- Email: [email protected]
Merlot wines from several countries vary in phenolic content
OXIDATIVE STRESS CHANGES DURING TRANSITION FROM RV HYPERTROPHY TO FAILURE Firoozeh Farahmand, Neelam Khaper 8 Pawan K. Singal. lnst of Cardlovasc Sci, Dept of Physiology, University of Manitoba, Wlnnlpeg, Canada. Changes in oxidative stress, an imPortant contributing factor in the transition between hypertrophy to heart failure were examined. Monocrotaline (MCT)-induced pulmonary hypertension (PH) and secondary right ventricular (RV) pressure overload and eventually failure was produced in rats by a single injection of MCT (60 mglkg, i. p.). Based on the hemodynamic, clinical and histopathological observations, three functional stages were observed at I-, 2- and 6-week
post-injection hypertrophy), increase
periods. In the l-week RV pressure overload, in antioxidant
enzymes
group (early stage of RV RV hypertrophy, a mild and
a decrease
in lipid
peroxidation were seen. In the 2-week group (compensated stage of RV failure), histopathologic evidence of pulmonary damage was associated with RV pressure overload, RV hypertrophy, reduced or no bulging of the interventricular septum (MS) into the lefl ventricle (LV), an increase in the activities of antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase and a decrease in lipid peroxidation. In the 6-week group (decompensated stage of RV failure), severe pneumotoxicity was associated with pulmonary edema; RV hypertrophy, dilation and failure; severe bulging of the IVS into the LV and liver congestion. In conclusion, the gradual worsening of RV function in the early stages was accompanied by hypertrophy and an increase in the antioxidant reserve, and in a later stage, RV failure was associated with a decreased antioxidant reserve. (Supported by the Canadian Institutes of Health Research.)
Ca” BINDING TO RAT NUCLEOPORINS OR PHOSPHORYLATION OF NUCLEOPORINS BY MAP KINASE DOES NOT REGULATE NUCLEAR IMPORT Randolph S. Faustlno, Mkhrel P. Czubryt, and Grant N. Plsrce, Division of Stroke and Vascular Disease, St. Bonifscs Research Centm, Dept. of Physiology, University of Manitoba, Winnipeg, Canada. Perinudear Ca** can modulate nudear import but its mechanism is unknown. Phosphorylatlon also alters import but it is unclear if ohosphorvlatlon of the nuclear oore wmolex (NPC) itself has anv effects. he examined the presence of Caz*-binding proteins as wsll as the effects of NPC modification by ~38, JNK, and ERK-2 phosphorylatfon on import. NPCs wre purified from isolated rat nuclei. Stains-All and Caz* overlays were used to determine the presence of calcium binding proteins. Substrates vmre identified by labeling NPC frrctfons tith QP, followed by SDS-PAGE and autoradiography. For nuclear import assays, SMCs were penneabiiized, incubated with import buffer containing ~38, JNK or ERK-2 kinase and then inverted on buffer containing a Ruorescanl substrate. Nudear fluorescence was quantitated using a SGI Wrkstation. No Ca2* binding proteins v.we detected. Autoradllraphy showed thal nucleoporins werephosphoryfated by endogenous kinas& Treatment 01 samples with apigenin and SB-202190 decreased phosphoryfation of a 7C kDa protein by 80% and 40%, respectively. Adding exogenous p38 and ERK-2 to NPCs resulted in phosphoryfation of the 70 kDa band. However, addition of these kinases to permeabilized SMCs had no effect on import. The NPC from rat hepatocytes does not contain significant Caz+ binding nucieoporins, but a p38 kinase may be associated with it. Phosphoryfation of the NPC by ~38, JNK and ERK-2 does not affecl nudear import in SMCs. Instead, it may be significant in relation to NPC structure or assembly. This wwk was supported by the Canadian Institutes of Health Research.
MAP KINASE INHIBITION OF NUCLEAR IMPORT
MERLOT
Randolph S. Faustlno, Grant N. Ftsrce, Dspt. of physioio(ly, Division of Stroks and Vascuier Msease, St. Boniface Ressarch Centre, University of Manitoba, Canadr
IN PHENOLIC CONTENT
The purpose of the present study was to determine whether import was affected by phosphoryfating a cytoplasmic component of the nuclear transport machinery through ~38, JNK or ERK-2 pathways in smooth muscle cells @MC). Nuclear import assays were carded out according to the procedure of Adam et al. ~38, JNK or ERK-2 was added to 5Opls of cytosolic import mix and incubated for 15 min. at 37’C. Coverdips containing SMCs were permeablized, rinsed with nuclear import buffer and inverted on the treated cytosoi plus 5pl.s of an ALEXA-conjugated, BSA-NLS import substrate and incubated for 30 min. Images ore acquired by wnfocal microscopy and nudear fluorescence measured using the SGI. Treatment of cytosol with MAP kinases at physiological levels showed
a dscrease in n&ear
transport. Incubating. l&l
of SE-202190 or 5-
WINES
FROM SEVERAL
COUNTRIES
VARY
Randolph S. Faustino, Andma E. Edsi, Sslisha Sobmttss, Gnnt N.
