Pseudomonas

Mol Biol Cell, 1998 Feb, 9(2), 387 - 402
Involvement of ATP-dependent Pseudomonas exotoxin translocation from a late recycling compartment in lymphocyte intoxication procedure; Alami M et al.; Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis . Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes . When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal 125I-labeled PE was transported after 2 h at 37 degrees C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated . Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell . Accordingly, when PE delivery to this structure was inhibited using a 20 degrees C endocytosis temperature, subsequent translocation from purified endosomes was impaired . Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements . No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form . ATP hydrolysis was found to directly provide the energy required for PE translocation . Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect 125I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient . Nevertheless, when 125I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal . When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired 125I-labeled PE translocation from purified endosomes . We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.

Biochemistry, 1998 Mar 17, 37(11), 3994 - 4000
The MCD and EPR of the heme centers of nitric oxide reductase from Pseudomonas stutzeri: evidence that the enzyme is structurally related to the heme-copper oxidases; Cheesman MR et al.; EPR spectra at liquid helium temperatures and MCD spectra at room temperature and 4.2 K are presented for fully oxidized nitric oxide reductase (NOR) from Pseudomonas stutzeri . The MCD spectra show that the enzyme contains three heme groups at equivalent concentrations but distinctive in their axial coordination . Two, in the low-spin ferric state at all temperatures, give rise to infrared charge-transfer transitions which show the hemes to have bis-histidine and histidine-methionine ligation, respectively . The EPR spectra show them to be magnetically isolated . The third heme has an unusual temperature-dependent spin state and spectroscopic features which are consistent with histidine-hydroxide coordination . No EPR signals have been detected from this heme . Together with its unusual near-infrared MCD, this suggests a spin-spin interaction between this heme and another paramagnet . The three hemes account for only 75% of the iron content, and it is concluded that the additional paramagnet is a mononuclear ferric ion . These results provide further evidence that NOR is indeed structurally related to heme-copper oxidases and that it contains a heme/non-heme iron spin-coupled pair at the active site.

Leukemia, 1998 Feb, 12(2), 182 - 91
Pediatric acute myelogenous leukemia cells express IL-6 receptors and are sensitive to a recombinant IL6-Pseudomonas exotoxin; Boayue KB et al.; We have studied IL-6 receptor (IL-6R) expression on AML cells from 15 pediatric patients by immunocytochemistry/flow cytometry, reverse-transcription polymerase chain reaction, and Scatchard analysis . High-affinity IL-6R were detected on leukemic cells from 12 (80%) patients . Binding sites per cell ranged from 140 to 3580 (median 920; mean 1240), with dissociation constants of 0.26 to 0.71 nM . We therefore assessed the in vitro sensitivity of IL-6R+ AML cells to treatment with a recombinant IL6-Pseudomonas exotoxin fusion protein (IL6-PE4E), using the XTT cytotoxicity assay . Leukemic cells from eight patients had ID50 values (concentration of IL6-PE4E producing a 50% decrease in cell viability) of <1000 ng/ml (median, 87 ng/ml; mean, 262 ng/ml) . Sensitivity to IL6-PE4E correlated significantly with receptor number . Normal bone marrow mononuclear cells had undetectable IL6-R expression (<20 receptors/cell) and were relatively resistant to IL6-PE4E . To test the efficacy of IL6-PE4E for ex vivo purging in an autologous stem cell transplantation setting, we incubated primary IL-6R+ AML cells with 10(3) ng/ml IL6-PE4E for 24 h, followed by inoculation into SCID mice . Mice receiving treated cells showed no leukemic engraftment, while all mice receiving untreated or control-treated cells developed leukemia with a median presymptomatic interval of 55 days . In recipients of IL6-PE4E treated cells, no evidence of occult leukemia was detected by PCR analysis of blood and bone marrow cells at 185 days postinoculation . These data suggest that IL-6R are expressed on leukemic cells from a substantial percentage of pediatric AML patients . Furthermore, leukemic cells expressing high numbers of IL6-R may be sensitive to IL6-PE4E in an ex vivo purging protocol.

Protein Expr Purif, 1998 Mar, 12(2), 233 - 8
Overproduction and purification of glutaryl 7-amino cephalosporanic acid acylase; Li Y et al.; The gene encoding glutaryl 7-amino cephalosporanic acid acylase (GL-7ACA acylase) from Pseudomonas sp . 130 has been cloned and expressed in Escherichia coli using a high-level expression system . The specific activity of the acylase in the crude extract of cells in this system is approximately 10 times that in the previous one . The overproduced enzyme can be easily isolated within 3 days to a purity of over 90% by a simple and inexpensive two-step preparative chromatographic method with an overall yield of nearly 50% . The deletion of the signal peptide and mutation in the alpha-subunit of the acylase have little influence on its posttranslational processing and its kinetic parameters.

Gene, 1998 Feb 16, 208(1), 37 - 42
Two aberrant mercury resistance transposons in the Pseudomonas stutzeri plasmid pPB; Reniero D et al.; The two mer operons of the Pseudomonas stutzeri OX plasmid pPB and their flanking regions have been sequenced and found to be part of two aberrant transposons . The narrow spectrum mer operon is almost identical to that of Tn501, but is associated with the remnants of Tn5053 tni genes rather than the Tn501 transposition module . The broad spectrum mer operon shows an overall homology with that of Tn5053, but differs from it in the presence of a merB gene, absent in Tn5053, and a merC gene instead of a merF . The pPB broad spectrum mer operon is associated with an incomplete Tn5053-like transposition module and with the Tn501 tnp genes, which are proximal, respectively, to the end and to the beginning of the mer operon . A hypothesis about pPB evolution is presented.

J Bacteriol, 1998 Mar, 180(6), 1360 - 7
Growth phase and temperature influence promoter activity, transcript abundance, and protein stability during biosynthesis of the Pseudomonas syringae phytotoxin coronatine; Budde IP et al.; The plant-pathogenic bacterium Pseudomonas syringae pv . glycinea PG4180.N9 synthesizes high levels of the polyketide phytotoxin coronatine (COR) at 18 degrees C, whereas no detectable toxin is produced at 28 degrees C . Previously, we reported that the temperature-sensitive activation of three promoters within the COR biosynthetic gene cluster might explain thermoregulation of COR biosynthesis . The present study was aimed at furthering our understanding of the transcriptional as well as the posttranslational effects of temperature on expression of cmaB, which encodes an enzyme involved in COR biosynthesis . Transcriptional fusions using a promoterless glucuronidase gene and Northern blot analyses were used to monitor promoter activities and transcript abundance for the cmaABT operon during bacterial growth at 18 and 28 degrees C . Promoter activity and transcription rates were maximal when cells were incubated at 18 degrees C and sampled at mid-logarithmic phase . Transcription declined moderately during the transition to stationary phase but remained higher at 18 C than at 28 degrees C . Western blot analysis indicated that CmaB accumulated in the late stationary phase of P . syringae cultures grown at 18 degrees C but not in cultures incubated at 28 degrees C . Temperature shift experiments indicated that CmaB stability was more pronounced at 18 degrees C than at 28 degrees C . Although temperature has a strong impact on transcription of COR biosynthetic genes, we propose that thermoregulation of protein stability might also control COR synthesis.

J Bacteriol, 1998 Mar, 180(6), 1354 - 9
Cloning and sequencing of a 2,5-dichlorohydroquinone reductive dehalogenase gene whose product is involved in degradation of gamma-hexachlorocyclohexane by Sphingomonas paucimobilis; Miyauchi K et al.; Sphingomonas (formerly Pseudomonas) paucimobilis UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH), a halogenated organic insecticide, as a sole carbon and energy source . In a previous study, we showed that gamma-HCH is degraded to 2,5-dichlorohydroquinone (2,5-DCHQ) (Y . Nagata, R . Ohtomo, K . Miyauchi, M . Fukuda, K . Yano, and M . Takagi, J . Bacteriol . 176:3117-3125, 1994) . In the present study, we cloned and characterized a gene, designated linD, directly involved in the degradation of 2,5-DCHQ . The linD gene encodes a peptide of 343 amino acids and has a low level of similarity to proteins which belong to the glutathione S-transferase family . When LinD was overproduced in Escherichia coli, a 40-kDa protein was found after sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Northern blot analysis revealed that expression of the linD gene was induced by 2,5-DCHQ in S . paucimobilis UT26 . Thin-layer chromatography and gas chromatography-mass spectrometry analyses with the LinD-overexpressing E . coli cells revealed that LinD converts 2,5-DCHQ rapidly to chlorohydroquinone (CHQ) and also converts CHQ slowly to hydroquinone . LinD activity in crude cell extracts was increased 3.7-fold by the addition of glutathione . All three of the Tn5-induced mutants of UT26, which lack 2,5-DCHQ dehalogenase activity, had rearrangements or a deletion in the linD region . These results indicate that LinD is a glutathione-dependent reductive dehalogenase involved in the degradation of gamma-HCH by S . paucimobilis UT26.

Protein Sci, 1998 Jan, 7(1), 178 - 84
Purification and characterization of the heteromeric transcriptional activator MvaT of the Pseudomonas mevalonii mvaAB operon; Rosenthal RS et al.; The mvaAB operon of Pseudomonas mevalonii encodes HMG-CoA reductase (EC 1.1.1.88) and HMG-CoA lyase (EC 4.1.3.4), enzymes that catalyze the initial reactions of mevalonate catabolism in this organism . Expression of this operon is regulated by the constitutively expressed transcriptional activator protein MvaT that binds in vitro to an upstream regulatory element . Mevalonate is essential for activation of transcription in vivo, and in vitro data demonstrated that MvaT binds to the mvaAB cis-regulatory element in the absence of mevalonate with a Kd,app of 2 nM . Purification of MvaT enriched for two polypeptides of approximate molecular mass 15 kDa and 16 kDa, designated P15 and P16 . MvaT, assayed by its DNA-binding activity, comigrated with P15 and P16 during DNA-affinity chromatography, size-exclusion chromatography, and sucrose density gradient centrifugation . P15 and P16 also comigrated during denaturing isoelectric focusing of purified MvaT . Treatment of MvaT with dimethylsuberimidate formed a 31-kDa polypeptide complex that contained N-terminal sequences from P15 and P16 . The apparent association of P15 and P16 in solution and their copurification with MvaT activity strongly suggests that MvaT is comprised of these two subunits . Size-exclusion chromatography gave an estimated molecular mass for MvaT of 33 kDa . A partial DNA sequence of the P16 gene was obtained using PCR employing degenerate primers directed against the N-termini of P15 and P16 . P16 appears to be comprised of at least 128 aminoacyl residues having a predicted molecular mass of 14.3 kDa.

Nippon Ganka Gakkai Zasshi, 1998 Feb, 102(2), 83 - 7
{The effect of matrix metalloproteinase inhibitor on pseudomonal proteinases}; Kosaku K et al.; We examined the effect of CS-610, a newly developed matrix metalloproteinase (MMP) inhibitor, on pseudomonal proteinase in vitro . Alkaline proteinase (1143-4977 unit/ml Type I collagenase equivalent) and elastase (13.6-22.6 unit/ml Type I collagenase equivalent) were obtained from strains of P . aeruginosa of IID-1117, IID-1030 and IID-1130 . Zymographic analysis of cultured broth of P . aeruginosa demonstrated that CS-610 inhibited alkaline proteinase with an IC50 (50% inhibition concentration) of 1.06-29.0 (x 10(-8)M) and elastase with an IC50 of 1.0-33.3 (x 10(-8)M) . CS-610 is a potent inhibitor of pseudomonal proteinases.

Biochim Biophys Acta, 1998 Jan 15, 1382(1), 1 - 4
Investigation of the potential active site of a cyanide dihydratase using site-directed mutagenesis; Watanabe A et al.; Cyanide dihydratase has conserved residues in the amino acid sequence with nitrilase, and cyanide hydratase . The conserved amino acid residues in the cyanide dihydratase from Pseudomonas stutzeri AK61 were altered by site-directed mutagenesis . The enzyme completely lost its activity of the cyanide hydrolysis by the replacement of cysteine-163 to serine . The replacement of tyrosine-53 to phenylalanine caused an increase of the K(m) value of the enzyme for cyanide . Substitution of nine other residues seemed to affect the structure of the enzyme.

J Gen Intern Med, 1998 Feb, 13(2), 131 - 6
Diagnosing HIV-related disease: using the CD4 count as a guide; Jung AC et al.; OBJECTIVE: To summarize current information on the relation between CD4 counts and the risk of different HIV-related diseases . MEASUREMENTS AND MAIN RESULTS: MEDLINE search of English language articles between 1985 and 1996 using the medical subject heading (MeSH) term "CD4 lymphocyte count" and searches using key words of multiple HIV-related diseases were conducted . Some HIV-related diseases can be stratified to different CD4 count levels . Regardless of their CD4 count, HIV-infected patients are susceptible to sinusitis, Kaposi's sarcoma, community-acquired pneumonia, and oral hairy leukoplakia . In advanced HIV, when CD4 is below 200/mm3, Pneumocystis carinii pneumonia, toxoplasmosis, progressive multifocal leukoencephalopathy, Mycobacterium avium complex, molluscum contagiosum, and bacillary angiomatosis all increase in incidence . In very advanced HIV disease, when CD4 counts are below 50/mm3, patients are at risk of pseudomonas pneumonia, cytomegalovirus retinitis, central nervous system lymphoma, aspergillosis, and disseminated histoplasmosis.

Biosci Biotechnol Biochem, 1998 Jan, 62(1), 1 - 9
Biochemical synthesis of several chiral insecticide intermediates and mechanisms of action of relevant enzymes; Hirohara H et al.; Efficient biochemical processes were developed for the synthesis of several chiral alcohol and acid intermediates of insecticides by a combination of strictly stereoselective hydrolytic enzyme-catalyzed reactions and subsequent chemical transformations with inversion or racemization of the chiral center of the undesired antipodes . The whole amounts of starting racemic mixtures are converted to desired stereoisomers in the processes, which are generally applicable to the industrial productions of various chiral secondary alcohols and alpha-substituted acids once a highly stereospecific enzyme is obtained for the target compounds . The alcohols reported here are: 1-(4-phenoxyphenoxy)-2-propanol, 1; 4-hydroxy-3-methyl-2-(2'-propynyl)-2-cyclopentenone, 2; and alpha-cyano-3-phenoxybenzyl alcohol, 3 . The acids are 2, 2-dimethyl-3-(2-methyl-1-propenyl)-cyclopropanecarboxylic acid (chrysanthemic acid), 4; and 2-(4-chlorophenyl)-3-methylbutyric acid, 5 . In addition, the mechanism of action of Pseudomonas cepacia lipase (PCL), the most effective enzyme for the resolution of 1, and the recombinant Arthrobacter globiformis esterase (AES) for 4 was studied from the reaction kinetics . The site-directed mutagenesis techniques were also used for AES . The results indicated that the stereoselectivity of PCL is caused by the position and direction of a medium-sized substituent at the chiral center of the alcohol moiety in the rate-determining breakdown of a tetrahedral intermediate in the acylation of the enzyme and that the catalytic site of AES has the characteristics of the penicillin-recognizing enzymes in which Ser 59 in the concensus motif Ser-X-X-Lys plays a vital role as a nucleophile during acylation and Lys 62 acts as a general base.

Cancer Res, 1998 Mar 1, 58(5), 968 - 75
Accumulation of a recombinant immunotoxin in a tumor in vivo: fewer than 1000 molecules per cell are sufficient for complete responses; Kreitman RJ et al.; Recombinant immunotoxins have been shown to cure human tumor xenografts in mice, but their biodistribution to both tumors and normal organs has not been reported . Anti-Tac(Fv)-PE38 is a single-chain recombinant immunotoxin composed of the variable heavy and light domains of the anti-Tac monoclonal antibody that reacts with the primate interleukin 2 (IL2) receptor alpha subunit (IL2R alpha or CD25) fused to a truncated form of Pseudomonas exotoxin (PE) . 125I-labeled anti-Tac(Fv)-PE38 was given i.v . to immunodeficient mice each bearing two A431 tumors, one that expresses IL2R alpha (ATAC-4) and one that does not (A431, parental) . A single i.v . dose of 4 microg/mouse caused complete regression of the IL2R alpha + tumor . At 6 h, over 6% of the injected dose/g was found in the ATAC-4 tumor, and 2% was in the A431 tumor . Uptake in the ATAC-4 tumor was higher than in any other tissue . Sections of tumor examined by autoradiography indicated that anti-Tac(Fv)-PE38 was distributed throughout the entire tumor, with some portions having higher uptake than others . By subtracting uptake in tumors without receptor (A431) from uptake in receptor-containing tumors (ATAC-4), we calculated that at least 400 molecules/cell specifically bound to IL2R alpha-positive tumor cells at 90 min and 750 molecules/cell bound at 360 min . This is similar to the 400-870 molecules/cell required for >99.9% killing of ATAC-4 cells growing as a monolayer . The results show that solid tumors in mice can be eradicated like cells in tissue culture, and that delivery of less than 1000 molecules/cell is sufficient to cause complete tumor regressions.

Blood, 1998 Mar 1, 91(5), 1820 - 7
Purging of mammary carcinoma cells during ex vivo culture of CD34+ hematopoietic progenitor cells with recombinant immunotoxins; Spyridonidis A et al.; Tumor cells have been found in autologous hematopoietic cell transplants used after high-dose chemotherapy . To specifically eliminate contaminating mammary tumor cells during ex vivo expansion of CD34+ hematopoietic progenitor cells, we used recombinant immunotoxins (ITs) directed against cell-surface antigens expressed on mammary carcinoma cells . ITs were expressed from fusion cDNAs combining a single-chain antibody fragment (scFv) directed against the Erb-B2 or epidermal growth factor (EGF) receptors with a truncated Pseudomonas exotoxin A fragment devoid of its cell-binding domain . CD34+ hematopoietic progenitor cells did not express Erb-B2 and EGF receptors as detected by Western blotting . Ex vivo expansion of total hematopoietic cells or of colony-forming cells from CD34+ progenitors in the presence of stem-cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (Epo) was not affected when ITs were added to the cultures . In contrast, MDA-MB 453 and MCF-7 mammary carcinoma cells were depleted in a dose- and time-dependent manner by more than 3 log in coculture with CD34+ cells over a period of 7 days in the presence of 100 to 1,000 ng/mL of anti-Erb-B2 IT . This included elimination of the subpopulations with regrowth potential . Similarly, addition of either anti-Erb-B2 or anti-EGF receptor ITs to primary breast cancer cells isolated from patients with metastatic disease resulted in elimination of cytokeratin-positive cells in seven of seven samples . ITs are highly efficient and convenient to use for the depletion of mammary tumor cells during ex vivo expansion of hematopoietic progenitor-cell autografts.

Int J Cancer, 1998 Mar 16, 75(6), 878 - 84
Expression of an oncogenic mutant EGF receptor markedly increases the sensitivity of cells to an EGF-receptor-specific antibody-toxin; Schmidt M et al.; EGFRvIII is a ligand-independent, constitutively active variant of the epidermal growth factor receptor (EGFR) that is specifically expressed in gliomas and various other human malignancies and has been proposed as a target for directed tumor therapy . We have recently constructed a highly potent single-chain antibody-toxin, scFv(14E1)-ETA, which consists of the variable domains of the antibody 14E1 specific for human full-length EGFR genetically fused to a truncated form of Pseudomonas exotoxin A . We demonstrate here binding of 14E1 antibody to both full-length and variant EGFR . In contrast to a recombinant toxin containing transforming growth factor-alpha (TGF-alpha) as a cell targeting domain, scFv(14E1)-ETA was highly active on cells expressing EGFRvIII . Surprisingly, scFv(14E1)-ETA displayed cell killing activity on EGFRvIII-expressing cells that was up to 100-fold higher than on control cells expressing full-length EGFR . No differences in the binding affinities of scFv(14E1)-ETA to full-length EGFR or EGFRvIII were observed, suggesting that events downstream of immunotoxin binding are responsible for the increased sensitivity of EGFRvIII-expressing cells . This might have implications for the development of therapeutic reagents simultaneously targeting different forms of the EGFR.

Int J Food Microbiol, 1997 Aug 19, 38(1), 55 - 63
Validation of a model describing the effects of temperature and water activity on the growth of psychrotrophic pseudomonads; Neumeyer K et al.; The reliability of the predictive model for the growth of psychrotrophic pseudomonads was evaluated both under controlled laboratory conditions and in industry conditions using various milks, milk-based products, meat and meat products . The validation process involved monitoring the growth of pseudomonads at various temperatures and comparing the observed generation times to those predicted by the model using bias and accuracy factors . For fluctuating temperatures bias and accuracy factors were used to compare the predicted and observed times to reach various population densities . The psychrotrophic pseudomonad model was shown to predict accurately the growth of pseudomonads in the products tested . In some instances knowledge of the lag phase duration was required to maximise the performance of the model.

Prostate, 1998 Mar 1, 34(4), 259 - 69
Identification of diphtheria toxin via screening as a potent cell cycle and p53-independent cytotoxin for human prostate cancer therapeutics; Rodriguez R et al.; BACKGROUND: Metastatic human prostate cancer requires novel therapeutic strategies in order to overcome its low proliferative rate and its resistance to conventional chemotherapeutic agents . To identify potential cytotoxin gene products for use in experimental therapeutics such as in vivo gene therapy, an in vitro screen was designed . METHODS: Eight recombinant cellular toxins were tested for activity against a spectrum of metastatic human prostate cancer cell phenotypes . Pseudomonas exotoxin A, ricin, tumor necrosis factor alpha (TNF-alpha), diphtheria toxin (DT), Crotalus durissus terrificus toxin, crotalus adamenteus toxin, Naja naja toxin, and Naja mocambique toxin were evaluated . Comparative survival distinguished the relative potencies of these cytotoxins for irreparable prostate cancer cell death . RESULTS: Of the phospholipase A2 toxins, Crotalus durissus terrificus and Naja mocambique are active against the PSA secreting LNCaP cell line; however, the effect is reversible, and no other hormone refractory prostate cell line tested is sensitive . Screening identified toxin-specific differences: dose-dependent cytotoxic activity against all human prostate cancer cell lines tested was only identified for ricin and diphtheria toxin (DT) as highly potent . DT has an IC50 in the range of 20-00 pM by clonogenic survival and kills irreversibly by both apoptosis as well as nonapototic pathways . Acquisition of p53 mutant status conferred no reduction in sensitivity to DT cytotoxicity . Cell cycle arrest by aphidicolin did not protect human prostate cells from irreversible DT-induced cell death . TNF-alpha had modest cytostatic activity in the screen; however, the combination of TNF-alpha and DT resulted in marked acceleration of the time to prostate cancer cell death . CONCLUSIONS: The rational screening of cytotoxins allows the identification of cell cycle-independent agents of variable potency against human prostate cancer . DT-mediated cell death is cell cycle independent, and p53 independent, making it particularly attractive for application to cytoreductive gene therapy, targeted monoclonal antibodies, and prodrug delivery of toxins applied to human prostate cancer therapeutics.

Microbiology, 1998 Feb, 144 ( Pt 2), 569 - 76
Natural genetic transformation of Pseudomonas stutzeri in a non-sterile soil; Sikorski J et al.; Natural transformation of the soil bacterium Pseudomonas stutzeri JM300 in a non-sterile brown earth microcosm was studied . For this purpose, the microcosm was loaded with purified DNA (plasmid or chromosomal DNA, both containing a high-frequency-transformation marker, his+, of the P . stutzeri genome), the non-adsorbed DNA was washed out with soil extract and then the soil was charged with competent cells (his-1) . Both chromosomal and plasmid transformants were found among the P . stutzeri cells recovered from the soil . The number of plasmid transformants increased in a linear fashion with the amount of DNA added {10-600 ng (0.7 g soil)-1} . The observed efficiency of transformation, the time course of transformant formation and the complete inhibition of transformation by DNase I, when added to the soil, were similar to that seen in optimized transformations in nutrient broth . Addition of cells as late as 3 d after loading the soil with plasmid DNA still yielded 3% of the initial transforming activity . This suggests that nucleases indigenous to the soil destroyed the transforming DNA, but at a rate allowing considerable DNA persistence . Transformants were also obtained when intact P . stutzeri cells were introduced into the soil to serve as plasmid DNA donors . Apparently, DNA was released from the cells, adsorbed to the soil material and subsequently taken up by recipient cells . The results indicate that competent cells of P . stutzeri were able to find access to and take up DNA bound on soil particles in the presence of micro-organisms and DNases indigenous to the soil.

Eur J Biochem, 1998 Feb 1, 251(3), 971 - 9
Structural investigation of the exopolysaccharide produced by Pseudomonas flavescens strain B62--degradation by a fungal cellulase and isolation of the oligosaccharide repeating unit; Cescutti P et al.; Pseudomonas flavescens strain B62 (NCPPB 3063) is a recently described bacterium isolated from walnut blight cankers . This strain has been designated as the type strain of a Pseudomonas rRNA group-I species . Strain B62 produced a mixture of two exopolysaccharides, differing in weight average relative molecular mass and composition . Only the most abundant exopolysaccharide (90% by mass), corresponding to the one with the lower molecular mass, was investigated by use of methylation analysis, partial acid hydrolysis, and NMR spectroscopy . The polysaccharide was depolymerised by the action of the cellulase produced by Penicillum funiculosum and the oligosaccharide obtained, corresponding to the repeating unit, was characterised by NMR spectroscopy and ion-spray mass spectrometry . The repeating unit of the B62 exopolysaccharide is {structure in text} where X is glucose (75%) or mannose (25%), and Lac is lactate . The O-acetyl groups are present only on 75% of the repeating units, and they are linked to the C6 of the hexose residues in non-stoichiometric amounts.

Mol Microbiol, 1998 Feb, 27(3), 651 - 9
Activation of the toluene-responsive regulator XylR causes a transcriptional switch between sigma54 and sigma70 promoters at the divergent Pr/Ps region of the TOL plasmid; Bertoni G et al.; The mechanism by which XylR, the toluene-responsive activator of the sigma54-dependent Pu and Ps promoters of the Pseudomonas TOL plasmid pWW0, downregulates its own sigma70 promoter Prhas been examined . An in vitro transcription system was developed in order to reproduce the repression of Probserved in cells of P . putida (pWW0) both in the presence and in the absence of the XylR inducer, benzyl alcohol . DNA templates bearing the two sigma70-RNA polymerase (RNAP) binding sites of Pr, which overlap the upstream activating sequences (UAS) for XylR in the divergent sigma54 promoter Ps, were transcribed in the presence of a constitutively active XylR variant deleted of its N-terminal domain (XylRdeltaA) . The addition of ATP, known to trigger multimerization of the regulator at the UAS, enhanced the repression of Pr by XylR . Furthermore, we observed activation of the divergent sigma54 promoter Ps during Pr downregulation by XylRdeltaA . These results support the notion that activation of XylR by aromatic inducers in vivo triggers a transcriptional switch between Pr and Ps . Such a switch is apparently caused by the ATP-dependent multimerization and strong DNA binding of the protein required for activation of the sigma54 promoter . This device could reset the level of XylR expression during activation of the sigma54 Pu and Ps promoters of the TOL plasmid.

Biochemistry (Mosc), 1997 Dec, 62(12), 1439 - 43
Formate dehydrogenase in a reversed micelle system: regulation of catalytic activity and oligomeric composition of the enzyme; Klyachko NL et al.; Formate dehydrogenases from the methylotrophic bacteria Pseudomonas sp . 101 and Mycobacterium vaccae N10 were studied in a system of Aerosol OT reversed micelles in octane . Three peaks of the catalytic activity were found on the plot of activity versus surfactant hydration degree (the size of the micellar inner cavity) which corresponded to functions of the enzyme in various oligomeric forms: monomeric, dimeric, and octameric . Kinetic data were confirmed by results of sedimentation analysis . The enzyme was chemically modified by a bifunctional reagent (dimethyl suberimidate) to obtain a catalytically active non-dissociating dimeric molecule of formate dehydrogenase . In the case of the covalently-linked non-dissociating dimeric enzyme, the peak which corresponded to the monomeric form of the enzyme was found to disappear from the catalytic activity curve.

Mikrobiol Z, 1997 Sep-Oct, 59(5), 28 - 33
{The properties of the proteins and nucleic acids of 3 Pseudomonas syringae phages}; Boiko AL et al.; The 9B, 123, 788/8 Pseudomonas syringae phages were investigated . PAAG electrophoretic profiles of phage proteins were identical for all three phages except the minor polypeptide having molecular weight 35,000 Da . The band corresponding to this protein was present only in 9B and 788/8 phage protein profiles . Amino acid composition of phage proteins varied insignificantly showing prevalence of Asp, Glu, Ala, Leu . Phage DNA fragments electrophoresis, carried out after processing with specific endonuclease Hind III, made it possible to evaluate common restriction sites in phage genomes . Genome molecular weight was equal to 15 mda for 9B phage and to 14 mDa for 123 and 788/8 phages . The analysis of phage growth cycle showed that latent period consisted of 50 min at 20 degrees C and the yield equalled to 70 virions per infected bacterium cell . The similarity of the phages' features suggests their broad spreading in the environment.

Appl Environ Microbiol, 1997 Dec, 63(12), 4839 - 43
Studies of the catabolic pathway of degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45: removal of the amino group from 2-aminomuconic semialdehyde; He Z et al.; Pseudomonas pseudoalcaligenes JS45 utilizes nitrobenzene as the sole source of nitrogen, carbon, and energy . Previous studies have shown that degradation of nitrobenzene involves the reduction of nitrobenzene to nitrosobenzene and hydroxylaminobenzene, followed by rearrangement to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde . In the present paper, we report the enzymatic reactions responsible for the release of ammonia after ring cleavage . 2-Aminomuconic semialdehyde was oxidized to 2-aminomuconate in the presence of NAD by enzymes in crude extracts . 2-Aminomuconate was subsequently deaminated stoichiometrically to 4-oxalocrotonic acid . No cofactors are required for the deamination . Two enzymes, 2-aminomuconic semialdehyde dehydrogenase and a novel 2-aminomuconate deaminase, distinguished by partial purification of the crude extracts, catalyzed the two reactions . 4-Oxalocrotonic acid was further degraded to pyruvate and acetaldehyde . The key enzyme, 2-aminomuconate deaminase, catalyzed the hydrolytic deamination that released ammonia, which served as the nitrogen source for growth of the organism.

Lipids, 1998 Jan, 33(1), 71 - 5
Studies of lipase-catalyzed esterification reactions of some acetylenic fatty acids; Lie Ken Jie MS et al.; Esterification of five positional isomers of acetylenic fatty acids {viz . 9:1(2a), 11:1(10a), 18:1(6a), 18:1(9a) and 22:1(13a)} of different chain lengths with n-butanol in n-hexane in the presence of eight different lipases {Lipozyme IM 20 (Rhizomucor miehei), Lipolase 100T (R . miehei), Novozyme 435 (Candida antarctica), PPL (porcine pancreatic lipase), CCL (C . cylindracea), PS-D (Pseudomonas cepacia), Lipase A-12 (Aspergillus niger) and Lipase AY-30 (C . rugosa)} was studied . 2-Nonynoic acid was not esterified except when catalyzed by the lipase from C . antarctica (Novozyme 435) to give 42% butyl ester after 48 h . The lipases from A . niger (Lipase A-12) and C . rugosa (Lipase AY-30) showed poor biocatalytic behavior in the esterification of the acetylenic fatty acids studied . 10-Undecynoic acid gave the highest conversion rate of esterification with each kind of lipase used . 6-Octadecynoic acid showed a marked degree of resistance to esterification carried out in the presence of C . cylindracea (CCL), P . cepacia (PS-D), or porcine pancreatic (PPL) lipase but not significantly in the presence of the lipases of R . miehei (Lipozyme IM 20), R . miehei (Lipolase 100T), or Novozyme 435 . 9-Octadecynoic acid and 13-docosynoic acid were not discriminated and were readily esterified by the remaining six lipases, but when compared to oleic acid the acetylenic fatty acids were comparatively much slower in conversion to the esters.

Appl Environ Microbiol, 1998 Feb, 64(2), 486 - 91
A cold-adapted lipase of an Alaskan psychrotroph, Pseudomonas sp . strain B11-1: gene cloning and enzyme purification and characterization; Choo DW et al.; A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a Pseudomonas strain . The lipase gene (lipP) was cloned from the strain and sequenced . The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714 . LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from Antarctic Moraxella TA144 (G . Feller, M . Thirty, J . L . Arpigny, and C . Gerday, DNA Cell Biol . 10:381-388, 1991) and the mammalian hormone-sensitive lipase (D . Langin, H . Laurell, L . S . Holst, P . Belfrage, and C . Holm, Proc . Natl . Acad . Sci . USA 90:4897-4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide . LipP was purified from an extract of recombinant Escherichia coli C600 cells harboring a plasmid coding for the lipP gene . The enzyme showed a 1,3-positional specificity toward triolein . p-Nitrophenyl esters of fatty acids with short to medium chains (C4 and C6) served as good substrates . The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8 . The activation energies for the hydrolysis of p-nitrophenyl butyrate and p-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35 degrees C . The enzyme was unstable at temperatures higher than 45 degrees C . The Km of the enzyme for p-nitrophenyl butyrate increased with increases in the assay temperature . The enzyme was strongly inhibited by Zn2+, Cu2+, Fe3+, and Hg2+ but was not affected by phenylmethylsulfonyl fluoride and bisnitrophenyl phosphate . Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.

Cell Struct Funct, 1997 Oct, 22(5), 545 - 54
Inhibitory effect of dideoxyforskolin on cell death induced by ricin, modeccin, diphtheria toxin, and Pseudomonas toxin in MDCK cells; Oda T et al.; We have found that 1,9-Dideoxyforskolin (DDF) strongly inhibited the cell death induced by ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in MDCK cells, suggesting that these protein toxins have a DDF-sensitive common pathway leading to cell death . However, no significant effect of forskolin on these toxins was observed, implying that cAMP-independent DDF specific mechanism is responsible for the inhibitory effect . The protective effect of DDF against ricin-induced cell death was significantly reversed by the increase in extracellular Ca2+ concentrations . The addition of brefeldin A (BFA) also reversed the protective effect of DDF, while BFA alone slightly increased the cytotoxicity of ricin . The protein synthesis inhibitory activity of modeccin was strongly inhibited by DDF, while only partial inhibition of the activities of ricin and diphtheria toxin was observed . However, the activity of Pseudomonas toxin was enhanced by DDF rather than inhibited . Thus, the process leading to cell death and protein synthesis inhibition by these toxins may be separately affected by DDF, and the protective effect of DDF against toxin-induced cell death is distinct from its effect on protein synthesis inhibition by toxins . Forskolin and DDF slightly increased rather than inhibited the binding and the internalization of ricin to MDCK cells . Despite the strong inhibitory effect of DDF on toxin-induced cell death, DDF did not block toxin-induced DNA fragmentation . These results suggest that DNA fragmentation and cell death may be triggered through separate pathways during apoptosis caused by these toxins, and that a DDF-sensitive specific step may be present in the pathway leading to cell death.

J Biol Chem, 1997 Dec 12, 272(50), 31707 - 11
Furin-mediated cleavage of Pseudomonas exotoxin-derived chimeric toxins; Chiron MF et al.; Pseudomonas exotoxin (PE) requires proteolytic cleavage to generate a 37-kDa C-terminal fragment that translocates to the cytosol and ADP-ribosylates elongation factor 2 . Cleavage within cells is mediated by furin, occurs between arginine 279 and glycine 280, and requires an arginine at both P1 and P4 residues . To study the proteolytic processing of PE-derived chimeric toxins, TGFalpha-PE38 (transforming growth factor fused to the domains II and III of PE) and a mutant form, TGFalpha-PE38gly279, were each produced in Escherichia coli . When assessed on various epidermal growth factor (EGF) receptor-positive cell lines, TGFalpha-PE38 was 100-500-fold more toxic than TGFalpha-PE38gly279 . In contrast to PE, where cleavage by furin is only evident at pH 5.5, furin cleaved TGFalpha-PE38 over a broad pH range, while TGFalpha-PE38gly279 was resistant to cleavage . TGFalpha-PE38 was poorly toxic for furin-deficient LoVo cells, unless it was first pretreated in vitro with furin . Furin treatment produced a nicked protein that was 30-fold more toxic than its unnicked counterpart . Using the single chain immunotoxin HB21scFv-PE40 as a substrate, furin-mediated processing of an antibody-based immunotoxin was also evaluated . HB21scFv-PE40, which targets cells expressing the transferrin receptor, was cleaved in a similar fashion to that of TGFalpha-PE38 and nicked HB21scFv-PE40 exhibited increased toxicity for LoVo cells . In short-term experiments, the rate of reduction in protein synthesis by furin-nicked immunotoxins was increased compared with unnicked protein, indicating that cleavage by furin can be a rate-limiting step . We conclude that furin-mediated cleavage of PE-derived immunotoxins is important for their cytotoxic activity.

Pneumologie, 1997 Aug, 51(8), 822 - 7
{Why do adults with mucoviscidosis refuse a medically recommended course of intravenous antibiotic therapy?}; Ullrich G et al.; BACKGROUND: Regular courses of intravenous antibiotics are recommended for the treatment of chronic Pseudomonas aeruglnosa (PA) infection in patients with cystic fibrosis . We report the results of interviews performed to evaluate why a subgroup of patients vote against regular intravenous (i.v.) antibiotic treatment . METHODS: Structured interviews covering a) the individual's perception of chronic PA infection, b) the patient's expectations regarding the effectiveness of i.v . treatment, c) the patient's personal reasons for refusal of i.v . treatment . STUDY COHORT: 16 out of 18 adult patients treated in the adult CF outpatient clinic at Hannover Medical School who had voted against the physician's recommendation to receive regular i.v . therapy twice a year . RESULTS: More than one half of the patients did not regard chronic PA infection as important due to the lack of specific symptoms . A subgroup of patients had no idea of what their clinical status should be if i.v . antibiotics would be necessary; these patients reported prior experience of treatment courses which had been ineffective and had been instituted after talking into the patients . The most frequent reasons against IV treatment were not being sick enough and fear of adverse drug effects . ASSESSMENT: The results are being discussed considering the physician-patient relationship . The reasons why patients refuse help should be extensively explored rather than simply addressing this attitude as "non-compliance" . Patients, too, come to reasonable decisions, and it is important to know their thoughts and reasoning if one intends to influence them.

Hautarzt, 1997 Aug, 48(8), 523 - 7
{Fluorescence with Wood's light . Current applications in dermatologic diagnosis, therapy follow-up and prevention}; Wigger-Alberti W et al.; The invisible long-wave ultraviolet radiation (340-450 nm, max.365 nm) produced by a Wood lamp can help to diagnose dermatoses with a characteristic fluorescence (tinea capitis, erythrasma, tinea versicolor, Pseudomonas infections, porphyrians, and pigmentary alterations) . It is also used in the detection of medications that are taken systemically (tetracycline) or that are applied to the skin . Recently, a fluorescence technique with Wood light has been used as a preventive measure to monitor and quantify skin protection at the workplace and to teach workers in high-risk occupations the proper use of protective creams.

Appl Biochem Biotechnol, 1998 Jan, 69(1), 53 - 67
Purification and stability of glutaryl-7-ACA acylase from Pseudomonas sp; Battistel E et al.; The enzyme glutaryl-7-ACA acylase from Pseudomonas sp . NCIMB 40474, produced by a recombinant Escherichia coli host, was purified to homogeneity . The enzyme is a tetramer composed of two couples of asymmetric dimers, each of them constituted of two subunits of mol wt 18 and 52 kDa, respectively . It was found that glutaric acid, one of the products of the substrate hydrolysis, is an effective acylase inhibitor . Between pH 6.0 and pH 10.0, the enzymatic activity is almost constant, but below pH 6.0 it progressively declines . The acylase activity decreased sharply as a function of guanidine HCl concentration . The loss is significant even at concentrations of denaturant lower than those causing unfolding, as suggested by UV spectroscopy and fluorescence emission studies . In these conditions (low denaturant concentration and low pH) the inactivation of the enzyme is caused by the tetramer dissociation into dimers . The lability of the quaternary structure of the enzyme is a key feature that must be taken into account for the improvement of the catalyst stability.

Arch Microbiol, 1998 Jan, 169(1), 29 - 35
Purification and characterization of 9-hexadecenoic acid cis-trans isomerase from pseudomonas sp . strain E-3
Okuyama H, Ueno A, Enari D, Morita N, Kusano T.
A 9-hexadecenoic acid cis-trans isomerase (9-isomerase) that catalyzed the cis-to-trans isomerization of the double bond of free 9-cis-hexadecenoic acid {16:1(9c)} was purified to homogeneity from an extract of Pseudomonas sp . strain E-3 and characterized . Electrophoresis of the purified enzyme on both incompletely denaturing and denaturing polyacrylamide gels yielded a single band of a protein with a molecular mass of 80 kDa, suggesting that the isomerase is a monomeric protein of 80 kDa . The 9-isomerase, assayed with 16:1(9c) as a substrate, had a specific activity of 22.8 &mgr;mol h-1 (mg protein)-1 and a Km of 117.6 mM . The optimal pH and temperature for catalysis were approximately pH 7-8 and 30 degrees C, respectively . The 9-isomerase catalyzed the cis-to-trans conversion of a double bond at positions 9, 10, or 11, but not that of a double bond at position 6 or 7 of cis-mono-unsaturated fatty acids with carbon chain lengths of 14, 15, 16, and 17 . Octadecenoic acids with a double bond at position 9 or 11 were not susceptible to isomerization . These results suggest that 9-isomerase has a strict specificity for both the position of the double bond and the chain length of the fatty acid . The enzyme catalyzed the cis-to-trans isomerization of fatty acids in a free form, and in the presence of a membrane fraction it was also able to isomerize 16:1(9c) esterified to phosphatidylethanolamine . The 9-isomerase was strongly inhibited by catecholic antioxidants such as alpha-tocopherol and nordihydroguaiaretic acid, but was not inhibited by 1, 10-phenanthroline or EDTA or under anoxic conditions . Based on these results, the possible mechanism of catalysis by this enzyme is discussed.

