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PubMed1995

Morphine tolerance and physical dependence: reversal of opioid inhibition to enhancement of cyclic AMP formation.

This laboratory has previously demonstrated that the mu-selective opiate receptor agonist sufentanil can produce a naloxone-reversible increase or decrease in the stimulated formation of cyclic AMP (cAMP) in the myenteric plexus, depending on the concentration of opioid used. On the basis of these results, it was suggested that mu-opiate receptors are positively as well as negatively coupled to adenylyl cyclase. In the present study, the effect of chronic morphine exposure, in vivo, on the magnitude of electrically stimulated formation of cAMP and its modulation by sufentanil was investigated. In chronic morphine-treated preparations, the magnitude of electrically stimulated cAMP formation, while in the presence of an inhibitory (10(-6) M) concentration of sufentanil, is indistinguishable from the formation that occurs in opiate-naive preparations (in the absence of exogenous opioid). This indicates that the negative modulation of stimulated enteric cAMP formation by sufentanil manifests tolerance. Paradoxically, however, in "addicted tissue" the magnitude of the increase in cAMP formation produced by electrical stimulation in the presence of a previously inhibitory concentration of sufentanil is significantly larger than in its absence. Thus, the equivalence between the magnitude of stimulation-induced increase in cAMP formation observed in naive versus tolerant/dependent tissue, while in the presence of sufentanil, is due to the ability of an originally inhibitory concentration of opioid to enhance or facilitate stimulated formation of cAMP. It is suggested that tolerance/dependence to the opioid inhibition of stimulated cAMP formation results not only from the loss of inhibitory potency but also from its reversal to enhancement.

Animals↗
PubMed1998

Rac1 mediates dendrite formation in response to melanocyte stimulating hormone and ultraviolet light in a murine melanoma model.

Melanocytes are pigment producing cells that reside in the basal layer of the epidermis, and form multiple long dendritic processes that transport melanosomes from the melanocyte cell body to the dendritic tips, and then to keratinocytes. Dendrite formation requires actin polymerization in the newly forming dendrite, and dendrite formation in melanocytes is stimulated by hormones and ultraviolet light. The rho-subfamily of monomeric guanosine triphosphate-binding proteins is implicated in remodeling the cellular actin cytoskeleton, resulting in the formation of filopodia, lamellipodia, and stress fibers, as well as in oncogenesis and activation of the Jun/p38 mitogen activated kinase cascade. In this paper we show that rac1 induces the formation of dendrite-like structures when activated mutants are transiently expressed in B16F1 murine melanoma cells and in four human melanoma cell lines. Activated mutants of cdc42 and rhoA induced the formation of filopodia and stress fibers, respectively, in B16F1 cells, but not dendrites. A dominant negative inhibitor of rac1 abrogated the ability of alpha-melanocyte stimulating hormone, a peptide hormone known to stimulate melanocyte dendrite formation, and ultraviolet light, to induce dendrite formation in B16F1 cells, and alpha-melanocyte stimulating hormone and ultraviolet light stimulated the localization of rac1 to dendrite cell membranes. These results suggest that rac1 is an important signaling intermediate in dendrite formation in B16F1 cells, and that rac1 mediates the well-known ability of alpha-melanocyte stimulating hormone and ultraviolet light to induce dendrite formation.

Actins↗
PubMedDate not supplied

The Academy of Managed Care Pharmacy Format for Formulary Submissions: an evolving standard--a Foundation for Managed Care Pharmacy Task Force report.

OBJECTIVE: The objective of this report is to provide, in detail, the evidentiary requirements contained in Version 2.0 of the Academy of Managed Care Pharmacy's (AMCP's) Format for Formulary Submissions and to elaborate on several key issues regarding the use of the Format that users, potential users, and pharmaceutical manufacturers have raised since AMCP published the first version of the guidelines in October 2000. BACKGROUND: The AMCP published its Format for Formulary Submissions in October 2000. The AMCP leadership and its members were motivated to develop these guidelines by a growing need to ensure that any increased utilization of medications, biopharmaceuticals, and vaccine products was appropriate and that newer products would bring added clinical and economic value to covered populations. Since publication of the Format, it has garnered nationwide publicity and attracted considerable positive and negative attention. As adoption of the AMCP Format has spread, manufacturers have begun to standardize the framework within which they present population-specific data. Since publication of the AMCP Format, the AMCP and the Foundation for Managed Care Pharmacy (FMCP) have continuously sought input from pharmaceutical manufacturers and health-system pharmacists through various venues to improve and clarify the process. Version 2.0 is the first attempt to address user's comments and concerns. METHODS: The majority of the article text is taken directly from Version 2.0 of the AMCP Format for Formulary Submissions published by the AMCP in October 2002. CONCLUSION: The AMCP and the FMCP believe that the AMCP Format is a tool that will help health systems establish a record of commitment to rational decision making, thus gaining the confidence of patients, clinicians, and members. While providing manufacturers a vehicle for communicating the scientifically based value of their product to a health system, the required evidence to substantiate that value argument allows a pharmacy and therapeutics committee to determine the clinical benefits of a drug, verify any cost savings the drug may generate, and determine the overall cost consequences to their health system. The AMCP Format for Formulary Submissions is an essential tool to evaluate medications, but requires thoughtful consideration as it is used.

