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The 2.6 A (1 A=0.1 nm) resolution structure has been determined for the glycosylated Fab (fragment antigen binding) of an IgM (Yvo) obtained from a subject with Waldenström's macroglobulinaemia. Dynamic light scattering was used to estimate the gel point and monitor the formation of an ordered hydroscopic gel of Yvo IgM upon cooling. If a cryoglobulin forms gels in peripheral tissues and organs, the associated swelling and damage to microvasculature can result in considerable morbidity and mortality. The three-dimensional structure of the branched N-linked oligosaccharide associated with the CH1 domain (first constant domain of heavy chain) is reported. The carbohydrate may act to shield part of the lateral surface of the CH1 domain and crowd the junction between the CH1 and CH2 domains, thereby limiting the segmental flexibility of the Fab arms in intact Yvo IgM, especially at low temperatures. Recently, Yvo IgM was shown to have the properties of a naturally occurring proteolytic antibody [Paul, Karle, Planque, Taguchi, Salas, Nishiyama, Handy, Hunter, Edmundson and Hanson (2004) J. Biol. Chem. 279, 39611-39619; Planque, Bangale, Song, Karle, Taguchi, Poindexter, Bick, Edmundson, Nishiyama and Paul (2004) J. Biol Chem. 279, 14024-14032]. The Yvo protein displayed the ability to cleave, by a nucleophilic mechanism, the amide bonds of a variety of serine protease substrates and the gp120 coat protein of HIV. An atypical serine, arginine and glutamate motif is located in the middle of the Yvo antigen-binding site and displays an overall geometry that mimics the classical serine, histidine and aspartate catalytic triad of serine proteases. Our present findings indicate that pre-existing or natural antibodies can utilize at least one novel strategy for the cleavage of peptide bonds.

We demonstrated previously that exposure of rats to 2-butoxyethanol (BE) was associated with morphological changes in red blood cells, hemolytic anemia, and disseminated thrombosis and infarction in different organs including the eyes. In order to elucidate the mechanism of thrombosis formation, we examined in this study the histology and immunohistochemical expression of vascular cell adhesion molecule-1 (VCAM-1), endothelial intercellular adhesion molecule-1 (ICAM-1), and P-selectin in the eyes of the female F344 rat exposed to 2, 3, or 4 daily doses of BE/250 mg/kg body weight. In this BE hemolysis and thrombosis model, positive VCAM-1 expression occurred only in eyes of rats exposed to 3 and 4 doses and was localized in the iris (epithelium lining the posterior surface, anterior mesenchymal epithelium), ciliary processes (lining epithelium, stromal cells), and retina (hypertrophic retinal pigment epithelium). Only weak immunolabeling was seen in eyes exposed to 2 doses. The appearance of VCAM-1 immunostaining correlated with the development of thrombosis located in the same structures. No change in ICAM-1 or P-selectin expression was seen. This immunolabeling distribution suggests that VCAM-1 functions in the pathogenesis of BE-related thrombosis by promoting adhesion of erythrocytes to the endothelium.

Riddelliineis a naturally occurring pyrrolizidine alkaloid found in certain poisonous rangeland plants of the western United States. In National Toxicology Program 2-year studies, riddelliine induced high incidences of hemangiosarcoma in the liver of F344/N rats (both sexes) and B6C3F1 mice (males). To understand this pathogenesis, we tested short-term effects of riddelliine. Three groups (control; 1.0 mg/kg/day, high dose used in the 2-year study; and 2.5 mg/kg/day) of seven male F344/N rats per group were terminated after 8 consecutive doses and 30 doses (6 weeks, excluding weekends). Serum vascular endothelial growth factor (VEGF), histological, immunohistochemical [factor VIII-related antigen/von Willebrand factor (fVIII-ra/vWf)], VEGF, VEGF receptor-2 (VEGFR2), glutathione S-transferase-pi, S-phase (BrdU), p53, apoptosis, and ultrastructural evaluations were performed on the liver. Following 8 doses of 1.0 and 2.5 mg/kg/day, increased numbers of apoptotic and S-phase nuclei appeared in hepatocytes and endothelial cells. Following 30 doses of 1.0 and 2.5 mg/kg/day, hepatocytes exhibited reduced mitosis, fewer S-phase nuclei, increased hypertrophy, and fatty degeneration, while endothelial cells showed karyomegaly, cytomegaly, decreased apoptosis, more S-phase nuclei, and p53 positivity. Hepatocytes of treated animals expressed higher VEGF immunopositivity. That altered endothelial cells were fVIII-ra/vWf and VEGFR2 positive confirmed their identity. These changes may have promoted hemangiosarcoma development upon long-term exposure through endothelial adduct formation, apoptosis, proliferation of endothelial cells having undamaged and/or damaged DNA, and mutation. Endothelial proliferation may also have been promoted through endothelial arrest at S phase, which was associated with endothelial karyo- and cytomegaly, resulting in hepatocytic hypoxia, triggering VEGF induction.