Biochem J, 1997 Dec 15, 328 ( Pt 3), 815 - 20
Reconstitution, morphology and crystallization of a fatty acid beta-oxidation multienzyme complex from Pseudomonas fragi; Ishikawa M et al.; The fatty acid beta-oxidation multienzyme complex from Pseudomonas fragi, HDT, exhibits predominantly the three enzymic activities of 2-enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16) . The HDT complex is encoded by the faoAB operon, consisting of the faoA and faoB genes that encode two individual constituents, the alpha-subunit and the beta-subunit . We have constructed Escherichia coli overexpression systems for the faoAB gene product (coexpression of the alpha- and beta-subunits), the alpha-subunit alone and the beta-subunit alone, and have purified the three respective products . Gel-filtration analysis revealed that the faoAB gene product forms a heterotetrameric structure, alpha2beta2, identical with the native HDT oligomeric state from P . fragi, whereas the alpha-subunit and beta-subunit individually form dimers . Electron microscopy demonstrated that each protein morphologically adopts the above oligomeric structures . The HDT complex, reconstituted in vitro from the isolated alpha- and beta-subunits, exhibits the three original enzymic activities and yields the same crystal as those from the native enzyme . CD measurements indicated that the alpha- and beta-dimers hardly alter their global conformations upon the formation of the HDT complex . Interestingly, the beta-dimer alone does not exhibit 3-oxoacyl-CoA thiolase activity, whereas the alpha-dimer alone exhibits both the 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities . These results suggest that the contact between the alpha- and beta-subunits is essential for the thiolase activity . We have identified several structurally important proteolytic sites within each subunit, which are protected in the intact heterotetrameric molecule . These findings allow the possible location of the interface between the two subunits, which should be crucial for the exhibition of thiolase activity.

J Ind Microbiol Biotechnol, 1997 Nov-Dec, 19(5-6), 401 - 7
Genetics of naphthalene and phenanthrene degradation by Comamonas testosteroni; Goyal AK et al.; Naphthalene and phenanthrene have long been used as model compounds to investigate the ability of bacteria to degrade polycyclic aromatic hydrocarbons . The catabolic pathways have been determined, several of the enzymes have been purified to homogeneity, and genes have been cloned and sequenced . However, the majority of this work has been performed with fast growing Pseudomonas strains related to the archetypal naphthalene-degrading P . putida strains G7 and NCIB 9816-4 . Recently Comamonas testosteroni strains able to degrade naphthalene and phenanthrene have been isolated and shown to possess genes for polycyclic aromatic hydrocarbon degradation that are different from the canonical genes found in Pseudomonas species . For instance, C . testosteroni GZ39 has genes for naphthalene and phenanthrene degradation which are not only different from those found in Pseudomonas species but are also arranged in a different configuration . C . testosteroni GZ42, on the other hand, has genes for naphthalene and phenanthrene degradation which are arranged almost the same as those found in Pseudomonas species but show significant divergence in their sequences.

J Ind Microbiol Biotechnol, 1997 Nov-Dec, 19(5-6), 355 - 9
Purification and properties of ferredoxinBPH, a component of biphenyl 2,3-dioxygenase of Pseudomonas sp strain LB400; Haddock JD et al.; The ferredoxin component (ferredoxinBPH) of biphenyl 2,3-dioxygenase was purified to homogeneity from crude cell extract of Pseudomonas sp strain LB400 using ion exchange, hydrophobic interaction and gel filtration column chromatography . The protein was a monomer with a molecular weight of 15,000 and contained 2 gram-atoms each of iron and acid-labile sulfur . Ultraviolet-visible absorbance spectroscopy showed peaks at 325 nm and 460 nm with a broad shoulder around 575 nm . The spectrum was partially bleached in the visible region upon reduction by reductaseBPH with NADPH as the source of electrons . Electron paramagnetic resonance spectrometry showed no signals for the oxidized protein . Upon reduction with sodium dithionite, signals with gx = 1.82, gy = 1.92 and gz = 2.02 were detected . These results indicate that the protein contains a Rieske-type (2Fe-2S) iron-sulfur center . FerredoxinBPH was required for the oxidation of biphenyl by the terminal oxygenase component of the enzyme and is probably involved in the transfer of reducing equivalents from reductaseBPH to the terminal oxygenase during catalysis.

Cytometry, 1998 Jan 1, 31(1), 20 - 8
Quantification of apoptotic and lytic cell death by video microscopy in combination with artificial neural networks; Weisser M et al.; Apoptosis is characterized by chromatin condensation and DNA fragmentation in the absence of release of cytosolic enzymes such as lactate dehydrogenase (LDH) . In contrast, necrosis is characterized by cell swelling, membrane disintegration with cytosolic enzyme release, and absence of chromatin condensation . Staining of cells with Hoechst H33342 dye is a routine method for identifying apoptotic nuclei . However, this process is tedious and prone to individual bias . Therefore, we investigated the suitability of an artificial neural network (ANN) to recognize and distinguish between apoptosis and necrosis . Using a human endothelial cell line (ECV304), we trained an ANN with DNA-stained apoptotic and necrotic nuclei obtained from cells exposed for 8 h to cycloheximide (Chx; 100 microM)/tumour necrosis factor-alpha (TNF; 50 ng/ml) or tert-butylhydroperoxide (t-BH; 2 mM), respectively . After this training step, the ANN correctly assigned necrosis induced by t-BH and apoptosis induced via Chx/TNF, Chx/CD95 activation, Pseudomonas exotoxin A/TNF, or X-rays . In all cases, apoptosis and necrosis as assigned by the ANN correlated with DNA fragmentation and LDH release.

Mol Plant Microbe Interact, 1998 Feb, 11(2), 156 - 62
Auxin production is a common feature of most pathovars of Pseudomonas syringae; Glickmann E et al.; We investigated indole-3-acetic acid (IAA) production by 57 pathovars of Pseudomonas syringae and related species . Most of those analyzed produced IAA, especially in the presence of tryptophan . Eight strains produced high IAA concentrations in the absence of Trp . The iaaM and iaaH genes of P . savastanoi pv . savastanoi were detected in a limited number of strains only, including the eight above-mentioned strains . Thus, IAA synthesis in most assayed strains of P . syringae and related species does not involve genes highly similar to iaaM and iaaH . In contrast, the iaaL gene encoding an IAA-lysine synthase was detected in most pathovars, and was often found on plasmids.

Plant Physiol, 1998 Jan, 116(1), 231 - 8
Accumulation of salicylic acid and 4-hydroxybenzoic acid in phloem fluids of cucumber during systemic acquired resistance is preceded by a transient increase in phenylalanine ammonia-lyase activity in petioles and stems; Smith-Becker J et al.; Cucumber (Cucumis sativa) leaves infiltrated with Pseudomonas syringae pv . syringae cells produced a mobile signal for systemic acquired resistance between 3 and 6 h after inoculation . The production of a mobile signal by inoculated leaves was followed by a transient increase in phenylalanine ammonia-lyase (PAL) activity in the petioles of inoculated leaves and in stems above inoculated leaves; with peaks in activity at 9 and 12 h, respectively, after inoculation . In contrast, PAL activity in inoculated leaves continued to rise slowly for at least 18 h . No increases in PAL activity were detected in healthy leaves of inoculated plants . Two benzoic acid derivatives, salicylic acid (SA) and 4-hydroxybenzoic acid (4HBA), began to accumulate in phloem fluids at about the time PAL activity began to increase, reaching maximum concentrations 15 h after inoculation . The accumulation of SA and 4HBA in phloem fluids was unaffected by the removal of all leaves 6 h after inoculation, and seedlings excised from roots prior to inoculation still accumulated high levels of SA and 4HBA . These results suggest that SA and 4HBA are synthesized de novo in stems and petioles in response to a mobile signal from the inoculated leaf.

Biochem Pharmacol, 1998 Jan 15, 55(2), 159 - 68
Combined effect of organophosphorus hydrolase and oxime on the reactivation rate of diethylphosphoryl-acetylcholinesterase conjugates; Ashani Y et al.; Reactivation of inhibited acetylcholinesterase (AChE) is essential for rapid recovery after organophosphate (OP) poisoning . However, following administration of an oxime reactivator, such as pralidoxime mesylate (P2S), in patients poisoned with certain diethylphosphorothioate pesticides, no reactivation is observed, presumably due to reinhibition by circulating anti-cholinesterase OPs . Pretreatment alone with organophosphorus hydrolases (OPH) that are capable of rapidly hydrolyzing OPs was demonstrated, in animals, to confer significant protection against OP toxicity . One strategy to augment the potentially therapeutic scope of OPHs is a combined post-exposure treatment consisting of a drug(s) commonly used against OP toxicity and a suitable hydrolase . In this study, we examined the in vitro ability of OPH from Pseudomonas sp . (OPHps) to prevent reinhibition of P2S-reactivated AChE by excess OPs . The kinetic parameters of the reactivation of a series of diethylphosphoryl-AChE (DEP--AChE) conjugates, obtained by the use of various diethylphosphates, were determined and compared with the rates of reactivation in the presence of OPHps, with and without the OP inhibitors in the reactivation medium . Extrapolation of the in vitro results to in vivo conditions suggests that an OPHps concentration as low as 1 microgram/mL blood would result in a 100-fold decrease in the concentration of circulating anti-AChE pesticides within less than one blood-circulation time, thereby minimizing reinhibition of the reactivated enzyme . Thus, for DEP-based pesticides, the combination of P2S-OPH treatment can significantly improve clinical recovery after OP intoxication . In addition, it is shown here for the first time that an OPH can effectively hydrolyze quaternary ammonium-containing OPs . This indicates that hydrolysis of phosphorylated oximes, toxic side products of oxime treatment, may also be accelerated by OPHs.

Med Klin (Munich), 1997 Oct 15, 92(10), 626 - 9
{Economic aspects in treatment of cystic fibrosis with chronic pulmonary pseudomonas infection . Ambulatory intravenous therapy in comparison with inpatient treatment}; Graf von der Schulenburg JM et al.; BACKGROUND: Due to limited resources within the health service and the continuous discussion on cost containment, economic criteria should also be considered when assessing therapy concepts . Particular results in terms of economic efficiency reserves are to be expected from a transfer of care from the in-patient to the out-patient sector . METHODS: In a prospective, direct cost recording of all relevant uses of resources, the direct and indirect costs of the treatment of 14 patients with cystic fibrosis (CF) were included in the cross-over-design . The quality of life was recorded at least once for each patient using the EuroQol . In-patient intravenous antibiotic therapy carried out during the block of out-patient care served as one of the disqualification criteria when selecting patients . RESULT: Over an observation period of nine months, the average direct cost recorded were DM 35,706 for out-patient and DM 40,143 for in-patient treatment (+15%) . As far as indirect costs are concerned, the losses of production in the national economy recorded for in-patient treatment were 80% higher . CONCLUSION: The direct and indirect costs for in-patient CF-therapy are in total higher than for out-patient care . Whether these cost advantages have to be "bought" with lower medical effectiveness needs to be demonstrated by further clinical studies . In the sense of the disease management approach, the results of this study should be used to help rationally weigh up the costs of out-patient care against alternative treatment concepts.

J Biol Chem, 1997 Dec 26, 272(52), 33015 - 22
Three-dimensional structure of L-2-haloacid dehalogenase from Xanthobacter autotrophicus GJ10 complexed with the substrate-analogue formate; Ridder IS et al.; The L-2-haloacid dehalogenase from the 1,2-dichloroethane degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids . Its crystal structure was solved by the method of multiple isomorphous replacement with incorporation of anomalous scattering information and solvent flattening, and was refined at 1.95-A resolution to an R factor of 21.3% . The three-dimensional structure is similar to that of the homologous L-2-haloacid dehalogenase from Pseudomonas sp . YL (1), but the X . autotrophicus enzyme has an extra dimerization domain, an active site cavity that is completely shielded from the solvent, and a different orientation of several catalytically important amino acid residues . Moreover, under the conditions used, a formate ion is bound in the active site . The position of this substrate-analogue provides valuable information on the reaction mechanism and explains the limited substrate specificity of the Xanthobacter L-2-haloacid dehalogenase.

Biochemistry, 1997 Dec 16, 36(50), 15650 - 9
Acyl-adenylate motif of the acyl-adenylate/thioester-forming enzyme superfamily: a site-directed mutagenesis study with the Pseudomonas sp . strain CBS3 4-chlorobenzoate:coenzyme A ligase; Chang KH et al.; 4-Chlorobenzoate:coenzyme A (4-CBA:CoA) ligase catalyzes 4-chlorobenzoyl-coenzyme A formation in a two-step reaction consisting of the adenylation of 4-chlorobenzoate with adenosine 5'-triphosphate followed by acyl transfer from the 4-chlorobenzoyl adenosine 5'-monophosphate diester intermediate to coenzyme A . In this study, two core motifs present in the Pseudomonas sp . strain CBS3 4-CBA:CoA ligase (motif I, 161T-S-G-T-T-G-L-P-K-G170, and motif II, 302Y-G-T-T-E306) and conserved among the sequences representing the acyl-adenylate/thioester-forming enzyme family (to which the ligase belongs) were tested for their possible role in substrate binding and/or catalysis . The site-directed mutants G163I, G166I, P168A, K169M, and E306Q were prepared and then subjected to steady-state and transient kinetic studies . The results, which indicate reduced catalysis of the adenylation of 4-chlorobenzoate in the mutant enzymes, are interpreted within the context of the three-dimensional structure of the acyl-adenylate/thioester-forming enzyme family member, firefly luciferase.

Biosci Biotechnol Biochem, 1997 Dec, 61(12), 1986 - 90
Expression of genes encoding membrane-bound hydrogenase in Pseudomonas hydrogenovora under autotrophic condition is dependent on two different promoters; Ohtsuki T et al.; Expression of a membrane-bound hydrogenase of the hydrogen-utilizing bacterium Pseudomonas hydrogenovora was induced specifically in autotrophic condition . Dot blot and primer extension analysis showed that the transcription of the hydrogenase gene started from the region upstream of a hydrogenase structural gene, hupS, which contained three putative sigma54-type promoter sequences (Phup1, Phup2, and Phup3) . The lacZ-fusion analysis of the hupS-upstream region combined with site-directed mutagenesis showed that Phup1 and Phup3 would be essential for a transcriptional initiation . It was also found that the region upstream of Phup sequences was concerned with regulation of transcription.

Lipids, 1997 Dec, 32(12), 1297 - 300
Lipase-based quantitation of triacylglycerols in cellular lipid extracts: requirement for presence of detergent and prior separation by thin-layer chromatography; Van Veldhoven PP et al.; A protocol, based on the use of Pseudomonas lipase, is presented to measure quantitatively the amount of triacylglycerols in extracts from cultured cells of tissues . Since the lipase also acts on di- and monoacylglycerols, separation of the extracts by thin-layer chromatography is recommended . In order to allow the lipase-catalyzed hydrolysis to proceed efficiently, lipid extracts or eluates from silica scrapings were mixed with the detergent Thesit {dodecylpoly(ethylene glycol ether)}, prior to drying . After dissolution of the dried residues in water, the amount of triacylglycerols was quantified using Pseudomonas sp . lipase, glycerol kinase, glycerol-phosphate oxidase, and peroxidase . The activity of the latter enzyme was followed either colorimetrically in the presence of 4-aminoantipyrine and 2,4,6-tribromo-3-hydroxybenzoic acid or fluorimetrically in the presence of homovanillic acid.

Proc Natl Acad Sci U S A, 1998 Jan 20, 95(2), 669 - 74
Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity; Chowdhury PS et al.; Mesothelin is a differentiation antigen present on the surface of ovarian cancers, mesotheliomas, and several other types of human cancers . Because among normal tissues, mesothelin is present only on mesothelial cells, it represents a good target for antibody-mediated delivery of cytotoxic agents . In the present study mice were immunized with an eukaryotic expression vector coding for mesothelin . When high serum antibody titers were obtained, a phage display library was made from the splenic mRNA of these mice . After three rounds of panning on recombinant mesothelin, a single-chain Fv (scFv)-displaying phage was selected that bound specifically to recombinant mesothelin and mesothelin-positive cells . The scFv was used to construct an immunotoxin by genetically fusing it with a truncated mutant of Pseudomonas exotoxin A . The purified immunotoxin binds mesothelin with high affinity (Kd 11 nm), is stable for over 40 hr at 37 degrees C and is very cytotoxic to cells expressing mesothelin . It also produces regressions of tumors expressing mesothelin . This combination of selective cytotoxicity, high activity, and stability makes the immunotoxin a good candidate for development as a therapeutic agent . This work also shows that DNA immunization can be used to isolate and clone antibodies against epitopes present on human proteins in their native conformation.

Biochem Biophys Res Commun, 1997 Dec 18, 241(2), 294 - 6
Enzymatic hydrolysis of Russian-VX by organophosphorus hydrolase; Rastogi VK et al.; The Russian-VX (R-VX) is the principle V-type nerve agent in the chemical warfare (CW) arsenal of the Former Soviet Union . We here report the enzymatic hydrolysis of the P-S bond of Russian-VX by organophosphorus hydrolase (OPH) from Pseudomonas diminuta . While the Michaelis constant, K(m) for R-VX (474 microM), was similar to that for VX (434 microM), the Vmax for R-VX (2.1 mumoles/mg/min) was about four-fold higher compared to that for VX (0.56 mumoles/mg/min) . A 50% inhibition in the rate of the enzymatic hydrolysis of R-VX was observed in the presence of 0.5% ethanol, isoamyl-alcohol, or isopropanol . The presence of acetonitrile, diethylene glycol, or methanol had marginal effects . These results comprise the first demonstration of enzymatic detoxification of R-VX.

Biochem J, 1997 Nov 15, 328 ( Pt 1), 131 - 6
Recombinant two-iron rubredoxin of Pseudomonas oleovorans: overexpression, purification and characterization by optical, CD and 113Cd NMR spectroscopies; Lee HJ et al.; The gene (alk G) encoding the two-iron rubredoxin of Pseudomonas oleovorans was amplified from genomic DNA by PCR and subcloned into the expression vector pKK223-3 . The vector directed the high-level production of rubredoxin in Escherichia coli . A simple three-step procedure was used to purify recombinant rubredoxin in the 1Fe form . 1Fe-rubredoxin was readily converted to the 2Fe, apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presence or absence of ferrous ammonium sulphate or CdCl2 respectively . Recombinant 1Fe and 2Fe rubredoxins are redox-active and able to transfer electrons from reduced spinach ferredoxin reductase to cytochrome c . The absorption spectrum and dichroic features of the CD spectrum for the cadmium-substituted protein are similar to those reported for cadmium-substituted Desulfovibrio gigas rubredoxin {Henehan, Poutney, Zerbe and Vasak (1993) Protein Sci . 2, 1756-1764} . Difference absorption spectroscopy of cadmium-substituted rubredoxin revealed the presence of four Gaussian-resolved maxima at 207, 228, 241 and 280 nm; the 241 nm band is attributable, from Jorgensen's electronegativity theory, to a CysS-CdII charge-transfer excitation . The 113Cd NMR spectrum of the 113Cd-substituted rubredoxin contains two 113Cd resonances with chemical shifts located at 732.3 and 730 p.p.m . The broader linewidth and high frequency shift of the resonance at 730 p . p.m . indicates that the Cd2+ ion is undergoing chemical exchange and, consistent with the difference absorption spectra, is bound less tightly than the Cd2+ ion, giving rise to the chemical shift at 732.3 p.p.m.

J Bacteriol, 1998 Jan, 180(1), 152 - 8
AtzC is a new member of the amidohydrolase protein superfamily and is homologous to other atrazine-metabolizing enzymes; Sadowsky MJ et al.; Pseudomonas sp . strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC . The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine . The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding N-isopropylammelide . In this study, the third gene in the atrazine catabolic pathway, atzC, was cloned from a Pseudomonas sp . strain ADP cosmid library as a 25-kb EcoRI DNA fragment in Escherichia coli . The atzC gene was further delimited by functional analysis following transposon Tn5 mutagenesis and subcloned as a 2.0-kb EcoRI-AvaI fragment . An E . coli strain containing this DNA fragment expressed N-isopropylammelide isopropylamino hydrolase activity, metabolizing N-isopropylammelide stoichiometrically to cyanuric acid and N-isopropylamine . The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids . AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily . The sequence of AtzC was compared to that of E . coli cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein . Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity . AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase . Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family . Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.

J Bacteriol, 1998 Jan, 180(1), 27 - 34
EpsR modulates production of extracellular polysaccharides in the bacterial wilt pathogen Ralstonia (Pseudomonas) solanacearum; Chapman MR et al.; Ralstonia solanacearum is the causal agent of bacterial wilt of many agriculturally important crops . Exopolysaccharide synthesized by products of the epsI operon is the major virulence factor for R . solanacearum . Expression of epsI has been demonstrated to be under the control of several proteins, including several two-component regulators . Overexpression of EpsR was found previously to reduce the amount of synthesis specifically from the epsI promoter . Here we present data that a single chromosomal copy of epsR activates the epsI promoter, suggesting that EpsR is a concentration-dependent effector of epsI gene expression . Furthermore, the ability of EpsR to modulate epsI expression is dependent on the phosphorylation state of EpsR . Gel mobility shift assays suggest that EpsR can specifically bind the epsI promoter and that this binding requires a phosphorylated form of EpsR.

Nat Biotechnol, 1997 Dec, 15(13), 1398 - 401
Genetic engineering of parthenocarpic plants; Rotino GL et al.; Transgenic tobacco and eggplants expressing the coding region of the iaaM gene from Pseudomonas syringae pv . savastanoi, under the control of the regulatory sequences of the ovule-specific DefH9 gene from Antirrhinum majus, showed parthenocarpic fruit development . Expression of the DefH9-iaaM chimeric transgene occurs during flower development in both tobacco and eggplant . Seedless fruits were produced by emasculated flowers . When pollinated, the parthenocarpic plants produced fruits containing seeds . In eggplant, the genetic manipulation allowed fruit set and growth under environmental conditions prohibitive for fruit setting in the untransformed line, which did not set fruit at all . Under normal environmental conditions, production of marketable fruits took place from pollinated and unpollinated transgenic flowers, while flowers of untransformed control plants did produce fruits of marketable size only from fertilized flowers.

Gene, 1997 Oct 24, 200(1-2), 163 - 72
Primary structure of an invertebrate dihydrolipoamide dehydrogenase with phylogenetic relationship to vertebrate and bacterial disulfide oxidoreductases; Pullikuth AK et al.; Dihydrolipoamide dehydrogenase (E3) is a flavoprotein component of multi-enzyme complexes catalyzing oxidative decarboxylation of alpha-ketoacids in the Krebs' cycle . We have cloned a 2.4-kb E3 cDNA from an arthropod, Manduca sexta, that codes for 497 amino acids and translates to a 51-kDa protein in vitro . Sequences at and around the dinucleotide binding domains, disulfide active site and the C-terminal interface domain involved in substrate binding are highly conserved in Manduca E3 . Phylogenetic analysis of protein sequences from the flavoprotein class of disulfide oxidoreductases family of enzymes suggests that in spite of the homologous nature of E3 and glutathione reductase (goR) in sequence and structure, E3 shares a common ancestor with mercuric reductase (merA), whereas goR is more related to trypanothione reductase (tryR) than to other members . All members, except goRs, seemed to be monophyletic . Plant goRs seemed to have arisen differently and are more closely related to tryRs than to bacterial and vertebrate goRs . Earlier speculation on the nature of origin of E3 in Pseudomonas is not supported by phylogenetic data . A possible structural relationship of Manduca E3 to other pyridine-binding proteins, such as the neurotransmitter transporters and channels, is proposed.

Med Pediatr Oncol, 1998 Feb, 30(2), 95 - 100
Home intravenous antibiotic treatment for febrile episodes in immune-compromised pediatric patients; Shemesh E et al.; The purpose of this work was to assess the feasibility of home intravenous antibiotic treatment (HIAT) for febrile episodes in immune-compromised (neutropenic, splenectomized), low-risk pediatric patients . Thirty hematology-oncology patients who presented to our emergency room from January 1993 to January 1995 and who suffered from a febrile episode and were considered at low risk for septic complications were immediately discharged on HIAT . Patients were followed for at least 3 weeks after recovery . Patients and parents were retrospectively questioned about adverse effects and about their degree of satisfaction with home treatment . Patients who required hospitalization during this period were considered unresponsive to HIAT and were analyzed for causes and adverse effects . Thirteen out of 60 (22%) febrile episodes, or eight out of 42 (19%) episodes of fever and neutropenia eventually led to hospitalization . Pseudomonas species infections were associated with the highest rate of unresponsiveness (88%) . A central venous catheter infection developed in two cases following HIAT (two cases out of 640 days of therapy) . No other complications were identified . No infection-related morbidity was observed . Patients and parents were highly satisfied with HIAT and wanted to use it again, if necessary . Immediate discharge on HIAT for low-risk pediatric immune-compromised patients suffering from a febrile episode is feasible, safe, and well accepted by patients and families . Patients who are found to have Pseudomonas infections should probably be hospitalized . Our results are preliminary and must be confirmed by a prospective, randomized trial before definite recommendations can be made.

J Bacteriol, 1997 Dec, 179(24), 7663 - 70
N-acyl-homoserine lactone-mediated regulation of phenazine gene expression by Pseudomonas aureofaciens 30-84 in the wheat rhizosphere; Wood DW et al.; Pseudomonas aureofaciens 30-84 is a soilborne bacterium that colonizes the wheat rhizosphere . This strain produces three phenazine antibiotics which suppress take-all disease of wheat by inhibition of the causative agent Gaeumannomyces graminis var . tritici . Phenazines also enhance survival of 30-84 within the wheat rhizosphere in competition with other organisms . Expression of the phenazine biosynthetic operon is controlled by the phzR/phzI N-acyl-homoserine lactone (AHL) response system (L . S . Pierson III et al., J . Bacterial 176:3966-3974, 1994; D . W . Wood and L . S . Pierson III, Gene 168:49-53, 1996) . By using high-pressure liquid chromatography coupled with high-resolution mass spectrometry, the AHL produced by PhzI has now been identified as N-hexanoyl-homoserine lactone (HHL) . In addition, the ability of HHL to serve as an interpopulation signal molecule in the wheat rhizosphere has been examined by using isogenic reporter strains . Disruption of phzI reduced expression of the phenazine biosynthetic operon 1,000-fold in the wheat rhizosphere . Coinoculation of an isogenic strain which produced the endogenous HHL signal restored phenazine gene expression in the phzI mutant to wild-type levels in situ . These results demonstrate that HHL is required for phenazine expression in situ and is an effective interpopulation signal molecule in the wheat rhizosphere.

Biosci Biotechnol Biochem, 1997 Nov, 61(11), 1853 - 7
Cloning and sequence analysis of the gene (alyII) coding for an alginate lyase of Pseudomonas sp . OS-ALG-9; Kraiwattanapong J et al.; Pseudomonas sp . OS-ALG-9 produces several kinds of alginate-degrading enzymes both intra- and extracellularly . As a second alginate lyase of this bacterium, the gene encoding alyII has been cloned in Escherichia coli JM109 by shotgun techniques and then sequenced . The alyII gene has an open reading frame of 2141 bp encoding 713 amino acid residues with a calculated molecular mass of 79,803 Da . The deduced amino acid sequence did not show any extensive similarity with those of other known alginate lyases, however, hydrophobic cluster analysis showed that alyII belonged to class 3 of alginate lyases . The alginate lyase from E . coli harboring the alyII gene showed a single active band, which coincided with one of four major alginate lyases from the crude cell extracts of Pseudomonas sp . OS-ALG-9 on a zymogram.

J Biochem (Tokyo), 1997 Oct, 122(4), 788 - 94
Resynthesis of reactive site peptide bond and temporary inhibition of Streptomyces metalloproteinase inhibitor; Seeram SS et al.; Streptomyces metalloproteinase inhibitor (SMPI) is a small proteinaceous inhibitor which inhibits metalloproteinases such as thermolysin (Ki =1.14 x 10(-10) M) . When incubated with the enzyme, it is gradually hydrolyzed at the Cys64-Val65 peptide bond, which was identified as the reactive site by mutational analysis . To achieve a further understanding of the inhibition mechanism, we attempted to resynthesize the cleaved reactive site by using the enzyme catalytic action . The native inhibitor was resynthesized from the modified inhibitor (Ki =2.18 x 10(-8) M) by incubation with a catalytic amount of thermolysin under the same conditions as used for hydrolysis (pH 7.5, 25 degrees C), suggesting that SMPI follows the standard mechanism of inhibition of serine proteinase inhibitors . Temporary inhibition was observed when the native inhibitor and thermolysin were incubated at a 1:100 (mol/mol) enzyme-inhibitor ratio at 37 degrees C . SMPI showed temporary inhibition towards all the enzymes it inhibited . The inhibitory spectrum of SMPI was analyzed with various metalloproteinases based on the Ki values and limited proteolysis patterns . Pseudomonas elastase and Streptomyces griseus metalloproteinase II formed more stable complexes and showed much lower Ki values (approximately 2 pM) than thermolysin . In the limited proteolysis experiments weak inhibitors were degraded by the enzymes . SMPI did not inhibit almelysin, Streptomyces caespitosus neutral proteinase or matrix metalloproteinases . SMPI specifically inhibits metalloproteinases which are sensitive to phosphoramidon.

Biochemistry, 1997 Nov 25, 36(47), 14577 - 82
An early step in Pseudomonas exotoxin action is removal of the terminal lysine residue, which allows binding to the KDEL receptor; Hessler JL et al.; During the intoxication process, Pseudomonas exotoxin (PE) and immunotoxins containing PE internalize into the target cell and become processed into two fragments, and the carboxyl fragment translocates into the cytosol where it inactivates elongation factor 2 . We have proposed that after internalization into cells the carboxyl-terminal fragment of PE (amino acids 280-613), which ends in REDLK, binds to the KDEL receptor (ERD2) which carries it to the endoplasmic reticulum, from which the PE fragment translocates to the cytosol . Earlier experiments showing that REDL but not REDLK binds to the KDEL receptor suggested that the terminal lysine is removed sometime during the intoxication process . To determine if and where this occurs, we exposed a peptide ending in REDLK to malignant cells in culture and found that binding to the KDEL receptor was restored . Restoration of receptor binding also occurred if a peptide or toxin ending in REDLK at its carboxyl terminus was incubated with plasma, indicating that the terminal lysine is removed prior to entry of the toxin into the cell . We conclude that plasma carboxypeptidase(s) cleave(s) the lysine residue from the carboxyl terminus of PE and PE-containing immunotoxins as an early and essential step in their cellular intoxication pathway.

Cornea, 1997 Nov, 16(6), 602 - 11
The Castroviejo Lecture . Prolonged eyelid closure is a risk to the cornea; Baum JL; PURPOSE: The author introduces new concepts and summarizes published evidence suggesting that prolonged eyelid closure puts the cornea at risk . METHODS: Significant clinical and experimental publications are reviewed, and the author's experience is applied relating to the pathogenesis and treatment of a variety of clinical entities thought to be induced, to some degree, by prolonged eyelid closure . RESULTS: Evidence from the scientific literature suggests that the hypoxia or reduced tear volume or both that result after prolonged eyelid closure, especially during sleep and when a soft contact lens is in place, serve as risk factors in the "sucked-on" contact lens syndrome, recurrent corneal erosion, chronic corneal deepithelialization, Pseudomonas keratitis, filamentary keratitis, superior limbic keratoconjunctivitis, sterile midperipheral corneal infiltrates, and corneal vascularization . The limbal stem cells under the upper eyelid are subjected to continuous hypoxic stress and are at special risk to other insults . CONCLUSIONS: Prolonged eyelid closure, such as in patching and in sleep, is a risk factor in the pathogenesis of a variety of corneal conditions.

FEBS Lett, 1997 Nov 10, 417(2), 168 - 72
Purified HrpA of Pseudomonas syringae pv . tomato DC3000 reassembles into pili; Roine E et al.; Pseudomonas syringae pv . tomato DC3000 produces Hrp pili under inducing in vitro conditions . A preparation of partially purified extracellular filaments contains HrpA, flagellin and some minor contaminants . HrpA was separated from the major contaminant, the flagellin, by gel filtration to a fraction containing HrpA as well as its three N-terminally truncated forms . These were further separated by two steps of reversed phase chromatography . HrpA and its degradation products were each shown to reassemble into filament structures after denaturation and renaturation showing that HrpA alone is sufficient for formation of filament structures.

J Bacteriol, 1997 Dec, 179(23), 7331 - 42
The cold shock response of the psychrotrophic bacterium Pseudomonas fragi involves four low-molecular-mass nucleic acid-binding proteins; Michel V et al.; The psychrotrophic bacterium Pseudomonas fragi was subjected to cold shocks from 30 or 20 to 5 degrees C . The downshifts were followed by a lag phase before growth resumed at a characteristic 5 degrees C growth rate . The analysis of protein patterns by two-dimentional gel electrophoresis revealed overexpression of 25 or 17 proteins and underexpression of 12 proteins following the 30- or 20-to-5 degrees C shift, respectively . The two downshifts shared similar variations of synthesis of 20 proteins . The kinetic analysis distinguished the induced proteins into cold shock proteins (Csps), which were rapidly but transiently overexpressed, and cold acclimation proteins (Caps), which were more or less rapidly induced but still overexpressed several hours after the downshifts . Among the cold-induced proteins, four low-molecular-mass proteins, two of them previously characterized as Caps (CapA and CapB), and heat acclimation proteins (Haps) as well as heat shock proteins (Hsps) for the two others (TapA and TapB) displayed higher levels of induction . Partial amino acid sequences, obtained by microsequencing, were used to design primers to amplify by PCR the four genes and then determine their nucleotide sequences . A BamHI-EcoRI restriction fragment of 1.9 kb, containing the complete coding sequence for capB, was cloned and sequenced . The four peptides belong to the family of small nucleic acid-binding proteins as CspA, the major Escherichia coli Csp . They are likely to play a major role in the adaptative response of P . fragi to environmental temperature changes.

Immunotechnology, 1996 Feb, 2(1), 47 - 57
In vitro and in vivo characterisation of a recombinant carboxypeptidase G2::anti-CEA scFv fusion protein; Michael NP et al.; BACKGROUND: There is considerable interest in the specific targeting of therapeutic agents to cancer cells . Of particular promise is a technique known as Antibody-Directed Enzyme Prodrug Therapy (ADEPT) . In this approach an enzyme is targeted to the tumour by its conjugation to a tumour specific-antibody tumour . After allowing sufficient time for the conjugate to localise at the tumour and clear from the circulatory system, a relatively non-toxic prodrug is administered . This prodrug is converted to a highly cytotoxic drug by the action of the targeted enzyme localised at the tumour site . OBJECTIVES: To construct gene fusions between the pseudomonad carboxypeptidase G2 (CPG2) gene and DNA encoding MFE-23 (an anti-carcinoembryonic antigen (CEA) single-chain Fv (scFv) molecule), derived from a phage display library . To overexpress the resultant gene fusions in Escherichia coli, and assess the in vitro and in vivo properties of the purified fusion proteins . STUDY DESIGN: To introduce unique cloning restriction sites into the 5'-end of the CPG2 gene by site-directed mutagenesis to facilitate fusion to the 3'-end of the gene encoding MFE-23 (constructs with or without a flexible (Gly4Ser)3 linker-encoding sequence were designed) . To overexpress the resultant gene fusions under transcriptional control of the lac promoter and to direct the fusion proteins produced to the periplasmic space of E . coli through translational coupling to the pelB signal peptide . RESULTS: Biologically active recombinant CPG2::MFE-23 scFv fusion proteins were produced in E . coli and shown to possess enzyme and anti-CEA activity . Affinity chromatography followed by size exclusion gel filtration yielded approximately 0.7-1.4 mg/l from shake flask culture . The fusion protein in which the enzyme and antibody moieties were joined by a linker peptide was shown to be effectively localised in nude mice bearing human colon tumour xenografts, giving favourable tumour to blood ratios . CONCLUSION: MFE-23 scFv serves as an ideal candidate for the antibody arm of a bacterially expressed fusion protein with CPG2 . The biological properties of this recombinant protein suggest that it may be employed for tumour specific prodrug activation . However, further assessment of its stability and pharmokinetics is required if genetic fusion is to be considered as an alternative to chemical conjugation.

J Cataract Refract Surg, 1997 Oct, 23(8), 1271 - 2
Delayed-onset Pseudomonas keratitis after radial keratotomy; Procope JA; A 62-year-old man had nonsimultaneous, four-incision radial and astigmatic keratotomy in both eyes . Both surgeries were uneventful . Twenty-one months later, the patient developed a corneal ulcer within one of the radial incisions in the left eye . Cultures were positive for Pseudomonas . Although Pseudomonas infections in post-RK eyes have been reported, late-onset Pseudomonas keratitis in a patient with four radial incisions and no history of contact lens wear is contrary to previous reports.

Biochem Biophys Res Commun, 1997 Nov 7, 240(1), 41 - 5
Nucleotide sequence of the Pseudomonas sp . DJ77 phnG gene encoding 2-hydroxymuconic semialdehyde dehydrogenase; Kim S et al.; The nucleotide sequence of a 1520 bp region, spanning the coding region for the meta-cleavage pathway enzyme, 2-hydroxymuconic semialdehyde dehydrogenase, was determined . This enzyme, encoded by the phnG, is the first of three sequential enzymes required for conversion of 2-hydroxymuconic semialdehyde, which is produced from catechol by the PhnE catechol 2,3-dioxygenase, to 2-hydroxypent-2,4-dienoate in the dehydrogenative branch of the pathway . The deduced protein sequence is 484 amino acid residues long with a M(r) of 51504 . The phnG has a high degree of homology with genes encoding isofunctional proteins from other Pseudomonas strains . We now show that the relative position of the phnG dehydrogenase gene in the phn operon is unique compared to the other meta-cleavage operons which have a dehydrogenative branch of the pathway.

Biochem Biophys Res Commun, 1997 Oct 29, 239(3), 884 - 8
Construction and enhanced cytotoxicity of a {cyanovirin-N}-{Pseudomonas exotoxin} conjugate against human immunodeficiency virus-infected cells; Mori T et al.; Cyanovirin-N (CV-N) is a novel 11-kDa anti-HIV(human immunodeficiency virus) protein that binds with high affinity to the viral envelope glycoprotein gp120 . In contrast to soluble CD4 and most known neutralizing antibodies that bind gp120, CV-N exerts potent anti-viral activity against primary clinical HIV isolates as well as laboratory-adapted strains of HIV . Here we describe the recombinant production, purification, and characterization of a chimeric toxin molecule, FLAG-CV-N-PE38, that contains CV-N as a gp120-targeting moiety linked to the translocation and cytotoxic domains of Pseudomonas exotoxin A . FLAG-CV-N-PE38 showed enhanced cytotoxicity to HIV-infected, gp120-expressing H9 cells compared to uninfected H9 cells . Competition experiments with free CV-N provided further support that the enhanced FLAG-CV-N-PE38-induced cytotoxicity was due to interactions of the CV-N moiety with cell surface gp120 . This study establishes the feasibility of use of CV-N as a gp120-targeting sequence for construction and experimental therapeutic investigations of unique new chimeric toxins designed to selectively destroy HIV-infected host cells.

Curr Opin Pulm Med, 1995 Nov, 1(6), 457 - 64
New treatment modalities for cystic fibrosis; Sexauer WP et al.; Refinements in standard therapy for cystic fibrosis have led to dramatic increases in survival and quality of life over the past three decades . Standard therapy has consisted of oral and intravenous antibiotics, chest percussion with postural drainage, and aerosol bronchodilator therapy . The discovery of the cystic fibrosis gene and elucidation of the underlying biochemical defect have broadened our understanding of the pathophysiology of cystic fibrosis and provided a rationale for many new and innovative therapies . Modulation of airway epithelial ion transport may improve mucociliary clearance and delay colonization by infective organisms . Anti-inflammatory therapy may decrease lung injury that results from the host's attempt to limit airway infection . Supplementation of airway antiproteases may limit the destructive effects of unopposed proteases on pulmonary architecture . Genetic biotechnology has already produced agents that preserve pulmonary function and decrease infectious exacerbations by altering the viscoelastic properties of sputum from patients with cystic fibrosis . Both active and passive immunotherapy are currently being investigated as a measure to delay or combat endobronchial infection with Pseudomonas spp . Aerosolized aminoglycoside antibiotics are being increasingly employed to control pulmonary infection while minimizing systemic toxicity . These treatment modalities, combined with the prospects for gene therapy, provide a brighter outlook for the patient with cystic fibrosis than ever before.

Curr Opin Pulm Med, 1995 May, 1(3), 216 - 22
Respiratory infections in patients with HIV; Rosen MJ; Pneumocystis carinii pneumonia has long been considered the predominant pulmonary disease in patients with HIV, but several factors are changing this perception . The population infected with HIV is increasingly composed of injection drug users, and racial and ethnic minorities, which represent groups that have a high incidence of bacterial pneumonia and tuberculosis . The increased longevity attributed to antiretroviral therapy and P . carinii pneumonia prophylaxis is accompanied by more profound immunosuppression, rendering patients susceptible to Pseudomonas, Aspergillus, and other opportunistic pneumonias . Trimetrexate and atovaquone are now available for the treatment of P . carinii pneumonia . Both are less effective than standard regimens of trimethoprim-sulfamethoxazole, but have fewer adverse effects . The diagnosis of respiratory infections complicating HIV usually depends on isolation of the pathogen . The routine use of transbronchial biopsy during bronchoscopy is controversial because the prevalence of P . carinii pneumonia is high in most centers caring for patients with AIDS, and bronchoalveolar lavage is usually diagnostic in this disease . However, biopsy enhances the yield of bronchoscopy, especially in the diagnosis of noninfectious pulmonary disorders and infections other than P . carinii pneumonia.