Pierce, Dept. of physiology,
Division of Stroke and Vascular
Disease, St. Soniface Research Centre, Univentty Canada
of Manitoba,
The purpose of our study was to compare quantities of catechin, epicatechin, rutin, transresveratrol and qua&in in selected f¬ tines from Chile, Canada and the United States. Catechin, epicatechin, rulin, transresveratrol and quercetin standards were separated by HPLC to i) determine the time of elukon from the wlumn and ii) to establish a standard catBraWn curve. lad aliquots of red tines wBre analyzed for pofyphenofii content. Concentration was determined for the phenoftc species and reported as mg!L. Catechin possessed the highest concentration in all Medot wines sampled (88% of total flavcnoid content). The remaining, in order from highest to io~st abundance, are e@catechin (26%) quercetin (5%). rutin
iodotubercidin (inhibits ~38 and ERK-2, respactively) and heat inactivated
(2%) and transresverstrol
JNK with the cytosolic &
and Canadian tines. American wines had the highest epkatechin and quercebn wntent. Rutin content was greatest in Chilean tines and transresveratrol occurred equally in tines from ail three countries. Catechin content had the dosest wrrelation with price (R = -0.5, PcO.05). Phenoiii wntent of the tines vanes and reflects differences in processing. harvesting or envimnment in which the grapes are grown. Catechin and epicatechin were the largest flavonoid components wiul the remaining three occurring at small percentages. Pricing of Me&t tines and polyphenoiic content w?re generally found to be poorfy wrrelated. This work was supported by the Canadian Institutes of Health Research.
restored nuclear impon-to near control levels.
JNK inhibited transport the most (nudear fluorescence was at 58.5% of control levels). ERK-2 displayed a concentration dependent effect. At physiological levels, ERK-2 treated cytosol inhibited nudear import. Higher concentrations increased levels of nuclear transport by as much as 50%. For p38 and JNK, the most pronounced effects on nuclear import
occurredafter 15 min. of phosphotyia5on and returned to baseline levels afler 1hr. A cytosolic component (or components) exists that can decrease or increase nuclear import once phosphoryfated by ~38, JNK or ERK-2. The attenuation in import can be reversed by the addition of specific pharmacological antagonists. This work was supported by the Canadian Institutes of Health Research.
(1%). Catschin content was hihest in Chilean
A33
post-injection hypertrophy), increase
periods. In the l-week RV pressure overload, in antioxidant
enzymes
group (early stage of RV RV hypertrophy, a mild and
a decrease
in lipid
peroxidation were seen. In the 2-week group (compensated stage of RV failure), histopathologic evidence of pulmonary damage was associated with RV pressure overload, RV hypertrophy, reduced or no bulging of the interventricular septum (MS) into the lefl ventricle (LV), an increase in the activities of antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase and a decrease in lipid peroxidation. In the 6-week group (decompensated stage of RV failure), severe pneumotoxicity was associated with pulmonary edema; RV hypertrophy, dilation and failure; severe bulging of the IVS into the LV and liver congestion. In conclusion, the gradual worsening of RV function in the early stages was accompanied by hypertrophy and an increase in the antioxidant reserve, and in a later stage, RV failure was associated with a decreased antioxidant reserve. (Supported by the Canadian Institutes of Health Research.)