The cyclooxygenase (COX)-2 enzyme has been implicated as an important mediator of pulmonary fibrosis. In this study, the lung fibrotic responses were investigated in COX-1 or COX-2-deficient (-/-) mice following vanadium pentoxide (V(2)O(5)) exposure. Lung histology was normal in saline-instilled wild-type and COX-deficient mice. COX-2(-/-), but not COX-1(-/-) or wild-type mice, exhibited severe inflammatory responses by 3 days following V(2)O(5) exposure and developed pulmonary fibrosis 2 weeks post-V(2)O(5) exposure. Western blot analysis and immunohistochemistry showed that COX-1 protein was present in type 2 epithelial cells, bronchial epithelial cells, and airway smooth muscle cells of saline or V(2)O(5)-exposed wild-type and COX-2(-/-) mice. COX-2 protein was present in Clara cells of wild-type and COX-1(-/-) terminal bronchioles and was strongly induced 24 hours after V(2)O(5) exposure. Prostaglandin (PG) E(2) levels in the bronchoalveolar lavage (BAL) fluid from wild-type and COX-1(-/-) mice were significantly up-regulated by V(2)O(5) exposure within 24 hours, whereas PGE(2) was not up-regulated in COX-2(-/-) BAL fluid. Tumor necrosis factor-alpha was elevated in the BAL fluid from all genotypes after V(2)O(5) exposure, but was significantly and chronically elevated in the BAL fluid from COX-2(-/-) mice above wild-type or COX-1(-/-) mice. These findings indicate that the COX-2 enzyme is protective against pulmonary fibrogenesis, and we suggest that COX-2 generation of PGE(2) is an important factor in resolving inflammation.

The X-ray structure of an immunoglobulin light-chain dimer isolated from the urine as a "Bence-Jones protein" from a patient with multiple myeloma and amyloidosis (Sea) was determined at 1.94 A resolution and refined to R and R(free) factors of 0.22 and 0.25, respectively. This "amyloidogenic" protein crystallized in the orthorhombic P2(1)2(1)2(1) space group with unit-cell parameters a = 48.28, b = 83.32, c = 112.59 A as determined at 100 K. In the vital organs (heart and kidneys), the equivalent of the urinary protein produced fibrillar amyloid deposits which were fatal to the patient. Compared with the amyloidogenic Mcg light-chain dimer, the Sea protein was highly soluble in aqueous solutions and only crystallized at concentrations approaching 100 mg ml(-1). Both the Sea and Mcg proteins packed into crystals in highly ordered arrangements typical of strongly diffracting crystals of immunoglobulin fragments. Overall similarities and significant differences in the three-dimensional structures and crystalline properties are discussed for the Sea and Mcg Bence-Jones proteins, which together provide a generalized model of abnormalities present in lambda chains, facilitating a better understanding of amyloidosis of light-chain origin (AL).

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Cytochrome P450 (P450) monooxygenases catalyze the epoxidation of arachidonic acid to form epoxyeicosatrienoic acids, which modulate bronchial smooth muscle tone and airway transepithelial ion transport. We recently described a new human P450 arachidonic acid epoxygenase (CYP2J2) and the corresponding rat homologue (CYP2J3). Northern analysis of lung RNA using CYP2J cDNA probes demonstrated that CYP2J2 and CYP2J3 mRNAs were expressed in the lung. Immunoblotting of microsomal fractions prepared from human and rat lungs using a polyclonal antibody raised against recombinant human CYP2J2 revealed a single 56-kDa band confirming abundant pulmonary CYP2J2 and CYP2J3 protein expression. Immunohistochemical analysis of formalin-fixed paraffin-embedded human and rat lung sections using the anti-human CYP2J2 IgG and avidin/biotin/peroxidase detection showed that CYP2J proteins were primarily expressed in ciliated epithelial cells lining the airway. Prominent staining was also noted in nonciliated airway epithelial cells, bronchial and pulmonary vascular smooth muscle cells, pulmonary vascular endothelium, and alveolar macrophages, whereas less intense staining was noted in alveolar epithelial cells. Endogenous epoxyeicosatrienoic acids were detected in both human and rat lung using gas chromatography/mass spectrometry, thus providing direct evidence for the in vivo human and rat pulmonary P450 metabolism of arachidonic acid. Based on these data, we conclude that CYP2J2 and CYP2J3 are abundant pulmonary arachidonic acid epoxygenases and that CYP2J products, the epoxyeicosatrienoic acids, are endogenous constituents of human and rat lung. In addition to known effects on airway smooth muscle tone and transepithelial electrolyte transport, the localization of CYP2J proteins to vascular smooth muscle and endothelium suggests that epoxyeicosatrienoic acids may also be involved in the modulation of pulmonary vascular tone.

