Article No. |
Title |
UH1021 |
Determination and Genotoxicity of Polycyclic Aromatic Hydrocarbons Isolated from Dreissina polymorpha (Zebra Mussels) Sampled from Hamilton Harbour |
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CHRIS H. MARVIN, BRIAN E. MCCARRY, DOUGLAS W. BRYANT |
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Polycyclic aromatic hydrocarbons (PAH) were determined in extracts of zebra mussels sampled from sites in Hamilton Harbour. The mussels were extracted using a tissue homogenizer method and the resulting solvent extracts were subjected to an alumina column and a Sephadex LH20 column clean-up procedure. Total PAH concentrations in Zebra mussel extracts ranged from 0.35 pg/g to 10.0 pg/g of mussel homogenate. The extracts were also found to exhibit significant mutagenic responses in the Ames Salmonella/microsome assay. The results indicate that zebra mussels might be used as biological indicators of PAH contamination in freshwater environments such as Hamilton Harbour. |
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UH1022 |
Inhibition of renal cell carcinoma angiogenesis and growth by antisense oligonucleotides targeting vascular endothelial growth factor |
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W SHI1 AND DW SIEMANN |
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Angiogenesis is critical for growth and metastatic spread of solid tumours. It is tightly controlled by specific regulatory factors. Vascular endothelial growth factor has been implicated as the key factor in tumour angiogenesis. In the present studies we evaluated the effects of blocking vascular endothelial growth factor production by antisense phosphorothioate oligodeoxynucleotides on the growth and angiogenic activity of a pre-clinical model of renal cell carcinoma (Caki-1). In vitro studies showed that treating Caki-1 cells with antisense phosphorothioate oligodeoxynucleotides directed against vascular endothelial growth factor mRNA led to a reduction in expressed vascular endothelial growth factor levels sufficient to impair the proliferation and migration of co-cultured endothelial cells. The observed effects were antisense sequence specific, dose dependent, and could be achieved at a low, non-toxic concentration of phosphorothioate oligodeoxynucleotides. When vascular endothelial growth factor antisense treated Caki-1 cells were injected into nude mice and evaluated for their angiogenic potential, the number of vessels initiated were approximately half that induced by untreated Caki-1 cells. To test the anti-tumour efficacy of vascular endothelial growth factor antisense, phosphorothioate oligodeoxynucleotides were administrated to nude mice bearing macroscopic Caki-1 xenografts. The results showed that the systemic administration of two doses of vascular endothelial growth factor antisense phosphorothioate oligodeoxynucleotides given 1 and 4 days after the tumours reached a size of *200 mm3 significantly increased the time for tumours to grow to 1000 mm3. |
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UH1023 |
Preparation of microparticulate B-glucan from Saccharomyces cerevisiae for use in immune potentiation |
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K.W. HUNTER JR, R.A. GAULT AND M.D. BERNER |
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Beta-glucans are polymers of -(1,3)-D-glucose [with or without -(1,6)-D-glucose side chains] found in the cell walls of many bacteria, plants and yeasts. There is an extensive literature describing the immunomodulating effects of both water soluble and insoluble -glucans, with macrophages as the principal target cells (Reynolds et al. 1980; DiLuzio 1983; Gallin1992; Cleary et al. 1999). While various soluble and particulate -glucans have been used in pharmaceutical applications (Williams et al. 1992; Chihara 1992; Babineau et al. 1994) particulate -glucan preparations derived from the yeast Saccharomyces cerevisiae are widely used as over-the-counter nutritional supplements. Examination of several commercially available products consistently revealed a predominant 'globular' morphology consisting of aggregated -glucan particles ranging in size from 5 to 100-µ diameter, with some unaggregated single particles in the 1-2-µ range. |
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UH1024 |
Suramin inhibits initiation of defense signaling by systemin, chitosan, and a b-glucan elicitor in suspension-cultured Lycopersicon peruvianumcells |
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JOHANNES STRATMANN, JUSTIN SCHEER, AND CLARENCE A. RYAN |
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Systemin-mediated defense signaling in tomato (Lycopersicon esculentum) plants is analogous to the cytokine-mediated inflammatory response in animals. Herein, we report that the initiation of defense signaling in suspension-cultured cells of Lycopersicon peruvianum by the peptide systemin, as well as by chitosan and b-glucan elicitor from Phytophtora megasperma, is inhibited by the polysulfonated naphtylurea compound suramin, a known inhibitor of cytokine and growth factor receptor interactions in animal cells. Using a radioreceptor assay, we show that suramin interfered with the binding of the systemin analog 125I-Tyr-2,Ala-15-systemin to the systemin receptor with an IC50 of 160 mM. Additionally, labeling of the systemin receptor with a photoaffinity analog of systemin was inhibited in the presence of suramin. Receptormediated tyrosine phosphorylation