The isoprostanes: Novel prostaglandin-like products of the free radical-catalyzed peroxidation of arachidonic acid

The isoprostanes (IsoPs) are a unique series of prostaglandin-like compounds formed in vivo from the free radical-catalyzed peroxidation of arachidonic acid. This review summarizes our current knowledge regarding these compounds. Novel aspects of the biochemistry and bioactivity of IsoPs are detailed and methods by which these compounds are analyzed are discussed. A considerable portion of this review deals with the utility of measuring IsoPs as markers of oxidant injury in human diseases particularly in association with risk factors that predispose to atherosclerosis, a condition in which excessive oxidative stress has been causally implicated.     

Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (+/-)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1

We have used V79MZ hamster lung fibroblasts stably transfected with human cytochrome P450-1A1 (hCYP1A1; cell line designated V79MZh1A1) or P450-1B1 (hCYP1B1; cell line designated V79MZh1B1) alone, or in combination with human glutathione-S-transferase (GST) alpha-1 (hGSTA1), in order to examine GST protection against cytotoxicity and mutagenicity of dibenzo[a,l]pyrene (DBP) and the intermediate dihydrodiol metabolite (+/-)-DBP-11,12-dihydrodiol (DBPD). At comparable expression levels of hCYP1A1 and hCYP1B1, both DBP and DBPD were more cytotoxic in V79MZ1A1 (IC(50)=2.7 and 0.7nM, respectively) than in V79MZh1B1 (IC(50)=6.0 and 4.8nM, respectively). In contrast, both DBP and DBPD were two- to four-fold more mutagenic in V79MZh1B1 than in V79MZ1A1. Co-expression of hGSTA1 with hCYP1A1 decreased DBP cytotoxicity two-fold compared to V79MZh1A1 with hCYP1A1 alone, and provided a small, yet still statistically significant, 1.3-fold protection against DBPD. Protection against mutagenicity of these compounds was comparable to that for cytotoxicity in cells expressing hCYP1A1. In V79MZh1B1 cells, co-expression of hGSTA1 conferred up to five-fold protection against DBP cytotoxicity, and up to nine-fold protection against the (+/-)-DBP-dihydrodiol cytotoxicity relative to the cells expressing hCYP1B1 alone. Co-expression of hGSTA1 also reduced mutagenicity of DBP or its dihydrodiol to a lesser extent (1.3-1.8-fold) than the protection against cytotoxicity in cells expressing hCYP1B1. These findings demonstrate that the protective efficacy of hGSTA1 against DBP and DBPD toxicity is variable, depending on the compound or metabolite present, the specific cytochrome P450 isozyme expressed, and the specific cellular damage endpoint examined.     

Dual embryonic origin of the mammalian otic vesicle forming the inner ear

The inner ear and cochleovestibular ganglion (CVG) derive from a specialized region of head ectoderm termed the otic placode. During embryogenesis, the otic placode invaginates into the head to form the otic vesicle (OV), the primordium of the inner ear and CVG. Non-autonomous cell signaling from the hindbrain to the OV is required for inner ear morphogenesis and neurogenesis. In this study, we show that neuroepithelial cells (NECs), including neural crest cells (NCCs), can contribute directly to the OV from the neural tube. Using Wnt1-Cre, Pax3(Cre/+) and Hoxb1(Cre/+) mice to label and fate map cranial NEC lineages, we have demonstrated that cells from the neural tube incorporate into the otic epithelium after otic placode induction has occurred. Pax3(Cre/+) labeled a more extensive population of NEC derivatives in the OV than did Wnt1-Cre. NEC derivatives constitute a significant population of the OV and, moreover, are regionalized specifically to proneurosensory domains. Descendents of Pax3(Cre/+) and Wnt1-Cre labeled cells are localized within sensory epithelia of the saccule, utricle and cochlea throughout development and into adulthood, where they differentiate into hair cells and supporting cells. Some NEC derivatives give rise to neuroblasts in the OV and CVG, in addition to their known contribution to glial cells. This study defines a dual cellular origin of the inner ear from sensory placode ectoderm and NECs, and changes the current paradigm of inner ear neurosensory development.     

