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Promoter elements involved in transcriptional regulation of human apolipoprotein AII gene
,J Mel Cell Cardiol
28
22 (Supplement
IV) (1990)
EFFECTS OF OXYGEN FREE RADICALS ON CARDIOVASCULAR LINE RELEASE. R. Chahine, X. Chen, N. Campeau, J.
FUNCTION
AND MYOCARDIAL
NORADRENA-
de Champlain, R. Nadeau. Centre de recherche, Ho^pital du Sacr&Coeur, Universitb de Montreal, Quebec, Canada. In our study, rat isolated hearts were perfused according to the Langendorff technique and oxygen free radicals (OFR) were generated by electrolysis (E) using a 10 mA constant DC current for 1 min. Left ventricular pressure (LVP), its first derivative dp/dt max, left ventricular end diastolic pressure (LVEDP), coronary flow (CF) were recorded and noradrenaline (NA) washout in the perfusion fluid was quantitated by radioenzymatic assay. Changes in ventricular function, CF and NA washout reached a maximum 5 min after E. The OFR scavengers superoxide dismutase (SOD) 100 U/ml. catalase 150 U/ml. and mannitol 50 mM orovided a sionificant (~~0.05) but not complete protection against the deleterious e'ffects of OFR: Data obta;ned w&-e: LVP LVEDP CF NA dp/dt E (n=8) -64?7% -7Oi9% 385+35% -7Oi25% 42+8% SOD -12?2% -17i2% 92?25% -27k5 % -5?3% CATALASE [!-:I -24?3% -2023% 5oi-7 % -28?4 % 10+2% MANNITOL (n=6) -13?1% -1623% 48?8 % -2323 "6 -6?2% Thus, electrolysis induced-OFR not only cause myocardial dysfunction but also Significant arrhythmias were not obneuronal injury and subsequently NA release. served, which suggest that OFR may not be arrhythmogenic in the absence of ischemia.
29
OXIDATION
OF OXYHEMOGLQBIN
BY HYDROGEN
PEROXIDE.
B. CHAILLOT”, B. GASSIER”, P. LABRUDE’. Facultts de Pharmacie de Dijou” et de Nancy’, FRANCE. We will remind first that oxyhemoglobin (HbOZ) undergoes different alterations consecutive to various oxidizing process such as chemical oxidation by potassium ferricyauide, autoxidatiou by air dioxygen, by hydrogen peroxide (H202) or by some organic compounds. The Hb02/H202 system is choice in agreement with the postulated mechanism of catalysis of lipid peroxydation by heme proteins, suggestmg that HbO2 interacts with H20 forming a reactive oxydant species. H202 effects are studied here in vitro according to Halliwel’s hypothesis (1) : t?ae Hb02/H202 system does not generate hydroxyl radicals OH* but ionized iron, catalytic species of oxidation. We estimate this iron by the Ferrozin method (2) and parallely we searched to quantify OH’ radicals by a calorimetric assay using dimethylsulfoxide (3). Our results show that the determination of OH* is negative. However, according to the weak sensibility of the calorimetric method, we cannot conclude that a generation of free radicals does not occur. Electron spin resonance studies appear to be necessary. Oppositely, our results point out that an iron release is induced in out cxpcrimental conditions. (1) A. XJPPO and B. HALLIWEL, Free Rad. Res. Comm., 1989,5,277-281 (2) P. CARTER, Anal. B&hem., 1971,40,45C-458 (3) C.F. BABBS and D.W. GRIFFIN, Free Rad. Biol. Med., 1989,6,493-m.
30 PROMOTER
ELEMENTS INVOLVED IN TRANSCRIPTIONAL REGULATION OF HUMAN APOLIPOPROTEIN AII GENE J. CHAMBAZ*, D. PASTIER’ G. BEREZIAT and V. ZANNIS+ * URA CNRS 1283, CHU St Antoine, and + Section of Molecular Genetics, Boston University Human apolipoprotein AII (apo AII) is the second most abundant apolipoprotein component of high density lipoproteins and is also a minor component of other lipoproteins. Apo AI1 is exclusively synthesized by the liver and to a lesser extent by the intestine, providing a good system to search for cisacting elements and trans-acting factors involved in tissue-specific regulation of mammalian genes. Apo AI1 gene expression is controlled by a complex set of distal and proximal cis-acting regulatory elements. In order to perform genetic analysis of the promoter, a 911 bp fragment of the 5’ flanking region of human apo AI1 gene has been cloned upstream of the CAT gene, and different recombinant plasmids were prepared by 5’ and internal deletions. Apo AII promotor (-91 l/+29) stimulates CAT expression to the same extent as the Rous Sarcoma virus promoter. Both the distal and proximal domains are required to CAT transcription in HepG2 and CaCo cells. The cis-acting regulatory elements have been mapped by fcwt-prilllitl;; .uialysis. Five overlapping DNA fragments ?Yl to 100 bp lnng were ~y~~ksi& by IWW~ of PCK amplification. Fourteen-protected regions have been identified by foot-printing analysis with nuclear extracts prepared from rat liver, designated A to N. Seven of these regions (C, D, F, G, H, K, and L) bind heat stable factors. The binding properties of these regions have been further studied by cotnpetition foot-printing and bandshift with the biding domains of hNF1 and different C/EBP-like factors as competitors. s.10
28
22 (Supplement
IV) (1990)
EFFECTS OF OXYGEN FREE RADICALS ON CARDIOVASCULAR LINE RELEASE. R. Chahine, X. Chen, N. Campeau, J.
