Anti-mitochondrial antibodies in patients with dilated cardiomyopathy (anti-M7) are directed against flavoenzymes with covalently bound FAD

Anti-mitochondrial antibodies (anti-M7) in sera from patients with dilated cardiomyopathy and myocarditis recognize, besides mitochondrial antigens, bacterial sarcosine dehydrogenase. The common target antigen was identified as the covalently bound FAD of mitochondrial and bacterial flavoenzymes. Thus, anti-M7-positive serum reacted on Western blots exclusively with covalently flavinylated enzymes. The antigenic specificity of anti-M7 sera was reproduced by an antiserum raised in rabbits with 6-hydroxy- D -nicotine oxidase. The heart mitochondrial membrane antigen recognized by anti-M7 serum was identified as the flavoprotein subunit of succinate dehydrogenase, the antigens in rat liver mitochondrial matrix as the flavoenzymes dimethylglycine dehydrogenase and sarcosine dehydrogenase. Anti-M7 serum contained a specific anti-flavoenzyme antibody fraction. Nanomolar concentrations of FAD and riboflavin inhibited the immune reaction on Western blots and in ELISA, and incubation with FAD-agarose depleted the anti-M7 activity of the serum. N-terminally deleted dimethylglycine dehydrogenase proteins were only immunoprecipitated by anti-M7 sera when the FAD was covalently incorporated. An affinity constant (K_D) of 10^?8 M was established for the anti-flavoenzyme antibodies by competitive ELISA. Of patients with cardiomyopathy and myocarditis, 36% and 25%, respectively, were anti-flavoenzyme-positive by Western blot and ELISA, but only two of 15 patients with other heart diseases and none of 50 healthy controls.     

Anti-mitochondrial flavoprotein autoantibodies of patients with myocarditis and dilated cardiomyopathy (anti-M7): interaction with flavin-carrying proteins, effect of vitamin B2 and epitope mapping

Vitamin B2 and flavin cofactors are transported tightly bound to immunoglobulin in human serum. We reasoned that anti-mitochondrial flavoprotein autoantibodies (alpha Fp-AB) present in the serum of patients with myocarditis and cardiomyopathy of unknown aetiology may form immunoglobulin aggregates with these serum proteins. However, immunodiffusion and Western blot assays demonstrated that the flavin-carrying proteins were not recognized by alpha Fp-AB. Apparently the flavin moiety in the native protein conformation was inaccessible to alpha Fp-AB. This conclusion was supported by the absence of an immunoreaction between the riboflavin-binding protein from egg white and alpha FP-AB. Intravenous application of vitamin B2 to rabbits immunized with 6-hydroxy-D-nicotine oxidase, a bacterial protein carrying covalently attached FAD, did not neutralize alpha Fp-AB which had been raised in the serum of the animals. FAD-carrying peptides generated from 6-hydroxy-D-nicotine oxidase by trypsin and chymotrypsin treatment were not recognized by the alpha Fp-AB, but those generated by endopeptidase Lys were. This demonstrates that the epitope recognized by alpha Fp-AB comprises, besides the flavin moiety, protein secondary structure elements.     

High dietary iron concentrations enhance the formation of cholesterol oxidation products in the liver of adult rats fed salmon oil with minimal effects on antioxidant status

