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P60 Silver sulfide nanoparticle based microplate assay for hydrogen sulfide
S38
Abstracts / Nitric Oxide 27 (2012) S11–S42
the interactions of hydrogen sulfide with hemeproteins and hemeperoxidases. The human hemoglobin (LeuB10, HisE7, ValE11), myoglobin (LeuB10, HisE7, ValE11) and lactoperoxidase (Arg, HisE7), in the presence hydrogen peroxide (H2O2), and hydrogen sulfide (H2S), form an inactive sulfheme derivate characterized by a covalent sulfur modification of the heme ‘‘B’’ pyrrole ring. The derivate formation causes a partial inactivation of myoglobin, human hemoglobin and lactoperoxidase. However, the invertebrate organism Lucina pectinata remains unaffected in the presence of H2S and O2. The hemoglobin I (PheB10, GlnE7, PheE11) of this clam is the sulfide reactive protein that binds H2S as ligand. It has a glutamine in the distal E7 position instead of typical histidine found in hemoglobin, myoglobin and lactoperoxidase. Studies with hemeproteins demonstrate that the distal residues play an important role in the sulfheme derivates formation since the reactivity of H2S with hemeproteins are controlled by the polarity of the distal heme cavity, and the stereo orientation of the distal side residues. To confirm it, HbI mutants that mimic the active site of myoglobin and lactoperoxidase were done by site-directed mutagenesis. Then UV–vis spectroscopic analyses were made on Mb, Hb, HbI, and HbI mutants protein samples upon the reaction with hydrogen peroxide/oxygen and hydrogen sulfide. So far mutants with HisE7 apparently are the only mutants to show the characteristic UV–vis band at 624 nm that indicates the sulfheme product formation. Disclosure: This presentation was supported in part by Grants from NSF, RISE TWO BEST program, and RCMI program. http://dx.doi.org/10.1016/j.niox.2012.08.059
P59 H2S ameliorates homocysteine mediated attenuation of miR-133a and b2-AR in diabetic hearts Paras Kumar Mishra, Vishalakshi Chavali, Pushpakumar Sathur, Natia Qipshidze, Suresh Tyagi University of Louisville, Physiology & Biophysics, Louisville, KY 40202, United States Previously we have reported that hydrogen sulfide (H2S) protects against chronic heart failure, and elevated level of homocysteine (Hcy) called hyperhomocysteinemia (HHcy, a co-morbid condition) exacerbates diabetic cardiomyopathy. The generation of H2S from Hcy requires cystathionine c-lyase (CBS) enzyme and mutation of CBS (CBS+/) causes pathological cardiac remodeling. Since miR133a (cardiac fibrosis) and b2-adrenergic receptors (b2-AR: contractile dysfunction) are attenuated in diabetic hearts and contribute to cardiac remodeling, we hypothesize that inhibition of CBS induces HHcy and attenuates miR-133a and b2-AR due to decreased production of H2S. To prove the hypothesis, we used C57BL/6 J (WT), CBS+/ with and without NaHS (a H2S donor, 30 lM/L in drinking water for 4 weeks) and measured the levels of miR-133a and b2-AR by individual miR-133a assay, multiplex RT-PCR and Western blotting. Additionally, to assess the role of H2S in mitigation of glucose mediated inhibition of b2-AR, we determined the levels of stimulatory G -protein coupled receptor (Gs: inducer of b2-AR) and b2-AR in HL1 cardiomyocytes treated with high glucose (25 mM D-glucose) and high glucose + H2S (30 lM) by RT-PCR flow cytometry and RT-PCR. The results revealed that both miR-133a and b2-AR are down regulated in CBS+/ hearts. However, treatment with H2S improves the levels of miR-133a and b2-AR. Similarly, Gs and b2-AR are down regulated in high glucose treated cardiomyocytes and H2S ameliorates both Gs and b2-AR. These findings elicit that H2S induces miR-133a and b2-AR in HHcy hearts and ameliorates diabetic cardiomyopathy. Disclosure: Financial supports from American Heart Association grant 11BGIA7690055 to Paras Kumar Mishra and NIH HL-74185 is acknowleged.
