The role of oxidative stress in indium phosphide-induced lung carcinogenesis in rats.

Indium phosphide (IP), widely used in the microelectronics industry, was tested for potential carcinogenicity. Sixty male and 60 female Fischer 344 rats were exposed by aerosol for 6 h/day, 5 days/week, for 21 weeks (0.1 or 0.3 mg/m(3); stop exposure groups) or 105 weeks (0 or 0.03 mg/m(3) groups) with interim groups (10 animals/group/sex) evaluated at 3 months. After 3-month exposure, severe pulmonary inflammation with numerous infiltrating macrophages and alveolar proteinosis appeared. After 2 years, dose-dependent high incidences of alveolar/bronchiolar adenomas and carcinomas occurred in both sexes; four cases of squamous cell carcinomas appeared in males (0.3 mg/m(3)), and a variety of non-neoplastic lung lesions, including simple and atypical hyperplasia, chronic active inflammation, and squamous cyst, occurred in both sexes. To investigate whether inflammation-related oxidative stress functioned in the pathogenesis of IP-related pulmonary lesions, we stained lungs of control and high-dose animals immunohistochemically for four markers indicative of oxidative stress: inducible nitric oxide synthase (i-NOS), cyclooxygenase-2 (COX-2), glutathione-S-transferase Pi (GST-Pi), and 8-hydroxydeoxyguanosine (8-OHdG). Paraffin-embedded samples from the 3-month and 2-year control and treated females were used. i-NOS and COX-2 were highly expressed in inflammatory foci after 3 months; at 2 years, all four markers were expressed in non-neoplastic and neoplastic lesions. Most i-NOS staining, mainly in macrophages, occurred in chronic inflammatory and atypical hyperplastic lesions. GST-Pi and 8-OHdG expression occurred in cells of carcinoma epithelium, atypical hyperplasia, and squamous cysts. These findings suggest that IP inhalation causes pulmonary inflammation associated with oxidative stress, resulting in progression to atypical hyperplasia and neoplasia.

Unique renal tubule changes induced in rats and mice by the peroxisome proliferators 2,4-dichlorophenoxyacetic acid (2,4-D) and WY-14643.

Peroxisome proliferators are non-mutagenic carcinogens in the liver of rodents, acting both as initiators and promoters. The National Toxicology Program (NTP) conducted a study of several peroxisome proliferators (PPs), including Wyeth (WY)-14643 as a prototypical PP and 2,4-dichlorophenoxyacetic acid (2,4-D) as a weak PP, in Sprague-Dawley rats. B6C3F1 mice, and Syrian hamsters. In the kidney, an unusual change was observed in the outer stripe of the outer medulla, especially in rats treated with 2,4-D or WY-14643. This change was characterized by foci of tubules that were partially or completely lined by basophilic epithelial cells with decreased cytoplasm and high nuclear density. Changes typical of chronic nephropathy such as interstitial fibrosis or basement membrane thickening were not associated with these foci. Results of immunohistochemical staining for catalase and cytochrome P-450 4A in the kidney indicated increased staining intensity in renal tubular epithelial cells primarily in the region where the affected tubules were observed: however, the altered cells were negative for both immunohistochemical markers. Ultrastructurally, affected cells had long brush borders typical of the P3 tubule segment. The most distinguishing ultrastructural change was a decreased amount of electronlucent cytoplasm that contained few differentiated organelles and, in particular, a prominent reduced volume and number of mitochondria; changes in peroxisomes were not apparent. In addition to the lesion in rats, mice treated with the highest dose of 2,4-D, but not WY-14643, manifested similar renal tubular changes as seen by light microscopy. Neither chemical induced renal tubular lesions in hamsters. Hepatocellular changes characteristic of PPs were present in all 3 species treated with WY-14643, but not 2,4-D. These results indicate that the rat is the species most sensitive to the nephrotoxic effects of PPs and there is a site specificity to this toxicity related to areas of PP-related enzyme induction. Although 2,4-D is considered a weak PP for the liver, it was the most effective at inducing renal lesions, indicating that the toxic potency of various PPs will depend on the target organ.