alpha -Catenin binds directly to spectrin and facilitates spectrin-membrane assembly in vivo

The anchorage of spectrin to biological membranes is mediated by protein and phosphoinositol phospholipid interactions. In epithelial cells, a nascent spectrin skeleton assembles in regions of cadherin-mediated cell-cell contact, and conversely, cytoskeletal assembly is required to complete the cell-adhesion process. The molecular interactions guiding these processes remain incompletely understood. We have examined the interaction of spectrin with alpha-catenin, a component of the adhesion complex. Spectrin (alphaIIbetaII) and alpha-catenin coprecipitate from extracts of confluent Madin-Darby canine kidney, HT29, and Clone A cells and from solutions of purified spectrin and alpha-catenin in vitro. By surface plasmon resonance and in vitro binding assays, we find that alpha-catenin binds alphaIIbetaII spectrin with an apparent K(d) of approximately 20-100 nm. By gel-overlay assay, alpha-catenin binds recombinant betaII-spectrin peptides that include the first 313 residues of spectrin but not to peptides that lack this region. Similarly, the binding activity of alpha-catenin is fully accounted for in recombinant peptides encompassing the NH(2)-terminal 228 amino acid region of alpha-catenin. An in vivo role for the interaction of spectrin with alpha-catenin is suggested by the impaired membrane assembly of spectrin and its enhanced detergent solubility in Clone A cells that harbor a defective alpha-catenin. Transfection of these cells with wild-type alpha-catenin reestablishes alpha-catenin at the plasma membrane and coincidentally recruits spectrin to the membrane. We propose that ankyrin-independent interactions of modest affinity between alpha-catenin and the amino-terminal domain of beta-spectrin augment the interaction between alpha-catenin and actin, and together they provide a polyvalent linkage directing the topographic assembly of a nascent spectrin-actin skeleton to membrane regions enriched in E-cadherin.     

Formation of reactive cyclopentenone compounds in vivo as products of the isoprostane pathway

Cyclopentenone prostaglandins A2 and J2 are reactive compounds that possess unique biological activities. However, the extent to which they are formed in vivo remains unclear. In this study, we explored whether D2/E2-isoprostanes undergo dehydration in vivo to form A2/J2-isoprostanes. Oxidation of arachidonic acid in vitro generated a series of compounds that were confirmed to be A2/J2-isoprostanes by mass spectrometric analyses. A2/J2-isoprostanes were detected in vivo esterified to lipids in livers from normal rats at a level of 5. 1 +/- 2.3 ng/g, and levels increased dramatically by a mean of 24-fold following administration of CCl4. An A2-isoprostane, 15-A2t-isoprostane, was obtained and found to readily undergo Michael addition with glutathione and to adduct covalently to protein. A2/J2-isoprostanes could not be detected in the circulation, even following CCl4 administration, which we hypothesized might be explained by rapid formation of adducts. This was supported by finding that essentially all the radioactivity excreted into the urine following infusion of radiolabeled 15-A2t-isoprostane into a human volunteer was in the form of a polar conjugate(s). These data identify a new class of reactive compounds that are produced in vivo as products of the isoprostane pathway that can exert biological effects relevant to the pathobiology of oxidant injury.     

