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Article No. Title
UH1011 Replication-Defective Adenovirus Infection Reduces Helicobacter felis Colonization in the Mouse in a Gamma Interferon- and Interleukin-12-Dependent Manner
PDF icon Helicobacter infection leads to chronic inflammation of the stomach. Although the infection persists in spite of an immune response, animal studies have shown that adjuvant-based oral vaccines can protect against infection and even eliminate established infection. These vaccines are thought to induce a Th2 immune response, counterbalancing the Th1 response seen with natural infections. As a prelude to using adenovirus vectors carrying cytokine genes to modulate the immune response to established Helicobacter felis infection, we first examined the effect of the replication-defective adenovirus (RDA) vector itself. C57BL/6 mice chronically infected with H. felis (8 to 10 weeks) received intramuscular injections of RDA. The effect of RDA on the severity of H. felis colonization and the degree of gastric inflammation was assessed 2 weeks later. RDA caused a significant decrease in H. felis colonization without significantly altering the associated inflammation. RDA did not alter the H. felis-specific immunoglobulin G1 (IgG1), IgG2a, and IgA responses in the serum but was associated with an increase in gamma interferon (IFN-g)-producing CD81 spleen cells. To determine if IFN-g or Th1 cytokines were involved in the response to RDA, we examined RDA treatment of H. felis infection in mice lacking either IFN-g or interleukin-12 (IL-12). RDA failed to alter H. felis colonization in either of these two mouse strains. Thus, viral infection of mice chronically infected with H. felis led to a significant decrease in H. felis colonization in an IFN-g- and IL-12-dependent manner.
UH1012 Characterization of maize (Zea mays) pollen profilin function in vitro and in live cells
PDF icon Profilin is a small, 12±15 kDa, actin-binding protein that interacts with at least three dfferent ligands. The 1:1 interaction of profilin with globular actin (G-actin) was originally thought to provide a mechanism for sequestering actin monomers in the cytoplasm. It has recently become clear that the role of profilin in the cell is more complex, perhaps due to interactions with polyphosphoinositides and proline-rich proteins, or due to the ability to lower the critical concentration for actin assembly at the fast-growing barbed end of actin filaments. Because actinbinding proteins have been shown to behave differently with heterologous sources of actin, we characterized the interaction between maize pollen profilins and plant G-actin. The equilibrium dissociation constants measured by tryptophan fluorescence quenching were similar to those of other CaATP-G-actin±profilin complexes (KdØ1.0±1.5 lM). The ability of maize profilin isoforms to bind poly-l-proline was analysed, and the Kd values for recombinant pollen and human profilins were similar when determined by two independent methods. However, the affinity of native maize pollen profilin for poly-l-proline was substantially lower than that of any of the recombinant proteins by one of these assays. The possibility of post-translational modification of profilin in the mature pollen grain is discussed. Finally, we quantified the effects of microinjection of each profilin isoform on the cytoarchitecture of Tradescantia stamen hair cells and show that the resultant disruption can be used to compare actinbinding proteins in living cells.
