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Effect of oxidative stress on rabbit nerve aldose reductase
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AntoxidantCompounds VITAMIN E IN MEMBRANES INVESTIGATED SPECTROSCOPY G. van Ginkel, A.A. van 't Veld, F. Y.K. Levine. Dept. of Molecular Biophysics, Laboratory, Princetonplein 5, Utrecht Netherlands.
WITH OPTICAL Siemssen and Buys Ballot 3584 CC, The
Vitamin E (~-tocopherol and other tocopherols) interacts with other membrane lipids in physiological processes. The question is how this interaction affects membrane organization. Answers can be obtained using the optical absorption and fluorescence emission of vitamin E itself and of added fluorescent probe molecules. The fluorescence emission and excitation spectra in both ethanol and cyclohexane show drastic changes in the concentration range 5xIO-3M - IO-4M. The fluorescence decay, however, does not change with concentration. In ethanol the decay is monoexponential, average lifetime = 2.04 ns. In cyelohexane and various lipids a biexponential decay is found, with between 1.3 and 2.0 ns. Angle-resolved fluorescence depolarization (AFD) measurements in planar bilayers (DOPC, POPC, DLPC or PLPC), using DPH as a probe, show that ~tocopherol hardly affects membrane structure (molecular order), but changes membrane dynamics (=fluidity) in a concentration dependent way. Time resolved anisotropy and AFD measurements show that at high concentrations (1:50 and i:i00 relative to DOPC) ~-tocopherol does not move within the time window of its fluorescence decay. At a i00:i ratio the ~-tocopherol chromophore is oriented perpendicular to the DOPC membrane plane, whereas at a 50:1 ratio it is oriented parallel.
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EFFECT OF OXIDATIVE STRESS ON RABBIT NERVE ALDOSE REDUCTASE Elsie Wong, Arnold Miller, Daniel Vellom, and Henry Powell Departments of Pathology, Neurosciences, and Chemistry, University of California at San Diego, La Jolla, CA 92093, U.S.A. Increased activity of aldose reductase has been implicated in the development of diabetic complications in nerve, eye, kidney and microvascular tissue. Bovine lens aldose reductase modified in vitro in the presence of high ionic strength and oxygen free radical generating systems has demonstrated enhanced activity and loss of sensitivity to inhibition by sorbinil [Del Corso et al., (1987) Biochem. Biophys. Res. Comm. 148:369; and (1989) Arch. Biochem. Biophys. 270:604]. We have examined the effect ol oxidative stress on the activity of aldose reductase in extracts of rabbit nerve. The activity of the enzyme is increased two-fold in the presence o! 0.4 M ammonium sullate, and three-fold alter a 3 h preincubalion with 0.4 M ammoniumsulfate and Fe II / EDTA (0.1 mM/0.3 mM). In addition, nerve aldose reductase modified in the presence of this oxygen free radical generating system is less susceptible to inhibition by sorbinil. Our preliminary results suggest that oxidative stress may play a role in the pathogenesis of diabetic neuropathy. [Supported by NIH Grants NS-07078, NS-14162 and NEY-07055].
EBSELEN PREVENTS THE SYSTEMIC ENDOTOXIN-INDUCED RELEASE OF TNF~ AND PROTECTS IN VIVO AGAINST TNF Albrecht Wendel and Gisa Tiegs Faculty of B i o l o g y , U n i v e r s i t y of Konstanz, D-7750 Konstanz, F.R.G.
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Mice pretreated w i t h 700 mg/kg galactosamine and 5 ~ / k g endotoxin, or a l t e r n a t i v e l y w i t h 15 ~g/kg rmu TNF~ instead of endotoxin developed a severe f u l m i n a n t h e p a t i t i s w i t h i n 9 hours. In previous studies we provided evidence t h a t endotoxin adm i n i s t r a t i o n leads to TNF release which in turn represents a f i n a l t o x i c mediator (1). The e f f i c a c y of ebselen as a p r o t e c t a n t in t h i s l i v e r i n j u r y model has been a t t r i b u t e d to i t s a n t i - i n f l a m m a t o r y pr p e r t i e s , e s p e c i a l l y the a b i l i t y to i n h i b i t l e u k o t r i e n e synthesis (2). This study shows t h a t mice protected by p.o. pretreatment with 600 mg/kg ebselen did not release detectable amounts of TNF~ i n t o the c i r c u l a t i o n w i t h i n the next hour a f t e r a d m i n i s t r a t i o n of end o t o x i n . Moreover, ebselen was shown to p r o t e c t as well in the galactosamine/TNF~-induced i n j u r y . These f i n d i n g s suggest t h a t ebselen might be a useful drug in septic shock. 1) Tiegs, G., Wolter, M., and Wendel, A. (1989) Biochem. Pharmacol. 34, 627-631. 2) Tiegs, G. and Wende~ A. (1986) Biochem. Pharmacol. 35, 2115-2118.
