Pseudomonas

Pediatr Med Chir, 1995 May-Jun, 17(3), 189 - 96
{How best to perform aerosol therapy . 2 . Drugs}; Battistini A; The individuation of a 5:1 ratio for the posology of beta 2-agonist drugs administered respectively by nebulizer or as a spray plus a spacer, allows the use of these drugs by the latter way also during an acute attack of bronchospasm . Other qualifying aspects of beta 2-agonist are the possibility of increasing the dose up to a continuous administration in case of a severe attack of bronchoconstriction and the demonstrated clinical efficacy of these drugs during the first year of life . The question of the correct dose to administer according to the age of the patient appears to be rather complex: in fact if on the one hand during the first months of life the low tidal volume (VT) can reduce the quantity of drug inhaled, on the other the low pulmonary volumes (FRC) of the first years of life determine a lesser dilution of the inhaled drug and therefore a greater pulmonary concentrations . Among corticosteroids for inhalation, the ones with the fewer side effects should be chosen: these may appear although in a subclinical fashion at relatively low doses of about 400 micrograms/day of beclomethasone . The most frequent indication for an antibiotic treatment by inhalation is represented by chronic pulmonary infection caused by bacteria sensible only to parenteral antibiotics (for ex . Pseudomonas infection in cystic fibrosis) . The extremely high cost of these treatments requires the use of devices with a very high efficiency, in other words capable of nebulizing very large percentages of drug into small particles (< 6 microns).(ABSTRACT TRUNCATED AT 250 WORDS)

Mikrobiol Z, 1995 May-Jun, 57(3), 53 - 6
{The isolation and characteristics of cloned strains of Pseudomonas pseudomallei phages}; Denisov II et al.; On the basis of museum strains of the melioidosis agent Pseudomonas pseudomallei, 14 pure lines of bacteriophages were isolated belonging to morphological groups IV and V according to the classification by A . S . Tikhonenko . These phages show a broad bacteriolytic range as far as museum P . pseudomallei cultures are concerned . It was determined that some phages isolated from the same melioidosis culture and belonging to the same morphological type display different bacteriolytic activity . It is demonstrated that some strains of melioidosis agent are characterized by polylysogeny.

Pharm Res, 1995 May, 12(5), 642 - 8
Interdomain interactions in the chimeric protein toxin sCD4(178)-PE40: a differential scanning calorimetry (DSC) study; Davio SR et al.; The thermal denaturation of the chimeric protein toxin known as sCD4(178)-PE40 (sCD4-PE40) was studied using differential scanning calorimetry (DSC) . sCD4-PE40 consists of HIV-binding domains of the T-cell membrane protein known as CD4 and the cytotoxic domains of Pseudomonas exotoxin A (PE40) . sCD4-PE40 undergoes two DSC transitions . An endothermic transition associated with unfolding of the CD4 and PE40 components occurs at approximately 46 degrees C in buffered saline at pH 6.5 . An exothermic transition associated with precipitation of unfolded protein occurs at higher temperatures . Both transitions are irreversible . DSC studies of solutions of pH 5.0 to 9.5 indicate that sCD4-PE40 shows maximal thermal stability at around pH 6.5 . Variable pH experiments are also presented on solutions of sCD4(183) and PE40 revealing how these components denature as independent structural entities . sCD4(183) denaturation occurs at significantly higher temperatures than does the CD4 component of sCD4-PE40 . PE40 denaturation occurs at the same temperatures as sCD4-PE40 . These results suggest that the native CD4 and PE40 components are independent and non-interacting entities in the chimeric sCD4-PE40 molecule and that unfolding of the less-stable PE40 component induces unfolding of the CD4 component . These destabilizing interdomain interactions of sCD4-PE40 are in contrast to the stabilizing interactions which apparently exist in wild-type Pseudomonas exotoxin A between its PE40 domains and the cell binding domain of the native toxin (analogous to the CD4 component in sCD4-PE40) . Reasons are discussed why the type of interdomain interactions observed for sCD4-PE40 might be the norm for chimeric proteins.

Biochem J, 1995 Apr 15, 307 ( Pt 2), 603 - 8
Tropine dehydrogenase: purification, some properties and an evaluation of its role in the bacterial metabolism of tropine; Bartholomew BA et al.; Tropine dehydrogenase was induced by growth of Pseudomonas AT3 on atropine, tropine or tropinone . It was NADP(+)-dependent and gave no activity with NAD+ . The enzyme was very unstable but a rapid purification procedure using affinity chromatography that gave highly purified enzyme was developed . The enzyme gave a single band on isoelectric focusing with an isoelectric point at approximately pH 4 . The native enzyme had an M(r) of 58,000 by gel filtration and 28,000 by SDS/PAGE and therefore consists of two subunits of equal size . The enzyme displayed a narrow range of specificity and was active with tropine and nortropine but not with pseudotropine, pseudonortropine, or a number of related compounds . The apparent Kms were 6.06 microM for tropine and 73.4 microM for nortropine with the specificity constant (Vmax/Km) for tropine 7.8 times that for pseudotropine . The apparent Km for NADP+ was 48 microM . The deuterium of {3-2H}tropine and {3-2H}pseudotropine was retained when these compounds were converted into 6-hydroxycyclohepta-1,4-dione, an intermediate in tropine catabolism, showing that the tropine dehydrogenase, although induced by growth on tropine, is not involved in the catabolic pathway for this compound . 6-Hydroxycyclohepta-1,4-dione was also implicated as an intermediate in the pathways for pseudotropine and tropinone catabolism.

Biochemistry, 1995 Apr 4, 34(13), 4441 - 7
Ultraviolet resonance Raman spectroscopy of delta 5-3-ketosteroid isomerase revisited: substrate polarization by active-site residues; Austin JC et al.; The delta 5-3-ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni promotes extremely rapid conversion of delta 5- to delta 4-3-ketosteroids by a conservative intramolecular proton transfer via an enolic intermediate . The competitive inhibitor 19-nortestosterone displays marked spectroscopic changes upon binding to the enzyme, but the mechanisms responsible for these changes have not been unequivocally established . Ultraviolet resonance Raman (UVRR) spectra are reported for 19-nortestosterone in acid solutions and for this ligand when bound to delta 5-3-ketosteroid isomerase, as well as to its D38N and Y14F/D38N mutants . The frequencies of UVRR bands associated with C = O and C = C stretching can be used to monitor the state of polarization of the enone fragment of the steroid and the effects of the catalytic side chains, Tyr-14 and Asp-38, on these polarizations . Strong polarization is indicated by marked frequency downshifts of the C = O and C = C bands in the native protein; the downshifts are diminished by the mutations of these catalytic residues . The lower polarizing effects of the Y14F and D38N single mutants and the Y14F/D38N double mutant indicate that most of the polarization of the conjugated ketone is attributable to hydrogen-bond donation by the hydroxyl group of Tyr-14 . A smaller contribution of Asp-38 is detected which is, in part, cooperative with that of Tyr-14 . Reference spectra of hydrogen-bonded and protonated forms of 19-nortestosterone are reassigned, on the basis of the species identification of D . C . Hawkinson and R . M . Pollack {(1993) Biochemistry 32, 694-698}.(ABSTRACT TRUNCATED AT 250 WORDS)

Can J Microbiol, 1995 Apr-May, 41(4-5), 438 - 43
Enzymes from Pseudomonas sp . strain NCIB 11097 participating in biotransformation of acetaldehyde and glycine to threonine isomers; Diaz-Diaz M et al.; Enzyme activities involved in L-threonine bioconversions present in cells of Pseudomonas sp . strain NCIB 11097 were separated by phenyl-Sepharose hydrophobic chromatography . The separation of the two main activity components was monitored by discontinuous polyacrylamide gel electrophoresis . Threonine aldolase catalyzed the conversion of glycine and acetaldehyde to a mixture of isomers, L-threonine and L-allothreonine, in a biotransformation reaction having pH and temperature optima of 7.5 and 25-30 degrees C, respectively . The fraction containing serine hydroxymethyltransferase converted acetaldehyde and glycine specifically to L-allothreonine in a biotransformation reaction having pH and temperature optima of 7.4 and 37 degrees C, respectively.

Can J Microbiol, 1995 Apr-May, 41(4-5), 372 - 7
Viability and respiratory activity of Pseudomonas syringae cells starved in buffer; Cabral JP; Pseudomonas syringae cells starved in buffer released orcinol-reactive molecules and materials that absorbed ultraviolet light . The number of cells culturable in nutrient medium decreased more rapidly than the number of intact particles determined by microscopy . The results suggested that starvation resulted in the lysis of an increasing number of cells, and that a fraction of the intact particles were not culturable . Starvation also resulted in a decrease in the rate of oxygen consumption with acetate, glycerol, and succinate, but at different levels . Whereas the respiration of acetate and glycerol decreased concomitantly with culturability, the respiration of succinate decreased to levels similar to the concentration of intact cells, suggesting that all intact particles respired the succinate, but only the culturable cells respired the acetate and glycerol . The results suggest that measuring the activity of the electron-transport system can overestimate the viability of starved bacterial cells, and that complex metabolic activities such as the respiration of acetate and glycerol are probably better suited for the evaluation of this parameter.

Z Lebensm Unters Forsch, 1995 Apr, 200(4), 293 - 6
Preservation of raw milk with CO2 . Sensory evaluation of heat-processed milks; Amigo L et al.; The effect of CO2 on the growth of psychrotrophic milk spoilage organisms was studied, both in raw fresh milk and in pure cultures of three species of Pseudomonas growing in sterilised milk . Changes of sensory properties of CO2-treated samples after heat treatment were also analysed . Inhibition of psychrotrophic growth at 7 degrees C in milk treated with CO2 to a pH 6.2 or 6.0 was impaired by a gradual reduction of the CO2 content during storage . Growth inhibition was considerably improved by pH adjustment at 24-h intervals . Sensory analysis showed significant differences between non-acidified and acidified samples after heat treatment at 75 degrees C for 20 s or 110 degrees C for 5 min . No sensory differences were found between non-acidified and acidified milks degassed before heat treatment.

Arch Microbiol, 1995 Apr, 163(4), 242 - 7
Organomercurial resistance determinants in Pseudomonas K-62 are present on two plasmids; Kiyono M et al.; Pseudomonas strain K-62 was found to contain six plasmids . A mutant derivative cured of the 26-kb plasmid showed a higher sensitivity to mercurials; however, the strain was still able to volatilize them . Loss of the 68-kb plasmid in addition to the 26-kb plasmid abolished the ability of mercury volatilization in this strain and led to a further decrease in the level of mercurial resistance . These results are the first to demonstrate that the organomercurial resistance of Pseudomonas strain K-62 is plasmid-based, and that both the 26- and 68-kb plasmids are required for full expression of the mercurial resistance . Probes specific for the mer genes merA, merB, and merR strongly hybridized with the 26-kb plasmid, but not with the 68-kb plasmid . Two fragments of the 26-kb plasmid that hybridized with the mer genes were cloned and expressed in Escherichia coli . One recombinant plasmid (pMRA17) inducibly encoded a typical broad-spectrum mercurial resistance, whereas the other recombinant plasmid (pMRB01) constitutively conferred hypersensitivity to phenylmercury in the absence of mercuric reductase activity . The results suggest that the two organomercurial lyases in the cells are transcribed from different operator-promoters.

Eur J Biochem, 1995 Apr 1, 229(1), 119 - 31
Spectroscopic characterisation of disaccharides derived from keratan sulfates; Huckerby TN et al.; Skeletal keratan sulfates have been degraded by three independent techniques and the resultant, borohydride-reduced, disaccharides have been characterised by NMR spectroscopy . The 1H and 13C (where available) chemical shifts are reported for the following substances, where GalNAc-ol represents N-acetyl-galactosaminitol, GlcNAc-ol represents N-acetyl-glucosaminitol, GlcNAc(6S)-ol represents N-acetyl-glucosaminitol 6-O-sulfate and 2,5AnMan(6S)-ol represents 2,5-anhydro-D-mannitol 6-O-sulfate . (a) GlcNAc(6S)beta(1-3)Gal-ol, isolated after keratanase (from Pseudomonas sp.) digestion . (b) Gal beta(1-4)GlcNAc(6S)-ol and Gal(6S)beta(1-4) GlcNAc(6S)-ol, the 1H chemical shifts have been reported previously {Brown, G . M., Huckerby, T . N., Morris, H . G., Abram, B . L . & Nieduszynski, I . A . (1994) Biochemistry 33, 4836-4846; Brown, G . M., Huckerby, T . N . & Nieduszynski, I . A . (1994) Eur . J . Biochem . 224, 281-308}, GlcNAc(6S)beta(1-6)GalNAc-ol, {formula: see text}, {formula: see text}, all isolated after keratanase II digestion . (c) Gal beta(1-4)2,5AnMan(6S)-ol and Gal(6S)beta(1-4)2,5AnMan(6S)-ol, isolated after hydrazinolysis and nitrous acid digestion . In addition, the model compounds Gal beta(1-4)GlcNAc-ol and Gal beta(1-6)GlcNAc-ol have also been examined by 1H and 13C NMR spectroscopy . The value of these data for microstructural analysis of keratan sulfate samples is discussed.

Cryobiology, 1995 Apr, 32(2), 129 - 38
Differential effects of growth temperature on ice nuclei active at different temperatures that are produced by cells of Pseudomonas syringae; Gurian-Sherman D et al.; The temperature at which ice-nucleating bacteria are grown causes differences of 100- to 10,000-fold in the fraction of cells that nucleate ice at a given temperature (ice nucleation frequency) . Ice nucleation frequencies of cells of Pseudomonas syringae grown at temperatures that ranged from 9 to 33 degrees C were examined in order to more accurately characterize physiological effects on ice nuclei active at temperatures of from about -2 to -10 degrees C, the temperature range for this phenotype . Large differences in ice nucleation frequency occurred at all but the lowest assay temperatures in cells of P . syringae grown in the temperature range of 15 to 33 degrees C . These differences in ice nucleation frequency may be attributed, at least in part, to post-translational factors . Because other studies have indicated that ice nuclei active at the lowest assay temperatures may reflect the amount of ice nucleation protein produced, while higher nucleation temperatures reflect aggregates of this ice nucleation protein, data was normalized to the frequency of ice nuclei active at the lowest ice nucleation temperatures (which also correspond to the most abundant nuclei) . This was done in order to develop a baseline of comparison for cells grown at different temperatures that more clearly shows possible post-translational effects such as aggregation of the nucleation protein . After this normalization was performed, and in contrast to the results noted above, the number of ice nuclei in cells grown at 9, 15, and 20 degrees C that were active at different assay temperatures was very similar . Differences in ice nucleation frequency that occurred over all assay temperatures in cells grown between 9 and 20 degrees C may be attributed to differences in the total number of nuclei present in the population of cells . The large effects of growth temperature on nucleation frequency have important implications for estimating numbers of ice nucleating bacteria in environmental samples by determining the number of bacterial ice nuclei in such samples.

Mol Ecol, 1995 Apr, 4(2), 221 - 30
Recombinant and wild-type Pseudomonas aureofaciens strains introduced into soil microcosms: effect on decomposition of cellulose and straw; England LS et al.; The effect of a genetically engineered Pseudomonas aureofaciens (Ps3732RNL11) strain (GEM) and the parental wild-type (Ps3732RN) on decomposition of cellulose paper, straw and calico cloth was assessed after 18 weeks incubation in laboratory soil microcosms . Effect(s) of inoculum density (10(3), 10(5), and 10(8) cells/g dry soil) and single versus multiple bacterial inoculations were also investigated . Cellulose paper was completely decomposed after 18 weeks in all treatments . There were no significant differences (95% level), between treatments, in percentage decomposition of either straw or calico cloth . Recovery of the GEM at 18 weeks, using viable plating, was limited to treatments originally receiving 10(8) cells/g dry soil . Log 1.8 CFU/g dry soil were recovered from the single dose treatment while log 4.2 CFU/g dry soil were recovered from the multiple dose treatment . Biolog metabolic tests were used to determine if the GEM or parental wild-type had any effect on overall carbon utilization in soil . Results suggested they did not . Detection of the recombinant lacZY gene sequence in soil using PCR suggested the possibility of viable but nonculturable cells and/or persistence of chromosomal DNA.

J Bacteriol, 1995 Apr, 177(8), 2204 - 8
Transformation of carbon tetrachloride via sulfur and oxygen substitution by Pseudomonas sp . strain KC; Lewis TA et al.; Pseudomonas sp . strain KC transforms carbon tetrachloride into carbon dioxide and nonvolatile products, without chloroform as an intermediate . To define the pathway for hydrolysis, nonvolatile products were analyzed . Condensation products containing the carbon atom of carbon tetrachloride as carbonyl and thioxo moieties were identified, indicating the intermediacy of phosgene and thiophosgene in the pathway.

Biochem J, 1995 Apr 1, 307 ( Pt 1), 29 - 37
Importance of the glutamate residue of KDEL in increasing the cytotoxicity of Pseudomonas exotoxin derivatives and for increased binding to the KDEL receptor; Kreitman RJ et al.; It was previously shown that amino acids 609-613 (REDLK) at the C-terminus of Pseudomonas exotoxin (PE) are necessary for cytotoxicity, presumably by directing the toxin to the endoplasmic reticulum (ER) {Chaudhary, Jinno, FitzGerald and Pastan (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 308-312} . Using the anti-{interleukin 2 receptor (IL2R)} immunotoxin anti-Tac(Fv)-PE38 (AT-PE38REDLK), it was found that removing the terminal lysine did not alter the activity, but replacing REDL with KDEL, the most common ER retention sequence, increased activity . To determine which amino acid in KDEL was responsible for the increase in activity, we tested eight C-terminal mutants of AT-PE38REDLK . Using IL2R-bearing MT-1 cells, we found that the glutamate residue of KDEL was required for high activity, as the cytotoxicity of AT-PE38 ending in KDEL, RDEL, KEEL or REEL was much greater than that of AT-PE38 ending in REDL, KEDL, RDDL or KDDL . Using freshly isolated lymphocytic leukaemia cells, AT-PE38 ending in KDEL, REEL or RDEL was more than 100-fold more cytotoxic than AT-PE38 ending in KEDL, REDL, RDDL or the native sequence REDLK . The RDEL sequence also improved the cytotoxic activity of an interleukin 4-PE38 toxin fusion protein . Improved cytotoxic activity correlated with improved binding of the C-termini to the KDEL receptor on rat Golgi membranes . These data indicate that the glutamate residue of KDEL improves the cytotoxicity of PE by increasing binding to a sorting receptor which transports the toxin from the transreticular Golgi apparatus to the ER, where it is translocated to the cytosol and inhibits protein synthesis.

Laryngoscope, 1995 Apr, 105(4 Pt 1), 354 - 8
Management of chronic sinusitis in cystic fibrosis; Davidson TM et al.; Chronic rhinosinusitis is extremely common in patients with cystic fibrosis . It causes numerous problems in these patients and can put them at risk for life-threatening illness . Potential problems include nasal obstruction, congestion, sinus pain and pressure, infection (usually with Pseudomonas organisms), hyposmia or anosmia, and the seeding of bacteria into the lower respiratory tract . Cystic fibrosis patients with chronically infected sinuses are at increased risk for pneumonia following lung transplantation . A prophylactic protocol has been developed for the management of chronic sinusitis in patients with cystic fibrosis . These patients are fully evaluated at the Nasal Dysfunction Clinic of the University of California, San Diego (UCSD), Medical Center . Based on the results of the evaluation, they are treated with endoscopic sinus surgery, partial middle turbinectomy, septoplasty, and a large middle meatal maxillary antrostomy . Surgery is followed by a rigorous regimen of pulsatile hypotonic saline nasal irrigation to wash away tenacious cystic secretions . Tobramycin (Nebcin) is given once daily in the nasal irrigant to inhibit the growth of Pseudomonas organisms . At the USCD Nasal Dysfunction Clinic, this prepulmonary transplantation protocol is now used in all cystic fibrosis patients with chronic sinusitis.

Biol Pharm Bull, 1995 Apr, 18(4), 615 - 7
In vitro antitumor activity of polymyxin acylase from Pseudomonas sp . M-6-3; Yasuda N et al.; Polymyxin acylase from Pseudomonas sp . M-6-3 can deacylate not only polymyxin group antibiotics, but also the long-chain fatty acyl group of proteins and peptides . We found the in vitro antitumor activity of polymyxin acylase against murine and human tumor cells, especially KB cells . The mechanism of the antitumor activity remains equivocal, but we speculate that it may result from the affinity of polymyxin acylase for long-chain fatty acyl proteins in human carcinoma cells.

J Biochem (Tokyo), 1995 Apr, 117(4), 856 - 62
Molecular cloning and sequencing of a cDNA encoding alanine-glyoxylate aminotransferase 2 from rat kidney; Lee IS et al.; Alanine-glyoxylate aminotransferase (AGT) 2 is a pyridoxal 5'-phosphate dependent, mitochondrial enzyme which, in the rat, is expressed at a high level in the kidney . The amino acid sequences of nine tryptic and seven CNBr peptides of the rat kidney AGT2 were determined . Three overlapping cDNAs encoding the AGT2 were cloned on the basis of its partial amino acid sequences by means of a polymerase chain reaction-based approach involving rat kidney poly(A)+ RNA . The complete cDNA sequence comprised 1,919 bases, and contained a 1,536-base open reading frame which encodes a polypeptide of 512 amino acid residues with a putative presequence consisting of 39 amino acid residues at the amino terminus, giving a precursor protein with a molecular mass of 57,150 Da . The sequence of AGT2 exhibits significant homology with neither peroxisomal AGT1 from human liver nor mitochondrial AGT1 from rat liver . However, the sequence of AGT2 exhibited 30.8, 29.2, and 27.1% identity with those of Escherichia coli 4-aminobutyrate aminotransferase, rat ornithine aminotransferase, and Pseudomonas cepacia 2,2-dialkylglycine decarboxylase, respectively . The active site sequences were also well conserved among these aminotransferases . AGT2, thus, is more similar to the other aminotransferases than to AGT1 . The results suggest that the rat kidney AGT2 may play a biological role in amino acid metabolism distinct from that of AGT1.

Appl Environ Microbiol, 1995 Apr, 61(4), 1201 - 7
Survival and activity of Pseudomonas sp . strain B13(FR1) in a marine microcosm determined by quantitative PCR and an rRNA-targeting probe and its effect on the indigenous bacterioplankton; Leser TD et al.; Genetically engineered Pseudomonas sp . strain B13(FR1) was released into laboratory-scale marine ecosystem models (microcosms) . Survival of the introduced population in the water column and the sediment was determined by plating on a selective medium and by quantitative competitive PCR . The activity of the released bacteria was determined by in situ hybridization of single cells with a specific rRNA-targeting oligonucleotide probe . Two microcosms were inoculated with 10(6) cells ml-1, while an uninoculated microcosm served as a control . The number of Pseudomonas sp . strain B13(FR1) cells decreased rapidly to ca . 10(2) cells ml-1 within 2 days after the release, which is indicative of grazing by protozoa . Three days after the introduction into seawater, cells were unculturable, but PCR continued to detect cells in low numbers . Immediately after the release, the ribosomal content of Pseudomonas sp . strain B13(FR1) corresponded to a generation time of 2 h . The growth rate decreased to less than 0.04 h-1 in 5 days and remained low, probably because of carbon limitation of the cells . Specific amendment of the microcosms with 10 mM 4-chlorobenzoate resulted in a rapid increase of the growth rate and an exponentially increasing number of cells detected by PCR, but not in resuscitation of the cells to a culturable state . The release of Pseudomonas sp . strain B13(FR1) into the microcosms seemed to affect only the indigenous bacterioplankton community transiently . Effects on the community were also apparent from the handling of water during filling of the microcosms and the amendment with 4-chlorobenzoate.

Wei Sheng Wu Xue Bao, 1995 Apr, 35(2), 86 - 90
{Isolation and identification of a pathogen of grasshoppers}; Liu S et al.; A pathogen was isolated from natural dead pests of Ceracris kiangsu in Geleshan farm of Chongqin . Its pathogenecity was confirmed by the law of KOCK . It was identified as Pseudomonas pseudoalcaligenes according to its physiological and biochemical properties as well as the G + C content of DNA (63.73mol%) . The results of preliminary bioassay show that the pathogen can infect the grasshoppers and Ceracris kiangsu, and also can infect other pests of grassland in a certain extent.

J Biol Chem, 1995 Mar 31, 270(13), 7625 - 30
HER4 expression correlates with cytotoxicity directed by a heregulin-toxin fusion protein; Siegall CB et al.; We have constructed, expressed, and purified a fusion protein, HAR-TX beta 2, consisting of heregulin-beta 2 fused to a binding-defective form of Pseudomonas exotoxin A, PE40 . The fusion protein was found to induce receptor tyrosine phosphorylation in CEM cells transfected with HER4 alone or in combination with HER2 but not in cells transfected with HER2 or HER1 alone . The phosphorylation of receptor tyrosines was both dose-dependent and saturable in amounts similar to those shown to be active for native heregulin . HAR-TX beta 2 was specifically cytotoxic toward a variety of carcinoma cell lines in the ng/ml range . However, some tumor cell lines were found to be insensitive to the cytotoxic action of the fusion protein even at > 2 micrograms/ml . Relative amounts of HER4, HER3, and HER2 were determined on seven cell lines sensitive and four cell lines insensitive to HAR-TX beta 2 . All lines that express HER4 were killed by HAR-TX beta 2, while none lacking HER4 were affected . HAR-TX beta 2 was able to bind to and signal via tyrosine phosphorylation in cell lines that co-express HER2 and HER3 in the absence of HER4 without inducing cytotoxicity . Thus HAR-TX beta 2 may prove to be a useful reagent for the targeting and elimination of HER4-positive tumor cells.

Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2765 - 9
Intrathecal administration of single-chain immunotoxin, LMB-7 {B3(Fv)-PE38}, produces cures of carcinomatous meningitis in a rat model; Pastan IH et al.; LMB-7 {B3(Fv)-PE38} is a single-chain immunotoxin constructed from the murine monoclonal antibody B3 and a truncated from of Pseudomonas exotoxin PE38 . Antibody B3 recognizes a carbohydrate epitope found on solid tumors that frequently invade the intrathecal space and cause neoplastic meningitis . We tested the therapeutic value of intrathecally administered LMB-7 by using a model of human neoplastic meningitis in athymic rats . This model is representative of a clinical situation in that antibody B3 cross-reacts with a number of normal tissues that can be used to monitor potential systemic toxicity . Treatment was begun 3 days after A431 tumor implantation . Without treatment, the animals median survival was 10 days . Intrathecal administration of 10 micrograms of LMB-7 in 40 microliters on days 3, 5, and 7 produced 4 of 10 and 8 of 10 long-term survivors (> 170 days) in two experiments . Of the long-term survivors, 2 of 4 and 7 of 8 survivors had no microscopic evidence of tumor and were considered histologic cures . Lack of significant toxicity in the effective dose range and specificity make LMB-7 an excellent candidate for intrathecal treatment of neoplastic meningitis in humans.