UH1013 Characterization of the Helicase Activity of the Escherichia coli RecBCD Enzyme Using a Novel Helicase Assay
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We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent K , values for recBCD enzyme helicase activity on linear M13 dsDNA molecules a t 25 OC are 0.6 nM dsDNA molecules and 130 pM ATP, respectively. The apparent turnover number for unwinding is approximately 15 pM base pairs s-l (pM recBCD enzyme)-'. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, k,, for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-l (functional recBCD complex)-' a t 25 O C . At 37 O C , the apparent K , value for dsDNA molecules was the same as that a t 25 OC, but the apparent turnover number became 56 pM base pairs s-l (pM recBCD enzyme)-' [or 930 base pairs s-l (functional recBCD complex)-' when corrected for observed stoichiometry]. With increasing NaCl concentration, k,, peaks a t 100 mM, and the apparent K , value for dsDNA increases by 3-fold a t 200 mM NaC1. In the presence of 5 mM calcium acetate, the apparent K , value is increased by 3-fold, and k,, decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.>
UH1014 Properties of Bacillus cereus hemolysin II: A heptameric transmembrane pore
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The gene encoding hemolysin II (HlyII) was amplified from Bacillus cereus genomic DNA and a truncated mutant, HlyII(ΔCT), was constructed lacking the 94 amino acid extension at the C terminus. The proteins were produced in an E. coli cell-free in vitro transcription and translation system, and were shown to assemble into SDS-stable oligomers on rabbit erythrocyte membranes and liposomes. The hemolytic activity of HlyII was measured with rabbit erythrocytes yielding an HC50 value of 1.64 ng mL-1, which is over 15 times more potent than staphylococcal Δ-hemolysin. HlyII(ΔCT) was about eight times less potent than HlyII in this assay. Limited proteolysis of the oligomers formed by HlyII and HlyII(ΔCT) on red cell membranes showed that the C-terminal extension is sensitive to digestion, while HlyII(ΔCT) is protease resistant and migrates with an electrophoretic mobility similar to that of digested HlyII. HlyII forms moderately anion selective, rectifying pores (I+80/I-80 = 0.57, 1 M KCl, pH 7.4) in planar lipid bilayers of diphytanoylphosphatidylcholine with a unitary conductance of 637 pS (1 M KCl, 5 mM HEPES, pH 7.4) and exhibits no gating over a wide range of applied potentials (-160 to +160 mV). In addition, it was demonstrated that HlyII forms a homoheptameric pore by using gel shift electrophoresis aided by a genetically encoded oligoaspartate tag.
UH1015 GTP binds to Rab3A in a complex with Ca2+/calmodulin
PDF icon Ras-like small GTP-binding proteins of the Rab family regulate traæcking of the secretory or endocytic pathways. Rab3 proteins within the Rab family are expressed at high levels in neurons and endocrine cells, where they regulate release of dense-core granules and synaptic vesicles (SVs). Rab3A is present as either the soluble or the SV membrane-bound form in neurons that are dependent on the GDP- or GTP-bound states respectively. GDP dissociation inhibitor (GDI) is known to induce the dissociation of Rab3A from synaptic membranes when GTP is depleted. In an earlier study, Ca 2+/calmodulin (CaM) was also shown to dissociate Rab3A from synaptic membranes by forming an equimolar complex with Rab3A in äitro. We have examined a possible role for Ca 2+/CaM in modulating both the binding of guanine nucleotides to Rab3A and the GTPase activity of Rab3A. The basal level of Rab3A GTPase activity was not aåected by an association with Ca 2+/CaM. Ca 2+/CaM±Rab3A complex that was formed in synaptic membranes was able to bind guanine nucleotides, whereas the Rab3A±GDI complex could not. In addition, Ca 2+/CaM led to the replacement of the GDP molecule in the Rab3A±GDI complex with GTP in Rab3A. Taken together, these results suggest that CaM may have a role in stimulating GTP binding to Rab3A that is complexed with GDI, which leads to the formation of an active GTP-bound form of the Rab3A±Ca 2+/CaM complex.
UH1016 HC fragment (C-terminal portion of the heavy chain) of tetanus toxin activates protein kinase C isoforms and phosphoproteins involved in signal transduction
PDF icon A recent report [Gil, Chaib-Oukadour, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177±182] describes activation of signal transduction pathways by tetanus toxin (TeTx), a Zn#+-dependent endopeptidase synthesized by the Clostridium tetani bacillus, which is responsible for tetanus disease. In the present work, specific activation of protein kinase C (PKC) isoforms and of intracellular signal-transduction pathways, which include nerve-growth-factor (NGF) receptor trkA, phospholipase C(PLC)c-1 and extracellular regulated kinases (ERKs) 1 and 2, by the recombinant C-terminal portion of the TeTx heavy chain (HC-TeTx) is reported. The activation of PKC isoforms was assessed through their translocation from the soluble (cytosolic) compartment to the membranous compartment, showing that clear translocation of PKC-a, -b, -c and -d isoforms exists, whereas PKC-e showed a slight decrease in its soluble fraction immunoreactivity. The PKC-f isoform showed no consistent response. Using immunoprecipitation assays against phosphotyrosine residues, time- and dose-dependent increases in tyrosine phosphorylation were observed in the trkA receptor, PLCc-1 and ERK-1}2. The effects shown by the HC-TeTx fragment on tyrosine phosphorylation were compared with the effects produced by NGF.