J Antibiot (Tokyo), 1997 Sep, 50(9), 742 - 9
Novel butyrolactones with antifungal activity produced by Pseudomonas aureofaciens strain 63-28; Gamard P et al.; The bacterium Pseudomonas aureofaciens 63-28 is antagonistic to several plant pathogenic fungi, including Pythium spp . The bacterium produced at least four antifungal metabolites active against Pythium ultimum and Phytophthora cryptogea when tested in culture for antifungal activity . Two of these compounds were identified as the novel butyrolactones (Z)-4-hydroxy-4-methyl-2-(1-hexenyl)-2-butenolide and (Z)-4-hydroxymethyl-2-(1-hexenyl)-2-butenolide, by using NMR and GC-MS . All compounds were different from other antibiotics produced by Pseudomonas spp., including pyoluteorin, pyrrolnitrin, and 2,4-diacetylphloroglucinol, as determined by HPLC . This is the first report of butyrolactones with antifungal activity produced by a saprophytic Pseudomonas spp.

Mol Plant Microbe Interact, 1997 Nov, 10(8), 947 - 60
The phtE locus in the phaseolotoxin gene cluster has ORFs with homologies to genes encoding amino acid transferases, the AraC family of transcriptional factors, and fatty acid desaturases; Zhang YX et al.; A cluster of genes involved in the production of phaseolotoxin, a phytotoxin produced by Pseudomonas syringae pv . phaseolicola, contains eight (phtA through phtH) complementation groups (Y . X . Zhang, K . B . Rowley, and S . S . Patil, J . Bacteriol., 175:6451-6458, 1993) . In this study, sequencing of the region encompassing the phtE locus revealed six putative open reading frames (ORFs), each preceded by a putative ribosomal binding site, and all oriented in the same direction . Reverse transcription-polymerase chain reaction suggested that the phtE locus is transcribed as one large (6.4 kb) transcript, indicating that the ORFs constitute an operon . Primer extension analysis showed that the transcript begins at a T, located 31 bp upstream of the ATG codon of ORF1 . Comparison of the sequences of the putative ORFs with the sequences of known genes revealed that ORF3, encoding a protein containing 395 amino acids, has 55% similarity to the acetylornithine aminotransferase gene from Escherichia coli, and the ornithine aminotransferase genes from other organisms . A lysine residue that is a binding site for pyridoxal phosphate and an arginine residue that is a binding site for the alpha-carboxylate group of the substrate are conserved in ORF3 . These data suggest that ORF3 encodes a protein involved in the biosynthesis of ornithine, a constituent of phaseolotoxin . ORF5, encoding a peptide of 378 amino acid residues, possesses a helix-turn-helix motif at the C-terminal end that is characteristic of the AraC family of transcriptional factors, and there is a possible leucine zipper at the N-terminal end of this peptide . ORF6, encoding a protein of 327 amino acids, has about 40% similarity with the fatty acid desaturase gene, desA, of Synechocystis Pcc6803 and considerable similarity with fatty acid desaturase genes from other organisms . ORF6 and desA show very similar hydropathy profiles and both contain a copper binding signature . Computer searches did not discover significant homologies in the data base for the other ORFs, but hydropathy analysis showed that all of them contain one to several hydrophobic domains, suggesting that the gene products of these ORFs may be membrane associated.

Lett Appl Microbiol, 1997 Oct, 25(4), 300 - 2
An immuno-dot blot assay for detection of thermostable protease from Pseudomonas sp . AFT-36 of dairy origin; Matta H et al.; A dot-ELISA technique for the detection of Pseudomonas protease was developed using IgG of anti-Pseudomonas AFT-36 protease as capture antibody . The detection limit of protease in buffer or milk was 1.01 ng ml-1 . The procedure was performed at room temperature, took about 2.5 h and was economical . Protease AFT-36 is immunologically related to five out of seven Pseudomonas spp . The results suggest that the assay could be used to detect proteases in dairy products.

Plant J, 1997 Sep, 12(3), 669 - 75
Enhanced tolerance to bacterial pathogens caused by the transgenic expression of barley lipid transfer protein LTP2; Molina A et al.; Purified lipid transfer protein LTP2 from barley applied on tobacco leaves eliminated symptoms caused by infiltration of Pseudomonas syringae pv . tabaci 153 . Growth of the pathogen in leaves of transgenic tobacco plants was retarded when compared with non-transformed controls . The percentage of inoculation points that showed necrotic lesions was greatly reduced in transgenic tobacco (17-38% versus 78%) and the average size of these lesions was 61-81% that of control . The average total lesion area (necrosis and chlorosis) in the transgenic plants was also reduced (38% of control) . Arabidopsis thaliana transgenic plants inoculated with P . syringae pv . tomato DC3000 also had lower percentages of necrotic lesions (22-38% versus 76%), a reduced average area for each lesion (53-67% of control), and a smaller total lesion area per inoculation (43% of control) . These results further support the assignment of a defense role for LTPs and highlight their biotechnological potential.

J Biol Chem, 1997 Oct 10, 272(41), 25596 - 601
Augmented hydrolysis of diisopropyl fluorophosphate in engineered mutants of phosphotriesterase; Watkins LM et al.; The phosphotriesterase from Pseudomonas diminuta hydrolyzes a wide variety of organophosphate insecticides and acetylcholinesterase inhibitors . The rate of hydrolysis depends on the substrate and can range from 6000 s-1 for paraoxon to 0.03 s-1 for the slower substrates such as diethylphenylphosphate . Increases in the reactivity of phosphotriesterase toward the slower substrates were attempted by the placement of a potential proton donor group at the active site . Distances from active site residues in the wild type protein to a bound substrate analog were measured, and Trp131, Phe132, and Phe306 were found to be located within 5.0 A of the oxygen atom of the leaving group . Eleven mutants were created using site-directed mutagenesis and purified to homogeneity . Phe132 and Phe306 were replaced by tyrosine and/or histidine to generate all combinations of single and double mutants at these two sites . The single mutants W131K, F306K, and F306E were also constructed . Kinetic constants were measured for all of the mutants with the substrates paraoxon, diethylphenylphosphate, acephate, and diisopropylfluorophosphate . Vmax values for the mutant enzymes with the substrate paraoxon varied from near wild type values to a 4-order of magnitude decrease for the W131K mutant . There were significant increases in the Km for paraoxon for all mutants except F132H . Vmax values measured using diethylphenylphosphate decreased for all mutants except for F132H and F132Y, whereas Km values ranged from near wild type levels to increases of 25-fold . Vmax values for acephate hydrolysis ranged from near wild type values to a 10(3)-fold decrease for W131K . Km values for acephate ranged from near wild type to a 5-fold increase . Vmax values for the mutants tested with the substrate diisopropylfluorophosphate showed an increase in all cases except for the W131K, F306K, and F306E mutants . The Vmax value for the F132H/F306H mutant was increased to 3100 s-1 . These studies demonstrated for the first time that it is possible to significantly enhance the ability of the native phosphotriesterase to hydrolyze phosphorus-fluorine bonds at rates that rival the hydrolysis of paraoxon.

Ugeskr Laeger, 1997 Sep 22, 159(39), 5790 - 4
{Improved prognosis for patients with cystic fibrosis . A result of aggressive center-based treatment}; Frederiksen B et al.; We report survival data for Danish centre-treated cystic fibrosis (CF) patients, covering the period 1974-1993 using cross-sectional cumulative survival probability based on annual age-specific mortality rates . No significant differences were noted in the survival probability when patients were grouped according to sex or absence/presence of meconium ileus . The annual mortality rate for 1989-1993 was 0-1.2% . Using the age-specific mortality rate for 1989-1993, we were unable to calculate the median survival probability because the curve did not fall below 50% (age up to 45 years) . It was, however, possible to show that the survival probability for a CF child born after 1989 to reach his or hers 45th birthday was 80.4% (95% confidence interval 76.5-84.6%) . The probability of surviving 40 years after the diagnosis of CF is made was 83.3% (95% confidence interval 80.1%-86.6%) . This is considerably higher than any other published survival probability . An aggressive anti-Pseudomonas aeruginosa treatment regimen seemed important in achieving the observed improved survival.

Biosci Biotechnol Biochem, 1997 Sep, 61(9), 1575 - 6
Purification and some properties of lignostilbene-alpha, beta-dioxygenase isozyme IV from Pseudomonas paucimobilis TMY1009; Kamoda S et al.; Lignostilbene-alpha, beta-dioxygenase isozyme IV was purified from ultrasonic extracts of Pseudomonas paucimobilis TMY1009 through four steps of column chromatography . The fraction obtained gave a single band on SDS-PAGE and a single peak on reversed-phase HPLC and DEAE-HPLC . The specific activity and Km of purified isozyme IV were 110 mu kat/g and 4.2 microM for 4.4'-dihydroxy-3,3'-dimethoxystilbene, 150 mu kat/g and 3.3 microM for 4,2'-dihydroxy-3,3'-dimethoxy-5'-(2"-carboxyvinyl)stilbene . The molecular mass of intact isozyme IV was estimated to be 94 kDa by gel permeation chromatography, and that of its subunits was 52 kDa by SDS-PAGE under denaturing conditions . The N-terminal amino acid sequence of isozyme IV differed slightly from that of other isozymes . Isozyme IV seemed to be composed of two identical subunits, gamma gamma.

Plant Cell, 1997 Sep, 9(9), 1573 - 84
The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance; Bowling SA et al.; The cpr5 mutant was identified from a screen for constitutive expression of systemic acquired resistance (SAR) . This single recessive mutation also leads to spontaneous expression of chlorotic lesions and reduced trichome development . The cpr5 plants were found to be constitutively resistant to two virulent pathogens, Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2; to have endogenous expression of the pathogenesis-related gene 1 (PR-1); and to have an elevated level of salicylic acid (SA) . Lines homozygous for cpr5 and either the SA-degrading bacterial gene nahG or the SA-insensitive mutation npr1 do not express PR-1 or exhibit resistance to P . s . maculicola ES4326 . Therefore, we conclude that cpr5 acts upstream of SA in inducing SAR . However, the cpr5 npr1 plants retained heightened resistance to P . parasitica Noco2 and elevated expression of the defensin gene PDF1.2, implying that NPR1-independent resistance signaling also occurs . We conclude that the cpr5 mutation leads to constitutive expression of both an NPR1-dependent and an NPR1-independent SAR pathway . Identification of this mutation indicates that these pathways are connected in early signal transduction steps and that they have overlapping functions in providing resistance.

Leukemia, 1997 Oct, 11(10), 1779 - 86
Expression of interleukin-6 receptors by pediatric acute lymphoblastic leukemia cells with the t(4;11) translocation: a possible target for therapy with recombinant IL6-Pseudomonas exotoxin; Gu L et al.; We have detected expression of interleukin-6 receptors (IL-6R) by primary leukemic cells from three of six patients with t(4;11)+ ALL . Scatchard analysis revealed from 960 to 2100 high-affinity IL-6R/cell on these cells (median, 1560; mean, 1540) . All three IL-6R+ cases also expressed CD33, which was not expressed on IL-6R-negative cases . To determine if these receptors could serve as a target for a recombinant ligand-toxin, we examined the sensitivity of primary IL-6R+ ALL cells to a recombinant IL6-Pseudomonas exotoxin (IL6-PE4E) fusion protein, in which the toxicity and specificity of the chimeric toxin was enhanced by substitution of four glutamine residues for naturally occurring amino acids in PE domain I . Primary cells from IL-6R+ cases were sensitive to IL6-PE4E in a 48-h cytotoxicity assay, with ID50 values (concentrations causing 50% decrease in viability) ranging from 23 ng/ml to 92 ng/ml (median, 61; mean, 58) . Furthermore, incubation of these cells with 10(3) ng/ml IL6-toxin for 24 h prevented their subsequent engraftment in SCID mice . Thus, IL6-PE4E may be useful for ex vivo purging of IL-6R+ leukemic cells in an autologous bone marrow transplantation setting and possibly for therapy of residual, chemotherapy-resistant disease.

Biosci Rep, 1997 Jun, 17(3), 343 - 6
The terminal oxidase in the marine bacterium Pseudomonas nautica 617; Simpson H et al.; The molecular properties of a novel membrane quinol oxidase from the marine bacterium Pseudomonas nautica 617 are presented . The protein contains 2b hemes/mole which may be distinguished by EPR spectroscopy but not by optical spectroscopy and electrochemistry . Respiration, though being cyanide insensitive, is not inhibited by carbon monoxide and oxygen reduction is carried out only half-way with production of hydrogen peroxide . The terminal oxidase represents, therefore, a unique example in the large family of terminal oxidases known up to date.

Int J Cancer, 1997 Sep 26, 73(1), 117 - 24
Targeted therapy of schwannoma cells in immunocompetent rats with an erbB2-specific antibody-toxin; Altenschmidt U et al.; Over-expression of the erbB2-receptor tyrosine kinase is frequently observed in many human tumors of epithelial origin . Due to its causal involvement in malignant transformation and its presence on the tumor cell surface erbB2 is an attractive target for directed tumor therapy . We earlier described the potent anti-tumoral activity of the recombinant single-chain antibody toxin scFv(FRP5)-ETA in vitro and in nude mouse tumor models in vivo . This molecule consists of the variable domains of the heavy and light chains of an erbB2-specific antibody genetically fused to a truncated Pseudomonas exotoxin A . Here we have investigated the in vivo effects of this immunotoxin on erbB2 expressing NV2Cd schwannoma cells growing as s.c . tumors in syngeneic BDIX rats . Established tumors were treated either locally by intra-tumoral injection of scFv(FRP5)-ETA or systemically by injection into the tail vein . Both routes of application resulted in pronounced inhibition of tumor growth with local treatment being more effective . Treatment with 25 micrograms/day of scFv(FRP5)-ETA for 10 days suppressed tumor growth almost completely . Antibodies directed mainly against the toxin domain of the fusion protein developed in all animals treated.

Int J Syst Bacteriol, 1997 Oct, 47(4), 1020 - 33
Taxonomy of Pseudomonas strains isolated from tomato pith necrosis: emended description of Pseudomonas corrugata and proposal of three unnamed fluorescent Pseudomonas genomospecies; Sutra L et al.; Thirty-three fluorescent Pseudomonas strains isolated from tomato pith necrosis (FPTPN strains) and 89 Pseudomonas corrugata strains were studied by numerical taxonomy . In the dendrogram of distances, the P . corrugata strains constituted a single phenon (phenon 1), whereas 17 of the 33 FPTPN strains clustered in a separate phenon (phenon 2) . The other 16 FPTPN strains were included in phena consisting of well-characterized fluorescent Pseudomonas species or were isolated phenotypes . Phena 1 and 2 were distinguished by fluorescence on King B medium, accumulation of poly-beta-hydroxybutyrate, production of levan, and assimilation of sorbitol . DNA-DNA hybridization showed that P . corrugata is a true genomic species (66 to 100% DNA relatedness) and that the FPTPN strains of phenon 2 were divided into three genomic groups . Genomic groups 1 and 2 were not distinct from each other phenotypically, and genomic group 3 could be distinguished from genomic groups 1 and 2 only on the basis of assimilation of citraconate and laevulinate . Genomic groups 1 and 2 are related to P . corrugata (40 to 55% DNA relatedness), whereas genomic group 3 is less closely related to P . corrugata (20 to 23% DNA relatedness) . The lipopolysaccharide patterns on electrophoresis gels and fatty acid profiles of strains belonging to genomic group 1 through 3 are different from each other and from the lipopolysaccharide patterns and fatty acid profiles of P . corrugata . However, cross-reactions were observed between P . corrugata and the FPTPN strain genomic groups, indicating that there are common epitopes of the lipopolysaccharides . The three FPTPN strain genomic groups were not named as species but were designated Pseudomonas genomospecies FP1, FP2, and FP3.

Protein Sci, 1997 Oct, 6(10), 2084 - 96
Unusual conformation of nicotinamide adenine dinucleotide (NAD) bound to diphtheria toxin: a comparison with NAD bound to the oxidoreductase enzymes; Bell CE et al.; The conformation of NAD bound to diphtheria toxin (DT), an ADP-ribosylating enzyme, has been compared to the conformations of NAD(P) bound to 23 distinct NAD(P)-binding oxidoreductase enzymes, whose structures are available in the Brookhaven Protein Data Bank . For the oxidoreductase enzymes, NAD(P) functions as a cofactor in electron transfer, whereas for DT, NAD is a labile substrate in which the N-glycosidic bond between the nicotinamide ring and the N-ribose is cleaved . All NAD(P) conformations were compared by (1) visual inspection of superimposed molecules, (2) RMSD of atomic positions, (3) principal component analysis, and (4) analysis of torsion angles and other conformational parameters . Whereas the majority of oxidoreductase-bound NAD(P) conformations are found to be similar, the conformation of NAD bound to DT is found to be unusual . Distinctive features of the conformation of NAD bound to DT that may be relevant to DT's function as an ADP-ribosylating enzyme include (1) an unusually short distance between the PN and N1N atoms, reflecting a highly folded conformation for the nicotinamide mononucleotide (NMN) portion of NAD, and (2) a torsion angle chi N approximately 0 degree about the scissile N-glycosidic bond, placing the nicotinamide ring outside of the preferred anti and syn orientations . In NAD bound to DT, the highly folded NMN conformation and torsion angle chi N approximately 0 degree could contribute to catalysis, possibly by orienting the C1'N atom of NAD for nucleophilic attack, or by placing strain on the N-glycosidic bond, which is cleaved by DT . The unusual overall conformation of NAD bound to DT is likely to reflect the structure of DT, which is unusual among NAD(P)-binding enzymes . In DT, the NAD binding site is formed at the junction of two antiparallel beta-sheets . In contrast, although the 24 oxidoreductase enzymes belong to at least six different structural classes, almost all of them bind NAD(P) at the C-terminal end of a parallel beta-sheet . The structural alignments and principal component analysis show that enzymes of the same structural class bind to particularly similar conformations of NAD(P), with few exceptions . The conformation of NAD bound to DT superimposes closely with that of an NAD analogue bound to Pseudomonas exotoxin A, an ADP-ribosylating toxin that is structurally homologous to DT . This suggests that all of the ADP-ribosylating enzymes that are structurally homologous to DT and ETA will bind a highly similar conformation of NAD.

J Biotechnol, 1997 Sep 16, 57(1-3), 59 - 69
Cellulose binding domains and linker sequences potentiate the activity of hemicellulases against complex substrates; Black GW et al.; To evaluate the role of the CBDs and linker sequences in Pseudomonas xylanase A (XYLA) and arabinofuranosidase C (XYLC), the catalytic activity of derivatives of these enzymes, lacking either the linker sequences or CBDs, was assessed . Removal of the CBDs or linker sequences did not affect the activity of either XYLA or XYLC against soluble arabinoxylan, while derivatives of XYLA, in which either the CBD or interdomain regions had been deleted, exhibited decreased activity against the xylan component of cellulose/hemicellulose complexes . Although a truncated derivative of XYLC (XYLC"'), lacking its CBD, was less active than the full-length enzyme against plant cell wall material containing highly substituted arabinoxylan, XYLC"' was more active than XYLC on complex substrates where the degree of substitution of arabinoxylan was very low . These data indicate that CBDs and linker sequences play an important role in the activity of hemicellulases against plant cell walls and other cellulose/hemicellulose complexes.

FEBS Lett, 1997 Sep 15, 414(3), 590 - 4
Molecular organisation of the ice nucleation protein InaV from Pseudomonas syringae; Schmid D et al.; A new ice nucleation gene from Pseudomonas syringae was isolated and overexpressed as a fully active protein in Escherichia coli in order to gain experimental data about the structure of ice nucleation proteins . No evidence of a signal sequence or secondary glycosylation was found . Differences in the extent of aggregation were shown to modulate the ice nucleation activity . The circular dichroism spectrum of the purified protein indicated the presence of beta-sheet structure . This finding supports a recently proposed hypothetical model for the structure of ice nucleation proteins, which provides a plausible explanation for their aggregation tendency.

Phytochemistry, 1997 Sep, 46(2), 289 - 92
Lipopolysaccharides from three phytopathogenic pseudomonads; Corsaro MM et al.; Analysis of the polysaccharide and lipid moieties of the lipopolysaccharides (LPSs) of the phytopathogenic bacteria, Pseudomonas amygdali and P . syringae pv . ciccaronei has demonstrated that for both bacteria, the O-chain consists of a tetrasaccharide repeating unit of three alpha-L-Rhap and one terminal nonreducing alpha-D-Fucp3NAc . Two of the rhamnosyl residues are 3-linked, the third one 2,3-linked . This structure had been previously found for the O-chains of three phytopathogenic strains of P . syringae subsp . savastanoi, but this is the first report on its occurrence in P . amygdali and P . syringae pv . ciccaronei . The results of the LPS lipid residue analysis made it possible to make some chemotaxonomic considerations and therefore classify P . amygdali as a chemotype, which is different from that of the other two bacteria examined.

FEMS Microbiol Lett, 1997 Sep 15, 154(2), 403 - 8
Novel organization of catechol meta-pathway genes in Sphingomonas sp . HV3 pSKY4 plasmid; Yrjala K et al.; Sphingomonas sp . strain HV3 (formerly Pseudomonas sp . HV3), which degrades aromatics and chloroaromatics, harbors a mega-plasmid, pSKY4 . A sequenced 4 kb fragment of the plasmid reveals a novel gene organization for catechol meta-pathway genes . The putative meta operon starts with the cmpF gene encoding a 2-hydroxymuconic semialdehyde hydrolase . The gene has a 6 bp overlap with the previously characterized ring-cleavage gene, catechol 2,3-dioxygenase, cmpE . Downstream of cmpE is a 429 bp open reading frame of unknown function . Gene cmpC, encoding a 2-hydroxymuconic semialdehyde dehydrogenase, starts 44 bp further downstream . It has the highest homology to 2-hydroxymuconic semialdehyde dehydrogenases of dmp and xyl pathways and to XylC from the marine oligotroph Cycloclasticus oligotrophus . The gene organization is different from other known meta pathways . This is the first report of organization of plasmid-encoded meta-pathway genes in the genus Sphingomonas.

J Biol Chem, 1997 Sep 26, 272(39), 24257 - 65
Purification and characterization of 2-hydroxybiphenyl 3-monooxygenase, a novel NADH-dependent, FAD-containing aromatic hydroxylase from Pseudomonas azelaica HBP1; Suske WA et al.; 2-Hydroxybiphenyl 3-monooxygenase (HbpA), the first enzyme of 2-hydroxybiphenyl degradation in Pseudomonas azelaica HBP1, was purified 26-fold with a yield of 8% from strain HBP1 grown on 2-hydroxybiphenyl . The enzyme was also purified from a recombinant of Escherichia coli JM109, which efficiently expressed the hbpA gene . Computer densitometry of scanned slab gels revealed a purity of over 99% for both enzyme preparations . Gel filtration, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the enzyme was a homotetramer with a molecular mass of 256 kDa . Each subunit had a molecular mass of 60 kDa containing one molecule of noncovalently bound FAD . The monooxygenase had a pI of 6.3 . It catalyzed the NADH-dependent ortho-hydroxylation of 2-hydroxybiphenyl to 2,3-dihydroxybiphenyl . Molecular oxygen was the source of the additional oxygen of the product . The enzyme hydroxylated various phenols with a hydrophobic side chain adjacent to the hydroxy group . All substrates effected partial uncoupling of NADH oxidation from hydroxylation with the concomitant formation of hydrogen peroxide . 2,3-Dihydroxybiphenyl, the product of the reaction with 2-hydroxybiphenyl, was a non-substrate effector that strongly facilitated NADH oxidation and hydrogen peroxide formation without being hydroxylated and also was an inhibitor . The apparent Km values (30 degrees C, pH 7.5) were 2.8 microM for 2-hydroxybiphenyl, 26.8 microM for NADH, and 29.2 microM for oxygen . The enzyme was inactivated by p-hydroxymercuribenzoate, a cysteine-blocking reagent . In the presence of 2-hydroxybiphenyl, the enzyme was partly protected against the inactivation, which was reversed by the addition of an excess of dithiothreitol . The NH2-terminal amino acid sequence of the enzyme contained the consensus sequence GXGXXG, indicative of the betaalphabeta-fold of the flavin binding site and shared homologies with that of phenol 2-hydroxylase from Pseudomonas strain EST1001 as well as with that of 2,4-dichlorophenol 6-hydroxylase from Ralstonia eutropha.

J Biotechnol, 1997 Aug 11, 56(2), 129 - 33
Lipase-catalysed resolution of gamma- and delta-lactones; Enzelberger MM et al.; Lipase-catalysed stereoselective hydrolysis of a family of saturated gamma- and delta-lactones was investigated . Increasing the chain length from delta-octalactone to delta-dodecalactone leads to higher enantiomeric excess . Best results were found using a lipase from Pseudomonas species (KW51) yielding highest enantiomeric excesses (greater than 99% ee at 50% conversion, E > 100) at short reaction times (10 h) for delta-undecalactone and delta-dodecalactone . In contrast, gamma-lactones were resolved less efficiently . Highest enantioselectivities (70%ee at 50% conversion, E = 11) were found for gamma-nonalactone . Optimum reaction conditions were found at pH 8 and 12.5 degrees C.

Vet Parasitol, 1997 Aug, 71(4), 283 - 300
Different organisms associated with heartwater as shown by analysis of 16S ribosomal RNA gene sequences; Allsopp M et al.; Cowdria ruminantium is a rickettsial parasite which causes heartwater, a economically important disease of domestic and wild ruminants in tropical and subtropical Africa and parts of the Caribbean . Because existing diagnostic methods are unreliable, we investigated the small-subunit ribosomal RNA (srRNA) gene from heartwater-infected material to characterise the organisms present and to develop specific oligonucleotide probes for polymerase chain reaction (PCR) based diagnosis . DNA was obtained from ticks and ruminants from heartwater-free and heartwater-endemic areas from Cowdria in tissue culture . PCR was carried out using primers designed to amplify only rickettsial srRNA genes, the target region being the highly variable V1 loop . Amplicons were cloned and sequenced; 51% were C . ruminantium sequences corresponding to four genotypes, two of which were identical to previously reported C . ruminantium sequences while the other two were new . The four different Cowdria genotypes can be correlated with different phenotypes . Tissue-culture samples yielded only Cowdria genotype sequences, but an extraordinary heterogeneity of 16S sequences was obtained from field samples . In addition to Cowdria genotypes we found sequences from previously unknown Ehrlichia spp., sequences showing homology to other Rickettsiales and a variety of Pseudomonadaceae . One Ehrlichia sequence was phylogenetically closely related to Ehrlichia platys (Group II Ehrlichia) and one to Ehrlichia canis (Group III Ehrlichia) . This latter sequence was from an isolate (Germishuys) made from a naturally infected sheep which, from brain smear examination and pathology, appeared to be suffering from heartwater; nevertheless no Cowdria genotype sequences were found in this isolate . In addition no Cowdria sequences were obtained from uninfected ticks . Complete 16S rRNA gene sequences were determined for two C . ruminantium genotypes and for two previously uncharacterised heartwater-associated Ehrlichia spp . Sequenced difference within the V1 loop were sufficient for the derivation of four Cowdria genotype-specific oligonucleotide probes . Four further probes were designed; one for the detection of any Cowdria genotype, one for the detection of any Group II Ehrlichia sp., one for any Group III Ehrlichia sp . and one for all Pseudomonadaceae . All the probes were specific except that for the Cowdria (Ball 3) genotype . The high prevalence (96%) in field samples of pseudomonad-like 16S sequences was the result of environmental contamination . The probes were used to screen DNA from goats in an area free of both Amblyomma ticks and clinical heartwater . A substantial proportion (42%) gave positive reactions for the apparently apathogenic Cowdria (Omatjenne), indicating that this genotype is relatively common.

Biochem Biophys Res Commun, 1997 Sep 8, 238(1), 56 - 60
Localization and sequence analysis of the phnH gene encoding 2-hydroxypent-2,4-dienoate hydratase in Pseudomonas sp . strain DJ77; Kim S et al.; The phnDEFG genes of Pseudomonas sp . DJ77, which are responsible for the degradation of polyaromatic hydrocarbons and chlorinated aromatics, were located previously on the 6.8 kb XhoI fragment of chromosomal DNA . Here, we sequenced a downstream region hitherto unknown and identified the phnH gene encoding a 2-hydroxypent-2,4-dienoate hydratase, which is required for the conversion of 2-hydroxypent-2,4-dienoate to 4-hydroxy-2-oxovalerate in the meta-cleavage pathway of catechols . The relative position of the hydratase gene in the phn operon is unique compared to the other meta-cleavage operons which have a dehydrogenative branch of the pathway . The PhnH hydratase contains 264 amino acids with a Mr of 28048 . The deduced amino acid sequence of the PhnH enzyme is 60.9-31.6% identical to those of homologous enzymes encoded by the todG, bphE, cmtF, bphH, bphX1, xylJ, dmpE, cumE, MTCY03C7.20 and etbE genes.

FEMS Microbiol Lett, 1997 Sep 1, 154(1), 65 - 72
Expression and analysis of coronafacate ligase, a thermoregulated gene required for production of the phytotoxin coronatine in Pseudomonas syringae; Rangaswamy V et al.; Coronafacic acid, the polyketide component of the phytotoxin coronatine, is activated and coupled to coronamic acid via amide bond formation, a biosynthetic step presumably catalyzed by the coronafacate ligase (cfl) gene product . In the present study, cfl was fused to the carboxy terminus of malE, which encodes the maltose-binding protein (MBP), and overexpressed in Escherichia coli . Immunoblot analysis indicated that Cfl contained an ATP-binding region, a motif conserved in enzymes which activate their substrates by adenylation . MBP-Cfl was overproduced and purified from Pseudomonas syringae and the protein fusion was used to generate antisera . Anti-MBP-Cfl antibodies and a transcriptional fusion of the cfl promoter to a promoterless glucuronidase gene were used to follow the temporal expression of coronafacate ligase . The results indicated that transcription of cfl is temperature-sensitive . Furthermore, a nonpolar mutation in cfl suggested that the gene may have a role in coronafacic acid biosynthesis.

J Bacteriol, 1997 Sep, 179(18), 5922 - 7
A possible role for acetylated intermediates in diaminopimelate and tabtoxinine-beta-lactam biosynthesis in Pseudomonas syringae pv . tabaci BR2.024; Liu L et al.; The deduced product of an open reading frame (ORF3) located in the tabtoxinine-beta-lactam (T beta L) biosynthetic region of Pseudomonas syringae pv . tabaci BR2.024 (BR2.024) has significant sequence homology to the dapD products of other bacteria . dapD encodes L-2,3,4,5-tetrahydrodipicolinate succinyl coenzyme A succinyltransferase (THDPA-ST), an enzyme in the diaminopimelate (DAP) and lysine biosynthetic pathway . Complementation studies, in vitro transcription-translation experiments, and enzymatic assays indicated that ORF3 encodes a product with THDPA-ST activity in Escherichia coli dapD mutant beta 274 . However, a BR2.024 mutant with an insert in ORF3 was prototrophic, and only basal THDPA-ST activity was detected in extracts of both parent and mutant . This finding suggested that ORF3 was not required for DAP biosynthesis and that it did not encode a product with THDPA-ST activity . The results of enzymatic studies, indicating that BR2.024 uses acetylated intermediates for DAP biosynthesis, are consistent with the hypothesis that BR2.024 does not need THDPA-ST for DAP biosynthesis . The ORF3 mutant produced reduced levels of tabtoxin, indicating that ORF3 may have a role in T beta L biosynthesis . We have named the gene tabB and have proposed a possible function for the gene product.

Appl Environ Microbiol, 1997 Sep, 63(9), 3553 - 60
Genetic and biochemical characterization of a new extracellular lipase from Streptomyces cinnamomeus; Sommer P et al.; Streptomyces cinnamomeus Tu89 secretes a 30-kDa esterase and a 50-kDa lipase . The lipase-encoding gene, lipA, was cloned from genomic DNA into Streptomyces lividans TK23 with plasmid vector pIJ702 . Two lipase-positive clones were identified; each recombinant plasmid had a 5.2-kb MboI insert that contained the complete lipA gene . The two plasmids differed in the orientation of the insert and the degree of lipolytic activity produced . The lipA gene was sequenced; lipA encodes a proprotein of 275 amino acids (29,213 Da) with a pI of 5.35 . The LipA signal peptide is 30 amino acids long, and the mature lipase sequence is 245 amino acids long (26.2 kDa) and contains six cysteine residues . The conserved catalytic serine residue of LipA is in position 125 . Sequence similarity of the mature lipases (29% identity, 60% similarity) was observed mainly in the N-terminal 104 amino acids with the group II Pseudomonas lipases; no similarity to the two Streptomyces lipase sequences was found . lipA was also expressed in Escherichia coli under the control of lacZ promoter . In the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), growth of the E . coli clone was severely affected, and the cells lysed in liquid medium . Lipase activity in the E . coli clone was found mainly in the pellet fraction . In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, three additional protein bands of 50, 29, and 27 kDa were visible . The 27-kDa protein showed lipolytic activity and represents the mature lipase; the 29- and 50-kDa forms showed no activity and very probably represent the unprocessed form and a dimeric misfolded form, respectively . For higher expression of lipA in S . lividans, the gene was cloned next to the strong aphII promoter . In contrast to the lipA-expressing E . coli clone, S . cinnamomeus and the corresponding S . lividans clone secreted only an active protein of 50 kDa . The lipase showed highest activity with C6 and C18 triglycerides; no activity was observed with phospholipids, Tween 20, or p-nitrophenylesters . Upstream of lipA and in the same orientation, an open reading frame, orfA, is found whose deduced protein sequence (519 amino acids) shows similarity to various membrane-localized transporters . Downstream of lipA and in the opposite orientation, an open reading frame, orfB (encoding a 199-amino-acid protein) is found, which shows no conspicuous sequence similarity to known proteins, other than an NAD and flavin adenine dinucleotide binding-site sequence.

Blood, 1997 Sep 1, 90(5), 2020 - 6
Recombinant RFB4 immunotoxins exhibit potent cytotoxic activity for CD22-bearing cells and tumors; Mansfield E et al.; Many B-cell malignancies express the CD22 antigen on their cell surface . To kill cells expressing this antigen, the RFB4 monoclonal antibody (MoAb) has been linked chemically with either deglycosylated ricin A chain or truncated versions of Pseudomonas exotoxin . These immunotoxins exhibited selective cytotoxic activity for CD22+ cells and antitumor activity in nude mouse models bearing human B-cell lymphomas . To construct a recombinant immunotoxin targeted to CD22, we first cloned the variable portions of the heavy and light chains of RFB4 . The cloned Fv fragments were joined by a newly created disulfide bond to form a disulfide stabilized (ds) construct . The RFB4 construct was combined by gene fusion with PE38, a truncated version of PE . The recombinant immunotoxin was then expressed in Escherichia coli, purified by column chromatography and tested for cytotoxicity activity . RFB4(dsFv)PE38 retained its binding activity for CD22, was very stable at 37 degrees C and exhibited selective cytotoxic activity for CD22+-cultured cell lines . Because of its favorable binding characteristics and potency for CD22-positive cell lines, RFB4(dsFv)PE38 was tested for antitumor activity in a nude mouse model of human lymphoma . CA46 cells were injected subcutaneously and then treated with the RFB4(dsFv)PE38 immunotoxin . Antitumor activity was dose responsive and was not evident when an irrelevant immunotoxin was administered on the same schedule.

Plast Reconstr Surg, 1997 Sep, 100(4), 862 - 8
Infections in craniofacial surgery: a combined report of 567 procedures from two centers; Fearon JA et al.; This retrospective review of infectious complications was undertaken at two craniofacial centers (Dallas and Philadelphia) . Fourteen infections were identified over a 6.5-year period in 567 intracranial procedures primarily for craniosynostosis . There were no infections in infants under 13 months of age and no cases of meningitis . The overall infection rate was 2.5 percent, and 85 percent of infections occurred in secondary reoperative cases . Tracheostomies were not identified as a risk factor for infection . No difference was found in infection rates between patients with shaved and unshaved scalps . Candida and Pseudomonas were the two most common organisms identified, and 28 percent of our infections involved yeast . The average time to diagnose infection was 11.5 days (excluding three patients who averaged 5 months) . Thirteen of the fourteen infections were treated surgically with placement of a subgaleal irrigation/drainage system . Initial bony debridement was kept to a minimum . Based on our findings, recommendations are made to further lower infection rates, particularly those caused by opportunistic organisms.

J Urol, 1997 Sep, 158(3 Pt 1), 948 - 53
Interleukin-13 receptors on human prostate carcinoma cell lines represent a novel target for a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin; Maini A et al.; We have discovered a new cell surface protein in the form of interleukin-13 receptor on several solid tumor cells, including human renal cell carcinoma cells (Obiri et al., 1995; Debinski et al., 1995) . This study reports that human prostate cancer cell lines also express high affinity IL-13 receptors (Kd = 159 pM) . These receptors are functional because IL-13 surprisingly increased proliferation of all three prostate cancer cell lines studied as determined by thymidine uptake and clonogenic assays . IL-13 receptors on prostate cancer cell lines were targeted using a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (PE38QQR) . This molecule, termed IL13-PE38QQR, has been found cytotoxic to all three prostate cancer cell lines as determined by the inhibition of protein synthesis . The IC50 ranged between 1 nmol/l, to 15 nmol/l . These data were confirmed by clonogenic assays in which IL13-PE38QQR almost completely inhibited colony formation at 10 nmol/l . IL13-PE38QQR was not cytotoxic to cells that express little or no IL-13R . Heat inactivated IL13-PE38QQR was not cytotoxic to prostate cancer cells indicating specificity . IL13-PE38QQR was also cytotoxic to colonies when they were allowed to form first for several days before the addition of toxins . Our data suggest that additional studies should be performed to target IL-13 receptor bearing prostate cancer.

J Mol Biol, 1997 Aug 29, 271(4), 619 - 28
Crystal structures of a mutant maltotetraose-forming exo-amylase cocrystallized with maltopentaose; Yoshioka Y et al.; The three-dimensional structures of the catalytic residue Glu219-->Gln mutant of Pseudomonas stutzeri maltotetraose-forming exo-alpha-amylase, and its complex with carbohydrate obtained by cocrystallization with maltopentaose were determined . Two crystal forms were obtained for the complexed enzyme, and a bound maltotetraose was found in each . The structures were analyzed at 2.2 A and 1.9 A resolution, respectively for the uncomplexed and complexed mutant . These structures were compared with the wild-type enzyme structure . In the complexed crystals, the maltotetraose was firmly bound, extensively interacting with the amino acid environments in the active cleft . The non-reducing end glucose unit was hydrogen bonded to the side-chain of Asp160 and the main-chain nitrogen of Gly158, which seem to be predominantly required for the recognition of the non-reducing end of the substrate that determines the exo-wise degradation of this enzyme . The reducing end glucose unit of bound maltotetraose showed clear deformation, adopting a half-chair conformation with extensive hydrogen bonds to surrounding polypeptides . The C1-atom of this deformed glucose unit lies very close to Asp193OD1 with a distance of 2.6 A . The catalytic residue Asp294 is firmly hydrogen-bonded to the O2 and O3-hydroxyl groups of the deformed reducing end glucose unit . Upon binding of the carbohydrate, small but significant induced fits were observed in the regions of Asp294, Phe156, Ile157, and Asp160 . Possible roles of the three catalytic residues are also discussed.

Biochemistry, 1997 Aug 19, 36(33), 10039 - 51
Structures of competitive inhibitor complexes of protocatechuate 3,4-dioxygenase: multiple exogenous ligand binding orientations within the active site; Orville AM et al.; Protocatechuate 3,4-dioxygenase (3,4-PCD) catalyzes the oxidative ring cleavage of 3,4-dihydroxybenzoate to produce beta-carboxy-cis, cis-muconate . Crystal structures of Pseudomonas putida3,4-PCD {quaternary structure of (alphabetaFe3+)12} complexed with seven competitive inhibitors {3-hydroxyphenylacetate (MHP), 4-hydroxyphenylacetate (PHP), 3-hydroxybenzoate (MHB), 4-hydroxybenzoate (PHB), 3-fluoro-4-hydroxybenzoate (FHB), 3-chloro-4-hydroxybenzoate (CHB), and 3-iodo-4-hydroxybenzoate (IHB)} are reported at 2.0-2.2 A resolution with R-factors of 0 . 0.159-0.179 . The inhibitors bind in a narrow active site crevasse lined with residues that provide a microenvironment that closely matches the chemical characteristics of the inhibitors . This results in as little as 20% solvent-exposed surface area for the higher-affinity inhibitors (PHB, CHB, and FHB) . In uncomplexed 3,4-PCD, the active site Fe3+ is bound at the bottom of the active site crevasse by four endogenous ligands and a solvent molecule (Wat827) . The orientations of the endogenous ligands are relatively unperturbed in each inhibitor complex, but the inhibitors themselves bind to or near the iron in a range of positions, all of which perturb the position of Wat827 . The three lowest-affinity inhibitors (MHP, PHP, and IHB) yield distorted trigonal bipyramidal iron coordination geometry in which the inhibitor C4-phenolate group displaces the solvent ligand . MHB binds within the active site, but neither its C3-OH group nor the solvent molecule binds to the iron . The C4-phenolate group of the three highest-affinity inhibitors (PHB, CHB, and FHB) coordinates the Fe3+ adjacent to Wat827, resulting in a shift in its position to yield a six-coordinate distorted octahedral geometry . The range of inhibitor orientations may mimic the mechanistically significant stages of substrate binding to 3, 4-PCD . The structure of the final substrate complex is reported in the following paper {Orville, A . M., Lipscomb, J . D., & Ohlendorf, D . H . (1997) Biochemistry 36, 10052-10066}.

