ABSTRACT
Activation of cyclic nucleotide dependent signaling pathways leads to relaxation of smooth muscle, alterations in the cytoskeleton of cultured cells, and increases in the phosphorylation of ...HSP20. To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, phosphopeptide analogs of HSP20 were synthesized. These peptides contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Treatment of Swiss 3T3 cells with phosphopeptide analogs of HSP20 led to loss of actin stress fibers and focal adhesion complexes as demonstrated by immunocytochemistry, interference reflection microscopy, and biochemical quantitation of globular‐actin. Treatment with phosphopeptide analogs of HSP20 also led to dephosphorylation of the actin depolymerizing protein cofilin. Pull‐down assays demonstrated that 14‐3‐3 proteins associated with phosphopeptide analogs of HSP20 (but not peptide analogs in which the serine was not phosphorylated). The binding of 14‐3‐3 protein to phosphopeptide analogs of HSP20 prevented the association of cofilin with 14‐3‐3. These data suggest that HSP20 may modulate actin cytoskeletal dynamics by competing with the actin depolymerizing protein cofilin for binding to the scaffolding protein 14‐3‐3. Interestingly, the entire protein was not needed for this effect, suggesting that the association is modulated by phosphopeptide motifs of HSP20. These data also suggest the possibility that cyclic nucleotide dependent relaxation of smooth muscle may be mediated by a thin filament (actin) regulatory process. Finally, these data suggest that protein transduction can be used as a tool to elucidate the specific function of peptide motifs of proteins.
Three novel recombinant dengue type 3 (DEN3) virus vaccine candidates have been generated from a DEN3 virus isolated from a mild outbreak of dengue fever in the Sleman area of central Java in ...Indonesia in 1978. Antigenic chimeric viruses were prepared by replacing the membrane precursor and envelope (ME) proteins of recombinant DEN4 (rDEN4) virus with those from DEN3 Sleman/78 in the presence (rDEN3/4Delta30(ME)) and the absence (rDEN3/4(ME)) of the Delta30 mutation, a previously described 30-nucleotide deletion in the 3' untranslated region. In addition, a full-length infectious cDNA clone was generated from the DEN3 isolate and used to produce rDEN3 virus and the vaccine candidate rDEN3Delta30. The chimeric viruses rDEN3/4(ME) and rDEN3/4Delta30(ME) appear to be acceptable vaccine candidates since they were restricted in replication in severe combined immune deficiency mice transplanted with human hepatoma cells, in rhesus monkeys, and in Aedes and Toxorynchites mosquitoes, and each was protective in rhesus monkeys against DEN3 virus challenge. The rDEN3/4(ME) and rDEN3/4Delta30(ME) viruses were comparable in all parameters evaluated, indicating that antigenic chimerization resulted in the observed high level of attenuation. Surprisingly, rDEN3Delta30 was not attenuated in any model tested when compared with wild-type rDEN3 and therefore, is not a vaccine candidate at present. Thus, the rDEN3/4(ME) and rDEN3/4Delta30(ME) antigenic chimeric viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.
