2(3)tert-Butyl4hydroxyanisole (BHA) is one of several widely used antioxidant food additives that protect against chemical carcinogenesis and toxicity. The present report concerns the enhancement of dicoumarolinhibited NAD(P)HL quinone reductase [NAD(P)H dehydrogenase (quinone); NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.21 activity in mouse tissues in response to dietary administration of BHA. Cytosolic quinone reductase specific activity was increased significantly in 10 of 15 tissues examined from BHA-fed mice. The greatest proportionate increase, to 10 times control levels, was observed in liver. BHA also increased the quinone reductase activities of kidney, lung, and the mucosa of the upper small intestine severalfold. The increases of quinone reductase activities in liver and digestive tissues in response to BHA were comparable to the increases previously observed in glutathione S-transferase (EC 2.5.1.18) and epoxide hydratase (EC 3.3.2.3) activities. Quinones are among the toxic products of oxidative metabolism of aromatic hydrocarbons. NAD(P)H:quinone reductase exhibits broad specificity for structurally diverse hydrophobic quinones an may facilitate the microsomal metabolism of quinones to readily excreted conjugates. The protectie effects of BHA appear to be due, at least in part, to the ability of this antioxidant to increase the activities in rodent tissues of several enzymes involved in the nonoxidative metabolism of a wide variety of xenobiotics.The widely used antioxidant food additive 2(3)-tert-butyl-4-hydroxyanisole (BHA) has a number of interesting and potentially important pharmacological properties. BHA Administration of BHA to mice has little or no effect on numerous other monooxygenase activities of hepatic microsomes (11,15,17), although ring hydroxylation of aniline is markedly enhanced (11). Liver microsomes from BHA-fed mice also produce altered patterns of metabolites from benzo[a]pyrene in vitro. Lam and Wattenberg (18) observed decreased epoxidation and increased formation of 3-hydroxybenzo[a]pyrene relative to controls, and these changes were accompanied by a 50% decrease in the binding of metabolites to DNA (19,20).The NADH-and NADPH-linked quinone oxidoreductase here called quinone reductase has also been designated variously as menadione reductase, DT-diaphorase, NAD(P)H dehydrogenase, and vitamin K reductase (21). Recent studies have shown that rat liver cytosol azoreductase activity for methyl red also reflects the action of quinone reductase (22, 23). Unusual features of this flavoprotein enzyme include: (i) comparable reaction with NADH and NADPH (24-26); (ti) potent and specific inhibition, in competition with reduced nicotinamide nucleotides, by dicoumarol and related vitamin K antagonists (24, 27); (iii) reversible stimulation of activity of the soluble cytosolic quinone reductase by serum albumin, neutral phospholipids, and a number of detergents (24,26,27); and (iv) broad specificity for a variety of hydrophobic quinones, including benzoquinones, na...
Using a family of synthetic tetradecamer oligonucleotides as a primer for cDNA synthesis and a second family of tetradecamers as a hybridization probe, we have prepared and isolated a cDNA clone of mouse myelin basic protein (MBP). The clone, pNZ111, corresponds to the region of the mRNA that codes for an amino acid sequence present in all four major forms of MBP (2)(3)(4)(5). Large amounts of the membrane are produced relative to the size of the myelin-forming cell. The process involves the synthesis of myelin-specific proteins followed by their integration into the growing membrane. During early myelinogenesis, membranes are formed that are loosely whorled around the axon. As myelin maturation proceeds, compaction of these membranes occurs to form the tight multilamellar structure characteristic of myelin.The myelin basic protein (MBP) constitutes about 33% of the protein of the myelin sheath in the central nervous system and 1-10% in the peripheral (6, 7). In most species, the predominant form of the MBP has a molecular mass of 18.5 kilodaltons (kDa), and the primary sequence of this protein is well conserved in animals as distantly related as the chicken, human, and rat (8-10). Two major forms of MBP predominate in rats and mice: one of these corresponds to the single major protein found in other species (18.5 kDa) and the other has a molecular mass of 14 kDa. In rats, the 14-kDa MBP is identical in sequence to the 18.5-kDa MBP, except for an internal deletion of residues 118-158 [numbered according to Martenson (11)] near the carboxyl terminus of the protein (10, 12). Two quantitatively minor forms of MBP have been found in rats and mice-a 21.5-kDa protein that is presumably identical to the 18.5-kDa MBP, except for the inclusion of a 25-30 amino acid sequence in the NH2-terminal half of the molecule and a 17-kDa MBP that bears the same relationship to the 14-kDa MBP (13).Myelination occurs postnatally in mouse brain beginning 8-10 days postpartum and continuing actively for 7-10 wk, with the maximal rate of myelin deposition occurring at about 18 days (4). Maximal synthesis of the 18.5-kDa and 14-kDa MBPs occurs at 18 days in vivo and coincides closely with the peak of myelin synthesis (14). During myelin maturation in the mouse, the proportions of the four MBPs in the membrane change (15-17) and this may be due to alterations in the relative rates of synthesis of the four proteins with age. With maturation, the proportion of the two minor MBPs (i.e., the 21.5 kDa and 17 kDa in myelin falls relative to the 18.5-kDa proteins and the 14-kDa/18.5-kDa MBP ratio increases dramatically. This latter change has been correlated with the relative rate of synthesis of these two proteins in vivo (14). Recently, Carson et al. (18) have identified trace amounts of larger forms of MBP that appear during the early stages of myelination. Identification of these larger forms has depended on immunoreagents and the relationships of the amino acid sequences to the major MBPs have, as yet, not been determined.We have r...