Biochemistry, 1997 Aug 5, 36(31), 9283 - 9
Changes in the regiospecificity of aromatic hydroxylation produced by active site engineering in the diiron enzyme toluene 4-monooxygenase; Pikus JD et al.; Pseudomonas mendocina KR1 toluene 4-monooxygenase is a multicomponent diiron enzyme . the diiron center is contained in the tmoA polypeptide of teh hydroxylase component {alphabetagamma)2,Mr approximately 212 kDa} . Product distribution studies reveal that the natural isoform is highly specific for para hydroxylation of toluene (kcat approximately 2 s-1 with respect to an alphabetagamma promoter), o-xylene (kcat approximately 0.8 s-1), m-xylene (kcat approximately 0.6 s-1), and other aromatic hydrocarbons . This degree of regioselectivity for methylbenzenes is unmatched by numerous other oxygenase enzymes . However, during the T4MO-catalyzed oxidation of p-xylene (kcat approximately 0.4 s-1), 4-methyl benzyl alcohol is the major product, showing that the enzyme could catalyze either aromatic or benzylic hydroxylation with the appropriate substrate . Site-directed mutagenesis has been used to study the contributions of tmoA active site residues Q141, I180, and F205 to the regiospecificity . Isoforms Q141C and F205I yielded shifts of regiospecificity away from p-cresol formation, with F205I giving an approximately 5-fold increase in the percentage of m-cresol formation relative to that of the natural isoform . The kcat of purified Q141C for toluene oxidation was approximately 0.2 s-1 . Isoform Q141C also functioned predominantly as an aromatic ring hydroxylase during the oxidation of p-xylene, in direct contrast to the predominant benzylic hydroxylation observed for the natural isoform, while isoform F205I gave nearly equivalent amounts of benzylic and phenolic products from p-xylene oxidation . Isoform I180F gave no substantial shift in product distributions relativeto the natural isoform for all substrates tested . Upon the basis of a proposed active site model, both Q141 anf F205 are suggested to lie in a hydrophobic region closer to the FeA iron site, while I180 will be closer to FeB . These studies reveal that changes in the hydrophobic region predicted to be nearest to FeA can influence the regiospecificity observed for toluene 4-monooxygenase.

Microbiology, 1997 Aug, 143 ( Pt 8), 2557 - 67
Association of newly discovered IS elements with the dichloromethane utilization genes of methylotrophic bacteria; Schmid-Appert M et al.; Dichloromethane (DCM) dehalogenases enable facultative methylotrophic bacteria to utilize DCM as sole carbon and energy source . DCM-degrading aerobic methylotrophic bacteria expressing a type A DCM dehalogenase were previously shown to share a conserved 4.2 kb BamHI DNA fragment containing the dehalogenase structural gene, dcmA, and dcmR, the gene encoding a putative regulatory protein . Sequence analysis of a 10 kb DNA fragment including this region led to the identification of three types of insertion sequences identified as IS1354, IS1355 and IS1357, and also two ORFs, orf353 and orf192, of unknown function . Two identical copies of element IS1354 flank the conserved 4.2 kb fragment as a direct repeat . The occurrence of these newly identified IS elements was shown to be limited to DCM-utilizing methylotrophs containing a type A DCM dehalogenase . The organization of the corresponding dcm regions in 12 DCM-utilizing strains was examined by hybridization analysis using IS-specific probes . Six different groups could be defined on the basis of the occurrence, position and copy number of IS sequences . All groups shared a conserved 5.6 kb core region with dcmA, dcmR, orf353 and orf192 as well as IS1357 . One group of strains including Pseudomonas sp . DM1 contained two copies of this conserved core region . The high degree of sequence conservation observed within the genomic region responsible for DCM utilization and the occurrence of clusters of insertion sequences in the vicinity of the dcm genes suggest that a transposon is involved in the horizontal transfer of the DCM-utilization character among methylotrophic bacteria.

Microbiology, 1997 Aug, 143 ( Pt 8), 2549 - 56
Tn5041: a chimeric mercury resistance transposon closely related to the toluene degradative transposon Tn4651; Kholodii GY et al.; This paper reports the discovery and characterization of Tn5041, a novel-type transposon vehicle for dissemination of mercury resistance in natural bacterial populations . Tn5041 (14876 bp), identified in a Pseudomonas strain from a mercury mine, is a Tn3 family mercury resistance transposon far outside the Tn21 subgroup . As in other Tn3 family transposons, Tn5041 duplicates 5 bp of the target sequence following insertion . Tn5041 apparently acquired its mer operon as a single-ended relic of a transposon belonging to the classical mercury resistance transposons of the Tn21 subgroup . The putative transposase and the 47 bp terminal inverted repeats of Tn5041 are closely related to those of the toluene degradative transposon Tn4651 and fall into a distinct subgroup on the fringe of the Tn3 family . The amino acid sequence of the putative resolvase of Tn5041 resembles site-specific recombinases of the integrase family . Besides the mer operon and putative transposition genes, Tn5041 contains a 4 kb region that accommodates a number of apparently defective genes and mobile elements.

FEMS Microbiol Lett, 1997 Aug 1, 153(1), 215 - 9
Pseudomonas tolaasii: extra-genomic factor mediates toxin production and efficiency; Mamoun M et al.; Pseudomonas tolaasii is responsible for brown blotch symptoms on Agaricus bisporus . Investigations among 15 P . tolaasii wild-type strains revealed the existence of an extra-genomic factor . Pseudomonas tolaasii CNBP2152D, a spontaneous derivative of the wild-type strain CNBP2152 which has lost the factor, exhibited a significant decline in pathogenicity . Comparison of blotch symptoms induced by toxins extracted from CNBP2152 and CNBP2152D showed that the extragenomic factor mediates toxin production and efficiency.

Appl Environ Microbiol, 1997 Aug, 63(8), 2989 - 96
Use of a promoterless lacZ gene insertion to investigate chitinase gene expression in the marine bacterium Pseudoalteromonas sp . strain S9; Techkarnjanaruk S et al.; Sequence data for genes encoding 16S rRNA indicated that the marine strain previously named Pseudomonas sp . strain S9 would be better identified as a Pseudoalteromonas sp . By use of transposon mutagenesis, a chitinase-negative mutant of S9 with a lacZ reporter gene insertion was isolated . Part of the interrupted gene was cloned and sequenced . The deduced amino acid sequence had homology to sequences of bacterial chitinases . Expression of the chitinase gene promoter was quantified by measuring the lacZ reporter gene product, beta-galactosidase, beta-Galactosidase production was induced 10-fold by N-acetylglucosamine and 3-fold by chitin in minimal medium . Repression of beta-galactosidase synthesis was observed in rich medium either with or without chitin but was not observed in minimal medium containing glucose . The chitinase gene promoter was induced by starvation and higher-than-ambient levels of carbon dioxide but not by cadmium ion, heat or cold shock, or UV exposure.

Mol Plant Microbe Interact, 1997 Aug, 10(6), 716 - 24
Differential induction of systemic resistance in Arabidopsis by biocontrol bacteria; Van Wees SC et al.; Selected nonpathogenic, root-colonizing bacteria are able to elicit induced systemic resistance (ISR) in plants . To elucidate the molecular mechanisms underlying this type of systemic resistance, an Arabidopsis-based model system was developed in which Pseudomonas syringae pv . tomato and Fusarium oxysporum f . sp . raphani were used as challenging pathogens . In Arabidopsis thaliana ecotypes Columbia and Landsberg erecta, colonization of the rhizosphere by P . fluorescens strain WCS417r induced systemic resistance against both pathogens . In contrast, ecotype RLD did not respond to WCS417r treatment, whereas all three ecotypes expressed systemic acquired resistance upon treatment with salicylic acid (SA) . P . fluorescens strain WCS374r, previously shown to induce ISR in radish, did not elicit ISR in Arabidopsis . The opposite was found for P . putida strain WCS358r, which induced ISR in Arabidopsis but not in radish . These results demonstrate that rhizosphere pseudomonads are differentially active in eliciting ISR in related plant species . The outer membrane lipopolysaccharide (LPS) of WCS417r is the main ISR-inducing determinant in radish and carnation, and LPS-containing cell walls also elicit ISR in Arabidopsis . However, mutant WCS417rOA-, lacking the O-antigenic side chain of the LPS, induced levels of protection similar to those induced by wild-type WCS417r . This indicates that ISR-inducing bacteria produce more than a single factor that trigger ISR in Arabidopsis . Furthermore, WCS417r and WCS358r induced protection in both wild-type Arabidopsis and SA-nonaccumulating NahG plants without activating pathogenesis-related gene expression . This suggests that elicitation of an SA-independent signaling pathway is a characteristic feature of ISR-inducing biocontrol bacteria.

J Bacteriol, 1997 Aug, 179(15), 4850 - 8
Identification and characterization of genes encoding carbazole 1,9a-dioxygenase in Pseudomonas sp . strain CA10; Sato SI et al.; Nucleotide sequence analysis of the flanking regions of the carBC genes of Pseudomonas sp . strain CA10 revealed that there were two open reading frames (ORFs) ORF4 and ORF5, in the upstream region of carBC . Similarly, three ORFs, ORF6 to ORF8, were found in the downstream region of carBC . The deduced amino acid sequences of ORF6 and ORF8 showed homologies with ferredoxin and ferredoxin reductase components of bacterial multicomponent dioxygenase systems, respectively . ORF4 and ORF5 had the same sequence and were tandemly linked . Their deduced amino acid sequences showed about 30% homology with large (alpha) subunits of other terminal oxygenase components . Functional analysis using resting cells harboring the deleted plasmids revealed that the products of ORF4 and -5, ORF6, and ORF8 were terminal dioxygenase, ferredoxin, and ferredoxin reductase, respectively, of carbazole 1,9a-dioxygenase (CARDO), which attacks the angular position adjacent to the nitrogen atom of carbazole, and that the product of ORF7 is not indispensable for CARDO activity . Based on the results, ORF4, ORF5, ORF6, and ORF8 were designated carAa, carAa, carAc, and carAd, respectively . The products of carAa, carAd, and ORF7 were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be polypeptides with molecular masses of 43, 36, and 11 kDa, respectively . However, the product of carAc was not detected in Escherichia coli . CARDO has the ability to oxidize a wide variety of polyaromatic compounds, including dibenzo-p-dioxin, dibenzofuran, biphenyl, and polycyclic aromatic hydrocarbons such as naphthalene and phenanthrene . Since 2,2',3-trihydroxydiphenyl ether and 2,2',3-trihydroxybiphenyl were identified as metabolites of dibenzo-p-dioxin and dibenzofuran, respectively, it was considered that CARDO attacked at the angular position adjacent to the oxygen atom of dibenzo-p-dioxin and dibenzofuran as in the case with carbazole.

J Bacteriol, 1997 Aug, 179(15), 4841 - 9
Cloning of genes involved in carbazole degradation of Pseudomonas sp . strain CA10: nucleotide sequences of genes and characterization of meta-cleavage enzymes and hydrolase; Sato SI et al.; The DNA fragment encoding meta-cleavage enzymes and the meta-cleavage compound hydrolase, involved in carbazole degradation, was cloned from the carbazole-utilizing bacterium Pseudomonas sp . strain CA10 . DNA sequence analysis of this 2.6-kb SmaI-SphI fragment revealed that there were three open reading frames (ORF1, ORF2, and ORF3, in this gene order) . ORF1 and ORF2 were indispensable for meta-cleavage activity for 2'-aminobiphenyl-2,3-diol and its easily available analog, 2,3-dihydroxybiphenyl, and were designated carBa and carBb, respectively . The alignment of CarBb with other meta-cleavage enzymes indicated that CarBb may have a non-heme iron cofactor coordinating site . On the basis of the phylogenetic tree, CarBb was classified as a member of the protocatechuate 4,5-dioxygenase family . This unique extradiol dioxygenase, CarB, had significantly higher affinity and about 20-times-higher meta-cleavage activity for 2,3-dihydroxybiphenyl than for catechol derivatives . The putative polypeptide encoded by ORF3 was homologous with meta-cleavage compound hydrolases in other bacteria, and ORF3 was designated carC . The hydrolase activity of CarC for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the meta-cleavage compound of 2,3-dihydroxybiphenyl, was 40 times higher than that for 2-hydroxy-6-oxohepta-2,4-dienoic acid, the meta-cleavage compound of 3-methylcatechol . Alignment analysis and the phylogenetic tree indicate that CarC has greatest homologies with hydrolases involved in the monoaromatic compound degradation pathway . These results suggest the possibility that CarC is a novel type of hydrolase.

Phytochemistry, 1997 Aug, 45(7), 1385 - 91
Biological activities of pseudomycin A, a lipodepsinonapeptide from Pseudomonas syringae MSU 16H; Di Giorgio D et al.; Similarly to other Pseudomonas lipodepsinonapeptides, pseudomycin A inhibits proton extrusion from maize roots, promotes closure of stomata in Vicia faba, necrosis of tobacco leaves, haemolysis of human erythrocytes, affects H(+)-ATPase activity and proton translocation in plasma membrane vesicles, and stimulates succinate respiration in pea mitochondria . In general, the biological activities of pseudomycin A are lower than those of syringomycin-E, the prototype member of this family of bacterial metabolities . This difference might depend on the diverse number and distribution of charged residues in the peptide moiety of these compounds.

Phytochemistry, 1997 Aug, 45(7), 1359 - 63
Pectinolytic enzymes from Pseudomonas marginalis MAFF 03-01173; Hayashi K et al.; Two pectinolytic enzymes were purified from the culture broth of Pseudomonas marginalis pv . marginalis MAFF 03-01173 with total 33% recovery of the initial activity . From the substrate specificities against pectin and polygalacturonic acid, the requirement of calcium ion for the enzymatic activity, and the N-terminal sequences, the enzymes were identified as pectin lyase and pectate lyase . The M,s of pectin lyase and pectate lyase were estimated to be 34,000 and 43,000, respectively, by SDS polyacrylamide gel electrophoresis . Both enzymes showed almost the same pH dependent activity curves with the highest activity at pH 8.3

Int J Cancer, 1997 Jul 29, 72(3), 512 - 7
Amphiregulin antisense oligonucleotide inhibits the growth of T3M4 human pancreatic cancer cells and sensitizes the cells to EGF receptor-targeted therapy; Funatomi H et al.; Human pancreatic cancers overexpress the epidermal growth factor (EGF) receptor (EGFR) and all 5 ligands that bind to this receptor, including amphiregulin . It is not known, however, whether amphiregulin contributes in an autocrine manner to enhance pancreatic cancer cell growth . Therefore, we used an amphiregulin antisense oligonucleotide (AR-AS) to suppress amphiregulin expression in T3M4 human pancreatic cancer cells . These cells express high levels of EGFR and amphiregulin . AR-AS abolished amphiregulin immunoreactivity in T3M4 cells, decreased amphiregulin release into the medium and inhibited cell growth in a dose-dependent manner . Exogenous amphiregulin reversed AR-AS-mediated growth inhibition . A random oligonucleotide (AR-R) did not alter either cell growth or cellular amphiregulin immunoreactivity . AR-AS also increased cellular EGFR protein levels and enhanced the growth-inhibitory actions of TP40, a chimeric protein consisting of transforming growth factor-alpha coupled to Pseudomonas exotoxin that internalizes into cells via EGFR . These findings indicate that there is an important EGFR/ amphiregulin autocrine loop in T3M4 cells and raise the possibility that modalities aimed at abrogating amphiregulin action may prove useful in pancreatic cancer, especially when used in conjunction with EGFR-targeted therapy.

Biochemistry, 1997 Jul 29, 36(30), 9120 - 35
Refined structure, DNA binding studies, and dynamics of the bacteriophage Pf3 encoded single-stranded DNA binding protein; Folmer RH et al.; The solution structure of the 18-kDa single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been refined using 40 ms 15N- and 13C-edited NOESY spectra and many homo- and heteronuclear J-couplings . The structures are highly precise, but some variation was found in the orientation of the beta-hairpin denoted the DNA binding wing with respect to the core of the protein . Backbone dynamics of the protein was investigated in the presence and absence of DNA by measuring the R1 and R2 relaxation rates of the 15N nuclei and the 15N-1H NOE . It was found that the DNA binding wing is much more flexible than the rest of the protein, but its mobility is largely arrested upon binding of the protein to d(A)6 . This confirms earlier hypotheses on the role of this hairpin in the function of the protein, as will be discussed . Furthermore, the complete DNA binding domain of the protein has been mapped by recording two-dimensional TOCSY spectra of the protein in the presence and absence of a small amount of spin-labeled oligonucleotide . The roles of specific residues in DNA binding were assessed by stoichiometric titration of d(A)6, which indicated for instance that Phe43 forms base stacking interactions with the single-stranded DNA . Finally, all results were combined to form a set of experimental restraints, which were subsequently used in restrained molecular dynamics calculations aimed at building a model for the Pf3 nucleoprotein complex . Implying in addition some similarities to the well-studied M13 complex, a plausible model could be constructed that is in accordance with the experimental data.

Biochim Biophys Acta, 1997 Jul 12, 1347(1), 75 - 81
Oxydation of oleic acid to (E)-10-hydroperoxy-8-octadecenoic and (E)-10-hydroxy-8-octadecenoic acids by Pseudomonas sp . 42A2; Guerrero A et al.; Biotransformation of oleic acid with Pseudomonas sp . 42A2 has been found to produce(E)-10-hydroxy-8-octadecenoic acid (2a), (E)-10-hydroperoxy-8-octadecenoic acid (3a), and (E)-7,10-dihydroxy-8-octadecenoic acid (4a) . Structures of the metabolites were fully characterized by infrared and 1H and 13C NMR spectra of the acids, by fast atom bombardment (FAB) and electron impact (EI) and chemical ionization (CI) mass spectrometry of the corresponding methyl esters . This is the first time that the two former compounds of trans stereochemistry have been described to have originated from a Pseudomonas sp . cell culture . Time course of products accumulation showed that biotransformation started with bacterial growth, the amount of products 2a (5.58 g/l) and 4a (2.63 g/l) being optimum after 24 h of incubation while hydroperoxide 3a (1.15 g/l) reached its maximum after 16 h of the biotransformation process . Experiments conducted to ascertain whether the conversion enzyme(s) was cell-bound or extracellular, showed that the enzyme(s) is cell bound, located in the periplasmic space and has lipoxygenase activity.

Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7233 - 8
Gene cloning, sequence analysis, and expression of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase; Chaiyen P et al.; The gene encoding 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO; EC 1.14.12.4) was cloned by using an oligonucleotide probe corresponding to the N terminus of the enzyme to screen a DNA library of Pseudomonas sp . MA-1 . The gene encodes for a protein of 379 amino acid residues corresponding to a molecular mass of 41.7 kDa, the same as that previously estimated for MHPCO . MHPCO was expressed in Escherichia coli and found to have the same properties as the native enzyme from Pseudomonas sp . MA-1 . This study shows that MHPCO is a homotetrameric protein with one flavin adenine dinucleotide bound per subunit . Sequence comparison of the enzyme with other hydroxylases reveals regions that are conserved among aromatic flavoprotein hydroxylases.

J Biotechnol, 1997 Jul 4, 55(3), 193 - 203
A stereo-inverting D-phenylglycine aminotransferase from Pseudomonas stutzeri ST-201: purification, characterization and application for D-phenylglycine synthesis; Wiyakrutta S et al.; D-phenylglycine aminotransferase (D-PhgAT) from a newly isolated soil bacterium, Pseudomonas stutzeri ST-201, was purified to electrophoretic homogeneity and characterized . The molecular weight (M(r)) of the native enzyme was estimated to be 92,000 . It is composed of two subunits identical in molecular weight (M(r)) = 47,500) . The isoelectric point (pI) of the native enzyme was 5.0 . The enzyme catalyzed reversible transamination specific for D-phenylglycine or D-4-hydroxyphenylglycine in which 2-oxoglutarate was an exclusive amino group acceptor and was converted into L-glutamic acid . Neither the D- nor L-isomer of phenylalanine, tyrosine, alanine, valine, leucine, isoleucine or serine could serve as a substrate . The enzyme was most active at alkaline pH with maximum activity at pH 9-10 . The temperature for maximum activity was 35-45 degrees C . The apparent K(m) values for D-phenylglycine and for 2-oxoglutarate at 35 degrees C, pH 9.5 were 1.1 and 2.4 mM, respectively . The enzyme activity was strongly inhibited by typical inhibitors of pyridoxal phosphate-dependent enzymes . Possible application of this enzyme for synthesis of enantiomerically pure D-phenylglycine was demonstrated.

Mol Microbiol, 1997 Jul, 25(2), 211 - 8
Spontaneous duplication of a 661 bp element within a two-component sensor regulator gene causes phenotypic switching in colonies of Pseudomonas tolaasii, cause of brown blotch disease of mushrooms; Han B et al.; Spontaneous sectoring of Pseudomonas tolaasii colonies results in a phenotypic switch from the smooth, pathogenic form (designated 1116S) to the rough non-pathogenic form (designated 1116R) . This phenotypic switch can also be induced by mutation of the pheN master regulatory locus, which encodes a 99 kDa protein with homology to the conserved family of sensor regulator proteins . Southern blot analysis of genomic DNA from 1116S and 1116R probed with a 3.4 kb Xhol-BamHI fragment containing the pheN gene has revealed restriction fragment length polymorphisms in the pheN locus of 1116R . In order to characterize the genetic basis of this variation, the pheN locus (designated pheN') was cloned from 1116R and its nucleotide sequence determined . A 661 bp duplication was identified within pheN' introducing a frameshift mutation in the predicted pheN open reading frame (ORF) . A resulting predicted ORF of pheN' designated ORF2 encodes a polypeptide of 706 amino acid residues, with a predicted molecular weight of 77 kDa, and which lacks part of the PheN sensor domain . Southern blot analysis of genomic DNA using a probe within the duplicated sequence revealed the presence of two bands in 1116R but only one band in the 1116S form . Polymerase chain reaction (PCR) analysis of 25 independently isolated 1116R sectors using primers flanking the duplication site in pheN confirmed the presence of the duplicated 661 bp sequence within this region in all of the sectors and the absence of the duplicated sequence in spontaneous revertants from 1116R to 1116S . Northern blot analysis of RNA from 1116S and 1116R using a pheN probe showed that ORF2 was transcribed in the 1116R form . The presence of a truncated PheN protein in 1116R was verified by Western blot analysis of total cell protein using a LemA antiserum, which revealed the presence of 99kDa and 77kDa cross-reactive bands in 1116S and 1116R respectively . It is concluded that the spontaneous colony-sectoring event that results in the 1116R phenotypic variant form of P . tolaasii arises owing to a 661 bp DNA duplication within the 5' end of the pheN gene, which results in loss of the periplasmic sensor domain of PheN and elimination of normal PheN function.

Arch Dis Child, 1997 Jul, 77(1), 50 - 1
Abnormal technetium labelled white cell scan in the colitis of chronic granulomatous disease; Hoare S et al.; A child with colitis was treated for Crohn's disease, diagnosed on history, clinical and colonoscopic findings, radiolabelled white cell bowel scan, and colonic histology . After septicaemia caused by an unusual organism, further investigation lead to a diagnosis of chronic granulomatous disease (CGD) . The granulomatous colitis of CGD is clinically, histologically, and on white cell scanning, indistinguishable from that in Crohn's disease and should be considered in atypical cases . Infection with unusual 'pseudomonads' should prompt the exclusion of this disorder.

Microbiol Res, 1997 Jul, 152(2), 129 - 35
A high level of accumulation of 2-hydroxymuconic 6-semialdehyde from aniline by the transpositional mutant Y-2 of Pseudomonas species AW-2; Aoki K et al.; Four transpositional mutants of aniline-assimilating Pseudomonas sp . AW-2 produced 2-hydroxymuconic 6-semialdehyde (HMS) from aniline and accumulated it in a cultural medium . Among the four mutants, strain Y-2 produced the greatest amount of HMS (0.77 mg/ml) from 1 mg/ml of aniline hydrochloride in a 15-h growing culture . The conversion rate of aniline to HMS was 70% on a molar basis . Resting cells of strain Y-2 produced 0.65 mg/ml of HMS during 4h of incubation in a reaction mixture containing 1 mg/ml of aniline hydrochloride (conversion rate, 60%) . Transposon Tn5-Mob was found to be inserted into the gene of HMS dehydrogenase in strain Y-2 by sequence analysis.

Pediatr Pulmonol, 1997 Jul, 24(1), 42 - 7
Effectiveness of home versus hospital care in the routine treatment of cystic fibrosis; Bosworth DG et al.; Many cystic fibrosis patients with Pseudomonas lung infections receive intravenous (IV) antibiotics and chest physiotherapy (CPT) at home . Previous studies have suggested that home care, in the setting of a clinical study, is as efficacious as hospital care . This report compares the outcomes of home care with minimal supervision to outcomes of hospital care . We compared two groups of similar age and severity of lung impairment . Patients met strict definitions for home or hospital treatment (27 home care courses/33 hospital care courses) . Five patients completed six courses of both home care and hospital treatment . Treatment in both groups included intravenous antibiotics and CPT . Primary outcome measures included changes in pulmonary function between the start of treatment and after 2 weeks of therapy, duration of treatment, and intervals between antibiotic courses . In hospitalized patients, forced vital capacity (FVC) increased by 17.4 +/- 3.1% (mean +/- SEM), and forced expiratory volume in one second (FEV1) increased by 23.3 +/- 4.1%, both significant at P < 0.001 . The FVC and FEV1 of patients treated at home increased by 10.2 +/- 2.0% and 13.7 +/- 2.6% respectively, neither of which was a significant improvement . Similar results were found in the five patients completing both home and hospital courses . The average duration of treatment was twice as long and time between IV antibiotic courses only two-thirds as long for those treated at home compared with the hospitalized patients . Previous reports have claimed that home care in the setting of a prospective study is as efficacious as hospital care . Our experience indicates that routine home care with minimal supervision of patients is less effective than hospital care . Furthermore, home care as delivered to patients in this report increased the overall cost of care by as much as 30% because of longer and more frequent courses of antibiotic therapy.

Lett Appl Microbiol, 1997 Jul, 25(1), 70 - 2
Isolation of a Pseudomonas species from fish intestine that produces a protease active at low temperature; Hoshino T et al.; A psychrotrophic bacterium producing a protease active at low temperatures was isolated from fish intestine and identified as a Pseudomonas species . Optimum growth and protease-producing temperatures of this strain were 15 degrees C and 10 degrees C, respectively . The maximum temperature for proteolytic activity was 25 degrees C, an unusually low temperature.

J Bacteriol, 1997 Jul, 179(14), 4464 - 72
Characterization of the alginate biosynthetic gene cluster in Pseudomonas syringae pv . syringae; Penaloza-Vazquez A et al.; Alginate, a copolymer of D-mannuronic acid and L-guluronic acid, is produced by a variety of pseudomonads, including Pseudomonas syringae . Alginate biosynthesis has been most extensively studied in P . aeruginosa, and a number of structural and regulatory genes from this species have been cloned and characterized . In the present study, an alginate-defective (Alg-) mutant of P . syringae pv . syringae FF5 was shown to contain a Tn5 insertion in algL, a gene encoding alginate lyase . A cosmid clone designated pSK2 restored alginate production to the algL mutant and was shown to contain homologs of algD, alg8, alg44, algG, algX (alg60), algL, algF, and algA . The order and arrangement of the structural gene cluster were virtually identical to those previously described for P . aeruginosa . Complementation analyses, however, indicated that the structural gene clusters in P . aeruginosa and P . syringae were not functionally interchangeable when expressed from their native promoters . A region upstream of the algD gene in P . syringae pv . syringae was shown to activate the transcription of a promoterless glucuronidase (uidA) gene and indicated that transcription initiated upstream of algD as described for P . aeruginosa . Transcription of the algD promoter from P . syringae FF5 was significantly higher at 32 degrees C than at 18 or 26 degrees C and was stimulated when copper sulfate or sodium chloride was added to the medium . Alginate gene expression was also stimulated by the addition of the nonionic solute sorbitol, indicating that osmolarity is a signal for algD expression in P . syringae FF5.

Appl Environ Microbiol, 1997 Jul, 63(7), 2771 - 8
Analysis of the syrP gene, which regulates syringomycin synthesis by Pseudomonas syringae pv . syringae; Zhang JH et al.; Syringomycin is a lipodepsinonapeptide phytotoxin synthesized by Pseudomonas syringae pv . syringae on multienzymatic peptide synthetases . Sequence analysis of the interval between the syrB and syrD genes of P . syringae pv . syringae strain B301D revealed a 1,059-bp open reading frame (ORF), designated syrP . The predicted product of this ORF was a 39.6-kDa protein consisting of 353 amino acid residues . Searches of protein sequence databases demonstrated that SyrP was most similar to histidine kinases such as the CheA regulatory protein of Escherichia coli . The predicted SyrP sequence was aligned with the N terminus of CheA, a region corresponding to the phosphotransfer and acceptor domains of CheA . The SyrP region that aligns with the phosphotransfer domain of CheA contained a His at position 101 which is flanked by a weak consensus sequence of the unorthodox sensory kinase subfamily of two-component regulatory systems . Strain B301D-31, obtained by site-directed insertional mutagenesis of the syrP gene, exhibited an unusual pleiotropic phenotype including a failure to produce syringomycin in liquid media in contrast to production of elevated levels of the toxin on agar media . The syrP mutant was relieved of the suppression of toxin production that accompanies inorganic phosphate concentrations of > 1 mM on agar media . Nevertheless, the syrP mutant was substantially less virulent than the wild-type strain in pathogenicity assays in cherry fruits . These results suggest that the syrP gene encodes a regulatory protein that participates in a phosphorylation cascade controlling syringomycin production and virulence in P . syringae pv . syringae.

Blood, 1997 Jul 1, 90(1), 252 - 9
Recombinant toxins containing human granulocyte-macrophage colony-stimulating factor and either pseudomonas exotoxin or diphtheria toxin kill gastrointestinal cancer and leukemia cells; Kreitman RJ et al.; The granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is a potential target for toxin-directed therapy, because it is overexpressed on many leukemias and solid tumors and apparently not on stem cells . To investigate the potential therapeutic use of GM-CSF toxins, we fused human GM-CSF to truncated forms of either Pseudomonas exotoxin (PE) or diphtheria toxin (DT) and tested the cytotoxicity of the resulting GM-CSF-PE38KDEL and DT388-GM-CSF on human gastrointestinal (GI) carcinomas and leukemias . Toward gastric and colon cancer cell lines, GM-CSF-PE38KDEL was much more cytotoxic than DT388-GM-CSF, with IC50s (concentration resulting in 50% inhibition of protein synthesis) of 0.5 to 10 ng/mL compared with 4 to 400 ng/mL, respectively . In contrast, toward leukemia lines and fresh bone marrow cells DT388-GM-CSF was more cytotoxic than GM-CSF-PE38KDEL . The cytotoxicity of both GM-CSF-PE38KDEL and DT388-GM-CSF toward the human cells was specific, because it could be competed by an excess of GM-CSF . Binding studies indicated that human GM-CSF receptors were present on all of the human GI and leukemic cell lines tested, at levels of 540 to 3,700 sites per cell (kd = 0.2 to 2 nmol/L), and the number of sites per cell did not correlate with the cell type . A similar pattern of cytotoxicity was found with recombinant immunotoxins binding to the transferrin receptor, in that anti-TFR(Fv)-PE38KDEL was much more cytotoxic than DT388-anti-TFR(Fv) toward GI cells, but both were similar in their cytotoxic activity toward leukemia cells . The fact that PE is more effective than DT in killing GI but not leukemic tumor cells targeted by GM-CSF indicates a fundamental difference in the way PE or DT gains access to the cytosol in these cells . GM-CSF-PE38KDEL and DT388-GM-CSF deserve further evaluation as possible treatments for selected tumors.

FEBS Lett, 1997 Jun 9, 409(2), 227 - 31
A hairpin-loop conformation in tandem repeat sequence of the ice nucleation protein revealed by NMR spectroscopy; Tsuda S et al.; The 1H-NMR spectrum of a synthetic 24-residue peptide (A1-G-V-D-S-S-L-I-A-G-Y-G-S-T-Q-T-S-G-S-D-S-A-L-T24; INP24), comprising three repeats of the 8-residue consensus sequence of Pseudomonas syringae ice nucleation protein, was fully assigned using 2-dimensional (2D) NMR spectroscopy at 4 degrees C and 30 degrees C . Close proximity of the aliphatic protons between Leu7, Ile8, Ala9, and the ring-protons of Tyr11 was indicated from the observation of the inter-molecular nuclear Overhauser enhancement (NOE) effect . Hydrogen-bonding was strongly suggested for the NH group of Leu7 from its extremely low-temperature coefficient estimated from the temperature dependence of the chemical shift . These results indicate the formation of a hairpin-loop conformation constructed by a hexapeptide segment of INP24, -Leu7-Ile8-Ala9-Gly10-Tyr11-Gly12.

J Biol Chem, 1997 Jun 6, 272(23), 14727 - 32
Novel genes encoding 2-aminophenol 1,6-dioxygenase from Pseudomonas species AP-3 growing on 2-aminophenol and catalytic properties of the purified enzyme; Takenaka S et al.; 2-Aminophenol 1,6-dioxygenase was purified from the cell extracts of Pseudomonas sp . AP-3 grown on 2-aminophenol . The product from 2-aminophenol by catalysis of the purified enzyme was identified as 2-aminomuconic 6-semialdehyde by gas chromatographic and mass spectrometric analyses . The molecular mass of the native enzyme was 140 kDa based on gel filtration . It was dissociated into molecular mass subunits of 32 (alpha-subunit) and 40 kDa (beta-subunit) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the dioxygenase was a heterotetramer of alpha2beta2 . The genes coding for the alpha- and beta-subunits of the enzyme were cloned and sequenced . Open reading frames of the genes (amnA and amnB) were 816 and 918 base pairs in length, respectively . The amino acid sequences predicted from the open reading frames of amnA and amnB corresponded to the NH2-terminal amino acid sequences of the alpha-subunit (AmnA) and beta-subunit (AmnB), respectively . The deduced amino acid sequences of AmnB showed identities to some extent with HpaD (25.4%) and HpcB (24.4%) that are homoprotocatechuate 2,3-dioxygenases from Escherichia coli W and C, respectively, belonging to class III in the extradiol dioxygenases . On the other hand, AmnA had identity (23.3%) with only AmnB among the enzymes examined.

EMBO J, 1997 Jun 2, 16(11), 3207 - 18
The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes; Zhou J et al.; In tomato, the Pto kinase confers resistance to bacterial speck disease by recognizing the expression of a corresponding avirulence gene, avrPto, in the pathogen Pseudomonas syringae pv . tomato . Using the yeast two-hybrid system, we have identified three genes, Pti4, Pti5 and Pti6, that encode proteins that physically interact with the Pto kinase . Pti4/5/6 each encode a protein with characteristics that are typical of transcription factors and are similar to the tobacco ethylene-responsive element-binding proteins (EREBPs) . Using a gel mobility-shift assay, we demonstrate that, similarly to EREBPs, Pti4/5/6 specifically recognize and bind to a DNA sequence that is present in the promoter region of a large number of genes encoding 'pathogenesis-related' (PR) proteins . Expression of several PR genes and a tobacco EREBP gene is specifically enhanced upon Pto-avrPto recognition in tobacco . These observations establish a direct connection between a disease resistance gene and the specific activation of plant defense genes.

Protein Eng, 1997 Jun, 10(6), 725 - 30
Design of a solubilization pathway for recombinant polypeptides in vivo through processing of a bi-protein with a viral protease; Perez-Martin J et al.; An artificial maturation pathway for increasing the solubility in vivo of recombinant proteins overproduced in Escherichia coli is reported, which is based on the proteolytic processing of viral polyproteins . The gene product of interest is expressed as a fusion to a heterologous moiety (i.e . the maltose binding protein, MalE) in order to increase the overall solubility of the hybrid . The hinge region between the two fusion partners contains a cleavage site for the NIa protein, a very specific protease from the plum pox potyvirus, as well as an affinity tag . After production, the soluble hybrid is cleaved in vivo by the protease, that is encoded by a plasmid harboured by a specialized E.coli host . The released protein remains soluble and can be purified from cell extracts by means of an affinity tag (a poly-His group) that becomes present after the cleavage . The solubilization and purification of XylR, a xylene-responsive transcriptional factor of Pseudomonas, with this method are reported.

Immunol Cell Biol, 1997 Jun, 75(3), 289 - 94
A recombinant GM-CSF-PE40 ligand toxin is functionally active but not cytotoxic to cells; O'Brien P et al.; A granulocyte/macrophage colony-stimulating factor (GM-CSF)-Pseudomonas exotoxin (PE) 40 fusion protein was constructed for potential use in the treatment of myeloid leukaemias, as a conditioning agent prior to allogeneic bone marrow transplantation or for ex vivo purging of malignant cells prior to autologous bone marrow transplantation . The GM-CSF-PE40 fusion protein successfully binds to the GM-CSF receptor and is capable of initiating a mitogenic signal similar to native GM-CSF in the GM-CSF-dependent TF1 cell line . The toxin component also appears to be fully functional as determined by an in vitro adenosine diphosphate-ribosylation assay . The GM-CSF-PE40 fusion protein, however, was not cytotoxic to a number of myeloid leukaemia cell lines . It is suggested that the mechanism of internalization of the GM-CSF receptor is not appropriate for the translocation of PE to the cytosol where it can fulfil its cytotoxic potential.

Appl Microbiol Biotechnol, 1997 Jun, 47(6), 630 - 5
Activity of Pseudomonas cepacia lipase in organic media is greatly enhanced after immobilization on a polypropylene support; Pencreac'h G et al.; The purified lipase from Pseudomonas cepacia was used as free and immobilized enzyme preparation for hydrolysis of p-nitrophenyl palmitate (pNPP) and p-nitrophenyl acetate (pNPA) in organic media . The free enzyme was mixed with bovine serum albumin and lyophilized . Immobilization was on porous polypropylene . Conditions where diffusional limitations of the substrate were not limiting the reaction rate were defined . The specific activity of the lipase was greatly enhanced upon immobilization: 16.5- and 7.8-fold for pNPP and pNPA respectively . Both the free and immobilized lipases followed Michaelis-Menten kinetics in organic solvent despite the heterogeneity (solid/liquid) of the reaction mixture . For pNPP, the activation factor upon immobilization came mainly from a reduction in Km)app while kcat was increased for pNPA.

Biochem Mol Med, 1997 Jun, 61(1), 114 - 20
Active form of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase; Rogers KS et al.; Based on multiple gel permeation chromatographic experiments, we report a Stokes radius of 59.7 A for Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.88) and its His381Asn, His381Gln, and His381Lys mutant enzymes . Comparison of this Stokes radius with the radius calculated from the crystal structure indicated that the active form of P . mevalonii HMG-CoA reductase was a hexamer and not a dimer as previously thought . The Stokes radius, an S26,w of 11.0, and an estimated V of 0.723 were used in the Svedberg equation to calculate an anhydrous molecular mass of 270,084 Da for P . mevalonii HMG-CoA reductase (monomer mass 45,538 Da), consistent with the enzyme being a hexamer in solution . The Stokes radii of all standard proteins examined correlated with the inverse error function complement of their partition coefficient, Kd . Kd did not correlate with logarithm of the standard protein's molecular weight . Eight nonstandard proteins had Stokes radii that matched their crystallographic radii of longest axis . This indicated that the frozen conformation of a protein in its crystal form can dictate restraints on its shape in solution.

Can J Microbiol, 1997 Jun, 43(6), 509 - 16
Soil isolates of Pseudomonas spp . that utilize inositol phosphates; Richardson AE et al.; Soil bacteria that utilize inositol hexaphosphate (IHP) were isolated from a range of soils using defined selection media . An analysis of 200 randomly selected isolates indicated that less than 0.5% of the culturable population of soil bacteria were capable of using IHP as a sole source of C and P . From a further 238 isolates obtained from enrichment culture, four unique organisms (identified by randomly amplified polymorphic DNA-polymerase chain reaction) were selected and characterized for their ability to specifically utilize IHP . These four organisms were putatively identified as either fluorescent Pseudomonas spp . (P . putida CCAR53 and CCAR59) or nonfluorescent Pseudomonas spp . (P . mendocina CCAR31 and CCAR60) as determined by partial DNA sequence analysis of 16S rRNA genes . The fluorescent Pseudomonas strains exhibited marked phytase activity and liberated up to 81% of the phosphate from IHP either in the absence or presence of arabinose as an additional C source . The nonfluorescent strains also exhibited an ability to liberate Pi from IHP but were effective only in the presence of added arabinose . Strains CCAR59 and CCAR60 could effectively utilize either Na-IHP or Ca-IHP at pH 7.0, whereas only strain CCAR59 could grow and utilize these substrates at pH 5.0.

Biosci Biotechnol Biochem, 1997 Jun, 61(6), 948 - 55
Heterologous protein production in Acremonium chrysogenum: expression of bacterial cephalosporin C acylase and human thrombomodulin genes; Honda G et al.; We have developed an efficient expression system for foreign genes in Acremonium chrysogenum . After inserting the foreign gene between the phosphoglycerate kinase (PGK) promoter and a terminator derived from A . chrysogenum, multiple copies of this expression unit are tandemly ligated into cosmids and the resultant cosmids are introduced into A . chrysogenum . We expressed Pseudomonas cephalosporin C acylase and a human thrombomodulin mutant protein containing the fourth, fifth, and sixth epidermal growth factor (EGF)-like structures (E456) . The acylase activity in the transformants obtained using our system was several times higher than that in the transformants without the use of the system . The acylase proteins expressed had enzymatic and immunochemical properties identical to those of authentic acylase . The transformants with the expression plasmid for E456 secreted biologically active E456 protein into the culture medium . The amino terminal sequence of the purified E456 was identical to that of recombinant E456 obtained using mammalian cells.

Eur J Immunol, 1997 Jun, 27(6), 1459 - 68
Identification of epitopes on a mutant form of Pseudomonas exotoxin using serum from humans treated with Pseudomonas exotoxin containing immunotoxins; Roscoe DM et al.; PE38 is a 38-kDa derivative of the 66-kDa Pseudomonas exotoxin (PE) in which the cell binding domain of PE (domain Ia, amino acids 1-252) and a portion of domain Ib (amino acids 365-380) are deleted . The immunotoxins LMB-1 and LMB-7 contain PE38 and kill cancer cells by exploiting the cytotoxic action of PE38 . The major human B cell epitopes of PE38 were mapped by measuring the reactivity of 45 serum samples from patients treated with the PE38-containing immunotoxins LMB-1 or LMB-7 to two panels of overlapping synthetic peptides representing the sequence of PE38 . One panel of peptides is ten amino acids long and overlap by seven amino acids, and the second panel of peptides is twenty amino acids long and overlap by ten . Five major epitopes were identified: amino acids 274-283, 470-492, 531-540, 555-564, and the C-terminal amino acids 596-609 . Two minor epitopes were identified as well: amino acids 501-510 and 582-589 . These epitopes are predominantly located on the surface of the protein . The amino acids believed to be critical for binding are highly solvent-accessible residues . The results of the human antibody response to peptides are compared to the pattern of reactivity previously identified with serum samples obtained from monkeys administered LMB-1 and LMB-7 . The epitopes between monkey and human are almost identical, demonstrating similarity in the response of antibody repertoires between the two species and providing further support that these are the immunodominant epitopes . This information is critical for genetically engineering less immunogenic immunotoxins and provides a foundation for the development of a vaccine against pseudomonal infections which plague immunocompromised individuals and individuals with cystic fibrosis.