Glutathione S-transferase pi expression in forestomach carcinogenesis process induced by gavage-administered 2,4-hexadienal in the F344 rat.

2,4-Hexadienal (2,4-Hx), an unsaturated aldehyde formed by in vivo and in vitro peroxidation of unsaturated lipid induced, in National Toxicology Program (NTP) gavage studies of F344 rats, forestomach hyperplasia in 13-week and 2-year exposures and squamous papilloma and carcinoma in 2-year studies. Hyperplasia was characterized by thickening of all layers of epithelium with particularly prominent proliferation of the basal cells. The present investigation describes the nature and potential significance of glutathione-S-transferase-Pi (GST-Pi) immunoexpression of normal forestomach epithelium, compared to that of 2,4-Hx-related basal cell hyperplasia and squamous cell papilloma and carcinoma. Paraffin-embedded forestomachs from these NTP studies were used to investigate possible correlations between the carcinogenic process and expression of GST-Pi, a physiological metabolic barrier and an inducible phase II detoxifying enzyme suggested to decrease the responsiveness of reactive oxygen species (ROS) and organic electrophilic compounds. The amount of immunopositive staining was graded on a scale of 0 (no staining) to 4 (marked staining). The simple basal epithelium of control rats showed strong immunopositivity. In cases of basal cell hyperplasia from the 13-week and 2-year studies, these cells usually expressed strong immunopositivity for GST-Pi (grade 3 to 4). In the 2-year treated animals only, occasional focal reduction (grade 0 to 2) in immunoreactivity for GST-Pi was noted. In papillomas and squamous cell carcinomas, a wide range of GST-Pi expression was observed, perhaps indicating irregularities in its induction or change in the phenotype of these cells compared to normal or hyperplastic ones. Reduced expression of GST-Pi by the foci of basal cell hyperplasia and in tumor cells may suggest changes in cellular protection from oxidative or electrophilic DNA damage; these changes may result in genetic alterations and be the precursor to clonal expansion.

Proteolytic cleavage of the Fe-S subunit hinge region of Rhodobacter capsulatus bc(1) complex: effects of inhibitors and mutations.

The three-dimensional structure of the mitochondrial bc(1) complex reveals that the extrinsic domain of the Fe-S subunit, which carries the redox-active [2Fe2S] cluster, is attached to its transmembrane anchor domain by a short flexible hinge sequence (amino acids D43 to S49 in Rhodobacter capsulatus). In various structures, this extrinsic domain is located in different positions, and the conformation of the hinge region is different. In addition, proteolysis of this region has been observed previously in a bc(1) complex mutant of R. capsulatus [Saribas, A. S., Valkova-Valchanova, M. B., Tokito, M., Zhang, Z., Berry E. A., and Daldal, F. (1998) Biochemistry 37, 8105-8114]. Thus, possible correlations between proteolysis, conformation of the hinge region, and position of the extrinsic domain of the Fe-S subunit within the bc(1) complex were sought. In this work, we show that thermolysin, or an endogenous activity present in R. capsulatus, cleaves the hinge region of the Fe-S subunit between its amino acid residues A46-M47 or D43-V44, respectively, to yield a protease resistant fragment with a M(r) of approximately 18 kDa. The cleavage was affected significantly by ubihydroquinone oxidation (Q(o)) and ubiquinone reduction (Q(i)) site inhibitors and by specific mutations located in the bc(1) complex. In particular, using either purified or detergent dispersed chromatophore-embedded R. capsulatus bc(1) complex, we demonstrated that while stigmatellin blocked the cleavage, myxothiazol hardly affected it, and antimycin A greatly enhanced it. Moreover, mutations in various regions of the Fe-S subunit and cyt b subunit changed drastically proteolysis patterns, indicating that the structure of the hinge region of the Fe-S subunit was modified in these mutants. The overall findings establish that protease accessibility of the Fe-S subunit of the bc(1) complex is a useful biochemical assay for probing the conformation of its hinge region and for monitoring indirectly the position of its extrinsic [2Fe2S] cluster domain within the Q(o) pocket.