http://dx.doi.org/10.1016/j.niox.2012.08.060
P60 Silver sulfide nanoparticle based microplate assay for hydrogen sulfide Artur Jarosz, Terence Yep, Jingyuan Wang, Bulent Mutus University of Windsor, Chemistry and Biochemistry, Windsor, Ontario, Canada N9G 2S1 Hydrogen sulfide (H2S) is a physiologically important gas synthesized by mammalian cells, having a vast number of effects, particularly in the cardiovascular, immune, and central-nervous systems. The chemistry of hydrogen sulfide is complex as it dissociates into hyposulfide (HS), and sulfide (S 2 Þ anions in solution, reacts with protein thiols, oxidizes to other sulfur species, and can quench endogenous oxidants such as peroxynitrite. This makes the mechanisms of hydrogen sulfide-mediated biological activity particularly difficult to study. Existing assays for H2S such as the methylene blue method do not differentiate between sulfur species and are prone to interferences. This makes measurement of H2S in biological settings difficult and reported literature concentrations somewhat controversial. To measure H2S gas specifically we have developed a microplate-based assay where H2S gas diffuses to react with silver (Ag+) immobilized in a polymer to produce Ag2S nanoparticles and a colorimetric response. The polymer is transparent in the visible range and produces a strong absorbance at 310 nm as H2S gas reacts with Ag+ to produce Ag2S nanoparticles. The microplate assay can be used to monitor H2S gas in real time or can be used for endpoint measurements and has a detection limit in low nmol range. The assay was used to monitor H2S gas production from recombinant cystathionine gamma-lyase (CSE) enzyme and determine the Km and Vmax values of the enzyme. The microplate-based assay represents an attractive low-cost method for specific H2S gas detection. Currently we are optimizing the assay for H2S gas measurement in live cells. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2012.08.061
P61 Hydrogen sulfide therapy rescues critical limb ischemia in aged diabetic mice through NO/VEGF dependent pathway Shyamal Bir, Gopi Krishna Kolluru, Xinggui Shen, Sibile Pardue, Christopher Pattillo, Christopher Kevil LSU Health Sciences Center, Pathology, Shreveport, LA 71130, United States Introduction: Hydrogen sulfide is a gaseous signaling molecule which is produced in vivo by three enzymes such as cystathionine c-lyase (CSE), cystathionine b- synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MST) from L-cysteine.CSE is the prime enzyme among others for sulfide production in vascular tissues of our body. Hydrogen sulfide is reported to have vasodilatory, anti–inflammatory, anti-oxidant, cytoprotective and pro-angiogenic effects. Sulfide production is decreased in diabetic condition. However, the molecular mechanisms of sulfide-mediated protection during chronic tissue ischemia in diabetic condition are completely unknown. Objective: To determine molecular mechanisms involved in hydrogen sulfide mediated cytoprotection during chronic tissue ischemia in diabetic condition. Methods: Hind limb ischemia was induced in 10 week old wild type, 42 week old Db/Db diabetic or 10–12 week old CSE knock out (CSEKO, n = 8, each group). PBS, 0.1, 0.5 and 1 mg/kg sodium sulfide (Na2S) was administered twice daily by retro-orbital injection. Hind limb perfusion was measured using a laser Doppler perfusion probe.
Abstracts / Nitric Oxide 27 (2012) S11–S42
the interactions of hydrogen sulfide with hemeproteins and hemeperoxidases. The human hemoglobin (LeuB10, HisE7, ValE11), myoglobin (LeuB10, HisE7, ValE11) and lactoperoxidase (Arg, HisE7), in the presence hydrogen peroxide (H2O2), and hydrogen sulfide (H2S), form an inactive sulfheme derivate characterized by a covalent sulfur modification of the heme ‘‘B’’ pyrrole ring. The derivate formation causes a partial inactivation of myoglobin, human hemoglobin and lactoperoxidase. However, the invertebrate organism Lucina pectinata remains unaffected in the presence of H2S and O2. The hemoglobin I (PheB10, GlnE7, PheE11) of this clam is the sulfide reactive protein that binds H2S as ligand. It has a glutamine in the distal E7 position instead of typical histidine found in hemoglobin, myoglobin and lactoperoxidase. Studies with hemeproteins demonstrate that the distal residues play an important role in the sulfheme derivates formation since the reactivity of H2S with hemeproteins are controlled by the polarity of the distal heme cavity, and the stereo orientation of the distal side residues. To confirm it, HbI mutants that mimic the active site of myoglobin and lactoperoxidase were done by site-directed mutagenesis. Then UV–vis spectroscopic analyses were made on Mb, Hb, HbI, and HbI mutants protein samples upon the reaction with hydrogen peroxide/oxygen and hydrogen sulfide. So far mutants with HisE7 apparently are the only mutants to show the characteristic UV–vis band at 624 nm that indicates the sulfheme product formation. Disclosure: This presentation was supported in part by Grants from NSF, RISE TWO BEST program, and RCMI program. http://dx.doi.org/10.1016/j.niox.2012.08.059