Crit Care Med, 1997 Jun, 25(6), 977 - 82
Prolonged extracorporeal life support (ECLS) for varicella pneumonia; Lee WA et al.; OBJECTIVE: To review the institutional experience of a national tertiary referral center for extracorporeal life support (ECLS) in severe varicella pneumonia . DATA SOURCES: Hospital records and ECLS flow sheets . STUDY SELECTION: All pediatric (nonneonatal) and adult patients who were treated for varicella pneumonia with ECLS at the University of Michigan Medical Center between 1986 and 1995 . DATA EXTRACTION: Diagnosis of varicella pneumonia was made by history of recent exposure to chickenpox, progressive dyspnea, fever, a characteristic diffuse, vesicular rash, and a supporting chest roentgenogram . Indications for ECLS included a shunt fraction of > 30% or PaO2/FlO2 ratio of < 80 despite maximal conventional therapy, which included aggressive diuresis, blood transfusions to optimize oxygen-carrying capacity, pressure-controlled/inverse-ratio ventilation, and intermittent prone positioning . DATA SYNTHESIS: Between 1986 and 1995, 191 patients were referred for ECLS . Among these patients, there were 51 (27%) cases of viral pneumonia, of which nine cases were due to acute varicella-zoster infection . Intravenous acyclovir was administered to eight of the nine patients . Of the nine patients, two patients improved using conventional ventilator management, and seven patients underwent ECLS . Overall survival on ECLS was 71% (5/7) . The mean (+/-SD) alveolar-arterial oxygen gradient and PaO2/FlO2 ratio were 533 +/- 101 torr (71.3 +/- 13.5 kPa) and 67 +/- 24, respectively . The median duration of mechanical ventilation before ECLS and the subsequent duration of ECLS were 4 and 12.8 days, respectively . One of the deaths was from progressive right heart failure secondary to pulmonary hypertension and the other death was from overwhelming Pseudomonas sepsis . CONCLUSIONS: Early recognition of imminent pulmonary failure and rapid institution of ECLS are critical in the successful management of severe, life-threatening varicella pneumonia.

J Bacteriol, 1997 Jun, 179(12), 3936 - 43
Functional analyses of a variety of chimeric dioxygenases constructed from two biphenyl dioxygenases that are similar structurally but different functionally; Kimura N et al.; The biphenyl dioxygenases (BP Dox) of strains Pseudomonas pseudoalcaligenes KF707 and Pseudomonas cepacia LB400 exhibit a distinct difference in substrate ranges of polychlorinated biphenyls (PCB) despite nearly identical amino acid sequences . The range of congeners oxidized by LB400 BP Dox is much wider than that oxidized by KF707 BP Dox . The PCB degradation abilities of these BP Dox were highly dependent on the recognition of the chlorinated rings and the sites of oxygen activation . The KF707 BP Dox recognized primarily the 4'-chlorinated ring (97%) of 2,5,4'-trichlorobiphenyl and introduced molecular oxygen at the 2',3' position . The LB400 BP Dox recognized primarily the 2,5-dichlorinated ring (95%) of the same compound and introduced O2 at the 3,4 position . It was confirmed that the BphA1 subunit (iron-sulfur protein of terminal dioxygenase encoded by bphA1) plays a crucial role in determining the substrate selectivity . We constructed a variety of chimeric bphA1 genes by exchanging four common restriction fragments between the KF707 bphA1 and the LB400 bphA1 . Observation of Escherichia coli cells expressing various chimeric BP Dox revealed that a relatively small number of amino acids in the carboxy-terminal half (among 20 different amino acids in total) are involved in the recognition of the chlorinated ring and the sites of dioxygenation and thereby are responsible for the degradation of PCB . The site-directed mutagenesis of Thr-376 (KF707) to Asn-376 (LB400) in KF707 BP Dox resulted in the expansion of the range of biodegradable PCB congeners.

Ophthal Plast Reconstr Surg, 1997 Jun, 13(2), 135 - 8
Pseudomonas dacryoadenitis secondary to a lacrimal gland ductule stone; Mawn LA et al.; Infectious dacryoadenitis is a rare condition . A case of Pseudomonas dacryoadenitis has not been reported previously . We treated a patient with Pseudomonas dacryoadenitis secondary to obstruction from a lacrimal gland ductule stone . Histologically, the calculus contained hairs.

J Bacteriol, 1997 Jun, 179(11), 3639 - 48
A two-component system in Ralstonia (Pseudomonas) solanacearum modulates production of PhcA-regulated virulence factors in response to 3-hydroxypalmitic acid methyl ester; Clough SJ et al.; Expression of virulence factors in Ralstonia solanacearum is controlled by a complex regulatory network, at the center of which is PhcA, a LysR family transcriptional regulator . We report here that expression of phcA and production of PhcA-regulated virulence factors are affected by products of the putative operon phcBSR(Q) . phcB is required for production of an extracellular factor (EF), tentatively identified as the fatty acid derivative 3-hydroxypalmitic acid methyl ester (3-OH PAME), but a biochemical function for PhcB could not be deduced from DNA sequence analysis . The other genes in the putative operon are predicted to encode proteins homologous to members of two-component signal transduction systems: PhcS has amino acid similarity to histidine kinase sensors, whereas PhcR and OrfQ are similar to response regulators . PhcR is quite unusual because its putative output domain strongly resembles the histidine kinase domain of a sensor protein . Production of the PhcA-regulated factors exopolysaccharide I, endoglucanase, and pectin methyl esterase was reduced 10- to 100-fold only in mutants with a nonpolar insertion in phcB {which express phcSR(Q) in the absence of the EF}; simultaneously, expression of phcA was reduced fivefold . Both a wild-type phenotype and phcA expression were restored by addition of 3-OH PAME to growing cultures . Mutants with polar insertions in phcB or lacking the entire phcBSR(Q) region produced wild-type levels of PhcA-regulated virulence factors . The genetic data suggest that PhcS and PhcR function together to regulate expression of phcA, but the biochemical mechanism for this is unclear . At low levels of the EF, it is likely that PhcS phosphorylates PhcR, and then PhcR interacts either with PhcA (which is required for full expression of phcA) or an unknown component of the signal cascade to inhibit expression of phcA . When the EF reaches a threshold concentration, we suggest that it reduces the ability of PhcS to phosphorylate PhcR, resulting in increased expression of phcA and production of PhcA-regulated factors.

Arch Microbiol, 1997 Jun, 167(6), 356 - 62
Application of autolysin and deoxyribonuclease profiles generated by renaturing SDS-PAGE in the comparison of selected Proteobacteria; Charnock C; SDS-PAGE of cell-free extracts in gels containing bacterial murein or DNA allowed, after enzyme renaturation and staining of nonhydrolysed substrate, the detection of multiple autolysin or deoxyribonuclease activities directly in the gel as zones of clearing . Enzyme profiles of Proteobacteria which are, or were at one time, classified in the genus Pseudomonas were compared . For each species, a relatively large number of autolysin and deoxyribonuclease activities were detected . The distribution, numbers and intensities of zones of clearing in the gel provided complex species-specific patterns . Extensive data from two fundamental, and presumably evolutionarily distinct classes of enzymes were thus generated for purposes of comparison . Neither analysis suggested that these bacteria could represent a single natural cluster of species, lending support to their present multigeneric status . Ethidium-bromide-stained gels could be subsequently stained with Coomassie blue . This allowed the mapping of many deoxyribonuclease activities to particular peptides in the cell-free extract . In addition, modification of the substrate or renaturation buffer enabled a preliminary characterisation of several deoxyribonucleases in terms of their stability, substrate specificity, and other parameters expected to affect enzyme activity . Individual deoxyribonucleases could be located and screened for desired properties without prior purification.

Curr Microbiol, 1997 Jun, 34(6), 378 - 81
Plasmid-encoded sequestration of copper by Pseudomonas pickettii strain US321; Gilotra U et al.; Pseudomonas pickettii strain US321 appeared to elicit a copper resistance mechanism upon exposure to copper . The bacterial colonies turned blue in the presence of this metal in a chemically defined medium, suggesting accumulation of copper . Prolonged exposure to copper resulted in the characteristic "copper sink" morphology of the colonies . Atomic absorption spectrophotometric analysis confirmed that this organism can accumulate copper . The strain US321 exhibited a high-molecular-weight plasmid, pUS321 . The plasmid-cured strain, PC25, is highly sensitive to copper owing to a poor copper management . A plasmid-encoded sequestration mechanism operating in the strain US321 is suggested.

Biochem Biophys Res Commun, 1997 May 29, 234(3), 578 - 81
Structure of catechol 2,3-dioxygenase gene encoded in TOM plasmid of Pseudomonas cepacia G4; Oh JM et al.; The catechol 2,3-dioxygenase is an extradiol-type dioxygenase which cleaves the C-C bond at the meta position of catechol to form 2-hydroxymuconic semialdehyde . A catechol 2,3-dioxygenase gene (tomB) in the TOM plasmid of P . cepacia G4 has been cloned and its nucleotide sequence was analyzed . The enzyme gene consisted of 945 base pairs with an ATG initiation codon and a TGA termination codon which can encode a polypeptide of molecular weight 35 kDa containing 314 amino acid residues, and a putative ribosome-binding sequence was identified at approximately 10 nucleotides upstream of the initiation codon . The deduced amino acid sequence of catechol 2,3-dioxygenase from P . cepacia G4 exhibited 79-82% homologies with those of 3-methylcatechol 2,3-dioxygenase of P . putida UCC2 and catechol 2,3-dioxygenase of P . pickettii PKO1.

Schweiz Med Wochenschr, 1997 May 24, 127(21), 905 - 10
{Inhalational antibiotic therapy in patients with cystic fibrosis and Pseudomonas infection}; Mordasini C et al.; Treating chronic Pseudomonas infection of the bronchial tree is a very important part of the treatment strategy in patients with cystic fibrosis . There are only a few antibiotics which are effective against pseudomonas . Many of them soon lead to bacterial resistance (e.g . fluoro-quinolones) . Inhaling antibiotics produces high sputum concentrations and low systemic toxicity . Tolerance is good and resistance rare . Several clinical studies, some of them doubleblind placebo controlled, have shown a positive effect of inhaled antibiotics on symptoms, on frequency of necessary i.v . therapies and also on pulmonary function . Most commonly aminoglycosides (tobramycin) and colistin, which is not yet registered in Switzerland, are used . The main indication is chronic therapy of Pseudomonas infection.

Farmaco, 1997 May, 52(5), 307 - 11
Chemoenzymatic synthesis of chiral biologically active heterocycles; De Amici M et al.; The chemoenzymatic approach to the preparation of some chiral biologically active heterocycles is discussed . Synthetic strategies took advantage of enantioselective bioconversion processes carried out on suitable reaction intermediates . Reductions of carbonyl compounds catalyzed by different alcohol dehydogenases (TBADH from Thermoanaerobium brockii, 20 beta-HSDH from Streptomyces hydrogenans, beta-HSDH from Pseudomonas testosteroni) allowed the preparation with high enantiomeric purity of the eutomer of broxaterol (a selective beta 2-adrenergic agonist) and six out of the eight muscarine stereoisomers . On the other hand, hydrolyses, catalyzed by lipase PS (from Pseudomonas cepacia), of racemic butyrates were the key step in the synthesis of both the enantiomers of two muscarinic antagonists . Finally, the preparation of acetyl cycloserine antipodes was attained by means of a highly enantioselective hydrolysis catalyzed by lipase from Chromobacterium viscosum.

Mikrobiologiia, 1997 May-Jun, 66(3), 378 - 82
{Genetic control of the destruction of surface-active agents}; Dubeikovskii AN et al.; Degradation of anionic, cationic, and zwitterionic surfactants, such as alkylbenzene sulfonate, ethonium, amidobetaine, and alkylaminobispropionate, by pseudomonads can be controlled by plasmid genes . The majority of plasmids of surfactant-degrading strains are capable of conjugal transfer.

Ginecol Obstet Mex, 1997 May, 65, 194 - 201
{Ultrastructure of the tracheal epithelium in preterm neonates treated with mechanical ventilation}; Villegas Castrejon H et al.; Tracheal epithelial damage, was evaluated in prematures that where intubated and with mechanical ventilation; biopsies were taken in seven preterm neonates; weight was 1100 to 2350; five were by cesarean section, y one via vaginal . Bronchial culture was negative in five, in one Lysteria monocitogena, and in another one Pseudomonas spiralis; biopsy was taken at different times o intubation . Five died because of pneumonia associated to other diseases . In optical and ultrastructural study it was found that with one day of intubation there was cilia loss; with three days of ventilation there was ciliary cells loss, and not ciliary cellular death and there were no cellular unions . In one case with three days of ventilation and 17 days of post-extubation with infection by Pseudomonas, ulcerated zones and edema were found . At 14 days there necrosis zones, increase in collagene . At 17 days there were ulcerated zones to the muscular layer with fibrosis and cellular rests at tracheal path . So, mechanical ventilation and the presence of a catheter, damage bronchial epithelium since the first 24 hours.

Singapore Med J, 1997 May, 38(5), 223 - 5
An unusual case of sorethroat: nasopharyngeal melioidosis; Tan NG et al.; A 14-year-old Chinese female presented with severe sorethroat and swinging fever for two weeks despite one course of oral amoxycillin followed by one course of Unasyn (combination of sultamicillin, sulbactam and ampicillin) . Throat swab grew Pseudomonas pseudomallel . Serology for its antibodies was very strongly positive (> 1:512) . In this part of the world the IHA titre of 1:16 or greater is significant . She was successfully treated with intravenous ceftazidime . The swinging fever settled within two days . The nasopharyngeal and oropharyngeal lesions cleared after a week of therapy . A further two weeks of Ceftazidime were given to ensure a complete resolution of the infection . Oral tetracycline was given for maintenance therapy . Melioidosis involving various organs have been reported particularly pulmonary melioidosis . Nasopharyngeal melioidosis has not been reported, as far as we know . This is the first reported case.

Mol Med, 1997 May, 3(5), 327 - 38
Interleukin-4 receptor expression on AIDS-associated Kaposi's sarcoma cells and their targeting by a chimeric protein comprised of circularly permuted interleukin-4 and Pseudomonas exotoxin; Husain SR et al.; BACKGROUND: AIDS-associated Kaposi's sarcoma (AIDS-KS) represents one of the most common malignancies associated with human immunodeficiency virus infection . To target effective therapeutic agents to AIDS-KS, we have identified a new target in the form of interleukin-4 receptors (IL-4R) . MATERIALS AND METHODS: The expression of IL-4R on AIDS-KS cells and their subunit structure was determined by radioligand receptor binding, cross-linking and Northern and RT-PCR analyses . The in vitro effect of IL-4 and recombinant fusion protein made up of circularly permuted IL-4 and a mutated form of Pseudomonas exotoxin, IL-4(38-37)-PE38KDEL, was examined by clonogenic and protein synthesis inhibition assays . RESULTS: Five AIDS-KS cell lines expressed high-affinity IL-4R with a Kd of 23.5-219 pM . IL-4 appeared to cross-link to one major protein corresponding to 140 kDa and a broad band corresponding to 60-70 kDa . Both cross-linked proteins were immunoprecipitated with an antibody to human IL-4R beta chain . AIDS-KS cells exhibited IL-4R beta-specific mRNA . IL-4 caused a modest inhibition (31-34%) of colony formation in two AIDS-KS cell lines tested . IL-4(38-37)-PE38KDEL was found to be highly effective in inhibiting the protein synthesis in all five AIDS-KS examined . The IC50 ranged from 32 to 1225 pM . The cytotoxic action of IL-4 toxin was blocked by an excess of IL-4, exhibiting the specificity of IL-4(38-37)-PE38KDEL . The cytotoxicity of IL-4 toxin observed by a clonogenic assay corroborated well with the IC50 obtained by protein synthesis inhibition assay . Normal human endothelial cells expressed a negligible number of IL-4R (< 50 sites/cell) and were less sensitive or not sensitive to IL-4(38-37)-PE38KDEL . CONCLUSION: The presence of a new plasma membrane protein in the form of IL-4R on AIDS-KS cells may be targeted by IL-4(38-37)-PE38KDEL for its potential implication in the treatment of AIDS-KS.

Am J Orthop, 1997 May, 26(5), 365 - 7
Pseudomonas osteomyelitis of the metatarsal sesamoid bones; Rahn KA et al.; It is unusual for osteomyelitis of the sesamoid bones to occur after a puncture wound to the foot . When the puncture occurs through tennis shoes, the risk of Pseudomonas infection is increased . Pseudomonas was the causative organism in 7 of 22 cases reported in the literature . This report will explore the causes and natural history, as well as the treatment, of these infections . Initially, basic wound care principles should be adhered to when treating these wounds . Patient awareness and close follow-up is important to ensure complete healing . Treatment of this case involved intravenous antibiotics and medial sesamoidectomy . Foot deformities can occur with sesamoid osteomyelitis and sesamoid excision . Hallux valgus deformity was noted to occur with the osteomyelitis and worsen with sesamoid excision . Preservation of surrounding structures during excision is important to prevent deformity.

Chem Pharm Bull (Tokyo), 1997 May, 45(5), 824 - 31
Simple approach to optically active drimane sesquiterpenes based on enzymatic resolution; Akita H et al.; When the beta-acyloxy esters (+/-)-10 and (+/-)-11 were exposed to the lipase OF-360 from Candida rugosa or immobilized lipase OF-360 in a water-saturated organic solvent, the hydrolyzed product (8aS)-6 was obtained in high chemical (40%) and optical (> 99% ee) yields . The absolute structure of (8aS)-6 was confirmed by the fact that (8aS)-6 was converted into an authentic sample gamma-keto nitrile (8aS)-17 . Treatment of the diol (+/-)-12 with isopropenyl acetate in the presence of the lipase Godo E-4 from Pseudomonas sp . provided the unchanged (8aR)-12 (89% ee) in 42% yield.

FEMS Microbiol Lett, 1997 May 1, 150(1), 127 - 33
The hupC gene product is a component of the electron transport system for hydrogen oxidation in Pseudomonas hydrogenovora; Ohtsuki T et al.; The hydrogenase gene cluster containing nine genes (hupSLCDFGHIJ) was identified by sequencing of an 8.8-kb DNA region from Pseudomonas hydrogenovora . To investigate the function of the hupC gene product, we isolated a hupC-null mutant (HID3) of P . hydrogenovora by introducing an in-frame deletion into the hupC . The mutant, HID3, could not grow autotrophically but retained half the level of hydrogenase activity of the wild-type strain . Results of the oxygen consumption test and Western blot analysis revealed that the hupC gene product is a b-type cytochrome but not involved in the hydrogenase maturation process.

Ophthalmology, 1997 May, 104(5), 838 - 43
Combination intravenous ceftazidime and aminoglycosides in the treatment of pseudomonal scleritis; Helm CJ et al.; BACKGROUND: Pseudomonal scleritis is a serious and potentially blinding infection that usually is resistant to medical management . METHODS: Results for three patients with pseudomonal scleritis who were treated with both topical anti-infectives and a combination of intravenous ceftazidime and aminoglycoside are presented in this case series . RESULTS: All three patients had a rapid response to the addition of combination intravenous drug therapy to topical therapy; eradication of the infection and healing of the ocular surface occurred within 8 weeks . Only one patient, in whom cystoid macular edema developed, lost useful vision as a result of the infection . CONCLUSIONS: Combination therapy with intravenous ceftazidime and aminoglycoside may be more effective than single-intravenous agents when used in addition to topical antibiotics and may obviate the need for adjunctive surgical procedures, such ascryotherapy, surgical extirpation, or conjunctival recession.

Australas J Dermatol, 1997 May, 38(2), 93 - 4
Pseudomonas folliculitis; Hogan PA; Previous reports of Pseudomonas folliculitis in children identified heated pools, hot tubs or spa baths as the source of the infection . This report presents a 4-year-old female with Pseudomonas folliculitis acquired from the family bath tub . The source of the infection was contaminated bath toys and bath plug.

Appl Environ Microbiol, 1997 May, 63(5), 2067 - 70
Stereospecific oxidation of (R)- and (S)-1-indanol by naphthalene dioxygenase from Pseudomonas sp . strain NCIB 9816-4; Lee K et al.; A recombinant Escherichia coli strain which expresses naphthalene dioxygenase (NDO) from Pseudomonas sp . strain NCIB 9816-4 oxidized (S)-1-indanol to trans-(1S,3S)-indan-1,3-diol (95.5%) and (R)-3-hydroxy-1-indanone (4.5%) . The same cells oxidized (R)-1-indanol to cis-1,3-indandiol (71%), (R)-3-hydroxy-1-indanone (18.2%), and cis-1,2,3-indantriol (10.8%) . Purified NDO oxidized (S)-1-indenol to both syn- and anti-2,3-dihydroxy-1-indanol.

Genetics, 1997 May, 146(1), 381 - 92
Phytoalexin-deficient mutants of Arabidopsis reveal that PAD4 encodes a regulatory factor and that four PAD genes contribute to downy mildew resistance; Glazebrook J et al.; We are working to determine the role of the Arabidopsis phytoalexin, camalexin, in protecting the plant from pathogen attack by isolating phytoalexin-deficient (pad) mutants in the accession Columbia (Col-0) and examining their response to pathogens . Mutations in PAD1, PAD2, and PAD4 caused enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv . maculicola strain ES4326 (PsmES4326), while mutations in PAD3 or PAD5 did not . Camalexin was not detected in any of the double mutants pad1-1 pad2-1, pad1-1 pad3-1 or pad2-1 pad3-1 . Growth of PsmES4326 in pad1-1 pad2-1 was greater than that in pad1-1 or pad2-1 plants, while growth in pad1-1 pad3-1 and pad2-1 pad3-1 plants was similar to that in pad1-1 and pad2-1 plants, respectively . The pad4-1 mutation caused reduced camalexin synthesis in response to PsmES4326 infection, but not in response to Cochliobolus carbonum infection, indicating that PAD4 has a regulatory function . PAD1, PAD2, PAD3 and PAD4 are all required for resistance to the eukaryotic biotroph Peronospora parasitica . The pad4-1 mutation caused the most dramatic change, exhibiting full susceptibility to four of six Col-incompatible parasite isolates . Interestingly, each combination of double mutants between pad1-1, pad2-1 and pad3-1 exhibited additive shifts to moderate or full susceptibility to most of the isolates.

Gene, 1997 Apr 29, 190(1), 5 - 10
Versatile vectors for direct cloning and ligation-independent cloning of PCR-amplified fragments for surface display on filamentous bacteriophages; Sampath A et al.; We have constructed phagemid and phage-based vectors which can be used for both direct (T/A) and ligation-independent cloning (LIC) of PCR products for surface display of encoded peptides/proteins fused with the gIII protein of the filamentous bacteriophages M13 and fd-tet . The vectors harbour a DNA cassette consisting of the lacZ alpha fragment inserted between the +2 and +3 codons of gIIIp . The lacZ alpha fragment is flanked by several restriction enzyme recognition sites which can be used for conventional blunt- and cohesive-end cloning in addition to T/A cloning and LIC . The cloning strategies lead to the loss of the lacZ alpha fragment facilitating the selection of the recombinants in XGal plates . The efficiency of direct (T/A) cloning and LIC for surface display in both vectors was evaluated using PCR-amplified fragments encoding a variety of different proteins which included the Fc-binding domain of protein A, the ADP-ribosylation domain of Pseudomonas exotoxin A and a single-chain antibody fragment . The cloning efficiency obtained was 75-85% using the two strategies as monitored by restriction enzyme analysis of the recombinant white colonies on XGal plates . The expression of encoded proteins in recombinants, which were displayed as gIIIp fusions, was found to be 10% in case of T/A cloning but more than 90% in case of LIC.

J Mol Biol, 1997 Apr 25, 268(1), 107 - 17
Stabilization of a recombinant Fv fragment by base-loop interconnection and V(H)-V(L) permutation; Brinkmann U et al.; We have developed a novel method to stabilize a recombinant antibody Fv fragment . The V(H) and V(L) domains of this Fv fragment, called pFv (permutated Fv), are covalently interconnected to each other at the two "base-loops" that normally connect V(H) beta strand 3 to 3b and V(L) beta strand 3 to 3b . To produce the base-loop stabilized Fv fragment, we connected the N-terminal half of the V(L) domain (V(L) 1-40) of murine antibody anti-Tac to the C-terminal half of V(H) (V(H) 42-115) . We also fused the C terminus of V(H) by a (Gly4Ser)3 linker to the N-terminal half of V(H) (V(H) 1-40, thereby generating a permutated V(H) domain) . Finally we connected the base loop of V(H) (N-terminal half) to the C-terminal half of V(L) (V(H) 42-115) . The anti-Tac pFv fragment was fused to a truncated form of Pseudomonas exotoxin to generate a pFv-immunotoxin . Fvs with the correct structure were produced by refolding of recombinant inclusion body protein using a renaturation protocol that was originally developed for Fab and scFv fragments . Due to the artificially connected and permutated primary sequence, the folding pathway for the pFv structure may possibly be different from the conventional folding of antibody domains . Analysis of antigen binding of anti-Tac pFv, and of the specific cytotoxicity of pFv-immunotoxin towards antigen expressing cancer cells demonstrated that the anti-Tac pFv retained most of its affinity and full specificity when compared to anti-Tac scFv . Also anti-Tac pFv was relatively stable, retaining 25% of its binding activity after a 24 hour incubation in human serum at 37 degrees C . This indicates that connection of base loops can be a useful alternative to linker or disulfide stabilization of Fv fragments.

J Biol Chem, 1997 Apr 25, 272(17), 11597 - 603
Adenocarcinoma cells are targeted by the new GnRH-PE66 chimeric toxin through specific gonadotropin-releasing hormone binding sites; Nechushtan A et al.; Luteinizing hormone-releasing hormone, also termed gonadotropin-releasing hormone (GnRH), accounts for the hypothalamic-pituitary gonadal control of human reproduction . The involvement of GnRH has been demonstrated in several carcinomas of hormone-responsive tissues . Exploiting this common feature, we constructed a Pseudomonas exotoxin (PE)-based chimeric toxin (GnRH-PE66) aimed at targeting those cancer cells bearing GnRH binding sites . We report here the strong growth inhibition and killing of a surprisingly wide variety of cancers, confined to the adenocarcinoma type . These cancer cells arising from hormone-responsive tissues, as well as non-responsive ones, express specific GnRH binding sites as indicated by the marked killing of ovarian, breast, endometrial, cervical, colon, lung, hepatic, and renal adenocarcinoma . This cytotoxicity is specific as it could be blocked upon addition of excess GnRH . The specificity of GnRH-PE66 chimeric toxin was also confirmed by GnRH binding assays, and its ability to prevent the formation of colon cancer xenografts in nude mice is presented . Although the functional role of specific GnRH binding sites in human carcinomas remains obscure, GnRH-PE66 displays considerable targeting potential and its use as a therapeutic agent for cancer should be considered.

Gene, 1997 Apr 21, 189(2), 151 - 7
Nucleotide sequence and expression of the organomercurial-resistance determinants from a Pseudomonas K-62 plasmid pMR26; Kiyono M et al.; pMRA17 cloned from Pseudomonas K-62 plasmid pMR26 specified the resistance to both organic and inorganic mercurials . DNA sequence of this broad-spectrum resistant mer operon was determined . The 5504-bp sequence includes six open reading frames (ORFs), five of which were identified as merR, merT, merP, merA and merB in order by analysis of deletion mutants and by comparison with the DNA and amino acid (aa) sequences of previously sequenced mer operons . The merB encoding organomercurial lyase showed a less identity than the other mer genes with those from other broad-spectrum resistance operons . The remaining ORF named merE, located between merA and merB, had no significant homology with the published mer genes and seemed to be a new gene which may involve in phenylmercury resistance . Induction experiments and maxicell analyses of the mer-polypeptides revealed that pMRA17 mer operon expressed mercurial-inducible phenotype and the merB and merE as well as the merA were under the control of MerR which could activate not only by mercuric ion but also by organomercurials.

Biochem Biophys Res Commun, 1997 Apr 17, 233(2), 502 - 6
Reductase gene sequences and protein structures: p-cymene methyl hydroxylase; Dutta TK et al.; Oxygenases are critical to cycling carbon in the biosphere and dependent on reductase action, principally from flavoprotein enzymes . Oxygenase diversity among organisms and strains carries a common theme of protein sequence and folding . p-Cymene (para-isopropyl toluene) was chosen as a point of convergence in terpene-aromatic mineralization to characterize a methyl hydroxylase electron transport system with the aerobe Pseudomonas aureofaciens . The cymA hydroxylase reductase gene was isolated and sequenced and the protein primary structure deduced . Optimized amino acid sequence alignments of flavoprotein reductases revealed major similarities over protein length, in the binding domains for NAD(P)H, and the flavine centers of pro- and eukaryote systems.

Biochim Biophys Acta, 1997 Apr 11, 1319(2-3), 311 - 8
Lack of copper insertion into unprocessed cytoplasmic nitrous oxide reductase generated by an R20D substitution in the arginine consensus motif of the signal peptide; Dreusch A et al.; Metal insertion into an engineered cytoplasmic form of the multicopper enzyme N2O reductase (N2OR) (EC 1.7.99.6) of Pseudomonas stutzeri was studied . The reductase has an unusually long presequence of 50 amino acids for translocation into the periplasm . The signal peptide of N2OR shares a conserved twin-arginine sequence motif with the signal peptides of other N2O reductases and a sizeable group of periplasmic or membrane-bound enzymes, requiring cofactor insertion or processing . A catalytically inactive reductase, N2ORR20D, that lacked Cu, accumulated in the cytoplasm on mutation of the first arginine of this motif . The CuA site of N2ORR20D could be reconstituted in vitro indicating that the lack of metal was not due to a serious conformational restraint . Our findings locate the event of in vivo Cu insertion into N2OR in the periplasm or allow it to take place concomitant with protein translocation.

Exp Cell Res, 1997 Apr 10, 232(1), 186 - 90
Permeabilization of mammalian cells to proteins: poliovirus 2A(pro) as a probe to analyze entry of proteins into cells; Novoa I et al.; Two hybrid protein molecules containing the poliovirus protease 2A (MBP-2A(pro)) (maltose-binding protein-2A(pro) and MBP-Pseudomonas exotoxin A-2A(pro)) have been constructed and purified . Both hybrid proteins efficiently cleave the translation initiation factor eIF-4G when they are co-internalized into cells with adenovirus particles . Almost no intact eIF-4G can be detected in cells incubated with these proteins following this method . Reovirus infectious subviral particles also promote the delivery of MBP-2A(pro) into cells, although less efficiently than adenovirus particles . None of the other methods employed to permeabilize cells to MBP-2A(pro) achieves the degree of eIF-4G cleavage observed with adenovirus particles . By comparison about 30% of cells electroporated with MBP-2A(pro) still contain intact eIF-4G . More drastic electroporation conditions lead to a significant decrease of cell survival . Osmotic lysis of pinocytic vesicles resulted in 30% of the eIF-4G being cleaved in cells treated in suspension . Delivery of MBP-2A(pro) by pH-sensitive liposomes leads to poor hydrolysis of eIF-4G . Taken together our results indicate that permeabilization of cells with adenovirus particles is the most efficient method for introducing MBP-2A(pro) into cells.

J Mol Biol, 1997 Apr 4, 267(3), 661 - 72
Crystal structure of a maltotetraose-forming exo-amylase from Pseudomonas stutzeri; Morishita Y et al.; The three-dimensional structure of an exo-type alpha-amylase from Pseudomonas stutzeri which degrades starch from its non-reducing end to produce maltotetraose has been determined by X-ray structure analysis . The catalytic domain of this enzyme (G4-2), whose structure was determined, is a product of spontaneous limited proteolysis in culture broth . It has 429 amino acid residues and a molecular mass of 47,200, and crystallizes in ammonium sulfate solution at pH 7.5 . The structure was elucidated by the multiple isomorphous replacement method and refined at 2.0 A resolution, resulting in a final R-factor of 0.178 for significant reflections with a root-mean-square deviation from ideality in bond distances of 0.013 A . The polypeptide chain of this molecule folds into three domains; the first with a (beta/alpha)8 barrel structure, the second with an excursed part from the first one, and the third with five-stranded antiparallel beta-sheets . The active cleft is formed on the C-terminal side of the beta-sheets in the (beta/alpha)8 barrel as in the known endo-type alpha-amylases . A histidine side-chain nitrogen ND1 is coordinated to one of the bound calcium ion . The recognition site of the non-reducing end of the amylose that determines exo-wise degradation is presumed to be at one end of this cleft where there is a disordered loop consisting of the 66th to 72nd residues, and a loop carrying an aspartic acid (Asp160) . These structural features may be responsible for the binding of the non-reducing end of the substrate amylose.

J Biomol NMR, 1997 Apr, 9(3), 245 - 58
Floating stereospecific assignment revisited: application to an 18 kDa protein and comparison with J-coupling data; Folmer RH et al.; We report a floating chirality procedure to treat nonstereospecifically assigned methylene or isopropyl groups in the calculation of protein structures from NMR data using restrained molecular dynamics and simulated annealing . The protocol makes use of two strategies to induce the proper conformation of the prochiral centres: explicit atom 'swapping' following an evaluation of the NOE energy term, and atom 'floating' by reducing the angle and improper force constants that enforce a defined chirality at the prochiral centre . The individual contributions of both approaches have been investigated . In addition, the effects of accuracy and precision of the interproton distance restraints were studied . The model system employed is the 18 kDa single-stranded DNA binding protein encoded by Pseudomonas bacteriophage Pf3 . Floating chirality was applied to all methylene and isopropyl groups that give rise to non-degenerate NMR signals, and the results for 34 of these groups were compared to J-coupling data . We conclude that floating stereospecific assignment is a reliable tool in protein structure calculation . Its use is beneficial because it allows the distance restraints to be extracted directly from the measured peak volumes without the need for averaging or adding pseudoatom corrections . As a result, the calculated structures are of a quality almost comparable to that obtained with stereospecific assignments . As floating chirality furthermore is the only approach treating prochiral centres that ensures a consistent assignment of the two proton frequencies in a single structure, it seems to be preferable over using pseudoatoms or (R(-6)) averaging.

Biochim Biophys Acta, 1997 Apr 1, 1345(2), 188 - 96
Adsorption of lipase on polypropylene powder; Gitlesen T et al.; Adsorption of different lipases by EP-100 polypropylene powder from crude and pure lipase preparations was studied . Langmuir isotherms described the adsorption equilibria well both for protein and lipase activity adsorption . Adsorption isotherms for five different proteins all gave a similar saturation level of 220 mg protein per g carrier . Twelve commercial lipase preparations were tested for selectivity in the adsorption of lipase . For all preparations the selectivity factor was larger than one . In a crude lipase preparation from Pseudomonas fluorescence, the specific activity in solution decreased by two orders of magnitude after adsorption . The adsorption was not significantly influenced by pH changes in the adsorption buffer, indicating that hydrophobic and not electrostatic interactions are the dominating adsorption forces . Adsorption of a crude lipase from Candida rugosa (Sigma) was fast and equilibrium was reached in 30 and 100 min for protein and lipase activity adsorption respectively . Desorption in aqueous solution was negligible . Investigations with seven different lipases showed no correlation between the specific lipolytic activity of dissolved enzyme in aqueous solution and the specific activity of adsorbed enzyme in an esterification reaction in organic solvent.

Int J Syst Bacteriol, 1997 Apr, 47(2), 577 - 83
Classification of "Pseudomonas azotocolligans" Anderson 1955, 132, in the genus Sphingomonas as Sphingomonas trueperi sp . nov; Kampfer P et al.; "Pseudomonas azotocolligans" ATCC 12417T (T = type strain), which was described as a diazotrophic bacterium, was reinvestigated to clarify its taxonomic position . 16S ribosomal DNA sequence comparisons demonstrated that this strain clusters phylogenetically with species of the genus Sphingomonas and represents a new species . The results of investigations of the fatty acid patterns, polar lipid profiles, and quinone system supported this placement . The substrate utilization profile and biochemical characteristics displayed no obvious similarity to the characteristics of any previously described species of the genus Sphingomonas . The new name Sphingomonas trueperi is proposed on the basis of these results and previously published data for the G + C content of the genomic DNA and the polyamine pattern.

Mol Plant Microbe Interact, 1997 Apr, 10(3), 416 - 22
Comparison of avrD alleles from Pseudomonas syringae pv . glycinea; Keith LW et al.; Avirulence gene D alleles resided on indigenous plasmids in races 0, 2, 3, 4, 5, and 6 of Pseudomonas syringae pv . glycinea (Psg), but the allele in race 1 appeared to be chromosomal . These were all nonfunctional avirulence genes because they neither induced the avirulence phenotype on Rpg4 soybean cultivars nor directed the production of syringolide elicitors when expressed in Escherichia coli cells . The predicted proteins encoded by the seven Psg avrD genes were very similar to that of a functional class II allele from P . syringae pv . phaseolicola G50 race 2, but contained mutations collectively affecting only nine amino acid positions . Despite these relatively small amino acid differences and the location of avrD from each isolate on a 5.6-kb HindIII restriction fragment, the flanking regions varied considerably among the Psg isolates . The presence of avrD alleles with few alterations but different locational contexts in all tested Psg races argues that they provide an important selected function in the bacteria but have been modified to escape defense surveillance in Rpg4 soybean plants.

Mol Plant Microbe Interact, 1997 Apr, 10(3), 347 - 54
Lipopeptide phytotoxins produced by Pseudomonas syringae pv . syringae: comparison of the biosurfactant and ion channel-forming activities of syringopeptin and syringomycin; Hutchison ML et al.; The phytopathogenic bacterium Pseudomonas syringae pv . syringae produces two classes of necrosis-inducing lipodepsipeptide toxins commonly referred to as the syringomycins and syringopeptins . Members of the syringomycins class are pore-forming cytotoxins that act by promoting passive transmembrane ion flux . In this study, we test the hypothesis that syringopeptin forms SP22A and SP22B likewise function as pore-forming cytotoxins and are similar in activity to syringomycin in artificial and plant membranes . Correspondingly, syringopeptin increased the conductance of black-lipid membranes in a manner indicative of ion channel formation . In tobacco protoplast assays, syringopeptin forms SP22A and SP22B were equivalent in activity causing lysis of protoplasts and measurable 45Ca2+ influx at a threshold concentration of 50 ng/ml . A mixture of three forms of syringomycin did not show cytotoxic activity appreciably different from that of SP22A or SP22B in tobacco protoplast assays . Both forms of syringopeptin also displayed potent biosurfactant properties demonstrated by lowering of the interfacial tension of high-pressure liquid chromatography-grade water to 36 and 34.5 nm/m, respectively; the critical micellar concentration was 0.8 mg/ml for both forms of toxin . These results demonstrate that both classes of pore-forming lipodepsipeptides secreted by P . syringae pv . syringae are cytotoxic to plant cells at nanomolar concentrations and cause necrosis by forming ion channels that are freely permeable to divalent cations.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 2981 - 6
Mössbauer studies of alkane omega-hydroxylase: evidence for a diiron cluster in an integral-membrane enzyme; Shanklin J et al.; The gene encoding the alkane omega-hydroxylase (AlkB; EC 1.14.15.3) from Pseudomonas oleovorans was expressed in Escherichia coli . The integral-membrane protein was purified as nearly homogeneous protein vesicles by differential ultracentrifugation and HPLC cation exchange chromatography without the detergent solubilization normally required for membrane proteins . Purified AlkB had specific activity of up to 5 units/mg for octane-dependent NADPH consumption . Mossbauer studies of AlkB showed that it contains an exchange-coupled dinuclear iron cluster of the type found in soluble diiron proteins such as hemerythrin, ribonucleotide reductase, methane monooxygenase, stearoyl-acyl carrier protein (ACP) delta9 desaturase, rubrerythrin, and purple acid phosphatase . In the as-isolated enzyme, the cluster contains an antiferromagnetically coupled pair of high-spin Fe(III) sites, with an occupancy of up to 0.9 cluster per AlkB . The diferric cluster could be reduced by sodium dithionite, and the diferrous state was found to be stable in air . When both O2 and substrate (octane) were added, however, the diferrous cluster was quantitatively reoxidized, proving that the diiron cluster occupies the active site . Mossbauer data on reduced AlkB are consistent with a cluster coordination rich in nitrogen-containing ligands . New sequence analyses indicate that at least 11 nonheme integral-membrane enzymes, including AlkB, contain the 8-histidine motif required for catalytic activity in stearoyl-CoA desaturase . Based on our Mossbauer studies of AlkB, we propose that the integral-membrane enzymes in this family contain diiron clusters . Because these enzymes catalyze a diverse range of oxygenation reactions, this proposal suggests a greatly expanded role for diiron clusters in O2-activation biochemistry.

J Bacteriol, 1997 Apr, 179(7), 2247 - 58
Multiple loci of Pseudomonas syringae pv . syringae are involved in pathogenicity on bean: restoration of one lesion-deficient mutant requires two tRNA genes; Rich JJ et al.; A mutational analysis of lesion-forming ability was undertaken in Pseudomonas syringae pv . syringae B728a, causal agent of bacterial brown spot disease of bean . Following a screen of 6,401 Tn5-containing derivatives of B728a on bean pods, 26 strains that did not form disease lesions were identified . Nine of the mutant strains were defective in the ability to elicit the hypersensitive reaction (HR) and were shown to contain Tn5 insertions within the P . syringae pv . syringae hrp region . Ten HR+ mutants were defective in the production of the toxin syringomycin, and a region of the chromosome implicated in the biosynthesis of syringomycin was deleted in a subset of these mutants . The remaining seven lesion-defective mutants retained the ability to produce protease and syringomycin . Marker exchange mutagenesis confirmed that the Tn5 insertion was causal to the mutant phenotype in several lesion-defective, HR+ strains . KW239, a lesion- and syringomycin-deficient mutant, was characterized at the molecular level . Sequence analysis of the chromosomal region flanking the Tn5 within KW239 revealed strong similarities to a number of known Escherichia coli gene products and DNA sequences: the nusA operon, including the complete initiator tRNA(Met) gene, metY; a tRNA(Leu) gene; the tpiA gene product; and the MrsA protein . Removal of sequences containing the two potential tRNA genes prevented restoration of mutant KW239 in trans . The Tn5 insertions within the lesion-deficient strains examined, including KW239, were not closely linked to each other or to the lemA or gacA genes previously identified as involved in lesion formation by P . syringae pv . syringae.