Effects of apocynin and natural antioxidant from spinach on inducible nitric oxide synthase and cyclooxygenase-2 induction in lipopolysaccharide-induced hepatic injury in rat.

The immunoreactivity of inducible nitric oxide synthase, and cyclooxygenase-2 was compared among groups of male Wistar rats comprising those injected with lipopolysaccharide following pretreatment with either natural antioxidant from spinach or the antioxidant apocynin, with lipopolysaccharide without pretreatment with antioxidants, with each of the two antioxidants alone, and untreated controls. The grade of staining of both inducible nitric oxide synthase and cyclooxygenase-2 increased with the severity of the inflammatory reaction in the lipopolysaccharide-treated animals, compared to the antioxidant-treated groups. Interpretation of the results of the immunostained tissues indicated that pretreatment with either antioxidant significantly (P<0.05) attenuated the lipopolysaccharide-stimulated inducible nitric oxide synthase induction. Analysis of the cycloxygenase-2-stained liver samples indicated that the pretreatment with the natural antioxidant NAO significantly (P<0.05) attenuated lipopolysaccharide-stimulated cycloxygenase-2 induction; whereas, in animals pretreated with apocynin, there was a trend of reduction in the cyclooxygenase-2 expression, but not statistically significant (P>0.05). The negative nitrotyrosine immunoreactivity of the lipopolysaccharide-related hepatic lesions may indicate that there was relatively low interaction between superoxide anions and nitric oxide to form peroxynitrite or that the expression levels of the nitrotyrosine were below the limit of detection. In all treatment groups a positive correlation (P<0.05, r=0.86) found between the inducible nitric oxide synthase and cyclooxygenase-2 scores suggests a strong relationship between these two parameters. The results indicate the possible therapeutic efficacy of NAO and apocynin in the prevention of liver damage related to clinical endotoxemia known to be associated with oxidative stress.

An unusual human IgM antibody with a protruding HCDR3 and high avidity for its peptide ligands.

The crystal structure of the Fv molecule from a human monoclonal IgM cryoglobulin (Mez) was determined at 2.6 A resolution. Amino acid sequences of framework regions (FR) of the Mez light (L) and heavy (H) chain variable domains (VL and VH) are highly similar to their counterparts in another human Fv (Pot) previously subjected to X-ray analysis in our laboratory. As expected, the three-dimensional (3-D) structures of FR are quite similar in the two proteins, as are four of the six complementarity-determining regions (CDRs): CDRs 1 and 2 for both L and H chains. Absence of Pro 95L from the LCDR3 loop in Mez VL (relative to Pot LCDR3) results in compression of this loop and creates more space in the VL-VH interface. In the two IgMs, HCDR3 conformations differ significantly from all previously defined conformations for these loops. Pot has a 12-residue HCDR3 that collapses to fill all available space in the VL-VH domain interface, resulting in the formation of a relatively flat platform for antigen binding. In Mez, the HCDR3 is two residues longer and is comprehensively different. A semi-rigid ascending segment dominated by a Pro-Pro-Tyr sequence protrudes out into solvent. The descending portion has the sequence Gly-Trp-Gly-Gly-Gly, which promotes high local flexibility. This segment folds across the VL-VH domain interface to interact with residues in LCDR3. These features partition the Mez active site into two compartments, a large cavity between VL and VH and a smaller cavity lined entirely by constituents of the three heavy chain CDRs. Such an unusual topographical feature indicates why the Mez IgM does not bind to the Fc portion of intact human IgG antibodies in immunoassays yet interacts with high avidity with many Fc-derived octapeptides. The cavities are expected to be the repositories for the Fc-derived peptides, while the semi-rigid protrusion of the Mez HCDR3 prevents the close approach of another macromolecule (e.g. intact IgG) to the active site.

Mutations of ras protooncogenes and p53 tumor suppressor gene in cardiac hemangiosarcomas from B6C3F1 mice exposed to 1,3-butadiene for 2 years.