J Mol Biol, 1995 Mar 24, 247(2), 211 - 23
Transcription of repA, the gene of the initiation protein of the Pseudomonas plasmid pPS10, is autoregulated by interactions of the RepA protein at a symmetrical operator; Garcia de Viedma D et al.; Transcription of the repA gene of the Pseudomonas plasmid pPS10 is initiated from a sigma 70 type promoter located 81 bp upstream from the repA gene, extends through the repA gene and the adjacent open reading frame, and ends 1114 nucleotides downstream . The repA promoter is repressed by interactions of the RepA protein with a region of 44 bp that extends from the -10 box of the promoter to the dnaA box of the origin of replication . The core of the repA operator region is formed by two in-phase invertedly repeated sequences of 8 bp, S1 and S2, that flank the -35 box of the promoter, and that share homology with the internal sequences of the iterons present in the origin of replication . RepA enters at the operator region first by protein-DNA interactions and subsequently by protein-protein interactions . These sequential interactions lead to the formation of high, medium and low-mobility electrophoretic complexes . Formation of the high-order complexes seems to be important for an efficient repression of the promoter . Interactions of RepA with the repA promoter region (repPO) occur more efficiently than with the origin of replication.

Int J Cancer, 1995 Mar 3, 60(5), 730 - 9
Targeted inhibition of tumor-cell growth by recombinant heregulin-toxin fusion proteins; Jeschke M et al.; Fusion of functional domains of proteins by in vitro recombination of gene fragments can be used to generate novel anti-tumor agents . The combination of tumor-cell-recognition functions and toxic functions results in cytotoxic molecules with a high specificity for tumor cells . Human adenocarcinomas are frequently characterized by over-expression of members of the epidermal-growth-factor (EGF) receptor family (ErbB-1, 2, 3 and 4), when compared with normal cells . These tumors are particularly suited to treatment with recombinant toxins . The human heregulins (HRG) and their rat counterparts (neu differentiation factor, NDF) have been identified as ligands for these receptors . Two chimeric heregulin-toxin fusions consisting of the EGF-like receptor recognition domain of the heregulin isoforms HRG alpha and HRG beta I, and the domains II, Ib and III of the Pseudomonas exotoxin A (ETA) were constructed . HRG beta I-ETA is highly cytotoxic for the mammary carcinoma cell lines SK-BR-3 and MDA-MB-453 . HRG alpha-ETA was less active than HRG beta I-ETA . The killing activity of the recombinant toxins correlated with the expression levels of ErbB-3 and/or ErbB-4 in the cell lines studied . High expression of ErbB-2 is not sufficient to confer sensitivity towards the HRG-ETA . Treatment of mice with 0.4 mg/kg/day of HRG beta I-ETA caused growth retardation of transplanted human breast tumor cells . Higher levels of HRG beta I-ETA administration resulted in acute hemorrhagic necrosis of the liver.

Biochem Pharmacol, 1995 Mar 1, 49(5), 711 - 5
Hydrolysis of tetriso by an enzyme derived from Pseudomonas diminuta as a model for the detoxication of O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX); Hoskin FC et al.; An enzyme termed organophosphorus hydrolase (OPH), derived from Pseudomonas diminuta, had been found previously to hydrolyze the powerful acetylcholinesterase (AChE) inhibitor O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX) . This enzyme has now been shown to be correlated with the loss of AChE inhibitory potency (detoxication) . OPH also hydrolyzed and detoxified the VX analogue, O,O-diisopropyl S-(2-diisopropylaminoethyl) phosphorothiolate (Tetriso), also a potent AChE inhibitor, about five times faster than VX . The Km for the hydrolysis of the P-S bond of Tetriso was 6.7 x 10(-3) M . OPH also hydrolyzed diisopropylphosphorofluoridate (DFP) 50-60 times faster than Tetriso, and 1,2,2-trimethylpropyl methylphosphonofluoridate (Soman) about seven times faster than Tetriso . DFP was a non-competitive inhibitor of Tetriso hydrolysis, Ki = 8.7 x 10(-4) M . The DFP hydrolysis product, diisopropyl phosphate, was a competitive inhibitor, Ki = 2.3 x 10(-4) M . The rate of detoxication of Tetriso compared with the rate of hydrolysis suggests that OPH may not be totally specific for P-S bond cleavage . OPH was inhibited completely by 1.5 x 10(-4) M 8-hydroxyquinoline-5-sulfonate or 1,10-phenanthroline, both transition element chelators, but inhibited only partially by EDTA, a much more potent chelator.

J Bacteriol, 1995 Mar, 177(5), 1259 - 67
A complex network regulates expression of eps and other virulence genes of Pseudomonas solanacearum; Huang J et al.; We have discovered an unusual and complex regulatory network used by the phytopathogen Pseudomonas solanacearum to control transcription of eps, which encodes for production of its primary virulence factor, the exopolysaccharide EPS I . The major modules of this network were shown to be three separate signal transduction systems: PhcA, a LysR-type transcriptional regulator, an dual two-component regulatory systems, VsrA/VsrD and VsrB/VsrC . Using lacZ fusions and RNA analysis, we found that both PhcA and VsrA/VsrD control transcription of another network component, xpsR, which in turn acts in conjunction with vsrB/vsrC to increase transcription of the eps promoter by > 25-fold . Moreover, gel shift DNA binding assays showed that PhcA specifically binds to the xpsR promoter region . Thus, the unique XpsR protein interconnects the three signal transduction systems, forming a network for convergent control of EPS I in simultaneous response to multiple environmental inputs . In addition, we demonstrate that each individual signaling system of the network also acts independently to divergently regulate other unique sets of virulence factors . The purpose of this complex network may be to allow this phytopathogen to both coordinately or independently regulate diverse virulence factors in order to cope with the dynamic situations and conditions encountered during interactions with plants.

J Bacteriol, 1995 Mar, 177(5), 1254 - 8
Lipase modulator protein (LimL) of Pseudomonas sp . strain 109; Ihara F et al.; Plasmids containing a Pseudomonas sp . strain 109 extracellular lipase gene (lipL) lacking NH2-terminal sequence and a lipase modulator gene (limL) lacking the NH2-terminal hydrophobic region were constructed and expressed independently in Escherichia coli by using the T7 promoter expression vector system . Recombinant LipL (rLipL) was produced as inclusion bodies, whereas recombinant LimL (rLimL) was present as a soluble protein . During in vitro renaturation of the purified rLipL inclusion bodies after they had been dissolved in 8 M urea, addition of rLimL was essential to solubilize and modulate rLipL . The solubility and activity of rLipL were influenced by the rLimL/rLipL molar ratio; the highest level of solubility was obtained at an rLimL/rLipL ratio of 4:5, whereas the highest activity level was obtained at an rLimL/rLipL ratio of 4:1 . After renaturation, rLipL and rLimL were coprecipitated with anti-rLipL antibody, indicating the formation of an rLipL-rLimL complex . Activity of the native lipase purified from Pseudomonas sp . strain 109 was also inhibited by rLimL . By Western blotting (immunoblotting) with anti-rLimL antibody, native LimL was detected in Pseudomonas cells solubilized by sarcosyl treatment . LimL was purified from Pseudomonas sp . strain 109, and the NH2-terminal amino acid sequence was determined to be NH2-Leu-Glu-Pro-Ser-Pro-Ala-Pro- . We propose that to prevent membrane degradation, LimL weakens lipase activity inside the cell, especially in the periplasm, in addition to modulating lipase folding.

Clin Chest Med, 1995 Mar, 16(1), 95 - 109
Ventilator-associated pneumonia caused by Pseudomonas infection; Dunn M et al.; So far, P . aeruginosa has matched each new therapeutic advance with an antibiotic resistance or toxin move of its own . The result has been a persistently high mortality rate for Pseudomonas pneumonia, which is unacceptable . Further research to define aspects of the organism important in infection and to determine more effective treatment and prevention strategies are clearly needed.

Clin Infect Dis, 1995 Mar, 20(3), 641 - 5
Elevated serum procalcitonin levels in patients with melioidosis; Smith MD et al.; The prognostic value of serum procalcitonin levels in 43 patients with acute melioidosis, an infection with a wide range of clinical manifestations, was assessed . In eight patients with mild localized infections, the median procalcitonin levels were 0.13 ng/mL (range, 0.02-0.46 ng/mL), which were similar to those in 19 healthy controls (median, 0.07 ng/mL; range, 0.03-0.15 ng/mL) . In the patients with severe infections, the initial procalcitonin levels were significantly higher in the patients who died (median, 350 ng/mL; range, 63-3,538 ng/mL) than in the survivors (median, 19 ng/mL; range, 0.55-387 ng/mL) (P < .0001); 16 of 19 patients with procalcitonin levels of > 100 ng/mL died, compared with 2 of 16 patients with levels of < 100 ng/mL (relative risk, 6.7; 95% confidence interval, 1.8-25; P = .0001) . In those patients who survived, the subsequent procalcitonin levels reflected closely the clinical course of their infection . The serum concentration of procalcitonin correlates well with the severity of Pseudomonas pseudomallei infection and is comparable with other acute-phase markers . However, this prognostic indicator and marker of continuing disease activity is not specific to melioidosis and could be applied to other severe infections.

Plant Cell, 1995 Mar, 7(3), 249 - 57
Use of a gene expression system based on potato virus X to rapidly identify and characterize a tomato Pto homolog that controls fenthion sensitivity; Rommens CM et al.; A novel transient gene expression system was used to study both the tomato disease resistance gene Pto and a Pto homolog designated Fen . The gene expression system was based on potato virus X (PVX) . Tomato plants that were both susceptible to strains of Pseudomonas syringae pv tomato carrying the corresponding avirulence gene avrPto and insensitive to the insecticide fenthion were infected with in vitro-generated transcripts of PVX derivatives containing either Pto or Fen . Expression of the Pto gene from the virus genome failed to elicit P.s . tomato resistance, indicating that the PVX system is not suitable for the study of Pto . However, expression of the Fen gene resulted in sensitivity to fenthion . The utility of the PVX gene expression system was further demonstrated through structure/function studies of the Fen gene . A correlation was shown between Fen protein kinase activity and the ability of this protein to confer fenthion sensitivity to tomato . Furthermore, it was demonstrated that mutation of a putative N-terminal myristoylation site, proposed to be involved in membrane targeting, rendered the Fen protein inactive . Analysis of a Pto-Fen chimeric gene allowed the fenthion sensitivity domain to be localized to the C-terminal part of the Fen protein . Interestingly, expression of the Fen kinase from the PVX genome in Nicotiana spp resulted in a fenthion-independent necrotic response . Our results support the involvement of the Fen gene in a signal transduction pathway(s).

Gene Ther, 1995 Mar, 2(2), 116 - 23
Design of a genetic immunotoxin to eliminate toxin immunogenicity; Chen SY et al.; Host antibody response to toxin molecules is a major obstacle to the use of immunotoxins as efficacious agents in the treatment of human cancer and other diseases . In this study, a genetic form of immunotoxin has been designed which should eliminate toxin immunogenicity by replacing the toxin protein moiety with weakly immunogenic or nonimmunogenic plasmid DNA . A recombinant bifunctional fusion protein, which consists of a human antibody Fab targeting moiety {directed against gp120, the envelope glycoprotein of human immunodeficiency virus (HIV)-1} and a human DNA binding moiety (protamine), is used as a gene carrier . Toxin plasmid DNAs expressing the catalytic fragment of Pseudomonas exotoxin A (PEA) statically interact with the fusion proteins to form soluble protein-DNA complexes . The complexes are specifically transferred into HIV-1-infected cells by receptor-mediated endocytosis, resulting in selective killing of the target cells . These 'genetic immunotoxins' may have significant advantages over protein immunotoxins for the treatment of a variety of human diseases.

Immunity, 1995 Mar, 2(3), 281 - 7
Construction of a functional disulfide-stabilized TCR Fv indicates that antibody and TCR Fv frameworks are very similar in structure; Reiter Y et al.; Disulfide-stabilized Fvs (dsFv) are recombinant Fv fragments of antibodies in which the inherently unstable VH-VL heterodimer is stabilized by an interchain disulfide bond engineered between structurally conserved framework positions . We now design and produce a disulfide-stabilized Fv of a T cell receptor . It is composed of V alpha and V beta variable domains of the 2B4 TCR stabilized by a disulfide bond between framework residues of the TCR Fv at a site corresponding to that used for disulfide stabilization of antibody Fvs . For ease of production and detection, the TCRdsFv was fused to a truncated form of Pseudomonas exotoxin (PE38) . The TCR(dsFv) retains its native conformation and is much more stable than a TCR scFv . Moreover, it is functional in biological assays . Because successful disulfide stabilization of the TCR Fv by the positions used for antibody Fv stabilization would not occur unless the mutated residues in TCR Fv are at positions closely similar to those in antibody Fvs, most likely within less than 1.5 A, these results provide very strong experimental evidence for the structural similarity between immunoglobulin and TCR antigen-binding variable domains.

Mikrobiol Z, 1995 Mar-Apr, 57(2), 80 - 5
{The biological activity of Pseudomonas solanacearum polysaccharide}; Varbanets LD et al.; Antitumour, antileukosis and antimetastatic effects of lipopolysaccharide (LPS) isolated from Pseudomonas solanacearum have been studied . It is established that LPS does not possess the antitumour effect on experimental animals with Lewis lung carcinoma, melanoma B-16 and sarcoma S-37 and vice versa, intensifies the tumour growth . The life time of animals with experimental leukoses lymphocytic leukemia P-388 and lymphoid leukemia L 1210 inconsiderably increases . At the same time LPS possesses the expressed antimetastatic effect that has manifested in the decrease of the volume (40 and 5 times) and of the amount (4-4.2 times) of metastases in mice with Lewis lung carcinoma and melanoma B-16, respectively . Study of the contribution of certain structural components of LPS molecule to the total biological activity has shown that O-specific polysaccharide and oligosaccharide of core take the expressed antimetastatic effect . Lipid A in the used dose weakly modified the development of Lewis lung carcinoma metastases.

Hua Xi Yi Ke Da Xue Xue Bao, 1995 Mar, 26(1), 120 - 2
{Study on the optimums for Pseudomonas maltophilia HCG receptor expression}; Zhang C et al.; According to the design of orthogonal experiment, the optimums for expression of Pseudomonas maltophilia with HCG receptor were studied by radioassay . The results showed that the optimums influencing the specific binding percentage of Pseudomonas maltophilia were the cultures of No . 3 solid medium added with 0.5% galactose and incubated at a temperature of 37 C for 72 hours . Under these conditions, the binding percentage of Pseudomonas maltophilia to HCG was higher than those under other conditions.

Am J Respir Crit Care Med, 1995 Mar, 151(3 Pt 1), 899 - 903
Normal sweat chloride values do not exclude the diagnosis of cystic fibrosis; Stewart B et al.; Since its introduction in 1959, the sweat test has remained the "gold standard" diagnostic test for cystic fibrosis (CF) . It is apparent that CF encompasses a wide spectrum of disease, from meconium ileus and severe respiratory compromise in infants to the presentation of mild pulmonary symptoms and no evidence of gastrointestinal disease in adults . In patients with lung disease that might otherwise be consistent with CF, normal sweat chloride (Cl-) values have tended to exclude the diagnosis . In this report we describe two patients from two families with the compound heterozygotic CF mutations delta F508/3849 + 10 kb C-->T . These patients had mild manifestations of disease, including clinical pancreatic sufficiency (normal growth without pancreatic enzyme supplementation) and absence of azoospermia . Sweat Cl- values were in the normal range . However, both patients developed bronchiectasis, progressive obstructive lung disease, and colonization with Pseudomonas . The diagnosis of CF was made using nasal transepithelial voltage measurements and genotyping . These cases emphasize the need to maintain a high index of suspicion of CF in atypical cases, and to pursue alternative diagnostic tests to confirm a diagnosis of CF suspected on clinical grounds, despite normal sweat test results.

Gene, 1995 Feb 27, 154(1), 65 - 70
Sequence analysis of the gene cluster encoding toluene-3-monooxygenase from Pseudomonas pickettii PKO1; Byrne AM et al.; The nucleotide (nt) sequence and gene organization of the locus encoding the initial step of the toluene-3-monooxygenase (Tbu) pathway from Pseudomonas pickettii PKO1 has been determined . This is the first reported nt sequence for a toluene monooxygenase which hydroxylates the C-3 position of toluene . Six tightly assembled structural genes encoding several Tbu were identified and were designated tbuA1, tbuU, tbuB, tbuV, tbuA2 and tbuC . Comparison of the deduced amino acid (aa) sequences of each open reading frame (ORF) with translated sequences from the GenBank database revealed significant overall homology to peptides from the toluene-4-monooxygenase (Tmo) from Pseudomonas mendocina KR1, the multicomponent phenol hydroxylase (Dmp) from Pseudomonas sp . strain CF600 and the methane monooxygenases (Mmo) from both Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b . Similarities in both size and aa sequence between the peptides from these multicomponent oxygenases and the putative peptides from Tbu suggested roles for the TbuA1, TbuB, TbuV, TbuA2 and TbuC proteins.

Gene, 1995 Feb 3, 153(1), 17 - 23
Sequence, expression and transcriptional analysis of the coronafacate ligase-encoding gene required for coronatine biosynthesis by Pseudomonas syringae; Liyanage H et al.; Pseudomonas syringae pv . glycinea PG4180 produces the chlorosis-inducing phytotoxin coronatine (COR), which consists of a polyketide component, coronafacic acid (CFA), ligated by an amide bond to coronamic acid (CMA), an ethylcyclopropyl amino-acid derived from isoleucine . We report the nucleotide sequence of a 2.37-kb region containing the coronafacate ligase-encoding gene (cfl) which is required for the amide linkage of CFA and CMA . The transcription start point for cfl was identified, and the Cfl protein was overproduced from the T7lac promoter in Escherichia coli . The deduced amino-acid sequence of Cfl showed homology to a variety of adenylate-forming enzymes which bind and hydrolyze ATP in order to activate their substrates for further ligation.

FEMS Microbiol Lett, 1995 Feb 1, 126(1), 19 - 23
Purification and characterization of the recombinant alginate lyase from Pseudomonas sp . leaked by Escherichia coli upon addition of glycine; Fujiyama K et al.; The plasmid pAL205 encodes an alginate lyase gene of Pseudomonas sp . OS-ALG-9, fused in frame to the beta-galactosidase alpha-peptide gene . The alginate lyase (Aly) expressed in Escherichia coli (pAL205) was significantly secreted into the medium by the addition of glycine . The extracellular enzyme isolated from the culture of E . coli JM109 (pAL205) was purified over 15,000-fold by successive chromatography and subjected to amino acid sequence analysis . The sequence determined was identical to that of the intracellular protein . Since the activity and molecular size of the extracellular Aly is identical to the intracellular protein and to the Aly isolated from Pseudomonas, the glycine does not affect or modify the Aly during its leakage into the medium.

FEMS Microbiol Lett, 1995 Feb 1, 126(1), 13 - 7
High gene expression in Escherichia coli of recombinant alginate lyase as a fused protein with beta-galactosidase alpha-peptide; Fujiyama K et al.; Escherichia coli LE392 (pAL28) was previously isolated as a positive clone harboring the alginate lyase gene (aly) from an alginate-degrading strain, Pseudomonas sp . OS-ALG-9 . The plasmid pAL205, one of the constructs obtained after successive subcloning of pAL28, gave the highest expression of aly in E . coli cells . A 8-fold increase in the alginate lyase (Aly) activity in E . coli JM109 (pAL205) was induced with isopropyl-beta-D-thiogalactoside, which was 210 times higher than that in E . coli LE392 (pAL28) . The highly significant increase in the expression of the Aly enzyme with pAL205 was investigated through the nucleotide sequence around the 5' region of aly as well as the N-terminal sequence of the purified enzyme . It was found that the Aly expressed in E . coli (pAL205) was a fused protein containing 7 residues from the N-terminus of beta-galactosidase alpha-peptide and the mature protein found in the Pseudomonas sp . except for three residues in the N-terminal.

Curr Eye Res, 1995 Feb, 14(2), 153 - 8
Ocular distribution of the chimeric protein IL2-PE40; BenEzra D et al.; A construct of IL-2 and pseudomonas exotoxin (PE40) has been genetically engineered . An aliquot of 100 microliter of the chimeric protein, radiolabelled with I125, was administered to healthy rats by various routes . At different intervals, ocular and non ocular tissues were removed and the levels of the radiolabelled chimeric protein IL-2-PE40 measured . Systemic administration of IL2-PE40 either intravenously (IV) or intraperitoneally (IP) leads to high levels of the drug in the blood, liver and spleen . Little or no radioactivity is observed within the ocular tissues using this route . On the other hand, local administration of the drug either as subtenon injection or as eye drops resulted in a very high concentration of the drug within the conjunctiva, cornea and sclera, with little radioactivity detected systemically . Subtenon injection induced a significant drug level within the optic nerve . With the drops, the chimeric protein was also detected, in low levels, intraocularly.

Kidney Int, 1995 Feb, 47(2), 603 - 10
Diffusive and convective transfer of cytokine-inducing bacterial products across hemodialysis membranes; Pereira BJ et al.; The widespread use of bicarbonate dialysate, and high-flux and high-efficiency dialyzers have raised concerns regarding the transmembrane passage of bacterial products from the dialysate into the blood compartment . To study the mechanisms as well as magnitude of the transmembrane transfer of bacterial products from the dialysate, we developed a computerized in vitro dialysis model which provides continuous pressure recording from the arterial, venous, dialysate inflow and outflow ports . By virtue of a computer controlled on-line infusion pump, this model permits control of ultrafiltration/backfiltration . Heparinized (10 U/ml) whole blood (150 ml) was circulated through the blood compartment for 120 minutes at 100 ml/min . Bicarbonate dialysate contaminated with Pseudomonas maltophilia filtrate was circulated through the dialysate compartment at 100 ml/min . A two-point pressure of -10 mm of Hg (ultrafiltration) was maintained for the first 60 minutes and -10 mm of Hg (backfiltration) for the next 60 minutes . Whole blood samples (10 ml) were drawn from the blood at 0, 60 and 120 minutes . Peripheral blood mononuclear cells (PBMC) harvested from these samples were incubated for 24 hours in tissue culture medium . In addition, 0.5 ml samples of dialysate were collected at 0, 60 and 120 minutes and incubated with PBMC from the same donor for 24 hours . After 24 hour incubation, total cell-associated IL-1Ra and IL-1 beta were measured by specific radioimmunoassay . Paired experiments were performed with eight high-flux synthetic membranes (polyamide) and eight low-flux cellulose membranes (hemophan) . Cytokine production is expressed as pg/2.5 million PBMC.(ABSTRACT TRUNCATED AT 250 WORDS)

Plant J, 1995 Feb, 7(2), 309 - 20
Differential in vitro DNA binding activity to a promoter element of the gn1 beta-1,3-glucanase gene in hypersensitively reacting tobacco plants; Alonso E et al.; In a hypersensitive reaction to pathogen infection, expression of the beta-1,3-glucanase gn1 gene is induced in cells surrounding the necrotic lesions . The 5'-flanking sequence of gn1 was examined to investigate the molecular basis controlling activation of gene expression during this plant defense response . Studies on transgenic tobacco plants containing gn1 promoter deletions fused to the beta-glucuronidase reporter gene revealed the presence of negative and positive regulatory sequences mediating both the level and the spatial distribution of gn1 expression . Promoter sequences to -138 bp were sufficient to confer increased gene expression around the necrotic lesions produced in response to Pseudomonas syringae pv . syringae inoculation . It is demonstrated by electrophoretic mobility shift assays that nuclear proteins in both healthy and hypersensitively reacting tobacco leaves interact with DNA sequences within the regulatory elements identified . Among the binding sequences characterized, the promoter region extending from -250 to -217 bp contained the DNA motif -GGCGGC- found to be conserved in most if not all promoters of genes encoding pathogenesis-related basic proteins . The activity bound by this promoter sequence was stronger in hypersensitively responding tissues than in healthy untreated tobacco leaves.

Appl Environ Microbiol, 1995 Feb, 61(2), 840 - 3
Bacterial degradation of m-nitrobenzoic acid; Nadeau LJ et al.; Pseudomonas sp . strain JS51 grows on m-nitrobenzoate (m-NBA) with stoichiometric release of nitrite . m-NBA-grown cells oxidized m-NBA and protocatechuate but not 3-hydroxybenzoate, 4-hydroxy-3-nitrobenzoate, 4-nitrocatechol, and 1,2,4-benzenetriol . Protocatechuate accumulated transiently when succinate-grown cells were transferred to media containing m-NBA . Respirometric experiments indicated that the conversion of m-NBA to protocatechuate required 1 mol of oxygen per mol of substrate . Conversions conducted in the presence of 18O2 showed the incorporation of both atoms of molecular oxygen into protocatechuate . Extracts of m-NBA-grown cells cleaved protocatechuate to 2-hydroxy-4-carboxymuconic semialdehyde . These results provide rigorous proof that m-NBA is initially oxidized by a dioxygenase to produce protocatechuate which is further degraded by a 4,5-dioxygenase.

Appl Environ Microbiol, 1995 Feb, 61(2), 538 - 43
Cloning and sequencing of the genes involved in glyphosate utilization by Pseudomonas pseudomallei; Penaloza-Vazquez A et al.; Thirty-four strains of Pseudomonas pseudomallei isolated from soil were selected for their ability to degrade the phosphonate herbicide glyphosate . All strains tested were able to grow on glyphosate as the only phosphorus source without the addition of aromatic amino acids . One of these strains, P . pseudomallei 22, showed 50% glyphosate degradation in 40 h in glyphosate medium . From a genomic library of this strain constructed in pUC19, we have isolated a plasmid carrying a 3.0-kb DNA fragment which confers to E . coli the ability to use glyphosate as a phosphorus source . This 3.0-kb DNA fragment from P . pseudomallei contained two open reading frames (glpA and glpB) which are involved in glyphosate tolerance and in the modification of glyphosate to a substrate of the Escherichia coli carbon-phosphorus lyase . glpA exhibited significant homology with the E . coli hygromycin phosphotransferase gene . It was also found that the hygromycin phosphotransferase genes from both P . pseudomallei and E . coli confer tolerance to glyphosate.