Biochem Biophys Res Commun, 1997 Mar 17, 232(2), 288 - 91
Sequence analysis of the phnD gene encoding 2-hydroxymuconic semialdehyde hydrolase in Pseudomonas sp . strain DJ77; Shin HJ et al.; The 6.8-kb XhoI fragment of chromosomal DNA of Pseudomonas sp . DJ77 contains the phnDEFG genes involved in the degradation of polyaromatic hydrocarbons and chlorinated aromatics . Here, we report the nucleotide sequence of the phnD gene encoding a 2-hydroxymuconic semialdehyde hydrolase and its substrate specificity . The PhnD hydrolase contains 286 amino acids with a M(r) of 31301 . The deduced amino acid sequence of the PhnD enzyme is 31.0-50.5% identical to those of homologous enzymes encoded by the dmp, tod, xyl, and bph operons . The PhnD enzyme is required for conversion of 2-hydroxymuconic semialdehyde, which is produced from catechol by the PhnE catechol 2,3-dioxygenase, to 2-hydroxypent-2,4-dienoate . We now confirm that the phnD gene is located immediately upstream of the catechol 2,3-dioxygenase gene (phnE) unlike other meta-cleavage operons.

J Natl Cancer Inst, 1997 Mar 5, 89(5), 365 - 73
Abrogation of cisplatin-induced programmed cell death in human breast cancer cells by epidermal growth factor antisense RNA; Dixit M et al.; BACKGROUND: Epidermal growth factor receptor (EGF-R) perturbation by receptor ligand(s), e.g., epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), or receptor-specific antibodies accentuates cisplatin-induced toxicity in tumor cells . This sensitization occurs only in tumor cells with high expression of EGF-R but not in those with low expression of EGF-R . PURPOSE: Therefore, we have studied the role of EGF-R expression on cisplatin-mediated cytotoxicity . METHODS: MDA-468 human breast cancer cells were stably transfected with a p-chloramphenicol acetyl transferase (pact{p}-CAT) vector containing a 4.1-kilobase full-length antisense EGF-R complementary DNA . EGF-R content was assessed by 125I-EGF binding and EGF-R immunoblot assays . Cisplatin sensitivity was evaluated by (a) colony-forming assay in vitro, (b) xenograft growth in nude mice, (c) cell cycle distribution of propidium iodide-labeled DNA, (d) DNA fragmentation in agarose gels, and (e) terminal deoxynucleotidyl transferase (Tdt) fluorescence in situ . Cisplatin uptake was measured by atomic absorption spectroscopy, and the levels of drug-DNA intrastrand adducts were determined by a dissociation-enhanced fluoroimmunoassay that utilizes an antibody against cisplatin-modified DNA . RESULTS: Selected clones (MDA-468/AS-EGFR) exhibited more than 90% loss of both 125I-EGF binding and receptor content determined by western blot analysis, whereas clones transfected with the vector alone (MDA-468/p-CAT) had EGF-R levels similar to those of the parent cells . By use of a colony-forming assay, the 1-hour IC50 (i.e., the concentration of drug required for 1 hour to achieve 50% cell kill) for cisplatin was 2 microM or less for parental and vector-transfected clones (n = 4), whereas it was 25 microM or more for all MDA-468/AS-EGFR clones (n = 3) . MDA-468/p-CAT clones exhibited internucleosomal DNA fragmentation, enhanced Tdt-end labeling in situ, and G2 arrest 48 hours after a 1-hour incubation with 3-30 microM cisplatin . Under these conditions, apoptosis and G2 arrest were undetectable in all MDA-468/AS-EGFR clones . An MDA-468 subline selected after long-term treatment with a TGF-alpha-Pseudomonas exotoxin A fusion protein 40 lacked EGF binding and also exhibited cisplatin resistance (1-hour IC50: > 30 microM) compared with parental cells . This EGF-R-dependent difference in cisplatin response was confirmed in a nude mouse xenograft model by use of high- and low-EGF-R-expressing cell clones . Total intracellular drug accumulation after a 1-hour cisplatin exposure, as measured by atomic absorption spectroscopy, was identical in both groups of cells . Intrastrand drug-DNA adducts, however, were statistically higher in high EGF-R expressors than in low-EGF-R-expressing clones . CONCLUSIONS: These data indicate that a critical level of EGF-R signaling, which is amplified in some common human cancers, is necessary for cisplatin-mediated apoptosis in tumor cells and suggest an inhibitory effect of this pathway on the repair of cisplatin-damaged DNA.

Curr Opin Pulm Med, 1997 Mar, 3(2), 120 - 4
Therapy for nosocomial pneumonia; Wunderink RG; Because of a lack of clinical trials, the American Thoracic Society published a consensus statement on nosocomial pneumonia that included recommendations for antibiotic therapy . Almost concurrently, a multicenter study of the need to modify antibiotic therapy in ventilator-associated pneumonia (VAP) and a report specifically studying Pseudomonas VAP independently demonstrated both the appropriateness and some of the inadequacies of the therapies recommended by the American Thoracic Society . Anaerobic involvement as a copathogen was documented in early-onset VAP but probably impacts antibiotic choices little because of the presence of aerobic pathogens . Potential improvements in aminoglycoside treatment were suggested by meta-analysis of once-daily dosing and aerosolization . The most provocative study used decision-analysis techniques to suggest that antibiotic therapy based on clinical diagnosis of VAP would result in greater mortality than withholding antibiotics . Diagnosis of VAP by bronchoscopic and nonbronchoscopic quantitative cultures resulted in improved outcome with antibiotic treatment, possibly the result of documentation of inappropriate empiric antibodies in approximately 40% of cases of late-onset VAP.

Leukemia, 1997 Mar, 11 Suppl 1, S32 - 4
Studies of decitabine with allogeneic progenitor cell transplantation; Giralt S et al.; The aim was to determine the efficacy and safety of decitabine in the settings of relapse post-allogeneic progenitor cell transplantation or as part of the conditioning regimen . Three patients (two AML, one ALL) received single agent decitabine 1000 mg/m2 total dose) for treatment of relapse post-transplant (group 1) . Median age was 32 years . Median time to relapse was 7 months . In another study four patients (three CML in an accelerated phase, one AMML) received decitabine 400 mg/m2, with busulfan 12 mg/kg and cyclophosphamide 100 mg/kg as conditioning for allogeneic stem cell transplantation (group 2) . Median age was 42 years; median time to transplant was 5 months . All patients received at least 4 x 10(6) CD34+ cells from their HLA compatible donors . All patients in group 1 achieved complete remissions after decitabine therapy . The median time to neutrophil and platelet recovery were 24 and 23 days, respectively . Two patients required reinfusion of donor cells because of delayed engraftment . One patient remains alive and in remission 160 days post-decitabine therapy . Two patients in group 2 engrafted on days 23 and 25 . Two patients required reinfusion of stem cells because of lack of neutrophil recovery by day 21 . Two patients achieved complete cytogenetic and hematologic remission . Three patients are alive at 167,129, and 109 days post-transplant . One patient died of progressive Pseudomonas cellulitis 54 days post-initial infusion . Decitabine therapy is well tolerated in the setting of allogeneic stem cell transplantation, initial results in patients relapsing after transplant are encouraging and warrant further studies . The causes of delayed engraftment after single agent or combination therapy need to be better explored . The existence of active metabolites of decitabine which may still be present in the blood at the time of stem cell infusions, and/or insufficient immunosuppression of the preparative regimen are being explored as possible explanations for this phenomenon.

Eur J Biochem, 1997 Mar 1, 244(2), 462 - 70
The AlkB monooxygenase of Pseudomonas oleovorans--synthesis, stability and level in recombinant Escherichia coli and the native host; Staijen IE et al.; We have studied the synthesis and stability of the monooxygenase AlkB of Pseudomonas oleovorans in its natural host and in recombinant Escherichia coli . Three strains were investigated: the prototype strain P . oleovorans and the E . coli alk+ recombinants HB101 (pGEc47) and W3110 (pGEc47) . Plasmid pGEc47 allows regulated expression of alkB and synthesis of active AlkB in E . coli . The E . coli strains were selected because E . coli HB101 (pGEc47) produces similar amounts of AlkB as P . oleovorans (1.5-2% of total cell protein), whereas E . coli W3110 (pGEc47) is able to make substantially (about fivefold) more AlkB . The AlkB synthesis and degradation rates in batch cultures of the three strains were determined by means of isotopic-labeling and immunological techniques . The mean specific AlkB synthesis rates in P . oleovorans, E . coli HB101 (pGEc47) and E . coli W3110 (pGEc47) were approximately 7, 12.5 and 45 microg x mg protein(-1) x h(-1), respectively . The half-lives of AlkB were estimated to be 80, 3 and 15 for P . oleovorans, E . coli HB101 (pGEc47) and E . coli W3110 (pGEc47), respectively . Thus, the intracellular AlkB level in each of the three strains was the result of their AlkB synthesis and degradation rates . The AlkB level during batch growth was modelled by means of experimentally derived parameters for AlkB synthesis and degradation, and showed good agreement with AlkB levels determined by means of immunoblotting in all strains investigated.

Cancer Immunol Immunother, 1997 Mar, 44(1), 1 - 9
A novel immunotoxin recognising the epithelial glycoprotein-2 has potent antitumoural activity on chemotherapy-resistant lung cancer; Zimmermann S et al.; Resistance to chemotherapy is a major cause for failure in the treatment of lung cancer . Compared to conventional cytotoxic drugs, immunotoxins act by different mechanisms and thus might be promising for the treatment of chemoresistant cancer . The monoclonal antibody MOC31 recognises the epithelial glycoprotein-2 (EGP-2), a cell-surface antigen associated with small-cell lung cancer (SCLC) and a major fraction of lung adenocarcinomas . An immunotoxin composed of MOC31 and a recombinant from of Pseudomonas exotoxin A lacking the cell-binding domain (ETA252-613) was prepared and its effect on lung cancer cell lines examined . MOC31 ETA252-613 was selectively cytotoxic to EGP-2-positive SCLC and adenocarcinoma cell lines inhibiting proliferation by 50% at concentrations ranging from 0.01 nM to 0.3 nM . Moreover, the immunotoxin reduced the number of clonogenic tumour cells from cultures by factors of 10(4) and 10(5) during a 24-h and a 3-week exposure respectively . In athymic mice, the immunotoxin, which revealed a serum half-life of approximately 4 h, caused substantial regression of small (40 mm3) chemoresistant tumour xenografts and significantly delayed the growth of larger tumours (120 mm3) . This finding indicates that MOC31-ETA252-613 may be useful for the treatment of lung cancer in the setting of chemoresistant minimal residual disease.

Biosci Biotechnol Biochem, 1997 Mar, 61(3), 530 - 2
Isolation of extradiol dioxygenase genes that are phylogenetically distant from other meta-cleavage dioxygenase genes; Takami H et al.; meta-Cleavage dioxygenase genes were isolated from the benzoate-assimilating bacteria Pseudomonas stutzeri A401 and Pseudomonas mendocina KC37 . The gene products had the enzymatic activity of 2,3-dihydroxy-biphenyl-1,2-dioxygenase . Phylogenetic analysis based on the alignment of amino acid sequences suggests that these enzymes are distantly related to other meta-cleavage enzymes and may be members of a new family of extradiol dioxygenases.

Plant Cell, 1997 Mar, 9(3), 305 - 16
Arabidopsis enhanced disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in PR-1 gene expression; Rogers EE et al.; To identify plant defense responses that limit pathogen attack, Arabidopsis eds mutants that exhibit enhanced disease susceptibility to the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 were previously identified . In this study, we show that each of four eds mutants (eds5-1, eds6-1, eds7-1, and eds9-1) has a distinguishable phenotype with respect to the degree of susceptibility to a panel of bacterial phytopathogens and the ability to activate pathogenesis-related PR-1 gene expression after pathogen attack . None of the four eds mutants exhibited observable defects in mounting a hypersensitive response . Although all four eds mutants were also capable of mounting a systemic acquired resistance response, enhanced growth of P . s . maculicola ES4326 was still apparent in the secondarily infected leaves of three of the eds mutants . These data indicate that eds genes define a diverse set of previously unknown defense responses that affect resistance to virulent pathogens.

Ophthal Plast Reconstr Surg, 1997 Mar, 13(1), 68 - 71
Osteomyelitis of the orbit; el-Toukhy E et al.; Although rare, orbital osteomyelitis secondary to sinusitis can be devastating . Early, aggressive ophthalmologic surgical intervention, as well as otorhinolaryngologic co-management, is necessary to obtain the best outcomes . We present two cases of orbital osteomyelitis . One patient remained infected with Pseudomonas meningitis even after extensive sinus and orbital surgery, rapidly declined, and is now deceased . The other patient, after multiple sinus procedures and a medial orbitotomy, was placed on hyperbaric oxygen and is still undergoing treatment.

South Med J, 1997 Mar, 90(3), 328 - 9
Bilateral non-Hodgkin's lymphoma of the breast mimicking mastitis; Dawn B et al.; We recently encountered a case of recurrent non-Hodgkin's lymphoma manifested after a long period of quiescence as bilateral involvement of the breasts . This 37-year-old woman had stage IVA nodular poorly differentiated lymphocytic lymphoma diagnosed 9 years previously and was followed up without treatment . She was lost to follow-up after 4 years but had been in good health until seen with malaise and fever and pain, swelling, and erythema involving both breasts . Biopsies of lymph node and bone marrow showed a high-grade non-Hodgkin's lymphoma (lymphoblastic lymphoma) of B cell origin with central nervous system involvement . Combination chemotherapy produced a dramatic remission, but the patient died of Pseudomonas septicemia.

Mol Plant Microbe Interact, 1997 Mar, 10(2), 247 - 56
Expression of avrPphB, an avirulence gene from Pseudomonas syringae pv . phaseolicola, and the delivery of signals causing the hypersensitive reaction in bean; Puri N et al.; Protein production encoded by the avirulence gene avrPphB from Pseudomonas syringae pv . phaseolicola was examined . Incorporation of {35S}-labeled methionine into the AvrPphB protein indicated processing of the full-length peptide in Escherichia coli to give a major 28-kDa product . The 28-kDa native peptide was isolated from E . coli following over-expression of avrPphB and found not to elicit the hypersensitive response (HR) after infiltration into bean leaves . Antiserum raised to the 28-kDa peptide allowed expression of avrPphB and processing of AvrPphB protein to be examined in P . syringae pv . phaseolicola; immunoreactive peptides of both 35 and 28-kDa were detected in races 3 and 4 (which contain avrPphB) only after induction in minimal medium + 10 mM sucrose . Antiserum raised to a synthetic peptide, derived from the sequence of the 62 amino acids found to be cleaved from the full-length AvrPphB protein, revealed the accumulation of peptides corresponding to the smaller cleavage products, in both E . coli and P . syringae pv . phaseolicola . Biochemical localization experiments showed that all AvrPphB peptides were cytoplasmic in P . syringae pv . phaseolicola . No AvrPphB peptides were produced in a hrpL mutant unless expression of the gene was directed by a strong vector promoter; induction kinetics similar to wild type were observed in a hrpY- strain, although it also failed to cause a confluent HR . Growth of P . syringae pv . phaseolicola under inducing conditions removed the requirement for rifampicin-sensitive mRNA synthesis by bacteria to allow HR development (the induction time) in bean and lettuce leaves . Constitutive expression of hrpL reduced but did not remove the induction time . Expression of the hrp gene cluster of P . syringae pv . phaseolicola from plasmid pPPY430 in E . coli enabled phenotypic expression of avrPphE (also carried by pPPY430) and avrPphB (if over-expressed from pPPY3031) . Despite constitutive expression of the hrp and avr genes in E . coli, a protein synthesis dependent induction time was still required for development of the HR in bean genotypes with matching resistance genes . The significance of processing for the function of AvrPphB peptides and the delivery of elicitors of the HR are discussed.

Appl Environ Microbiol, 1997 Mar, 63(3), 983 - 9
Bacterial diversity among small-subunit rRNA gene clones and cellular isolates from the same seawater sample; Suzuki MT et al.; Numerous investigations applying the cloning and sequencing of rRNA genes (rDNAs) to the study of marine bacterioplankton diversity have shown that the sequences of genes cloned directly from environmental DNA do not correspond to the genes of cultured marine taxa . These results have been interpreted as support for the hypothesis that the most abundant heterotrophic marine bacterioplankton species are not readily culturable by commonly used methods . However, an alternative explanation is that marine bacterioplankton can be easily cultured but are not well represented in sequence databases . To further examine this question, we compared the small-subunit (SSU) rDNAs of 127 cellular clones isolated from a water sample collected off the Oregon coast to 58 bacterial SSU rDNAs cloned from environmental DNAs from the same water sample . The results revealed little overlap between partial SSU rDNA sequences from the cellular clones and the environmental clone library . An exception was the SSU rDNA sequence recovered from a cellular clone belonging to the Pseudomonas subgroup of the gamma subclass of the class Proteobacteria, which was related to a single gene cloned directly from the same water sample (OCS181) (similarity, 94.6%) . In addition, partial SSU rDNA sequences from three of the cultured strains matched a novel rDNA clone related to the gamma subclass of the Proteobacteria found previously in an environmental clone library from marine aggregates (AGG53) (similarity, 94.3 to 99.6%) . Our results support the hypothesis that many of the most abundant bacterioplankton species are not readily culturable by standard methods but also show that heterotrophic bacterioplankton that are culturable on media with high organic contents include many strains for which SSU rDNA sequences are not available in sequence databases.

Appl Environ Microbiol, 1997 Mar, 63(3), 916 - 23
The atzB gene of Pseudomonas sp . strain ADP encodes the second enzyme of a novel atrazine degradation pathway; Boundy-Mills KL et al.; We previously reported the isolation of a 21.5-kb genomic DNA fragment from Pseudomonas sp . strain ADP, which contains the atzA gene, encoding the first metabolic step for the degradation of the herbicide atrazine (M . de Souza, L . P . Wackett, K . L . Boundy-Mills, R . T . Mandelbaum, and M . J . Sadowsky, Appl . Environ . Microbiol . 61:3373-3378, 1995) . In this study, we show that this fragment also contained the second gene of the atrazine metabolic pathway, atzB . AtzB catalyzed the transformation of hydroxyatrazine to N-isopropylammelide . The product was identified by use of high-performance liquid chromatography, mass spectrometery, and nuclear magnetic resonance spectroscopy . Tn5 mutagenesis of pMD1 was used to determine that atzB was located 8 kb downstream of atzA . Hydroxyatrazine degradation activity was localized to a 4.0-kb ClaI fragment, which was subcloned into the vector pACYC184 to produce plasmid pATZB-2 . The DNA sequence of this region was determined and found to contain two large overlapping divergent open reading frames, ORF1 and ORF2 . ORF1 was identified as the coding region of atzB by demonstrating that (i) only ORF1 was transcribed in Pseudomonas sp . strain ADP, (ii) a Tn5 insertion in ORF2 did not disrupt function, and (iii) codon usage was consistent with ORF1 being translated . AtzB had 25% amino acid identity with TrzA, a protein that catalyzes a hydrolytic deamination of the s-triazine substrate melamine . The atzA and atzB genes catalyze the first two steps of the metabolic pathway in a bacterium that rapidly metabolizes atrazine to carbon dioxide, ammonia, and chloride.

Curr Microbiol, 1997 Mar, 34(3), 138 - 43
Transformation of pBR322-Derived Plasmids in PhytopathogenicPseudomonas avenae and Enhanced Transformation in ItsProline-Auxotrophic Mutant
Fukumoto F, Sato M, Minobe Y.
Efficient transformation of pBR322 and its derivedplasmids, which have been widely used as cloning vectors in Escherichiacoli, was observed in Pseudomonas avenae (K1), the pathogen ofleaf blight disease in cereals . Moreover, there was a 10- to 50-foldtransformation efficiency (1.3-3.0 x 10(6)/&mgr;g DNA) in theproline-auxotrophic mutant (Pr47), whose virulence to rice seedlingsdecreased . Similar enhancement of the frequency of transfer by mobilizationof RSF1010, a broad host range plasmid, was observed in the recipient Pr47strain in mating with donor Pseudomonas syringae . The plasmidsharbored in these strains were maintained very stably after subcultures.Thus, a highly efficient transformation system with pBR322-derived plasmidsused as a vector and Pseudomonas as a host bacterium was developed.

Cancer Lett, 1997 Feb 26, 113(1-2), 153 - 8
In vitro effects of a recombinant toxin, mSCF-PE40, targeting c-kit receptors ectopically expressed in small cell lung cancers; Nishida K et al.; Most small cell lung cancers (SCLCs) ectopically express high levels of the c-kit receptor . We have examined if the receptor can serve as a target for a chimeric toxin, mSCF-PE40 composed of murine stem cell factor (SCF) genetically fused to the N terminus of a modified form of Pseudomonas exotoxin (PE) lacking its cell recognition domain . Selective cytotoxicity was found for human c-kit receptor-negative cells . This agent thus warrants further evaluation for therapy of human CSLCs.

Biochemistry, 1997 Feb 25, 36(8), 2173 - 7
Protein engineering of the HMG-CoA reductase of Pseudomonas mevalonii . Construction of mutant enzymes whose activity is regulated by phosphorylation and dephosphorylation; Friesen JA et al.; The activity of Pseudomonas mevalonii HMG-CoA reductase (EC 1.1.1.88) is not regulated by phosphorylation, presumably due to the absence of a suitable target serine and protein kinase recognition motif . We have engineered P . mevalonii HMG-CoA reductase to a form whose activity, like that of mammalian HMG-CoA reductases, is regulated by phosphorylation/dephosphorylation . We substituted serine for arginine 387, the residue that corresponds to the regulatory serine of the HMG-CoA reductases of higher eukaryotes . A recognition motif for cAMP-dependent protein kinase was added by replacing leucine 384 by histidine (enzyme L384H/R387S) and also valine 391 by leucine (enzyme L384H/R387S/V391L) . The activity of P . mevalonii HMG-CoA reductase mutant enzymes L384H/R387S and L384H/R387S/V391L was attenuated by phosphorylation . Restoration of activity accompanied subsequent dephosphorylation catalyzed by lambda protein phosphatase . Incorporation and subsequent release of phosphate paralleled the attenuation and restoration of catalytic activity . Incorporation of 0.5 mol of phosphate per subunit was accompanied by an approximately 50% decrease in initial activity . As in the analogous Syrian hamster mutant enzyme S871D, P . mevalonii mutant enzyme R387D exhibited 10% wild-type activity, suggesting that the attenuation of activity that accompanies phosphorylation results at least in part from the introduction of negative charge . Engineering of P . mevalonii HMG-CoA reductase to forms whose activity is reversibly regulated by phosphorylation/dephosphorylation provides an attractive model for future structure-based mechanistic studies . Solution of the X-ray structure of phosphorylated and dephosphorylated forms of engineered P . mevalonii HMG-CoA reductase should then reveal interactions of the active site phosphoseryl residue that result in attenuation of catalytic activity.

EMBO J, 1997 Feb 17, 16(4), 726 - 38
Rapid stimulation of a soybean protein-serine kinase that phosphorylates a novel bZIP DNA-binding protein, G/HBF-1, during the induction of early transcription-dependent defenses; Droge-Laser W et al.; The G-box (CACGTG) and H-box (CCTACC) cis elements function in the activation of phenylpropanoid biosynthetic genes involved in the elaboration of lignin precursors, phytoalexins and the secondary signal salicylic acid as early responses to pathogen attack . We have isolated a soybean cDNA encoding a novel bZIP protein, G/HBF-1, which binds to both the G-box and adjacent H-box in the proximal region of the chalcone synthase chs15 promoter . While G/HBF-1 transcript and protein levels do not increase during the induction of phenylpropanoid biosynthetic genes, G/HBF-1 is phosphorylated rapidly in elicited soybean cells, almost exclusively on serine residues . Using recombinant G/HBF-1 as a substrate, we identified a cytosolic protein-serine kinase that is rapidly and transiently stimulated in cells elicited with either glutathione or an avirulent strain of the soybean pathogen Pseudomonas syringae pv . glycinea . Phosphorylation of G/HBF-1 in vitro enhances binding to the chs15 promoter and we conclude that stimulation of G/HBF-1 kinase activity and G/HBF-1 phosphorylation are terminal events in a signal pathway for activation of early transcription-dependent plant defense responses.

Structure, 1997 Feb 15, 5(2), 187 - 202
The open conformation of a Pseudomonas lipase; Schrag JD et al.; BACKGROUND: . The interfacial activation of lipases results primarily from conformational changes in the enzymes which expose the active site and provide a hydrophobic surface for interaction with the lipid substrate . Comparison of the crystallization conditions used and the structures observed for a variety of lipases suggests that the enzyme conformation is dependent on solution conditions . Pseudomonas cepacia lipase (PCL) was crystallized in conditions from which the open, active conformation of the enzyme was expected . Its three-dimensional structure was determined independently in three different laboratories and was compared with the previously reported closed conformations of the closely related lipases from Pseudomonas glumae (PGL) and Chromobacterium viscosum (CVL) . These structures provide new insights into the function of this commercially important family of lipases . RESULTS: . The three independent structures of PCL superimpose with only small differences in the mainchain conformations . As expected, the observed conformation reveals a catalytic site exposed to the solvent . Superposition of PCL with the PGL and CVL structures indicates that the rearrangement from the closed to the open conformation involves three loops . The largest movement involves a 40 residue stretch, within which a helical segment moves to afford access to the catalytic site . A hydrophobic cleft that is presumed to be the lipid binding site is formed around the active site . CONCLUSIONS: . The interfacial activation of Pseudomonas lipases involves conformational rearrangements of surface loops and appears to conform to models of activation deduced from the structures of fungal and mammalian lipases . Factors controlling the conformational rearrangement are not understood, but a comparison of crystallization conditions and observed conformation suggests that the conformation of the protein is determined by the solution conditions, perhaps by the dielectric constant.

Structure, 1997 Feb 15, 5(2), 173 - 85
The crystal structure of a triacylglycerol lipase from Pseudomonas cepacia reveals a highly open conformation in the absence of a bound inhibitor; Kim KK et al.; BACKGROUND: . Lipases, a family of enzymes which catalyze the hydrolysis of triglycerides, are widely distributed in many organisms . True lipases are distinguished from esterases by the characteristic interfacial activation they exhibit at an oil-water interface . Lipases are one of the most frequently used biocatalysts for organic reactions performed under mild conditions . Their biotechnological applications include food and oil processing and the preparation of chiral intermediates for the synthesis of enantiomerically pure pharmaceuticals . Recent structural studies on several lipases have provided some clues towards understanding the mechanisms of hydrolytic activity, interfacial activation, and stereoselectivity . This study was undertaken in order to provide structural information on bacterial lipases, which is relatively limited in comparison to that on the enzymes from other sources . RESULTS: . We have determined the crystal structure of a triacylglycerol lipase from Pseudomonas cepacia (PcL) in the absence of a bound inhibitor using X-ray crystallography . The structure shows the lipase to contain an alpha/beta-hydrolase fold and a catalytic triad comprising of residues Ser87, His286 and Asp264 . The enzyme shares several structural features with homologous lipases from Pseudomonas glumae (PgL) and Chromobacterium viscosum (CvL), including a calcium-binding site . The present structure of PcL reveals a highly open conformation with a solvent-accessible active site . This is in contrast to the structures of PgL and PcL in which the active site is buried under a closed or partially opened 'lid', respectively . CONCLUSIONS: . PcL exhibits some structural features found in other lipases . The presence of the Ser-His-Asp catalytic triad, an oxyanion hole, and the opening of a helical lid suggest that this enzyme shares the same mechanisms of catalysis and interfacial activation as other lipases . The highly open conformation observed in this study is likely to reflect the activated form of the lipase at an oil-water interface . The structure suggests that the interfacial activation of bacterial lipases involves the reorganization of secondary structures and a large movement of the lid to expose the active site . This is similar to the mechanism described for other well characterized fungal and mammalian lipases.

J Biol Chem, 1997 Feb 7, 272(6), 3363 - 8
Paracatalytic inactivation of L-2-haloacid dehalogenase from Pseudomonas sp . YL by hydroxylamine . Evidence for the formation of an ester intermediate; Liu JQ et al.; Asp10 of L-2-haloacid dehalogenase from Pseudomonas sp . YL was proposed to act as a nucleophile to attack the alpha-carbon of L-2-haloalkanoic acids to form an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon (Liu, J.-Q, Kurihara, T., Miyagi, M., Esaki, N., and Soda, K . (1995) J . Biol . Chem . 270, 18309-18312) . We have found that the enzyme is paracatalytically inactivated by hydroxylamine in the presence of the substrates monochloroacetate and L-2-chloropropionate . Ion spray mass spectrometry demonstrated that the molecular mass of the enzyme inactivated by hydroxylamine during the dechlorination of monochloroacetate is about 74 Da greater than that of the native enzyme . To determine the increase of the molecular mass more precisely, we digested the inactivated enzyme with lysyl endopeptidase and measured the molecular masses of the peptide fragments . The molecular mass of the hexapeptide Gly6-Lys11 was shown to increase by 73 Da . Tandem mass spectrometric analysis of this peptide revealed that the increase is due to a modification of Asp10 . When the enzyme was paracatalytically inactivated by hydroxylamine during the dechlorination of L-2-chloropropionate, the molecular mass of the hexapeptide was 87 Da higher . Hydroxylamine is proposed to attack the carbonyl carbon of the ester intermediate and form a stable aspartate beta-hydroxamate carboxyalkyl ester residue in the inactivated enzyme.

Biochemistry, 1997 Feb 4, 36(5), 1157 - 62
Identification of elements critical for phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by adenosine monophosphate-activated protein kinase: protein engineering of the naturally nonphosphorylatable 3-hydroxy-3-methylglutaryl coenzyme A reductase from Pseudomonas mevalonii; Friesen JA et al.; The initially nonphosphorylatable 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of Pseudomonas mevalonii (E.C . 1.1.1.88) was engineered to phosphorylatable forms in order to identify elements critical for phosphorylation of HMG-CoA reductase by AMP-activated protein kinase . P . mevalonii, mutant enzymes phosphorylatable by AMP-activated protein kinase were engineered by substituting cognate residues from the kinase recognition sequence of Syrian hamster HMG-CoA reductase (E.C . 1.1.1.34) . Various combinations of residues 381-391, which correspond to the kinase recognition sequence of the hamster enzyme, were mutated . P . mevalonii mutant enzyme R387S, in which a serine had been inserted at position P, which corresponds to that of the regulatory serine of the hamster enzyme, was only weakly phosphorylated . Genes that encoded thirty-six additional mutant enzymes containing various portions of the hamster kinase recognition sequence were constructed . Following expression, purified mutant enzymes were assayed as substrates for AMP-activated protein kinase . Identified as critical for phosphorylation was the simultaneous presence of aspartate or asparagine at position P+3 and of leucine at position P+4, three and four residues on the C-terminal side of the phosphorylatable serine, respectively . Two basic residues at positions P-1, P-2, or P-3 also appeared to be critical for phosphorylation when present in combination with aspartate or asparagine at P+3 and leucine at P+4.

J Mol Cell Cardiol, 1997 Feb, 29(2), 799 - 811
Partial inhibition of protein synthesis by Pseudomonas exotoxin A deranges catecholamine sensitivity of cultured rat heart myocytes; Muller-Werdan U et al.; To elucidate cellular mechanisms of myocardial depression in Pseudomonas sepsis the effects of sublethal concentrations of P . aeruginosa exotoxin A--a main virulence factor--were studied in cultured neonatal rat cardiomyocytes . It is known that this toxin exerts its pathogenic effect by inhibition of protein synthesis via ADP-ribosylation and thereby inactivation of elongation factor 2 (EF-2) . Within 48 72 h, half maximal inhibition of protein synthesis occurs at 4-10 ng/ml . The toxin prevents the beta-adrenoceptor(AR)-mediated myosin heavy chain isozyme shift (V3/V1), while the T3-induced myosin shift is not suppressed . While beta 1-AR-downregulation by excess of norepinephrine (NE) is not affected, protein synthesis-dependent receptor upregulation in the recover period after removal of NE is completely suppressed by P . aeruginosa exotoxin A . Thus, a non-lethal, partial inhibition of global cellular protein synthesis by P . aeruginosa exotoxin A: (1) completely prevents beta 1-AR-mediated myosin isozyme shift and beta-AR upregulation: (2) sustains the cardiomyocytes in a catecholamine-refractory contractile state in the recovery period after catecholamine desensitization: (3) suggests cellular mechanisms by which P . aeruginosa exotoxin A might impair heart function in Pseudomonas sepsis: and (4) may help reveal the possible influence of endogenous inhibitors of EF-2.

Plant Cell Physiol, 1997 Feb, 38(2), 194 - 200
Purification and characterization of wall-bound exo-1,3-beta-D-glucanase from barley (Hordeum vulgare L.) seedlings; Kotake T et al.; A beta-D-glucanase activity hydrolyzing 1,3:1,4-beta-D-glucan was released from the cell walls of barley by 3 M LiCl treatment . It was purified by sequential cation-exchange, gel-filtration and hydrophobic chromatography . The molecular mass of the glucanase was 66 kDa as determined by SDS-polyacrylamide gel electrophoresis . Sequence determination of the first thirty amino acids of the N-terminus revealed a high homology of this enzyme to the Pseudomonas 1,4-beta-D-glucosidase (56.5%) . The purified beta-D-glucanase has a pH optimum at 5.0, and hydrolyzes oligosaccharides containing beta-D-1,3 or beta-D-1,4 linkage . The glucanase showed maximum hydrolytic activity toward laminaritetraose, the rate being about two times that of cellotetraose and about four times that of gentiobiose . Polysaccharides such as lichenan, 1,3:1,4-beta-D-glucan (from barley), laminarin and pustulan are also hydrolyzed, but not carboxylmethyl-curdlan, carboxymethyl-cellulose, xyloglucan and maltose . The purified beta-D-glucanase yielded monomeric glucose from laminarihexaose, and exhibited characteristics of an exo-1,3-beta-D-glucanase (EC 3.2.1.58) . The activity and biochemical characteristics of this enzyme suggest that it is an exo-1,3-beta-D-glucanase involved in the rapid turnover of 1,3:1,4-beta-D-glucan in barley cell walls during seedling growth.

Plant Cell, 1997 Feb, 9(2), 261 - 70
Salicylic acid potentiates an agonist-dependent gain control that amplifies pathogen signals in the activation of defense mechanisms; Shirasu K et al.; The phenylpropanoid-derived natural product salicylic acid (SA) plays a key role in disease resistance . However, SA administered in the absence of a pathogen is a paradoxically weak inductive signal, often requiring concentrations of 0.5 to 5 mM to induce acquired resistance or related defense mechanisms or to precondition signal systems . In contrast, endogenous SA accumulates to concentrations of < 70 microM at the site of attempted infection . Here, we show that although 10 to 100 microM SA had negligible effects when administered to soybean cell suspensions in the absence of a pathogen, physiological concentrations of SA markedly enhanced the induction of defense gene transcripts, H2O2 accumulation, and hypersensitive cell death by an avirulent strain of Pseudomonas syringae pv glycinea, with optimal effects being at approximately 50 microM . SA also synergistically enhanced H2O2 accumulation in response to the protein phosphatase type 2A inhibitor cantharidin in the absence of a pathogen . The synergistic effect of SA was potent, rapid, and insensitive to the protein synthesis inhibitor cycloheximide, and we conclude that SA stimulates an agonist-dependent gain control operating at an early step in the signal pathway for induction of the hypersensitive response . This fine control mechanism differs from previously described time-dependent, inductive coarse control mechanisms for SA action in the absence of a pathogen . Induction of H2O2 accumulation and hypersensitive cell death by avirulent P . s . glycinea was blocked by the phenylpropanoid synthesis inhibitor alpha-aminooxy-beta-phenylpropionic acid, and these responses could be rescued by exogenous SA . Because the agonist-dependent gain control operates at physiological levels of SA, we propose that rapid fine control signal amplification makes an important contribution to SA function in the induction of disease resistance mechanisms.

Eur J Immunol, 1997 Feb, 27(2), 486 - 94
Targeted elimination of cells expressing the high-affinity receptor for IgE (Fc epsilon RI) by a Pseudomonas exotoxin-based chimeric protein; Fishman A et al.; The interaction between IgE and its high-affinity receptor Fc epsilon RI found on mast cells and basophils is the primary effector pathway in allergic response . To achieve a targeted elimination of cells expressing Fc epsilon RI receptors, we constructed a chimeric protein in which a Fc fragment of mouse IgE is attached to a truncated form of Pseudomonas exotoxin (PE) . To prepare the targeting moiety, we used a DNA sequence corresponding to amino acids 301-437, representing 30 residues of domain 2 and domain 3 of the mouse IgE constant region . This sequence was fused at the 5' of a cDNA encoding PE40, a truncated form of PE lacking the cell binding domain . The chimeric protein, termed Fc(2'-3)-PE40, was expressed in Escherichia coli and partially purified . The protein is highly cytotoxic to mouse mast cell lines and bone marrow-derived primary mast cells . This cytotoxicity is specific, as it could be blocked upon addition of whole IgE . Moreover, the protein had no effect on other cell lines of hemopoietic origin . The Fc(2'-3)-PE40 chimeric protein offers a new approach to the treatment of allergic disorders.

Schweiz Med Wochenschr, 1997 Feb 1, 127(5), 158 - 64
Aerosol therapy in patients with cystic fibrosis; Schoni MH et al.; Aerosol therapy is one of the mainstays of treatment, together with regular physiotherapy, in patients with cystic fibrosis . Inhalation can contribute to hydration of the epithelial lining fluid as well as delivering different drugs directly to the lungs . Topically administered antibiotics can protect the lungs from Pseudomonas infection, recombinant DNase, amiloride and beta-agonists can have a positive effect on the mucociliary clearance, and steroid inhalations can reduce inflammation . Therefore, all these drugs are part of a comprehensive treatment strategy contributing to improvement in lung function and quality of life . Gene therapy and pharmacological correction of the chloride channel defect are perspectives for the future . Aerosol therapy, however, is somewhat cumbersome and requires strict patient education.

Ann Thorac Surg, 1997 Feb, 63(2), 529 - 31
Double mycotic aneurysms of the ascending aorta; Chen YF et al.; Infection in the vascular tree has been proved to be one of the greatest challenges for cardiovascular surgeons . Of these, mycotic aneurysms of the ascending aorta were considered to be almost always lethal until recently . A thorough survey of the literature indicates that only 42 cases of mycotic aneurysm of the ascending aorta have been reported . All the reported cases of mycotic aneurysm of the ascending aorta were a single lesion in the ascending aorta except a case reported in 1993 . This report describes an additional case of double mycotic aneurysms of the ascending aorta caused by Pseudomonas infection.

Appl Environ Microbiol, 1997 Feb, 63(2), 498 - 505
Assessment of genetic diversity among strains of Pseudomonas syringae by PCR-restriction fragment length polymorphism analysis of rRNA operons with special emphasis on P . syringae pv . tomato; Manceau C et al.; Phylogenetic relationships among 77 bacterial strains belonging to Pseudomonas syringae and Pseudomonas viridiflava species were assessed by analysis of the PCR-restriction fragment length polymorphism (RFLP) patterns of three DNA fragments corresponding to rrs and rrl genes and the internal transcribed spacer, ITS1 . No difference among all strains in rrs and rrl genes was observed with 14 restriction enzymes, which confirms the close relationships existing between these two species . The nucleotidic sequence of the internal transcripted spacer (ITS1) between rrs and rrl for the P . syringae pv . syringae strain CFBP1392 was determined . Restriction maps of the PCR-amplified ITS1 region were prepared and compared for all 77 strains . Seventeen RFLP patterns, forming three main clusters, were distinguished . One contained all strains of P . syringae pv . tomato and of other pathovars which had been previously described as closely related by either pathogenicity studies or biochemical analyses . This cluster was equally far from P . viridiflava and from other P . syringae pathovars . These other pathovars of P . syringae formed a less coherent taxon.

J Bacteriol, 1997 Feb, 179(4), 1044 - 50
Enzymology of oxidation of tropic acid to phenylacetic acid in metabolism of atropine by Pseudomonas sp . strain AT3; Long MT et al.; Pseudomonas sp . strain AT3 grew with dl-tropic acid, the aromatic component of the alkaloid atropine, as the sole source of carbon and energy . Tropic acid-grown cells rapidly oxidized the growth substrate, phenylacetaldehyde, and phenylacetic acid . Crude cell extracts, prepared from dl-tropic acid-grown cells, contained two NAD+-linked dehydrogenases which were separated by ion-exchange chromatography and shown to be specific for their respective substrates, dl-tropic acid and phenylacetaldehyde . Phenylacetaldehyde dehydrogenase was relatively unstable . The stable tropic acid dehydrogenase was purified to homogeneity by a combination of ion-exchange, molecular-sieve, and affinity chromatography . It had a pH optimum of 9.5 and was equally active with both enantiomers of tropic acid, and at this pH, phenylacetaldehyde was the only detectable product of tropic acid oxidation . The formation of phenylacetaldehyde from tropic acid requires, in addition to dehydrogenation, a decarboxylation step . By analogy with NAD+-specific isocitrate and malate dehydrogenases, phenylmalonic semialdehyde, a 3-oxoacid, would be expected to be the precursor of phenylacetaldehyde . Other workers have established that isocitrate and malate dehydrogenases catalyze the decarboxylation of enzyme-bound or added 3-oxoacid intermediates, a reaction that requires Mn2+ or Mg2+ ions . Studies with tropic acid dehydrogenase were hampered by lack of availability of phenylmalonic semialdehyde, but in the absence of added divalent metal ions, both enantiomers of tropic acid were completely oxidized and we have not, by a number of approaches, found any evidence for the transient accumulation of phenylmalonic semialdehyde.