The effects of diet composition and ration on biotransformation enzymes and stress parameters in rainbow trout, Oncorhynchus mykiss

Juvenile rainbow trout (Oncorhynchus mykiss) were fed one of three isoenergetic diets varying in protein (35-55%) and lipid content (8-18%), at full satiation levels or half rations for 6 weeks in order to investigate the effects of diet on baseline stress parameters and biotransformation enzyme activity. Growth was greatest in fish fed to satiation on a low protein and high lipid diet. Stress parameters, including plasma lactate, glucose and cortisol concentrations were not significantly affected by dietary treatment or ration. Basal biotransformation enzymes, including glutathione S-transferase (GST) and ethoxyresorufin O-deethylase (EROD) activity, were also unaffected by dietary treatment. Fish exposed to the biotransformation enzyme inducer beta-naphthoflavone did not exhibit an alteration in stress indicators or GST activity; however, EROD activity was increased (87- to 210-fold) in fish receiving all diets and rations. The results of the present study indicate that, unlike mammals, fish may be more recalcitrant to different levels of ingestion of isoenergetic diets varying in protein and lipid concentration with respect to stress responses and the maintenance of basal titers of biotransformation enzymes and their induction.     

Prevalence of antibodies to Neospora caninum, Sarcocystis neurona, and Toxoplasma gondii in wild horses from central Wyoming

Sarcocystis neurona, Neospora caninum, N. hughesi, and Toxoplasma gondii are 4 related coccidians considered to be associated with encephalomyelitis in horses. The source of infection for N. hughesi is unknown, whereas opossums, dogs, and cats are the definitive hosts for S. neurona, N. caninum, and T. gondii, respectively. Seroprevalence of these coccidians in 276 wild horses from central Wyoming outside the known range of the opossum (Didelphis virginiana) was determined. Antibodies to T. gondii were found only in 1 of 276 horses tested with the modified agglutination test using 1:25, 1:50, and 1:500 dilutions. Antibodies to N. caninum were found in 86 (31.1%) of the 276 horses tested with the Neospora agglutination test--the titers were 1:25 in 38 horses, 1:50 in 15, 1:100 in 9, 1:200 in 8, 1:400 in 4, 1:800 in 2, 1:1,600 in 2, 1:3,200 in 2, and 1:12,800 in 1. Antibodies to S. neurona were assessed with the serum immunoblot; of 276 horses tested, 18 had antibodies considered specific for S. neurona. Antibodies to S. neurona also were assessed with the S. neurona direct agglutination test (SAT). Thirty-nine of 265 horses tested had SAT antibodies--in titers of 1:50 in 26 horses and 1:100 in 13. The presence of S. neurona antibodies in horses in central Wyoming suggests that either there is cross-reactivity between S. neurona and some other infection or a definitive host other than opossum is the source of infection. In a retrospective study, S. neurona antibodies were not found by immunoblot in the sera of 243 horses from western Canada outside the range of D. virginiana.     

Direct activation of AMP-activated protein kinase stimulates nitric-oxide synthesis in human aortic endothelial cells

Recent studies have indicated that endothelial nitric-oxide synthase (eNOS) is regulated by reversible phosphorylation in intact endothelial cells. AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and activate eNOS at Ser-1177 in vitro, yet the function of AMPK in endothelium is poorly characterized. We therefore determined whether activation of AMPK with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) stimulated NO production in human aortic endothelial cells. AICAR caused the time- and dose-dependent stimulation of AMPK activity, with a concomitant increase in eNOS Ser-1177 phosphorylation and NO production. AMPK was associated with immunoprecipitates of eNOS, yet this was unaffected by increasing concentrations of AICAR. AICAR also caused the time- and dose-dependent stimulation of protein kinase B phosphorylation. To confirm that the effects of AICAR were indeed mediated by AMPK, we utilized adenovirus-mediated expression of a dominant negative AMPK mutant. Expression of dominant negative AMPK attenuated AICAR-stimulated AMPK activity, eNOS Ser-1177 phosphorylation and NO production and was without effect on AICAR-stimulated protein kinase B Ser-473 phosphorylation or NO production stimulated by insulin or A23187. These data suggest that AICAR-stimulated NO production is mediated by AMPK as a consequence of increased Ser-1177 phosphorylation of eNOS. We propose that stimuli that result in the acute activation of AMPK activity in endothelial cells stimulate NO production, at least in part due to phosphorylation and activation of eNOS. Regulation of endothelial AMPK therefore provides an additional mechanism by which local vascular tone may be controlled.