of a 48-kDa mitogen-activated protein kinase and alkalinization of the medium of suspensioncultured cells in response to systemin and carbohydrate elicitors were also inhibited by suramin. The inhibition of medium alkalinization by suramin was reversible in the presence of high concentrations of systemin and carbohydrate elicitors. Calyculin A and erythrosin B, intracellular inhibitors of phosphatases and plasma membrane proton ATPases, respectively, both induce medium alkalinization, but neither response was inhibited by suramin. The polysulfonated compound heparin did not inhibit systemininduced medium alkalinization. NF 007, a suramin derivative, induced medium alkalinization, indicating that neither NF 007 nor heparin interact with elicitor receptors like suramin. The data indicate that cell-surface receptors in plants show some common structural features with animal cytokine and growth factor receptors that can interact with suramin to interfere with ligand binding. |
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UH1025 |
Effects of Microstructure on the Compressive Yield Stress |
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GLENN M. CHANNELL, KELLY T. MILLER, AND CHARLES F. ZUKOSKI |
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The effects of microstructure on the compressive properties of aggregated alumina suspensions are determined by intentionally introducing heterogeneities into the suspension. Suspensions are prepared at a high volume fraction and diluted with low shear hand mixing to a series of initial concentrations. As the initial concentration is increased, larger heterogeneities are introduced, and the suspension becomes more compressible relative to the compressive yield stress of the uniform suspension. A simple model is proposed in which the heterogeneous suspensions compress by rearrangement (of the dense aggregates until a critical concentration, which coincides with the) volume fraction prior to dilution is reached. Above, the suspensions consolidate (identically to the uniform suspension. With a single fitting parameter the size of the) heterogeneities , the model shows semiquantitativeagreement with the experimental data. |
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UH1026 |
Microsomal long chain fatty acyl-CoA transacylation: di°erential effect of sterol carrier protein-2 |
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HSU CHAO A, JE°REY T. BILLHEIMER B, ANN B. KIER A, FRIEDHELM SCHROEDER |
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The recent discovery that sterol carrier protein-2 (SCP-2) binds long chain fatty acyl-CoA (LCFA-CoA) with high affinity (A. Frolov et al., J. Biol. Chem. 271 (1997) 31878^31884) suggests new possible functions of this protein in LCFA-CoA metabolism. The purpose of the present investigation was to determine whether SCP-2 differentially modulated microsomal LCFA-CoA transacylation to cholesteryl esters, triacylglycerols, and phospholipids in vitro. Microsomal acyl- CoA:cholesterol acyltransferase (ACAT) activity measured with liposomal membrane cholesterol donors depended on substrate LCFA-CoA level, mol% cholesterol in the liposomal membrane, and total amount of liposomal cholesterol. As compared to basal activity without liposomes, microsomal ACAT was inhibited 30^50% in the presence of cholesterol poor (1.4 mol%) liposomes. In contrast, cholesterol rich (s25 mol%) liposomes stimulated ACAT up to 6.4-fold compared to basal activity without liposomes and nearly 10-fold as compared to cholesterol poor (1.4 mol%) liposomes. Increasing oleoyl-CoA reversed the inhibition of microsomal ACAT by cholesterol poor (1.4 mol%) liposomes, but did not further stimulate ACAT in the presence of cholesterol rich (35 mol%) liposomes. In contrast, high (100 WM) oleoyl-CoA inhibited ACAT nearly 3-fold. This inhibition was reversed by LCFA-CoA binding proteins, bovine serum albumin (BSA) and SCP-2. SCP-2 was 10-fold more effective (mole for mole) than BSA in reversing LCFA-CoA inhibited microsomal ACAT. Concomitantly, under conditions in which SCP-2 stimulated ACAT it equally enhanced transacylation of oleoyl-CoA into phospholipids, and 5.2-fold enhanced oleoyl-CoA transacylation to triacylglycerols. In summary, SCP-2 appeared to exert its greatest effects on microsomal transacylation in vitro by reversing LCFA-CoA inhibition of ACAT and by differentially targeting LCFACoA to triacylglycerols. These data suggest that the high affinity interaction of SCP-2s with LCFA-CoA may be physiologically important in microsomal transacylation reactions. |
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UH1027 |
Molecular Cloning of the nahG Encoding Salicylate Hydroxylase from Pseudomonas fluorescens |
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YOU-SUN CHUNG, NA-RI LEE, CHOONG-ILL CHEON, EUN-SOOK SONG, MYEONG-SOK LEE, YOUNGSOO KIM, AND KYUNG-HEE MIN |
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A gene encoiding the salicylate hydroxylase was cloned from the genomic DNA of Pseudomonas fluorescens SME11. The DNA fragment containing the nahG gene for the salicylate hydroxylase was mapped with restriction endonucleases and sequenced. The DNA fragment contained an ORF of 1,305 bp encoding a polypeptide of 434 amino acid residues. The nucleotide and amino acid sequences of the salicylate hydroxylase revealed several conserved regions with those of the enzyme encoded in P. putida PpG7: The homology of the nucleotide sequence is 83% and that of amino acid sequence is 72%. We found large conserved regions of the amino acid sequence at FAD and NADH binding regions. The FAD binding site is located at the amino terminal region and a lysine residue functions as a NADH-binding site. |