Northern blot and "dot" blot analyses using a myelin basic protein (MBP) specific cDNA probe and in vitro translation techniques were utilized to estimate the relative levels of myelin basic protein messenger RNA (mRNA) in the brains of C57BL/6J control mice, three dysmyelinating mutants (qk/qk, jp/Y, and shi/shi), and three heterozygote controls (qk/+, jp/+, and shi+) during early postnatal development. In general, the MBP mRNA levels measured directly by Northern blot and "dot" blot analyses correlated well with the indirect in vitro translation measurements. The Northern blots indicated that the size of MBP mRNAs in quaking and jimpy brain polysomes appeared to be similar to controls. Very low levels of MBP mRNAs were observed in shi/shi brain polyribosomes throughout early postnatal development. Compared to C57BL/6J controls, accumulation of MBP mRNAs in qk/qk and qk/+ brain polyribosomes was delayed by several days. That is, whereas MBP mRNA levels were below normal between 12 and 18 days, normal levels of message had accumulated in both qk/qk and qk/+ brain polyribosomes by 21 days. Furthermore, normal levels of MBP mRNAs were observed to be maintained until at least 27 days. MBP mRNA levels remained well below control levels in jp/Y brain polyribosomes throughout early postnatal development. The levels did, however, fluctuate slightly and peaked at 15 days in both jp/Y and jp/+ brains, 3 days earlier than in normal mice. Thus, it appears that jimpy and quaking mice exhibit developmental patterns of MBP expression different from each other and from C57BL/6J control mice.
GSH transferase isoenzymes of class Mu (two forms), class Pi (one form) and class Alpha (two forms) were purified from liver cytosols of female CD-1 mice pretreated with an anticarcinogenic inducer, 2(3)-t-butyl-4-hydroxyanisole. GSH transferases GT-8.7, GT-8.8a and GT-8.8b, GT-9.0, GT-9.3, GT-10.3 and GT-10.6 contained a minimum of six types of subunits distinguishable by structural, catalytic and immunological characteristics. H.p.l.c. analysis of the subunit compositions of affinity-purified GSH transferases from liver cytosols of induced and non-induced male and female CD-1 mice showed that two anticarcinogenic compounds, 2(3)-t-butyl-4-hydroxyanisole and bisethylxanthogen, differed markedly in their specificities as inducers of GSH transferase.
Mice ranging in age from 16 to 44 days were injected intracerebrally with 3H-leucine, and incorporation into total brain proteolipids and the myelin proteolipid protein was measured. All proteolipids were isolated from whole brain by ether precipitation and separated into their individual components by SDS polyacrylamide gel electrophoresis. Two major proteolipids with apparent molecular weights of 20,700 and 25,400 were observed in these preparations, and their proportion increased over the developmental period examined. A Ferguson plot analysis comparing these proteins with those of isolated myelin showed that the 25,400-dalton proteolipid component from whole brain was the myelin proteolipid protein. Rates of incorporation of 3H-leucine into total brain proteolipids peaked at 22 days of age. Synthesis of the myelin proteolipid protein increased rapidly to a maximum value at 22 days and decreased rather slowly until at 44 days it was about 83% of its maximum rate of synthesis. The data indicate that the developmental pattern of synthesis of the myelin proteolipid protein is unlike that of the myelin basic proteins. Synthesis of the major myelin proteins is developmentally asynchronous in that peak synthesis of the myelin proteolipid appears to occur several days later than the basic proteins. In addition, it maintains its maximum rate of synthesis over a longer period of time than do the basic proteins.
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