J Bacteriol, 1997 Feb, 179(3), 762 - 8
Determinants for overproduction of the Pseudomonas oleovorans cytoplasmic membrane protein alkane hydroxylase in alk+ Escherichia coli W3110; Nieboer M et al.; The Pseudomonas oleovorans alkB gene is expressed in alk+ Escherichia coli W3110 to 10 to 15% of the total cell protein, which is exceptional for a (foreign) cytoplasmic membrane protein . In other E . coli recombinants such as alk+ HB101, AlkB constitutes 2 to 3% of the total protein . In this study, we have investigated which factors determine the expression level of alkB in alk+ W3110 . In particular, we have investigated the role of AlkB-induced stimulation of phospholipid synthesis . Blocking phospholipid synthesis in alk+ W3110 did not specifically alter the expression of alkB, and we conclude that stimulation of phospholipid synthesis is not a prerequisite for high-level expression of the membrane protein . W3110 is able to produce exceptionally high levels of alkane monooxygenase, because the rate of alkB mRNA synthesis in W3110 is an order of magnitude higher than that in HB101 . This may be due in part to the higher copy number of pGEc47 in W3110 in comparison with HB101.

Biochemistry, 1997 Jan 28, 36(4), 923 - 31
Ion binding induces closed conformation in Pseudomonas 7A glutaminase-asparaginase (PGA): crystal structure of the PGA-SO4(2-)-NH4+ complex at 1.7 A resolution; Jakob CG et al.; Pseudomonas 7A glutaminase-asparaginase (PGA) catalyzes the hydrolysis of D- and L-isomers of glutamine and asparagine . X-ray quality type-1 crystals of PGA have been obtained from 2.0 M ammonium sulfate . The space group is C222(1) with unit-cell dimensions a = 78.62, b = 135.80, and c = 137.88 A . The tetrameric molecule is located on a crystallographic 2-fold axis, and two subunits form the asymmetric portion of the unit cell . The structure was solved by the molecular replacement method and refined at 1.7 A resolution to an R = 19.9% with a good geometry of the model, G = 0.05 . The resultant electron density maps enabled us to resolve individual constituent atoms of most residues and introduce minor revisions to the amino acid sequence . The catalytic loop, Thr20-Gly40, is in the closed conformation with excellent electron density in both subunits . A sulfate ion and an ammonium ion are bound in the substrate binding site and interect with the loop . This interaction appears to be responsible for the observed closed conformation . New arguments supporting Thr20 as the catalytic nucleophile in the asparaginase activity are proposed.

Biochemistry, 1997 Jan 21, 36(3), 495 - 504
Solution structure of phenol hydroxylase protein component P2 determined by NMR spectroscopy; Qian H et al.; Phenol hydroxylase from Pseudomonas sp . CF600 is a member of a family of binuclear iron-center-containing multicomponent oxygenases, which catalyzes the conversion of phenol and some of its methyl-substituted derivatives to catechol . In addition to a reductase component which transfers electrons from NADH, optimal turnover of the hydroxylase requires P2, a protein containing 90 amino acids which is readily resolved from the other components . The three-dimensional solution structure of P2 has been solved by 3D heteronuclear NMR spectroscopy . On the basis of 1206 experimental constraints, including 1060 distance constraints obtained from NOEs, 70 phi dihedral angle constraints, 42 psi dihedral angle constraints, and 34 hydrogen bond constraints, a total of 12 converged structures were obtained . The atomic root mean square deviation for the 12 converged structure with respect to the mean coordinates is 2.48 A for the backbone atoms and 3.85 A for all the heavy atoms . This relatively large uncertainty can be ascribed to conformational flexibility and exchange . The molecular structure of P2 is composed of three helices, six antiparallel beta-strands, one beta-hairpin, and some less ordered regions . This is the first structure among the known multicomponent oxygenases . On the basis of the three-dimensional structure of P2, sequence comparisons with similar proteins from other multicomponent oxygenases suggested that all of these proteins may have a conserved structure in the core regions.

J Immunol, 1997 Jan 15, 158(2), 756 - 64
The IL-13 receptor structure differs on various cell types and may share more than one component with IL-4 receptor; Obiri NI et al.; We have reported on the expression and characteristics of IL-13R and have demonstrated that IL-13 competes for IL-4 binding while IL-4 did not compete for the IL-13 binding on some cell types . Based on these observations, and the size of IL-13 and IL-4 cross-linked proteins, we concluded that the receptor for IL-13 is complex and shares a subunit with the receptor for IL-4 . To explore the complexity of the IL-13R, a wide variety of cell types was examined for IL-13 and IL-4 binding . We report in this work that IL-4 does not always bind well to cells that bind IL-13, but the reverse is also true . We also found that IL-4 can compete more effectively for IL-13 binding than IL-13 itself . Cross-linking studies support these observations and demonstrate that 125I-labeled IL-13 bound exclusively to a single 65- to 70-kDa protein in MA-RCC and U251 cells, while in TF-1 cells it cross-linked to two membrane proteins of 65 to 70 kDa and 140 kDa . Furthermore, by using a chimeric protein composed of IL-13 and Pseudomonas exotoxin A, we observed that IL-4 neutralized the cytotoxicity of the IL-13 toxin on COS-7 cells by blocking a common form of the two cytokine receptors . We propose that the 65- to 70-kDa form of the IL-13R is the predominant common component shared between IL-13 and IL-4R . However, the primary IL-4 binding (p140) protein also participates in the formation of the IL-13R complex in some cell types . In addition, the gamma(c) or another interactive subunit may influence IL-13 binding to its receptor complex . Thus, we propose that there are at least four forms of IL-13R.

J Biol Chem, 1997 Jan 10, 272(2), 945 - 51
On the role of DmpK, an auxiliary protein associated with multicomponent phenol hydroxylase from Pseudomonas sp . strain CF600; Powlowski J et al.; DmpK from Pseudomonas sp . strain CF600 represents a group of proteins required by phenol-degrading bacteria that utilize a multicomponent iron-containing phenol hydroxylase . DmpK has been overexpressed in Escherichia coli and purified to homogeneity; it lacks redox cofactors and was found to strongly inhibit phenol hydroxylase in vitro . Chemical cross-linking experiments established that DmpK binds to the two largest subunits of the oxygenase component of the hydroxylase; this may interfere with binding of the hydroxylase activator protein, DmpM, causing inhibition . Since expression of DmpK normally appears to be much lower than that of the components of the oxygenase, inhibition may not occur in vivo . Hence, the interaction between DmpK and the oxygenase manifested in the inhibition and cross-linking results prompted construction of E . coli strains in which the oxygenase component was expressed in the presence and absence of a low molar ratio of DmpK . Active oxygenase was detected only when expressed in the presence of DmpK . Furthermore, inactive oxygenase could be activated in vitro by adding ferrous iron, in a process that was dependent on the presence of DmpK . These results indicate that DmpK plays a role in assembly of the active form of the oxygenase component of phenol hydroxylase.

Vestn Ross Akad Med Nauk, 1997, (6), 37 - 40
{Effect of TRA-system of plasmids RP4 and R68.45 on pseudomonas mallei virulence}; Verevkin VV et al.; In Pseudomonas mallei, spontaneous mutants (Tra- mutants) of the plasmids RP4 and R68.45, losing the ability to transfer at conjugation are formed . The plasmids RP4 and R68.45 with Tra(+)-phenotype caused a decrease in P . mallei virulence for laboratory animals . At the same time, Tra- mutants of these plasmids do not affect P . mallei virulence . The insertion of DNA fragment of about 1900 bp into the plasmid transfer gene regions (tra-2-tra-3) gave rise to RP4 and R68.45 tra mutations detectable on examination.

Antibiot Khimioter, 1997, 42(5), 29 - 34
{Homeostatic changes in monkeys in a model of glanders}; Manzeniuk IN et al.; Some aspects of homeostasis impairment in monkeys infected with Pseudomonas mallei were investigated . The levels of the hormonal shifts, complement components, beta 2-microglobulin and prostaglandins evident of the infection severity were estimated . The pathomorphological profiles of various forms of malleus in the primates were studied.

Br J Cancer, 1997, 75(11), 1575 - 84
Construction and functional characterization of scFv(14E1)-ETA - a novel, highly potent antibody-toxin specific for the EGF receptor; Schmidt M et al.; Epidermal growth factor (EGF) receptor-overexpression is characteristic of many human tumours of epithelial origin and has been correlated with unfavourable patient prognosis . Its involvement in the malignant process, its elevated expression in tumours and its accessibility on the tumour cell surface make the EGF receptor a potential target for directed tumour therapy . We have previously characterized a recombinant antibody - Pseudomonas exotoxin A fusion protein, scFv(225)-ETA, which displayes antitumoral activity towards EGF receptor-overexpressing tumour cells but is less potent in tumour cell killing than TGF-alpha-ETA, a recombinant toxin using the natural EGF receptor ligand transforming growth factor alpha (TGF-alpha) as a targeting domain . Here, we describe the construction and functional characterization in vitro of a novel single-chain antibody-toxin, scFv(14E1)-ETA, based on the independently isolated EGF receptor-specific monoclonal antibody 14E1 . ScFv(14E1)-ETA binds to an EGF receptor epitope that is very similar or identical to that of scFv(225)-ETA with nine times higher affinity than the latter and displays more than tenfold higher cytotoxic activity on EGF receptor-overexpressing tumour cells . ScFv(14E1)-ETA cell killing activity was very similar to that of TGF-alpha-ETA on receptor-overexpressing cells but, in contrast to the latter, scFv(14E1)-ETA was much more selective and did not display significant cytotoxic activity on cells expressing moderate EGF receptor levels.

Antibiot Khimioter, 1997, 42(4), 21 - 3
{Effect of medium temperature and pH on the sensitivity of pathogenic pseudomonads to chemotherapeutic agents}; Iliukhin VI et al.; Antibiotic susceptibility of four Pseudomonas species i.e . P.aeruginosa, P.cepacia, P.mallei and P.pseudomallei was studied under conditions of different temperature and pH of the medium (within the physiologically possible deviations) . It was shown that along with the general tendencies (lowered MICs at the medium alkalization) there were obvious species differences in the effect of the physicochemical factors on the Pseudomonas antibiotic resistance . The results of the study may to a certain extent serve as an explanation of the lack of coincidence of the chemotherapy efficacy with the data on the drug in vitro estimation.

Rozhl Chir, 1997 Jan, 76(1), 36 - 9
{Bilateral nephrectomy--a life-saving procedure}; Schraml J et al.; Authors report a rare case of complications that occurred after an invasive urodynamic examination . In order to save the life of a patient with a bilateral acute abscessing pyelonefritidis due to pseudomonad uroseption and accompanied by a shock status with multi-organ failure it was necessary to perform the complete bilateral nefrectomy . After removing of the infection zone, i.e . -both kidneys, -the status of the patient dramatically improved . The patient survived and is taking part in dialysis-transplantation programme.

J Infect, 1997 Jan, 34(1), 35 - 40
Central venous line infections in AIDS; Moore DA et al.; The relative infection rates of Port-a-Cath and Hickman lines used for the maintenance treatment of cytomegalovirus retinitis were studied by retrospective casenote review . Seventy-five lines were inserted over an 18 month period . The overall infection rates were significantly greater for Hickman lines: 0.97/100 line-days compared to 0.39/100 line-days for Port-a-Caths, with a trend towards higher infection rates in those prescribed ganciclovir . A total of 56 episodes of septicaemia were recorded with a significantly greater incidence of septicaemia within 1 month of line insertion in those with a neutrophil count < 1.0 x 10(3)/mm3 . Ten infectious episodes were associated with death, six of which were due to Pseudomonas spp . CONCLUSIONS: In HIV seropositive patients, use of a Port-a-Cath is associated with a significantly lower rate of line associated sepsis than use of a Hickman line . Insertion of long term central venous catheters should be avoided if possible when the neutrophil count is < 1.0 x 10(3)/mm3.

Mol Gen Mikrobiol Virusol, 1997, (1), 17 - 22
{Creation of genomic DNA libraries for Pseudomonas mallei and Pseudomonas pseudomallei}; Abaev IV et al.; The genomic libraries of P . mallei and P . pseudomallei species were constructed in Escherichia coli . The chromosomal DNA of P . pseudomallei C-141 strain has been cloned into the cosmid vector pHC79 and the broad host range plasmid vector pES154 . The chromosomal DNA of P . mallei {symbol: see text}-5 strain has been cloned into the plasmid vector pSUP202 . The recombinant clones of the genomic libraries were screened by the enzyme-linked immunoadsorbent assay (ELISA) to detect the production of Pseudomonas antigens: 28 clones were positive . Twelve recombinant strains demonstrated specific antigenic determinants of P . mallei and P . pseudomallei by immunoblotting . Cloned proteins of P . mallei and P . pseudomallei have molecular weights from 30 to 70 kD . A new method for introducing foreign genes into Pseudomonas genomes is offered . P . mallei strains with the chromosomally integrated plasmids pSM are universal recipients for ColEI-based cloning vectors.

Mol Gen Mikrobiol Virusol, 1997, (1), 14 - 7
{Insertion mutations in Pseudomonas pseudomallei genome induced by transposons Tn10 . Tn9, and Tn5}; Merinova LK et al.; Tn10 and Tn9 from temperature-sensitive replicons RPI::Tn10 Rep is and RP1::Tn9 Rep ts and Tn5 from the "suicidal" vector pSUP5011 may integrate in the P . pseudomallei chromosome . Transposons induced auxotrophic mutations of different spectrum, depending on the strain, Tn element, and defects in the genes responsible for the mobility sign, function of some extracellular elements (lecithinase and lipase), hemolytic activity, and antigen 8 synthesis, which are considered as the potential factors of the pathogenicity of melioidosis agent . No secondary restructuring induced by Tn elements were detected in the domains adjacent to insertion sites . Plasmids RP1::Tn10 Rep ts and RP1::Tn9 Rep ts were stable in insertion mutant cells, mediating the variable TR-TS phenotype probably indicating that plasmid integration with the chromosome is unstable.

Biosci Biotechnol Biochem, 1997 Jan, 61(1), 185 - 7
Mechanism of stereospecific conversion of DL-5-substituted hydantoins to the corresponding L-amino acids by Pseudomonas sp . strain NS671; Ishikawa T et al.; The mechanism of stereospecific conversion of DL-5-substituted hydantoins to the corresponding L-amino acids by Pseudomonas sp . strain NS671 was studied . The results indicated that the hydantoinase catalyzed the hydrolysis reaction of both D- and L-5-(2-methylthioethyl)hydantoin, and that the hydrolysis of the L-enantiomer proceeded preferentially compared with that of the D-enantiomer . On the basis of these findings, the mechanism was speculated to be as follows: DL-5-substituted hydantoins are converted exclusively to the L-forms of the corresponding N-carbamyl-amino acids by the hydantoinase in combination with hydantoin racemase . The N-carbamyl-L-amino acids are then converted to L-amino acids by N-carbamyl-L-amino acid amidohydrolase.

Biosci Biotechnol Biochem, 1997 Jan, 61(1), 146 - 51
Purification and characterization of dipeptidyl aminopeptidase from Aureobacterium sp . WO26; Ogasawara W et al.; We isolated a bacterial strain with an enzyme which releases dipeptide from Gly-Arg-p-nitroanilide . The bacterium was tentatively identified as Aureobacterium sp . The enzyme, named AuDAP, was purified and characterized . It was homogenous by SDS-PAGE and IEF, and had a molecular mass of 90,000 Da by SDS-PAGE and 88,000 Da by gel filtration, so it may be a monomer . The isoelectric point was 3.8 and the optimum pH was 10.0 . The purified enzyme hydrolyzed Gly-Arg-pNA, a model substrate for DAP I, and Arg-Arg-MNA, a model substrate for DAP III . However, this enzyme did not hydrolyze Gly-Phe pNA, also a model substrate for DAP I . These results suggested that this enzyme did not fall under the classification of mammalian DAPs and was similar to DAP BI from Pseudomonas sp . WO24 and dDAP from Dictyostelium discoideum, although several differences were observed between them . The N-terminal amino acid sequence of this enzyme showed no significant homology to any enzyme and protein, except only for DAP BI.

Biosci Biotechnol Biochem, 1997 Jan, 61(1), 102 - 4
Hesperidin as an inhibitor of lipases from porcine pancreas and Pseudomonas; Kawaguchi K et al.; In the course of our screening work for new types of lipase inhibitors, hesperidin was identified as a simple and small molecular weight inhibitor in the peels of Citrus unshiu . Hesperidin inhibited lipase activity from porcine pancreas and that from Pseudomonas, and their IC50 was 32 and 132 micrograms/ml, respectively . Hesperidin, neohesperidin, narirutin, and naringin are known as the main flavonoids in Citrus unshiu . Neohesperidin also inhibited the lipase from procine pancreas, but did not have any effect on Pseudomonas . Narirutin and naringin did not show this effect on lipases from porcine pancreas and Pseudomonas . In animal experiments, the concentration of plasma triglyceride in rats fed a diet containing 10% hesperidin were significantly lower than that fed the control diet.

Plant Cell, 1997 Jan, 9(1), 61 - 73
Alleles of Pto and Fen occur in bacterial speck-susceptible and fenthion-insensitive tomato cultivars and encode active protein kinases; Jia Y et al.; The Pto gene was derived originally from the wild tomato species Lycopersicon pimpinellifolium and confers resistance to Pseudomonas syringae pv tomato strains expressing the avirulence gene avrPto . The Fen gene is also derived from L . pimpinellifolium and confers sensitivity to the insecticide fenthion . We have now isolated and characterized the alleles of Pto and Fen from cultivated tomato, L . esculentum, and designated them pto and fen . High conservation of genome organization between the two tomato species allowed us to identify the pto and fen alleles from among the cluster of closely related Pto gene family members . The pto and fen alleles are transcribed and have uninterrupted open reading frames that code for predicted proteins that are 87 and 98% identical to the Pto and Fen protein kinases, respectively . In vitro autophosphorylation assays revealed that both the pto and fen alleles encode active kinases . In addition, the pto kinase phosphorylates a previously characterized substrate of Pto, the Pto-interacting Pti1 serine/threonine kinase . However, the pto kinase shows impaired interaction with Pti1 and with several previously isolated Pto-interacting proteins in the yeast two-hybrid system . The observation that pto and fen are active kinases and yet do not confer bacterial speck resistance or fenthion sensitivity suggests that the amino acid substitutions distinguishing them from Pto and Fen may interfere with recognition of the corresponding signal molecule or with protein-protein interactions involved in the Pto- and Fen-mediated signal transduction pathways.

Biol Pharm Bull, 1997 Jan, 20(1), 107 - 9
Phenylmercury transport mediated by merT-merP genes of Pseudomonas K-62 plasmid pMR26; Uno Y et al.; The merB-merA-deleted plasmid pMRD141 which contains the intact merT-merP genes of pMRA17 conferred bacterial hypersensitivity not only to Hg2+ but also to C6H5Hg+ . The bacterium with pMRD141 took up significantly more C6H5Hg+ than its isogenic strain with the cloning vector Bluescript II . The hypersensitivity to C6H5Hg+ seems to be based on hyperaccumulation of toxic C6H5Hg+ in the absence of detoxifying enzymes encoded by merB and merA . Our results show that bacterial transport of C6H5Hg+ into the cytoplasm is regulated by merT-merP genes.

Hear Res, 1997 Jan, 103(1-2), 131 - 41
Antibodies to the sulphated, high molecular mass mouse tectorin stain hair bundles and the olfactory mucus layer; Killick R et al.; Polyclonal antibodies were raised in chickens to the glycosylated forms of the high (H), medium (M) and low (L) molecular mass (MM) mouse tectorins . In the mouse cochlea, all three antibodies stained the tectorial membrane . Antibodies raised to HMM tectorin also stained the hair bundles of both inner and outer hair cells . A number of other mouse tissues were screened with the anti-tectorin antibodies to look for similar or antigenically related molecules . Staining was not observed in any other tissue type with the antibodies directed against the MMM and LMM tectorins . In the nose, the anti-HMM tectorin antibodies stained Bowman's glands and the mucus layer overlying the olfactory epithelium . The surface of the adjacent respiratory epithelium was not stained by these antibodies . HMM tectorin can be specifically radiolabelled by injecting neonatal mice with 35SO4 and undergoes a shift in electrophoretic mobility following treatment with keratanase, an endo-beta-galactosidase from Pseudomonas . However, when centrifuged on shallow CsCl gradients HMM tectorin has a buoyant density similar to that of glycoproteins and does not behave as a typical cartilage type proteoglycan . HMM tectorin does not react with mab 5D4, a monoclonal antibody that recognises keratan sulphate glycosaminoglycan from corneal and skeletal muscle proteoglycan . Unlike antibodies to HMM tectorin, mab 5D4 selectively stains the upper surface of the tectorial membrane, Hensen's stripe and the mucus layer overlying the respiratory epithelium . These studies indicate that the MMM and LMM tectorins may be unique to the cochlea, and that HMM may be a "light' keratan sulphate proteoglycan that is antigenically related to either the mucins or a more specific component of the olfactory mucus layer.

Int J Syst Bacteriol, 1997 Jan, 47(1), 132 - 43
Genome organization of Pseudomonas stutzeri and resulting taxonomic and evolutionary considerations; Ginard M et al.; In order to determine the genome variability within Pseudomonas stutzeri, 20 strains representing the seven described genomovars and strain JM300 were analyzed by using various resolution levels of rare cutting enzymes . XbaI and SpeI fingerprints revealed a high degree of heterogeneity of restriction patterns that did not correlate with the division into genomovars . However, a fragment pattern comparison led to the establishment of several groups of clonal variants within genomovars . One circular chromosome ranging in size from 3.75 to 4.64 Mb constitutes the genome of P . stutzeri strains . The I-CeuI, PacI, and SwaI low-resolution map of P . stutzeri type strain CCUG 11256 shows the locations of 12 genes, including rrn operons and the origin of replication . I-CeuI digests of the 20 strains studied plus the positions of six genes allowed a comparison of the rrn backbone organization within genomovars; the four rrn operons seemed to be at similar locations with respect to the origin of replication, as did the rest of the genes . However, a comparison of I-CeuI cleavage maps of the genomovar reference strains revealed a diverse genome organization in the genomovars relative to rrn operons and gene locations . In most genomovars, rrn operons are not arranged around the origin of replication but are equally distributed on the chromosome . Strain JM300 does not belong to any described genomovar, as determined from the organization of its genome . Large chromosomal rearrangements seem to be responsible for the differences in superordinate genome structure and must have played an important role in P . stutzeri diversification and niche colonization . An ancestral chromosome is suggested, and some plausible pathways for the generation of the various genome structures are proposed.

Arch Biochem Biophys, 1997 Jan 1, 337(1), 103 - 14
L-Malate dehydrogenase from Pseudomonas stutzeri: purification and characterization; Labrou NE et al.; L-Malate dehydrogenase (MDH) from Pseudomonas stutzeri was purified to homogeneity by a two-step procedure comprising anion-exchange chromatography and affinity chromatography on immobilized anthraquinone alpha-ketocarboxyl biomimetic dye . The enzyme has molecular mass of 66,500 Da and consists of two identical subunits of molecular mass of approximately 34,000 Da . Initial velocity, product inhibition, and binding studies were consistent with an ordered Bi-Bi mechanism for the enzyme action and the formation of a ternary complex . The enzyme is susceptible to activation and inhibition by its substrates . Thermodynamic analysis and kinetic inhibition studies were performed for determining basic equilibrium and kinetic constants . Malate dehydrogenase was covalently inactivated by a dichlorotriazine dye, Vilmafix Blue A-R (VBAR) . The inactivation process follows first-order kinetics, and the results from kinetic analysis suggested the formation of a noncovalent enzyme-dye complex prior to the covalent reaction, with Kd 84.6 microM and a maximum rate constant 0.16 min(-1) . The enzyme inactivation process was partially inhibited by substrates and inhibitors . Quantitatively inactivated MDH contained approximately 1 mole of dye per mole of enzyme subunit . The denatured enzyme contains 10 sulfhydryl groups per subunit, as shown after reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid), of which 5 can be titrated also in the native enzyme, exhibiting time-dependent reactivity . One sulfhydryl group is located in the coenzyme binding site . This study shows that the physical and catalytic properties of P . stutzeri MDH strongly resemble those of the mitochondrial eukariotic enzyme . This finding strengthens the existing view that, in the evolution process, the mitochondrial MDH might have appeared before the cytoplasmic.

J Bacteriol, 1997 Jan, 179(1), 202 - 8
Purification and characterization of 2,4,6-trichlorophenol-4-monooxygenase, a dehalogenating enzyme from Azotobacter sp . strain GP1; Wieser M et al.; The enzyme which catalyzes the dehalogenation of 2,4,6-trichlorophenol (TCP) was purified to apparent homogeneity from an extract of TCP-induced cells of Azotobacter sp . strain GP1 . The initial step of TCP degradation in this bacterium is inducible by TCP; no activity was found in succinate-grown cells or in phenol-induced cells . NADH, flavin adenine dinucleotide, and O2 are required as cofactors . As reaction products, 2,6-dichlorohydroquinone and Cl- ions were identified . Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of O2 per mol of TCP and the formation of 1 mol of Cl- ions . No evidence for membrane association or for a multicomponent system was obtained . Molecular masses of 240 kDa for the native enzyme and 60 kDa for the subunit were determined, indicating a homotetrameric structure . Cross-linking studies with dimethylsuberimidate were consistent with this finding . TCP was the best substrate for 2,4,6-trichlorophenol-4-monooxygenase (TCP-4-monooxygenase) . The majority of other chlorophenols converted by the enzyme bear a chloro substituent in the 4-position . 2,6-Dichlorophenol, also accepted as a substrate, was hydroxylated in the 4-position to 2,6-dichlorohydroquinone in a nondehalogenating reaction . NADH and O2 were consumed by the pure enzyme also in the absence of TCP with simultaneous production of H2O2 . The NH2-terminal amino acid sequence of TCP-4-monooxygenase from Azotobacter sp . strain GP1 revealed complete identity with the nucleotide-derived sequence from the analogous enzyme from Pseudomonas pickettii and a high degree of homology with two nondehalogenating monooxygenases . The similarity in enzyme properties and the possible evolutionary relatedness of dehalogenating and nondehalogenating monooxygenases are discussed.

Appl Environ Microbiol, 1997 Jan, 63(1), 282 - 8
Comparative analysis of Pseudomonas syringae pv . actinidiae and pv . phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences; Sawada H et al.; Pseudomonas syringae pv . phaseolicola, which causes halo blight on various legumes, and pv . actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin . We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index . It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P . syringae pv . phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P . syringae pv . phaseolicola . Moreover, the kiwifruit and tara vine isolates of P . syringae pv . actinidiae were also found to possess the identical argK . On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P . syringae pv . actinidiae and pv . phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development . The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P . syringae pv . actinidiae and/or pv . phaseolicola.

Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15497 - 502
Molecular recognition of pathogen attack occurs inside of plant cells in plant disease resistance specified by the Arabidopsis genes RPS2 and RPM1; Leister RT et al.; The Arabidopsis thaliana disease resistance genes RPS2 and RPM1 belong to a class of plant disease resistance genes that encode proteins that contain an N-terminal tripartite nucleotide binding site (NBS) and a C-terminal tandem array of leucine-rich repeats . RPS2 and RPM1 confer resistance to strains of the bacterial phytopathogen Pseudomonas syringae carrying the avirulence genes avrRpt2 and avrB, respectively . In these gene-for-gene relationships, it has been proposed that pathogen avirulence genes generate specific ligands that are recognized by cognate receptors encoded by the corresponding plant resistance genes . To test this hypothesis, it is crucial to know the site of the potential molecular recognition . Mutational analysis of RPS2 protein and in vitro translation/translocation studies indicated that RPS2 protein is localized in the plant cytoplasm . To determine whether avirulence gene products themselves are the ligands for resistance proteins, we expressed the avrRpt2 and avrB genes directly in plant cell using a novel quantitative transient expression assay, and found that expression of avrRpt2 and avrB elicited a resistance response in plants carrying the corresponding resistance genes . This observation indicates that no bacterial factors other than the avirulence gene products are required for the specific resistance response as long as the avirulence gene products are correctly localized . We propose that molecular recognition of P . syringae in RPS2- and RPM1-specified resistance occurs inside of plant cells.

J Biol Chem, 1996 Dec 20, 271(51), 32803 - 9
Purification and characterization of a membrane-bound deglycating enzyme (1-deoxyfructosyl alkyl amino acid oxidase, EC 1.5.3) from a Pseudomonas sp . soil strain; Saxena AK et al.; Searching for novel approaches for uncoupling glycation from hyperglycemia as a cause of diabetic complications, a Pseudomonas sp . soil strain containing a membrane-bound enzyme that deglycates amino acids under release of free fructosamine was isolated (Gerhardinger, C., Marion, S . M., Rovner, A., Glomb, M., and Monnier, V . M . (1995) J . Biol . Chem . 270, 218-224) . This enzymatic activity was found to be very sensitive to inactivation by most detergents . From the plasma membrane ( approximately 3 mg/ml protein concentration), the enzyme could be solubilized in active form using 10 mM 3-{(3-chlolamidopropyl) dimethylammonio}-2-hydroxy-1-propanesulfonate aided by 2 M NaCl and 10% glycerol (27% optimal solubilization yield) . The supernatant from a 55% saturation (NH4)2SO4 cut was fractionated onto a phenyl-Superose HR 5/5 column and enzymatic activity was eluted with a inverse gradient of (NH4)2SO4 . Following removal of (NH4)2SO4 with PD-10 columns and fractionation with a Mono Q HR 5/5 column, a sharp peak of enzyme activity was eluted . Analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major band at 106 kDa and, on isoelectrofocusing gel, a pI of 5.1 . The activity was completely inhibited by CN- and N3-, suggestive of copper as a likely cofactor . Identification of the protein was confirmed by affinity labeling with 14CN- and isoelectrofocusing . The "amadoriase" activity was also inhibited by Hg2+, Ag2+, Cu2+, and Zn2+ and had Km and Vmax values of 0.14 mM and 0.48 unit/ml (16 units/mg of protein), respectively, for epsilon-(1-deoxyfructosyl) aminocaproate . Significant activity was noted toward many glycated amino acids (highest with epsilon-fructosyl lysine) but not with glycated proteins . The sequence of the first 16 NH2-terminal amino acids and a search in various data bases revealed that this amadoriase enzyme is a novel protein . Based on its properties, this deglycating enzyme, which degrades Amadori products oxidatively into free fructosamine, is classified as fructosyl aminocaproate:oxygen oxidoreductase (EC 1.5.3).

Science, 1996 Dec 20, 274(5295), 2063 - 5
Molecular Basis of Gene-for-Gene Specificity in Bacterial Speck Disease of Tomato
Scofield SR, Tobias CM, Rathjen JP, Chang JH, Lavelle DT, Michelmore RW, Staskawicz BJ.
Transient expression of the Pseudomonas syringae avirulence gene avrPto in plant cells resulted in a Pto-dependent necrosis . The AvrPto avirulence protein was observed to interact directly with the Pto resistance protein in the yeast two-hybrid system . Mutations in the Pto and avrPto genes which reduce in vivo activity had parallel effects on association in the two-hybrid assay . These data suggest that during infection the pathogen delivers AvrPto into the plant host cell and that resistance is specified by direct interaction of Pto with AvrPto.

Science, 1996 Dec 20, 274(5295), 2060 - 3
Initiation of Plant Disease Resistance by Physical Interaction of AvrPto and Pto Kinase
Tang X, Frederick RD, Zhou J, Halterman DA, Jia Y, Martin GB.
Resistance to bacterial speck disease in tomato occurs when the Pto kinase in the plant responds to expression of the avirulence gene avrPto in the Pseudomonas pathogen . Transient expression of an avrPto transgene in plant cells containing Pto elicited a defense response . In the yeast two-hybrid system, the Pto kinase physically interacted with AvrPto . Alterations of AvrPto or Pto that disrupted the interaction in yeast also abolished disease resistance in plants . The physical interaction of AvrPto and Pto provides an explanation of gene-for-gene specificity in bacterial speck disease resistance.

Cancer Res, 1996 Dec 15, 56(24), 5631 - 7
Preclinical development of a recombinant toxin containing circularly permuted interleukin 4 and truncated Pseudomonas exotoxin for therapy of malignant astrocytoma; Puri RK et al.; Effective treatment is lacking for malignant glioblastoma/astrocytoma . We have identified interleukin-4 receptors (IL-4R) on human malignant astrocytoma . We demonstrate that 16 of 21 surgical samples of high-grade astrocytoma and glioblastoma but not normal brain tissues expressed IL-4R as assessed by reverse transcriptase PCR . We further demonstrate that human malignant astrocytoma cell lines express high-affinity IL-4R . Using a chimeric protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin A, we observed that this toxin IL4(38-37)-PE38KDEL) is highly cytotoxic to IL-4R-bearing glioblastoma cells . Compared with a previously reported IL4-PE chimeric protein (IL-PE4E), IL4(38-37)-PE38KDEL bound with higher affinity and was 3-30-fold more cytotoxic to glioblastoma cell lines . Upon intrathecal administration in monkeys, high cerebrospinal fluid IL4(38-37)-PE38KDEL levels were achieved using 2- and 6-microg/kg doses without any central nervous system or other abnormalities . IL4(38-37)-PE38KDEL levels were not detectable in the serum of any monkey studied . When IL4(38-37)-PE38KDEL was injected into the right frontal cortex of rats, localized necrosis was observed at 1000-ng/ml doses but not at < or = 100-ng/ml doses . We conclude that by localized administration, nontoxic levels of IL4(38-37)-PE38KDEL can be achieved, which may have significant cytotoxic activity against malignant astrocytoma.

Gene, 1996 Dec 12, 183(1-2), 167 - 73
Characterisation of genes involved in biosynthesis of coronafacic acid, the polyketide component of the phytotoxin coronatine; Penfold CN et al.; Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant pathogen, Pseudomonas syringae . In the present study we have determined the nucleotide sequence of a 3.92-kb DNA fragment involved in CFA biosynthesis . Analysis of the sequence revealed four complete open reading frames (ORFs) designated cfa1 to cfa4 and one incomplete ORF (cfa5), all transcribed in the same direction . The predicted translation products of cfa1, cfa2 and cfa3 showed relatedness to acyl carrier proteins, fatty acid dehydrases and beta-ketoacylsynthases, respectively, which are required for polyketide synthesis . cfa1 was subcloned, its sequence was confirmed, and it was overexpressed in E . coli to yield a peptide with an apparent molecular mass of 6 kDa.

Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14972 - 7
Isolation and characterization of a tobacco mosaic virus-inducible myb oncogene homolog from tobacco; Yang Y et al.; Salicylic acid (SA) plays an important role in signaling the activation of plant defense responses against pathogen attack including induction of pathogenesis-related (PR) proteins . To gain further insight into the SA-mediated signal transduction pathway, we have isolated and characterized a tobacco mosaic virus (TMV)-inducible myb oncogene homolog (myb1) from tobacco . The myb1 gene was induced upon TMV infection during both the hypersensitive response and development of systemic acquired resistance in the resistant tobacco cultivar following the rise of endogenous SA, but was not activated in the susceptible cultivar that fails to accumulate SA . The myb1 gene was also induced by incompatible bacterial pathogen Pseudomonas syringae pv . syringae during the hypersensitive response . Exogenous SA treatment rapidly (within 15 min) activated the expression of myb1 in both resistant and susceptible tobacco cultivars with the subsequent induction of PR genes occurring several hours later . Biologically active analogs of SA and 2,6-dichloroisonicotinic acid (a synthetic functional analog of SA), which induce PR genes and enhanced resistance, also activated the myb1 gene . In contrast, biologically inactive analogs were poor inducers of myb1 gene expression . Furthermore, the recombinant Myb1 protein was shown to specifically bind to a Myb-binding consensus sequence found in the promoter of the PR-1a gene . Taken together, these results suggest that the tobacco myb1 gene encodes a signaling component down-stream of SA that may participate in transcriptional activation of PR genes and plant disease resistance.

Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14815 - 20
Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: targeting with a single chain antibody variable domain isolated by phage display; Lorimer IA et al.; EGFRvIII is a mutant epidermal growth factor receptor found in glioblastoma, and in carcinoma of the breast, ovary, and lung . The mutant receptor has a deletion in its extracellular domain that results in the formation of a new, tumor-specific extracellular sequence . Mice were immunized with a synthetic peptide corresponding to this sequence and purified EGFRvIII . A single chain antibody variable domain (scFv) phage display library of 8 x 10(6) members was made from the spleen of one immunized mouse . A scFv specific for EGFRvIII was isolated from this library by panning with successively decreasing amounts of synthetic peptide . This was used to make an immunotoxin by fusing the scFv DNA sequence to sequences coding for domains II and III of Pseudomonas exotoxin A . Purified immunotoxin had a Kd of 22 nM for peptide and a Kd of 11 nM for cell-surface EGFRvIII . The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC50 of 1 ng/ml (16 pM) on mouse fibroblasts transfected with EGFRvIII and an IC50 of 7-10 ng/ml (110-160 pM) on transfected glioblastoma cells . There was no cytotoxic activity at 1000 ng/ml on the untransfected parent glioblastoma cell line . The immunotoxin was completely stable upon incubation at 37 degrees C for 24 h in human serum . The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinical evaluation.

J Mol Graph, 1996 Dec, 14(6), 338 - 40, 369-73
Prediction of high-frequency electron paramagnetic resonance spectra of spin S = 3/2, 5/2 systems; Wu H; By the use of the universal EPR simulation program created by the author, spin S = 3/2 and S = 5/2 systems are studied and their simulated EPR spectra at high frequencies (Q-band for 35 GHz and W-band for 95 GHz) are presented here . The mononuclear Fe3+ in rubredoxin, isolated from Pseudomonas oleovorans (which is an S = 5/2 system with D = 1.76 cm-1 and E/D = 0.28), is extensively studied by EPR spectrum simulation at the Q-band, W-band, and "Z"-band . The molybdenum- and iron-containing protein (MoFe protein), which has g values at g = 4.32, 3.65, and 2.01 (S = 3/2, D = 6.0 cm-1, and E/D = 0.055) at the X-band, is also studied by EPR spectrum simulation at high frequencies.

Antimicrob Agents Chemother, 1996 Dec, 40(12), 2710 - 3
In vitro antifungal and fungicidal activities and erythrocyte toxicities of cyclic lipodepsinonapeptides produced by Pseudomonas syringae pv . syringae; Sorensen KN et al.; Recent increases in fungal infections, the few available antifungal drugs, and increasing fungal resistance to the available antifungal drugs have resulted in a broadening of the search for new antifungal agents . Strains of Pseudomonas syringae pv . syringae produce cyclic lipodepsinonapeptides with antifungal activity . The in vitro antifungal and fungicidal activities of three cyclic lipodepsinonapeptides (syringomycin E, syringotoxin B, and syringostatin A) against medically important isolates were evaluated by a standard broth microdilution susceptibility method . Erythrocyte toxicities were also evaluated . All three compounds showed broad antifungal activities and fungicidal actions against most of the fungi tested . Overall, the cyclic lipodepsinonapeptides were more effective against yeasts than against the filamentous fungi . Syringomycin E and syringostatin A had very similar antifungal activities (2.5 to > 40 micrograms/ml) and erythrocyte toxicities . Syringotoxin B was generally less active (0.8 to 200 micrograms/ml) than syringomycin E and syringostatin A against most fungi and was less toxic to erythrocytes . With opportunities for modification, these compounds are potential lead compounds for improved antifungal agents.

Gene, 1996 Nov 28, 181(1-2), 57 - 61
Cloning and sequencing of the genes encoding 2-nitrotoluene dioxygenase from Pseudomonas sp . JS42; Parales JV et al.; The first step in the metabolism of 2-nitrotoluene (2NT) by Pseudomonas sp . JS42 (JS42) is the addition of dioxygen to the aromatic nucleus of 2NT to form 3-methylcatechol with concomitant release of nitrite . This reaction is catalyzed by the three-component dioxygenase system 2-nitrotoluene 2,3-dioxygenase (2NTDO) . We report here the cloning and nucleotide (nt) sequence of a 4912-basepair (bp) SacI DNA fragment from JS42 encoding all of the genes required for 2NTDO activity . Sequence analysis of the 4912-bp SacI DNA fragment revealed five open reading frames (ORFs) . The amino acid (aa) sequences of the predicted polypeptides from these ORFs exhibit high homology to the aa sequences of polypeptides from other three-component dioxygenase systems . Based on aa sequence analyses, four of the peptides were designated Reductase2NT, Ferredoxin2NT, ISP alpha 2NT and ISP beta 2NT (ISP for iron-sulfur protein) with gene designations ntdAaAbAcAd . The predicted aa sequence from the remaining ORF (ORF2) had identity to ISP alpha subunits from other three-component dioxygenase systems but had a calculated molecular weight (M(r)) of 21,259, which is uncharacteristically small for ISP alpha subunits.