1,3-Butadiene is a multisite carcinogen in rodents. Incidences of cardiac hemangiosarcomas were significantly increased in male and female B6C3F1 mice that inhaled 1,3-butadiene (BD) for 2 years. Eleven BD-induced cardiac hemangiosarcomas were examined for genetic alterations in ras protooncogenes and in the p53 tumor suppressor gene. Nine of 11 (82%) BD-induced hemangiosarcomas had K-ras mutations and 5 of 11 (46%) had H-ras mutations. All of the K-ras mutations were G-->C transversions (GGC-->CGC) at codon 13; this pattern is consistent with reported results in BD-induced lung neoplasms and lymphomas. Both K-ras codon 13 CGC mutations and H-ras codon 61 CGA mutations were detected in 5 of 9 (56%) hemangiosarcomas. The 11 hemangiosarcomas stained positive for p53 protein by immunohistochemistry and were analyzed for p53 mutations using cycle sequencing of polymerase chain reaction (PCR) amplified DNA isolated from paraffin-embedded sections. Mutations in exons 5 to 8 of the p53 gene were identified in 5 of 11 (46%) hemangiosarcomas, and all of these were from the 200- or 625-ppm exposure groups that also had K-ras codon 13 CGC mutations. Our data indicate that K-ras, H-ras, and p53 mutations in these hemangiosarcomas most likely occurred as a result of the genotoxic effects of BD and that these mutations may play a role in the pathogenesis of BD-induced cardiac hemangiosarcomas in the B6C3F1 mouse.

Airway fibrosis in rats induced by vanadium pentoxide.

Vanadium pentoxide (V(2)O(5)) is a cause of occupational asthma and bronchitis. We previously reported that intratracheal instillation of rats with V(2)O(5) causes fibrosis of the lung parenchyma (J. C. Bonner, P. M. Lindroos, A. B. Rice, C. R. Moomaw, and D. L. Morgan. Am. J. Physiol. Lung Cell. Mol. Physiol. 274: L72-L80, 1998). In this report, we show that intratracheal instillation of V(2)O(5) induces airway remodeling similar to that observed in individuals with asthma. These changes include airway smooth muscle cell thickening, mucous cell metaplasia, and airway fibrosis. The transient appearance of peribronchiolar myofibroblasts, which were desmin and vimentin positive, coincided with a twofold increase in the thickness of the airway smooth muscle layer at day 6 after instillation and preceded the development of airway fibrosis by day 15. The number of nuclear profiles within the smooth muscle layer also increased twofold after V(2)O(5) instillation, suggesting that hyperplasia accounted for thickening of the smooth muscle layer. The majority of cells incorporating bromodeoxyuridine at day 3 were located in the connective tissue surrounding the airway smooth muscle wall that was positive for vimentin and desmin. These data suggest that myofibroblasts are the principal proliferating cell type that contributes to the progression of airway fibrosis after V(2)O(5) injury.

The 21- and 23-kD forms of TCR zeta are generated by specific ITAM phosphorylations.

The T cell receptor (TCR) zeta subunit contains three immunoreceptor tyrosine-based activation motifs (ITAMs) that translate effective extracellular ligand binding into intracellular signals by becoming phosphorylated into 21- and 23-kD forms. We report here that the 21-kD form of TCR zeta is generated by phosphorylation of the tyrosines in the second and third ITAMs, whereas the 23-kD form is formed by the additional phosphorylation of the membrane-proximal ITAM tyrosines. The stable formation of the 21- and 23-kD species requires the binding of the tandem SH2 domains of ZAP-70. We also report that TCR-mediated signaling processes can proceed independently of either the 21- or 23-kD species of TCR zeta.

Specific inhibitors of platelet-derived growth factor or epidermal growth factor receptor tyrosine kinase reduce pulmonary fibrosis in rats.

