Identification of a pharmacologically distinct prostaglandin H synthase in cultured epithelial cells.

Nonsteroidal anti-inflammatory drugs inhibit the action of prostaglandin H synthase (PGH synthase), and this effect may constitute the basis for therapeutic and idiosyncratic responses to these agents. We found that aspirin treatment of cultured ovine tracheal epithelial cells blocked PGH synthase-catalyzed formation of PG as expected but also caused a dose-dependent increase in 15-hydroxyeicosatetraenoic acid (15-HETE) production from arachidonic acid. In contrast, aspirin caused only inhibition of PG production without enhancing 15-HETE formation in ovine seminal vesicle and other tissues. The 15-HETE formed by aspirin-treated ovine tracheal epithelial cells was generated by a PGH synthase-dependent mechanism because: (i) the 15-HETE forming activity was just as sensitive as PG forming activity to selective inhibition by indomethacin; (ii) both 15-HETE and PG forming activities were quantitatively immunoprecipitated (depleted from supernatants and recovered in immune complex pellets) by a specific anti-PGH synthase antiserum. Additional immunoprecipitation experiments indicated that anti-PGH synthase monoclonal antibodies (cyo-1 and cyo-5) raised against the aspirin-inhibited form of the enzyme (contained in seminal vesicle) did not recognize the aspirin-stimulated 15-HETE-forming PGH synthase (contained in cultured epithelial cells). Thus, sequential immunoprecipitation of cultured epithelial cell material first with excess cyo-1 followed by anti-PGH synthase antiserum indicated that two isoforms of PGH synthase were expressed in these cells. SDS-polyacrylamide gel electrophoresis of immunoprecipitated PGH synthase from cultured epithelial cells revealed distinct protein bands for each form of the enzyme (M(r) = 70,000 and 72,000). The identification of a distinct PGH synthase which may be modified by aspirin so that selective oxygenation of fatty acid substrate is enhanced (while PG formation is inhibited) indicates that isozymes of PGH synthase exist which are pharmacologically distinct.     

Identification of clathrin and clathrin adaptors on tubulovesicles of gastric acid secretory (oxyntic) cells

-Adaptin and clathrin heavy chain were identified ontubulovesicles of gastric oxyntic cells with the anti--adaptinmonoclonal antibody (MAb) 100/3 and an anti-clathrin heavy chain MAb(MAb 23), respectively. In Western blots, crude gastric microsomes fromrabbit and rat and density gradient-purified, H-K-ATPase-rich microsomes from these same species were immunoreactive for-adaptin and clathrin. In immunofluorescent labeling of isolatedrabbit gastric glands, anti--adaptin and anti-clathrin heavy chainimmunoreactivity appeared to be concentrated in oxyntic cells. Inprimary cultures of rabbit oxyntic cells, the immunocytochemicaldistribution of -adaptin immunoreactivity was similar to that of thetubulovesicular membrane marker in oxyntic cells, the H-K-ATPase.Further biochemical characterization of the tubulovesicular-adaptin-containing complex suggested that it has a subunitcomposition that is typical of that for a clathrin adaptor: in additionto the -adaptin subunit, it contains a -adaptin subunit and othersubunits of apparent molecular masses of 50 kDa and 19 kDa. Fromsolubilized gastric microsomes from rabbit, -adaptin could becopurified with the major cargo protein of tubulovesicles, theH-K-ATPase. Thus this tubulovesicular coat may bind directly to theH-K-ATPase and may thereby mediate the regulated trafficking of theH-K-ATPase at the apical membrane of the oxyntic cell during thegastric acid secretory cycle. Given the similarities of the regulatedtrafficking of the H-K-ATPase with recycling of cargo through theapical recycling endosome of many epithelial cells, we propose thattubulovesicular clathrin and adaptors may regulate some part of anapical recycling pathway in other epithelial cells.