Human liver microsomal fractions metabolized arachidonic acid in the presence of NADPH yielding epoxyeicosatrienoic acids and their hydration products, dihydroxyeicosatrienoic acids, as the principal reaction products. Inhibition studies using polyclonal antibodies prepared against recombinant CYP2C8, an abundant human liver cytochrome P450 epoxygenase, demonstrated 85-90% inhibition of arachidonic acid epoxide formation. Both epoxyeicosatrienoic acids and dihydroxyeicosatrienoic acids were detected in large amounts in human liver using gas chromatography/mass spectrometry. Chiral analysis of the endogenous liver epoxides demonstrated a preference for the 14(R),15(S)-, 11(R),12(S)-, and 8(S),9(R)-epoxyeicosatrienoic acid enantiomers. Importantly, the chirality of liver epoxyeicosatrienoic acids matched that previously reported for recombinant CYP2C8 (Zeldin et al. (1995) Arch. Biochem. Biophys. 322, 76-86). Incubations of both human liver cytosolic and microsomal fractions with synthetic epoxyeicosatrienoic acids revealed a 10-fold higher rate of dihydroxyeicosatrienoic acid formation with cytosolic relative to microsomal fractions. Recombinant human liver cytosolic epoxide hydrolase rapidly and regioselectively hydrated epoxyeicosatrienoic acids. We conclude, based on these data, that CYP2C8 is one of the primary, constitutive hepatic arachidonic acid epoxygenases responsible for formation of epoxyeicosatrienoic acids and that cytosolic epoxide hydrolase is the principal liver enzyme which forms the dihydroxyeicosatrienoic acids. We speculate that these biologically active eicosanoids may be important in maintaining homeostasis in the liver.

A cDNA encoding a human cytochrome P450 arachidonic acid epoxygenase was isolated from a human liver cDNA library. Sequence analysis revealed that this 1,876-base pair cDNA contained an open reading frame and encoded a new 502-amino acid protein designated CYP2J2. Blot hybridization analysis of RNA prepared from human tissues revealed that CYP2J2 was highly expressed in the heart. Recombinant CYP2J2 protein was prepared using the baculovirus expression system and purified to near electrophoretic homogeneity. The enzyme metabolized arachidonic acid predominantly via olefin epoxidation to all four regioisomeric cis-epoxyeicosatrienoic acids (catalytic turnover 65 pmol of product formed/nmol of cytochrome P450/min at 30 degrees C). Epoxidation of arachidonic acid by CYP2J2 at the 14,15-olefin was highly enantioselective for (14R, 15S)-epoxyeicosatrienoic acid (76% optical purity). Immunoblotting of microsomal fractions prepared from human tissues using a polyclonal antibody raised against the recombinant hemoprotein confirmed primary expression of CYP2J2 protein in human heart. The in vivo significance of CYP2J2 was suggested by documenting the presence of epoxyeicosatrienoic acids in the human heart using gas chromatography/mass spectroscopy. Importantly, the chirality of CYP2J2 products matched that of the epoxyeicosatrienoic acid enantiomers present, in vivo, in human heart. We propose that CYP2J2 is one of the enzymes responsible for epoxidation of endogenous arachidonic acid pools in human heart and that epoxyeicosatrienoic acids may, therefore, play important functional roles in cardiac physiology.

The activity of the intracellular protease, the proteasome, is modulated by a number of specific regulatory proteins. One such regulator, PA700, is a 700,000-Da multisubunit protein that activates hydrolytic activities of the proteasome via a mechanism that involves the ATP-dependent formation of a proteasome-PA700 complex. Four subunits of PA700 have been shown previously to be members of a protein family that contains a consensus sequence for ATP binding, and purified PA700 expresses ATPase activity. We report here the identification, purification, and initial characterization of a new modulator of the proteasome. The modulator has no direct effect on the activity of the proteasome, but enhances PA700 activation of the proteasome by up to 8-fold. This activation is associated with the formation of a proteasome/PA700-containing complex that is significantly larger than that formed in its absence. The modulator has a native Mr of approximately 300,000, as determined by gel filtration chromatography, and is composed of three electrophoretically distinct subunits with Mr values of 50,000, 42,000, and 27,000 (p50, p42, and p27, respectively). Amino acid sequence analysis of the subunits shows that p50 and p42 are members of the same ATP-binding protein family found in PA700. The p50 subunit is identical to TBP1, a protein previously reported to interact with human immunodeficiency virus Tat protein (Nelbock, P., Dillion, P. J., Perkins, A., and Rosen, C. A. (1990) Science 248, 1650-1653), while the p42 subunit seems to be a new member of the family. The p27 subunit has no significant sequence similarity to any previously described protein. Both p50 and p42, but not p27, were also identified as components of PA700, increasing the number of ATP-binding protein family members in this complex to six. Thus, p50 and p42 are subunits common to two protein complexes that regulate the proteasome. The PA700-dependent proteasome activator represents a new member of a growing list of proteins that regulate proteasome activity.