The proliferation of myofibroblasts is a central feature of pulmonary fibrosis. In this study we have used tyrosine kinase inhibitors of the tyrphostin class to specifically block autophosphorylation of the platelet-derived growth factor receptor (PDGF-R) or epidermal growth factor receptor (EGF-R). AG1296 specifically inhibited autophosphorylation of PDGF-R and blocked PDGF-stimulated [3H]thymidine uptake by rat lung myofibroblasts in vitro. AG1478 was demonstrated as a selective blocker of EGF-R autophosphorylation and inhibited EGF-stimulated DNA synthesis in vitro. In a rat model of pulmonary fibrosis caused by intratracheal instillation of vanadium pentoxide (V2O5), intraperitoneal delivery of 50 mg/kg AG1296 or AG1478 in dimethylsulfoxide 1 hour before V2O5 instillation and again 2 days after instillation reduced the number of epithelial and mesenchymal cells incorporating bromodeoxyuridine (Brdu) by approximately 50% at 3 and 6 days after instillation. V2O5 instillation increased lung hydroxyproline fivefold 15 days after instillation, and AG1296 was more than 90% effective in preventing the increase in hydroxyproline, whereas AG1478 caused a 50% to 60% decrease in V2O5-stimulated hydroxyproline accumulation. These data provide evidence that PDGF and EGF receptor ligands are potent mitogens for collagen-producing mesenchymal cells during pulmonary fibrogenesis, and targeting tyrosine kinase receptors could offer a strategy for the treatment of fibrotic lung diseases.

Molecular cloning, enzymatic characterization, developmental expression, and cellular localization of a mouse cytochrome P450 highly expressed in kidney.

A cDNA encoding a new cytochrome P450 was isolated from a mouse liver library. Sequence analysis reveals that this 1,886-base pair cDNA encodes a 501-amino acid polypeptide that is 69-74% identical to CYP2J subfamily P450s and is designated CYP2J5. Recombinant CYP2J5 was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 cells using a baculovirus system. Microsomal fractions of CYP2J5/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolize arachidonic acid to 14,15-, 11,12-, and 8, 9-epoxyeicosatrienoic acids and 11- and 15-hydroxyeicosatetraenoic acids (catalytic turnover, 4.5 nmol of product/nmol of cytochrome P450/min at 37 degrees C); thus CYP2J5 is enzymologically distinct. Northern analysis reveals that CYP2J5 transcripts are most abundant in mouse kidney and present at lower levels in liver. Immunoblotting using a polyclonal antibody against a CYP2J5-specific peptide detects a protein with the same electrophoretic mobility as recombinant CYP2J5 most abundantly in mouse kidney microsomes. CYP2J5 is regulated during development in a tissue-specific fashion. In the kidney, CYP2J5 is present before birth and reaches maximal levels at 2-4 weeks of age. In the liver, CYP2J5 is absent prenatally and during the early postnatal period, first appears at 1 week, and then remains relatively constant. Immunohistochemical staining of kidney sections with anti-human CYP2J2 IgG reveals that CYP2J protein(s) are present primarily in the proximal tubules and collecting ducts, sites where the epoxyeicosatrienoic acids are known to modulate fluid/electrolyte transport and mediate hormonal action. In situ hybridization confirms abundant CYP2J5 mRNA within tubules of the renal cortex and outer medulla. Epoxyeicosatrienoic acids are endogenous constituents of mouse kidney thus providing direct evidence for the in vivo metabolism of arachidonic acid by the mouse renal epoxygenase(s). Based on these data, we conclude that CYP2J5 is an enzymologically distinct, developmentally regulated, protein that is localized to specific nephron segments and contributes to the oxidation of endogenous renal arachidonic acid pools. In light of the well documented effects of epoxyeicosatrienoic acids in modulating renal tubular transport processes, we postulate that CYP2J5 products play important functional roles in the kidney.

Induction of PDGF receptor-alpha in rat myofibroblasts during pulmonary fibrogenesis in vivo.

Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells. Induction of the PDGF receptor-alpha (PDGF-R alpha) in vitro enhances PDGF-induced mitogenesis and chemotaxis. Thus we investigated whether the PDGF-R alpha is induced in vivo during pulmonary fibrogenesis using a vanadium pentoxide (V2O5) model of lung injury. PDGF-R alpha mRNA expression was induced 24 h postinstillation. PDGF-R beta mRNA was constitutively expressed and did not increase. Western blotting showed upregulation of PDGF-R alpha protein by 48 h, and immunohistochemical analysis localized PDGF-R alpha primarily in mesenchymal cells residing within fibrotic lesions. Upregulation of PDGF-R alpha in vivo preceded mesenchymal cell hyperplasia (3-7 days) and collagen deposition by day 15. Supernatants from alveolar macrophages treated with V2O5 in vitro released upregulatory activity for PDGF-R alpha on cultured lung myofibroblasts, and this activity was blocked by the interleukin-1-receptor antagonist. These data suggest that interleukin-1 beta-mediated induction of PDGF-R alpha in vivo is important to lung myofibroblast hyperplasia during fibrogenesis.

Novel HY peptide antigens presented by HLA-B27.

We have identified two peptides corresponding to the male-specific HY minor histocompatibility Ags presented by HLA-B27 in transgenic rodents, isolated from whole cell extracts and from immunoprecipitated B27 molecules of male B27 rat spleen cells. HPLC peptide fractions that sensitized female B27 targets for lysis by B27-restricted anti-HY CTL were analyzed by electrospray tandem mass spectrometry using a new highly sensitive quadrupole/time-of-flight instrument. Two peptide sequences were obtained, KQYQKSTER and AVLNKSNREVR. Synthetic peptides corresponding to these sequences bound B27 in vitro and were recognized by distinct B27-restricted anti-HY CTL populations. Neither peptide sequence entirely matches known protein sequences or shows a resemblance to known Y chromosome genes, but both show homology to known autosomally encoded proteins. Both peptides were shown to be controlled by the Sxr(b) segment of the short arm of the mouse Y chromosome, a segment known to contain all previously identified HY Ags. Taken together, these findings suggest that the two peptides arise as a result of Y chromosome-regulated control of one or more autosomal gene products. Although arginine at position 2 is a dominant anchor residue for peptides bound to B27, neither B27-presented HY sequence contains this residue. These studies, employing sensitive new methodology for identification of MHC-bound peptides, significantly extend the complexity of the genetic basis of HY Ags and expand the repertoire of antigenically active peptides bound to B27.

CYP2J subfamily cytochrome P450s in the gastrointestinal tract: expression, localization, and potential functional significance.

Our laboratory recently described a new human cytochrome P450 arachidonic acid epoxygenase (CYP2J2) and the corresponding rat homologue (CYP2J3), both of which were expressed in extrahepatic tissues. Northern analysis of RNA prepared from the human and rat intestine demonstrated that CYP2J2 and CYP2J3 mRNAs were expressed primarily in the small intestine and colon. In contrast, immunoblotting studies using a polyclonal antibody raised against recombinant CYP2J2 showed that CYP2J proteins were expressed throughout the gastrointestinal tract. Immunohistochemical staining of formalin-fixed, paraffin-embedded intestinal sections using anti-CYP2J2 IgG and avidin-biotin-peroxidase detection revealed that CYP2J proteins were present at high levels in nerve cells of autonomic ganglia, epithelial cells, intestinal smooth muscle cells, and vascular endothelium. The distribution of this immunoreactivity was confirmed by in situ hybridization using a CYP2J2-specific antisense RNA probe. Microsomal fractions prepared from human jejunum catalyzed the NADPH-dependent metabolism of arachidonic acid to epoxyeicosatrienoic acids as the principal reaction products. Direct evidence for the in vivo epoxidation of arachidonic acid by intestinal cytochrome P450 was provided by documenting, for the first time, the presence of epoxyeicosatrienoic acids in human jejunum by gas chromatography/mass spectrometry. We conclude that human and rat intestine contain an arachidonic acid epoxygenase belonging to the CYP2J subfamily that is localized to autonomic ganglion cells, epithelial cells, smooth muscle cells, and vascular endothelium. In addition to the known effects on intestinal vascular tone, we speculate that CYP2J products may be involved in the release of intestinal neuropeptides, control of intestinal motility, and/or modulation of intestinal fluid/electrolyte transport.

Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes.