The aim of this study was to investigate the effect of high dietary iron concentrations on the antioxidant status of rats fed two different types of fat. Four groups of male adult Sprague-Dawley rats were fed diets with adequate (50 mg iron supplemented per kg diet) or high (500 mg iron supplemented per kg diet) iron concentrations with either lard or salmon oil as dietary fat at 100 g/kg for 12 wk. The antioxidant status of the rats was profoundly influenced by the type of fat. Rats fed salmon oil diets had higher concentrations of thiobarbituric acid-reactive substances (TBARS) (P < 0.001), various cholesterol oxidation products (COP) (P < 0.001), total and oxidized glutathione (P < 0.05) and a lower concentration of alpha-tocopherol (P < 0.05) in liver and plasma than rats fed lard diets. The iron concentration of the diet did not influence the concentrations of TBARS, the activities of superoxide dismutase and glutathione peroxidase or the concentration of alpha-tocopherol in plasma or liver. The activity of catalase (P < 0.01) and the concentrations of total, oxidized and reduced glutathione (P < 0.05) in liver were slightly but significantly higher in rats fed high iron diets than in rats fed adequate iron diets, irrespective of the dietary fat. Rats fed the high iron diets with salmon oil, moreover, had higher concentrations of various COP in the liver (P < 0.001) than rats fed adequate iron diets with salmon oil. These results suggest that feeding a high iron diet does not generally affect the antioxidant status of rats but enhances the formation of COP, particularly if the diet is rich in polyunsaturated fatty acids.     

Effects of peroxidation products in thermoxidised dietary oil in female rats during rearing, pregnancy and lactation on their reproductive performance and the antioxidative status of their offspring

The present study was performed to investigate whether lipid peroxidation products in thermoxidised dietary oil fed during rearing, pregnancy and lactation influences the reproductive performance of female rats and the antioxidant status of their offspring. Twenty-four female rats were divided into two groups at 4 weeks of age. They were fed diets containing fresh or oxidised oil (the latter prepared by heating at a temperature of 50 degrees C for 16 d) for 14 weeks. At the age of 12 weeks female rats were mated. The number of total pups and pups born alive was not different between both groups. However, individual pups and litters of dams fed oxidised oil were lighter at birth and gained less weight during the suckling period than those of dams fed fresh oil (P < 0.05). Pups of dams fed oxidised oil contained less protein and more fat in their carcasses than those of dams fed fresh oil (P < 0.05). The milk of dams fed oxidised oil had a lower concentration of triacylglycerols and a lower energy content than that of dams fed the fresh oil (P < 0.05). The pups of dams fed oxidised oil had higher concentrations of lipid peroxidation products in the liver at birth and day 19 of lactation than those of dams fed fresh oil (P < 0.05). In conclusion, the present study shows that feeding oxidised oil with a high concentration of lipid peroxidation products to female rats during rearing, pregnancy and lactation influences the development and antioxidant status of fetus and suckling pups.     

Influence of enhancers on the absorption and on the pharmacokinetics of cefodizime using in-vitro and in-vivo models

In the development of novel antibiotics, more and more compounds have been found that cannot be absorbed orally and, therefore, must be administered intravenously or intramuscularly. Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability has received much attention in pharmaceutical research. Cefodizime, a novel third-generation cephalosporin with significant advantages in the parenteral treatment of common infections, was used as a model drug. Cefodizime behaves as a highly hydrophilic compound, as shown from its extremely low partition coefficient. The effect of cationic absorption enhancers (hexadecyldimethylbenzylammonium chloride, N-hexadecylpyridinium bromide, dodecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide) on the lipophilicity of cefodizime was investigated by means of the n-octanol/water system. Results showed that the counter-ions had a positive influence on the solubility of cefodizime. These results on partitioning coefficients in the n-octanol/buffer system were confirmed using an in-vitro transport model with artificial and biological membranes (Caco-2-cells). Furthermore, the physiological compatibility of the absorption enhancers was investigated using the active D-glucose transport. The pharmacokinetic profile of cefodizime was evaluated in rabbits after intraduodenal administration with and without an absorption enhancer.     

Effects of a dietary oxidized fat on cholesterol in plasma and lipoproteins and the susceptibility of low-density lipoproteins to lipid peroxidation in guinea pigs fed diets with different concentrations of vitamins E and C