The nin1-1 mutant of Saccharomyces cerevisiae cannot perform the G1/S and G2/M transitions at restrictive temperatures. At such temperatures, nin1-1 strains fail to activate histone H1 kinase after release from alpha factor-imposed G1 block and after release from hydroxyurea-imposed S block. The nin1-1 mutation shows synthetic lethality with certain cdc28 mutant alleles such as cdc28-IN. Two lines of evidence indicate that Nin1p is a component of the 26S proteasome complex: (i) Nin1p, as well as the known component of the 26S proteasome, shifted to the 26S proteasome peak in the glycerol density gradient after preincubation of crude extract with ATP-Mg2+, and (ii) nin1-1 cells accumulated polyubiquitinated proteins under restrictive conditions. These results suggest that activation of Cdc28p kinase requires proteolysis. We have cloned a human cDNA encoding a regulatory subunit of the 26S proteasome, p31, which was found to be a homolog of Nin1p.

We have purified the citrate synthase from Azotobacter vinelandii and have determined that the size of the subunit is 48,000 Da and the structure of the holoenzyme is a hexamer. This contrasts with earlier estimates that indicate a 58,000 Da subunit and a tetrameric structure. In addition, the enzyme is allosteric with a Hill coefficient of 1.5 and is inhibited by NADH. The Hill coefficient is changed to about 1 by high ionic strength and AMP. The enzyme is thus similar to the citrate synthases of many other Gram-negative, facultative, anaerobic organisms. In addition, the amino acid sequence of about 100 residues has been determined and found to be highly similar to the sequence of Pseudomonas aeruginosa citrate synthase.

Three rat brain cDNA clones approximately 3500, 1465, and 1135 base pairs in length encoding succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.24) were isolated from two cDNA libraries using a polymerase chain reaction derived probe. Restriction mapping and DNA sequencing revealed that the 3.5-kilobase clone contained an 84-base pair (28 amino acid) insert in the coding region. Composite clones encoding mature SSADH predicted proteins with 488 amino acids (M(r) = 52,188) when including the insert and 460 amino acids (M(r) = 48,854) without the insert. The cDNA clones were confirmed by expression of enzyme activity in bacteria and protein sequence data obtained from sequencing purified rat brain SSADH. Two human liver SSADH cDNA clones of 1091 and 899 base pairs were also isolated. Human and rat SSADH share 83 and 91% identity in nucleotide and protein sequence, respectively. Northern blot analysis revealed two differentially expressed SSADH transcripts of approximately 2.0 and 6.0 kilobases in both rat and human tissues. Human genomic Southern blots indicate that the two SSADH transcripts are encoded by a greater than 20-kilobase single copy gene. Mammalian SSADH contains significant homology to bacterial NADP(+)-succinic semialdehyde dehydrogenase (EC 1.2.1.16) and conserved regions of general aldehyde dehydrogenases (EC 1.2.1.3), suggesting it is a member of the aldehyde dehydrogenase superfamily of proteins.

PA28, one of a series of a positive allosteric regulators of the 20 S proteasome, stimulates the enzyme's peptidase activities in an ATP-independent manner by binding to the terminal rings of the 20 S complex. PA28 has a native molecular mass of 180,000 Da and contains at least six subunits of approximately 28,000 Da. In this study we show that PA28 prepared from bovine heart contains two different subunits separable by reverse phase high performance liquid chromatography and that these subunits occur in approximately equal abundance. The subunits display mass values of 27,290 +/- 3.7 and 28,606 +/- 2.8 Da by electrospray mass spectrometry, showing that they differ in covalent structure. Partial amino acid sequence analysis of the subunits indicates that the subunits are the products of two different but homologous genes. A pair of subunits has also been isolated from rabbit heart, and partial amino acid sequence analysis shows each to be homologous to the corresponding subunit in bovine tissues. This indicates that the genes encoding two different polypeptide components of PA28 have been conserved during evolution and suggests the possibility that the two subunits play functionally distinct roles. Isolation of complexes formed between purified PA28 and the 20 S proteasome using density gradient centrifugation reveals that both PA28 subunits bind to the proteasome, indicating that both are components of functional PA28 molecules. These results are consistent with two alternative models for the subunit structure of PA28. There may exist two different PA28 molecules that are homooligomers of the 27,290- and 28,606-Da subunits, respectively. Alternatively, PA28 oligomers may contain mixtures of the 27,290- and 28,606-Da subunits either of fixed or variable stoichiometry.

