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Article No. Title
UH1031 Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-Monooxygenase Constitutively
  DENNIS C. YEE, JENNIFER A. MAYNARD, AND THOMAS K. WOOD

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Trichloroethylene (TCE) was removed from soils by using a wheat rhizosphere established by coating seeds with a recombinant, TCE-degrading Pseudomonas fluorescens strain that expresses the tomA1 (toluene o-monooxygenase) genes from Burkholderia cepacia PR123(TOM23C). A transposon integration vector was used to insert tomA1 into the chromosome of P. fluorescens 2-79, producing a stable strain that expressed constitutively the monooxygenase at a level of 1.1 nmol/min z mg of protein (initial TCE concentration, 10 mM, assuming that all of the TCE was in the liquid) for more than 280 cell generations (36 days). We also constructed a salicylate-inducible P. fluorescens strain that degraded TCE at an initial rate of 2.6 nmol/min z mg of protein in the presence of 10 mM TCE [cf. B. cepacia G4 PR123(TOM23C), which degraded TCE at an initial rate of 2.5 nmol/min z mg of protein]. A constitutive strain, P. fluorescens 2-79TOM, grew (maximum specific growth rate, 0.78 h21) and colonized wheat (3 3 106 CFU/cm of root) as well as wild-type P. fluorescens 2-79 (maximum specific growth rate, 0.77 h21; level of colonization, 4 3 106 CFU/cm of root). Rhizoremediation of TCE was demonstrated by using microcosms containing the constitutive monooxygenase-expressing microorganism, soil, and wheat. These closed microcosms degraded an average of 63% of the initial TCE in 4 days (20.6 nmol of TCE/day z plant), compared to the 9% of the initial TCE removed by negative controls consisting of microcosms containing wild-type P. fluorescens 2-79-inoculated wheat, uninoculated wheat, or sterile soil.
 
UH1032 Seasonal changes in temperature and light drive acclimation of photosynthetic physiology and macromolecular content in Lobaria pulmonaria
  TYLER D.B.MACKENZIE, TARA M. MACDONALD, LUC A . DUBOIS, AND DOUGLAS A. CAMPBELL

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Lobaria pulmonaria (L.) Hoffm. is an epephytic lichen common to temperate deciduous forests where it copes with large changes in temperature and lights levels through repeated annual cycles. Samples of L. pulmonaria were taken from a deciduous forest in southeastern Canada at 35-day intervals from February 1999 to February 2000 and also from a rare population in an evergreen forest in March and August 1999. At fieldambient temperatures and light levels, the realised photosystem II (PSII) electron transport was low both in the summer and winter, with transient peaks in the spring and autumn. In contrast, the seasonal pattern of potential electron transport measured at a fixed 20*C peaked in winter, showing the importance of temperature in driving photosynthesis to low levels in the witner despite an acclimation of electron-transport potential to exploit the high ambient light. Realised gross CO2 uptake was correlated with PSII electron transport at mechanistically plausible rates at all sampling sites in the summer but not in the winter, indicating electron diversion away from CO2 fixation in the winter. Cholrophyll content was highest in the dark summer months. The amount of ribulose-1, 5-bisphospate carboxylaseoxygenase (RuBisCO) large subunit (LSU) was highest in spring. Changes in the level of this hyperabundant protein and in the activity of PSII maintained a relatively constatnt rate of maximum CO2 uptake per RuBisCO LSU from April through November, despite great changes in the seasonal light and temperature. L. pulmonaria acclimates between light and temperature stress in the winter months to light-limitaion in the dark summer months. Transition intervals in the spring and autumn, with warm, bright and wet conditions, are likely the most amenable times for growth.
 
UH1033 Selective enhancement of emotional, but not motor, learning in monoamine oxidase A-deficient mice
  JEANSOK J. KIM, JEAN C. SHIH, KEVIN CHEN, LUCHEN, SHAOWEN BAO, STEPHEN MAREN, STEPHAN G. ANAGNOSTARAS, MICHAEL S. FANSELOW, EDWARD DE MAEYER, ISABELLE SEIF, AND RICHARD F. THOMPSON
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Mice deficient in monoamine oxidase A (MAOA), an enzyme that metabolizes monoamines such as norepinephrine and serotonin, have elevated norepinephrine and serotonin levels in the frontal cortex, hippocampus, and cerebellum, compared with normal wild-type mice. Since monoamines in these areas are critically involved in a variety of behaviors, we examined learning and memory (using emotional and motor tasks) in MAOA mutant mice. The MAOAdeficient mice exhibited significantly enhanced classical fear conditioning (freezing to both tone and contextual stimuli) and step-down inhibitory avoidance learning. In contrast, eyeblink conditioning was normal in these mutant mice. The female MAOA-deficient mice also displayed normal speciestypical maternal behaviors (nesting, nursing, and pup retrieval). These results suggest that chronic elevations of monoamines, due to a deletion of the gene encoding MAOA, lead to selective alterations in emotional behavior.
 