Appl Environ Microbiol, 1995 Feb, 61(2), 443 - 7
A meta cleavage pathway for 4-chlorobenzoate, an intermediate in the metabolism of 4-chlorobiphenyl by Pseudomonas cepacia P166; Arensdorf JJ et al.; Bacterial degradation of biphenyl and polychlorinated biphenyls proceeds by a well-studied pathway which produces benzoate and 2-hydroxypent-2,4-dienoate (or, in the case of polychlorinated biphenyls, the chlorinated derivatives of these compounds) . Pseudomonas cepacia P166 utilizes 4-chlorobiphenyl for growth and produces 4-chlorobenzoate as a central intermediate . In this study we found that strain P166 further transforms 4-chlorobenzoate to 4-chlorocatechol, which is mineralized by a meta cleavage pathway . Key metabolites which we identified include the meta cleavage product (5-chloro-2-hydroxymuconic semialdehyde), 5-chloro-2-hydroxymuconate, 5-chloro-2-oxopent-4-enoate, 5-chloro-4-hydroxy-2-oxopentanoate, and chloroacetate . Chloroacetate accumulated transiently, and slow but stoichiometric dehalogenation was observed.

Singapore Med J, 1995 Feb, 36(1), 60 - 2
Comparison of Pseudomonas pseudomallei from humans, animals, soil and water by restriction endonuclease analysis; Yap EH et al.; Pseudomonas pseudomallei isolates from 62 human, 17 animal, 3 soil and 3 water samples were examined by genomic DNA digestion with PstI . Five major (RE I, II, III, IV, V) reproducible restriction patterns were observed, with most (56/62) of the human isolates displaying RE I (30/62), II (5/62), III (15/62), IV (4/62), V (2/62), and the animal (16/17), soil (2/3), water (3/3) isolates showing predominantly RE II profiles . Six human and one soil isolates showed patterns different from those of RE I to V . Restriction endonuclease analysis may be applied in epidemiological studies of melioidosis.

J Thorac Cardiovasc Surg, 1995 Feb, 109(2), 224 - 34; discussion 234-5
Improved results of lung transplantation for patients with cystic fibrosis; Egan TM et al.; Patients with cystic fibrosis pose particular challenges for lung transplant surgeons . Earlier reports from North American centers suggested that patients with cystic fibrosis were at greater risk for heart-lung or isolated lung transplantation than other patients with end-stage pulmonary disease . During a 3 1/2 year period, 44 patients with end-stage lung disease resulting from cystic fibrosis underwent double lung transplantation at this institution . During the same interval, 18 patients with cystic fibrosis died while waiting for lung transplantation . The ages of the recipients ranged from 8 to 45 years, and mean forced expiratory volume in 1 second was 21% predicted . Seven patients had Pseudomonas cepacia bacteria before transplantation . Bilateral sequential implantation with omentopexy was used in all patients . There were no operative deaths, although two patients required urgent retransplantation because of graft failure . Cardiopulmonary bypass was necessary in six procedures in five patients and was associated with an increased blood transfusion requirement, longer postoperative ventilation, and longer hospital stay . Actuarial survival was 85% at 1 year and 67% at 2 years . Infection was the most common cause of death within 6 months of transplantation (Pseudomonas cepacia pneumonia was the cause of death in two patients), and bronchiolitis obliterans was the most common cause of death after 6 months . Actuarial freedom from development of clinically significant bronchiolitis obliterans was 59% at 2 years . Results of pulmonary function tests improved substantially in survivors, with forced expiratory volume in 1 second averaging 78% predicted 2 years after transplantation . Double lung transplantation can be accomplished with acceptable morbidity and mortality in patients with cystic fibrosis.

Biochemistry, 1995 Jan 17, 34(2), 426 - 34
Enzymatic and nonenzymatic polarizations of alpha,beta-unsaturated ketosteroids and phenolic steroids . Implications for the roles of hydrogen bonding in the catalytic mechanism of delta 5-3-ketosteroid isomerase; Zhao Q et al.; Ketosteroids (e.g., 19-nortestosterone) and phenolic steroids (e.g., 17 beta-estradiol and 17 beta-dihydroequilenin), which are potent competitive inhibitors of delta 5-3-ketosteroid isomerase (isomerase, EC 5.3.3.1) of Pseudomonas testosteroni, undergo significant polarization upon binding to the active site of the enzyme . The 10 nm red shift of the UV absorption maximum of the enone chromophore of 19-nortestosterone, which occurs in the enzyme-steroid complex, resembles that observed when this steroid is exposed to strong acid . The UV and fluorescence spectral changes of 17 beta-estradiol and 17 beta-dihydroequilenin in the enzyme-bound complex resemble the spectra of ionized phenolate species in aqueous basic solutions . Since most enzymes bind their substrates and competitive inhibitors in a solvent-inaccessible hydrophobic environment, and the generation of charges in such nonpolar environments is unfavorable, we investigated the possibility that the spectral perturbations of the steroids might arise from strong hydrogen bonding in nonpolar environments . For this purpose, the spectral properties of model compounds capable of forming intramolecular hydrogen bonds were studied in nonpolar solvents . Thus, 4-hydroxyandrost-4-ene-3,17-dione, in which the 4-hydroxyl group is intramolecularly hydrogen-bonded to the 3-carbonyl group through a five-membered ring, exhibits a lambda max of 276.0 nm, while the corresponding 4-methyl ether, 4-methoxyandrost-4-ene-3,17-dione, which cannot form an internal hydrogen bond, shows a lambda max of 258.5 nm in aqueous solution.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1995 Jan 13, 270(2), 541 - 4
Conservation of a common motif in enzymes catalyzing ADP-ribose transfer . Identification of domains in mammalian transferases; Takada T et al.; Bacterial toxin ADP-ribosyltransferases, e.g . diphtheria toxin (DT) and pertussis toxin, have in common consensus sequences involved in catalytic activity, which are localized to three regions . Region I is notable for a histidine or arginine; region II, approximately 50-75 amino acids downstream, is rich in aromatic/hydrophobic amino acids; and region III, further downstream, has a glutamate and other acidic amino acids . A similar motif was observed in the sequence of the glycosylphosphatidylinositol-linked muscle ADP-ribosyltransferase . Site-directed mutagenesis was performed to verify the role of this motif . Proteins were expressed in rat adenocarcinoma cells, released from the cell with phosphatidylinositol-specific phospholipase C, and quantified with polyclonal antibodies . Transferase His114 in region I aligned with His21 of DT; as with DT, the H114N mutant was active . Aromatic/hydrophobic amino acids (region II) were found approximately 30-50 amino acids downstream of this histidine . Although transferase has a Glu278-Tyr-Ile sequence characteristic of region III in DT, Glu278 was not critical for activity . In an alternative region III containing Glu238-Glu239-Glu240, Glu238 and Glu240 but not Glu239 were critical . Glu240 aligned with critical glutamates in DT, Pseudomonas exotoxin, and C3 transferase . Thus, the mammalian ADP-ribosyltransferases have motifs similar to toxin ADP-ribosyltransferases, suggesting that these sequences are important in ADP-ribose transfer reactions.

Arch Biochem Biophys, 1995 Jan 10, 316(1), 241 - 8
Purification and characterization of the flavoprotein tryptophan 2-monooxygenase expressed at high levels in Escherichia coli; Emanuele JJ et al.; Tryptophan 2-monooxygenase from Pseudomonas savastanoi is a flavoprotein which catalyzes the formation of indoleacetamide from tryptophan . This is the first step in a two-step pathway for the formation of indoleacetic acid during infection of plants and subsequent gall formation by this and other bacteria . The enzyme has been expressed in Escherichia coli at high levels, and a purification procedure has been developed which generates micromolar amounts of protein . The purified enzyme contains tightly bound indoleacetamide; a method involving dialysis against 20% methanol has been developed for removing the indoleacetamide without significant loss of enzyme activity . Amino acids with large hydrophobic side chains are the best substrates; N-substituted phenylalanines will also act as substrates . N-ethylmaleimide, methyl methanethiol-sulfonate, and diethylpyrocarbonate act as active site-directed reagents, consistent with a histidine and a cysteine at or near the enzyme active site . Vinylglycine partially inactivates the enzyme, while propargylglycine has no effect.

J Biol Chem, 1995 Jan 6, 270(1), 218 - 24
Novel degradation pathway of glycated amino acids into free fructosamine by a Pseudomonas sp . soil strain extract; Gerhardinger C et al.; A Pseudomonas sp . soil strain, selected for its ability to grow on epsilon-(1-deoxyfructosyl) aminocaproic acid, was induced to express a membrane-bound enzymatic activity which oxidatively degrades Amadori products into free fructosamine . Apparent Km values for fructosyl aminocaproate, epsilon-fructosyl lysine, fructosyl glycine, and ribated lysine were 0.21 mM, 2.73 mM, 3.52 mM, and 1.57 mM, respectively . The enzyme was also active against alpha-fructosyl lysine and borohydride-reduced Amadori product, weakly active with ribated and glycated polylysine, and inactive with reducing sugars, amino acids, and glycated proteins . The enzymatic activity was highest at pH 6.5 and 25 degrees C in 0.1 M sodium phosphate, while over 80% of the activity was lost above 65 degrees C . Complete inhibition was observed by HgCl2, NaN3, and NaCN suggesting a role for SH groups and copper in the enzymatic activity . The reaction products were characterized by 1H NMR, 13C NMR, and GC/MS and found to correspond to 1-deoxy-1-aminofructose, i.e . free "fructosamine," and adipic acid . Confirmation of the free fructosamine structure was based on the complete spectroscopic identity of the borohydride reduction product with commercially available glucamine (1-amino-1-deoxyglucitol) . The new enzyme is provisorily classified as fructosyl N-alkyl amino acid oxidase (EC 1.5.3) (fructosyl-amino acid:oxygen oxidoreductase) and may thus belong to a novel class of "Amadoriases" which deglycate Amadori products oxidatively . In contrast, however, the new enzyme acts on the alkylamine bond rather than the ketoamine bond of the Amadori product.

Bull Soc Belge Ophtalmol, 1995, 259, 45 - 52
A peculiar case of necrotizing sclerokeratitis with Pseudomonas infection; Hanssens M; A 71-year old woman complains of painless inflammation, discharge and decreased vision at the right eye since two weeks . Eight years before she was operated at this eye for a nasally located pterygium and received adjuvant radiotherapy . On examination a necrotizing scleritis was seen which rapidly involved the cornea and was infected by Pseudomonas . Despite intensive antibiotherapy the sclerokeratitis extended further and eventually lead to enucleation . Surgically induced scleritis-especially after pterygium surgery with adjuvant radiotherapy-and complicating Pseudomonas infection are discussed.

Acta Microbiol Pol, 1995, 44(3-4), 255 - 66
Formation of tomatine in tomato plants infected with Streptomyces species and treated with herbicides, correlated with reduction of Pseudomonas solanacearum and Fusarium oxysporum f . sp . lycopersici; El-Raheem A et al.; Pretreatment of tomato seeds with pendimethalin or metribuzin and inoculation of seedlings with the antagonistic Streptomyces corchorusii or/and Streptomyces mutabilis were tested for the formation of tomatine in roots and stems of tomato, infested with Pseudomonas solancearum or/and Fusarium oxysporum f . sp . lycopersici . All treatments induced the formation of variable quantities of tomatine, compared with untreated control . The variation was proportional to: the pathogen, Fusarium was more stimulating than Pseudomonas; the antagonistic organism, S . corchorusii being more eliciting than S . mutabilis; the herbicide and its concentration, pendimethalin at 2 x 10(-3) M being the most eliciting of tomatine; and according to the soil, plants grown in non-sterilized soil accumulated more tomatine than did these grown in sterilized soil . In all treatments, stems had more tomatine than roots and non-sterilized soil was better than sterilized soil . The antagonistic streptomycetes induced accumulation of tomatine more than did the herbicides . The highest amounts of tomatine were detected in plants pretreated with pendimethalin at 2 x 10(-3) M, grown in non-sterilized soil, infested with F . oxysporum, and inoculated with S . corchorusii and S . mutabilis . The effect of the extracted tomatine on the growth of Fusarium and Pseudomonas was examined in vitro . The crude extract of tomatine from all treatments reduced growth and sporulation of F . oxysporum and growth of P . solanacearum in defined media . The reduction varied according to the treatment and was proportional to the quantities of extracted tomatine, the highest amounts being the most effective . The mechanism of phytoalexins in controlling tomato wilt pathogens was also discussed.

Folia Microbiol (Praha), 1995, 40(5), 454 - 6
Metabolism of 2-chlorobenzoic acid in Pseudomonas stutzeri; Kozlovsky SA et al.; 3-Chloropyrocatechol is formed as a result of oxidation of 2-chlorobenzoate by Pseudomonas stutzeri . 2-Chloro-cis,cis-muconic acid is the product of oxidation of 3-chloropyrocatechol . A catabolic pathway for the degradation of 2-chlorobenzoate by a newly isolated strain of P . stutzeri is proposed.

Int Urol Nephrol, 1995, 27(5), 563 - 5
Use of fibrin sealant in the treatment of prostatic cutaneous fistula in a case of Pseudomonas prostatitis; Felipetto R et al.; Authors report on a case of Pseudomonas prostatitis complicated with a prostatoperineal fistula in continuity with the urethra that has been successfully treated with human fibrin sealant (Tissucol).

Hum Antibodies Hybridomas, 1995, 6(4), 145 - 52
A human monoclonal antibody to carbohydrate moiety of carcinoembryonic antigen; Tsukazaki K et al.; Hybridoma BSRF-S-97, secreting a human monoclonal antibody of IgG1 subclass reactive to the carcinoembryonic antigen, was generated by fusing the regional lymph node lymphocytes from a cervical cancer patient with RF-S1 human-mouse heteromyeloma fusion line . This monoclonal antibody was found specifically reactive to carinoembryonic antigen-producing cell lines, including those of cervical cancer (SKG-II), mucinous type ovarian cancer (RMUG-L), stomach cancer (MKN-45), and lung cancer (PC-10) . The monoclonal antibody reactivity with pepsin- and periodate-treated carcinoembryonic antigen demonstrated that this monoclonal antibody recognizes the carbohydrate moiety of carcinoembryonic antigen specifically . Possibilities of the monoclonal antibody reaction with mucin and blood-group antigens were excluded by the comparative studies with a placental mucin-containing protein which reacted with carcinoembryonic antigen-specific rabbit polyclonal antibody . The monoclonal antibody conjugated with Pseudomonas exotoxin showed potent regression effects on the growth of the MKN-45 cell line in both the dish culture and xenografted nude mice, indicating potential usefulness of this human monoclonal antibody as a promising tumor targeting vehicle.

C R Seances Soc Biol Fil, 1995, 189(5), 705 - 12
{Cloning of genes coding for 3-alpha-hydroxysteroid dehydrogenase and for (3-17)-beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni}; Floch HH et al.; A genomic library of Pseudomonas testosteroi total DNA constructed from SauIIIA digests ligated to a lambda gt11 vector was probed with different polyclonal antibodies raised against purified 3 alpha-HSD and (3 beta-17 beta)-HSD . Two different clones reacting with one antibody were selected . The clone reacting with (3-17)beta-HSD antibody contained a 2,661-base pair insert and was found to contained an open reading frame of 765 base pair that corresponds to a protein of 254 amino-acid residues . A 1,492-base pair was inserte in pBR 322 plasmid vector; the recombinant bacterie over expressed the (3-17)beta-HSD gene . The clone reacting with 3 alpha-HSD antibody contained a 1746 base pair insert which contained an open reading frame of 696 base pairs that corresponds to a protein of 231 amino-acid residues . A search for homologous proteins was performed . Distant similarities were found between (3-17)beta-HSD and members of the short-chain alcool dehydrogenase (SCAD) family but no similarity was observed between 3 alpha-HSD and proteins of this family.

Microbiol Immunol, 1995, 39(7), 437 - 42
Characterization of Pseudomonas paucimobilis FP2001 which forms flagella depending upon the presence of rhamnose in liquid medium; Miake F et al.; A bacterial strain FP2001 isolated from the exudate of land reclaimed for municipal waste was identified as Pseudomonas paucimobilis . Cells of strain FP2001 were mobile by means of polar monotrichous flagellum, only when rhamnose was added as a carbon source in the liquid medium . The replacement of rhamnose by arabinose, galactose, glucose or xylose did not lead to the formation of flagella.

Mol Med, 1995 Jan, 1(2), 206 - 16
Expression cloning of cDNAs that render cancer cells resistant to Pseudomonas and diphtheria toxin and immunotoxins; Brinkmann U et al.; BACKGROUND: Several immunotoxins in which antibodies are coupled to plant or bacterial toxins are now in clinical trials for the treatment of cancer . One of these is B3-LysPE38 in which MAb B3 which reacts with many human cancers, is coupled with a genetically modified form of Pseudomonas exotoxin (PE) . MATERIALS AND METHODS: To investigate how cells can become resistant to PE-derived immunotoxins, we constructed an immunotoxin-sensitive MCF-7 breast cancer cell line that contains SV40 T antigen and allows episomal replication of SV40 origin containing plasmids . We transfected a pCDM8/HeLa cDNA expression library into these cells, thereby causing over-expression of the plasmid-encoded genes . The transfected cells were treated with immunotoxin to select for resistance-mediating plasmids, which were reisolated from these cells and amplified in Escherichia coli . The resulting plasmid pool was transfected into cells for two further rounds of selection and plasmid reisolation . RESULTS: Several plasmids that caused immunotoxin resistance were enriched by this selection procedure . Four plasmids were stably transfected into MCF-7 cells and found to increase their resistance to PE-derived immunotoxins by 5- to 20-fold . These plasmids also confer resistance to native PE and to diphtheria toxin but not to ricin or cycloheximide . Thus, they appear to specifically interfere with the action of ADP-ribosylating toxins . CONCLUSION: Cancer cells can become resistant to immunotoxins by deregulated expression of normal genes . The clinical significance of this type of resistance will be evaluated in clinical trials.

J Bacteriol, 1995 Jan, 177(1), 20 - 6
Dihydroxylation and dechlorination of chlorinated biphenyls by purified biphenyl 2,3-dioxygenase from Pseudomonas sp . strain LB400; Haddock JD et al.; Oxidation of biphenyl and nine chlorinated biphenyls (CBs) by the biphenyl 2,3-dioxygenase from Pseudomonas sp . strain LB400 was examined . The purified terminal oxygenase required the addition of partially purified electron transport components, NAD(P)H, and ferrous iron to oxidize biphenyl and CBs . cis-Biphenyl 2,3-dihydrodiol was produced with biphenyl as the substrate . Dihydrodiols were produced from all CBs, and more than one compound was produced with most substrates . Catechols were produced when the dioxygenase-catalyzed reaction occurred at the 2,3 position of a 2-chlorophenyl ring, resulting in dechlorination of the substrate . Oxidation at the 3,4 position of a 2,5-dichlorophenyl ring produced a 3,4-dihydrodiol . Compounds resulting from both types of reaction were produced during oxidation of 2,5,2'-trichlorobiphenyl . The broad substrate specificity and the ability to oxidize at different ring positions suggest that the biphenyl 2,3-dioxygenase is responsible for the wide range of CBs oxidized by Pseudomonas sp . strain LB400.

J Virol, 1995 Jan, 69(1), 75 - 81
Unique insertion sequence and pattern of CD4 expression in variants selected with immunotoxins from human immunodeficiency virus type 1-infected T cells; Fang H et al.; To study the variability of human immunodeficiency virus type 1 (HIV-1), we used immunotoxins to select for variants within a population of H9 cells persistently infected with a molecular clone of HIV-1 designated NL4-3 . Chimeric immunotoxin CD4-PE40 (a chimeric fusion protein consisting of the amino-terminal two domains of CD4 and the carboxy-terminal domains of Pseudomonas exotoxin A) was used to select for cells lacking cell surface expression of HIV Env (envelope proteins gp160, gp120, and gp41) . The cells described here (A1, A7, C9, and E9) fail to express HIV proteins because they have markedly diminished transcription of the integrated provirus (A1, A7, and E9) or no HIV provirus (C9) . Analysis demonstrated that two different cloned variants, A1 and E9, contain the complementary sequence of tRNA(3Lys) (45 bp) inserted 3' to the primer-binding site, following by a 169-bp deletion through the start of the gag gene . No HIV mRNA was detected by Northern (RNA) blotting, but PCR demonstrated the presence of the viral message . These variants were found very infrequently in the unselected H9/NL4-3 cell population, and they contained proviruses distinct from that found in the dominant population . In addition, all of these variants had similar patterns of CD4 surface expression that allowed them to escape reinfection within the tissue culture . The data are discussed with regard to mechanisms and errors of HIV reverse transcription, as well as the evolution of mutants within a population of persistently infected cells.

J Virol, 1995 Jan, 69(1), 513 - 6
Isolation of the measles virus hemagglutinin protein in a soluble form by protease digestion; Sato TA et al.; The hemagglutinin (H) glycoprotein was isolated in a soluble form by digesting measles virus particles with an endoproteinase, Asp-N (from a Pseudomonas fragi mutant) . Digestion of H with Asp-N brought about glycopeptides in three different forms, depending on the cleaving site: AHD, which has an M(r) of 66,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and which formed a disulfide-linked homodimer with an M(r) of 132,000, and two monomeric digestion products, AHM-1 (with an M(r) of 64,000) and AHM-2 (with an M(r) of 58,000) . The susceptibility of the H glycoprotein to the protease depended on the enzyme concentration . AHD was readily formed at a low concentration of Asp-N, while AHM-1 and AHM-2 required higher and even higher protease concentrations, respectively . All of the cleavage products reacted with monoclonal antibodies to various epitopes of the H protein; however, only AHD showed a significant hemagglutinin activity on African green monkey erythrocytes . The hemagglutinin activities of AHM-1 and AHM-2 were restored after a monoclonal antibody lacking the hemagglutination-inhibiting activity was added to the reaction mixture . AHDs purified by size-exclusion high-pressure liquid chromatography had two associating forms; one had an M(r) higher than and the other an M(r) as high as that of a tetramer . The former was associated noncovalently in addition to having two intermolecular disulfide bonds, and the latter was associated covalently with a single intermolecular disulfide bond and was also duplicated through a noncovalent association . In addition, both AHM-1 and AHM-2, having no intermolecular disulfide bond, were in a dimer form . These results suggest that AHM-1 and AHM-2 are monovalent in the hemagglutinin activity, while AHDs are divalent . Comparative analyses of the N termini of these soluble glycopeptides with the sequence of H suggested that the cysteine residue at position 139 was responsible for the intermolecular disulfide bonding between the monomeric H glycoproteins . The cysteine at position 154 was also suggested to participate in the forming of the intermolecular disulfide bond.

Eur J Haematol, 1995 Jan, 54(1), 18 - 20
Outpatient supportive therapy after induction to remission therapy in adult acute myelogenous leukaemia (AML) is feasible: a multicentre study; Ruiz-Arguelles GJ et al.; Twenty-four adult patients with AML were treated with standard "7 + 3" chemotherapy . After administering the myeloablative drugs in the hospital, patients were instructed to continue their supportive treatment on an outpatient basis; they received ciprofloxacin, cotrimoxasole and itraconazole vo until the absolute granulocyte count rose above 1 x 10(9)/l . Platelet concentrates were given every other day until the platelet count rose above 20 x 10(9)/l . Complete remission (CR) was obtained in 87% . Fever developed in 29% and 2 cases were complicated by indwelling-catheter-related Pseudomona aeruginosa septicaemia, 1 Entamoeba hystolytica enteritis and 1 Pneumocystis carinii pneumonia; these patients were hospitalized to treat these infections specifically . In no case was the infection fatal . The median disease free-survival (DFS) was 17 months, 12-month DFS was 66%, and 30-month DFS was 17% . Our calculations have shown that 1700 USD/patient were saved by avoiding prolonged hospitalization; this may provide not only economical, but also psychological advantages to patients.

Cell Immunol, 1995 Jan, 160(1), 65 - 70
GM-CSF is required for the Pseudomonas exotoxin A-induced proliferation of immature T cells in athymic mice; Dixon DM et al.; Previous studies have demonstrated that Pseudomonas exotoxin A stimulated the proliferation of immature T lymphocytes within the splenocytes of athymic mice . These studies were performed to determine which lymphokines were involved in the proliferation of the immature T cells . The results of this study indicate that exotoxin A does not induce the production of interleukin-2 or tumor necrosis factor from B cell-depleted splenotypes from athymic mice . However, exotoxin A does induce the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from B cell-depleted splenocytes . Furthermore, the GM-CSF was shown to be produced by a Thy1+, CD4-, CD8- T lymphocyte . The addition of anti-GM-CSF antibody abrogates the exotoxin A-induced proliferation of B cell-depleted splenocytes from athymic mice . Thus, these data indicate that exotoxin A induces the production of GM-CSF from immature T lymphocytes within the splenocytes of athymic mice and the exotoxin A-induced proliferation of these immature T cells is dependent on the presence of GM-CSF.

Medicine (Baltimore), 1995 Jan, 74(1), 42 - 7
Diagnostic criteria for cystic fibrosis in men with congenital absence of the vas deferens; Durieu I et al.; The high frequency of cystic fibrosis (CF) mutations in males with absence of vas deferens supported the hypothesis of a primarily genital phenotype of CF disease . To consider the idea of an attenuated form of CF, we investigated 14 men with congenital bilateral aplasia of the vasa deferentia . All patients were consulting for infertility and none was known to have CF . The median age was 30.5 years (range, 20-38 yr) . DNA analysis for 22 CF mutations showed at least 1 mutation in 10 patients (71%), whereas the CF carrier frequency is only 4% in the general population . Three compound heterozygotes were identified, all carriers of the R117H mutation . The sweat test was considered positive in 6 patients (43%), and a high frequency of radiologic evidence of sinus disease (8 patients) and of elevated antibodies to Pseudomonas (8 patients) was found . Only 2 patients were free of all these criteria for CF disease . This study strengthens the hypothesis that absence of vas deferens is an attenuated form of CF . We propose a combination of tests including DNA study, computerized tomographic scan of the paranasal sinuses, and testing of anti-Pseudomonas antibodies when the sweat test is inconclusive.

