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 conforma