     

Guanidinoacetate methyltransferase activity in tissues and cultured cells

Guanidinoacetate methyltransferase, the enzyme catalyzing the last step in creatine biosynthesis, has previously been considered to be restricted to a few tissues, but it has been found to occur in the cultured cells H4Az C2 rat hepatoma, N4TG1 mouse neuroblastoma, and IMR-90 human fetal lung fibroblast, as well as in skeletal and cardiac muscle of the rat. Activity was highest in the hepatoma, but tissues and cultured cells of nonhepatic origin had 5-20% of the activity of rat liver. Dialyzed 100,000g supernatants prepared from cultured cells or skeletal muscle tissue yielded values for apparent Km in the range of 1.2-3.4 microM for S-adenosylmethionine and 0.050-0.096 mM for guanidinoacetate. Intact monolayers of the three types of cultured cells converted labeled guanidinoacetate in the culture medium to creatine, which was identified by chromatographic behavior and by reaction with creatine kinase. The amounts of guanidinoacetate converted to creatine by fibroblasts and neuroblastoma cells during an 18-h period of incubation suggested that synthesis was proceeding at rates approaching Vmax, even in medium containing the relatively low physiological concentrations of guanidinoacetate. Fibroblast and neuroblastoma cell monolayers also have the capacity to take up creatine provided in the culture medium. The amounts of creatine taken up by monolayers of those cells were measured under the same conditions that were used for measurement of creatine synthesis. Comparison of the amounts of creatine synthesized with the amounts taken up showed that synthesis can make a significant contribution to intracellular pools of creatine plus phosphocreatine in fibroblasts and neuroblastoma cells.     

Ultrastructural immunocytochemical localization of clathrin in cultured fibroblasts

Using an affinity-purified antibody prepared against the major coat protein of brain-coated vesicles, clathrin, we have localized this protein by ultrastructural immunocytochemistry in Swiss 3T3 cultured fibroblasts. Fixation, permeabilization, and labeling were performed using the EGS and ferritin bridge labeling techniques. Localization of clathrin was detected on the coated regions of both the plasma membrane and the GERL apparatus. Almost no clathrin was found in the cytosol or in association with any other organelles. A very low concentration of labeling was occasionally seen randomly distributed on the inner surface of the plasma membrane and reticular membranous structures near the plasma membrane. The significance of these results in the role of the clathrin-coated regions in receptor-mediated endocytosis and Golgi function are discussed.     

Meta-vinculin distribution in adult human tissues and cultured cells

Meta-vinculin distribution in adult human tissue was studied by immunoblotting technique. Meta-vinculin was found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, kidney and cultured cells - macrophages, foreskin fibroblasts, peripheral blood lymphocytes and vascular endothelial cells. In the primary culture of smooth muscle cells from human aorta the meta-vinculin/vinculin ratio was reduced, and on the onset of cell division meta-vinculin could hardly be detected. Subcultured smooth muscle cells from human aorta did not contain meta-vinculin. The data show that the presence of meta-vinculin is characteristic of 'contractile' smooth muscle cells rather than of proliferating in vitro.     

Stable expression of an active soluble recombinant form of human fucosyltransferase IX in Spodoptera frugiperda Sf9 cells

A secretory form of human α3-fucosyltransferase IX (sFUT9) was overexpressed in Spodoptera frugiperda (Sf9) insect cells using the stable expression vector pIB/V5-His-TOPO and the signal sequence of human interleukin 2 for efficient secretion. sFUT9 was active and its three potential N-glycosylation sites were occupied. sFUT9 efficiently fucosylated the type II acceptors Galbeta4GlcNAC-R and Fucalpha2Galbeta4GlcNAc-R (R = (CH2)3NHCO(CH2)5–NH-biotin) but not the corresponding sialylated acceptor, and only very poorly the type I (Galbeta3GlcNAc-R) related acceptors. sFUT9 showed a clear preference for glycoproteins containing type II acceptors, with values of 121, 113 and 110 microU/million cell for asialofetuin, erythropoietin and asialoerythropoietin, respectively, values approximately 11-fold higher than those obtained for the small acceptors.     