Transplantation, 1996 Nov 27, 62(10), 1441 - 50
Cytokine pattern during rejection and infection after liver transplantation--improvements in postoperative monitoring?
Platz KP, Mueller AR, Rossaint R, Steinmuller T, Lemmens HP, Lobeck H, Neuhaus P.
Despite improvements in immunosuppression, rejection occurs in 50% of liver transplant patients and may cause significant morbidity . The most frequent cause of death after liver transplantation is severe infection . Determination of the cytokine network may lead to earlier detection of patients at risk for severe rejection and infection . For this purpose, 81 patients with 85 liver transplants were monitored for cytokines and neopterin on a daily basis . During the first postoperative month, 28 patients (34.6%) developed acute rejection; 14 patients were successfully treated with methylprednisolone (steroid-sensitive rejection), while 14 patients required additional treatment with FK506 and OKT3 (steroid-resistant rejection) . Ten patients developed severe infections, and 11 patients experienced asymptomatic cholangitis . Patients with an uneventful postoperative course (n=37) were the control group . One-year patient survival was 88.9%: 1 patient died because of chronic rejection and Pseudomonas urosepsis; a further 4 patients died of aspergillus pneumonia and bacterial sepsis . Soluble TNF-RII, sIL-2R-, and IL-10 levels were significantly elevated 3 days prior to or at the onset of acute steroid-resistant rejection (P < or = 0.01 versus steroid-sensitive rejection and on uneventful postoperative course) . An increase in IL-8, neopterin, and sTNF-RII was indicative of severe infection 3 days prior to onset of infection . In this group of patients, a simultaneous increase in IL-10 indicated a lethal outcome of severe infection . During the second week of acute steroid-resistant rejection and lethal infection, a significant rise in IL-1beta, IFN-gamma, and IL-6 was observed (P < or = 0.01 versus control groups) . The different patterns in neopterin- and cytokine-increase could differentiate between severe rejection and severe infection . Furthermore, the increase in these parameters indicated severe rejection--i.e., steroid resistance at the onset of acute rejection--which could prompt us to initiate rescue therapy immediately . The ability to detect patients at risk for severe or lethal infection may result in intensified infectious screening and more aggressive antiinfectious treatment . Therefore, routine monitoring of these parameters may lead to changes in therapeutic management of severe acute rejection and infection after liver transplantation.

Lett Appl Microbiol, 1996 Nov, 23(5), 299 - 302
Aerobic degradation of 2-sulphobenzoic acid by Pseudomonas strain SB(W); Goncalves JA et al.; A 2-sulphobenzoic acid-degrading bacterium, Pseudomonas sp . strain SB(W), was isolated from creosote-contaminated soil . It used this compound as its sole carbon, sulphur and energy source, and gave a nearly stoichiometric release of sulphate from 2-sulphobenzoic acid . It did not grow on 3- to 4- sulphobenzoic acids . Isolated SB(W) produced two transient metabolites . The first to appear, and the more abundant metabolite, was identified as 2,3-dihydroxybenzoic acid . The second metabolite was identified as salicylic acid . Both of these compounds served as growth substrates for the isolates.

Plant Cell, 1996 Nov, 8(11), 2033 - 46
Characterization of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes; Parker JE et al.; The interaction between Arabidopsis and the biotrophic oomycete Peronospora parasitica (downy mildew) provides an attractive model pathosystem to identify molecular components of the host that are required for genotype-specific recognition of the parasite . These components are the so-called RPP genes (for resistance to P . parasitica) . Mutational analysis of the ecotype Wassilewskija (Ws-0) revealed an RPP-nonspecific locus called EDS1 (for enhanced disease susceptibility) that is required for the function of RPP genes on chromosomes 3 (RPP1/RPP14 and RPP10) and 4 (RPP12) . Genetic analyses demonstrated that the eds1 mutation is recessive and is not a defective allele of any known RPP gene, mapping to the bottom arm of chromosome 3 (approximately 13 centimorgans below RPP1/RPP14) . Phenotypically, the Ws-eds1 mutant seedlings supported heavy sporulation by P . parasitica isolates that are each diagnostic for one of the RPP genes in wild-type Ws-0; none of the isolates is capable of sporulating on wild-type Ws-0 . Ws-eds1 seedlings exhibited enhanced susceptibility to some P . parasitica isolates when compared with a compatible wild-type ecotype, Columbia, and the eds1 parental ecotype, Ws-0 . This was observed as earlier initiation of sporulation and elevated production of conidiosporangia . Surprisingly, cotyledons of Ws-eds1 also supported low sporulation by five isolates of P . parasitica from Brassica oleracea . These isolates were unable to sporulate on > 100 ecotypes of Arabidopsis, including wild-type Ws-0 . An isolate of Albugo candida (white blister) from B . oleracea also sporulated on Ws-eds1, but the mutant exhibited no alteration in phenotype when inoculated with several oomycete isolates from other host species . The bacterial resistance gene RPM1, conferring specific recognition of the avirulence gene avrB from Pseudomonas syringae pv glycinea, was not compromised in Ws-eds1 plants . The mutant also retained full responsiveness to the chemical inducer of systemic acquired resistance, 2,6-dichloroisonicotinic acid; Ws-eds1 seedlings treated with 2,6-dichloroisonicotinic acid became resistant to the Ws-0-compatible and Ws-0-incompatible P . parasitica isolates Emwa1 and Noco2, respectively . In summary, the EDS1 gene appears to be a necessary component of the resistance response specified by several RPP genes and is likely to function upstream from the convergence of disease resistance pathways in Arabidopsis.

Mol Microbiol, 1996 Nov, 22(4), 769 - 78
Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine-rich loop of diphtheria toxin; Chiron MF et al.; To be toxic for mammalian cells, Pseudomonas exotoxin (PE) requires proteolytic cleavage between Arg-279 and Gly-280 . Cleavage, which is mediated by the cellular protease furin, generates an active C-terminal fragment which translocates to the cytosol and inhibits protein synthesis . In vitro, furin-mediated cleavage is optimal at pH 5.5 with a relatively slow turnover rate . Within cells, only 5-10% of cell-associated PE is cleaved . To investigate the reasons for this inefficient cleavage, the amino acid composition near the cleavage site was altered to resemble more closely the arginine-rich sequence from the functionally similar region of diphtheria toxin (DT) . Four PE-DT mutants were generated, whereby 1, 5, 6 or 8 amino acids at the PE-cleavage site were changed to amino acids found at the DT-cleavage site . Mutant proteins were expressed in Escherichia coli, purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to cell-mediated cleavage, and cytotoxic activity relative to wild-type PE . At pH 5.5, the rate of both furin-mediated cleavage and trypsin-mediated cleavage increased dramatically when amino acids in PE were altered to resemble the DT sequence . This increase did not alter the pH optimum for furin-mediated cleavage of PE toxins, which remained at pH 5.0-5.5 . When radioactive versions of selected PE-DT proteins were added to intact cells, an increase in the percentage of molecules that were cleaved relative to wild-type PE was also seen . However, changes that favoured increased proteolysis apparently interfered with other important toxin functions because none of the PE-DT proteins exhibited enhanced toxicity for cells when compared with the activity of wild-type PE.

J Appl Bacteriol, 1996 Nov, 81(5), 531 - 7
Bioassay methods for the detection of antifungal activity by Pseudomonas antimicrobica against the grey mould pathogen Botrytis cinerea; Walker R et al.; Antagonism against the grey mould pathogen Botrytis cinerea by Pseudomonas antimicrobica was demonstrated in vitro and in vivo . Cell-free filtrates showed activity against B . cinerea growing on Potato Dextrose Agar (PDA) in a media-dependent manner with the most distinct antagonism being produced in Czapek Dox Broth (CDB) . Cell-free filtrates of CDB-grown cultures also significantly reduced conidial germination of B . cinerea . An assay based on the inhibition of conidial germination was compared with two assays measuring the antagonism of mycelial growth on PDA . The conidial germination bioassay was more sensitive in the detection of this antifungal activity than the Petri dish bioassay while a bioassay using Microdetection plates did not detect antagonism due to the small loading capacity of the latter . The conidial germination bioassay was modified for detection of antibiosis on the surface of strawberry leaves . Significant reductions in percentage conidial germination were recorded on the surface of leaves of both micropropagated and glasshouse grown strawberry plants when the antifungal compounds of Ps . antimicrobica were applied to the leaf tissue with the conidia . In addition, antifungal compounds were also detectable when conidia were applied to leaf tissue which had previously been sprayed with cells of Ps . antimicrobica . These tests indicate that Ps . antimicrobica would be a suitable biocontrol agent for the control of B . cinerea.

J Bacteriol, 1996 Nov, 178(22), 6459 - 65
Immunochemical characterization of O polysaccharides composing the alpha-D-rhamnose backbone of lipopolysaccharide of Pseudomonas syringae and classification of bacteria into serogroups O1 and O2 with monoclonal antibodies; Ovod V et al.; Murine monoclonal antibodies (MAbs) reacting with Pseudomonas syringae lipopolysaccharide (LPS) O polysaccharides (OPS) composed of tetra- and tri-alpha-D-rhamnose repeats in the backbone {3)D-Rha(alpha1-3)D-Rha(alpha1-2)D-Rha(alpha1-2)D-Rha(alpha1} and {3)D-Rha(alpha1-3)D-Rha(alpha1-2)D-Rha(alpha1} were generated and used for immunochemical analysis and for serological classification of the bacteria . A total of 195 of 358 P . syringae strains tested representing 21 pathovars were shown to share a common epitope, 1a, and were classified into serogroup O1 . All strains with pathovars aptata, glycinea, japonica, phaseolicola, and pisi, most of the strains with pathovars atrofaciens and striafaciens, and half of the strains with pathovar syringae were classified into serotypes O1a', O1b, O1c, and O1d within serogroup O1 . Serogroup-specific epitope 1a was inferred to be related to the (alpha1-2)D-Rha(alpha1-3) site of the OPS backbone . The serotype-specific epitopes 1b, 1c, 1d, and 1a' were inferred as relating to the immunodominant lateral (alpha1-3)D-Rha, (beta1-4)D-GlcNAc, and (alpha1-4)D-Fuc substituents and backbone-located site (alpha1-3)D-Rha(alpha1-2), respectively, of OPSs that share the common tetra-D-rhamnose repeats in the backbone . A total of 7.3% of the strains studied, all with pathovars morsprunorum and lapsa, were classified as serotypes O2a and O2d within serogroup 02 . Serotype-specific epitope 2a was inferred as being related to the backbone-located site D-Rha(alpha1-3)D-Rha and epitope 2d to the immunodominant lateral (alpha1-4)D-Fuc residue of OPS consisting of tri-D-rhamnose repeats in the backbone . Epitope 2d alternated with 2a within the same LPS molecule and did not cross-react with epitope 1d . Serotypes O2a and O2d were observed in some strains correlating with the coexpression of the two chemotypes of OPS by the same strain . The serogroup O1-specific MAb Ps1a reacted weakly but definitely with all strains from serogroup 02 . We propose serological formulas for serogroups O1 and 02 as well as for individual strains within these serogroups.

Appl Environ Microbiol, 1996 Nov, 62(11), 4073 - 80
Regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene by naphthalene dioxygenase from Pseudomonas sp . strain NCIB 9816-4; Resnick SM et al.; The regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas sp . strain NCIB 9816-4 . The initial oxidation products were isolated and identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry . Salicylate-induced cells of Pseudomonas sp . strain 9816/11 and isopropyl-beta-D-thiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized fluorene to (+)-(3S,4R)-cis-3,4-dihydroxy-3,4-dihydrofluorene (80 to 90% relative yield; > 95% enantiomeric excess {ee}) and 9-fluorenol (< 10% yield) . The same cells oxidized dibenzofuran to (1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzofuran (60 to 70% yield; > 95% ee) and (3S,4R)-cis-3, 4-dihydroxy-3,4-dihydrodibenzofuran (30 to 40% yield; > 95% ee) . Induced cells of both strains, as well as the purified dioxygenase, also oxidized dibenzothiophene to (+)-(1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzothiophene (84 to 87% yield; > 95% ee) and dibenzothiophene sulfoxide (< 15% yield) . The major reaction catalyzed by naphthalene dioxygenase with each substrate was stereospecific dihydroxylation in which the cis-dihydrodiols were of identical regiochemistry and of R configuration at the benzylic center adjacent to the bridgehead carbon atom . The regiospecific oxidation of dibenzofuran differed from that of the other substrates in that a significant amount of the minor cis-3,4-dihydrodiol regioisomer was formed . The results indicate that although the absolute stereochemistry of the cis-diene diols was the same, the nature of the bridging atom or heteroatom influenced the regiospecificity of the reactions catalyzed by naphthalene dioxygenase.

Cornea, 1996 Nov, 15(6), 566 - 70
Trends in contact lens-associated corneal ulcers; Cohen EJ et al.; Charts of 320 patients with corneal ulcers seen on the Cornea Service of Wills Eye Hospital from July 1, 1992, to June 30, 1995, were reviewed retrospectively . Of these cases, 96 (30%) were associated with contact lens use . Ulcers in contact lens users accounted for 36% of cases in the last 6 months of 1992 and all of 1993, 20% of cases in 1994, and 29% in the first 6 months of 1995 . The contact lenses most commonly associated with ulcers were disposable extended-wear lenses . They were used in 33% of contact lens-associated ulcers in 1992, 27% in 1993, 39% in 1994, and 44% in 1995 . Pseudomonas was the predominant organism prior to 1993 (1-4) . From 1993 to 1995, however, the number of Pseudomonas ulcers steadily decreased . Two or three Acanthamoeba infections continue to be treated each year . There has been a significant decrease in the number of contact lens-related ulcers treated at our institution compared with previous years (p < 0.01) (3, 4).

J Bacteriol, 1996 Nov, 178(21), 6399 - 402
The Pseudomonas syringae Hrp regulation and secretion system controls the production and secretion of multiple extracellular proteins; Yuan J et al.; Pseudomonas syringae pv . tomato DC3000 produces seven to eight major extracellular proteins (EXPs) in a minimal medium inducing hrp genes . Using a polyclonal antibody against DC3000 EXPs, we have determined that the production and secretion of five EXPs (EXP-60, EXP-45, EXP-43, EXP-22, and EXP-10) are under the control of the Hrp regulation and secretion system.

J Bacteriol, 1996 Nov, 178(21), 6288 - 95
Two types of novel dipeptidyl aminopeptidases from Pseudomonas sp . strain WO24; Ogasawara W et al.; Two kinds of dipeptidyl aminopeptidase I (DAP I {cathepsin C})-like activities which hydrolyze Gly-Phe-p-nitroanilide (Gly-Phe-pNA) were detected in Pseudomonas sp . strain WO24 . They were purified and characterized . The isolated enzymes, named DAP BII and DAP BIII, were revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing . DAP BII was estimated to have a molecular mass of 150,000 Da by gel filtration and a subunit size of 73,000 Da by SDS-PAGE, indicating it to be a homodimer . The molecular mass of DAP BIII was evaluated to be approximately 60,000 Da by gel filtration and 69,000 Da by SDS-PAGE, indicating that it is monomeric . The isoelectric points of DAP BII and DAP BIII were 6.1 and 5.0, and their optimal pHs were 8.0 and 8.5 to 9.0, respectively . The result of peptide mapping for DAP BII and DAP BIII showed that these enzymes consist of different components . Both enzymes were completely inhibited by diisopropylphosphofluoride but not by general thiol inhibitors, indicating that they are serine proteases . DAP BII and DAP BIII hydrolyzed Gly-Phe-pNA but not Gly-Arg-pNA, both of which are model substrates for mammalian DAP I . Despite these shared activities toward DAP I, DAP BII released dipeptides from Ala-Ala-pNA and Lys-Ala-4-methylcoumarinamide (a substrate for DAP II), whereas DAP BIII did not hydrolyze either of these compounds and was presumed to prefer substrates composed of bulky, hydrophobic amino acids at P1 and P1' positions . In addition, DAP BII showed no endopeptidase activity, whereas DAP BIII possessed the activity on N-terminally blocked peptide derivatives besides exopeptidase activity . Assays performed with bioactive peptides such as angiotensin I and neuromedin N as substrates indicate that DAP BII has a considerably broader substrate specificity than DAP BIII and is able to hydrolyze an X-Pro bond, an imido bond that few peptidases and no known DAPs can cleave . These characteristics, namely, substrate specificities, molecular mass, pI, peptide mapping, pH optimum, and effect of inhibitors, suggested that the two DAPs purified in this work are distinct enzymes and do not belong to any of the previously reported DAP classes.

J Bacteriol, 1996 Nov, 178(21), 6227 - 32
2-aminophenol 1,6-dioxygenase: a novel aromatic ring cleavage enzyme purified from Pseudomonas pseudoalcaligenes JS45; Lendenmann U et al.; Most bacterial pathways for the degradation of aromatic compounds involve introduction of two hydroxyl groups either ortho or para to each other . Ring fission then occurs at the bond adjacent to one of the hydroxyl groups . In contrast, 2-aminophenol is cleaved to 2-aminomuconic acid semialdehyde in the nitrobenzene-degrading strain Pseudomonas pseudoalcaligenes JS45 . To examine the relationship between this enzyme and other dioxygenases, 2-aminophenol 1,6-dioxygenase has been purified by ethanol precipitation, gel filtration, and ion exchange chromatography . The molecular mass determined by gel filtration was 140,000 Da . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two subunits of 35,000 and 39,000 Da, which suggested an alpha2beta2 subunit structure . Studies with inhibitors indicated that ferrous iron was the sole cofactor . The Km values for 2-aminophenol and oxygen were 4.2 and 710 microM, respectively . The enzyme catalyzed the oxidation of catechol, 6-amino-m-cresol, 2-amino-m-cresol, and 2-amino-4-chlorophenol . 3-Hydroxyanthranilate, protocatechuate, gentisate, and 3- and 4-methylcatechol were not substrates . The substrate range and the subunit structure are unique among those of the known ring cleavage dioxygenases.

Leukemia, 1996 Nov, 10(11), 1796 - 803
Treatment of acute myelocytic leukemia with interleukin-6 Pseudomonas exotoxin fusion protein in a rat leukemia model; Rozemuller H et al.; We studied the applicability of interleukin-6 Pseudomonas exotoxin fusion protein (IL-6PE4E) for treatment of acute myelocytic leukemia (AML) . Leukemic cells from five out of 10 AML patients studied expressed IL-6 receptor (IL-6R) and proliferation in vitro was inhibited in four of these cases . The potential of this approach in vivo was tested in a pre-clinical model for AML; the Brown Norway acute myelocytic leukemia (BNML) . To obtain IL-6R expression levels on BNML cells comparable to the numbers expressed on human AML, human IL-6R gene transfectants of the BNML sub-line LT12 (LT12/IL-6R) were generated . IL-6PE4E is cytotoxic in vitro to LT12/IL-6R expressing 1400 high affinity IL-6R per cell with 50% inhibition of DNA synthesis at 1 ng/ml . In vivo treatment of leukemic rats carrying LT12/IL-6R leukemia indicated that the maximal tolerated dose of IL-6PE4E was 275 +/- 25 microg/kg/day, when continuously administered for 7 days and resulted in a 90% reduction in leukemic cell load . At this dose level of IL-6PE4E no reduction of normal hemopoietic progenitors was seen in non-leukemic rats . At higher dose levels (350-1050 microg/kg/day) severe systemic toxicity was encountered . On the basis of these pre-clinical studies the feasibility of growth factor-toxins for selective in vivo targeting to AML cells is evaluated.

Mol Plant Microbe Interact, 1996 Nov, 9(8), 713 - 9
Defense reaction in Medicago sativa: a gene encoding a class 10 PR protein is expressed in vascular bundles; Breda C et al.; Infiltration of Medicago sativa leaves with a suspension of Pseudomonas syringae pv . pisi elicits the accumulation of several mRNA classes . A clone, designated as MsPR10-1, encoding a polypeptide exhibiting strong similarity to the class 10 PR protein was isolated and characterized from a cDNA library prepared from leaf mRNA . The corresponding gene was shown to be developmentally regulated: Except in roots, its expression was not detectable in other analyzed organs of healthy plants (hypocotyls, cotyledons, stems, leaves, and flower buds) . MsPR10-1 transcript accumulation was especially high in leaf blades during an incompatible interaction: It was already detectable 3 h after infection, reached its maximum level 24 h postinfection, and remained at a high level over a period of at least 72 h . In addition, the expression of this gene was induced by salicylic acid treatment of the leaves . Southern hybridizations showed that this gene belongs to a multigene family . Using a 5' extension technique for cDNA, we demonstrated that during the incompatible interaction with P . syringae pv . pisi several genes or allelic variants of this class were expressed . Measurements of transcript accumulation in both the infiltrated and noninfiltrated zones by Northern and in situ hybridization allowed to demonstrate the "systemic" expression pattern of the MsPR10-1 . In situ hybridizations indicated that MsPR10-1 was expressed in the vascular bundles adjacent to and distant from the infection site.

Gene, 1996 Oct 24, 177(1-2), 77 - 81
Resistance to ultraviolet light in Pseudomonas syringae: sequence and functional analysis of the plasmid-encoded rulAB genes; Sundin GW et al.; The indigenous plasmids, pPSR1 and pPSR5, were each shown to confer resistance to ultraviolet light (UV) in Pseudomonas syringae (Ps) pv . syringae FF5 . The UV-resistance (UVR) determinant was subcloned from a cosmid library of pPSR1, and sequence analysis revealed the presence of two ORFs, designated rulAB which are homologous to the Escherichia coli umuDC mutagenic DNA repair systems and other plasmid-encoded UVR operons . Amino acid (aa) alignments indicated that RulAB are most closely related to the RumAB proteins from plasmid R391, sharing 40.5% and 48.6% aa identity with RumA and RumB, respectively . UV sensitivity assays with the cloned rulAB genes indicated that the expression of UVR in Ps required a functional recA gene.

J Biol Chem, 1996 Oct 18, 271(42), 26170 - 3
Translocation of full-length Pseudomonas exotoxin from endosomes is driven by ATP hydrolysis but requires prior exposure to acidic pH; Taupiac MP et al.; We attached human transferrin to Pseudomonas exotoxin A (PE) to specifically localize this toxin to the endosomal compartment and study its translocation from purified endosomes using a cell-free assay . Transferrin was linked to PE via a disulfide bond . Chemical derivatization inactivated the PE cell-binding domain, and transferrin-PE was found to be endocytosed via the transferrin receptor only . Transferrin was also conjugated to a truncated PE with no receptor-binding domain (PE46) . After labeling mouse lymphocytes with radiolabeled transferrin-PE or transferrin-PE46 and endosome isolation, selective translocation of the full-sized toxin portion of the conjugate was observed in a cell-free system . This translocation was strictly dependent upon ATP hydrolysis and was not affected when the acidity of the endosome lumen was neutralized using weak bases, protonophores, or bafilomycin A1 . Nevertheless, when present during cell labeling, inhibitors of endosome acidification prevented PE from acquiring translocation competence . Similar inhibition was observed when endocytosis was performed in the presence of brefeldin A, a drug known to interfere with the delivery of endocytic tracers to acidic endosomes . Our data indicate that full-length PE can be transferred to the cytosol directly from endosomes during intoxication by PE conjugates and that, although exposure to acidic pH is a prerequisite for translocation, ATP hydrolysis directly provides the energy required for PE translocation.

Gene, 1996 Oct 17, 176(1-2), 177 - 84
Cloning, nucleotide sequence and characterization of the rpoD gene encoding the primary sigma factor of Rhodobacter capsulatus; Pasternak C et al.; A synthetic oligodeoxynucleotide probe based on a highly conserved region of the sigma factors was used to identify and clone the rpoD gene encoding the principal sigma factor of R . capsulatus . The deduced polypeptide contains 674 amino acids and has a predicted molecular mass of 75,942 Da . The deduced amino acid sequence of R . capsulatus RpoD protein exhibits 46.2% and 45.7% identity with housekeeping sigma factors of Pseudomonas and E . coli, respectively . Unsuccessful attempts to inactivate the single chromosomal rpoD gene of R . capsulatus by gene replacement technique indicate that this gene is essential for cell survival, as expected for the primary sigma factor . The rpoD transcript 5'-end was mapped by primer extension analysis, 74 bp upstream of the initiation codon and DNA sequence analysis has identified a motif resembling the delta 70 E . coli consensus promoter sequences at the expected distance from this proposed transcription start site . rpoD gene expression, as measured by the activity of the phi (rpoD'-lacZ+) translational fusion, was found to be constant throughout exponential and early plateau phases, but significantly increased at later times of the stationary phase.

Eur J Biochem, 1996 Oct 15, 241(2), 691 - 6
Overproduction of a foreign membrane protein in Escherichia coli stimulates and depends on phospholipid synthesis; Nieboer M et al.; When the Pseudomonas oleovorans alk system, consisting of the alkBFGHJKL and alkST genes, is expressed in Escherichia coli W3110, significant changes in phospholipid metabolism of the host are observed . A major role seems to be played by the cytoplasmic membrane protein alkane hydroxylase (AlkB), which is synthesized as up to 10-15% of the total protein in this strain {Nieboer, M., Kingma, J . & Witholt, B . (1993) The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110{pGEc47} affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subfraction, Mol . Microbiol . 8, 1039-1051} . In the present paper, we have studied the link between synthesis of the membrane protein and the synthesis of phospholipids and fatty acids by examining the kinetics of these processes . Using {35S}methionine labeling, it was shown that induction of AlkB was maximal within 30-60 min after addition of inducer, when up to 15% of all newly synthesized protein is AlkB . Phospholipid synthesis was followed by measuring the incorporation of 14C-labeled acetate and 33P-labeled phosphoric acid into phospholipids . Despite a negative effect of the inducer on the growth rate of W3110{pGEc47}, net phospholipid synthesis was significantly enhanced as a result of the expression of alkB . Synthesis of all three major phospholipids were stimulated to comparable extents by the induction of alkB . Induction did not increase 33P incorporation into lipids in the control recombinant alk+ strain which lacked alkB . Simultaneous with AlkB synthesis, the conversion of unsaturated 9-hexadecenoic acid (C16:1) into 9,10-methylene hexadecanoic acid (C17:ocyc) was reduced in the alk+ recombinant . Overall, these data show that the production of a foreign membrane protein in E . coli can engender a response of the phospholipid-synthesizing system of the host . In the absence of such a response, induction of the alk system would be much more toxic to the cells . Apparently, the increased phospholipid synthesis plays an important role in enabling the AlkB overproducing strain to grow.

Biochemistry, 1996 Oct 15, 35(41), 13478 - 84
Determination of the chemical pathway for 4-chlorobenzoate:coenzyme A ligase catalysis; Chang KH et al.; 4-Chlorobenzoate:coenzyme A ligase (4-CBA:CoA ligase) catalyzes the first step of the 4-CBA degradation pathway of Pseudomonas sp . strain CBS3 . In this reaction, 4-CBA-CoA thioester synthesis is coupled to ATP cleavage . The studies described in this paper examine the intermediacy of 4-chlorobenzoyl-adenosine 5'phosphate diester (4-CBA-AMP) in the ligase reaction . The 4-CBA-AMP adduct was isolated from the ligase reaction mixture generated from magnesium adenosine 5-triphosphate (MgATP) and 4-CBA in the absence of CoA . The structure of the 4-CBA-AMP was verified by 1H- 13C-, and 31P-nuclear magnetic resonance analysis . Single-turnover reactions carried out with 14C-labeled 4-CBA in a rapid quench apparatus demonstrated formation of the enzyme . 4-CBA-AMP.MgPPi complex from the enzyme.4-CBA.MgATP complex at a rate of 135 s-1 . The rate of ligand release from the enzyme.4-CBA-AMP.MgPPi complex was measured at 0.013 s-1 . Single-turnover reactions of {14C}-4-CBA, MgATP, and CoA catalyzed by the ligase revealed that the 4-CBA-AMP intermediate formed reaches a maximum level of 25% of the starting 4-CBA within 10 ms and then declines with the formation of the 4-CBA-CoA . The rates of the adenylation and thioesterification partial reactions, determined by kinetic simulation of the rate data, are nearly equal (135 and 100 s-1) . Substitution of CoA with the slow substrate pantetheine did not significantly alter the rate of the adenylation step but did reduce the rate of the thioesterification step to 2 s-1 . The maximum level of 4-CBA-AMP reached during the single-turnover reaction of 4-CBA, MgATP, and pantetheine corresponded to one-half of the starting 4-CBA.

Mol Cell Biochem, 1996 Oct-Nov, 163-164, 291 - 303
Elucidating molecular mechanisms of septic cardiomyopathy--the cardiomyocyte model; Werdan K et al.; In the multiple organ dysfunction syndrome of sepsis and septic shock the heart is one of the organs subject to failure . Many new insights into the mechanisms underlying septic cardiomyopathy were gained in the last years . Experimental work with neonatal and adult cardiomyocytes considerably contributed to this progress, facilitating the documentation of direct attenuation of the contractions of the heart muscle cell by toxins and mediators, as well as investigating the underlying cellular mechanisms . With this respect, contractile-depressant effects have been found in cardiomyocytes for many toxins and sepsis mediators, with endotoxin, Pseudomonas exotoxin A, tumor necrosis factor alpha, interleukin-1 and nitric oxide being the most relevant ones identified . These substances interfere at clinically relevant concentrations with several main inotropic axes, not only with the beta-adrenoceptor/adenylyl cyclase and with the NO-cGMP-system-on which most of the interest is focused at present-but also with the alpha 1-adrenoceptor/phosphoinositide pathway and the Ca2+ homeostasis of the cardiomyocyte, the latter representing the common final inotropic pathway . Not a single cardiodepressant factor, but more likely a total bunch of toxins and mediators with different attack mechanisms seem to contribute to the picture of septic cardiomyopathy.

Int J Biol Macromol, 1996 Oct, 19(3), 177 - 83
Intracellular depolymerase and polyhydroxyoctanoate granule integrity in Pseudomonas oleovorans; Foster LJ et al.; When polyhydroxyoctanoate (PHO) was produced by Pseudomonas oleovorans during a regimen of intermittent feeding on octanoic acid, there was a significant change in both the polymer associated proteins and the composition of the enclosed polymer . The polymer granules were isolated with their protein coat intact and the enzymatic hydrolysis of the polymer within this cell free system was determined . The degradation rate for the PHO in these native granules reached a maximum of 1.17 mg/h at an optimum pH of 9 when incubated at 30 degrees C . A study of the effect of various inhibitors on depolymerase activity suggested that the enzyme most likely has disulfide linkages and serine residues at its active site . Ultrastructure studies suggested this loss of enzyme activity was correlated with significant organizational degeneration in the proteins associated with the PHO inclusion body . Once solubilized from the granule, the depolymerase itself remained enzymatically active, and addition of this released material to other granule preparations increased the rate of polymer granule degradation . Similarly, when colloidal suspensions of purified, amorphous PHO were placed in contact with that depolymerase, they also underwent rapid degradation . In contrast, when crystalline solvent-cast PHO films were placed in contact with this enzyme, no degradative activity was observed.

Int J Syst Bacteriol, 1996 Oct, 46(4), 1138 - 44
DNA relatedness among Pseudomonas strains isolated from natural mineral waters and proposal of Pseudomonas veronii sp . nov; Elomari M et al.; The taxonomic position of eight strains isolated from mineral water and previously grouped in the authentic pseudomonads on the basis of a phenotypic analysis (cluster Ib of M . Elomari, L . Coroler, D . Izard, and H . Leclerc {J . Appl . Bacteriol . 78:71-81, 1995}) has been further studied by DNA-DNA hybridizations . Using the S1 nuclease method at 60 degrees C and labeled reference DNA from a representative strain, CFML 92-134, we showed that members of cluster Ib constituted a homogeneous group with a relative binding ratio of greater than 80% and changes in melting temperature of less than 1 degree C . With a total of 67 strains representing known or partially characterized species of the genus Pseudomonas, only 4 to 47% DNA hybridization and changes in melting temperature of between 8 and 20 degrees C were found, the highest hybridization values being measured with members of the saprophytic fluorescent pseudomonads . Since cluster Ib could also be clearly differentiated from members of the latter group and from other phenotypic clusters containing isolates from mineral water, we designated the Ib strains members of a new Pseudomonas species for which the name Pseudomonas veronii sp . nov . has been proposed . Members of this species grew on alpha-aminobutyrate, sucrose, butyrate, isobutyrate, erythritol, L-tryptophan, and trigonelline as sole sources of carbon and energy . The average G+C content of the DNA of the eight strains of P . veronii was 61.5 +/- 0.5 mol% . The type strain is CFML 92-134T (CIP 104663T), with a G+C content of 61 mol% . The clinical significance of P . veronii is unknown.

FEMS Microbiol Lett, 1996 Oct 1, 143(2-3), 191 - 4
Poly(3-hydroxybutyrate) depolymerases bind to their substrate by a C-terminal located substrate binding site; Behrends A et al.; Binding of (i) purified wild-type poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ4 of Pseudomonas lemoignei, (ii) a purified truncated form of PhaZ4, which lacked 55 C-terminal amino acids and (iii) commercial lactate dehydrogenase to aqueous suspensions of PHB, chitin or cellulose was studied . Only the wild-type PHB depolymerase was specifically able to bind to PHB granules . No other combination of protein and polymeric substrate resulted in polymer-bound protein . Similar results were obtained for other PHB depolymerases . We concluded that the C-terminal amino acids of PHB depolymerases represent a PHB-specific binding domain or at least an essential part of it.

Biochemistry, 1996 Sep 17, 35(37), 11945 - 50
Structural determinants of nucleotide coenzyme specificity in the distinctive dinucleotide binding fold of HMG-CoA reductase from Pseudomonas mevalonii; Friesen JA et al.; The 102-residue small domain of the 428-residue NAD(H)-dependent HMG-CoA reductase of Pseudomonas mevalonii (EC 1.1.1.88) binds NAD(H) at a distinctive, non-Rossmann dinucleotide binding fold . The three-dimensional structure reveals that Asp146 lies close to the 2'-OH of NAD- . To investigate the role of this residue in determination of coenzyme specificity, Asp146 was mutated to Ala, Gly, Ser, and Asn . The mutant enzymes were analyzed for their ability to catalyze the oxidative acylation of mevalonate to HMG-CoA using either the natural coenzyme NAD+ or the alternate coenzyme NADP+ . Mutation of Asp146 to Ala or Gly increased the specificity for NADP+, expressed as the ratio of kcat/K(m) for NADP+ to kcat/K(m) for NAD+, 1200-fold (enzyme D146G) and 6700-fold (enzyme D146A) . Mutation of Asp146 was accompanied by 565-fold (D146G) and 330-fold (D146A) increases in kcat/K(m) for NADP+ and 2-fold (D146G) and 20-fold (D146A) decreases in kcat/K(m) for NAD+ . To further improve NADP+ specificity, Gln147, Leu148, Leu149, or Thr192 of enzyme D146G or D146A was replaced by lysine or arginine, which could stabilize the 2'-phosphate of NADP+ . Enzymes D146G/T192K, D146G/T192R, D146G/L148K, D146A/L148K, and D146A/L148R exhibited 3200-, 4500-, 56000-, 72000-, and 83000-fold increases in the specificity for NADP+ relative to the wild-type enzyme.

J Biol Chem, 1996 Sep 13, 271(37), 22428 - 33
Receptor for interleukin (IL) 13 does not interact with IL4 but receptor for IL4 interacts with IL13 on human glioma cells; Debinski W et al.; Recently, we have demonstrated that human (h) glioma cell lines express large number of receptors (R) for interleukin 13 (IL13) (Debinski, W., Obiri, N . I., Powers, S . K., Pastan, I., and Puri, R . K . (1995) Clin . Cancer Res . 1, 1253-1258) . These cells are extremely sensitive to a chimeric protein composed of hIL13 and a derivative of Pseudomonas exotoxin (PE), PE38QQR . We have found that the cytotoxicity of hIL13-PE38QQR was blocked by hIL13 but not by hIL4 on the U-251 MG and U-373 MG cells, contrary to what was observed on several adenocarcinoma cell lines . In the present study, we further explored interactions between receptor for IL13 and IL4 on glioma cells . Established human glioma cell lines, such as DBTRG MG, Hs 683, U-87 MG, SNB-19, and A-172, are very susceptible to hIL13-PE38QQR, and the action of the chimeric toxin is not blocked by hIL4 on all these cells either . Also, hIL4 is not a competitor for 125I-hIL13 binding sites on glioma cells . Of interest, a corresponding hIL4-based chimeric toxin, hIL4-PE38QQR, is poorly active or not active on all the tested glioma cell lines . When active, however, hIL4 toxin action was blocked by hIL13 . hIL13 is a competitor for 125I-hIL14 binding in a competitive binding assay on glioma cells . hIL13 and hIL4 did not affect the growth of the tested glioma cell lines . Human glioblastoma multiforme explant cells exhibited similar responses to the chimeric toxins and interleukins when compared with that found in established glioma cultures . Our results suggest that the hIL13R on glioma cells is expressed in one predominant form, the form that does not interact with IL4 . Thus, this type of hIL13R is apparently different from the one demonstrated previously on several adenocarcinoma cell lines.

Biochem Biophys Res Commun, 1996 Sep 4, 226(1), 15 - 20
Structure of the pcbC gene encoding 2,3-dihydroxybiphenyl dioxygenase of Pseudomonas sp . P20; Kim CK et al.; Pseudomonas sp . P20 is a soil bacterium growing in biphenyl or 4-chlorobiphenyl as the sole carbon and energy source, where the initial catabolism of biphenyl to form benzoate is catalyzed by four enzymes encoded in the pcbABCD gene cluster . The nucleotide sequence of the 2,3-dihydroxybiphenyl dioxygenase gene corresponding to the pcbC gene was determined . The pcbC gene was composed of 921 base pairs with an ATG initiation codon and a TGA termination codon, which can encode a polypeptide of molecular mass 34 kDa containing 306 amino acids . A promoter-like sequence and ribosome-binding sequence were identified upstream of the initiation codon . The deduced amino acid sequence of 2,3-dihydroxybiphenyl dioxygenase exhibited 47% identity with that of corresponding enzyme of Pseudomonas sp . DJ-12, and less than 35% identity with those of other extradiol-type dioxygenases.

Mikrobiol Z, 1996 Sep-Oct, 58(5), 18 - 24
{The surface-active compounds of a Pseudomonas sp . S-27 culture}; Karpenko EV et al.; Pseudomonas species S-27 was grown on various substrates . It was established that the Pseudomonas species S-27 strain can produce biosurfactants of ramnolipid nature decreasing the surface and interfacial tension to 29.2 and 0.05 mN/m . respectively, as well as a biopolymer stabilizing the emulsions with hydrocarbons and oils . The biosurfactant and bioemulsifier synthesis is shown to depend of the substrate nature.

Acta Otorrinolaringol Esp, 1996 Sep-Oct, 47(5), 404 - 6
{Vasculitis caused by Pseudomonas: a case report}; Escamilla Y et al.; Pseudomona vasculitis is an exceptional disease . Only a few cases have been reported, non with oropharyngeal involvement . The case of a 30-year-old, HIV-positive man who suddenly developed septicemia and necrotizing lesions with tissue destruction of the oropharynx is reported . Histological study confirmed vasculitis . Pseudomona aeruginosa was isolated in peripheral blood and in the biopsy of the palatal lesion . Antibiotic treatment produced satisfactory results.

Bioconjug Chem, 1996 Sep-Oct, 7(5), 557 - 63
Characterization of RFB4-Pseudomonas exotoxin A immunotoxins targeted to CD22 on B-cell malignancies; Mansfield E et al.; To develop an immunotoxin for the treatment of B-cell malignancies, we constructed several candidate conjugates with RFB4, a B-cell specific anti-CD22 IgG1, and truncated forms of Pseudomonas exotoxin (PE) . The four versions of PE included PE35 and PE35KDEL, which were linked to RFB4 via a disulfide bond, and PE38 and PE38KDEL, which were linked via a thioether bond . The PE35 truncated forms, which are fully active in ADP ribosylation and lack receptor binding sequences, do not require intracellular proteolytic cleavage in order to be active . PE35KDEL has the consensus endoplasmic reticulum retention signal, KDEL, replacing the wild type PE C-terminal sequence, REDLK . The PE38 forms retain all of domain II and therefore require cleavage to be active within cells . Cytotoxicity experiments on CD22-positive cell lines revealed that the PE35 conjugates were more active than the PE38 versions and the presence of the KDEL sequence generally enhanced toxicity by 5-10-fold compared to that of REDLK . The RFB4-PE35KDEL immunotoxin was most active in cytotoxicity assays against Burkitt's lymphoma cell lines such as Daudi and CA46 (IC50 = 0.2 ng/mL) and displayed little cytotoxicity toward human vascular endothelial cells (IC50 > 20 micrograms/mL) . Results of experiments conducted in nude mice showed that both RFB4-PE35KDEL and RFB4-PE35 could inhibit the development of subcutaneous CA46 tumors.

Br J Cancer, 1996 Sep, 74(6), 853 - 62
Targeted inhibition of tumour cell growth by a bispecific single-chain toxin containing an antibody domain and TGF alpha; Schmidt M et al.; Overexpression of the epidermal growth factor receptor (EGFR) and ErbB-2 has been observed in a variety of human tumours, making these receptors promising targets for directed tumour therapy . Since many tumour cells express both ErbB-2 and EGFR and these receptors synergise in cellular transformation, therapeutic reagents simultaneously binding to ErbB-2 and EGFR might offer advantages for tumour therapy . We have previously described the potent anti-tumoral activity of a bispecific antibody toxin that contains ErbB-2- and EGFR-specific single-chain Fv (scFv) domains . Here we report the construction and functional characterisation of a novel bispecific recombinant toxin, scFv(FRP5)-TGF alpha-ETA . The fusion protein consists of the antigen-binding domain of the ErbB-2-specific MAb, FRP5, and the natural EGFR ligand, TGF alpha, inserted at different positions in truncated Pseudomonas exotoxin A . ScFv(FRP5)-TGF alpha-ETA protein displayed binding to EGFR and ErbB-2, thereby inducing activation of the receptors, which was dependent on the cellular context and the level of EGFR and ErbB-2 expression . The bispecific molecule was cytotoxic in vitro for tumour cells expressing various levels of the target receptors . In vivo scFv(FRP5)-TGF alpha-ETA potently inhibited the growth of established A431 tumour xenografts in nude mice.

Mol Plant Microbe Interact, 1996 Sep, 9(7), 637 - 41
Use of translational fusions to the maltose-binding protein to produce and purify proteins in Pseudomonas syringae and assess their activity in vivo; Penaloza-Vazquez A et al.; A simple approach is described for the production and purification of proteins in Pseudomonas syringae . The strategy involves the use of the tac promoter, the maltose-binding protein, and the broad-host-range vector, pRK415 . This approach was used to partially purify two proteins involved in coronatine biosynthesis from P . syringae . The activity of the fusions was demonstrated in vivo in complementation experiments using the appropriate mutants.

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