Protein kinase C isoenzymes can be subdivided into two classes, based on their requirement for calcium. Protein kinase C-alpha, beta I, -beta II, and -gamma are calcium dependent, whereas protein kinase C-gamma, -epsilon, -zeta, -eta, and -theta are calcium independent. We have examined the expression, translocation, downregulation, and activation of calcium-dependent and -independent protein kinase C isoenzymes in human skin keratinocytes and fibroblasts. Human keratinocytes and fibroblasts expressed protein kinase C-alpha, -delta, -epsilon, and -zeta mRNA and protein, whereas protein kinase C-eta (L) was detected only in keratinocytes. Protein kinase C-beta I, -beta II, -gamma, and -theta were not detected in either cell type. The protein kinase C activators 12-0-tetradecanoylphorbol 13-acetate and bryostatin-1 (50 nM, for 5 min) induced translocation of protein kinase C-alpha and -epsilon cytosol to membrane in both keratinocytes and fibroblasts. 12-0-tetradecanoylphorbol 13-acetate and bryostatin-1, for 18 h, induced complete downregulation (i.e., loss) of protein kinase C-alpha and -epsilon in keratinocytes, but only partial downregulation was observed in fibroblasts. The subcellular distribution of protein kinase C-delta, -zeta or protein kinase C-eta, in keratinocytes or fibroblasts, did not change in response to 12-0-tetradecanoylphorbol 13-acetate or bryostatin-1. These data indicate differential expression, subcellular distribution, and regulation of protein kinase C isoenzymes in human skin cells.

PA700 is a 700,000-dalton multisubunit protein that activates multiple proteolytic activities of the 20 S proteasome by a mechanism dependent upon ATP hydrolysis (Ma, C.-P., Vu, J.H., Proske, R.J., Slaughter, C.A., and DeMartino, G.N. (1994) J. Biol. Chem. 269, 3539-3547). In order to determine the identities of and structural relationships among the subunits of PA700, individual PA700 subunits were isolated by a combination of reverse phase high performance liquid chromatography (HPLC) and SDS-polyacrylamide gel electrophoresis. Seven of the 16 subunits of PA700 so isolated were subjected to solid phase protease digestion followed by reverse phase HPLC. Selected peptides from each protein were sequenced by automated Edman degradation. Comparison of the resulting amino acid sequences with those in current data bases indicated that three of the subunits represented novel proteins, whereas four subunits were homologous to previously describe proteins. Three subunits of the latter group were, in turn, homologous to one another and are members of a large family of proteins containing a consensus sequence for ATP binding. Purified PA700 demonstrated ATPase activity. Treatment of PA700 with alkylating agents, such as N-ethylmaleimide, inhibited with similar kinetics both proteasome activation and ATPase activity, suggesting that these two activities are functionally linked. Thus, PA700 is composed of multiple members of a protein family that may function in the ATP-dependent regulation of proteasome activity.

The identification of genes in the class II region of the MHC that are homologous to genes encoding subunits of the proteasome has led to intense interest in the possible role of this enzyme in the proteolytic processing of polypeptide Ags. We have tested the ability of the 20S proteasome to produce peptides that can be presented by class I molecules as targets for killing by OVA-specific and beta-galactosidase-specific CTL clones. Samples of intact OVA and beta-galactosidase were subjected to digestion in vitro by 20S proteasome purified from bovine red cells and the resulting peptide mixtures were fractionated by reverse-phase HPLC. The fractions were tested for their ability to sensitize appropriate mouse target cells for lysis by specific CTL clones. In both cases, components that under all chromatographic conditions eluted with retention times indistinguishable from synthetic peptides representing known epitopes of the naturally processed proteins were found to be able to sensitize the target cells. Moreover, in the case of OVA, the presence of the expected target peptides was demonstrated directly by amino acid sequence and mass spectrometric analysis. The results demonstrate that the pure 20S proteasome is capable of generating antigenic peptides from two proteins for presentation by class I molecules without the participation of additional components of the protein degradation system. This finding is consistent with the hypothesis of proteasome involvement in Ag processing in vivo.