UH1034 DNA binding by DctD and DctD-NifA chimeras
  BAOHUA GU, BRYAN S. WANG, MICHAEL LIANG, DEAN SCHOLL, JOON LEE, TIMOTHY R. HOOVER, AND B. TRACY NIXON
PDF icon DctD is a óN- or ó54-dependent transcriptional activator of Rhizobium meliloti and Rhizobium leguminosarum that regulates expression of DctA, a C4-dicarboxylate transport protein. Based on sequence analysis, DctD was predicted to possess three separate functional domains. Prior results described an N-terminal regulatory domain, homologous to two-component regulators, that inhibits transcriptional activation but not sequence-specific DNA binding. Here we confirm that C-terminal deletions eliminate both UAS-directed transcriptional activation and sequence specific DNA binding. Such deletion proteins retained some ability to activate the dctA promoter, but this activity was UAS-independent. One mutant protein lacking the HtH region was purified and found to still bind DNA nonspecifically. Positive evidence for a sequence-specific, C-terminal DNA binding module was observed in chimeric DctD-NifA proteins, in which various C-terminal deletions of R. leguminosarum DctD were fused to C-terminal fragments of R. meliloti NifA. While in vivo activity levels were very low for these chimeric proteins, two were regulated by the dct pathway, preferentially stimulating transcription of nifH rather than that of dctA in response to succinate as the sole carbon source. Fusions of selected DctD-NifA chimeras and maltose binding protein were made, purified and used in gel-shift assays to provide direct evidence for altered DNA binding specificities. These results confirm predictions of sequence specific DNA binding by the HtH motif regions of DctD and NifA, and raise the possibility that during activation the central domain of DctD binds DNA nonspecifically.
 
UH1035 Structural Studies of Polymer-Cushioned Lipid Bilayers
  J. MAJEWSKI, J. Y. WONG, C. K. PARK, M. SEITZ, J. N. ISRAELACHVILI, AND G. S. SMITH
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The structure of softly supported polymer-cushioned lipid bilayers, prepared in two different ways at the quartz-solution interface, were determined using neutron reflectometry. The polymer cushion consisted of a thin layer of branched, cationic polyethyleneimine (PEI), and the bilayers were formed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles. When vesicles were first allowed to adsorb to a bare quartz substrate, an almost perfect bilayer formed. When the polymer was then added to the aqueous solution, it appeared to diffuse beneath this bilayer, effectively lifting it from the substrate. In contrast, if the polymer layer is adsorbed first to the bare quartz substrate followed by addition of vesicles to the solution, there is very little interaction of the vesicles with the polymer layer, and the result is a complex structure most likely consisting of patchy multilayers or adsorbed vesicles.
 
UH1036 Characterization of maize (Zea mays) pollen profilin function in vitro and in live cells
  BRYAN C. GIBBON, HAIYUN REN AND CHRISTOPHER J. STAIGER
PDF icon Profilin is a small, 12±15 kDa, actin-binding protein that interacts with at least three diåerent 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 diåerently 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 profiins were similar when determined by two independent methods. However, the aænity 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. The results are discussed in relation to a recent model of the interphase actin array in these plant cells.
 
UH1037 Characterization and purification of neutrophil ecto-phosphatidic acid phosphohydrolase
  DENIS ENGLISH, MARGARET MARTIN, KEVIN A. HARVEY, LUKE P. AKARD, RUTH ALLENÃ, THEODORE S. WIDLANSKIÅ, JOE G. N. GARCIAÇ AND RAFAT A. SIDDIQUI
PDF icon Phosphatidic acid and its derivatives play potentially important roles as extracellular messengers in biological systems. An ectophosphatidic acid phosphohydrolase (ecto-PAPase) has been identified which effectively regulates neutrophil responses to exogenous phosphatidic acid by converting the substrate to diacylglycerol. The present study was undertaken to characterize this ecto-enzyme on intact cells and to isolate the enzyme from solubilized neutrophil extracts. In the absence of detergent, short chain phosphatidic acids were hydrolysed most effectively by neutrophil plasma membrane ecto-PAPase; both saturated and unsaturated long chain phosphatidic acids were relatively resistant to hydrolysis. Both long (C18:1) and short (C8) chain lysophosphatidic acids were hydrolysed at rates comparable with those observed for short chain (diC)) phosphatidic acid. Activity of the ecto-enzyme accounted for essentially all of the Nethylmaleimide-insensitive, Mg2+-independent PAPase activity recovered from disrupted neutrophils. At 37 °C and pH 7.2, the apparent Km for dioctanoyl phosphatidic acid (diC)PA) was 1.4x10-3 M. Other phosphatidic acids and lysophosphatidic acids inhibited hydrolysis of [32P]diC8 PA in a rank order that correlated with competitor solubility, lysophosphatidic acids and unsaturated phosphatidic acids being much more effective inhibitors than long chain saturated phosphatidic acids. Dioleoyl (C18:1) phosphatidic acid was an unexpectedly strong inhibitor of activity, in comparison with its ability to act as a direct substrate in the absence of detergent. Other inhibitors of neutrophil ecto-PAPase included sphingosine, dimethyl- and dihydrosphingosine, propranolol, NaF and MgCl2. Of several leucocyte populations isolated from human blood by FACS, including T cells, B cells, NK lymphocytes and monocytes, ecto-PAPase was most prevalent on neutrophils; erythrocytes were essentially devoid of activity. A non-hydrolysable, phosphonate analogue of phosphatidic acid, phosphonate 1, eæciently solubilized catalytic activity from intact neutrophils without causing cell disruption or increasing permeability. Enzyme activity in solubilized extracts was purified in the absence of detergent by successive heparin±Sepharose, gel filtration and anion exchange chromatography. By assaying activity in renatured SDS}polyacrylamide gel slices, the molecular mass of neutrophil ecto-PAPase was estimated to be between 45 and 52 kDa, similar to the molecular mass of previously purified plasma membrane PAPases. Since a large portion of neutrophil plasma membrane PAPase is available for hydrolysis of exogenous substrates, ecto-PAPase may play an important role in regulating inflammatory cell responses to extracellular phosphatidic acid in biological systems.
 