Methionine adenosyltransferase activity in cultured cells and in human tissues

We have investigated methionine adenosyltransferase activity (MAT) in extracts of a variety of normal and malignant human tissues and cultured cell lines. MAT activity assayed from 17 different cultured cell lines varied to a great extent. Ramos (human, Burkitt's lymphoma) and EL4 (mouse, T cell lymphoma) cell showed MAT activity near 300 pmol/mg per min. Daudi (human, Burkitt's lymphoma) and almost all monolayer cells had MAT activity below 100 pmol/mg per min. Human peripheral blood lymphocytes had MAT activity of 36 pmol/mg permin. The MAT activity of the cell lines can be related to doubling time: cell lines with short doubling times have much higher MAT activity than other cell lines. A large variation in MAT activity in different human tissues was observed. In autopsy samples MAT activity was highest in the brain and in the colon. Malignant tissue samples gave much higher MAT activity than normal tissues. Lung cancer (carcinoma squamocellulare pulmonis) had MAT activity of 30.7 pmol/mg per min, while in normal lung it was 2.4 pmol/mg per min.     

Subcellular distribution of clathrin in cultured hypothalamic neurons.

The subcellular distribution of clathrin has been examined in developing hypothalamic neurons cultured in a chemically defined medium up to synapse formation (12-13 days in vitro) and exposed, or not, to a depolarizing concentration of KCl (60 mM for 3 min) followed, or not, by a return to control KCl concentration (3 mM KCl for 3 min). Previous studies have shown that such treatments induce in synaptic boutons a rapid vesicle depletion followed by massive restoration. Using an enzyme immunoassay, we have compared the relative proportion of assembled and unassembled pools of clathrin as a function of exposure to depolarizing or repolarizing concentrations of KCl. In parallel we have localized clathrin at the electron microscopic level using immunoperoxidase. Clathrin concentration in culture is lower (0.36 vs 0.75%) and the proportion of unassembled clathrin is much higher than in the adult brain (82 vs 14%). These proportions were not affected by depolarizing or repolarizing treatments. Morphologically clathrin was exclusively detected in two neuron compartments: perikarya and synaptic boutons. In perikarya clathrin was localized as a thick coat on plasma membrane coated pits and in the Golgi zone on coated buds and vesicles, presumably located in a trans compartment. In synaptic boutons clathrin immunoreaction was found as an irregular thin rim around synaptic vesicles, whatever the polarization state of the cells, but coated vesicles were extremely rare. Taken together these findings raise the problem of the functional meaning and localization of the large unassembled pool of clathrin in such neurons and question its role in vesicular traffic in synaptic boutons.     

Identification of novel and distinct binding sites within tenascin-C for soluble and fibrillar fibronectin

Interactions between fibronectin and tenascin-C within the extracellular matrix provide specific environmental cues that dictate tissue structure and cell function. The major binding site for fibronectin lies within the fibronectin type III-like repeats (TNfn) of tenascin-C. Here, we systematically screened TNfn domains for their ability to bind to both soluble and fibrillar fibronectin. All TNfn domains containing the TNfn3 module interact with soluble fibronectin. However, TNfn domains bind differentially to fibrillar fibronectin. This distinct binding pattern is dictated by the fibrillar conformation of FN. TNfn1-3, but not TNfn3-5, binds to immature fibronectin fibrils, and additional TNfn domains are required for binding to mature fibrils. Multiple binding sites for distinct regions of fibronectin exist within tenascin-C. TNfn domains comprise a binding site for the N-terminal 70-kDa domain of fibronectin that is freely available and a binding site for the central binding domain of fibronectin that is cryptic in full-length tenascin-C. The 70-kDa and central binding domain regions are key for fibronectin matrix assembly; accordingly, binding of several TNfn domains to these regions inhibits fibronectin fibrillogenesis. These data highlight the complexity of protein-protein binding, the importance of protein conformation on these interactions, and the implications for the physiological assembly of complex three-dimensional matrices.     

DNA repair synthesis in cultured fish and human cells exposed to fish S9-activated aromatic hydrocarbons