Academies and Institutes↗
PubMed1996

Interleukin 1 beta markedly stimulates nitric oxide formation in the absence of other cytokines or lipopolysaccharide in primary cultured rat hepatocytes but not in Kupffer cells.

To investigate whether a single inflammatory cytokine could stimulate nitric oxide formation in the absence of other cytokines or lipopolysaccharide (LPS), NO was measured by the redox chemiluminescence method in primary cultured rat hepatocytes and in rat Kupffer cells. Interleukin (IL) 1 beta, but neither IL-6, tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), nor LPS stimulated NO formation in a dose-dependent manner and induced half-maximal effects at 30 pmol/L. Maximal stimulation was achieved at 12 to 16 hours after the addition of 1I nmol/L of IL-1 beta, and was 50- to 60-fold above basal levels in rat hepatocytes. The combined effect of these cytokines with LPS or IFN-gamma on NO formation was also examined. Neither LPS nor IFN-gamma affected the IL-1 beta-induced NO formation. TNF-alpha, however, stimulated IL-1 beta-induced NO formation, while IL-6 inhibited it, although independently these cytokines had no effect on NO formation. None of the cytokines tested stimulated NO formation in cultured rat Kupffer cells. In hepatocytes, the NO formation induced by IL-l beta was blocked by both the NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA) and by IL-1 receptor antagonist (IL-1ra). Furthermore, IL-1 beta markedly increased NOS activity, and this increase in activity was accompanied by the expression of inducible NOS (iNOS) messenger RNA (mRNA). This study clearly demonstrated that IL-1 beta markedly stimulates NO formation in hepatocytes, in the absence of other cytokines or LPS.

Animals↗
PubMed2002

The enthalpy of the alanine peptide helix measured by isothermal titration calorimetry using metal-binding to induce helix formation.

The goal of this study is to use the model system described earlier to make direct measurements of the enthalpy of helix formation at different temperatures. For this we studied model alanine peptides in which helix formation can be triggered by metal (La(3+)) binding. The heat of La(3+) interaction with the peptides at different temperatures is measured by isothermal titration calorimetry. Circular dichroism spectroscopy is used to follow helix formation. Peptides of increasing length (12-, 16-, and 19-aa residues) that contain a La(3+)-binding loop followed by helices of increasing length, are used to separate the heat of metal binding from the enthalpy of helix formation. We demonstrate that (i) the enthalpy of helix formation is -0.9 +/- 0.1 kcal/mol; (ii) the enthalpy of helix formation is independent of the peptide length; (iii) the enthalpy of helix formation does not depend significantly on temperature in the range from 5 to 45 degrees C, suggesting that the heat capacity change on helix formation is very small. Thus, the use of metal binding to induce helix formation has an enormous potential for measuring various thermodynamic properties of alpha-helices.

Alanine↗
PubMed2004

Tying synaptonemal complex initiation to the formation and programmed repair of DNA double-strand breaks.