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UH1028 |
Polycyclic Aromatic Compound Profiles From Extracts of Dreissenid Mussels and Gammarid Amphipods Coexisting in Hamilton Harbour |
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CHRIS H. MARVIN, BRIAN E. MCCARRY, LAURIE ALLAN AND DOUGLAS W. BRYANT |
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Aggregates of dreissenid mussels were collected in Hamilton Harbour (western Lake Ontario) from a south shore site (Randle Reef) in an area characterized by coal tarcontaminated sediments, and from a site on the north shore exposed to particulate circulating in the harbour water column. Samples were separated into three components: dreissenid mussels, gammarid amphipods (Gammarus fasciatus), and particulate material. The samples were freeze-dried, and extracted using ultrasonication in dichloromethane. The organic solvent extracts were subjected to an open-column alumina and Sephadex LH-20 gel column clean-up procedure, and characterized by gas chromatography-mass spectrometry (GC-MS). The chromatographic profiles of all sample extracts were dominated by polycyclic aromatic hydrocarbons (PAH). The concentrations of the individual compounds were normalized for contaminant profile comparison of the extracts of dreissenids, amphipods, and particulate associated with aggregates of dreissenid mussels. These profiles were also compared with extracts of coal tar-contaminated sediment from the Randle Reef area, and extracts of suspended particulate obtained from sediment traps. The similarities in the PAH profiles provide evidence of exposure to a common source of contaminants. These data also show that PAH associated with suspended particulate in Hamilton Harbour are being accumulated by dreissenid mussels and gammarid amphipods. |
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UH1029 |
Polymer-Cushioned Bilayers. I. A Structural Study of Various Preparation Methods Using Neutron Reflectometry |
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J. Y. WONG J. MAJEWSKI,# M. SEITZ, C. K. PARK, J. N. ISRAELACHVILI, ANDG. S. SMITH |
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This neutron reflectometry study evaluates the structures resulting from different methods of preparing polymer-cushioned lipid bilayers. Four different techniques to deposit a dimyristoylphosphatidylcholine (DMPC) bilayer onto a polyethylenimine (PEI)-coated quartz substrate were examined: 1) vesicle adsorption onto a previously dried polymer layer; 2) vesicle adsorption onto a bare substrate, followed by polymer adsorption; and 3, 4) Langmuir-Blodgett vertical deposition of a lipidmonolayer spreadover a polymer-containing subphase to form a polymer-supportedlipidmonolayer, followedby formation of the outer lipidmonolayer by either 3) horizontal deposition of the lipidmonolayer or 4) vesicle adsorption. We show that the initial conditions of the polymer layer are a critical factor for the successful formation of our desired structure, i.e., a continuous bilayer atop a hydratedPEI layer. Our desiredstructure was foundfor all methods investigatedexcept the horizontal deposition. The interaction forces between these polymer-supportedbilayers are investigatedin a separate paper (Wong, J. Y., C. K. Park, M. Seitz, andJ. Israelachvili. 1999. Biophys. J. 77:1458-1468), which indicate that the presence of the polymer cushion significantly alters the interaction potential. These polymer-supportedbilayers couldserve as model systems for the study of transmembrane proteins under conditions more closely mimicking real cellular membrane environments. |
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UH1030 |
Reconstitution of the signal recognition particle of the halophilic archaeon Haloferax volcanii |
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IRIT TOZIK, QIAOJIA HUANG, CHRISTIAN ZWIEB AND JERRY EICHLER |
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The signal recognition particle (SRP) is a ribonucleoprotein complex involved in the recognition and targeting of nascent extracytoplasmic proteins in all three domains of life. In Archaea, SRP contains 7S RNA like its eukaryal counterpart, yet only includes two of the six protein subunits found in the eukaryal complex. To further our understanding of the archaeal SRP, 7S RNA, SRP19 and SRP54 of the halophilic archaeon Haloferax volcanii have been expressed and puriÆed, and used to reconstitute the ternary SRP complex. The availability of SRP components from a haloarchaeon offers insight into the structure, assembly and function of this ribonucleoprotein complex at saturating salt conditions. While the amino acid sequences of H.volcanii SRP19 and SRP54 are modiÆed presumably as an adaptation to their saline surroundings, the interactions between these halophilic SRP components and SRP RNA appear conserved, with the possibility of a few exceptions. Indeed, the H.volcanii SRP can assemble in the absence of high salt. As reported with other archaeal SRPs, the limited binding of H.volcanii SRP54 to SRP RNA is enhanced in the presence of SRP19. Finally, immunolocalization reveals that H.volcanii SRP54 is found in the cytosolic fraction, where it is associated with the ribosomal fraction of the cell. |