The 59-kDa dystrophin-associated protein triplet (59-DAP) is a component of the dystrophin-glycoprotein complex which may directly associate with dystrophin. The cDNA encoding one component (59-1 DAP) of the 59-DAP triplet has now been cloned from rabbit skeletal muscle. The deduced amino acid sequence of 59-1 DAP predicts a 505-amino acid polypeptide containing nine potential phosphorylation sites and no predicted transmembrane domains. This is consistent with the 59-1 DAP being a peripheral membrane protein associated with the cytoplasmic face of the dystrophin-glycoprotein complex. Affinity-purified antibodies against rabbit 59-1 DAP fusion proteins only recognize the lowest band of the 59-DAP triplet in skeletal muscle sarcolemma and isolated dystrophin-glycoprotein complex. The tissue-specific expression of 59-1 DAP mRNA, which is most prominent in skeletal and cardiac muscle and is also detected in brain, parallels that of dystrophin but not of utrophin. Levels of 59-1 DAP mRNA are unaffected in mdx mouse skeletal and cardiac muscles, although all dystrophin-associated proteins, including 59-DAP, are greatly reduced in mdx mouse skeletal muscle. However, in mdx mouse cardiac muscle, the up-regulation of utrophin preserves all dystrophin-associated proteins except 59-DAP. Our results suggest that the 59-DAP triplet may contain different protein species and that the 59-1 DAP may associate more specifically with dystrophin than with utrophin.

The hydrolysis of phosphatidylcholine by phospholipase D (PLD) results in the production of phosphatidic acid and choline. An assay that uses an exogenous substrate was developed to measure this activity in membranes and solubilized preparations from HL60 cells. A cytosolic factor markedly enhanced PLD activity in membranes and was essential for GTP gamma S-dependent stimulation of an enriched preparation of PLD. The factor was purified to homogeneity from bovine brain cytosol and identified as a member of the ADP-Ribosylation Factor (ARF) subfamily of small G proteins. Subsequently, recombinant myristoylated ARF1 was found to be a better activator of PLD activity than was the nonmyristoylated form. ARF proteins have been implicated recently as factors for regulation of intracellular vesicle traffic. The current finding suggests that PLD activity plays a prominent role in the action of ARF and that ARF may be a key component in the generation of second messengers via phospholipase D.

The primary amino acid sequence for a highly abundant junctional sarcoplasmic reticulum glycoprotein (triadin) has been deduced from the cDNA sequence. Based on both biochemical analysis and the predicted amino acid sequence we suggest that this protein is an intrinsic membrane glycoprotein containing a single transmembrane domain that separates the protein into cytoplasmic and luminal domains. The cytoplasmic domain is proposed to contain the amino-terminal 47 amino acids. The remainder of the protein including the carboxyl terminus is proposed to be found within the lumen of the sarcoplasmic reticulum and contains an extremely high concentration of basic residues. Protease analysis of intact triads was consistent with the topological predictions. Western and Northern blots suggest that the protein is specifically expressed in skeletal muscle and not cardiac muscle or brain. The abundance and localization of this protein suggest that it plays an important regulatory or structural role in excitation-contraction coupling in skeletal muscle.

Chemical modification of the proteasome with N-ethylmaleimide (NEM) was performed for the purpose of identifying amino acid residues that play a role in the enzyme's proteolytic function. Modification of the proteasome with NEM specifically and irreversibly suppressed one of the three peptidase activities of the enzyme, viz., the "trypsin-like" activity. Leupeptin, a reversible competitive inhibitor of this activity, protected the activity from NEM inactivation, suggesting that NEM modifies a residue in the leupeptin binding site. Comparisons of enzyme samples labeled with [14C]NEM either in the presence or in the absence of leupeptin allowed the identification of a proteasome subunit containing an NEM-modified, leupeptin-protected cysteinyl residue. The leupeptin protection experiments suggest that residues of this subunit contribute to the active site responsible for the proteasome's trypsin-like activity. This subunit was purified by reverse-phase high-performance liquid chromatography. Peptide mapping and N-terminal amino acid sequencing were employed to acquire information about the primary structure of the subunit, including the sequence surrounding the leupeptin-protected cysteinyl residue. The sequencing data suggest that this proteasome subunit is evolutionarily related to other proteasome subunits that have been sequenced, which show no homology to other known proteases. The assignment of a catalytic function to a member of the proteasome family supports the hypothesis that proteasome subunits represent a structurally and possibly mechanistically novel group of proteases.