A cDNA encoding a P450 monooxygenase was amplified from reverse transcribed rat heart and liver total RNA by polymerase chain reaction using primers based on the 5'- and 3'-end sequences of two rat pseudogenes, CYP2J3P1 and CYP2J3P2. Sequence analysis revealed that this 1,778-base pair cDNA contained an open reading frame and encoded a new 502 amino acid protein designated CYP2J3. Based on the deduced amino acid sequence, CYP2J3 was approximately 70% homologous to both human CYP2J2 and rabbit CYP2J1. Recombinant CYP2J3 protein was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect cells using a baculovirus expression system. Microsomal fractions of CYP2J3/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolized arachidonic acid to 14,15-, 11,12-, and 8, 9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid as the principal reaction products (catalytic turnover, 0.2 nmol of product/nmol of cytochrome P450/min at 37 degrees C). Immunoblotting of microsomal fractions prepared from rat tissues using a polyclonal antibody raised against recombinant CYP2J2 that cross-reacted with CYP2J3 but not with other known rat P450s demonstrated abundant expression of CYP2J3 protein in heart and liver. Immunohistochemical staining of formalin-fixed paraffin-embedded rat heart tissue sections using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection localized expression of CYP2J3 primarily to atrial and ventricular myocytes. In an isolated-perfused rat heart model, 20 min of global ischemia followed by 40 min of reflow resulted in recovery of only 44 +/- 6% of base-line contractile function. The addition of 5 microM 11, 12-epoxyeicosatrienoic acid to the perfusate prior to global ischemia resulted in a significant 1.6-fold improvement in recovery of cardiac contractility (69 +/- 5% of base line, p = 0.01 versus vehicle alone). Importantly, neither 14,15-epoxyeicosatrienoic acid nor 19-hydroxyeicosatetraenoic acid significantly improved functional recovery following global ischemia, demonstrating the specificity of the biological effect for the 11, 12-epoxyeicosatrienoic acid regioisomer. Based on these data, we conclude that (a) CYP2J3 is one of the predominant enzymes responsible for the oxidation of endogenous arachidonic acid pools in rat heart myocytes and (b) 11,12-epoxyeicosatrienoic acid may play an important functional role in the response of the heart to ischemia.

Cytochrome c(y) of Rhodobacter capsulatus is attached to the cytoplasmic membrane by an uncleaved signal sequence-like anchor.

During the photosynthetic growth of Rhodobacter capsulatus, electrons are conveyed from the cytochrome (cyt) bc1 complex to the photochemical reaction center by either the periplasmic cyt c2 or the membrane-bound cyt c(y). Cyt c(y) is a member of a recently established subclass of bipartite c-type cytochromes consisting of an amino (N)-terminal domain functioning as a membrane anchor and a carboxyl (C)-terminal domain homologous to cyt c of various sources. Structural homologs of cyt c(y) have now been found in several bacterial species, including Rhodobacter sphaeroides. In this work, a C-terminally epitope-tagged and functional derivative of R. capsulatus cyt c(y) was purified from intracytoplasmic membranes to homogeneity. Analyses of isolated cyt c(y) indicated that its spectral and thermodynamic properties are very similar to those of other c-type cytochromes, in particular to those from bacterial and plant mitochondrial sources. Amino acid sequence determination for purified cyt c(y) revealed that its signal sequence-like N-terminal portion is uncleaved; hence, it is anchored to the membrane. To demonstrate that the N-terminal domain of cyt c(y) is indeed its membrane anchor, this sequence was fused to the N terminus of cyt c2. The resulting hybrid cyt c (MA-c2) remained membrane bound and was able to support photosynthetic growth of R. capsulatus in the absence of the cyt c(y) and c2. Therefore, cyt c2 can support cyclic electron transfer during photosynthetic growth in either a freely diffusible or a membrane-anchored form. These findings should now allow for the first time the comparison of electron transfer properties of a given electron carrier when it is anchored to the membrane or is freely diffusible in the periplasm.

Predominant expression of an arachidonate epoxygenase in islets of Langerhans cells in human and rat pancreas.

