M-lamlnin is a major member of the laminin family of basement membrane proteins. It is prominently expressed in striated muscle and peripheral nerve. M-laminin is deficient in patients with the autosomal recessive FuLkuyama congenital muscular dystrophy but is normal in patients with the sex-linked Duchenne and Becker muscular dystrophies. We have examined M-laminin expression in mice with autosomal recessive muscular dystrophy caused by the mutation dy. The heavy chain of M-laminin was undetectable in skeletal muscle, heart muscle, and peripheral nerve by immunofluorescence and immunoblotting in homozygous dystrophic dy/dy mice but was normal in heterozygous and wild-type nondystrophic mice. Immunofluorescence confirmed the presence of other major basement membrane proteins in the dystrophic mice. Very low levels of M-laminin heavy chain mRNA were detected by Northern blotting of muscle and heart tissue from dy/dy mice, suggesting that M-laminin heavy-chain mRNA may be produced at very low levels or is unstable. Information about the chromosomal localization of the M heavy-chain in human and mouse suggests that a mutation in the M-chain gene causes the muscular dystrophy in dy/dy mice. The dy mouse may provide a model for autosomal muscular dystrophies in humans and facilitate studies of functions of M-laminin.

An extensive body of research on the structural properties of cytochrome P450 enzymes has established that these proteins possess a b-type heme prosthetic group which is noncovalently bound at the active site. Coordinate, electrostatic, and hydrogen bond interactions between the protein backbone and heme functional groups are readily overcome upon mild acid treatment of the enzyme, which releases free heme from the protein. In the present study, we have used a combination of HPLC, LC/ESI-MS, and SDS-PAGE techniques to demonstrate that members of the mammalian CYP4B, CYP4F, and CYP4A subfamilies bind their heme in an unusually tight manner. HPLC chromatography of CYP4B1 on a POROS R2 column under mild acidic conditions caused dissociation of less than one-third of the heme from the protein. Moreover, heme was not substantially removed from CYP4B1 under electrospray or electrophoresis conditions that readily release the prosthetic group from other non-CYP4 P450 isoforms. This was evidenced by an intact protein mass value of 59,217 +/- 3 amu for CYP4B1 (i.e., apoprotein plus heme) and extensive staining of this approximately 60 kDa protein with tetramethylbenzidine/H(2)O(2) following SDS-PAGE. In addition, treatment of CYP4B1, CYP4F3, and CYP4A5/7 with strong base generated a new, chromatographically distinct, polar heme species with a mass of 632.3 amu rather than 616.2 amu. This mass shift is indicative of the incorporation of an oxygen atom into the heme nucleus and is consistent with the presence of a novel covalent ester linkage between the protein backbone of the CYP4 family of mammalian P450s and their heme catalytic center.

Organization of leukotriene and prostaglandin synthesis As described in the introduction to this Perspective series (1), signaling by arachidonic acid represents a paradigm for the use of oxygen in the transmission of information. At the same time, arachidonic acid signaling can also contribute to the propagation of cellular damage. This duality is typified by a signaling cascade that (a) prevents the activation of 5-lipoxygenase (5-LO) in resting cells and (b) results in the formation and release of leukotrienes (LTs), which requires the sequential activation and interaction of at least eight different proteins. In fact, all lipoxygenases require membrane translocation to exert activity. In the case of the formation of COX products, particularly prostaglandin E 2 (PGE 2 ) and PGD 2 , humans have evolved two sets of biosynthetic enzymes that differ not only in their cell-and tissue-specific localization, but also in their subcellular localization and requirement for reduced glutathione, a cellular defense against oxidative damage. This review will focus on three aspects of arachidonic acid biology. First, the compartmentalization and organization of eicosanoid synthesis, specifically LTs and PGs, will be discussed. This will illustrate the elaborate mechanisms that keep unwanted lipoxygenation at arm's length and also show that the enzymes such as glutathione-Stransferases, epoxide hydrolases, and carrier proteins that are commonly thought of as biosynthetic also belong to families that are generally considered to play a role in detoxification. Second, the potential cellular oxidative damage that is produced as a by-product of the use of oxygen and lipid substrates is examined. Finally, mechanisms that are used to amplify signaling diversity from a core of LTs and PGs are discussed. The role of leukotrienes C 4 and D 4 in diseaseLTs are the products of the 5-LO pathway of arachidonic acid metabolism (Figure 1). The initial interest in LTs followed largely from their association with the pathogenesis of asthma (2). LTC 4 , identified as the parent molecule of the sulfidopeptide LTs, is generated from eosinophils and mast cells in large amounts, and also from monocytes and macrophages (2-7). However, it is not formed by polymorphonuclear leukocytes (2-5). When released from cells, LTC 4 is converted to LTD 4 (2-7), and both exert their biological effects via G protein-coupled receptors (8, 9). LTD 4 and LTC 4 cause the constriction of smooth muscle, and the clinical correlate is bronchial smooth muscle constriction in asthma (2, 10, 11). The role of LTD 4 as a major contributor to asthmatic bronchospasm has been firmly established, and aerosolized LTD 4 and LTC 4 cause bronchospasm when taken by inhaler (10, 11). The metabolic product of LTD 4 , 5(S)-hydroxy,6(R)-cysteinyl-7,9,11-trans,14-cis-eicosatetraenoic acid, (LTE 4 ), has been found at high levels in the serum and urine of patients with asthma and allergic rhinitis (12). When cold-induced bronchoconstriction, allergen-induced asthma, and exercise-induced as...