Tumor-associated antigens are typically nonimmunogenic in cancer patients, "immune surveillance" having manifestly failed. The fact that most tumor antigens are normal human proteins presents ...significant obstacles to current cancer immunization approaches that researchers are presently striving to overcome. An alternative strategy bypasses immunization altogether by direct genetic alteration of autologous patient T cells, to create "designer T cells" specific to a particular antigen. Chimeric immunoglobulin-T cell receptors (IgTCR) with a specificity for carcinoembryonic antigen (CEA) were created to evaluate the optimal IgTCR structure for cancer therapy. Antigen-binding domains of a humanized antibody were combined with TCR signaling chains to yield four different chimeric IgTCR: single chain Fv fragment (sFv)-zeta, fragment antigen-binding (Fab)-zeta, sFv-epsilon, and Fab-epsilon. All of the IgTCR were well expressed on T cells, and all showed specific binding and activation, as demonstrated by IL-2 production on contact with immobilized or cellular CEA, excepting sFv-epsilon alone which was inert solely against cellular targets for steric reasons unique to this construct. In contrast to prior studies of isolated TCR chains that related increased tyrosine-based activation motifs in zeta as a reason for superior signaling potency, these tests are the first to show that epsilon and zeta are indistinguishable for T cell signaling when assayed in the context of the intact TCR complex. Further, Fab was equivalent to sFv as an IgTCR component for expression and antigen binding, establishing an important alternative for IgTCR antigen recognition because sFvs may often lose antigen affinity. When IgTCR was expressed on normal human T cells, cytotoxic potency was demonstrated at low E:T ratios, with T cell recycling and progressive tumor cell destruction. Contrary to recent speculations, these observations prove that high affinity TCR interactions are not an impediment to serial target engagement and disengagement by cytotoxic T cells. The multivalent intercellular interactions of target cell binding, activation, and cytotoxicity were resistant to inhibition by soluble CEA. These studies establish a potentially important new immunotherapeutic modality for the treatment of CEA-expressing tumors.
Transforming growth factor-β-1 (TGF-β1) is secreted by cells in a latent form (L-TGF-β1) noncovalently bound to a latency-associated peptide. Activated alveolar macrophages obtained from rat lungs ...after bleomycin-induced pulmonary injury released increased amounts of active TGF-β1 as well as plasmin, a protease, and thrombospondin-1 (TSP-1), a trimeric glycoprotein. Previously we had demonstrated that plasmin was critical to the activation of L-TGF- β1. In the present study we demonstrated that TSP-1 is also important for the activation of L-TGF- β1 because the activation can be inhibited by anti-TSP-1 monoclonal antibody. Proteins obtained from alveolar macrophage cell lysates immunoprecipitated with antibodies specific for TSP-1 were identified on immunoblots as LAP and TGF-β1, indicating that TSP-1/L-TGF-β1 complexes are present on alveolar macrophages. However, in the presence of plasmin both latency-associated peptide and TGF-β1 were decreased in the same cell lysates, indicating that L-TGF-β1 associated with TSP-1 is released by plasmin. Using immunofluorescence and antibodies to TGF-β1 and CD36, a receptor for TSP-1, there was colocalization of TGF-β1 with CD36. Because TSP-1 but not TGF-β1 is a natural ligand for CD36, these findings suggest that the L-TGF-β1 in a complex with TSP-1 localizes to the macrophage cell surface when TSP-1 interacts with its receptor, CD36. Furthermore, the association of TSP-1/L-TGF-β1 complex with CD36 is necessary to the activation of L-TGF-β1 because antibodies to CD36 prevent the colocalization of TGF-β1 with CD36 as observed by immunofluorescence and inhibit activation of the L-TGF-β1 by explanted alveolar macrophages. These findings suggest that activation of L-TGF-β1 by plasmin occurs at the cell surface of activated alveolar macrophages and requires a TSP-1/CD36 interaction.