TAUR1NE: PROTECTION FROM HYPOHALOUS ANIONS Charles E. Wright, Costas Ioannides, and Bruce M. Gillam Department of Developmental Biochemistry, N. Y. S. Institute for Basic Research, Staten Island, NY 10314, USA
Taurine is the major amino acid found in most animal cells. Reported concentrations (20 to < 140 mM) far exceed those of any other amino containing compound. Such amounts may be underestimated since 60-80% of cellular taurine is released during isolation. It is well documented that taurine reacts with hypohalous anions (HOX-) to form N-halogenated taurine (NXT). This reduces the oxidizing potential of HOX" and sequesters this oxidizing anion into a more stable lipophobic molecule. The significance of taurine as a natural antioxidant and NXT as a modulator of oxidant production has not been well recognized. In vitro experiments using cultured and/or isolated cells were used to examine the antioxidant properties of taurine and the biochemical effects of NXT. Compared with haloamine and other amino acid haloamines, NXT was found to be nontoxic at high concentrations. In fact, taurine would reduce the toxicity of all other haloamines. Rapid halide exchange was noted between the haloamine and taurine to form NXT. In competitive reactions, taurine reacted with HOX- and haloamines 5-6 times faster than the ammonium cation. The reaction had equal stoichometry, and exchange may be due to the anchimeric assistance of the sulfonate anion. NXT exposure to the oxidant metabolizing enzymes CAT, GPX, SOD, MPO, and XO resulted in covalent binding of 14[C]-taurine to the enzyme In all cases binding reduced enzyme hydrophobicity and activity. Because of its high cellular concentrations and its ability to rapidly compete with other amino groups for HOX', taurine is the prevalent antioxidant for the protection and modulation of ceils generating or exposed to oxidized halogens.
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AntoxidantCompounds VITAMIN E IN MEMBRANES INVESTIGATED SPECTROSCOPY G. van Ginkel, A.A. van 't Veld, F. Y.K. Levine. Dept. of Molecular Biophysics, Laboratory, Princetonplein 5, Utrecht Netherlands.
WITH OPTICAL Siemssen and Buys Ballot 3584 CC, The
Vitamin E (~-tocopherol and other tocopherols) interacts with other membrane lipids in physiological processes. The question is how this interaction affects membrane organization. Answers can be obtained using the optical absorption and fluorescence emission of vitamin E itself and of added fluorescent probe molecules. The fluorescence emission and excitation spectra in both ethanol and cyclohexane show drastic changes in the concentration range 5xIO-3M - IO-4M. The fluorescence decay, however, does not change with concentration. In ethanol the decay is monoexponential, average lifetime
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EFFECT OF OXIDATIVE STRESS ON RABBIT NERVE ALDOSE REDUCTASE Elsie Wong, Arnold Miller, Daniel Vellom, and Henry Powell Departments of Pathology, Neurosciences, and Chemistry, University of California at San Diego, La Jolla, CA 92093, U.S.A. Increased activity of aldose reductase has been implicated in the development of diabetic complications in nerve, eye, kidney and microvascular tissue. Bovine lens aldose reductase modified in vitro in the presence of high ionic strength and oxygen free radical generating systems has demonstrated enhanced activity and loss of sensitivity to inhibition by sorbinil [Del Corso et al., (1987) Biochem. Biophys. Res. Comm. 148:369; and (1989) Arch. Biochem. Biophys. 270:604]. We have examined the effect ol oxidative stress on the activity of aldose reductase in extracts of rabbit nerve. The activity of the enzyme is increased two-fold in the presence o! 0.4 M ammonium sullate, and three-fold alter a 3 h preincubalion with 0.4 M ammoniumsulfate and Fe II / EDTA (0.1 mM/0.3 mM). In addition, nerve aldose reductase modified in the presence of this oxygen free radical generating system is less susceptible to inhibition by sorbinil. Our preliminary results suggest that oxidative stress may play a role in the pathogenesis of diabetic neuropathy. [Supported by NIH Grants NS-07078, NS-14162 and NEY-07055].