J Bacteriol, 1995 Jan, 177(2), 320 - 5
Maleylacetate reductase of Pseudomonas sp . strain B13: specificity of substrate conversion and halide elimination; Kaschabek SR et al.; Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channelling maleylacetate and some chlorinated derivatives into the 3-oxoadipate pathway . Several substituted maleylacetates were prepared in situ by alkaline or enzymatic hydrolysis of dienelactones as the precursor . The conversion of these methyl-, chloro-, fluoro-, and bromo-substituted maleylacetates by malelacetate reductase from 3-chlorobenzoate-grown cells of Pseudomonas sp . strain B13 was studied . Two moles of NADH per mole of substrate was consumed for the conversion of maleylacetates which contain a halogen substituent in the 2 position . In contrast, only 1 mol of NADH was necessary to convert 1 mol of substrates without a halogen substituent in the 2 position . The conversion of 2-fluoro-, 2-chloro-, 2,3-dichloro-, 2,5-dichloro-, 2,3,5-trichloro-, 2-bromo-, 2,3-dibromo-, 2,5-dibromo-, 2-bromo-5-chloro-, 2-chloro-3-methyl-, and 2-chloro-5-methylmaleylacetate was accompanied by the elimination of halide from the 2 position and the temporary occurrence of the corresponding dehalogenated maleylacetate as an intermediate consuming the second mole equivalent of NADH . The properties of the halogen substituents influenced the affinity to the enzyme in the following manner . Km values increased with increasing van der Waals radii and with decreasing electronegativity of the halogen substituents (i.e., low steric hindrance and high electronegativity positively influenced the binding).The Km values obtained with 2-methyl-,3-methyl-, and 5-methylmaleylacetate showed that a methyl substituent negatively affected the affinity in the following order: 2 position >/ = 3 position >> 5 position . A reaction mechanism explaining the exclusive elimination of halogen substituents from the 2 position is proposed.

Cancer Res, 1995 Jan 1, 55(1), 71 - 7
Cytotoxic activity of chimeric toxins containing the epidermal growth factor-like domain of heregulins fused to PE38KDEL, a truncated recombinant form of Pseudomonas exotoxin; Kihara A et al.; The EGF-like domains of heregulin alpha, beta 1, beta 2, and beta 3 were fused to a truncated form of Pseudomonas exotoxin (PE38KDEL), which contains a modified carboxyl-terminal sequence, KDEL, that increases that toxin activity . The resulting chimeric toxins were produced in Escherichia coli, purified to near homogeneity, and shown to be cytotoxic to target cells with very high activity on HTB20, N-87 MCF-7, and HepG2 cells; high activity on A431 and MDA-MB468 cells; and low activity toward SK-OV3, L929, and KB cells . The fact that cytotoxicity did not correlate with the levels of erbB2 expression indicated that another receptor in the erb family might be involved . Accordingly, cytotoxicity assays were performed on NIH/3T3 cell lines transfected with EGFR, ErbB2, ErbB3, or ErbB4 . The results indicate that the heregulin toxins target ErbB4 or possibly ErbB3 but not ErbB2.

Mol Plant Microbe Interact, 1995 Jan-Feb, 8(1), 49 - 57
Characterization of avrE from Pseudomonas syringae pv . tomato: a hrp-linked avirulence locus consisting of at least two transcriptional units; Lorang JM et al.; Cosmid clone pPT10E9 from Pseudomonas syringae pv . tomato caused P . s pv . glycinea to elicit the HR on leaves of all tested soybean cultivars . The avirulence function of pPT10E9, called avrE, occurred on an 11.3-kb DNA fragment located immediately adjacent to the P . s . pv . tomato hrp gene cluster . Tn3-gus saturation mutagenesis of the avrE locus and adjacent DNA revealed at least four transcriptional units occurring immediately adjacent to the hrpRS locus that were all regulated in a manner similar to hrp genes (induced only in minimal induction media or in planta and required the hrpL and hrpRS loci for expression) . Transcriptional units III and IV, but not II or V, were required for avrE function . P . s . pv . tomato DC3000 carrying mutations in each of the four transcripts retained full virulence on tomato leaves and elicited the HR on tobacco and soybean plants . This was unlike strain PT23, where mutation of avrE greatly decreased virulence on tomato leaves . The promoter regions for three of the investigated transcriptional units contained a consensus sequence occurring in the promoter regions of several other P . syringae avirulence and hrp genes . The promoter region of transcriptional unit IV, required for avrE function, did not contain such a sequence, but included an element which may function as a sigma-54 promoter . Introduction of the cloned P . s . pv . tomato avrE locus into five other P . syringae pathovars did not cause them to elicit the HR on their normal host plants.

Mol Microbiol, 1995 Jan, 15(1), 155 - 65
The hrpRS locus of Pseudomonas syringae pv . phaseolicola constitutes a complex regulatory unit; Grimm C et al.; The right part of the hrp cluster of Pseudomonas syringae pv . phaseolicola contains two regulatory genes, the previously described hrpS gene and an adjacent locus, hrpR . In this study we determined the sequence of hrpR and analysed the functional organization of the two genes . HrpR and HrpS show high sequence similarities to each other and to other response regulators of the two-component regulatory system . This has recently also been described for the hrpRS system of the closely related pathogen Pseudomonas syringae pv . syringae . The results of our genetic analyses strongly indicate that hrpS expression is regulated by the hrpR gene product . DNA-protein binding studies and site-directed mutagenesis of the hrpR sequence provided further evidence that HrpR activates hrpS transcription by binding to an activator site . This HrpR binding site was mapped in a fragment which is located 378-609 nucleotides upstream of the hrpS transcription start site . The hrpS transcription start site maps 179 nucleotides upstream of the initiation codon ATG, as determined by primer extension analysis, and is preceded by a typical -12/-24 promoter motif.

Acta Derm Venereol, 1995 Jan, 75(1), 59 - 61
Protein-losing enteropathy in a child with junctional epidermolysis bullosa and pyloric atresia; Meldgaard Lund A et al.; We report on a newborn boy with junctional epidermolysis bullosa and pyloric atresia . Blisters were found on his skin at birth, especially in places exposed to pressure, and appeared later on his mucous membranes . Epidermolysis bullosa was confirmed by electron microscopy . Radiography revealed pyloric atresia, and a gastroduodenostomy was carried out at 7 days of age . A connective tissue septum was found between his ventricle and duodenum . The skin changes were mild, and the clinical course was determined by his protein-losing enteropathy . He died at 66 days of age from pseudomonas sepsis.

Electrophoresis, 1995 Jan, 16(1), 43 - 5
A new method for the detection of proteolytic activity in Pseudomonas lundensis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Lundy FT et al.; A new method for the visualization of proteolytic activity in cell culture supernatant from Pseudomonas lundensis after sodium dodecyl sulfate (SDS)--gel electrophoresis is described . Following conventional electrophoresis, the gel is washed in a methanol-containing buffer to facilitate partial removal of SDS . After incubation with 0.5% casein the gel is stained for protein with Coomassie Brilliant Blue R-250 . Bands with proteolytic activity appear as clear areas in the gel against a blue-stained background . Molecular weight standards electrophoresed in the same gel stain more intensely than the background and allow determination of the molecular weights of the proteolytic components . The sensitivity of post-electrophoretic reactivation in SDS-gels was determined using trypsin as standard . A slight modification of the technique allowed detection of proteolytic activity in nondenaturing and in isoelectric focusing gels.

Naunyn Schmiedebergs Arch Pharmacol, 1995 Jan, 351(1), 27 - 34
Chronic muscarinic cholinoceptor stimulation increases adenylyl cyclase responsiveness in rat cardiomyocytes by a decrease in the level of inhibitory G-protein alpha-subunits; Reithmann C et al.; Exposure of neonatal rat cardiomyocytes for 3 days to the muscarinic cholinoceptor agonist carbachol led to a concentration-dependent increase in adenylyl cyclase stimulation by the beta-adrenoceptor agonist isoproterenol by up to 115% (at 1 mmol/l carbachol) . In addition, direct adenylyl cyclase stimulation by forskolin was increased in carbachol (1 mmol/l)-treated cells by 32% . Pretreatment of the rat cardiomyocytes with pertussis toxin, which enhances adenylyl cyclase activity by a functional inactivation of the inhibitory G-protein (Gi), was performed to investigate the possible role of Gi-proteins in carbachol-induced sensitization of adenylyl cyclase stimulation . After pretreatment of the cells with pertussis toxin, the carbachol-mediated increase in forskolin-stimulated adenylyl cyclase activity was lost and the carbachol-mediated increase in beta-adrenoceptor-stimulated adenylyl cyclase activity was attenuated . Labelling of the 40 kDa pertussis toxin substrates in cardiomyocyte membranes was decreased by carbachol in a concentration-dependent manner by up to 34% (at 1 mmol/l carbachol) . The number and affinity of beta 1-adrenoceptors was unaltered following the chronic carbachol treatment . The specific protein synthesis inhibitor Pseudomonas exotoxin A was used to study whether the carbachol-induced decrease in the level of pertussis toxin-sensitive G-proteins and increase in adenylyl cyclase activity depend on de-novo protein synthesis . Pseudomonas exotoxin A inhibits peptide chain elongation by ADP-ribosylating elongation factor 2 . Treatment of the cells with 1 ng/ml Pseudomonas exotoxin A for 3 days led to a reduction in the subsequent ADP-ribosylation of elongation factor 2 in the cytosol of the heart muscle cells by 57%.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Microbiol, 1995 Jan, 163(1), 35 - 41
Cloning, nucleotide sequence, and expression of the gene encoding a novel dioxygenase involved in metabolism of carboxydiphenyl ethers in Pseudomonas pseudoalcaligenes POB310; Dehmel U et al.; Pseudomonas pseudoalcaligenes strain POB310 degrades 3- and 4-carboxydiphenyl ether . The initial reaction involves an angular dioxygenation yielding an unstable hemiacetal that spontaneously decays to phenol and protocatechuate . We cloned a DNA fragment containing the gene encoding the initial dioxygenase from an unstable, self-transmissible plasmid . Sequence analysis revealed two open reading frames encoding proteins with putative molecular masses of 46.3 and 33.6 kDa . The deduced amino acid sequences showed homologies to oxygenase and reductase subunits of aromatic ring-activating dioxygenases, and contained regions identical to consensus sequences that bind chloroplast-like and Rieske-type {2Fe2S} clusters, suggesting that the initial dioxygenase is a class IA aromatic ring-activating dioxygenase system . Initial dioxygenase activity was induced in bacteria grown in M9 minimal medium containing 3- or 4-carboxydiphenyl ether or phenol as carbon source, indicating that the regulation is dependent on the phenol pathway . The maximal specific activity was measured at the beginning of the exponential growth phase.

Can J Microbiol, 1995, 41 Suppl 1, 160 - 9
Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase of Comamonas sp . and of its structural gene; Jendrossek D et al.; The poly(3-hydroxybutyrate) (PHB) depolymerase structural gene of Comamonas sp . (phaZCsp) was cloned in Escherichia coli and identified by halo formation on PHB-containing solid medium . The nucleotide sequence of a 1719 base pair MboI fragment was determined and contained one large open reading frame (ORF1, 1542 base pairs) . This open reading frame encoded the precursor of the PHB depolymerase (514 amino acids; Mr, 53,095), and the deduced amino acid sequence was in agreement with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 26 onwards . Analysis of the deduced amino acid sequence revealed a domain structure of the protein: a signal peptide that was 25 amino acids long was followed by a catalytic domain of about 300 amino acids, a fibronectin type III (Fn3) modul sequence, and a putative PHB-specific substrate-binding site . By comparison of the primary structure with that of other polyhydroxyalkanoate (PHA) depolymerases, the catalytic domain apparently contained a catalytic triad of serine, histidine, and aspartate . In addition, a conserved region resembling the oxyanion hole of lipases was present . The catalytic domain was linked to a C-terminal putative substrate-binding site by a sequence about 90 amino acids long resembling the Fn3 modul of fibronectin and other eukaryotic extracellular matrix proteins . A threonine-rich region, which was found in four of five PHA depolymerases of Pseudomonas lemoignei, was not present in the Comamonas sp . depolymerase . The similarities with and differences from other PHA depolymerases are discussed.

Somat Cell Mol Genet, 1995 Jan, 21(1), 1 - 18
Analysis of mutations in alleles of the fur gene from an endoprotease-deficient Chinese hamster ovary cell strain; Spence MJ et al.; RPE.40 mutant cells differ from wild-type Chinese hamster ovary (CHO-K1) cells in their increased resistance to Pseudomonas exotoxin A and their inability to process the insulin proreceptor and certain viral envelope proproteins . Northern analysis revealed that RPE.40 cells maintained a substantially lower steady-state level of 4.0 kb fur mRNA than did CHO-K1 cells . Analysis of fur cDNAs showed that RPE.40 cells were diploid at the fur locus, and RPE.40 cells had a Cys (TGC) to Tyr (TAC) mutation in codon 196 of one allele (allele I) . Approximately 25-30% of the CHO-K1 cells were also heterozygous (Tyr/Cys) at codon 196, and pre-mRNAs transcribed from the second allele (allele II) in RPE.40 cells were defectively spliced . All other pre-mRNAs were correctly spliced . Rapid turnover of defectively spliced transcripts may account for the reduced steady-state level of fur mRNA observed in RPE.40 cells . Our results provide a mechanistic basis for the endoprotease-deficient phenotype of RPE.40 cells.

Mol Gen Mikrobiol Virusol, 1995 Jan-Mar, (1), 28 - 36
{Pseudomonas mallei and Pseudomonas pseudomallei: introduction and maintenance of natural and recombinant plasmid replicons}; Abaev IV et al.; Comparative analysis of recipient activity of Pseudomonas mallei, Pseudomonas pseudomallei, and Pseudomonas cepacia strains towards naturally occurring and recombinant plasmid replicons was carried out . Autonomic broad host range vector plasmids based on RSF1010(IncQ) and pSa(IncW) replicons as well as integrative vectors based on pSUP202(Co1E1) replicon have been constructed . The study has shown that naturally occurring plasmids RSF1010(IncQ), pSa(IncW), R15(IncN), and RP4(IncP) are being efficiently transferred and stably maintained in investigated Pseudomonas strains . However, recombinant plasmids with the mini-replicon pSa which are stable in Escherichia coli have shown segregative instability in Pseudomonas strains, whereas derivatives of plasmid RSF1010 demonstrated different stability depending on the type of insertion . Plasmid pSUP202 derivative integrative vector pSM525 is efficiently introduced and stably maintained in P . mallei C-5 strain . Two vector systems for genetic manipulations in P.mallei and P.pseudomallei cells have been developed.

Mol Plant Microbe Interact, 1995 Jan-Feb, 8(1), 165 - 71
Role of the phytotoxin coronatine in the infection of Arabidopsis thaliana by Pseudomonas syringae pv . tomato; Mittal S et al.; The role of the phytotoxin coronatine in the virulence of Pseudomonas syringae pv . tomato in Arabidopsis thaliana was evaluated by comparing symptom development, in planta bacterial multiplication, and the induction of defense-related genes in Arabidopsis plants inoculated with the coronatine-producing (Cor+) P . s . pv . tomato strain DC3000 and the coronatine-defective (Cor-) strain DC3661 by either infiltration or dipping methods . The Cor+ strain, P . s . pv . tomato DC3000, caused severe disease symptoms and multiplied by 4-6 logs after inoculation by either infiltration or dipping . P . s . pv . tomato DC3661 failed to produce any disease symptoms and multiplied by only 1-1.5 logs in dipped plants, whereas it caused mild symptoms and multiplied 6 logs over the 4-day experimental period in plants inoculated by infiltration . Parallel experiments using a natural host, tomato, yielded similar results . Analysis of the accumulation of mRNAs encoded by several distinct defense-related genes in Arabidopsis leaves infiltrated with either DC3000 or DC3661 demonstrated that the Cor- strain consistently induced higher levels of these transcripts . These results demonstrate that coronatine production is required under more natural inoculation conditions for the successful infection of Arabidopsis by DC3000, and that coronatine may play a critical role during the early stages of infection by suppressing the activation of defense-related genes.

Trans R Soc Trop Med Hyg, 1995 Jan-Feb, 89(1), 41 - 3
Isolation of Pseudomonas pseudomallei from soil in north-eastern Thailand; Wuthiekanun V et al.; In order to optimize the recovery from soil of Pseudomonas pseudomallei, the cause of melioidosis, 3 selective broths were compared . A basal salt solution containing L-threonine (TBSS) performed significantly better than trypticase soy broth containing crystal violet and colistin 50 mg/L (CVC50), both in isolation rate and suppression of overgrowth of other organisms, but the addition of colistin to TBSS gave the best results overall . In a survey in north-eastern Thailand, P . pseudomallei was recovered from 114 (68%) of the 167 sites tested . A detailed study of a single rice farm showed that the isolation rate increased with depth of soil sample, and P . pseudomallei could still be isolated during the dry season, although only from moist soil in areas where other crops were cultivated and around the water source.

Infect Control Hosp Epidemiol, 1995 Jan, 16(1), 30 - 2
Recovery of Pseudomonas cepacia and other Pseudomonas species from the environment; Mortensen JE et al.; Nine hundred sixteen cultures were obtained from homes of patients with cystic fibrosis, control homes, salad bars, and food markets, and analyzed for the presence of Pseudomonas cepacia and related bacteria . P cepacia was recovered from 5 (18%) of 27 homes, and from 20 (4%) of 509 cultures collected outside of homes . Relative to other pseudomonads, P cepacia is found infrequently in the environment . It is not clear how frequently these sources contribute to acquisition of this bacteria by persons with cystic fibrosis.

J Bacteriol, 1995 Jan, 177(2), 307 - 11
Metabolism of polychlorinated phenols by Pseudomonas cepacia AC1100: determination of the first two steps and specific inhibitory effect of methimazole; Tomasi I et al.; Resting cells of 2,4,5-trichlorophenoxyacetic acid-grown Pseudomonas cepacia AC1100 metabolize both dichlorophenols, such as 2,4-dichlorophenol, 2,6-dichlorophenol, 3,4-dichlorophenol, and 3,5-dichlorophenol, and more highly substituted phenols, such as 2,4,6-trichlorophenol and pentachlorophenol, to the corresponding chlorohydroquinones . The first hydroxylation occurs in the para position of the phenol regardless of whether this position is replaced by a chlorine substituent . The first evidence leading to the characterization of para-hydroxylase as a flavin-containing enzyme is provided by the inhibitory effect of methimazole, an alternate substrate for this monooxygenase, on the degradative ability of the strain . In a second step, with tetrachlorohydroquinone, trichlorohydroxyquinone was isolated and completely characterized . Trichlorohydroxyquinone was also obtained from tetrachloroquinone . Incubation of the cells in the presence of an external source of NADPH prevents the further degradation of tetrachlorohydroquinone, suggesting that the quinone derived from the two-electron oxidation of the hydroquinone is more likely the substrate for the second hydroxylation.

Biochim Biophys Acta, 1994 Dec 30, 1196(2), 145 - 53
Solubilization of the overexpressed integral membrane protein alkane monooxygenase of the recombinant Escherichia coli W3110{pGEc47}; Peters J et al.; The integral membrane-bound alkane monooxygenase (AlkB) from Pseudomonas oleovorans has been overexpressed in the recombinant Escherichia coli strain W3110{pGEc47} and expression levels of 10 to 15% relative to the total cell protein were reached . The amount of phospholipids in induced cells is about 3-fold higher compared to the wild-type and AlkB has been shown to be located in small membrane vesicles . We present here a study on the solubilization of these AlkB containing membrane vesicles by different detergents with special emphasis on structural requirements for a surfactant preserving the activity of AlkB . Moreover, the effects of the detergents used on the complete alkane hydroxylase system was studied.

Biochim Biophys Acta, 1994 Dec 30, 1188(3), 432 - 8
DNA sequence of the cut A, B and C genes, encoding the molybdenum containing hydroxylase carbon monoxide dehydrogenase, from Pseudomonas thermocarboxydovorans strain C2; Pearson DM et al.; Pseudomonas thermocarboxydovorans strain C2 is capable of using carbon monoxide as the sole source of carbon and energy . The key enzyme for CO utilisation is the molybdenum containing iron-flavoprotein carbon monoxide dehydrogenase (CODH) . This paper reports the DNA sequencing of a 4.7 kb region of the C2 genome which appears to encode the CODH enzyme . The genes for the three subunits of CODH, which we have named cut A, B and C, have been identified and they appear to form an operon . The predicted protein sequences of the three subunits have homology to the structurally related protein, xanthine dehydrogenase, from Drosophila melanogaster . By comparison with xanthine dehydrogenase it can be predicted that the molybdenum cofactor binds to the large subunit of CODH, the small subunit of CODH contains the iron-sulphur centers and the medium subunit binds FAD/NAD+.

J Biol Chem, 1994 Dec 16, 269(50), 31831 - 5
Furin activates Pseudomonas exotoxin A by specific cleavage in vivo and in vitro; Inocencio NM et al.; We have demonstrated that the native proenzymatic form of Pseudomonas exotoxin A can be cleaved at its specific activation site by furin in intact Chinese hamster ovary cells or in vitro by furin in isolated membrane fractions from these cells . We have compared the activity of furin in cell membrane fractions with that of purified, recombinant human furin . We have verified that RPE.40, a Pseudomonas toxin-resistant mutant cell strain, is mutant in the fur gene, and we have demonstrated that these cells are deficient in cleavage of the toxin . We have also determined that this cleavage of Pseudomonas toxin by furin takes place at the authentic activation site to release the 37-kDa active fragment.

Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 12051 - 5
Rapid humanization of the Fv of monoclonal antibody B3 by using framework exchange of the recombinant immunotoxin B3(Fv)-PE38; Benhar I et al.; B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv region of carcinoma-specific antibody B3 is fused to a truncated form of Pseudomonas exotoxin (PE) . The efficacy of monoclonal antibody B3 and B3 immunotoxins in cancer therapy and diagnosis may be limited by the human anti-mouse response . Here we describe the humanization of the Fv of B3(Fv)-PE38 by "framework exchange." The variable domains of the heavy (VH) and light (VL) chains were aligned with their best human homologs to identify framework residues that differ . Initially, 11 framework residues in VH and six in VL were changed by site-specific mutagenesis to human residues and introduced simultaneously into a preassembled single-chain Fv expression cassette . Six VH and five VL residues that differ were not changed because they were buried, in the interdomain interface, or previously found to result in decreased affinity when mutated . This basic design resulted in some 20-fold loss of activity . Changing VL residues at the interdomain interfacial position 100 and at the buried position 104 to the human sequence increased the activity 8-fold . Changing VH residue at position 82b from the human sequence back to that of the mouse restored the activity 2- to 3-fold to the full binding and cytotoxic activity of the mouse sequence . Humanized B3(Fv)-PE38 lost immunogenic epitopes recognized by sera from monkeys that had been immunized with B3(Fv)-PE38.

J Bacteriol, 1994 Dec, 176(24), 7757 - 62
Organization and evolution of naphthalene catabolic pathways: sequence of the DNA encoding 2-hydroxychromene-2-carboxylate isomerase and trans-o-hydroxybenzylidenepyruvate hydratase-aldolase from the NAH7 plasmid; Eaton RW; The sequence of a 2,437-bp DNA segment from the naphthalene upper catabolic pathway operon of plasmid NAH7 was determined . This segment contains three large open reading frames designated nahQ', nahE, and nahD . The first of these is the 3' end of an open reading frame that has no known function, the second (993 bp) encodes trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (deduced molecular weight, 36,640), and the third (609 bp) encodes 2-hydroxychromene-2-carboxylate isomerase (deduced molecular weight, 23,031) . This DNA has a high degree of sequence homology (greater than 91% for the first 2161 bp) with a DNA segment from the dox (dibenzothiophene oxidation) operon of Pseudomonas sp . strain C18, which encodes a pathway analogous to that encoded by NAH7 . However, 84 bp downstream from nahD, the last gene in the nah operon, this homology ends . This 84-bp sequence at the downstream end of nah and dox homology has 76% homology to a sequence that occurs just upstream of the nah promoter in NAH7 . These directly repeated 84-bp sequences thus encompass the upper-pathway nah operon and constitute the ends of a highly conserved region.

J Bacteriol, 1994 Dec, 176(24), 7574 - 86
The biosynthetic gene cluster for coronamic acid, an ethylcyclopropyl amino acid, contains genes homologous to amino acid-activating enzymes and thioesterases; Ullrich M et al.; Coronamic acid (CMA), an ethylcyclopropyl amino acid derived from isoleucine, functions as an intermediate in the biosynthesis of coronatine, a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv . glycinea PG4180 . The DNA required for CMA biosynthesis (6.9 kb) was sequenced, revealing three distinct open reading frames (ORFs) which share a common orientation for transcription . The deduced amino acid sequence of a 2.7-kb ORF designated cmaA contained six core sequences and two conserved motifs which are present in a variety of amino acid-activating enzymes, including nonribosomal peptide synthetases . Furthermore, CmaA contained a spatial arrangement of histidine, aspartate, and arginine residues which are conserved in the ferrous active site of some nonheme iron(II) enzymes which catalyze oxidative cyclizations . The deduced amino acid sequence of a 1.2-kb ORF designated cmaT was related to thioesterases of both procaryotic and eucaryotic origins . These data suggest that CMA assembly is similar to the thiotemplate mechanism of nonribosomal peptide synthesis . No significant similarities between a 0.9-kb ORF designated cmaU and other database entries were found . The start sites of two transcripts required for CMA biosynthesis were identified in the present study . pRG960sd, a vector containing a promoterless glucuronidase gene, was used to localize and study the promoter regions upstream of the two transcripts . Data obtained in the present study indicate that CMA biosynthesis is regulated at the transcriptional level by temperature.

J Pediatr, 1994 Dec, 125(6 Pt 1), 992 - 7
Effects of pentoxifylline on sputum neutrophil elastase and pulmonary function in patients with cystic fibrosis: preliminary observations; Aronoff SC et al.; High concentrations of free human neutrophil elastase in bronchial epithelial fluid are believed to be a major factor in the evolution of pulmonary injury in cystic fibrosis (CF) . To test this hypothesis, we studied pentoxifylline, a compound that inhibits tumor necrosis factor alpha transcription and its stimulatory effect on polymorphonuclear neutrophils, in patients with CF who had chronic Pseudomonas bronchitis . Subjects older than 11 years of age randomly received placebo or pentoxifylline (1600 mg/day) orally, in a double-blind fashion, for 6 months . Pulmonary function and sputum elastase concentrations were determined before therapy and bimonthly during therapy; compliance was determined by measuring serum drug concentrations . Of the 16 patients who completed the study, 9 received pentoxifylline . The sputum elastase concentrations among placebo recipients were significantly increased from baseline at 4 and 6 months (F = 3.44; p < 0.05); the values remained unchanged in the treatment group . The mean forced vital capacity for the placebo group decreased from 59.2% +/- 15.4% predicted at baseline to 52.0% +/- 12.9% predicted at 6 months; the values in the treatment group remained largely unchanged . The forced vital capacity improved between baseline and 6 months for four of nine pentoxifylline recipients and none of the seven control patients (p = 0.09) . During the study, four of seven placebo recipients experienced a significant pulmonary exacerbation compared with one of nine treated patients (p = 0.077) . These findings support the hypothesis that polymorphonuclear neutrophil elastase is a factor in the evolution of CF lung disease; further studies are needed to define the role of pentoxifylline in the treatment of CF.