1. Vascular endothelial cells release a variety of substances which affect the membrane potential and tone of underlying vascular
smooth muscle. In the presence of N omega-nitro-L-arginine to inhibit ...nitric oxide synthase and indomethacin to inhibit cyclo-oxygenase,
acetylcholine (ACh; EC50 approximately 1 microM) elicited the release of an endothelium-derived hyperpolarizing factor (EDHF)
in rabbit mesenteric arteries. 2. The hyperpolarization due to EDHF was blocked by apamin (IC50 approximately 0.3 nM), and
by other inhibitors of the apamin-sensitive K+ channel (10 nM scyllatoxin, 100 microM d-tubocurarine, 300 microM gallamine)
in the presence of indomethacin and N omega-nitro-L-arginine. The hyperpolarization was not blocked by glibenclamide (5 microM),
iberiotoxin (10 nM), tetraethylammonium (1 mM), barium (500 microM), 4-aminopyridine (500 microM), ouabain (10 microM), bumetanide
(10 microM), or nimodipine (100 nM). 3. In the presence of apamin and N omega-nitro-L-arginine, but the absence of indomethacin,
ACh triggered a hyperpolarization that was blocked by glibenclamide, an inhibitor of ATP-sensitive K+ (KATP) channels. A similar
glibenclamide-sensitive hyperpolarization was caused by Iloprost, a stable analogue of prostacyclin. 4. In experiments which
distinguished the effects of EDHF, prostanoids and nitric oxide, hyperpolarizations and/or relaxations triggered by ACh were
antagonized by muscarinic antagonists, the relative potencies (atropine approximately 4-DAMP > pirenzepine) of which indicated
that the release of all three endothelium-derived factors was mediated by M3 receptors. 5. Our results suggest that ACh stimulates
M3 receptors on endothelial cells, triggering the release of nitric oxide and prostanoids, which hyperpolarize underlying
smooth muscle by activation of KATP channels, and the release of an EDHF, which hyperpolarizes smooth muscle through the activation
of apamin-sensitive K+ (KAS) channels.
This article provides a review of experiments conducted over a six-year period to develop a biological control system for insect-transmitted diseases in vegetables based on induced systemic ...resistance (ISR) mediated by plant growth-promoting rhizobacteria (PGPR). Initial experiments investigated the factors involved in treatment with PGPR led to ISR to bacterial wilt disease in cucumber caused by Erwinia tracheiphila. Results demonstrated that PGPR-ISR against bacterial wilt and feeding by the cucumber beetle vectors of E. trachiphiela were associated with reduced concentrations of cucurbitacin, a secondary plant metabolite and powerful beetle feeding stimulant. In other experiments, PGPR induced resistance against bacterial wilt in the absence of the beetle vectors, suggesting that PGPR-ISR protects cucumber against bacterial wilt not only by reducing beetle feeding and transmission of the pathogen, but also through the induction of other plant defense mechanisms after the pathogen has been introduced into the plant. Additional greenhouse and field experiments are described in which PGPR strains were selected for ISR against cucumber mosaic virus (CMV) and tomato mottle virus (ToMoV). Although results varied from year to year, field-grown tomatoes treated with PGPR demonstrated a reduction in the development of disease symptoms, and often a reduction in the incidence of viral infection and an increase in tomato yield. Recent efforts on commercial development of PGPR are described in which biological preparations containing industrial formulated spores of PGPR plus chitosan were formulated and evaluated for use in a transplant soil mix system for developing plants that can withstand disease attack after transplanting in the field.PUBLICATION ABSTRACT
Background: Solar ultraviolet radiation (UVR) is the principal etiological factor in skin carcinogenesis. In vivo and in vitro studies have demonstrated previously that oxidative DNA damage, ...mitochondrial mass and mitochondrial membrane potential (MMP) changes are associated with skin cell response to UVR stress.
Methods: Spontaneously immortalized human skin keratinocytes were irradiated with increasing sub‐lethal doses of simulated sunlight irradiation (SSI) using a Q‐Sun solar simulator. The effects of SSI on reactive oxygen species (ROS) formation, mitochondrial mass and MMP were then determined.
Results: SSI induced mitochondrial mass increase post low SSI (0.25–2.5 J/cm2), whereas higher SSI doses (5.0 and 7.5 J/cm2) decreased mitochondrial mass. Mitochondrial mass increased with time post 5.0 J/cm2 irradiation and all changes in mass were independent of cell density status. Changes in ROS and MMP were cell density dependent. Additionally, an inverted dose‐dependent decrease in ROS formation was observed 3 h post SSI with the lower SSI dose (0.25 J/cm2).
Conclusions: Observations from the present study suggest that changes in the cell's microenvironment (modeled through varying cell density) influence changes in MMP and ROS detoxifying responses in sun‐exposed skin cells.
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