During meiosis, homologous chromosomes recombine and become closely apposed along their lengths within the synaptonemal complex (SC). In part because Spo11 is required both to make the double-strand breaks (DSBs) that initiate recombination and to promote normal SC formation in many organisms, it is clear that these two processes are intimately coupled. The molecular nature of this linkage is not well understood, but it has been proposed that SC formation initiates locally at the sites of ongoing recombination and in particular at the subset of sites that will eventually give rise to crossovers. To test this hypothesis, we examined further the relationship between DSBs and SC formation in Saccharomyces cerevisiae. SCs were monitored in a series of spo11 missense mutants with varying DSB frequencies. Alleles that blocked DSB formation gave SC phenotypes indistinguishable from a deletion mutant, and partial loss-of-function mutations with progressively more severe DSB defects caused corresponding defects in SC formation. These results strongly correlate SC formation with Spo11 catalytic activity per se. Numbers of Zip3 complexes on chromosomes, thought to represent the sites of SC initiation, also declined when Spo11 activity decreased, but in a markedly nonlinear fashion: hypomorphic spo11 alleles caused larger defects in DSB formation than in Zip3 complex formation. This nonlinear response of Zip3 closely paralleled the response of crossover recombination products. The quantitative relationship between Zip3 foci, SC formation, and crossing over strongly implicates crossover-designated recombination intermediates as the sites of SC initiation.

Chromatin↗
PubMed1979

Regulation of protein synthesis in rabbit reticulocyte lysates: additional initiation factor required for formation of ternary complex (eIF-2.GTP.Met-tRNAf) and demonstration of inhibitory effect of heme-regulated protein kinase.

Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA(f) and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA(f) complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978) Proc. Natl. Acad. Sci. USA 75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg(2+), ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA(f). In the absence of Mg(2+), however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity-hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.

Animals↗
PubMed1990

Osteoinductive factor inhibits formation of human osteoclast-like cells.

Osteoinductive factor (OIF) is a glycoprotein in bone that induces ectopic bone formation. Implantation of OIF plus transforming growth factor beta (TGF-beta) type 1 or 2 into subcutaneous tissues of rats induces formation of bone at the implantation site. Since TGF-beta is also present in bone matrix and inhibits formation of multinucleated cells that express an osteoclast phenotype in long-term human marrow cultures, we tested the effects of OIF on formation of these osteoclast-like cells to determine the effects of OIF on cells in the osteoclast lineage. We found that OIF inhibited total multinucleated cell (MNC) formation in a dose-dependent fashion and preferentially inhibited formation of MNCs that react with monoclonal antibody 23c6 (23c6-positive MNCs), an antibody that identifies osteoclasts. In addition, low concentrations of OIF in combination with low concentrations of TGF-beta acted synergistically to inhibit 23c6-positive MNC formation. The inhibition of 23c6-positive MNC formation by OIF was not mediated by prostaglandin synthesis. These data suggest that regulatory growth factors, such as OIF or TGF-beta, that are stored within the bone matrix and released when bone is resorbed can serve as natural inhibitors of osteoclast activity by inhibiting osteoclast formation.

Alprostadil↗
PubMed1994

Pseudouridine formation in U2 small nuclear RNA.

U2 small nuclear RNA contains 13 pseudouridine (psi) nucleotides, of which 11 are clustered in 5' regions involved in base-pairing interactions with other RNAs in the spliceosome. As a first step toward understanding the psi formation pathway in U2 RNA, we investigated psi formation on unmodified human U2 RNA in a HeLa cell extract system. Psi formation was found to occur specifically within only those RNase T1 oligonucleotide fragments of U2 RNA known to contain psi in vivo. Using 5-fluorouridine (FUrd)-containing U2 RNAs as specific inhibitors of psi formation in non-FUrd-substituted substrate U2 RNA, we found that wild-type FUrd-containing U2 RNA as well as several FUrd-containing mutant U2 RNAs completely inhibited psi formation. In contrast, certain other mutant U2 RNAs containing FUrd displayed reduced inhibitory capacity. In these cases psi modifications occurred in specific RNase T1 fragments of the substrate U2 RNA only if the FUrd-containing competitor RNA was mutated at or near this site. Formation of psi at one site in U2 RNA appeared to be neither dependent on prior psi formation at another site or sites nor required for subsequent psi formation elsewhere in the molecule. This autonomous mode of psi formation may be driven by multiple psi synthase enzymes acting independently at different sites in U2 RNA.

Animals↗
PubMed1996

Leukocyte lipid body formation and eicosanoid generation: cyclooxygenase-independent inhibition by aspirin.