Infect Control Hosp Epidemiol, 1994 Dec, 15(12), 757 - 8
A pseudo-epidemic involving bone allografts; Mermel LA et al.; Preimplantation cultures of four sterile bone allograft specimens grew Comomonas acidovorans and Pseudomonas species . An epidemiological investigation, including molecular subtyping methods, revealed that the allograft specimens were contaminated in a microbiology laboratory sonicator water bath.

Microbiology, 1994 Dec, 140 ( Pt 12), 3217 - 23
Phosphorylation of membrane proteins in response to temperature in an Antarctic Pseudomonas syringae; Ray MK et al.; Temperature-dependent phosphorylation and dephosphorylation of membrane proteins was studied in vitro in a number of psychrotrophic Antarctic bacteria which grow between 0 and 30 degrees C . One of them, a Pseudomonas syringae isolate, was studied in detail and was found to have three membrane proteins of molecular mass 30, 65 and 85 kDa which were phosphorylated differently in response to low and high temperatures . The 65 kDa protein was phosphorylated only at lower temperatures (between 0 and 15 degrees C) . The 30 kDa protein was phosphorylated more at higher temperatures and was possibly a histidine kinase . This protein was present in all the psychrotrophic Pseudomonas species studied and in Sphingobacterium antarcticus . A possible role for these proteins in sensing environmental temperature is proposed.

Plant Cell, 1994 Dec, 6(12), 1845 - 57
A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance; Bowling SA et al.; Systemic acquired resistance (SAR) is a nonspecific defense response in plants that is associated with an increase in the endogenous level of salicylic acid (SA) and elevated expression of pathogenesis-related (PR) genes . To identify mutants involved in the regulation of PR genes and the onset of SAR, we transformed Arabidopsis with a reporter gene containing the promoter of a beta-1,3-glucanase-encoding PR gene (BGL2) and the coding region of beta-glucuronidase (GUS) . The resulting transgenic line (BGL2-GUS) was mutagenized, and the M2 progeny were scored for constitutive GUS activity . We report the characterization of one mutant, cpr1 (constitutive expressor of PR genes), that was identified in this screen and shown by RNA gel blot analysis also to have elevated expression of the endogenous PR genes BGL2, PR-1, and PR-5 . Genetic analyses indicated that the phenotype conferred by cpr1 is caused by a single, recessive nuclear mutation and is suppressed in plants producing a bacterial salicylate hydroxylase, which inactivates SA . Furthermore, biochemical analysis showed that the endogenous level of SA is elevated in the mutant . Finally, the cpr1 plants were found to be resistant to the fungal pathogen Peronospora parasitica NOCO2 and the bacterial pathogen Pseudomonas syringae pv maculicola ES4326, which are virulent in wild-type BGL2-GUS plants . Because the cpr1 mutation is recessive and associated with an elevated endogenous level of SA, we propose that the CPR1 gene product acts upstream of SA as a negative regulator of SAR.

Br J Surg, 1994 Dec, 81(12), 1752 - 6
Multi-agent therapy in the treatment of sepsis-induced microvascular injury; Carey PD et al.; Cyclo-oxygenase inhibition (with ibuprofen) combined with histamine (H1, H2) receptor antagonism (with diphenhydramine and cimetidine) attenuates microvascular leak injury in sepsis syndromes . Ibuprofen reduces microvascular injury by limiting oxygen radical production by neutrophils . Histamine is known to inhibit this oxygen radical production, an effect antagonized by cimetidine . In the present study neutrophils isolated from pigs made septic with Pseudomonas organisms exhibited a significant (P < 0.05) increase in the production of the oxygen radicals, superoxide anion (O2-, 133 per cent) and hypochlorous acid (HOCl, 38 per cent) . Ibuprofen used alone attenuated this sepsis-stimulated overproduction . Addition of the antihistamines cimetidine and diphenhydramine produced a significant increase in oxygen radical production (P < 0.05), by 122 per cent (O2-) and 47 per cent (HOCl), equivalent to that in untreated septic animals . This coincided with a significant deterioration in pulmonary compliance (P < 0.05) compared with that found in control animals and those treated with ibuprofen alone, and a significant accumulation of extravascular lung water (P < 0.05) at 240 and 300 min versus baseline . Histamine receptor antagonism may inadvertently enhance microvascular injury in sepsis.

J Appl Bacteriol, 1994 Dec, 77(6), 613 - 20
Metabolic activities of pseudomonads in batch cultures in extract of minced lamb; Drosinos EH et al.; Pseudomonas fragi, Ps . lundensis and Ps . fluorescens were studied in axenic batch cultures growing in a lamb juice (pH 6.0) aerobically or in an atmosphere (Ps . fragi only) enriched with carbon dioxide at 4 degrees C . With all but a glucose dehydrogenase-deficient strain of Ps . fluorescens there was a sequential catabolism of glucose and lactate . Diauxic growth was observed in a nutrient-deficient meat juice supplemented with glucose and lactate . A transient peak in the concentration of gluconate and pyruvate was associated with the catabolism of glucose and lactate respectively . With Ps . fluorescens deficient in glucose dehydrogenase there was simultaneous catabolism of glucose and lactate . The stereoisomers of lactate were catabolized simultaneously in a laboratory medium . Glucose-6-phosphate was oxidized to 6-phosphogluconate by Ps . fragi and Ps . lundensis under aerobic conditions only . Creatine and creatinine were catabolized by Ps . fragi under aerobic conditions only . There was a slight decrease in the concentration of total amino acids (ninhydrin-reactive material) during the exponential phase of growth . The results suggest that the dominance of Ps . fragi in the climax populations in meat is due to catabolism of amino acid related substrates, creatine and creatinine.

Appl Environ Microbiol, 1994 Dec, 60(12), 4587 - 91
Pseudomonas sp . strain HBP1 Prp degrades 2-isopropylphenol (ortho-cumenol) via meta cleavage; Reichlin F et al.; Pseudomonas sp . strain HBP1 Prp grew on 2-isopropylphenol as the sole carbon and energy source with a maximal specific growth rate of 0.14 h-1 and transient accumulation of isobutyric acid . Oxygen uptake experiments with resting cells and enzyme assays with crude-cell extracts showed that 2-isopropylphenol was catabolized via a broad-spectrum meta cleavage pathway . These findings were confirmed by experiments with partially purified enzymes . Identification of 3-isopropylcatechol and 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid as the products of the initial monooxygenase reaction and the subsequent extradiol ring cleavage dioxygenase reaction, respectively, was based on gas chromatography-mass spectrometry analysis of the corresponding trimethylsilyl derivatives . The meta cleavage product hydrolase hydrolyzed 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid (meta cleavage product of 2-isopropylphenol) to isobutyric acid and 2-hydroxypent-2,4-dienoic acid.

Appl Environ Microbiol, 1994 Dec, 60(12), 4421 - 31
Genetic and plasmid diversity within natural populations of Pseudomonas syringae with various exposures to copper and streptomycin bactericides; Sundin GW et al.; We examined the genetic and plasmid diversity within natural populations of Pseudomonas syringae isolated from three ornamental pear nurseries in eastern Oklahoma . The bactericide spray regimen differed at each nursery; copper and streptomycin, only copper, and no bactericides were applied at nurseries I, II, and III respectively . Resistance to copper (Cur) and resistance to streptomycin (Smr) were determined for 1,938 isolates of P . syringae; isolates from nurseries I and II were generally Cur Sms; whereas most isolates from nursery III were Cus Sms . The plasmid profiles of 362 isolates were determined, and six, one, seven, and four plasmid profiles were obtained for Cur, Smr, Cur Smr, and Cus Sms isolates, respectively . All Smr plasmids contained sequences homologous to the strA and strB Smr genes from broad-host-range plasmid RSF1010 and were associated with Smr transposon Tn5393 . Plasmids were placed into two groups on the basis of hybridization to the oriV and par sequences from pOSU900, a cryptic plasmid in P . syringae pv . syringae . A total of 100 randomly chosen P . syringae isolates from nurseries I and III were analyzed for genetic diversity by using the arbitrarily primed PCR (AP-PCR) technique . An analysis of chromosomal genotypes by AP-PCR revealed a high degree of genetic diversity among the isolates, and the results of this analysis indicated that the isolates could be clustered into two distinct groups . The plasmid profiles were specific to isolates belonging to particular AP-PCR groups . Within each AP-PCR group, identical plasmid profiles were produced by isolates that had different chromosomal genotypes, implying that plasmid transfer has played an important role in the dissemination of Cur and Smr within the populations studied.

Biodegradation, 1994 Dec, 5(3-4), 289 - 300
Molecular genetics and evolutionary relationship of PCB-degrading bacteria; Furukawa K; Biphenyl-utilizing soil bacteria are ubiquitously distributed in the natural environment . They cometabolize a variety of polychlorinated biphenyl (PCB) congeners to chlorobenzoic acids through a 2,3-dioxygenase pathway, or alternatively through a 3,4-dioxygenase system . The bph genes coding for the metabolism of biphenyl have been cloned from several pseudomonads . The biochemistry and molecular genetics of PCB degradation are reviewed and discussed from the viewpoint of an evolutionary relationship.

Biodegradation, 1994 Dec, 5(3-4), 259 - 76
On the origins and functions of the enzymes of the 4-chlorobenzoate to 4-hydroxybenzoate converting pathway; Dunaway-Mariano D et al.; This review examines the enzymes of 4-chlorobenzoate to 4-hydroxybenzoate converting pathway found in certain soil bacteria . This pathway consists of three enzymes: 4-chlorobenzoate: Coenzyme A ligase, 4-chlorobenzoyl-Coenzyme A dehalogenase and 4-hydroxybenzoyl-Coenzyme A thioesterase . Recent progress made in the cloning and expression of the pathway genes from assorted bacterial strains is described . Gene order and sequence found among these strains are compared to reveal independent enzyme recruitment strategies . Sequence alignments made between the Pseudomonas sp . strain CBS3 4-chlorobenzoate pathway enzymes and structurally related proteins contained within the protein sequence data banks suggest possible origins in preexisting beta-oxidation pathways . The purification and characterization of the physical and kinetic properties of the pathway enzymes are described . Where possible a comparison of these properties between like enzymes from different bacterial sources are made.

Biodegradation, 1994 Dec, 5(3-4), 219 - 36
Genetics and biochemistry of phenol degradation by Pseudomonas sp . CF600; Powlowski J et al.; Pseudomonas sp . strain CF600 is an efficient degrader of phenol and methylsubstituted phenols . These compounds are degraded by the set of enzymes encoded by the plasmid located dmpoperon . The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified . In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised . The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase . Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25) . The other enzymes encoded by the operon are those of the well-known meta-cleavage pathway for catechol, and include the recently discovered meta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10) . The known properties of these meta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised . Analysis of the sequences of the pathway proteins, many of which are unique to the meta-pathway, suggests new approaches to the study of these generally little-characterised enzymes . Furthermore, biochemical studies of some of these enzymes suggest that physical associations between meta-pathway enzymes play an important role . In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.

Biodegradation, 1994 Dec, 5(3-4), 195 - 217
The evolution of pathways for aromatic hydrocarbon oxidation in Pseudomonas; Williams PA et al.; The organisation and nucleotide sequences coding for the catabolism of benzene, toluene (and xylenes), naphthalene and biphenyl via catechol and the extradiol (meta) cleavage pathway in Pseudomonas are reviewed and the various factors which may have played a part in their evolution are considered . The data suggests that the complete pathways have evolved in a modular way probably from at least three elements . The common meta pathway operons, downstream from the ferredoxin-like protein adjacent to the gene for catechol 2,3-dioxygenase, are highly homologous and clearly share a common ancestry . This common module may have become fused to a gene or genes the product(s) of which could convert a stable chemical (benzoate, salicylate, toluene, benzene, phenol) to catechol, thus forming the lower pathway operons found in modern strains . The upper pathway operons might then have been acquired as a third module at a later stage thus increasing the catabolic versatility of the host strains.

Biosci Biotechnol Biochem, 1994 Dec, 58(12), 2297 - 8
Expression of a pectin lyase gene in Escherichia coli from Pseudomonas marginalis N6301; Nikaidou N et al.; We constructed deletion mutant clones of a pectin lyase gene, and measured their pectin lyase activities in Escherichia coli . Pectin lyase activities were detected only in a recA+ strain but not in a recA- strain of E . coli . We also cloned and sequenced recA from Pseudomonas marginalis N6301 . The recA from P . marginalis N6301 can complement recA- to form recA+ in the phenotype of E . coli . Highly conserved sequences of recA are observed among E . coli, P . fluorescens, and P . marginalis . From these results, we presume that recA is required for the expression of the pectin lyase gene in P . marginalis N6301.

Biosci Biotechnol Biochem, 1994 Dec, 58(12), 2281 - 2
Cloning and sequencing of the L-fucose dehydrogenase gene from Pseudomonas sp . No . 1143; Yamamoto-Otake H et al.; The gene coding for L-fucose dehydrogenase (FDH) which is specific to NADP+ as a hydrogen acceptor, was cloned, sequenced, and expressed in Escherichia coli from Pseudomonas sp . No . 1143 . FDH was shown to be composed of 329 amino acid residues.

Hybridoma, 1994 Dec, 13(6), 469 - 76
Chimerization of LL2, a rapidly internalizing antibody specific for B cell lymphoma; Leung SO et al.; LL2 is a murine monoclonal antibody (MAb) that has been shown to be effective for the diagnosis and treatment of patients with non-Hodgkin's B cell lymphoma . Studies have also shown that radiolabeled murine LL2 (mLL2) or mLL2 and fragments thereof coupled to Pseudomonas exotoxin (PE) can effectively target human B cell lymphoma in mice . We have obtained the DNA sequences encoding the VK and VH domains of mLL2, an IgG2a MAb, which were combined with their respective human kappa and IgG1 constant region domains and expressed in SP2/0 cells . Like its murine counterpart, the chimeric LL2 (cLL2) antibody is glycosylated in the light chain variable region . Chimerization did not interfere with the immunoreactivity of the antibody, as determined by a competitive binding assay, where either antibody shows equivalent inhibition of the binding of its counterpart to the Raji cell membrane surface antigen, CD22 . Both antibodies bind and are rapidly internalized by Raji cells, whereas an irrelevant humanized antibody did not bind and was not internalized under similar conditions . The internalization rates of the bound murine or chimeric antibodies were nearly identical, with Ke values of 0.106 and 0.118 min-1 for mLL2 and cLL2, respectively . The observed close equivalence between the murine and chimeric antibodies suggests potential advantages of the latter as a less immunogenic agent . Studies are currently underway to evaluate the chimeric antibody as a potential therapeutic immunoconjugate.

Protein Eng, 1994 Dec, 7(12), 1509 - 15
Cloning, expression and characterization of the Fv fragments of the anti-carbohydrate mAbs B1 and B5 as single-chain immunotoxins; Benhar I et al.; The mAbs B1 (IgG1 kappa) and B5 (IgM kappa) recognize carbohydrate epitopes on human carcinoma cells . The Fv regions of these antibodies were separately cloned from hybridoma RNA using reverse transcription and PCR with oligonucleotide primers designed according to the amino acid sequences of the N-termini . The Fv regions also provide sequences for translation initiation in Escherichia coli (Fr1 oligos) and sequences of the constant region of the heavy and light domains (CH1 or C-kappa oligos) . Following the determination of the DNA sequence of the Fvs, primers were designed according to the 3' ends of the VH and VL domains . These also provided for a peptide linker at the C-terminus of the VH and a short connector at the C-terminus of the VL (Fr4 oligos) . The VH and VL were then each PCR-amplified using their corresponding Fr1 and phosphorylated Fr4 oligos . The resulting PCR products were annealed as 'mutagenic primers' to a uracil-containing single-stranded template obtained from an expression plasmid encoding a single-chain immunotoxin in which the B3 single-chain Fv is fused to a truncated form of Pseudomonas exotoxin (PE) . Thus, the B1 and B5 variable domains replaced their corresponding B3 domains in the expression plasmid by 'variable domain shuffling' without subcloning . The resulting B1(Fv)-PE38 and B5(Fv)-PE38 were expressed in E . coli and purified to near homogeneity . Both show specific cytotoxicities to human carcinoma cell lines, but B1(Fv)-PE38 is much more active, reflecting its higher affinity to the target cells.

Diagn Microbiol Infect Dis, 1994 Dec, 20(4), 181 - 6
Comparison by extended ribotyping of Pseudomonas cepacia isolated from cystic fibrosis patients with acute and chronic infections; Rozee KR et al.; Multiple isolates of Pseudomonas cepacia, from two cystic fibrosis (CF) patients who were chronically infected and two others who suffered acute fatal lung infections, were examined by multilocus enzyme electrophoresis and four-enzyme ribotyping . The strains isolated from the fatalities belonged to a clone, electropherotype 12 (ET12) that is endemic in the Ontario patients' province of origin . ET12 strains have also been isolated from outbreaks in CF patients in the United Kingdom, where they are considered to be strains of high virulence and transmissibility and epidemiologically related to Ontario strains . Four-enzyme ribotyping (EcoRI, Xho, PstI, and ClaI) established the close genetic relationship of the Ontario ET12 isolates and those from the United Kingdom, particularly an isolate from Manchester . In addition, four enzyme ribotypes of the sequential isolates taken during life and at autopsy of the ET12 clone were highly variable in comparison with the stability of the ribotypes of clone ET16 isolated sequentially from living chronic carriers . This extreme ribotype variability may be indicative of a highly virulent strain and poor prognosis . Isolates from our chronically infected CF patients belonged to a different clone, ET16, and it is also endemic in its region, 1000 miles east of ET12, in Nova Scotia . In both endemic circumstances, person-to-person transmission was easily demonstrated by four-enzyme ribotyping . The ET12 clone was found to be transmitted among summer campers and during a nosocomial outbreak, whereas an E16 strain was found to infect a sibling of a chronically infected patient; both infections were of the same ribotype.

Nippon Rinsho, 1994 Dec, 52(12), 3177 - 83
{LDL-receptor-related protein}; Ishibashi S; The LDL-receptor-related protein is a multifunctional endocytotic receptor that clear apo E- and/or lipoprotein lipase-enriched lipoproteins, proteases such as alpha 2-macroglobulin plasminogen and activator inhibitor-1 (PAI-1), Pseudomonas exotoxin A and probably vitellogenin from blood circulation . Its role in plasma chylomicron remnant clearance has been postulated, and recently has been demonstrated by using its inhibitor; receptor-associated protein (RAP) in LDL receptor knockout mice.

J Antibiot (Tokyo), 1994 Dec, 47(12), 1406 - 16
Cepacidine A, a novel antifungal antibiotic produced by Pseudomonas cepacia . II . Physico-chemical properties and structure elucidation; Lim Y et al.; Cepacidine A is a novel glycopeptide with a potent antifungal activity, which is produced by Pseudomonas cepacia AF 2001 . Its molecular weight was determined by FAB-MS (m/z 1215) . The compound is comprised of glycine (1), serine (2), 2,4-diaminobutyric acid (1), aspartic acid (1), beta-hydroxy tyrosine (1), beta-hydroxy asparagine (1), xylose (1) and 5,7-dihydroxy-3,9-diamino-octadecanoic acid (1) . Unfortunately, cepacidine A is a mixture of A1 and A2, either of which is barely distinguishable . Cepacidine A2 includes asparagine (1) instead of beta-hydroxy asparagine (1) of cepacidine A1 . The MS data and the NOESY, TOCSY and HMBC spectra show that cepacidine A is a cyclic peptide and xylose is connected to 5,7-dihydroxy-3,9-diaminooctadecanoic acid.

Mol Gen Genet, 1994 Dec 1, 245(5), 556 - 64
Chaperone-mediated activation in vivo of a Pseudomonas cepacia lipase; Aamand JL et al.; An extracellular Pseudomonas cepacia lipase, LipA, is inactive when expressed in the absence of the product of the limA gene . Evidence has been presented that LimA is a molecular chaperone . The lipA and limA genes have been cloned in separate and independently inducible expression systems in Escherichia coli . These systems were used to test the molecular chaperone hypothesis by investigating whether LimA could activate presynthesized prelipase and whether presynthesized LimA could activate newly synthesized prelipase . The results show that LimA cannot activate presynthesized prelipase and that presynthesized LimA can activate only a limited number of de novo synthesized prelipase molecules . Co-immunoprecipitation of prelipase/lipase with LimA generated a 1:1 complex of prelipase/lipase and LimA . The results suggest that a 1:1 complex of LipA and LimA is required for prelipase processing and secretion of active lipase.

Med Clin (Barc), 1994 Nov 26, 103(18), 681 - 3
{Cystic fibrosis in Asturias: an elevated frequency of the delta F508 mutation}; Coto E et al.; BACKGROUND: Cystic fibrosis (CF) is the most common autosomic-recessive inherited disorder . More than 300 different mutations in the CF gene (CFTR) have been described, being delta F508 and G542X the most common in the Spanish population . The frequencies of these mutations vary between the different European populations . METHODS: We have studied the delta F508 and G542X mutations in 20 CF-patients from Asturias . These mutations were analysed through the polymerase chain reaction (PCR) . RESULTS: The frequency of the delta F508 mutation in Asturias was 77.5%, higher than those found in most of the other Spanish populations . The frequency found in Asturias is close to the frequency described for the Basque Country population . Patients homozygous for the delta F508 mutation showed clinical symptoms similar to those described in studies on other populations . CONCLUSIONS: The high frequency of two mutations in the CFTR gene in Asturias makes possible the direct diagnostic in most families . The delta F508 mutation is associated to severe clinical manifestations, like early pancreatic insufficiency and Pseudomonas infection.

FEBS Lett, 1994 Nov 21, 355(1), 96 - 100
Novel bioactive lipodepsipeptides from Pseudomonas syringae: the pseudomycins; Ballio A et al.; The covalent structure and most of the stereochemistry of the pseudomycins, bioactive metabolites of a transposon-generated mutant of a Pseudomonas syringae wild-type strain proposed for the biological control of Dutch elm disease, have been determined . While two pseudomycins are identical to the known syringopeptins 25-A and 25-B, pseudomycins A, B, C, C' are new lipodepsinonapeptides . For all of these the peptide moiety corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH) -L-Thr (4-Cl) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N-terminal Ser . This is in turn N-acylated by 3,4-dihydroxytetradecanoate in pseudomycin A, by 3-hydroxytetradecanoate in pseudomycin B, by 3,4-dihydroxyhexadecanoate in pseudomycin C, and by 3-hydroxyhexadecanoate in pseudomycin C' . Some preliminary data on the biological activity of pseudomycin A are reported.

J Mol Biol, 1994 Nov 4, 243(4), 799 - 801
Preliminary crystallographic analysis of 4-oxalocrotonate tautomerase reveals the oligomeric structure of the enzyme; Roper DI et al.; Crystals of recombinant 4-oxalocrotonate tautomerase from Pseudomonas sp . strain CF600 have been obtained in a form suitable for X-ray analysis . The enzyme is a highly efficient catalyst and is unusual in that it consists of subunits of only 62 amino acids . It crystallises in the triclinic space group, P1, with unit cell dimensions a = 39.6 A, b = 51.5 A, c = 51.6 A, alpha = 60.0 degrees, beta = 81.4 degrees, gamma = 69.6 degrees . The crystals diffract to beyond 1.9 A resolution and are stable to irradiation with X-rays . Preliminary crystallographic data are not consistent with the previously suggested pentameric structure, but indicate that the complex is in fact a hexamer with 32 symmetry.

J Infect Dis, 1994 Nov, 170(5), 1180 - 8
Use of recombinant soluble CD4 Pseudomonas exotoxin, a novel immunotoxin, for treatment of persons infected with human immunodeficiency virus; Davey RT Jr et al.; Single and multiple doses of sCD4-PE40, a soluble recombinant fusion toxin selectively toxic to gp120-expressing cells, were evaluated in persons infected with human immunodeficiency virus type 1 (HIV-1) . Seventeen of 24 patients who completed a single-dose safety trial were given either 1, 5, 10, or 15 micrograms/kg of sCD4-PE40 by intravenous bolus once a month for 2 months, then weekly for 6 weeks . The weekly maximally tolerated dose was 10 micrograms/kg . The major toxicity was a transient dose-dependent elevation in hepatic aminotransferases peaking 48 h after infusion . Anti-Pseudomonas exotoxin antibody developed in 58% of recipients, and sera from 13 of 17 showed neutralizing activity against sCD4-PE40 . No consistent changes in immunologic or virologic markers were observed . Weekly infusions of < or = 10 micrograms/kg of sCD4-PE40 are generally well tolerated, but additional studies correlating optimal dosing and frequency of administration with efficacy will be needed to define the role of this novel agent in the management of HIV-1-infected patients.

J Neurochem, 1994 Nov, 63(5), 1635 - 45
Agonist and cyclic AMP-mediated regulation of beta 1-adrenergic receptor mRNA and gene transcription in rat C6 glioma cells; Hosoda K et al.; Exposure of rat C6 glioma cells to either agonists or agents that increase cyclic AMP levels leads to down-regulation of beta 1-adrenergic receptors (beta 1 AR) as measured by loss of radioligand binding sites . The present study examines the influence of isoproterenol and forskolin treatment on levels of beta 1 AR mRNA, mRNA stability, and gene transcription rate . Isoproterenol treatment of C6 cells altered beta 1 AR mRNA levels in a biphasic manner; i.e., short-term exposure (30-60 min) increased by 50%, whereas longer exposure (2-6 h) decreased by 50% the levels of beta 1 AR mRNA . The extent of both the up- and down-regulation was dependent on agonist concentration . Similar regulation of beta 1 AR mRNA was observed in forskolin-treated cells . Pretreatment of the cells with Pseudomonas exotoxin A, a potent inhibitor of protein synthesis, completely blocked isoproterenol- and forskolin-mediated down-regulation of beta 1 AR mRNA, and thereby potentiated the increase in receptor mRNA up to fourfold over the 6-h time course . The mechanisms underlying beta 1 AR mRNA down-regulation were examined . The half-life of beta 1 AR mRNA was slightly increased (from 61 to 77 min) after a 2-h exposure of the cells to either isoproterenol or forskolin . Nuclear run-on analysis demonstrated that the rate of beta 1 AR gene transcription was increased after isoproterenol incubation for 60 min, but then decreased after 90-240 min, consistent with the time course for up- and down-regulation of beta 1 AR mRNA . Isoproterenol treatment (120 min) also decreased the level of beta 1 AR nascent transcripts, purified by affinity chromatography of RNA isolated from 4-thiouridine-pulsed cells . The results demonstrate that beta 1 AR mRNA has a relatively short half-life and that agonist regulation of beta 1 AR mRNA is mediated by activation of the cyclic AMP system . Moreover, the results indicate that agonist regulation of beta 1 AR mRNA occurs at the level of beta 1 AR gene transcription, not mRNA stability . Finally, the observed requirement for protein synthesis indicates that beta 1 AR mRNA down-regulation may be mediated by the induction of a repressor of the beta 1 AR gene.