To investigate the effect of a dietary oxidized fat on the concentrations of cholesterol in liver, plasma, and lipoproteins and the susceptibility of low-density lipoproteins (LDL) to lipid peroxidation, and to explore the effects of vitamins E and C, male guinea pigs were divided into five groups. Four groups were fed diets with an oxidized fat supplemented with 35 or 175 mg alpha-tocopherol equivalents/kg and 300 or 1000 mg of vitamin C/kg for 29 days. One group, used as a control, was fed the same basal diet with fresh fat with 35 mg alpha-tocopherol equivalents/kg and 300 mg of vitamin C/kg. Guinea pigs fed the oxidized-fat diets, irrespective of dietary vitamin E and C concentrations, had significantly lower concentrations of total cholesterol in the liver and a lower concentration of cholesterol in LDL than the control animals fed the fresh fat. According to the lag time before onset of lipid peroxidation, LDL of guinea pigs fed the oxidized-fat diet with 35 mg alpha-tocopherol equivalents and 300 mg vitamin C/kg were significantly more susceptible to copper-induced lipid peroxidation than those of guinea pigs fed the fresh fat diet. Within the groups fed the oxidized fat diets, increasing the dietary vitamin E concentration from 35 to 175 mg/kg significantly (p < 0.05) and increasing the dietary vitamin C concentration from 300 to 1000 mg/kg in tendency (p < 0.10) reduced the susceptibility of LDL to oxidation. LDL of guinea pigs fed the oxidized fat diets with 175 mg alpha-tocopherol equivalents/kg were even more resistant to oxidation than LDL of guinea pigs fed the fresh diet. In conclusion, the study shows that dietary oxidized fat influences the cholesterol metabolism and the susceptibility of LDL to lipid peroxidation; the latter can be modified by dietary vitamins E and C.     

Homologous gene clusters of nicotine catabolism, including a new ω-amidase for α-ketoglutaramate, in species of three genera of Gram-positive bacteria

Gram-positive soil bacteria Arthrobacter nicotinovorans, Nocardioides sp. JS614 and Rhodococcus opacus were shown to contain similarly organized clusters of homologous genes for nicotine catabolism. An uncharacterized gene of a predicted nitrilase within these gene clusters was cloned from A. nicotinovorans and biochemical data unexpectedly showed that the protein exhibited ω-amidase activity toward α-ketoglutaramate. Structural modelling of the protein suggested the presence of the catalytic triad Cys-Glu-Lys, characteristic of this class of enzymes, and supported α-ketoglutaramate as substrate. A-ketoglutaramate could be generated by hydrolytic cleavage of the C-N bond of the trihydroxypyridine ring produced by nicotine catabolism in these bacteria. This ω-amidase, together with glutamate dehydrogenase, may form a physiologically relevant enzyme couple, leading to transformation of metabolically inert α-ketoglutaramate derived from trihydroxypyridine into glutamate, a central compound of nitrogen metabolism.     

Interaction of 31 beta-lactam antibiotics with the H+/peptide symporter PEPT2: analysis of affinity constants and comparison with PEPT1.

The activity of the renal peptide transporters PEPT2 and PEPT1 determines-among other factors such as metabolic stability in liver and plasma-the circulatory half-life of penicillins and cephalosporins during therapy. This study was initiated to examine systematically the interaction of beta-lactam antibiotics with PEPT2. Interaction of 31 cephalosporins and penicillins with the carrier protein was characterized by measuring their ability to inhibit the uptake of [(14)C]Gly-Sar into renal SKPT cells. Cefadroxil, cefaclor, cyclacillin, cephradine, cephalexin and moxalactam were recognized by PEPT2 with very high affinity comparable to that of natural dipeptides (K(i)=3-100microM). Ceftibuten, dicloxacillin, amoxicillin, metampicillin, cloxacillin, ampicillin, cefixime, cefamandole, oxacillin and cefmetazole interacted with PEPT2 with medium affinity (K(i)=0.1-5mM). For the other beta-lactam antibiotics studied interaction was very low or not measurable (K(i)>5mM). The affinity constants of beta-lactam antibiotics at rPEPT2 and hPEPT1 are significantly correlated, but the rank orders are not identical. Decisive differences between PEPT1 and PEPT2 recognition of the N-terminal part of the compounds became evident. Moreover, this large data set of affinity constants of beta-lactam antibiotics will be useful for structure-transport (binding) analyses of PEPT2.