Ca2+/calmodulin-dependent myosin light chain kinase phosphorylates the regulatory light chain of myosin. Rabbit skeletal muscle myosin light chain kinase also catalyzes a Ca2+/calmodulin-dependent autophosphorylation with a rapid rate of incorporation of 1 mol of 32P/mol of kinase and a slower rate of incorporation up to 1.52 mol of 32P/mol. Autophosphorylation was inhibited by a peptide substrate that has a low Km value for myosin light chain kinase. Autophosphorylation at both rates was concentration-independent, indicating an intramolecular mechanism. There were no significant changes in catalytic properties toward light chain and MgATP substrates or in calmodulin activation properties upon autophosphorylation. After digestion with V8 protease, phosphopeptides were purified and sequenced. Two phosphorylation sites were identified, Ser 160 and Ser 234, with the former associated with the rapid rate of phosphorylation. Both sites are located amino terminal of the catalytic domain. These results indicate that the extended "tail" region of the enzyme can fold into the active site of the kinase.

Each subunit of calpain (EC 3.4.22.17) is proteolytically modified when the enzymes are exposed to calcium. These cleavages appear to be important for regulating the proteolytic activity and calcium-sensitivity of the proteinases. We have synthesized peptides that correspond to the sites of autoproteolytic modification within the catalytic subunit of each calpain. Polyclonal antisera raised against these peptides are highly specific for the unmodified catalytic subunit of each calpain. The antiserum specific for the N-terminal epitope of milli-calpain was used to demonstrate an inverse relationship between the presence of this N-terminal peptide and casein hydrolysis. The antiserum specific for the N-terminal epitope of micro-calpain was used to demonstrate proteolytic modification of the catalytic subunit of mu-calpain in rat erythrocytes treated with ionomycin and calcium.

The molecular cloning and eukaryotic cell expression of the complementary DNA for human neutrophil acyloxyacyl hydrolase (AOAH) are described. AOAH is a leukocyte enzyme that selectively removes the secondary (acyloxyacyl-linked) fatty acyl chains from the lipid A region of bacterial lipopolysaccharides (endotoxins), thereby detoxifying the molecules. The two disulfide-linked subunits of the enzyme are encoded by a single mRNA. The amino acid sequence of the protein contains a lipase consensus sequence in the large subunit and a region in the small subunit that is similar to the saposins, cofactors for sphingolipid hydrolases. The recombinant enzyme, like native AOAH, hydrolyzes secondary acyl chains from more than one position on the lipopolysaccharide backbone. Acyloxyacyl hydrolase is a novel two-component lipase that, by deacylating lipopolysaccharides, may modulate host inflammatory responses to Gram-negative bacterial invasion.

Macropain (proteasome) is a high-molecular-weight proteinase complex composed of at least 13 electrophoretically distinct subunits. Previous work, including peptide mapping and limited amino acid sequencing, suggested that most of the subunits belong to an evolutionarily related group of different gene products (Lee et al. (1990) Biochim. Biophys. Acta. 1037, 178-185). In order to define the extent and pattern of subunit relatedness, and to determine the structural basis for possible similarities and differences in subunit functions, we are deducing the primary structures of macropain subunits by cDNA cloning and DNA sequence analysis. We report here the primary structures of four subunits. The data clearly demonstrate that the proteins represent different, but homologous gene products. Surprisingly, no evidence for homology with any other protein, including proteinases, was obtained. These results suggest that macropain is comprised of a previously unidentified family of evolutionarily related polypeptides. Because biochemical data indicate that macropain contains several different proteinase activities, the current results raise the possibility that the macropain complex is composed of a group of novel proteinases, distinct from those of other structurally identifiable proteinase families.

We examined the heat shock response of the pathogenic spirochetes Treponema pallidum, Borrelia burgdorferi, and Leptospira interrogans and certain saprophytic spirochetes. Cellular proteins synthesized after shifts to higher temperatures were [35S]methionine labeled and analyzed by gel electrophoresis and fluorography. Only T. pallidum failed to exhibit an obvious heat shock response. GroEL and DnaK homologs were identified in the various species, although these proteins were not thermoinducible in T. pallidum or Treponema denticola. DNA hybridization studies indicate that spirochetal groEL and dnaK genes are highly conserved.

The peptides generated from the degradation of the oxidized B chain of bovine insulin by the multiproteinase complex macropain (proteasome) have been analyzed by reverse-phase peptide mapping and identified by N-terminal amino acid sequencing and composition analysis. Six of the 29 peptide bonds in the insulin B chain were found to be rapidly cleaved by macropain. The catalytic center that cleaves the Gln4-His5 bond could be distinguished from the center or centers that cleave the other preferred bonds by its specific susceptibility to inhibition by leupeptin, antipain, chymostatin, and pentamidine, suggesting that macropain utilizes at least two distinct catalytic centers for the degradation of this model polypeptide. The same effectors simultaneously enhance the rate of cleavage at the other susceptible sites in insulin B. The quantitative characteristics of this effect indicate that different catalytic centers of the complex may be functionally coupled, possibly by an allosteric mechanism or possibly by a mechanism in which binding to the catalytic centers is preceded by a rate-limiting binding of the substrate to a site or sites on the enzyme distinct from the catalytic centers. The kinetics of insulin B chain degradation indicate that macropain can catalyze sequential hydrolysis of peptide bonds in a single substrate molecule via a reaction pathway that involves channeling of peptide intermediates between different catalytic centers within the multienzyme complex. This capacity for channeling may confer potential physiological advantages of increasing the efficiency of amino acid recycling and reducing the pool sizes of peptide intermediates that are generated during the degradation of polypeptides in the intracellular milieu.