Infect Immun, 1994 Nov, 62(11), 5055 - 65
Primate antibody response to immunotoxin: serological and computer-aided analysis of epitopes on a truncated form of Pseudomonas exotoxin; Roscoe DM et al.; NLysPE38 is a 38-kDa derivative of Pseudomonas exotoxin (PE) in which domain Ia (amino acids 1 to 252) and part of domain Ib (365 to 380) are deleted and an 11-amino-acid N-terminal peptide is added . LMB-1 is an immunotoxin in which the monoclonal antibody B3 is coupled to NLysPE38 near its N terminus . LMB-7 is a single-chain immunotoxin in which the Fv fragment of B3 is fused to PE38 . To identify the antigenic regions of PE38, 12 polyclonal serum samples from monkeys immunized with the immunotoxins LMB-1 (six monkeys) and LMB-7 (six monkeys) were tested for their reactivity to a panel of 120 synthetic, overlapping peptides representing the amino acid sequence of NLysPE38 . The antibody responses to peptides were similar among the 12 serum specimens, identifying several major immunodominant B-cell epitopes . Predominant reactivity was seen in six locations: amino acids 272 to 287, 341 to 359, 504 to 516, 540 to 564, and 573 to 591 and the C-terminal amino acids 591 to 613 . The sera did not react with approximately 75% of the peptides . Furthermore, a computer-aided analysis was done to predict the immunologically relevant areas and revealed the same antigenic regions defined by serum reactivity to peptides . Competition enzyme-linked immunosorbent assays and neutralization assays were performed with domain II, III, or III plus Ib of PE38 and confirmed the immunodominance of domain III . To analyze the role of specific amino acids in antibody binding, individual amino acids of PE38 with large accessible surface areas were altered by site-directed mutagenesis . These results also show that the predicted areas of immunogenicity agree with the reactivity of the anti-PE38 antibodies to peptides and to the mutants of PE.

Plasmid, 1994 Nov, 32(3), 262 - 9
IS204: an insertion sequence from Nocardia asteroides (mexicana) YP21; Yao W et al.; An insertion sequence was found in Nocardia asteroides YP21 . The element, designated IS204, is 1452 bp long and has 19/23 bp imperfect inverted repeats at its ends . The sequences of IS204 terminal inverted repeats have high homology with those of IS1096 from Mycobacterium smegmatis . An 8-bp duplication of target sequence was found at the insertion site . Sequence analysis revealed that IS204 contains an open reading frame of 1134 bp, which encodes a putative transposase similar to those found in IS1096 and in Tn4652 from Pseudomonas sp . EST1001 . At least nine copies of IS204 are present in the genome of N asteroides YP21 . The plasmid pCY 104::IS204 could be inserted with another copy of IS204 at a different site . The possible mechanisms are discussed.

J Biochem (Tokyo), 1994 Nov, 116(5), 1096 - 104
Analysis of DNA bend structure of promoter regulatory regions of xylene-metabolizing genes on the Pseudomonas TOL plasmid; Gomada M et al.; The transcription of both the upper operon (OP1) coding for m-xylene-degrading enzymes and the positive regulatory gene xylS on the TOL plasmid depends on sigma 54-RNA polymerase and requires the activator protein XylR that binds to the cis-acting upstream regulatory sequence of each promoter . For transcription of OP1 in Escherichia coli, integration host factor (IHF) is also required . IHF binds to DNA between the upstream regulatory sequence and the promoter sequence of OP1 . We showed that IHF induced a DNA bend in the promoter regulatory region of OP1 upon its binding, supporting the DNA-loop model for the activation of OP1 transcription . In contrast to OP1, the transcriptional activation of xylS does not require IHF . In the absence of IHF, the promoter regulatory region of xylS promoter was shown to have a weak but significant intrinsic DNA bend, which may be involved in stabilizing the DNA-loop structure for the activation of xylS transcription . When IHF was highly produced in E . coli, the xylS transcription was repressed . We found two weak binding sites for IHF, each overlapping with the promoter sequence and the upstream regulatory sequence . The IHF binding to these sites might result in repression of xylS expression by overproduced IHF . Evidence is presented that binding of IHF to these sites induces a DNA bend.

Klin Lab Diagn, 1994 Nov-Dec, (6), 46 - 8
{Specific immune complexes and neutrophil injury test in the diagnosis of experimental glanders and melioidosis}; Dunaev GS et al.; The authors have demonstrated the possibility of using solid-phase enzyme immunoassay to detect specific circulating immune complexes without their preliminary separation in guinea pigs infected with Pseudomonas mallei and Pseudomonas pseudomallei . They have examined the prospects of using neutrophil injury test and solid-phase enzyme immunoassay for the detection of specific immune complexes as additional tests used to predict the outcome of an infectious process in glanders and melioidosis.

Mol Plant Microbe Interact, 1994 Nov-Dec, 7(6), 726 - 39
Characterization of avrPphE, a gene for cultivar-specific avirulence from Pseudomonas syringae pv . phaseolicola which is physically linked to hrpY, a new hrp gene identified in the halo-blight bacterium; Mansfield J et al.; The avirulence gene matching the R2 gene for resistance to halo-blight disease in Phaseolus was cloned and sequenced from race 4 strain 1302A of Pseudomonas syringae pv . phaseolicola . The predicted 41-kDa AvrPphE protein is hydrophilic, has no features that indicate function, and no similarity to other protein sequences . The promoter region of avrPphE contains a "harp box" motif . The gene was expressed more strongly in minimal than in nutrient-rich media . Lower concentrations of the phytoalexin phaseollin accumulated in tissue undergoing the hypersensitive reaction (HR) determined by avrPphE than by avrPphB . Homologs of avrPphE were detected in strains representing eight races of P . s . pv . phaseolicola including those virulent on cultivars with the R2 resistance gene, and in P . s . pv . tabaci but not in P . cichorii or P . s . pvs . coronafaciens, glycinea, maculicola, pisi, or syringae . Disruption of avrPphE prevented induction of the HR but did not appear to affect basic pathogenicity . Transposon mutagenesis and DNA sequencing showed that avrPphE was linked to hrpY a hrp locus identified at the left end of the hrp gene cluster . Sequence analysis showed that the region linked to avrPphE was very similar to DNA containing hrp genes from P . s . pv . syringae including hrpJ, hrpL, and hrpK.

Bioconjug Chem, 1994 Nov-Dec, 5(6), 532 - 8
Analysis of sequences required for the cytotoxic action of a chimeric toxin composed of Pseudomonas exotoxin and transforming growth factor alpha; Kihara A et al.; Chimeric toxins composed of transforming growth factor alpha fused to mutant forms of Pseudomonas exotoxin bind to the EGF receptor and kill cells bearing these receptors . In early experiments, the binding domain of Pseudomonas exotoxin was deleted and replaced with TGF alpha to make TGF alpha-PE40 . This chimeric toxin required proteolytic processing within the target cell to be converted to its active form (Siegall et al . (1989) FASEB J . 3, 2647-2652) . Subsequently, recombinant toxins that do not require proteolytic processing were constructed by inserting TGF alpha near the carboxyl terminus of domain III and deleting toxin residues up to the processing site at position 280 . In addition, the carboxyl terminus of this toxin was converted from REDLK to KDEL to increase its activity . Recombinant toxins of this type, termed PE37/TGF alpha/KDEL, are about 100-fold more potent than TGF alpha-PE40 . To determine if other sequences can be removed from such chimeric toxins to make a smaller molecule that can penetrate tissues better, we have carried out a deletion analysis of sequences present within domains II and Ib . We find that all of domain Ib and a portion of domain II can be deleted without significant loss of cytotoxic activity, but larger deletions extending further into domain II lose cytotoxic activity . We also find that inserting a small linking peptide (Gly)4Ser between residual sequences in domain II and domain III, in molecules with diminished cytotoxic activity, enhances cytotoxicity suggesting that one role of domain Ib is to prevent undesirable interactions between domains II and III.(ABSTRACT TRUNCATED AT 250 WORDS)

Plant Cell, 1994 Nov, 6(11), 1543 - 52
A member of the tomato Pto gene family confers sensitivity to fenthion resulting in rapid cell death; Martin GB et al.; Leaves of tomato cultivars that contain the Pto bacterial resistance locus develop small necrotic lesions within 24 hr after exposure to fenthion, an organophosphorous insecticide . Recently, the Pto gene was isolated and shown to be a putative serine/threonine protein kinase . Pto is one member of a multigene family that is clustered within a 400-kb region on chromosome 5 . Here, we report that another member of this gene family, termed Fen, is responsible for the sensitivity to fenthion . Fen was isolated by map-based cloning using closely linked DNA markers to identify a yeast artificial chromosome clone that spanned the Pto region . After transformation with the Fen gene under control of the cauliflower mosaic virus (CaMV) 35S promoter, tomato plants that are normally insensitive to fenthion rapidly developed extensive necrotic lesions upon exposure to fenthion . Two related insecticides, fensulfothion and fenitrothion, also elicited necrotic lesions specifically on Fen-transformed plants . Transgenic tomato plants harboring integrated copies of the Pto gene under control of the CaMV 35S promoter displayed sensitivity to fenthion but to a lesser extent than did wild-type fenthion-sensitive plants . The Fen protein shares 80% identity (87% similarity) with Pto but does not confer resistance to Pseudomonas syringae pv tomato . These results suggest that Pto and Fen participate in the same signal transduction pathway.

J Pediatr Orthop, 1994 Nov-Dec, 14(6), 755 - 9
Pseudomonas osteochondritis complicating puncture wounds in children; Jarvis JG et al.; Pseudomonas osteochondritis is an uncommon complication of puncture wounds . It can have a particularly devastating affect in the growing child, often resulting in significant permanent sequelae . To assess the current approach to diagnosis and treatment of this condition in children, 15 such cases seen at the Children's Hospital of Eastern Ontario between 1975 and 1991 were studied retrospectively . Case presentations were similar, with delayed onset of localized pain, swelling, and elevated erythrocyte sedimentation rate following a puncture wound . All patients had previously received oral antibiotics . Initial radiographic changes were rare . All patients were treated with i.v . antibiotics: although most required surgical debridement . Complications including recurrence, chronic pain, and deformities required sequestrectomies, angular osteotomies, and leg-lengthening procedures . A high index of suspicion, coupled with aggressive medical and surgical treatment, is required for a satisfactory outcome.

Biosci Biotechnol Biochem, 1994 Nov, 58(11), 2096 - 8
Promoter screening from Pseudomonas species by a promoter probe transposon and its structure; Hosoya H et al.; By use of the promoter probe transposon Tn5-B21, two promoter fragments were isolated . One promoter (822 bp) (GC = 63.5%) isolated from P . putida loses all of its promoter activity by exonuclease or restriction enzyme deletion . Another promoter (264 bp) (GC = 49.2%) isolated from P . fluorescens could be shortened to 154 bp by exonuclease deletion without any effect on its promoter activity in several Pseudomonas species.

J Bacteriol, 1994 Nov, 176(21), 6550 - 7
Structural studies of the side chain of outer membrane lipopolysaccharide from Pseudomonas syringae pv . coriandricola W-43; Das S et al.; The lipopolysaccharide (LPS) was isolated from Pseudomonas syringae pv . coriandricola W-43 by hot phenol-water extraction . Rhamnose and 3-N-acetyl-3-deoxyfucose were found to be the major sugar constituents of the LPS together with N-acetylglucosamine, N-acetylgalactosamine, heptose, and 3-deoxy-D-manno-octulosonic acid (Kdo) . The main fatty acids of lipid A of the LPS were 3-OH-C:10, C12:0, 2-OH-C12:0, and 3-OH-C12:0 . The O-specific polysaccharide liberated from the LPS by mild-acid hydrolysis was purified by gel permeation chromatography . The compositional analysis of the O-specific polysaccharide revealed the presence of L-rhamnose and 3-N-acetyl-3-deoxy-D-fucose in a molar ratio of 4:1 . The primary structure of the O-specific polysaccharide was established by methylation analysis together with 1H and 13C nuclear magnetic resonance spectroscopy, including two-dimensional shift-correlated and one-dimensional nuclear Overhauser effect spectroscopy . The polysaccharide moiety was found to consist of a tetrasaccharide rhamnan backbone, and 3-N-acetyl-3-deoxy-D-fucose constitutes the side chain of the branched pentasaccharide repeating unit of the polysaccharide.

Biochem Biophys Res Commun, 1994 Oct 28, 204(2), 912 - 7
Substitution of the ISP alpha subunit of biphenyl dioxygenase from Pseudomonas results in a modification of the enzyme activity; Tan HM et al.; We have constructed a hybrid multicomponent dioxygenase gene cluster in which the bphA1 gene, coding for ISP alpha subunit of biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707, has been replaced by the bedC1 gene encoding the corresponding subunit of benzene dioxygenase from P . putida ML2 . Escherichia coli cells containing the chimeric dioxygenase acquired the novel capability of producing indigo from indole . Furthermore, when compared to biphenyl dioxygenase, the hybrid dioxygenase enzyme was half as active towards benzene but exhibited only 4% activity when biphenyl was the substrate . The results implicate the ISP alpha subunit to be involved in substrate specificity and activity of the dioxygenase enzyme.

Biochem Biophys Res Commun, 1994 Oct 28, 204(2), 983 - 93
Identification of a meta-cleavage pathway for metabolism of phenoxyacetic acid and phenol in Pseudomonas cepacia AC1100; Ghadi SC et al.; Pseudomonas cepacia strain AC1100 grows luxuriantly on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) but does not utilize phenoxyacetic acid . After long-term selective pressure on phenoxyacetic acid, mutants designated as strain PAA, capable of utilizing phenoxyacetic acid as well as phenol, emerged spontaneously at a frequency of 1 x 10(-8) . A deletion mutant strain PT88, which is devoid of a part of 2,4,5-T metabolic pathway, generated neither phenoxyacetic acid utilizing nor phenol-utilizing mutants . The wild type (Wt) strain AC1100 and all its mutants utilized benzoate via ortho-cleavage pathway . However, only mutant strain PAA harbored the whole set of enzymes required for utilization of phenol via meta-cleavage pathway . The results suggest that Wt strain AC1100 carries silent genes for meta-cleavage pathway which are expressed in strain PAA enabling it to utilize phenoxyacetic acid and phenol . Gene activation is presumed to be due to the translocation of insertion elements.

N Engl J Med, 1994 Oct 13, 331(15), 981 - 7
Transmissibility of Pseudomonas cepacia infection in clinic patients and lung-transplant recipients with cystic fibrosis; Steinbach S et al.; BACKGROUND . In patients with cystic fibrosis, infection with Pseudomonas cepacia is associated with poor outcomes . However, the extent of person-to-person transmission and the source of P . cepacia infection after lung transplantation are not well defined . Using DNA-based typing systems, we sought to determine the genetic relatedness of P . cepacia infection at one cystic fibrosis center . METHODS . We analyzed 65 P . cepacia isolates gathered over a period of eight years at a single cystic fibrosis center from 17 clinic patients and from 5 patients who underwent double-lung transplantation . The isolates were analyzed by ribotyping and chromosomal fingerprinting based on pulsed-field gel electrophoresis . RESULTS . Analyses of serial isolates revealed that each clinic patient and transplant recipient harbored a different P . cepacia clone that was persistent . In the transplant recipients, the preoperative and postoperative isolates were identical . In the two patients with disseminated infection after lung transplantation, isolates from multiple sites were identical and indicated clonal expansion of the previous respiratory P . cepacia strain . Pulsed-field gel electrophoresis proved both more discriminative and more practical than ribotyping as a means of defining the genetic relatedness of the P . cepacia isolates . CONCLUSIONS . Our serial analyses in patients with cystic fibrosis at one center found distinct strains of P . cepacia persistently infecting each patient and no evidence of person-to-person transmission of this organism . P . cepacia infection after lung transplantation was due to the persistence of the strain present before transplantation.

Arch Dis Child, 1994 Oct, 71(4), 349 - 52
Liver transplantation for hepatic cirrhosis in cystic fibrosis; Noble-Jamieson G et al.; Five children with cystic fibrosis complicated by hepatic cirrhosis received liver grafts . They all had portal hypertension with varices and three had variceal bleeding; respiratory function was only moderately impaired, but four were colonised with pseudomonas and one with aspergillus . Liver transplantation was well tolerated and there was no increase in respiratory or other early postoperative complications . Four of the children were fully well from 14 to 35 months after transplantation; the most recently transplanted had problems from a biliary stricture . In spite of the need for immunosuppression there was no increase in infection and respiratory function improved or remained stable . Once the children were stabilised after transplantation their nutrition and general health were greatly improved.

Plant Mol Biol, 1994 Oct, 26(1), 515 - 21
Structural organization of str 246C and str 246N, plant defense-related genes from Nicotiana tabacum; Froissard D et al.; We have previously identified a cDNA clone, pNt246, whose corresponding transcripts accumulate in leaves in response to inoculation by compatible and incompatible isolates of the phytopathogenic bacterium Pseudomonas solanacearum {19} . We now describe the nucleotide sequence of a genomic clone, str 246C, corresponding to this cDNA species, and of a related genomic clone, str 246N, which appears to be a pseudogene with a 5'-end deletion . The nucleotide sequence of the str 246C gene was found to be identical to that of the parA gene, previously shown to be regulated by auxin {28, 29} . Upstream of the str 246N gene, sequences homologous to a Bam HI repetitive element described in Vicia faba {15} are present within an ORF showing significant homologies to an integrase-encoding gene of several retroviruses . This observation indicates that this highly repetitive DNA originates from sequences present in transposable mobile elements.

J Infect Dis, 1994 Oct, 170(4), 1009 - 13
Failure of short-term CD4-PE40 infusions to reduce virus load in human immunodeficiency virus-infected persons; Ramachandran RV et al.; The safety, immunologic, and antiviral effects of a recombinant biologic product that combines the second and third domains of the CD4 molecule and Pseudomonas exotoxin A (PE40) were evaluated in 21 human immunodeficiency virus (HIV)-infected subjects in a phase III open-label dose-ranging study . Subjects with CD4+ lymphocyte counts of 100-500/mm3 received CD4-PE40 at 40, 80, or 160 micrograms/m2 by infusion three to seven times over 10 days . At the maximum tolerated dose (80 micrograms/m2), peak CD4-PE40 levels were 65-130 ng/mL with a serum half-life of 3.6 +/- 1.5 h . Toxicity, primarily increased hepatic transaminases, was dose-related and reversible . HIV DNA proviral levels in peripheral blood mononuclear cells and plasma HIV RNA remained stable during and after CD4-PE40 infusions . The relative resistance of clinical isolates of HIV, limits of the tolerated dose, and the immunogenicity and short half-life of the protein may explain the lack of in vivo antiviral effect of CD4-PE40.

Cancer Res, 1994 Oct 1, 54(19), 5154 - 9
Small chimeric toxins containing only transforming growth factor alpha and domain III of Pseudomonas exotoxin with good antitumor activity in mice; Kihara A et al.; Chimeric toxins composed of transforming growth factor alpha (TGF alpha) fused to mutant forms of Pseudomonas exotoxin (PE) bind to the epidermal growth factor receptor and kill cells bearing epidermal growth factor receptors . Initially, the binding domain (Ia; amino acids 1-252) of PE was deleted and replaced with TGF alpha to make TGF alpha-PE40 in which TGF alpha is fused to domains II, Ib, and III of PE (amino acids 253-613) . That drug is currently undergoing clinical study for the intravesical therapy of bladder cancer . To generate smaller molecules that would have increased tumor penetration, several deletion mutants were constructed . In one of these, TGF alpha was inserted near the carboxyl terminus of PE, and residues in domains II and Ib of PE (amino acids 253-279 and 365-380) were deleted so that the chimeric toxin did not need to be cleaved by an intracellular protease to be activated (Theuer et al., J . Biol . Chem., 267: 16872-16877, 1992) . We have now constructed chimeric toxins which contain only domain III, yet still exhibit high cytotoxic activity on epidermal growth factor receptor-containing cells and produce substantial tumor regressions in mice bearing a human xenograft . The high cytotoxic activity of these severely truncated toxins provides new insights on the proposed functions of domains II and III of PE.

Int J Epidemiol, 1994 Oct, 23(5), 1082 - 90
The epidemiology of melioidosis in Ubon Ratchatani, northeast Thailand; Suputtamongkol Y et al.; BACKGROUND . Melioidosis, or infection with Pseudomonas pseudomallei is an important cause of morbidity and mortality in South East Asia and Northern Australia . The epidemiology of melioidosis in Ubon Ratchatani, Northeast Thailand was studied over a 5-year period from 1987 to 1991 . METHODS . Rates and, when possible, the risks of developing melioidosis were calculated . The numerator was the number of culture-proven cases of melioidosis seen in the 1000-bed referral hospital of the province . The denominators were obtained from the population census, a survey of Health, Welfare and Use of Traditional Medicine, and the North Eastern Meterological Centre, Thailand . RESULTS . The average incidence of human melioidosis was 4.4 (95% confidence interval {CI}: 3.8-5.0) per 100,000 . The disease affected all ages with the highest incidence in 40-60 years olds . Melioidosis was 1.4 (95% CI: 0.4-5.3) times more common in males than females . The disease showed a significant seasonal variation in incidence, and a strong linear correlation with rainfall (r = 0.7, 95% CI: 0.5-0.9) Adults exposed to soil and water in their work (most were rice farmers) had an increased risk of melioidosis (in the 40-59 year age group, relative risk = 4.1, 95% CI: 2.4-6.9) . Most adult patients had an underlying disease (mainly diabetes mellitus) predisposing them to this infection . CONCLUSION . Melioidosis may result from either acute exposure to the organism in the soil and water, or 're-activation' of an asymptomatic childhood infection (by an unidentified possibly infective seasonal cofactor) . The results from this analysis are consistent with both hypotheses . Further epidemiological studies are needed to identify risk factors so that optimal strategies for control of melioidosis may be developed.

Trends Microbiol, 1994 Oct, 2(10), 347 - 53
Resistance to aminoglycosides in Pseudomonas . Aminoglycoside Resistance Study Groups; Characterization of alpha-glucosyltransferase from Pseudomonas mesoacidophila MX-45; Food Laboratory, Mitsui Sugar Co., Ltd., Kanagawa, Japanalpha-Glucosyltransferase was purified from Pseudomonas mesoacidophila MX-45 . The molecular weight was estimated to be 63,000 by SDS-PAGE, and the isoelectric point was pI 5.4 . For enzyme activity based on sucrose decomposition, the optimum pH and the optimum temperature were pH 5.8 and 40 degrees C, respectively . The ranges of stable pH and temperature were pH 5.1-6.7 and below 40 degrees C, respectively . The purified enzyme of MX-45 converted sucrose into trehalulose (1-O-alpha-D-glucopyranosyl- D-fructose) and isomaltulose (palatinose, 6-O-alpha-D-glucopyranosyl-D-fructose) simultaneously, and the ratio of trehalulose to isomaltulose increased at lower reaction temperatures . Therefore, optimum conditions for trehalulose production were pH 5.5-6.5 at 20 degrees C . The yield of trehalulose from sucrose (20-40% solution) was 91% . The Km for sucrose was 19.2 +/- 3.3 mM estimated by the Hanes-Woolf plot . Product inhibition was observed, and the product inhibition constant was 0.17 M . Hg2+, Fe3+, Cu2+, Mg2+, Ag+, Pb2+, glucono-1,5-lactone, and Tris(hydroxymethyl)aminomethane inhibited the reaction.

Singapore Med J, 1994 Oct, 35(5), 475 - 6
Fatal sepsis due to Pseudomonas cepacia; Saha SK et al.; Pseudomonas cepacia is known as an opportunistic pathogen in the patients with altered host defence . We report a case of hospital-acquired fatal sepsis due to P . cepacia in a child with no known defect in immune system . Virulence of the organism was indicated by the high magnitude of septicaemia . The organism was successfully eliminated by ceftriaxone but the patient died of renal failure.

Clin Genet, 1994 Oct, 46(4), 287 - 90
Molecular and clinical analyses of cystic fibrosis in the south of Spain; Borrego S et al.; We report molecular and clinical analyses in 71 unrelated patients with cystic fibrosis (CF) from Andalusia (South of Spain) . Direct mutation analysis of six mutations of the CFTR gene (delta F508, G542X, R1162X, N1303K, W1182X and 1949de184) was performed . The proportion of CF chromosomes with the above-mentioned mutations was 58.5% . Haplotype analysis was performed with the marker/enzyme pairs XV2C/TaqI and KM19/PstI . A particular haplotype has been found associated with each of the studied mutations, while the pooled data for the unknown mutations are not associated with any particular haplotype . This lack of association indicates that there will not be a single predominant mutation amongst the other CF chromosomes . To assess the relationship between genotype and phenotype in these patients, we correlated the pancreatic status and the occurrence of chronic Pseudomona aeruginosa infection with the observed genotype . Pancreatic insufficiency was present in all patients in whom the analyzed mutations were found to be homozygous or compound heterozygous . We also found a higher rate of Pseudomonas colonization in the group of patients in whom the genotype was homozygous or compound heterozygous for the analysed mutations when compared with the group of patients with a different genotype, but the difference was not statistically significant.

FEMS Microbiol Lett, 1994 Oct 1, 122(3), 251 - 6
Isolation and partial purification of a carbapenem-hydrolysing metallo-beta-lactamase from Pseudomonas cepacia; Baxter IA et al.; A metallo-beta-lactamase has been isolated from a clinical strain of Pseudomonas cepacia and partially purified using Cibacron blue F3GA coupled agarose . The resulting preparation showed a single band of beta-lactamase activity (pI 8.45) after analytical isoelectric focusing . The enzyme was particularly effective in the hydrolysis of imipenem . Meropenem, biapenem, cephaloridine, ceftazidime, benzylpenicillin, ampicillin and carbenicillin were also hydrolysed, although at a lower rate . An unusual inhibition profile was noted . Inhibition by the metal ion chelators ethylenediaminetetraacetic acid and o-phenanthroline was reversed by addition of zinc, indicating a metallo-enzyme, whilst > 90% inhibition was attainable with 0.1 mM concentrations of tazobactam and clavulanic acid . A study of 8 other clinical isolates showed the enzyme to be present and inducible by imipenem in each case . This enzyme was assigned PCM-I (Pseudomonas cepacia metalloenzyme I).