Our laboratory recently described a new human cytochrome P450 arachidonic acid epoxygenase (CYP2J2) and the corresponding rat homolog (CYP2J3). Immunoblotting studies using a polyclonal antibody raised against recombinant human CYP2J2 confirmed CYP2J protein expression in human and rat pancreatic tissues. Immunohistochemical staining of formalin-fixed paraffin-embedded rat and human pancreas using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection revealed that CYP2J2 protein expression was highly localized to cells in the islets of Langerhans, with minimal staining in pancreatic exocrine cells. Colocalization studies using antibodies to the glucagon, insulin, somatostatin, and pancreatic polypeptide as markers for alpha-, beta-, delta-, and PP cells, respectively, showed that CYP2J protein expression was abundantly present in all four cell types, but was highest in the glucagon-producing alpha-cells. Direct evidence for the epoxidation of arachidonic acid by pancreatic cytochrome P450 was provided by documenting, for the first time, the presence of epoxyeicosatrienoic acids in vivo in human and rat pancreas by gas chromatography/mass spectrometry. Importantly, the levels of immunoreactive CYP2J2 in different human pancreatic tissues were highly correlated with endogenous epoxyeicosatrienoic acid concentrations. We conclude that human and rat pancreas contain an arachidonic acid epoxygenase belonging to the CYP2J subfamily that is highly localized to islet cells. These data together with previous work showing effects of epoxyeicosatrienoic acids in stimulating insulin and glucagon secretion from isolated rat pancreatic islets support the hypothesis that epoxygenase products may be involved in stimulus-secretion coupling in the pancreas.

Characterization of delta-sarcoglycan, a novel component of the oligomeric sarcoglycan complex involved in limb-girdle muscular dystrophy.

The sarcoglycan complex is known to be involved in limb-girdle muscular dystrophy (LGMD) and is composed of at least three proteins: alpha-, beta-, and gamma-sarcoglycan. delta-Sarcoglycan has now been identified as a second 35-kDa sarcolemmal transmembrane glycoprotein that shares high homology with gamma-sarcoglycan and is expressed mainly in skeletal and cardiac muscle. Biochemical analysis has demonstrated that gamma- and delta-sarcoglycan are separate entities within the sarcoglycan complex and that all four sarcoglycans exist in the complex on a stoichiometrically equal basis. Immunohistochemical analysis of skeletal muscle biopsies from patients with LGMD2C, LGMD2D, and LGMD2E demonstrated a reduction of the entire sarcoglycan complex in these muscular dystrophies. Furthermore, we have mapped the human delta-sarcoglycan gene to chromosome 5q33-q34 in a region overlapping the recently linked autosomal recessive LGMD2F locus.

Identification of a cysteine residue essential for activity of protein farnesyltransferase. Cys299 is exposed only upon removal of zinc from the enzyme.

Protein farnesyltransferase (FTase) is a zinc metalloenzyme that performs a post-translational modification on many proteins that is critical for their function. The importance of cysteine residues in FTase activity was investigated using cysteine-specific reagents. Zinc-depleted FTase (apo-FTase), but not the holoenzyme, was completely inactivated by treatment with N-ethylmaleimide (NEM). Similar effects were detected after treatment of the enzyme with iodoacetamide. The addition of zinc to apo-FTase protects it from inactivation by NEM. These findings indicated the presence of specific cysteine residue(s), potentially located at the zinc binding site, that are required for FTase activity. We performed a selective labeling strategy whereby the cysteine residues exposed upon removal of zinc from the enzyme were modified with [3H]NEM. The enzyme so modified was digested with trypsin, and four labeled peptides were identified and sequenced, one peptide being the major site of labeling and the remaining three labeled to lesser extents. The major labeled peptide contained a radiolabeled cysteine residue, Cys299, that is in the beta subunit of FTase and is conserved in all known protein prenyltransferases. This cysteine residue was changed to both alanine and serine by site-directed mutagenesis, and the mutant proteins were produced in Escherichia coli and purified. While both mutant proteins retained the ability to bind farnesyl diphosphate, they were found to have lost essentially all catalytic activity and ability to bind zinc. These results indicate that the Cys299 in the beta subunit of FTase plays a critical role in catalysis by the enzyme and is likely to be one of the residues that directly coordinate the zinc atom in this enzyme.