A gene encoding PMP47, a peroxisomal integral membrane protein of the methylotrophic yeast Candida boidinii, was isolated from a genomic library. DNA sequencing of PMP47 revealed an open reading frame of 1269 base pairs capable of encoding a protein of 46,873 Da. At least two membrane-spanning regions in the protein are predicted from the sequence. Since the 3 amino acids at the carboxyl terminus are -AKE, PMP47 lacks a typical peroxisomal sorting signal. No significant similarities in primary structure between PMP47 and known proteins were observed, including PMP70, a rat peroxisomal membrane protein whose sequence has recently been reported (Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T. (1990). J. Biol. Chem. 265, 4534-4540). In order to study the import and assembly of PMP47 into peroxisomes by genetic approaches, the gene was expressed in the yeast Saccharomyces cerevisiae. When PMP47 was expressed in cells grown on oleic acid to induce peroxisomes, the protein was observed exclusively in peroxisomes as determined by marker enzyme analysis of organelle fractions. Most of the PMP47 co-purified with the endogenous peroxisomal membrane proteins on isopycnic sucrose gradients. Either in the native host or when expressed in S. cerevisiae, PMP47 was not extractable from peroxisomal membranes by sodium carbonate at pH 11, indicating an integral membrane association. These results indicate that PMP47 is competent for sorting to and assembling into peroxisomal membranes in S. cerevisiae.

A protein of approximately 40 kDa was the major Ca2(+)-binding protein purified by Ca2(+)-dependent hydrophobic affinity chromatography from the cell lysates and conditioned media of RAW macrophages. Other Ca2(+)-binding proteins, including several annexins (calelectrins), S100-like proteins, and calmodulin, were less abundant and preferentially found in the cell lysates. Amino acid sequences of tryptic fragments from the purified 40-kDa protein revealed its identity to gCap39, an actin-binding protein encoded by a cDNA isolated on the basis of its homology with gelsolin. When an expression vector containing the gCap39 coding region was transfected into COS cells, high levels of gCap39 were found in both the cells and conditioned media, whereas annexins were only present in the cells. gCap39 could also be purified from human plasma where it appeared to be a minor component. No signal sequence was detected in the primary structure of gCap39 and the secreted and intracellular forms of gCap39 are of identical size, suggesting that unlike gelsolin, the mechanism of gCap39 secretion may not depend on a signal sequence. The high concentration of gCap39 in macrophages and its constitutive secretion as well as intracellular retention suggest that this protein may have a dual role in macrophage function, namely that of a Ca2(+)- and polyphosphoinositide-regulated intracellular modulator of the cytoskeleton as well as that of a secreted protein involved in the clearance of actin from the extracellular environment.

Growth cone particle (GCP) membranes prepared from fetal day 17 rat brain are comprised of 5 major polypeptides as analyzed by SDS-PAGE: tubulin (p52), actin (p42), pp46/GAP-43 and two unidentified species, p38 and p34. Antibodies specific for the alpha- and beta-subunits of G proteins recognize p38 and p34, respectively, on immunoblots following one- and two-dimensional electrophoretic separation. That G protein subunits comprise major species of GCP membrane-associated polypeptides suggests a role for G proteins in transmembrane signaling in nerve growth cones.

The methanol-insoluble, heat-stable enterotoxin of Escherichia coli synthesized by clinical strains or strains that harbor the cloned gene was shown to be an extracellular polypeptide. The toxin (STB) was first detected as an 8,100-Mr precursor (pre-STB) that was converted to a transiently cell-associated 5,200-Mr form. Proteolytic conversion of pre-STB to STB was shown to be inhibited by the proton motive force uncoupler carbonyl cyanide m-chlorophenylhydrazone and did not occur in a secA background. After STB was detected as a cell-associated molecule, an extracellular form with identical electrophoretic mobility became apparent. The results suggest that there is no proteolytic processing during the mobilization of STB from the periplasm to the culture supernatant. The determined amino acid sequence of STB coincides fully with the 48 carboxy-terminal amino acids inferred from the DNA sequence. The 23 amino-terminal residues inferred from the DNA sequence were absent in the mature toxin.