharomyces pombe under oxidative stress conditions http://dx.doi.org/10.1016/j.niox.2014.09.050 Rika Indri Astuti, Hiroshi Takagi Nara Institute of Science and Technology
Nitric oxide (NO) confers oxidative stress tolerance by enhancing cellular antioxidative activity in mammals and plants. In the unicellular eukaryote yeast cells, NO may be involved in stress response pathways, but the synthetic mechanism and the physiological role of NO is poorly understood due to the lack of mammalian and bacterial NO synthase (NOS) orthologues in the genome. Our lab recently revealed the novel antioxidative mechanism mediated by NO in the budding yeast Saccharomyces cerevisiae, indicating that increased conversion of l-proline into l-arginine led to NO production in response to elevated temperature that induces intracellular reactive oxygen species generation. Here, we found that the fission yeast Schizosaccharomyces pombe cells possess both high mitochondrial respiratory activity with the nitrosyl thiolate compound accumulation and low activity which consequently induces the expression of a NO detoxification enzyme, S-nitrosoglutathione reductase (GSNOR). In addition to NOS-like activity, the mitochondrial activity, in particular cytochrome-c oxidase, is partially involved in NO synthesis. KCN and l-NAME treatments indeed abolish NO signaling, as confirmed by the loss of GSNOR expression. It should be noteworthy that NO is regulated by the constitutive expression of NO deoxygenase (NOD) through growth phases, presumably for NO homeostasis. Interestingly, l-arginine treatment enhances NO generation, most likely via NOS-like activity, simultaneously in response to H2O2-induced oxidative stress, which confers tolerance on yeast cells. Treatment with the exogenous NO donor (DetaNONOate) increased cell viability under oxidative stress conditions, while NO scavenger (c-PTIO) resulted in the contrary phenotype. Furthermore, NO-mediated stress tolerant mechanism is likely to occur via the pathway of the transcription factor Pap1 that has a role in the S. pombe stress response. Taken together, the fission yeast cells endogenously produce NO potentially involved in oxidative stress tolerance. Keywords: Fission yeast; Schizosaccharomyces pombe; NO; GSNOR; NOS; Oxidative stress.
115
P103. Restoring perivascular nitric oxide gradients normalizes breast cancer vasculature http://dx.doi.org/10.1016/j.niox.2014.09.051 Suboj Babykutty, Takahiro Heishi, Kosuke Tsukada, Yuhui Huang, Sergey Kozin, David Conner, Qingcong Lin, Raju Kucherlapati, Rakesh K. Jain, Dai Fukumura Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
Objective: Impaired blood supply hinders the delivery and efficacy of therapeutics in solid tumors. Normalization of aberrant tumor vasculature can overcome these physiological barriers. We have reported that nitric oxide (NO) produced by vascular endothelial cells is crucial for angiogenesis and vessel maturation, while NO from non-vascular sources such as neuronal NO synthase in glioma cells interferes with these processes. Breast cancers often overexpress inducible nitric oxide synthase (iNOS). We hypothesized that heterogeneous NO gradients in breast cancer tissues contribute to the formation of abnormal tumor vasculature and resultant microenvironment. Here, we determined the role of iNOS in murine breast cancers on their vasculature and response to radiation therapy. Methods: We grew murine breast cancers (MCaIV and E0771) orthotopically in the mammary fat pad in syngeneic C3H and C57BL/6 mice, respectively. To determine vessel maturation, we visualized perivascular cells using fluorescent protein reporter mice (a-smooth muscle actin promoter driven green fluorescent protein or DsRed). We then determined tissue oxygenation using a redox marker pimonidazole. To block iNOS in these tumors, we silenced iNOS expression in tumor cells by shRNA and also used an iNOS selective inhibitor 1400 W (10 mg/ kg/day). Fractionated radiation therapy (5 Gy/day × 3 days) was performed when mammary fat pad tumors reached 100 mm3. Results: Murine breast cancers express high levels of iNOS. Tumor cells were the major source of iNOS in the tumor microenvironment. We found that genetic and pharmacological inhibition of iNOS resulted in improved morphology and function of tumor vasculature such as an increase in functional blood vessels and their maturation, and a decrease in the abnormal vessel dilation and vascular hyperpermeability. Finally, iNOS silencing in the tumor alleviated tumor hypoxia and that was translated into improved tumor response to radiation therapy. Conclusion: We found that restoring perivascular NO gradients could normalize tumor vasculature both morphologically and functionally in murine breast cancers, followed by the improvements in tissue oxygenation and efficacy of radiation therapy. These data suggest that targeting non-vascular sources of NO is a potential mean to potentiate anti-tumor therapies. (This work was supported by NIH grant CA096915.) Keywords: Nitric oxide; Tumor microenvironment; Vessel normalization. P104. A systemically-injected targeted nitric oxide-delivery vehicle durably inhibits neointimal hyperplasia after arterial injury http://dx.doi.org/10.1016/j.niox.2014.09.052 Edward Bahnson, Tyson Moyer, Hussein Kassam, Janet Vercammen, Samuel Stupp, Melina Kibbe Northwestern University
Introduction: Vascular interventions continue to fail from restenosis secondary to neointimal hyperplasia. We developed a novel, systemically delivered, targeted therapy that will prevent restenosis following all cardiovascular interventions, using a highly customizable peptide amphiphile (PA). We previously showed that a PA targeted to a unique collagen-binding sequence (since collagen is exposed to the circulation only after endothelial