P59 H2S ameliorates homocysteine mediated attenuation of miR-133a and b2-AR in diabetic hearts Paras Kumar Mishra, Vishalakshi Chavali, Pushpakumar Sathur, Natia Qipshidze, Suresh Tyagi University of Louisville, Physiology & Biophysics, Louisville, KY 40202, United States Previously we have reported that hydrogen sulfide (H2S) protects against chronic heart failure, and elevated level of homocysteine (Hcy) called hyperhomocysteinemia (HHcy, a co-morbid condition) exacerbates diabetic cardiomyopathy. The generation of H2S from Hcy requires cystathionine c-lyase (CBS) enzyme and mutation of CBS (CBS+/) causes pathological cardiac remodeling. Since miR133a (cardiac fibrosis) and b2-adrenergic receptors (b2-AR: contractile dysfunction) are attenuated in diabetic hearts and contribute to cardiac remodeling, we hypothesize that inhibition of CBS induces HHcy and attenuates miR-133a and b2-AR due to decreased production of H2S. To prove the hypothesis, we used C57BL/6 J (WT), CBS+/ with and without NaHS (a H2S donor, 30 lM/L in drinking water for 4 weeks) and measured the levels of miR-133a and b2-AR by individual miR-133a assay, multiplex RT-PCR and Western blotting. Additionally, to assess the role of H2S in mitigation of glucose mediated inhibition of b2-AR, we determined the levels of stimulatory G -protein coupled receptor (Gs: inducer of b2-AR) and b2-AR in HL1 cardiomyocytes treated with high glucose (25 mM D-glucose) and high glucose + H2S (30 lM) by RT-PCR flow cytometry and RT-PCR. The results revealed that both miR-133a and b2-AR are down regulated in CBS+/ hearts. However, treatment with H2S improves the levels of miR-133a and b2-AR. Similarly, Gs and b2-AR are down regulated in high glucose treated cardiomyocytes and H2S ameliorates both Gs and b2-AR. These findings elicit that H2S induces miR-133a and b2-AR in HHcy hearts and ameliorates diabetic cardiomyopathy. Disclosure: Financial supports from American Heart Association grant 11BGIA7690055 to Paras Kumar Mishra and NIH HL-74185 is acknowleged.
http://dx.doi.org/10.1016/j.niox.2012.08.060
P60 Silver sulfide nanoparticle based microplate assay for hydrogen sulfide Artur Jarosz, Terence Yep, Jingyuan Wang, Bulent Mutus University of Windsor, Chemistry and Biochemistry, Windsor, Ontario, Canada N9G 2S1 Hydrogen sulfide (H2S) is a physiologically important gas synthesized by mammalian cells, having a vast number of effects, particularly in the cardiovascular, immune, and central-nervous systems. The chemistry of hydrogen sulfide is complex as it dissociates into hyposulfide (HS), and sulfide (S 2 Þ anions in solution, reacts with protein thiols, oxidizes to other sulfur species, and can quench endogenous oxidants such as peroxynitrite. This makes the mechanisms of hydrogen sulfide-mediated biological activity particularly difficult to study. Existing assays for H2S such as the methylene blue method do not differentiate between sulfur species and are prone to interferences. This makes measurement of H2S in biological settings difficult and reported literature concentrations somewhat controversial. To measure H2S gas specifically we have developed a microplate-based assay where H2S gas diffuses to react with silver (Ag+) immobilized in a polymer to produce Ag2S nanoparticles and a colorimetric response. The polymer is transparent in the visible range and produces a strong absorbance at 310 nm as H2S gas reacts with Ag+ to produce Ag2S nanoparticles. The microplate assay can be used to monitor H2S gas in real time or can be used for endpoint measurements and has a detection limit in low nmol range. The assay was used to monitor H2S gas production from recombinant cystathionine gamma-lyase (CSE) enzyme and determine the Km and Vmax values of the enzyme. The microplate-based assay represents an attractive low-cost method for specific H2S gas detection. Currently we are optimizing the assay for H2S gas measurement in live cells. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2012.08.061
P61 Hydrogen sulfide therapy rescues critical limb ischemia in aged diabetic mice through NO/VEGF dependent pathway Shyamal Bir, Gopi Krishna Kolluru, Xinggui Shen, Sibile Pardue, Christopher Pattillo, Christopher Kevil LSU Health Sciences Center, Pathology, Shreveport, LA 71130, United States Introduction: Hydrogen sulfide is a gaseous signaling molecule which is produced in vivo by three enzymes such as cystathionine c-lyase (CSE), cystathionine b- synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MST) from L-cysteine.CSE is the prime enzyme among others for sulfide production in vascular tissues of our body. Hydrogen sulfide is reported to have vasodilatory, anti–inflammatory, anti-oxidant, cytoprotective and pro-angiogenic effects. Sulfide production is decreased in diabetic condition. However, the molecular mechanisms of sulfide-mediated protection during chronic tissue ischemia in diabetic condition are completely unknown. Objective: To determine molecular mechanisms involved in hydrogen sulfide mediated cytoprotection during chronic tissue ischemia in diabetic condition. Methods: Hind limb ischemia was induced in 10 week old wild type, 42 week old Db/Db diabetic or 10–12 week old CSE knock out (CSEKO, n = 8, each group). PBS, 0.1, 0.5 and 1 mg/kg sodium sulfide (Na2S) was administered twice daily by retro-orbital injection. Hind limb perfusion was measured using a laser Doppler perfusion probe.