Unscheduled DNA repair synthesis was measured autoradiographically in cultured rainbow trout gonad (RTG) and human fibroblast (HF) cells following exposure to aflatoxin B1 (AFB1), 3,4-benzopyrene (BP), 1,2,5,6-dibenzanthracene (DBA), 1,2-benzanthracene (BA) and pyrene (PY) activated with S9 prepared from rainbow trout liver. S9 from rainbow trout injected with Arochlor 1254 or an oil extract was compared with S9 from Fischer rats injected with Arochlor 1254 for the ability to activate AFB1 and cause DNA repair in RTG and HF cells. All three types of S9 activated AFB1, but the measured DNA repair response was greater in the HF cells. A significant grain count response was found following exposure of HF cells to fish S9-activated BP. Using assay conditions which enhance fish cell grain counts, a significant level of DNA repair was also found in RTG cells exposed to fish S9-activated BP. Marginal but statistically significant amounts of DNA repair were elicited in HF and RTG cells exposed to rainbow trout S9-activated BA and DBA, but no response was detected following PY exposure. Fish S9 was found to be able to activate a series of polycyclic aromatic hydrocarbons (PAH) and cause DNA repair synthesis in both fish and mammalian cells. The magnitude of the repair response roughly parallels the carcinogenic potential of the PAHs. These results elicit trans species and phyla comparisons which help to validate fish as models for aquatic carcinogenesis research, and also demonstrate PAH DNA-damaging effects on fish DNA, adding further credence for studying the effects of these chemicals on aquatic organisms.     

Deep-etch visualization of 27S clathrin: a tetrahedral tetramer

It has recently been reported that 8S clathrin trimers or "triskelions" form larger 27S oligomers upon dialysis into low ionic strength buffers (Prasad, K., R. E. Lippoldt, H. Edelhoch, and M. S. Lewis, 1986, Biochemistry, 25:5214-5219). Here, deep-etch electron microscopy of the 27S species reveals that they are closed tetrahedra composed of four clathrin triskelions. This was determined by two approaches. First, standard quick-freezing and freeze-etching of unfixed 27S species suspended in 2 mM 2-(N-morpholino)ethane sulfonic acid (MES) buffer, pH 5.9, yielded unambiguous images of tetrahedra that measured 33 nm on each edge. Second, the technique of freeze-drying molecules on mica (Heuser, J. E., 1983, J. Mol. Biol., 169:155-195) was modified to overcome the low affinity of mica in 2 mM MES, by pretreating the mica with polylysine. Thereafter, 27S species adsorbed avidly to it and collapsed into characteristic configurations containing four globular domains, each linked to the others by three approximately 33-nm struts. The globular domains look like vertices of deep-etched clathrin triskelions and the links, numbering 12 in all, look like four sets of triskelion legs. New light scattering and equilibrium centrifugation data confirm that 27S polymer is four times as massive as one clathrin triskelion. We conclude that in conditions that do not favor the formation of standard clathrin cages, low affinity interactions lead to closed, symmetrical assemblies of four triskelions, each of which assumes a unique puckered, straight-legged configuration to create the edges of a tetrahedron. Tetrahedra are similar in construction to the cubic octomers of clathrin recently found in ammonium sulfate solutions (Sorger, P. K., R. A. Crowther, J. T. Finch, and B. M. F. Pearse, 1986, J. Cell Biol., 103:1213-1219) but are still smaller, involving only half as many clathrin triskelions.     

Identification of a major soluble protein in mitochondria from nonphotosynthetic tissues as NAD-dependent formate dehydrogenase.

In many plant species, one of the most abundant soluble proteins (as judged by two-dimensional polyacrylamide gel electrophoresis) in mitochondria from nongreen tissues is a 40-kD polypeptide that is relatively scarce in mitochondria from photosynthetic tissues. cDNA sequences encoding this polypeptide were isolated from a lambda gt11 cDNA expression library from potato (Solanum tuberosum L.) by screening with a specific antibody raised against the 40-kD polypeptide. The cDNA sequence contains an open reading frame of 1137 nucleotides whose predicted amino acid sequence shows strong homology to an NAD-dependent formate dehydrogenase (EC 1.2.1.2) from Pseudomonas sp. 101. Comparison of the cDNA sequence with the N-terminal amino acid sequence of the mature 40-kD polypeptide suggests that the polypeptide is made as a precursor with a 23-amino acid presequence that shows characteristics typical of mitochondrial targeting signals. The identity of the polypeptide was confirmed by assaying the formate dehydrogenase activity in plant mitochondria from various tissues and by activity staining of mitochondrial proteins run on native gels combined with antibody recognition. The abundance and distribution of this protein suggest that higher plant mitochondria from various nonphotosynthetic plant tissues (tubers, storage roots, seeds, dark-grown shoots, cauliflower heads, and tissues grown in vitro) might contain a formate-producing fermentation pathway similar to those described in bacteria and algae.     