Epidemiol Infect, 1994 Oct, 113(2), 307 - 12
Pseudomonas pseudomallei isolates collected over 25 years from a non-tropical endemic focus show clonality on the basis of ribotyping; Currie B et al.; Between 1966 and 1991, melioidosis, a disease caused by Pseudomonas pseudomallei that is mostly confined to tropical regions, occurred in farm animals and a farmer in temperate south-west Western Australia . Using an Escherichia coli probe containing a ribosomal RNA operon, P . pseudomallei DNA from isolates from 8 animals, a soil sample and the human case showed an identical ribotype on Southern blotting . The ribotype was different from the 3 commonest ribotypes seen in tropical Australia . This molecular typing supports the theory of clonal introduction of P . pseudomallei into a non-endemic region, with environmental contamination, local dissemination and persistence over 25 years . As melioidosis is often fatal in humans, such persistence in a temperate region is cause for concern.

Biochim Biophys Acta, 1994 Sep 28, 1201(1), 55 - 60
Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization; Bornscheuer U et al.; Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose . The eluted lipase crystallized spontaneously at 4 degrees C in the eluent, containing 58-69% 2-propanol . The yield of the lipase was 87-100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein . This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . Its purity was determined by SDS-PAGE and capillary zone electrophoresis to be > or = 99% . Immobilization on Sepharose increased its stability in organic solvents . This lipase of P . cepacia differs from that of other Pseudomonas strains in respect to substrate specificity and during crystallization . It exhibits a high stability in organic solvents and supercritical carbon dioxide.

Biochemistry, 1994 Sep 27, 33(38), 11637 - 44
Site-specific conjugation to interleukin 4 containing mutated cysteine residues produces interleukin 4-toxin conjugates with improved binding and activity; Kreitman RJ et al.; Fusion of a ligand to another protein frequently impairs the binding of the ligand . Recombinant toxins composed of mutants of Pseudomonas exotoxin (PE) fused to the C-terminus of human interleukin 4 (IL4) are cytotoxic to IL4 receptor- (IL4R-) bearing tumor cells but bind to the IL4R with only 1% the affinity of IL4 . We have developed a method to connect a toxin to a ligand which allows the junction to be moved to a location on the ligand which would minimize the binding impairment . We designed mutants of IL4 in which residue 28, 38, 68, 70, 97, or 105 was substituted with cysteine . All purified mutants bound to the IL4R with 60-100% the affinity of IL4, indicating that the IL4 structure was essentially unchanged . The IL4 mutants were then each conjugated through a disulfide bond to PE35, a truncated form of PE which contains a single cysteine . IL4 conjugated to PE35 at residue 28, 38, or 105 of IL4 bound with 10-fold improved affinity and was 10-fold more cytotoxic than the recombinant IL4-toxin in which PE is fused to position 129 at the C-terminus of IL4 . IL4 containing PE35 conjugated at position 68, 70, or 97 had lower binding affinity and cytotoxic activity . These results indicate that the location of the ligand-protein junction can be selectively moved to enhance conjugate effectiveness, and implications could be made regarding which regions of IL4 are important for binding.

Science, 1994 Sep 23, 265(5180), 1856 - 60
RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes; Bent AF et al.; Plant disease resistance genes function is highly specific pathogen recognition pathways . PRS2 is a resistance gene of Arabidopsis thaliana that confers resistance against Pseudomonas syringae bacteria that express avirulence gene avrRpt2 . RPS2 was isolated by the use of a positional cloning strategy . The derived amino acid sequence of RPS2 contains leucine-rich repeat, membrane-spanning, leucine zipper, and P loop domains . The function of the RPS2 gene product in defense signal transduction is postulated to involve nucleotide triphosphate binding and protein-protein interactions and may also involve the reception of an elicitor produced by the avirulent pathogen.

J Immunol, 1994 Sep 15, 153(6), 2497 - 505
Mechanisms of dichotomous action of IL-2-Pseudomonas exotoxin 40 (IL-2-PE40) on cell-mediated and humoral immune response; Volk HD et al.; IL-2-PE40 is a chimeric protein composed of human IL-2 genetically fused to the amino terminus of a modified form of Pseudomonas exotoxin lacking its cell recognition domain . The immunosuppressive efficacy of IL-2-PE40 was demonstrated in several experimental murine transplant and autoimmune models . However, some observations suggested that IL-2-PE40 could not inhibit the humoral response . In this report, we describe the dichotomous effects of IL-2-PE40 on humoral and cell-mediated immune response in a simple, well characterized in vivo model . Although IL-2-PE40 inhibited the cell-mediated delayed type hypersensitivity reaction to SRBC, it increased the humoral immune response to the same Ag . To understand the mechanism of dichotomous action of IL-2-PE40 on the immune response, IL-2R-bearing T cells were treated with IL-2-PE40 in vitro and the cytokine expression was studied at mRNA and protein level . Similar to IL-2, IL-2-PE40 promoted the expression of T helper 1-like (IFN-gamma) as well as T helper 2-like (IL-4, IL-10) cytokines . These in vitro studies show that IL-2-PE40 can induce signal transduction in activated T cells through the IL-2R before exerting its cytotoxic effect . In contrast to DTH reaction, humoral immune response requires T cell help only for a limited period . Therefore, the short-term stimulation of T helper cells by IL-2-PE40 may be sufficient in vivo to mediate a B cell response in the local environment, whereas the DTH reaction and other cell-mediated immune responses are inhibited by the toxin moiety of the chimeric protein.

Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 8955 - 9
Isolation of phytoalexin-deficient mutants of Arabidopsis thaliana and characterization of their interactions with bacterial pathogens; Glazebrook J et al.; A genetic approach was used to assess the extent to which a particular plant defense response, phytoalexin biosynthesis, contributes to Arabidopsis thaliana resistance to Pseudomonas syringae pathogens . The A . thaliana phytoalexin, camalexin, accumulated in response to infection by various P . syringae strains . No correlation between pathogen avirulence and camalexin accumulation was observed . A biochemical screen was used to isolate three mutants of A . thaliana ecotype Columbia that were phytoalexin deficient (pad mutants) . The mutations pad1, pad2, and pad3 were found to be recessive alleles of three different genes . pad1 and pad2 were mapped to chromosome IV and pad3 was mapped to chromosome III . Infection of pad mutant plants with strains carrying cloned avirulence genes revealed that the pad mutations did not affect the plants' ability to restrict the growth of these strains . This result strongly suggests that in A . thaliana, phytoalexin biosynthesis is not required for resistance to avirulent P . syringae pathogens . Two of the pad mutants displayed enhanced sensitivity to isogenic virulent P . syringae pathogens, suggesting that camalexin may serve to limit the growth of virulent bacteria.

Int J Cancer, 1994 Sep 1, 58(5), 744 - 8
Interleukin-4 receptors expressed on tumor cells may serve as a target for anticancer therapy using chimeric Pseudomonas exotoxin; Debinski W et al.; Interleukin-4 receptors (IL4R) are present on a wide variety of human cancer cells derived from both hematopoietic and epithelial malignancies . We have targeted IL4R on a human solid tumor xenograft with chimeric proteins composed of human IL4 (hIL4) and 2 different mutant forms of a powerful bacterial toxin, Pseudomonas exotoxin A (PE) . The 2 chimeric toxins, termed hIL4-PE4E and hIL4-PE38QQR, showed specific, hIL4R-dependent and dose-dependent antitumor activities . Neither of the chimeric toxins showed antitumor potency when the ADP-ribosylation activity of the toxin was inactivated by mutagenesis . One of the chimeras, hIL4-PE38QQR, caused a complete although transient regression of established solid tumors . These observations indicate that hIL4-PE chimeric proteins should be further evaluated for the treatment of human malignancies.

J Bacteriol, 1994 Sep, 176(17), 5233 - 43
Cloning and characterization of an autonomous replication sequence from Coxiella burnetii; Suhan M et al.; A Coxiella burnetii chromosomal fragment capable of functioning as an origin for the replication of a kanamycin resistance (Kanr) plasmid was isolated by use of origin search methods utilizing an Escherichia coli host . The 5.8-kb fragment was subcloned into phagemid vectors and was deleted progressively by an exonuclease III-S1 technique . Plasmids containing progressively shorter DNA fragments were then tested for their capability to support replication by transformation of an E . coli polA strain . A minimal autonomous replication sequence (ARS) was delimited to 403 bp . Sequencing of the entire 5.8-kb region revealed that the minimal ARS contained two consensus DnaA boxes, three A + T-rich 21-mers, a transcriptional promoter leading rightwards, and potential integration host factor and factor of inversion stimulation binding sites . Database comparisons of deduced amino acid sequences revealed that open reading frames located around the ARS were homologous to genes often, but not always, found near bacterial chromosomal origins; these included identities with rpmH and rnpA in E . coli and identities with the 9K protein and 60K membrane protein in E . coli and Pseudomonas species . These and direct hybridization data suggested that the ARS was chromosomal and not associated with the resident plasmid QpH1 . Two-dimensional agarose gel electrophoresis did not reveal the presence of initiating intermediates, indicating that the ARS did not initiate chromosome replication during laboratory growth of C . burnetii.

Cancer Res, 1994 Sep 1, 54(17), 4703 - 9
Epidermal growth factor receptors in human breast carcinoma cells: a potential selective target for transforming growth factor alpha-Pseudomonas exotoxin 40 fusion protein; Arteaga CL et al.; Epidermal growth factor (EGF) receptors are expressed in high levels by some poor prognosis breast tumors . We have examined the cytotoxic effect of the tumor growth factor alpha (TGF alpha)-delta Cys-Pseudomonas exotoxin (PE40) recombinant fusion protein on normal and tumorigenic human breast epithelial cells in vitro and in vivo . The MDA-468, MDA-231, BT-20, and MCF-7ADR estrogen receptor-negative, EGF receptor-rich breast cancer lines were exquisitely sensitive in vitro to TGF alpha-delta Cys-PE40 with a 50% inhibitory concentration of < or = 0.02 nM . The estrogen receptor-positive, low EGF receptor MCF-7, ZR75-1, and T47D cells were less sensitive to the fusion toxin with a 50% inhibitory concentration of > 0.2 nM . The nontumorigenic cell lines 184, 184A1, and 184B5 were relatively resistant to TGF alpha-delta Cys-PE40 despite exhibiting high levels of EGF receptors . Continuous i.p . administration of TGF alpha-delta Cys-PE40 via an osmotic minipump at a dose of 0.4 microgram/g/day over 7 days inhibited MDA-468, MA-231, and BT-20 but not MCF-7 tumor growth in female athymic mice . Host tissue toxicity was not observed with this dose of TGF alpha-delta Cys-PE40 . Mixed MDA-468/MCF-7 tumors were established in nude mice after coinoculation of both cell types in estrogen-supplemented animals . EGF receptor immunohistochemistry and immunoblot procedures indicated that TGF alpha-PE40 eliminated the MDA-468 cells while sparing the adjacent MCF-7 cells . By immunoblot, EGF receptors were consistently more abundant in tumor tissue than in adjacent nontumor tissue from the same mastectomy specimen (n = 7) . These data support the notion that EGF receptors can be selectively targeted in human breast cancer cells for the delivery of antitumor agents . Further clinical studies with TGF alpha-delta Cys-PE40 and other chimeric toxins using the same cellular target will address this possibility.

Virology, 1994 Sep, 203(2), 221 - 8
A new small low-abundant nonstructural protein encoded by the L segment of the dsRNA bacteriophage phi 6; Casini G et al.; A new small low-abundant protein encoded by the large genome segment of bacteriophage phi 6 has been detected in extracts of Escherichia coli bearing phi 6 cDNA clones and in extracts of phage-infected Pseudomonas phaseolicola . This 62 amino acid protein, designated P14, has a net charge of +2 at pH 7.0 . Gene 14 has been located by deletion analysis and N-terminal protein sequence . Expression of P14 is down-regulated in E . coli when the complete L segment clone is expressed . Polyclonal antibodies to P14 detected the new protein in small amounts in phage-infected cells but not in phi 6 virions or nucleocapsids, or in procapsids assembled in E . coli . Procapsids assembled in E . coli after expression of phi 6 L segment clones with or without gene 14 had essentially similar protein composition and activity in an in vitro packaging and replication system . Thus P14 does not appear to be essential for the structure or assembly of functional procapsids . P14 might, however, facilitate packaging in vivo . Alternatively this protein could play in a role in repair of host membranes following viral penetration.

Appl Environ Microbiol, 1994 Sep, 60(9), 3375 - 80
Comparative studies of genes encoding thermostable L-2-halo acid dehalogenase from Pseudomonas sp . strain YL, other dehalogenases, and two related hypothetical proteins from Escherichia coli; Nardi-Dei V et al.; We have determined the nucleotide sequence of the gene encoding thermostable L-2-halo acid dehalogenase (L-DEX) from the 2-chloroacrylate-utilizable bacterium Pseudomonas sp . strain YL . The open reading frame consists of 696 nucleotides corresponding to 232 amino acid residues . The protein molecular weight was estimated to be 26,179, which was in good agreement with the subunit molecular weight of the enzyme . The gene was efficiently expressed in the recombinant Escherichia coli cells: the amount of L-DEX corresponds to about 49% of the total soluble proteins . The predicted amino acid sequence showed a high level of similarity to those of L-DEXs from other bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp . strain B (38 to 57% identity) but a very low level of similarity to those of haloacetate dehalogenase H-1 from Moraxella sp . strain B (10%) and haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (12%) . By searching the protein amino acid sequence database, we found two E . coli hypothetical proteins similar to the Pseudomonas sp . strain YL L-DEX (21 to 22%).

Appl Environ Microbiol, 1994 Sep, 60(9), 3323 - 8
Regiospecific and stereoselective hydroxylation of 1-indanone and 2-indanone by naphthalene dioxygenase and toluene dioxygenase; Resnick SM et al.; The biotransformation of 1-indanone and 2-indanone to hydroxyindanones was examined with bacterial strains expressing naphthalene dioxygenase (NDO) and toluene dioxygenase (TDO) as well as with purified enzyme components . Pseudomonas sp . strain 9816/11 cells, expressing NDO, oxidized 1-indanone to a mixture of 3-hydroxy-1-indanone (91%) and 2-hydroxy-1-indanone (9%) . The (R)-3-hydroxy-1-indanone was formed in 62% enantiomeric excess (ee) (R:S, 81:19), while the 2-hydroxy-1-indanone was racemic . The same cells also formed 2-hydroxy-1-indanone from 2-indanone . Purified NDO components oxidized 1-indanone and 2-indanone to the same products produced by strain 9816/11 . P . putida F39/D cells, expressing TDO, oxidized 2-indanone to (S)-2-hydroxy-1-indanone of 76% ee (R:S, 12:88) but did not oxidize 1-indanone efficiently . Purified TDO components also oxidized 2-indanone to (S)-2-hydroxy-1-indanone of 90% ee (R:S, 5:95) and failed to oxidize 1-indanone . Oxidation of 1- and 2-indanone in the presence of {18O}oxygen indicated that the hydroxyindanones were formed by the incorporation of a single atom of molecular oxygen (monooxygenation) rather than by the dioxygenation of enol tautomers of the ketone substrates . As alternatives to chemical synthesis, these biotransformations represent direct routes to 3-hydroxy-1-indanone and 2-hydroxy-1-indanone as the major products from 1-indanone and 2-indanone, respectively.

Eur J Biochem, 1994 Sep 1, 224(2), 663 - 76
Secondary structure of the single-stranded DNA binding protein encoded by filamentous phage Pf3 as determined by NMR; Folmer RH et al.; Nuclear magnetic resonance spectroscopy was employed to study the single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 . The protein is 78 amino acids long and occurs in solution predominantly as a homodimer with a molecular mass of 18 kDa . Sequence-specific 1H and 15N resonance assignments have been obtained using homo- and heteronuclear two- and three-dimensional experiments . The secondary structure of the protein monomer was determined from a qualitative interpretation of nuclear Overhauser enhancement spectra and amide exchange data . It consists of a five-stranded antiparallel beta-sheet and three beta-hairpins . Problems caused by the protein's tendency to aggregate at concentrations needed for NMR spectroscopy were largely overcome by designing a mutant (Phe36-->His) which exhibits significantly improved solubility characteristics over the wild-type protein . It is shown that this mutation only locally affects the structure of the protein; the chemical shifts of the wild-type and mutant species differ only for a few residues near the site of the mutation, and the secondary structures of the proteins are identical . The secondary structure of the Pf3 single-stranded DNA binding protein is compared to that of the Ff gene V protein, the only single-stranded DNA binding protein for which the complete three-dimensional structure is known to date {Folkers, P . J . M., Nilges, M., Folmer, R . H . A., Konings, R . N . H . & Hilbers, C . W . (1994) J . Mol . Biol . 236, 229-246; Skinner, M . M., Zhang, H., Leschnitzer, D . H., Guan, Y., Bellamy, H., Sweet, R . M., Gray, C . W., Konings, R . N . H., Wang, A . H.-J . & Terwilliger, T . C . (1994) Proc . Natl Acad . Sci . USA 91, 2071-2075} . It is found that the secondary structures of the two proteins are very similar which supports the hypothesis that a five-stranded antiparallel beta-sheet with protruding beta-hairpins is a common motif in a certain class of single-stranded DNA binding proteins . In addition, the sequence and folding predicted earlier for the DNA binding wing in the single-stranded DNA binding protein of phage Pf3 {de Jong, E . A . M., van Duynhoven, J . P . M., Harmsen, B . J . M., Tesser, G . I., Konings, R . N . H . & Hilbers, C . W . (1989) J . Mol . Biol . 206, 133-156} is borne out by the present study . It closely resembles that in the single-stranded DNA binding protein of phage Ff, which may indicate that such a wing is a recurrent motif as well.

Mol Cell Biochem, 1994 Sep, 138(1-2), 131 - 3
Cellular ADP-ribosylation of elongation factor 2; Iglewski WJ; A cellular ADP-ribosyltransferase activity has been found in a variety of animals and tissues . The enzyme transfers ADP-ribose from NAD to elongation factor 2, inactivating the factor and thus inhibiting in vitro protein synthesis . Although, the mechanism of action of the cellular enzyme appears similar to diphtheria toxin and Pseudomonas exotoxin A, it differs from the toxins in that only a fraction of the EF-2 pool is modified . The endogenously ADP-ribosylated EF-2 has been detected by a variety of methods including two-dimensional electrophoresis and immunoprecipitation with elongation factor 2 antibody . The nature of the cellular ADP-ribosyltransferase and its physiological significance are unknown.

Mol Microbiol, 1994 Sep, 13(6), 1057 - 64
Gene expression in mycobacteria: transcriptional fusions based on xylE and analysis of the promoter region of the response regulator mtrA from Mycobacterium tuberculosis; Curcic R et al.; Understanding promoter regulation and signal-transduction systems in pathogenic mycobacteria is critical for uncovering the processes that govern interactions of these bacteria with the human host . In order to develop additional genetic tools for analysis of mycobacterial promoters, the xyIE gene from Pseudomonas was tested as a transcriptional fusion reporter in fast- and slow-growing mycobacteria . Initially, its utility was demonstrated by expression behind the hsp60 promoter in Mycobacterium smegmatis and Mycobacterium bovis BCG . The presence of an active promoter in front of the promoterless xyIE cassette on a plasmid was scored by development of a bright yellow colour upon spraying of mycobacterial colonies on plates with a solution of catechol . The gene product of xyIE, catechol 2,3 dioxygenase, was measurable in sonic extracts and whole cells, permitting quantitative determination of promoter activity in both fast- and slow-growing mycobacteria . The xyIE-based mycobacterial transcriptional fusion plasmid pRCX3 was constructed and used to assess promoter activity within the sequences located upstream of the newly characterized Mycobacterium tuberculosis H37Rv response regulator mtrA, a member of the superfamily of bacterial signal-transduction systems.

J Cell Sci, 1994 Sep, 107 ( Pt 9), 2599 - 608
The heparin-binding domain of heparin-binding EGF-like growth factor can target Pseudomonas exotoxin to kill cells exclusively through heparan sulfate proteoglycans; Mesri EA et al.; Heparin-binding EGF-like growth factor (HB-EGF) is a smooth muscle cell mitogen composed of both EGF receptor and heparin-binding domains . To better understand the function of its domains, intact HB-EGF or its heparin-binding (HB) domain (amino acids 1-45) were fused to a mutant Pseudomonas exotoxin with an inactivated cell-binding domain . The resulting chimeric toxins, HB-EGF-PE* and HB-PE*, were tested on tumor cells, proliferating smooth muscle cells and a mutant Chinese hamster ovary cell line deficient in heparan sulfate proteoglycans (HSPGs) . Two targets were found for HB-EGF-PE* . Cells were killed mainly through EGF receptors, but the HB domain was responsible for killing via HSPGs . HB-PE* did not bind to the EGF receptor and thus was cytotoxic by interacting exclusively with HSPGs . We conclude that the HB domain of HB-EGF is able to mediate internalization through HSPGs, without requiring the EGF receptor.

Biomed Chromatogr, 1994 Sep-Oct, 8(5), 236 - 41
Fluidized-bed receptor-affinity chromatography; Spence C et al.; A multipurpose fluidized-bed receptor-affinity purification system based upon the biological recognition between an immobilized receptor and its soluble protein ligands is described . The fluidized affinity sorbent consists of a soluble form of interleukin-2 receptor chemically bonded to an aldehyde derivative of controlled pore glass beads, which have a pore diameter of 1000 A and a particle density of 1.2-1.3 g/mL . The fluidized-bed separation device used in this study consists of a specially designed column fitted at the inlet end with a perforated distributor plate covered with a screen and the top outlet with an adjustable piston . The fluidized-bed consisting of a loose gel matrix permits the unimpeded passage of cell debris and particulate matter, while the target protein is captured by the affinity beads . Purification of the humanized-anti-Tac monoclonal antibody is used as a model system to determine the operational parameters . Also, fluidized-bed receptor-affinity chromatography has been successfully employed in the purification of recombinant interleukin-2 and single chain anti-Tac(Fv)-Pseudomonas exotoxin immunotoxin from unclarified inclusion body extracts . Overall, fluidized-bed receptor-affinity chromatography is found to be a productive affinity method suitable for the purification of recombinant human interleukin-2 and related molecules.

Biophys J, 1994 Sep, 67(3), 1207 - 15
Solution conformation of cytochrome c-551 from Pseudomonas stutzeri ZoBell determined by NMR; Cai M et al.; 1H NMR spectroscopy and solution structure computations have been used to examine ferrocytochrome c-551 from Pseudomonas stutzeri ZoBell (ATCC 14405) . Resonance assignments are proposed for all main-chain and most side-chain protons . Stereospecific assignments were also made for some of the beta-methylene protons and valine methyl protons . Distance constraints were determined based upon nuclear Overhauser enhancements between pairs of protons . Dihedral angle constraints were determined from estimates of scalar coupling constants and intra-residue NOEs . Twenty structures were calculated by distance geometry and refined by energy minimization and simulated annealing on the basis of 1012 interproton distance and 74 torsion angle constraints . Both the main-chain and side-chain atoms are well defined except for two terminal residues, and some side-chain atoms located on the molecular surface . The average root mean squared deviation in the position for equivalent atoms between the 20 individual structures and the mean structure obtained by averaging their coordinates is 0.56 +/- 0.10 A for the main-chain atoms, and 0.95 +/- 0.09 A for all nonhydrogen atoms of residue 3 to 80 plus the heme group . The average structure was compared with an analogous protein, cytochrome c-551 from pseudomonas stutzeri . The main-chain folding patterns are very consistent, but there are some differences, some of which can be attributed to the loss of normally conserved aromatic residues in the ZoBell c-551.

Anal Biochem, 1994 Sep, 221(2), 362 - 7
A sensitive assay of acyl-coenzyme A oxidase by coupling with beta-oxidation multienzyme complex; Souri M et al.; A highly sensitive and reliable method for assaying acyl-CoA oxidase (EC 1.3.99.3) activity was developed . An acyl-CoA oxidase-dependent {1-14C}palmitoyl-CoA degradation to acetyl-CoA, acid-soluble products, was measured by coupling with the multienzyme complex for fatty acid oxidation from Pseudomonas fragi . The activity, more than 2 pmol/min, could be assessed using this method . The activity was dependent on the coupling enzyme (multienzyme complex), coenzymes such as NAD+ and CoA, and oxygen, and the interference of acyl-CoA dehydrogenases was excluded . The activity in human samples of cultured skin fibroblasts and lymphocytes was compatible with the expected activity calculated from the amount of acyl-CoA oxidase protein estimated by immunoblot analysis . The method which was verified in several experiments can be used for clinical diagnosis of acyl-CoA oxidase deficiency and for determination of activity in samples with a low level of acyl-CoA oxidase.

Antibiot Khimioter, 1994 Sep-Oct, 39(9-10), 45 - 8
{Effectiveness of treatment of experimental glanders after aerogenic infection}; Iliukhin VI et al.; The in vitro studies revealed a number of promising drugs including gentamicin, doxycycline and minocycline for the treatment of malleus . The malleus causative agent was found to be highly susceptible to sulfamonomethoxine and biseptol as well as to imipenem and ofloxacin . Cephalosporins were less active but in the therapeutic concentrations they inhibited the growth of Pseudomonas mallei . In the treatment of golden hamsters infected subcutaneously by P . mallei ofloxacin proved to be the most active drug, then followed biseptol, doxycycline and minocycline . None of the tested drugs cured the animals infected aerogenically by 160 LD50 of P . malleus . When the infective dose was lower (16 LD50) only doxycycline provided a 70-percent protection of the animals.

Southeast Asian J Trop Med Public Health, 1994 Sep, 25(3), 436 - 42
Separation of antigenic glycoprotein fractions from cell-free homogenate of Pseudomonas pseudomallei and characterization as tyrosine phosphatase; Kondo E et al.; Cell-free extracts were prepared from Pseudomonas pseudomallei cells by freezing-thawing, sonication, and differential ultracentrifugation . The extracts were subjected to column chromatography with DEAE-sepharose to obtain glycoprotein fractions . The fractions showed acid phosphatase activity to p-nitrophenyl phosphate, tyrosine phosphate, serine phosphate, but not to threonine phosphate . They were highly antigenic when tested by immunofluorescence assay with the sera of melioidosis patients.

Appl Environ Microbiol, 1994 Sep, 60(9), 3368 - 74
Cometabolic degradation of trichloroethylene by Pseudomonas cepacia G4 in a chemostat with toluene as the primary substrate; Landa AS et al.; Pseudomonas cepacia G4 is capable of cometabolic degradation of trichloroethylene (TCE) if the organism is grown on certain aromatic compounds . To obtain more insight into the kinetics of TCE degradation and the effect of TCE transformation products, we have investigated the simultaneous conversion of toluene and TCE in steady-state continuous culture . The organism was grown in a chemostat with toluene as the carbon and energy source at a range of volumetric TCE loading rates, up to 330 mumol/liter/h . The specific TCE degradation activity of the cells and the volumetric activity increased, but the efficiency of TCE conversion dropped when the TCE loading was elevated from 7 to 330 mumol/liter/h . At TCE loading rates of up to 145 mumol/liter/h, the specific toluene conversion rate and the molar growth yield of the cells were not affected by the presence of TCE . The response of the system to varying TCE loading rates was accurately described by a mathematical model based on Michaelis-Menten kinetics and competitive inhibition . A high load of 3,400 mumol of TCE per liter per h for 12 h caused inhibition of toluene and TCE conversion, but reduction of the TCE load to the original nontoxic level resulted in complete recovery of the system within 2 days . These results show that P . cepacia can stably and continuously degrade toluene and TCE simultaneously in a single-reactor system without biomass retention and that the organism is more resistant to high concentrations and shock loadings of TCE than Methylosinus trichosporium OB3b.