Lipid bodies, cytoplasmic inclusions that develop in cells associated with inflammation, are inducible structures that might participate in generating inflammatory eicosanoids. Cis-unsaturated fatty acids (arachidonic and oleic acids) rapidly induced lipid body formation in leukocytes, and this lipid body induction was inhibited by aspirin and nonsteroidal antiinflammatory drugs (NSAIDs). Several findings indicates that the inhibitory effect of aspirin and NSAIDs on lipid body formation was independent of cyclooxygenase (COX) inhibition. First, the non-COX inhibitor, sodium salicylate, was as potent as aspirin in inhibiting lipid body formation elicited by cis-fatty acids. Second, cis-fatty acid-induced lipid body formation was not impaired in macrophages from COX-1 or COX-2 genetically deficient mice. Finally, NSAIDs inhibited arachidonic acid-induced lipid body formation likewise in macrophages from wild-type and COX-1- and COX-2-deficient mice. An enhanced capacity to generate eicosanoids developed after 1 hr concordantly with cis-fatty acid-induced lipid body formation. Arachidonic and oleic acid-induced lipid body numbers correlated with the enhanced levels of leukotrienes B4 and C4 and prostaglandin E2 produced after submaximal calcium ionophore stimulation. Aspirin and NSAIDs inhibited both induced lipid body formation and the enhanced capacity for forming leukotrienes as well as prostaglandins. Our studies indicate that lipid body formation is an inducible early response in leukocytes that correlates with enhanced eicosanoid synthesis. Aspirin and NSAIDs, independent of COX inhibition, inhibit cis-fatty acid-induced lipid body formation in leukocytes and in concert inhibit the enhanced synthesis of leukotrienes and prostaglandins.

Adult↗
PubMed2002

Stromal cell-derived factor-1alpha induces tube-like structure formation of endothelial cells through phosphoinositide 3-kinase.

Stromal cell-derived factor-1alpha (SDF-1alpha) is a CXC chemokine, which induces tube formation of endothelial cells. Although SDF-1alpha transduces signals via CXC receptor 4 (CXCR4), resulting in activating a panel of downstream signaling molecules, such as phosphoinositide 3-kinase (PI3-kinase), little is known about the SDF-1alpha-mediated signaling pathways leading to tube formation. Here we examined the signal transduction pathway involved in SDF-1alpha-mediated tube formation by primary human umbilical endothelial cells and murine brain capillary endothelial cell line (IBE (immortalized murine brain capillary endothelial) cells). SDF-1alpha stimulated tube formation by IBE cells, which was blocked by LY294002 and pertussis toxin, suggesting that PI3-kinase and G(i) protein were involved in this process. SDF-1 also stimulated tube formation of human umbilical endothelial cells, and the response was LY294002-sensitive. SDF-1alpha activated PI3-kinase in IBE cells. In stable IBE cell lines expressing either the mutant p85 subunit of PI3-kinase (denoted Deltap85-8 cells), which lacks association with the p110 subunit, or kinase-inactive c-Fes (denoted KEFes 5-15 cells), SDF-1alpha failed to activate PI3-kinase and to stimulate tube formation. SDF-1alpha-induced tube formation was inhibited by an antibody against murine vascular endothelial cadherin. The antibody as well as LY294002 attenuated SDF-1alpha-mediated compact cell-cell contact, which proceeded to tube formation. Taken together, SDF-1alpha induces compact cell-cell contact through PI3-kinase, resulting in tube formation of endothelial cells.

Animals↗
PubMed2003

Kinetics control preferential heterodimer formation of platelet-derived growth factor from unfolded A- and B-chains.

The folding and assembly of platelet-derived growth factor (PDGF), a potent mitogen involved in wound-healing processes and member of the cystine knot growth factor family, was studied. The kinetics of the formation of disulfide-bonded dimers were investigated under redox reshuffling conditions starting either from unfolded and reduced PDGF-A- or B-chains or an equimolar mixture of both chains. It is shown that in all cases the formation of disulfide-bonded dimers is a very slow process occurring in the time scale of hours with a first-order rate-determining step. The formation of disulfide-bonded PDGF-AA or PDGF-BB homodimers displayed identical kinetics, indicating that both monomeric forms as well as the dimerized homodimer have similar folding and assembly pathways. In contrast, the formation of the heterodimer occurred three times more rapidly compared with the formation of the homodimers. As both monomeric forms revealed similar renaturation kinetics, it can be concluded that the first-order rate-determining folding step does not occur during monomer folding but must be attributed to conformational rearrangements of the dimerized, not yet disulfide-bonded protein. These structural rearrangements allow a more rapid formation of intermolecular disulfide bonds between the two different monomers of a heterodimer compared with the formation of the disulfide bonds between two identical monomers. The preferential formation of disulfide-bonded heterodimers from an equimolar mixture of unfolded A- and B-chains is thus a kinetically controlled process. Moreover, similar activation enthalpies for the formation of all different isoforms suggest that faster heterodimerization is controlled by entropic factors.

