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The mouse cell line MOPC 315 is an IgA (lambda II)-producing myeloma. We have studied a derivative of MOPC 315 that secretes normal lambda II chains but no heavy chain. This derivative, MOPC 315-26, was found to contain a rearranged lambda I gene in addition to a rearranged lambda II gene. The rearranged lambda I gene was cloned into bacteriophage lambda DNA and its structure was studied. The lambda I gene was found to have arisen by an aberrant recombination event that resulted in a single base insertion at the site of V-J region joining. In addition, the gene contained numerous point mutations in the vicinity of the junction of the V and J regions. Two point mutations occurred in the donor splice sequence normally used for the removal of the intron between the J and C regions, suggesting that the RNA synthesized from the aberrantly rearranged lambda I gene would be unable to undergo proper RNA splicing.

In vitro studies examining the metabolic transformation of prostaglandin D2 (PGD2) by human liver were conducted. PGD2 was found to be converted by a NADPH-dependent enzyme in the 100,000 X g supernatant of human liver exclusively to a single more polar compound that had a mass spectrum essentially the same as that of prostaglandin F2 alpha (PGF2 alpha). However, this compound could be chromatographically separated from PGF2 alpha and failed to form a butylboronate derivative. The structure of this compound was established as 9 alpha, 11 beta-(15S)-trihydroxyprosta-(5Z, 13E)-dien-1-oic acid (9 alpha, 11 beta-PGF2) by comparison of its chromatographic and mass spectral characteristics with authentic 9 alpha, 11 beta-PGF2. This compound was found to be biologically active by demonstrating increases in blood pressure in rats in a dose-related fashion following intravenous administration. By using a mass spectrometric assay, levels of this compound in plasma and urine from a normal volunteer were 6 pg/ml and 982 ng/24 hr, respectively. In a patient with systemic mastocytosis associated with overproduction of PGD2, urinary excretion of 9 alpha, 11 beta-PGF 2 was 6634 ng/24 hr and a circulating plasma level as high as 490 ng/ml was found during a severe episode of systemic mast cell activation. 9 alpha, 11 beta-PGF2 is structurally a unique prostaglandin, is enzymatically formed, is produced in vivo in humans, and is biologically active.

Prostaglandin D2 (PGD2) was recently found to be stereospecifically converted to the compound (5Z,13E)-(15S)-9 alpha,11 beta,15-trihydroxyprosta-5,13-dien-1-oic acid (9 alpha,11 beta-PGF2) by a human liver cytosolic NADPH-dependent 11-ketoreductase enzyme. Because PGD2 is a potent bronchoconstrictor and is released into bronchoalveolar lavage fluid after allergen stimulation in patients with allergic asthma, the ability of human lung to metabolize PGD2 to 9 alpha,11 beta-PGF2 and the contractile effects of 9 alpha,11 beta-PGF2 on human bronchial smooth muscle were investigated. The 100,000 X g supernatant of human lung converted PGD2 in the presence of an NADPH-generating system stereospecifically to 9 alpha,11 beta-PGF2 at a rate of 3.46 +/- 0.94 pmol per min per mg of protein. 9 alpha,11 beta-PGF2 was found to contract human bronchial rings in a dose-dependent fashion with a potency virtually identical with that of both PGD2 and PGF2 alpha, known potent bronchial constrictors. PGD2 was found to be a very poor substrate for human lung 15-hydroxyprostaglandin dehydrogenases and to be preferentially metabolized by lung to 9 alpha,11 beta-PGF2. 9 alpha,11 beta-PGF2 was also found to be a very poor substrate for the lung 15-hydroxyprostaglandin dehydrogenases. Thus, once formed, 9 alpha,11 beta-PGF2 would not be expected to be rapidly inactivated in situ by these metabolic enzymes. These results suggest that 9 alpha,11 beta-PGF2 may participate along with other putative mediators in the pulmonary allergic response in humans.

Increasing attention has focused on the role of free radicals derived from oxygen in the pathophysiology of a wide variety of disorders. One of the well-recognized targets of free radical-induced injury is peroxidation of lipids. Using a variety of approaches, we have found that a series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase mechanism involving free radical-catalyzed peroxidation of arachidonic acid. Levels of these compounds in normal human plasma and urine range from 5 to 40 pg/ml and 500 to 4000 pg/mg of creatinine, respectively. In rats, their formation was found to increase as much as 200-fold in association with marked free radical-catalyzed lipid peroxidation induced by administration of CCl4 and diquat. To explore whether these prostanoids can exert biological activity, the effects of one of the compounds formed by this mechanism, 8-epi-prostaglandin F2 alpha, was examined in the kidney in the rat. Infusion of 8-epi-prostaglandin F2 alpha into a peripheral vein (5 micrograms/kg per min) or intrarenally (0.5-2.0 micrograms/kg per min) resulted in marked parallel reductions in renal blood flow and glomerular filtration rate. That the formation of these prostanoids is catalyzed by free radicals and that they can exert potent biological activity suggest that these prostanoids may participate as pathophysiological mediators in oxidant injury. Quantification of these compounds may also provide a noninvasive approach to assess oxidant status in humans. That the formation of these prostanoids occurs independent of the catalytic activity of the cyclooxygenase enzyme suggests that there may be limitations at times regarding the reliability of the use of cyclooxygenase inhibitors to assess the role of prostaglandins in certain pathophysiological processes.