UH1038 Trichloroethylene degradation and mineralization by pseudomonads and Methylosinus trichosporiumOB3b
  A. K. SUN · T. K. WOOD
PDF icon To examine the trichloroethylene (C2HCl3)- degrading capability of five microorganisms, the maximum rate, extent, and degree of C2HCl3 mineralization were evaluated for Pseudomonas cepacia G4, Pseudomonas cepacia G4 PR1, Pseudomonas mendocina KR1, Pseudomonas putida F1, and Methylosinus trichosporium OB3b using growth conditions commonly reported in the literature for expression of oxygenases responsible for C2HCl3 degradation. By varying the C2HCl3 concentration from 5 uM to 75uM, V max and KM values for C2HCI3 degradation were calculated as 9 nmol/(minmg protein) and 4 uM for P. cepacia G4, 18 nmol/(minmgprotein) and 29 uM for P. cepacia G4 PR1, 20 nmol/(minmgprotein) and 10uM for P. mendocina KR1, and 8 nmol/(minmgprotein) and 5 uM for P. putida F1. This is the first report of these Michaelis-Menten parameters for P. mendocina KR1, P. putida F1, and P. cepacia G4 PR1. At 75 lM, the extent of C2HCl3 that was degraded after 6 h of incubation with resting cells was 61%–98%; the highest degradation being achieved by toluene-induced P. mendocina KR1. The extent of C2HCl3 mineralization in 6 h (as indicated by concentration of chloride ion) was also measured and varied from 36% for toluene-induced P. putida F1 to 102% for M. trichosporium OB3b. Since C2HCl3 degradation requires new bio-mass, the specific growth rate (u max) of each of the C2HCl3-degradation microorganisms was determined and varied from 0.080/h (M. trichosporium OB3b) to 0.864/h (P. cepacia G4PR1).
 
UH1039 The fate of Mycobacterium tuberculosis in activated human macrophages
  V. VISHWANATH, SUJATHA NARAYANAN AND P. R. NARAYANAN

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Human peripheral blood monocytes, that are unstimulated in vitro, permit free multiplication of intracellular Mycobacterium tuberculosis after 72 h in culture. There was no killing of bacilli in the intracellular environment even after in vitro activation of monocytes with a cocktail of lipopolysaccharide, phorbol myristate acetate, interferon gamma and tumour necrosis factor-alpha. We also tested the ability of adenosine triphosphate (ATP) in reducing the intracellular viability of mycobacteria. Infected monocytes upon ATP treatment underwent cell death, but no loss in the intracellular viability of M. tuberculosis or M. smegmatis could be observed.
 
UH1040 The hyaluronan lyase of Streptococcus pyogenes bacteriophage H4489A
  JOHN R. BAKER1, SHENGLI DONG AND DAVID G. PRITCHARD
PDF icon Many pathogenic streptococci produce extracellular hyaluronan lyases which are thought to aid the spread of the organism in host tissues. In addition, several phages of group A streptococci are known to synthesize a bound form of hyaluronidase. It has been suggested that the function of this hyaluronidase is to facilitate penetration of the hyaluronan capsule by phage and thus to gain access for the phage to the cell surface of the host streptococcus [Hynes, Hancock and Ferretti (1995) Infect. Immun. 63, 3015±3020]. In the present work, the hyaluronidase of Streptococcus pyogenes bacteriophage H4489A, expressed in E. coli, has been purified and characterized. The enzyme was shown to be a lyase with a distributive action pathway. Unlike most bacterial hyaluronidases that have been characterized, the phage enzyme was found to specifically cleave hyaluronan, which adds credence to the view that its function is to digest the hyaluronan capsule of the host organism. This bacteriophage lyase may provide a practical alternative to the lyase from Streptomyces hyalurolyticus as a reagent for the specific cleavage of hyaluronan.
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