EBSELEN PREVENTS THE SYSTEMIC ENDOTOXIN-INDUCED RELEASE OF TNF~ AND PROTECTS IN VIVO AGAINST TNF Albrecht Wendel and Gisa Tiegs Faculty of B i o l o g y , U n i v e r s i t y of Konstanz, D-7750 Konstanz, F.R.G.
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Mice pretreated w i t h 700 mg/kg galactosamine and 5 ~ / k g endotoxin, or a l t e r n a t i v e l y w i t h 15 ~g/kg rmu TNF~ instead of endotoxin developed a severe f u l m i n a n t h e p a t i t i s w i t h i n 9 hours. In previous studies we provided evidence t h a t endotoxin adm i n i s t r a t i o n leads to TNF release which in turn represents a f i n a l t o x i c mediator (1). The e f f i c a c y of ebselen as a p r o t e c t a n t in t h i s l i v e r i n j u r y model has been a t t r i b u t e d to i t s a n t i - i n f l a m m a t o r y pr p e r t i e s , e s p e c i a l l y the a b i l i t y to i n h i b i t l e u k o t r i e n e synthesis (2). This study shows t h a t mice protected by p.o. pretreatment with 600 mg/kg ebselen did not release detectable amounts of TNF~ i n t o the c i r c u l a t i o n w i t h i n the next hour a f t e r a d m i n i s t r a t i o n of end o t o x i n . Moreover, ebselen was shown to p r o t e c t as well in the galactosamine/TNF~-induced i n j u r y . These f i n d i n g s suggest t h a t ebselen might be a useful drug in septic shock. 1) Tiegs, G., Wolter, M., and Wendel, A. (1989) Biochem. Pharmacol. 34, 627-631. 2) Tiegs, G. and Wende~ A. (1986) Biochem. Pharmacol. 35, 2115-2118.
TAUR1NE: PROTECTION FROM HYPOHALOUS ANIONS Charles E. Wright, Costas Ioannides, and Bruce M. Gillam Department of Developmental Biochemistry, N. Y. S. Institute for Basic Research, Staten Island, NY 10314, USA
Taurine is the major amino acid found in most animal cells. Reported concentrations (20 to < 140 mM) far exceed those of any other amino containing compound. Such amounts may be underestimated since 60-80% of cellular taurine is released during isolation. It is well documented that taurine reacts with hypohalous anions (HOX-) to form N-halogenated taurine (NXT). This reduces the oxidizing potential of HOX" and sequesters this oxidizing anion into a more stable lipophobic molecule. The significance of taurine as a natural antioxidant and NXT as a modulator of oxidant production has not been well recognized. In vitro experiments using cultured and/or isolated cells were used to examine the antioxidant properties of taurine and the biochemical effects of NXT. Compared with haloamine and other amino acid haloamines, NXT was found to be nontoxic at high concentrations. In fact, taurine would reduce the toxicity of all other haloamines. Rapid halide exchange was noted between the haloamine and taurine to form NXT. In competitive reactions, taurine reacted with HOX- and haloamines 5-6 times faster than the ammonium cation. The reaction had equal stoichometry, and exchange may be due to the anchimeric assistance of the sulfonate anion. NXT exposure to the oxidant metabolizing enzymes CAT, GPX, SOD, MPO, and XO resulted in covalent binding of 14[C]-taurine to the enzyme In all cases binding reduced enzyme hydrophobicity and activity. Because of its high cellular concentrations and its ability to rapidly compete with other amino groups for HOX', taurine is the prevalent antioxidant for the protection and modulation of ceils generating or exposed to oxidized halogens.
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