Ca” BINDING TO RAT NUCLEOPORINS OR PHOSPHORYLATION OF NUCLEOPORINS BY MAP KINASE DOES NOT REGULATE NUCLEAR IMPORT Randolph S. Faustlno, Mkhrel P. Czubryt, and Grant N. Plsrce, Division of Stroke and Vascular Disease, St. Bonifscs Research Centm, Dept. of Physiology, University of Manitoba, Winnipeg, Canada. Perinudear Ca** can modulate nudear import but its mechanism is unknown. Phosphorylatlon also alters import but it is unclear if ohosphorvlatlon of the nuclear oore wmolex (NPC) itself has anv effects. he examined the presence of Caz*-binding proteins as wsll as the effects of NPC modification by ~38, JNK, and ERK-2 phosphorylatfon on import. NPCs wre purified from isolated rat nuclei. Stains-All and Caz* overlays were used to determine the presence of calcium binding proteins. Substrates vmre identified by labeling NPC frrctfons tith QP, followed by SDS-PAGE and autoradiography. For nuclear import assays, SMCs were penneabiiized, incubated with import buffer containing ~38, JNK or ERK-2 kinase and then inverted on buffer containing a Ruorescanl substrate. Nudear fluorescence was quantitated using a SGI Wrkstation. No Ca2* binding proteins v.we detected. Autoradllraphy showed thal nucleoporins werephosphoryfated by endogenous kinas& Treatment 01 samples with apigenin and SB-202190 decreased phosphoryfation of a 7C kDa protein by 80% and 40%, respectively. Adding exogenous p38 and ERK-2 to NPCs resulted in phosphoryfation of the 70 kDa band. However, addition of these kinases to permeabilized SMCs had no effect on import. The NPC from rat hepatocytes does not contain significant Caz+ binding nucieoporins, but a p38 kinase may be associated with it. Phosphoryfation of the NPC by ~38, JNK and ERK-2 does not affecl nudear import in SMCs. Instead, it may be significant in relation to NPC structure or assembly. This wwk was supported by the Canadian Institutes of Health Research.
MAP KINASE INHIBITION OF NUCLEAR IMPORT
MERLOT
Randolph S. Faustlno, Grant N. Ftsrce, Dspt. of physioio(ly, Division of Stroks and Vascuier Msease, St. Boniface Ressarch Centre, University of Manitoba, Canadr
IN PHENOLIC CONTENT
The purpose of the present study was to determine whether import was affected by phosphoryfating a cytoplasmic component of the nuclear transport machinery through ~38, JNK or ERK-2 pathways in smooth muscle cells @MC). Nuclear import assays were carded out according to the procedure of Adam et al. ~38, JNK or ERK-2 was added to 5Opls of cytosolic import mix and incubated for 15 min. at 37’C. Coverdips containing SMCs were permeablized, rinsed with nuclear import buffer and inverted on the treated cytosoi plus 5pl.s of an ALEXA-conjugated, BSA-NLS import substrate and incubated for 30 min. Images ore acquired by wnfocal microscopy and nudear fluorescence measured using the SGI. Treatment of cytosol with MAP kinases at physiological levels showed
a dscrease in n&ear
transport. Incubating. l&l
of SE-202190 or 5-
WINES
FROM SEVERAL
COUNTRIES
VARY
Randolph S. Faustino, Andma E. Edsi, Sslisha Sobmttss, Gnnt N.
Pierce, Dept. of physiology,
Division of Stroke and Vascular
Disease, St. Soniface Research Centre, Univentty Canada
of Manitoba,
The purpose of our study was to compare quantities of catechin, epicatechin, rutin, transresveratrol and qua&in in selected f¬ tines from Chile, Canada and the United States. Catechin, epicatechin, rulin, transresveratrol and quercetin standards were separated by HPLC to i) determine the time of elukon from the wlumn and ii) to establish a standard catBraWn curve. lad aliquots of red tines wBre analyzed for pofyphenofii content. Concentration was determined for the phenoftc species and reported as mg!L. Catechin possessed the highest concentration in all Medot wines sampled (88% of total flavcnoid content). The remaining, in order from highest to io~st abundance, are e@catechin (26%) quercetin (5%). rutin
iodotubercidin (inhibits ~38 and ERK-2, respactively) and heat inactivated
(2%) and transresverstrol
JNK with the cytosolic &
and Canadian tines. American wines had the highest epkatechin and quercebn wntent. Rutin content was greatest in Chilean tines and transresveratrol occurred equally in tines from ail three countries. Catechin content had the dosest wrrelation with price (R = -0.5, PcO.05). Phenoiii wntent of the tines vanes and reflects differences in processing. harvesting or envimnment in which the grapes are grown. Catechin and epicatechin were the largest flavonoid components wiul the remaining three occurring at small percentages. Pricing of Me&t tines and polyphenoiic content w?re generally found to be poorfy wrrelated. This work was supported by the Canadian Institutes of Health Research.
restored nuclear impon-to near control levels.
JNK inhibited transport the most (nudear fluorescence was at 58.5% of control levels). ERK-2 displayed a concentration dependent effect. At physiological levels, ERK-2 treated cytosol inhibited nudear import. Higher concentrations increased levels of nuclear transport by as much as 50%. For p38 and JNK, the most pronounced effects on nuclear import
occurredafter 15 min. of phosphotyia5on and returned to baseline levels afler 1hr. A cytosolic component (or components) exists that can decrease or increase nuclear import once phosphoryfated by ~38, JNK or ERK-2. The attenuation in import can be reversed by the addition of specific pharmacological antagonists. This work was supported by the Canadian Institutes of Health Research.
(1%). Catschin content was hihest in Chilean
A33