Dimerization↗
PubMed2000

DNA damage formation, DNA repair, and survival after exposure of DNA repair-proficient and nucleotide excision repair-deficient human lymphoblasts to UVA1 and UVB.

PURPOSE: The comet assay has been used to visualize DNA damage in single cells after exposure to UV light. These comets are commonly thought to reflect transient, repair-induced DNA breaks. The goal of the work presented here was to further characterize the nature of UV-induced comets and to further elucidate DNA damage formation by different wavelengths of ultraviolet light. MATERIALS AND METHODS: Detailed dose-response and time-course experiments with comet formation were carried out with normal and nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) lymphoblasts. Irradiation was carried out with low, intermediate, or high doses of UVA1 or UVB, comet formation was observed, cell survival and viability were determined, and UV-induced apoptosis was measured. RESULTS: All responses were dose-dependent. With the intermediate dose of UVA1, a pronounced comet formation was observed without subsequent growth inhibition. Raising levels of porphyrins, which act as photosensitizers, by preincubation with 5-amino-levulinic acid increased comet formation with UVA1, but not with UVB. UVA1-sensitivity and comet formation in XP cells was not significantly different from the normal cells. With UVB no comet formation was seen without subsequent apoptotic cell death. XP cells exhibited the known UVB-hypersensitivity, but their comet formation was not significantly different from that of normal cells. CONCLUSIONS: The findings are compatible with the hypothesis that UV-induced comets represent transient repair-induced DNA breaks. Both, the NER of dimers and the base excision repair of oxidative DNA modifications are thought to contribute to comet formation.

Apoptosis↗
PubMed1995

Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants.

Cell plate formation in tobacco root tips and synchronized dividing suspension cultured tobacco BY-2 cells was examined using cryofixation and immunocytochemical methods. Due to the much improved preservation of the cells, many new structural intermediates have been resolved, which has led to a new model of cell plate formation in higher plants. Our electron micrographs demonstrate that cell plate formation consists of the following stages: (1) the arrival of Golgi-derived vesicles in the equatorial plane, (2) the formation of thin (20 +/- 6 nm) tubes that grow out of individual vesicles and fuse with others giving rise to a continuous, interwoven, tubulo-vesicular network, (3) the consolidation of the tubulo-vesicular network into an interwoven smooth tubular network rich in callose and then into a fenestrated plate-like structure, (4) the formation of hundreds of finger-like projections at the margins of the cell plate that fuse with the parent cell membrane, and (5) cell plate maturation that includes closing of the plate fenestrae and cellulose synthesis. Although this is a temporal chain of events, a developing cell plate may be simultaneously involved in all of these stages because cell plate formation starts in the cell center and then progresses centrifugally towards the cell periphery. The "leading edge" of the expanding cell plate is associated with the phragmoplast microtubule domain that becomes concentrically displaced during this process. Thus, cell plate formation can be summarized into two phases: first the formation of a membrane network in association with the phragmoplast microtubule domain; second, cell wall assembly within this network after displacement of the microtubules. The phragmoplast microtubules end in a filamentous matrix that encompasses the delicate tubulo-vesicular networks but not the tubular networks and fenestrated plates. Clathrin-coated buds/vesicles and multivesicular bodies are also typical features of the network stages of cell plate formation, suggesting that excess membrane material may be recycled in a selective manner. Immunolabeling data indicate that callose is the predominant lumenal component of forming cell plates and that it forms a coat-like structure on the membrane surface. We postulate that callose both helps to mechanically stabilize the early delicate membrane networks of forming cell plates, and to create a spreading force that widens the tubules and converts them into plate-like structures. Cellulose is first detected in the late smooth tubular network stage and its appearance seems to coincide with the flattening and stiffening of the cell plate.

Cell Division↗
PubMed1996

armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila.