Processing of soluble elastin in cultured neonatal rat smooth muscle cells

The synthesis and extracellular deposition of elastin by cultured neonatal rat aorta smooth muscle cells has been followed. The addition of beta-aminopropionitrile to the culture medium promotes accumulation of soluble precursors of elastin. Under such conditions, a protein possessing characteristics of a soluble elastin precursor with an apparent molecular weight of 77,000 was detected and partially purified. Pulse-chase studies suggested that this 77-kDa protein undergoes an extracellular, enzymatically catalyzed process to a 71-kDa protein. This 71-kDa protein is strikingly similar to tropoelastins isolated from other tissue systems, in which no evidence for higher molecular weight soluble precursors is at present available. Data presented in this communication suggest that the 77-kDa protein, which we have designated protropoelastin, represents a precursor to the tropoelastin moiety produced in the neonatal rat smooth muscle cell culture.     

Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast

Yeast produce two classes of secretory vesicles (SVs) that differ in both density and cargo protein content. In late-acting secretory mutants (e.g. snc1(ala43) and sec6-4), both low- (LDSV) and high-density (HDSV) classes of vesicles accumulate at restrictive temperatures. Here, we have found that disruptions in the genes encoding a dynamin-related protein (VPS1) or clathrin heavy chain (CHC1) abolish HDSV production, yielding LDSVs that contain all secreted cargos. Interestingly, disruption of the PEP12 gene, which encodes the t-SNARE that mediates all Golgi to pre-vacuolar compartment (PVC) transport, also abolishes HDSV production. In contrast, deletions in genes that selectively confer vacuolar hydrolase sorting to the PVC or protein transport to the vacuole (i.e. VPS34 and VAM3, respectively) have no effect. Thus, one branch of the secretory pathway in yeast involves an intermediate sorting compartment and has a specific requirement for clathrin and a dynamin-related protein in SV biogenesis.     

Th1 and Th2 cells form morphologically distinct immunological synapses

The arrangement of molecules at the interface between T cells and APCs is known as the immunological synapse (IS). We conducted experiments with supported planar bilayers and transfected fibroblast APC to examine the IS formed by polarized Th1 and Th2 cells. Th1 cells formed typical "bull's-eye" IS with a ring of adhesion molecules surrounding MHC/TCR interactions at all Ag concentrations tested, while Th2 cells formed multifocal IS at high concentrations of Ag. At low Ag concentrations, the majority of Th2 cells formed IS with a compact, central accumulation of MHC/TCR, but ICAM-1 was not excluded from the center of the IS. Additionally, CD45 was excluded from the center of the interface between Th1 cells and APC, while CD45 was found at the center of the multifocal IS formed by Th2 cells. Finally, phosphorylated signaling molecules colocalized with MHC/TCR to a greater extent in Th2 IS. Together, our results indicate that the IS formed by Th1 and Th2 cells are distinct in structure, with Th2 cells failing to form bull's-eye IS.     

A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice

Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.     

Identification of both Gi2 and a novel, immunologically distinct, form of Go in rat myometrial membranes

Immunoblotting of rat myometrial membranes with an antiserum (SG1) which recognizes the alpha-subunits of both Gi1 and Gi2 indicated the presence of detectable levels of an apparently single form of some 40 kDa. A second antiserum (LE2) specific for Gi2 also recognized this protein, confirming its identity. Immunoblotting of the myometrial membranes with a series of antipeptide (OC1, IM1, ON1) and polyclonal (RV3) antisera, all of which recognize rat brain Go, produced a more complex pattern. Antisera OC1 and ON1 recognized a single polypeptide which essentially comigrated with rat brain Go. In contrast, antisera RV3 and IM1 did not recognize the myometrial protein. These data provide evidence for the presence of Gi2 and of a novel G-protein, related immunologically to Go, in rat myometrial membranes.     

Expression of intermediate filaments (IF) in tissues and cultured cells

Intermediate filaments are found in most nucleated cells as part of their cytoskeleton. Intermediate filaments are formed by different proteins in cells of major tissues types. Therefore, antibodies against intermediate filaments can be used in tissue typing, in the analysis of cell lineages during development and in the elucidation of the origin of unknown tumors.