FUNCTION
AND MYOCARDIAL
NORADRENA-
de Champlain, R. Nadeau. Centre de recherche, Ho^pital du Sacr&Coeur, Universitb de Montreal, Quebec, Canada. In our study, rat isolated hearts were perfused according to the Langendorff technique and oxygen free radicals (OFR) were generated by electrolysis (E) using a 10 mA constant DC current for 1 min. Left ventricular pressure (LVP), its first derivative dp/dt max, left ventricular end diastolic pressure (LVEDP), coronary flow (CF) were recorded and noradrenaline (NA) washout in the perfusion fluid was quantitated by radioenzymatic assay. Changes in ventricular function, CF and NA washout reached a maximum 5 min after E. The OFR scavengers superoxide dismutase (SOD) 100 U/ml. catalase 150 U/ml. and mannitol 50 mM orovided a sionificant (~~0.05) but not complete protection against the deleterious e'ffects of OFR: Data obta;ned w&-e: LVP LVEDP CF NA dp/dt E (n=8) -64?7% -7Oi9% 385+35% -7Oi25% 42+8% SOD -12?2% -17i2% 92?25% -27k5 % -5?3% CATALASE [!-:I -24?3% -2023% 5oi-7 % -28?4 % 10+2% MANNITOL (n=6) -13?1% -1623% 48?8 % -2323 "6 -6?2% Thus, electrolysis induced-OFR not only cause myocardial dysfunction but also Significant arrhythmias were not obneuronal injury and subsequently NA release. served, which suggest that OFR may not be arrhythmogenic in the absence of ischemia.
29
OXIDATION
OF OXYHEMOGLQBIN
BY HYDROGEN
PEROXIDE.
B. CHAILLOT”, B. GASSIER”, P. LABRUDE’. Facultts de Pharmacie de Dijou” et de Nancy’, FRANCE. We will remind first that oxyhemoglobin (HbOZ) undergoes different alterations consecutive to various oxidizing process such as chemical oxidation by potassium ferricyauide, autoxidatiou by air dioxygen, by hydrogen peroxide (H202) or by some organic compounds. The Hb02/H202 system is choice in agreement with the postulated mechanism of catalysis of lipid peroxydation by heme proteins, suggestmg that HbO2 interacts with H20 forming a reactive oxydant species. H202 effects are studied here in vitro according to Halliwel’s hypothesis (1) : t?ae Hb02/H202 system does not generate hydroxyl radicals OH* but ionized iron, catalytic species of oxidation. We estimate this iron by the Ferrozin method (2) and parallely we searched to quantify OH’ radicals by a calorimetric assay using dimethylsulfoxide (3). Our results show that the determination of OH* is negative. However, according to the weak sensibility of the calorimetric method, we cannot conclude that a generation of free radicals does not occur. Electron spin resonance studies appear to be necessary. Oppositely, our results point out that an iron release is induced in out cxpcrimental conditions. (1) A. XJPPO and B. HALLIWEL, Free Rad. Res. Comm., 1989,5,277-281 (2) P. CARTER, Anal. B&hem., 1971,40,45C-458 (3) C.F. BABBS and D.W. GRIFFIN, Free Rad. Biol. Med., 1989,6,493-m.
30 PROMOTER
ELEMENTS INVOLVED IN TRANSCRIPTIONAL REGULATION OF HUMAN APOLIPOPROTEIN AII GENE J. CHAMBAZ*, D. PASTIER’ G. BEREZIAT and V. ZANNIS+ * URA CNRS 1283, CHU St Antoine, and + Section of Molecular Genetics, Boston University Human apolipoprotein AII (apo AII) is the second most abundant apolipoprotein component of high density lipoproteins and is also a minor component of other lipoproteins. Apo AI1 is exclusively synthesized by the liver and to a lesser extent by the intestine, providing a good system to search for cisacting elements and trans-acting factors involved in tissue-specific regulation of mammalian genes. Apo AI1 gene expression is controlled by a complex set of distal and proximal cis-acting regulatory elements. In order to perform genetic analysis of the promoter, a 911 bp fragment of the 5’ flanking region of human apo AI1 gene has been cloned upstream of the CAT gene, and different recombinant plasmids were prepared by 5’ and internal deletions. Apo AII promotor (-91 l/+29) stimulates CAT expression to the same extent as the Rous Sarcoma virus promoter. Both the distal and proximal domains are required to CAT transcription in HepG2 and CaCo cells. The cis-acting regulatory elements have been mapped by fcwt-prilllitl;; .uialysis. Five overlapping DNA fragments ?Yl to 100 bp lnng were ~y~~ksi& by IWW~ of PCK amplification. Fourteen-protected regions have been identified by foot-printing analysis with nuclear extracts prepared from rat liver, designated A to N. Seven of these regions (C, D, F, G, H, K, and L) bind heat stable factors. The binding properties of these regions have been further studied by cotnpetition foot-printing and bandshift with the biding domains of hNF1 and different C/EBP-like factors as competitors. s.10