Cellularization of the Drosophila embryo results in the formation of a cell monolayer with many characteristics of a polarized epithelium. We have used antibodies specific to cellular junctions and nascent plasma membranes to study the formation of the zonula adherens (ZA) in relation to the establishment of basolateral membrane polarity. The same approach was then used as a test system to identify X-linked zygotically active genes required for ZA formation. We show that ZA formation begins during cellularization and that the basolateral membrane domain is established at mid-gastrulation. By creating deficiencies for defined regions of the X chromosome, we have identified genes that are required for the formation of the ZA and the generation of basolateral membrane polarity. We show that embryos mutant for both stardust (sdt) and bazooka (baz) fail to form a ZA. In addition to the failure to establish the ZA, the formation of the monolayered epithelium is disrupted after cellularization, resulting in formation of a multilayered cell sheet by mid-gastrulation. SEM analysis of mutant embryos revealed a conversion of cells exhibiting epithelial characteristics into cells exhibiting mesenchymal characteristics. To investigate how mutations that affect an integral component of the ZA itself influence ZA formation, we examined embryos with reduced maternal and zygotic supply of wild-type Arm protein. These embryos, like embryos mutant for both sdt and baz, exhibit an early disruption of ZA formation. These results suggest that early stages in the assembly of the ZA are critical for the stability of the polarized blastoderm epithelium.

Animals↗
PubMed2002

ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat.

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPgammaS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.

ADP-Ribosylation Factors↗
PubMed1997

Interleukin-18 (interferon-gamma-inducing factor) is produced by osteoblasts and acts via granulocyte/macrophage colony-stimulating factor and not via interferon-gamma to inhibit osteoclast formation.

We have established by differential display polymerase chain reaction of mRNA that interleukin (IL)-18 is expressed by osteoblastic stromal cells. The stromal cell populations used for comparison differed in their ability to promote osteoclast-like multinucleated cell (OCL) formation. mRNA for IL-18 was found to be expressed in greater abundance in lines that were unable to support OCL formation than in supportive cells. Recombinant IL-18 was found to inhibit OCL formation in cocultures of osteoblasts and hemopoietic cells of spleen or bone marrow origin. IL-18 inhibited OCL formation in the presence of osteoclastogenic agents including 1alpha,25-dihydroxyvitamin D3, prostaglandin E2, parathyroid hormone, IL-1, and IL-11. The inhibitory effect of IL-18 was limited to the early phase of the cocultures, which coincides with proliferation of hemopoietic precursors. IL-18 has been reported to induce interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF) production in T cells, and both agents also inhibit OCL formation in vitro. Neutralizing antibodies to GM-CSF were able to rescue IL-18 inhibition of OCL formation, whereas neutralizing antibodies to IFN-gamma did not. In cocultures with osteoblasts and spleen cells from IFN-gamma receptor type II-deficient mice, IL-18 was found to inhibit OCL formation, indicating that IL-18 acted independently of IFN-gamma production: IFN-gamma had no effect in these cocultures. Additionally, in cocultures in which spleen cells were derived from receptor-deficient mice and osteoblasts were from wild-type mice and vice versa, we identified that the target cells for IFN-gamma inhibition of OCL formation were the hemopoietic cells. The work provides evidence that IL-18 is expressed by osteoblasts and inhibits OCL formation via GM-CSF production and not via IFN-gamma production.

Animals↗
PubMed2004

Promotion of neurite and filopodium formation by CD47: roles of integrins, Rac, and Cdc42.

Axon extension during development is guided by many factors, but the signaling mechanisms responsible for its regulation remain largely unknown. We have now investigated the role of the transmembrane protein CD47 in this process in N1E-115 neuroblastoma cells. Forced expression of CD47 induced the formation of neurites and filopodia. Furthermore, an Fc fusion protein containing the extracellular region of the CD47 ligand SHPS-1 induced filopodium formation, and this effect was enhanced by CD47 overexpression. SHPS-1-Fc also promoted neurite and filopodium formation triggered by serum deprivation. Inhibition of Rac or Cdc42 preferentially blocked CD47-induced formation of neurites and filopodia, respectively. Overexpression of CD47 resulted in the activation of both Rac and Cdc42. The extracellular region of CD47 was sufficient for the induction of neurite formation by forced expression, but the entire structure of CD47 was required for enhancement of filopodium formation by SHPS-1-Fc. Neurite formation induced by CD47 was also inhibited by a mAb to the integrin beta3 subunit. These results indicate that the interaction of SHPS-1 with CD47 promotes neurite and filopodium formation through the activation of Rac and Cdc42, and that integrins containing the beta3 subunit participate in the effect of CD47 on neurite formation.

Androstadienes↗