A sequence of epithelial cell proliferation, allocation to four principal lineages, migration-associated differentiation, and cell loss occurs along the crypt-villus axis of the mouse intestine. The sequence is completed in a few days and is recapitulated throughout the life-span of the animal. We have used an intestine-specific fatty acid binding protein gene, Fabpi, as a model for studying regulation of gene expression in this unique developmental system. Promoter mapping studies in transgenic mice identified a 20-bp cis-acting element (5'-AGGTGGAAGCCATCACACTT-3') that binds small intestinal nuclear proteins and participates in the control of Fabpi's cephalocaudal, differentiation-dependent, and cell lineage-specific patterns of expression. Immunocytochemical studies using confocal and electron microscopy indicate that it does so by acting as a suppressor of gene expression in the distal small intestine/colon, as a suppressor of gene activation in proliferating and nonproliferating cells located in the crypts of Lieberkühn, and as a suppressor of expression in the growth factor and defensin-producing Paneth cell lineage. The 20-bp domain has no obvious sequence similarities to known transcription factor binding sites. The three functions modulated by this compact element represent the types of functions required to establish and maintain the intestine's remarkably complex spatial patterns of gene expression. The transgenes described in this report also appear to be useful in characterizing the crypt's stem cell hierarchy.

We recently reported the discovery of a series of bioactive prostaglandin F2-like compounds (F2-isoprostanes) that are produced in vivo by free radical-catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase enzyme. Inasmuch as phospholipids readily undergo peroxidation, we examined the possibility that F2-isoprostanes may be formed in situ on phospholipids. Initial support for this hypothesis was obtained by the finding that levels of free F2-isoprostanes measured after hydrolysis of lipids extracted from livers of rats treated with CCl4 to induce lipid peroxidation were more than 100-fold higher than levels in untreated animals. Further, increased levels of lipid-associated F2-isoprostanes in livers of CCl4-treated rats preceded the appearance of free compounds in the circulation, suggesting that the free compounds arose from hydrolysis of peroxidized lipids. This concept was supported by demonstrating that free F2-isoprostanes were released after incubation of lipid extracts with bee venom phospholipase A2 in vitro. When these lipid extracts were analyzed by HPLC, fractions that yielded large quantities of free F2-isoprostanes after hydrolysis eluted at a much more polar retention volume than nonoxidized phosphatidylcholine. Analysis of these polar lipids by fast atom bombardment mass spectrometry established that they were F2-isoprostane-containing species of phosphatidylcholine. Thus, unlike cyclooxygenase-derived prostanoids, F2-isoprostanes are initially formed in situ on phospholipids, from which they are subsequently released preformed, presumably by phospholipases. Molecular modeling of F2-isoprostane-containing phospholipids reveals them to be remarkably distorted molecules. Thus, the formation of these phospholipid species in lipid bilayers may contribute in an important way to alterations in fluidity and integrity of cellular membranes, well-known sequelae of oxidant injury.

A prostaglandin F (PGF) synthase was recently purified from bovine lung that catalyzed the reduction of both PGH2 and PGD2 but at different active sites on the enzyme. In view of the recent finding that PGD2 is stereospecifically reduced to a unique biologically active compound, (5Z, 13E)-(15S)-9 alpha, 11 beta, 15-trihydroxyprosta-5,13-dien-1-oic acid (9 alpha,11 beta-PGF2 or 11-epi-PGF2 alpha), by a human liver cytosolic enzyme, detailed characterization of the products formed from PGH2 and PGD2 by the bovine lung PGF synthase was carried out. Chromatographic characteristics of the products formed and stereochemical analysis procedures using mass spectrometry indicated that the enzyme stereospecifically reduces PGH2 to PGF2 alpha, whereas PGD2 is stereospecifically converted to 9 alpha,11 beta-PGF2. The finding that this enzyme catalyzes the formation of both C-11 hydroxy epimers of PGF2, albeit from different substrates, is of interest in that these two compounds may exert different biological actions.