UH1017 Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice
PDF icon The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyerís patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.
UH1018 Cofactor Dynamics and Sufficiency in Estrogen Receptor–Regulated Transcription
PDF icon Many cofactors bind the hormone-activated estrogen receptor (ER), yet the specific regulators of endogenous ER-mediated gene transcription are unknown. Using chromatin immunoprecipitation (ChIP), we find that ER and a number of coactivators rapidly associate with estrogen responsive promoters following estrogen treatment in a cyclic fashion that is not predicted by current models of hormone activation. Cycles of ER complex assembly are followed by transcription. In contrast, the anti-estrogen tamoxifen (TAM) recruits corepressors but not coactivators. Using a genetic approach, we show that recruitment of the p160 class of coactivators is sufficient for gene activation and for the growth stimulatory actions of estrogen in breast cancer supporting a model in which ER cofactors play unique roles in estrogen signaling.
UH1019 Cooperative Binding of DctD to the dctA Upstream Activation Sequence of Rhizobium meliloti Is Enhanced in a Constitutively Active Truncated Mutant*
PDF icon DctD, a o54-dependent, two-component regulator, binds to promoter distal (A) and promoter proximal (B) sites in an activation sequence located upstream of the dctA promoter. We report gel filtration and quantitative DNase I footprint experiments supporting a model in which DctD2 binds to these sites cooperatively. The global analysis of upstream activation sequences containing sites A and B, A and B one-half helical turn out of phase, and only B yielded values for the intrinsic and cooperative binding free energies of (FORMULA) A separate analysis of data from upstream activation sequences containing site A and a point mutant of site B, and site A and mutant site B one-half helical turn out of phase confirmed the estimate of cooperativity, yielding free energy values of (FORMULA) We previously showed that removing the two-component receiver domain from DctD, making DctD (Delta symbol) (1–142), yields a constitutively active truncated protein. Global analysis of binding data for DctD (Delta symbol) (1–142) showed that this constitutively active mutant has intrinsic binding energies equal to that of the inactive DctD protein, but that it displays significantly higher cooperativity (FORMULA).
UH1020 Immunogenicity of a killed whole Neospora caninum tachyzoite preparation formulated with di€erent adjuvants
PDF icon A killed whole Neospora caninum tachyzoite preparation was formulated with various adjuvants and tested for its immunogenicity in cattle. The adjuvants used were: Havlogen, a polymer of acrylic acid cross-linked with polyallylsucrose; Polygen, a non-particulate copolymer; a mixture of Havlogen and Bay R-1005, which is a preparation of free base synthetic glycolipids; and Montanide ISA 773, a water-in-oil emulsion made with a mixture of metabolisable and mineral oils. Immune responses in immunised cattle were compared with those of cattle experimentally infected with culture-derived N. caninum tachyzoites. The overall mean serum IFAT titres were significantly higher (P<0.05) in experimentally infected cattle compared with all immunised cattle. Nonetheless, the maximum antibody titres of the immunised cattle, which were obtained following the third immunisation, were within the range of titres previously described for naturally infected cattle. The overall mean serum IFAT titres were significantly higher (P<0.05) in cattle immunised with the killed tachyzoite preparation formulated with Polygen and with the mixture of Havlogen and Bay R-1005, compared with cattle immunised with the Havlogen- and Montanide- based preparations. Two of the four adjuvant preparations were able to induce cell-mediated immune responses similar to those of the experimentally infected cattle. The Havlogen-adjuvanted tachyzoite preparation elicited N. caninum- specific proliferation of peripheral blood mononuclear cells statistically similar (P=0.095) to that of the infected animals.
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