Biochemistry, 1994 Aug 30, 33(34), 10257 - 65
Structural characterization of Pseudomonas 7A glutaminase-asparaginase; Lubkowski J et al.; The amino acid sequence and a 2-A-resolution crystallographic structure of Pseudomonas 7A glutaminase-asparaginase (PGA) have been determined . PGA, which belongs to the family of tetrameric bacterial amidohydrolases, deamidates glutamine and asparagine . The amino acid sequence of PGA has a high degree of similarity to the sequences of other members of the family . PGA has the same fold as other bacterial amidohydrolases, with the exception of the position of a 20-residue loop that forms part of the active site . In the PGA structure presented here, the active site loop is observed clearly in only one monomer, in an open position, with a conformation different from that observed for other amidohydrolases . In the other three monomers the loop is disordered and cannot be traced . This phenomenon is probably a direct consequence of a very low occupancy of product(s) of the enzymatic reaction bound in the active sites of PGA in these crystals . The active sites are composed of a rigid part and the flexible loop . The rigid part consists of the residues directly involved in the catalytic reaction as well as residues that assist in orienting the substrate . Two residues that are important for activity residue on the flexible loop . We suggest that the flexible loops actively participate in the transport of substrate and product molecules through the amidohydrolase active sites and participate in orienting the substrate molecules properly in relation to the catalytic residues.

Mol Gen Genet, 1994 Aug 15, 244(4), 341 - 51
Purification and characterization of CopR, a transcriptional activator protein that binds to a conserved domain (cop box) in copper-inducible promoters of Pseudomonas syringae; Mills SD et al.; The copper resistance (cop) operon promoter (Pcop) of Pseudomonas syringae is copper-inducible, and requires the regulatory genes copRS . Sequence analysis revealed that CopR has significant homology with other known activator proteins from bacterial two-component regulatory systems . In the present study we characterized Pcop and its interaction with CopR . We found that crude protein extracts from copper-resistant and -sensitive strains of P . syringae contain a Pcop-specific DNA-binding protein . We hypothesized that this DNA-binding protein was the product of copR . A 27-kDa protein, which corresponded to the predicted copR product, was expressed from this gene in Escherichia coli . CopR was purified, and the first eight amino acids were sequenced to confirm its relationship to copR . Specific binding of purified CopR to the plasmid-borne Pcop and the chromosomally encoded cop homolog promoter (PcopH), identified in this report, was demonstrated using specific and non-specific promoter competitors in DNA mobility shift assays . DNAse I footprinting identified a conserved CopR binding region (cop box) on Pcop and PcopH . The cop box contains an inverted repeat within a stretch of 16 bp, which shares approximately 75% identity with the PhoB binding region from several phosphate regulon gene promoters in E . coli . Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of copA, and the transcriptional initiation site of PcopH 88 bp 5' to the translational start site of the chromosomal homolog of copA . The cop box was localized to between positions -54 and -35 relative to the transcriptional initiation site of Pcop and PcopH . Deletion analysis of Pcop delimited copper-inducible activity to a 104-bp region . Pcop and PcopH do not share a sequence consensus with other characterized promoters from P . syrinagae or E . coli . The results presented delineate important regions on two copper-inducible promoters form P . syringae.

Int J Cancer, 1994 Aug 15, 58(4), 574 - 81
Human neurological cancer cells express interleukin-4 (IL-4) receptors which are targets for the toxic effects of IL4-Pseudomonas exotoxin chimeric protein; Puri RK et al.; Glioblastoma, glioma or neuroblastoma cells were examined for the expression of IL-4 receptors (IL-4R) by flow cytometric analysis and 125I-IL-4 binding . These cancer cell lines expressed IL-4R which were of high affinity (KD = 700 x 10(-12) M) on glioblastoma cells . To investigate the function of these receptors and to target potent cytotoxic antitumor agents to human neurological cancers, we utilized IL4-PE4E, which is composed of IL-4 and mutant Pseudomonas exotoxin (IL4-PE4E) . This chimeric molecule was cytotoxic toward human glioblastoma, neuroblastoma and glioma tumor cells in a dose-dependent manner . The cytotoxicity of IL4-PE4E was specific, since it was neutralized by excess IL-4, and by an anti-IL-4 monoclonal antibody in all types of brain tumor tested . IL2-PE4E and IL6-PE4E were not cytotoxic, nor was an IL4-PE4E mutant lacking ADP-ribosylating activity, indicating the IL4-PE4E-mediated cytotoxicity of the brain tumor cells required both IL-4R binding and enzymatic toxin activity . These data indicate that human neurological cancer cells express IL-4R which are targets for the cytotoxic effects of IL4-toxin . In addition, our data also suggest that IL4-PE4E should be studied further as a potential treatment for human neurological cancers.

Vet Microbiol, 1994 Aug 15, 41(4), 391 - 7
Potency of partially purified malleo-proteins for mallein test in the diagnosis of glanders in equines; Verma RD et al.; Malleo-proteins from synthetic broth mallein of six strains of Pseudomonas mallei were separated by trichloroacetic acid precipitation, ammonium sulfate precipitation and Ultrogel AcA 34 gel filtration chromatography . When tested comparatively with Dutch PPD mallein as standard on P . mallei-sensitized and normal horses all the strains were found to be malleinogenic, trichloroacetic acid precipitated proteins were comparable to Dutch PPD mallein in potency and innocuity whereas ammonium sulfate-precipitated proteins elicited non-specific reactions . Ultrogel AcA 34 chromatographed high molecular weight proteins (MW range > 350,000) were having equal or higher potency and without adverse reactions and low molecular weight proteins (MW range 120,000-170,000) nearly inactive . Ultrogel AcA 34 column fractionation studies revealed that mallein activity was associated with high molecular weight proteins and represented a purer sensitin than PPD mallein for the purpose of mallein test.

Appl Environ Microbiol, 1994 Aug, 60(8), 2931 - 8
Cloning of a phenazine biosynthetic locus of Pseudomonas aureofaciens PGS12 and analysis of its expression in vitro with the ice nucleation reporter gene; Georgakopoulos DG et al.; Pseudomonas aureofaciens PGS12 produces three phenazine antibiotics, in addition to siderophores, hydrogen cyanide, pyrrolnitrin, and indoleacetic acid . Tn5-259.7 transposon mutagenesis was carried out to identify and clone a chromosomal locus involved in phenazine biosynthesis . Three classes of mutants were obtained: mutants deficient in phenazine production (Phz-), mutants deficient in hydrogen cyanide production (HCN-), and mutants deficient in the production of both compounds . EcoRI DNA fragments that contained the transposon and flanking regions were cloned from three mutants with single-transposon insertions, one from each phenotypic class . Phenazine and hydrogen cyanide production was restored by complementation of Phz- or HCN- mutants with selected cosmids from a PGS12 genomic library . No cosmids that complemented the doubly deficient Phz-HCN- mutant were obtained . A promoterless ice nucleation reporter gene was inserted in a phenazine biosynthetic locus by Tn3-spice transposon mutagenesis of a cosmid which complemented a phenazine-minus mutant . Reporter gene fusions that expressed the ice nucleation phenotype and no longer complemented phenazine production were introduced into the PGS12 chromosome by marker exchange . The expression of this locus was then monitored under different culture conditions . Expression decreased at pH levels below 7, and it was not affected by iron . Shikimic acid and phenylalanine favored higher expression levels . Expression was reduced in media with low substrate concentrations, indicating the importance of nutrient availability.

Eur J Biochem, 1994 Aug 1, 223(3), 783 - 9
An unusual conformation of the methionine haem ligand in cytochrome cL established by two-dimensional 1H-NMR; Costa HS et al.; A complete relaxation-matrix analysis of NOESY cross-peak intensities was used to determine the conformation of the methionine ligand to the haem group in two ferrocytochromes cL from Methylophilus methylotrophus and Methylobacterium extorquens, including the configuration at the sulphur . The conformation of the axial methionine is of a type reported only for the cytochromes c5 from Pseudomonas mendocina and Azotobacter vinelandii . Although the conformation of the methionine is unusual, the paramagnetic shifts of the haem methyl proton resonances in the oxidized proteins indicate that the electronic structure of the haem groups is similar to that found in the mitochondrial type of cytochrome c.

J Am Acad Dermatol, 1994 Aug, 31(2 Pt 1), 210 - 2
Eosinophilic pustular folliculitis: a sterile folliculitis of unknown cause?
Brenner S, Wolf R, Ophir J.
BACKGROUND: Eosinophilic pustular folliculitis (EPF) was initially defined as a sterile folliculitis of unknown cause . Because attempts to demonstrate bacterial organisms have been unsuccessful, and antibiotic therapy is usually ineffective, a bacterial infection is not considered a plausible causative factor for this disease . OBJECTIVE: Our purpose was to describe five patients with the clinical and histologic characteristics of EPF and to report the results of bacterial cultures . METHODS: Biopsy specimens were examined and pustules were cultured . RESULTS: In three of the five patients, Pseudomonas infection of the hair follicle was the cause of the disease as proven by repeated cultures and the response to specific therapy . Three patients had a systemic disorder known to cause immunologic alteration: AIDS in one and a myeloproliferative disorder in two . CONCLUSION: Although EPF was initially defined as a sterile folliculitis of unknown origin, three of our patients had an identifiable and treatable cause . We believe that these cases warrant the diagnosis of EPF.

J Appl Bacteriol, 1994 Aug, 77(2), 195 - 207
Evaluation of determinative tests for pathovars of Pseudomonas syringae van Hall 1902; Young JM et al.; The utility of 36 presumptive determinative tests for 32 pathovars of Pseudomonas syringae was investigated . A total of 395 strains was examined . Most strains of 12 of these pathovars (Ps . syringae pv . cannabina, Ps . syr . delphinii, Ps . syr . glycinea, Ps . syr . helianthi, Ps . syr . lachrymans, Ps . syr . mori, Ps . syr . morsprunorum, Ps . syr . phaseolicola, Ps . syr . 'porri', Ps . syr . papulans, Ps . syr . savastanoi and Ps . syr . tabaci) formed clusters when test data were compared by centroid analysis . Pseudomonas syr . syringae, Ps . syr . aptata, Ps . syr . atrofaciens, Ps . syr . dysoxyli and Ps . syr . japonica formed a single cluster, indicating their possible synonymy . Strains of Ps . syr . antirrhini and Ps . syr . tomato were indistinguishable, as were those of Ps . syr . garcae and Ps . syr . oryzae . Strains of Ps . syr . berberidis, Ps . syr . coronafaciens, Ps . syr . eriobotryae, Ps . syr . maculicola, Ps . syr . passiflorae, Ps . syr . pisi and Ps . syr . striafaciens and Ps . syr . tagetis did not form distinguishable clusters . The tests which reliably differentiated pathovars are recorded in a determinative scheme.

Appl Environ Microbiol, 1994 Aug, 60(8), 2924 - 30
Identification and relatedness of coronatine-producing Pseudomonas syringae pathovars by PCR analysis and sequence determination of the amplification products; Bereswill S et al.; Production of the chlorosis-inducing phytotoxin coronatine in the Pseudomonas syringae pathovars atropurpurea, glycinea, maculicola, morsprunorum, and tomato has been previously reported . DNA hybridization studies previously indicated that the coronatine biosynthetic gene cluster is highly conserved among P . syringae strains which produce the toxin . In the present study, two 17-bp oligonucleotide primers derived from the coronatine biosynthetic gene cluster of P . syringae pv . glycinea PG4180 were investigated for their ability to detect coronatine-producing P . syringae strains by PCR analysis . The primer set amplified diagnostic 0.65-kb PCR products from genomic DNAs of five different coronatine-producing pathovars of P . syringae . The 0.65-kb products were not detected when PCR experiments utilized nucleic acids of nonproducers of coronatine or those of bacteria not previously investigated for coronatine production . When the 0.65-kb PCR products were digested with ClaI, PstI, and SmaI, fragments of identical size were obtained for the five different pathovars of P . syringae . A restriction fragment length polymorphism was detected in the amplified region of P . syringae pv . atropurpurea, since this pathovar lacked a conserved PvuI site which was detected in the PCR products of the other four pathovars . The 0.65-kb PCR products from six strains comprising five different pathovars of P . syringae were cloned and sequenced . The PCR products from two different P . syringae pv . glycinea strains contained identical DNA sequences, and these showed relatedness to the sequence obtained for the pathovar morsprunorum . The PCR products obtained from the pathovars maculicola and tomato were the most similar to each other, which supports the hypothesis that these two pathovars are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Biochem Biophys, 1994 Aug 1, 312(2), 467 - 73
P . mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: electron paramagnetic resonance investigation of the copper binding site; Narasimhan C et al.; The copper binding site in Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase has been investigated by multifrequency electron spin resonance spectroscopy . Methodology has been developed to introduce copper in vitro into the isolated apoenzyme . The X-band EPR of Cu2+ (mixed isotopes or 63Cu2+) introduced in this way is very similar to the EPR spectra of samples in which copper is introduced during protein expression . g parallel and A parallel values in the X-band spectra support the prediction of nitrogen ligands to the tightly bound copper . In the g parallel region of S-band EPR spectra (mI = -1/2) of lyase-bound 63Cu2+, superhyperfine interactions due to nitrogen ligands are observed . Computer simulation with appropriate g values and A values and strain parameters was used to satisfactorily model both the X-band and the S-band spectra . The g parallel value of 2.282 and the A parallel value of 470 MHz are consistent with two nitrogen and two oxygen donor atoms for a square planar type 2 copper center . By simulating the mI = -1/2 line of the S-band spectrum, the superhyperfine features could be well modeled . This simulation approach was also used to distinguish between two and three nitrogen donor atoms . Based on the intensity patterns of the superhyperfine lines and the estimated coupling constants, it is concluded that at least two (and probably only two) nitrogen donor atoms are liganded to the tightly bound copper in HMG-CoA lyase . Additionally, kinetic experiments demonstrate that a spin-labeled substrate analog (R.CoA) is a competitive inhibitor of HMG-CoA lyase (KI = 98 microM) . ESR titration experiments indicate that R.CoA binds to lyase with an equilibrium dissociation constant of 103 microM . Bound spin label exhibits a rotational correlation time, tau c, of 20 ns, in agreement with the value predicted for immobilization on a protein composed of two 32-kDa subunits.

J Biochem (Tokyo), 1994 Aug, 116(2), 248 - 9
Reconsideration of the essential role of a histidine residue of L-2-halo acid dehalogenase; Liu JQ et al.; His20 of L-2-halo acid dehalogenase from Pseudomonas cepacia MBA4 was suggested to serve as a catalytic base {Biochem . J . (1993) 292, 69-74} . In this study, we substituted Asn or Leu for His19 of L-2-halo acid dehalogenase from Pseudomonas sp . YL, which corresponds to His20 of the P . cepacia enzyme . Although the substrate specificity was affected by the substitution, the susceptibilities of substrate halo acids were not substantially diminished, and the Km and kcat values of the mutant enzymes for L-2-chloropropionate were not significantly different from those of the wild-type enzyme . In addition, the wild-type and mutant enzymes showed the same pH optimum . Accordingly, His19 is not essential for catalysis of L-2-halo acid dehalogenase.

Lett Appl Microbiol, 1994 Aug, 19(2), 88 - 91
CCD-monitoring of bioluminescence during the induction of the cell wall-deficient, L-form state of a genetically modified strain of Pseudomonas syringae pv . phaseolicola; Waterhouse RN et al.; Bioluminescence from developing L-form colonies of the plant pathogen, Pseudomonas syringae pv . phaseolicola, was monitored using the enhanced light-detecting capabilities of a charge-coupled device . During L-form induction, the bacteria entered a prolonged period during which the level of light output and hence metabolic activity, was very low . A relatively small number of highly bioluminescent L-form colonies were then observed to develop against a background of non-bioluminescent bacteria . When these colonies were sub-cultured and examined microscopically, typical L-form morphology was observed and continued high bioluminescence was detectable from derived colonies.

Bioseparation, 1994 Aug, 4(4), 279 - 84
Use of hydrophobic chromatography for purification of the membrane-located choline dehydrogenase from a Pseudomonas strain; Russell R et al.; Choline dehydrogenase has been purified using hydrophobic chromatography 250-fold from a Pseudomonas strain . Although the enzyme is associated with the cell membrane and could be extracted from membrane preparations, it was best purified from a complete cell extract made with a non-ionic detergent . Only phenazine methosulfate was able to act as electron acceptor; there was no evidence of bound flavin, but there was evidence of pyrroloquinoline quinone cofactor . The purified enzyme had a specific activity of up to 67 units/mg, which is at least ten times higher than the values reported for mitochondrial choline dehydrogenases, and up to 100 times higher than previous reports for the Pseudomonas enzyme . The estimated subunit size of 66 kDa, which corresponds with the native size, is close to that deduced from the gene sequence of the Escherichia coli betA gene, and preliminary N-terminal sequencing shows homology with this deduced sequence . The next enzyme in the degradation pathway of choline, betaine aldehyde dehydrogenase, was also purified from the same extract.

Antibiot Khimioter, 1994 Aug, 39(8), 30 - 3
{In vitro development of fluoroquinolone resistance in the glanders pathogen}; Stepanshin IuG et al.; To improve the scheme for the therapy of malleus, the possible development of fluoroquinolone resistance in the cells of Pseudomonas mallei was studied . The frequency of the mutations determining the resistance to oxolinic acid, norfloxacin, enoxacin, ofloxacin and ciprofloxacin was detected . The spectrum of the cross resistance in the strains was investigated . The influence of the resistance mutations on the culture virulence was studied . Recommendations on the rational use of fluoroquinolones in the treatment of malleus are presented.

Ther Immunol, 1994 Aug, 1(4), 197 - 204
Degradation of ricin A chain by endosomal and lysosomal enzymes--the protective role of ricin B chain; Bilge A et al.; We investigated the role of intracellular processing of ricin A chain (RTA) by proteolytic enzymes on the expression of its ribosome inhibitory activity . Endosomal and lysosomal proteases extracted from Jurkat cells and purified cathepsins B, D and G were incubated with RTA, resulting in generation of a 28-kDa fragment by proteolytic cleavage . This process was reminiscent of the nicking of Pseudomonas exotoxin and Diphtheria toxin by intracellular proteases to produce functionally active toxin fragments . However, the ribosome inhibitory activity of the purified 28-kDa fragment of RTA was 11,000-fold less than that of native RTA, suggesting that such cleavage is not an essential step in the cytotoxic activity of the toxin . Addition of ricin B chain (RTB) in degradation assays resulted in the protection of RTA from proteolytic activities of lysosomes and cathepsins . However, RTB did not protect another RNA acting protein, RNAase; nor did excess amounts of unlabeled RTA or IgG protect labelled RTA from degradation, suggesting that the protective effect of RTB was specific to its interaction with RTA . Such a protective role for RTB may partially account for the higher toxicity of immunotoxins (ITs) containing whole ricin compared to ITs containing only RTA.

Bioorg Khim, 1994 Aug-Sep, 20(8-9), 984 - 93
{Antigenic bacterial polysaccharides . Structure of O-specific polysaccharides from Pseudomonas cepacia serogroups C, I, O1, and O4}; Paramonov NA et al.; Like some Pseudomonas cepacia serogroups studied earlier, serogroups C, I (Nakamura), O1 and O4 (Heidt) are characterized by the presence of at least two structurally different O-antigenic polysaccharide chains in cell-wall lipopolysaccharides . On the basis of acid hydrolysis, methylation, 1H- and 13C-NMR spectroscopy, including computer-assisted 13C-NMR-based analysis, the complete structures of the predominant polysaccharides of serogroups I (I), C and O4 (III) and the minor polysaccharides of serogroups I (II) and O1 (V) were established, and the structure of the predominant polysaccharide of serogroup O1 (IV) established earlier (Cox A . D., Wilkinson S . G.@Carbohydr . Res . 1990 . V . 195 . No 2 . P . 295-301) was confirmed.

Pediatr Pulmonol, 1994 Aug, 18(2), 108 - 13
Epidemiology of Pseudomonas cepacia colonization among patients with cystic fibrosis; John M et al.; Colonization with Pseudmonas cepacia in patients with cystic fibrosis (CF) has been associated with increased morbidity and early death, compared with colonization by P . aeruginosa . The mode of acquisition of P . cepacia is not fully understood, although person-to-person spread appears likely . Recent epidemiologic studies support the importance of social contact in the spread of P . cepacia among patients with CF . This study was undertaken to investigate the epidemiology of P . cepacia colonization among patients with CF attending the CF clinic at our center . Isolates of P . cepacia were collected from patients at two CF treatment centers, including ours . Additional isolates were collected from patients without CF in the hospital ICU, from other teaching hospitals, and from the environment . Profiles of enzymes were obtained by ultrathin polyacrylamide gel electrophoresis of P . cepacia extracts . A predominant electromorphic type (ET) was found among P . cepacia isolates from patients at both centers, suggesting a common source or person-to-person transmission . The majority of hospital isolates fell into a single, different ET . Surveillance swabs of respiratory equipment at our CF clinic did not grow P . cepacia . Attendance of patients at CF summer camp correlated strongly with P . cepacia colonization (P < 0.0001).

Thorax, 1994 Aug, 49(8), 803 - 7
Effect of antibiotic treatment on inflammatory markers and lung function in cystic fibrosis patients with Pseudomonas cepacia; Peckham D et al.; BACKGROUND--The acquisition of Pseudomonas cepacia in patients with cystic fibrosis is associated with increasing deterioration in lung function and more frequent hospital admissions . Pseudomonas cepacia is usually resistant to several antibiotics in vitro, but the response of patients colonised with the organism has not been extensively studied in vivo . METHODS--A three month prospective study was performed to investigate the response of 14 Ps cepacia positive patients and 10 Ps cepacia negative patients to a two week course of intravenous antibiotics . All those who were Ps cepacia negative and six of the 14 Ps cepacia positive patients had Ps aeruginosa in their sputum which was sensitive to the prescribed therapy . The inflammatory markers C-reactive protein, white blood cell count, serum lactoferrin, neutrophil elastase/alpha 1-antitrypsin complex, and tumour necrosis factor alpha were measured at the start and end of each antibiotic course . RESULTS--The median (range) % improvement in baseline FEV1 and FVC following treatment in the group as a whole was 15.2% (-23.5% to 156.3%) and 23.9% (-36.8% to 232.7%) respectively . There was no statistical difference in improvement in lung function, body weight, or inflammatory markers between individuals who were Ps cepacia positive and those who were Ps cepacia negative . CONCLUSIONS--Patients who are Ps cepacia positive appear to respond as well to intravenous antibiotics as those who are Ps cepacia negative, despite having lower lung function and a bacterium in their sputum which is resistant in vitro to the antibiotics used.

Appl Environ Microbiol, 1994 Aug, 60(8), 2884 - 9
Formation of chlorocatechol meta cleavage products by a pseudomonad during metabolism of monochlorobiphenyls; Arensdorf JJ et al.; Pseudomonas cepacia P166 was able to metabolize all monochlorobiphenyls to the respective chlorobenzoates . Although they transiently accumulated, the chlorobenzoate degradation intermediates were further metabolized to chlorocatechols, which in turn were meta cleaved . 2- and 3-Chlorobiphenyl both produced 3-chlorocatechol, which was transformed to an acyl halide upon meta cleavage . 3-Chlorocatechol metabolism was toxic to the cells and impeded monochlorobiphenyl metabolism . In the case of 2-chlorobiphenyl, toxicity was manifested as a diminished growth rate, which nevertheless effected rapid substrate utilization . In the case of 3-chlorobiphenyl, which generates 3-chlorocatechol more rapidly than does 2-chlorobiphenyl, toxicity was manifested as a decrease in viable cells during substrate utilization . 4-Chlorobenzoate was transformed to 4-chlorocatechol, which was metabolized by a meta cleavage pathway leading to dehalogenation . Chloride release from 4-chlorocatechol metabolism, however, was slow and did not coincide with rapid 4-chlorocatechol turnover . Growth experiments with strain P166 on monochlorobiphenyls illustrated the difficulties of working with hydrophobic substrates that generate toxic intermediates . Turbidity could not be used to measure the growth of bacteria utilizing monochlorobiphenyls because high turbidities were routinely measured from cultures with very low viable-cell counts.

J Med Microbiol, 1994 Aug, 41(2), 106 - 11
Application of pyrolysis mass spectroscopy and SDS-PAGE in the study of the epidemiology of Pseudomonas cepacia in cystic fibrosis; Corkill JE et al.; Representative isolates of Pseudomonas cepacia from 15 cystic fibrosis (CF) patients attending the Respiratory Unit of Alder Hey Childrens' Hospital were investigated by SDS-PAGE of whole-cell polypeptides and by pyrolysis mass spectroscopy (PMS) . SDS-PAGE was less discriminatory than PMS . Eleven isolates were indistinguishable by PMS and considered to represent re-isolates of an endemic strain; four isolates were distinct from this group, and from one another . P . cepacia was first isolated on the unit in July 1989 from a patient who had attended a UK selection meeting for a Canadian CF camp . A ward and outpatient segregation policy was introduced, but colonisation of further patients occurred . In August 1991, the Adult CF Association recommended that all social activities involving colonised patients should cease . This, and an increased awareness amongst older CF patients of the risks of person-to-person transmission, was associated with a marked decline in new cases . Social activity and hospital admissions were compared for colonised patients during the year before colonisation with P . cepacia, and matched patients who did not acquire the endemic strain . This showed a significantly higher attendance at CF social events for colonised patients, but no significant association between colonisation and hospital admission . These results are strong indirect evidence that transmission of P . cepacia occurs through social contact outside the hospital environment.

Arch Pediatr Adolesc Med, 1994 Aug, 148(8), 805 - 12
Possible nosocomial transmission of Pseudomonas cepacia in patients with cystic fibrosis; Pegues DA et al.; OBJECTIVE: To determine whether nosocomial transmission of Pseudomonas cepacia occurred at a hospital with endemic P cepacia infection of patients with cystic fibrosis . DESIGN: Two retrospective case-control studies . SETTING: A large pediatric cystic fibrosis center . PARTICIPANTS: To assess risk factors for acquisition of P cepacia, 18 cases, defined as any patient with cystic fibrosis with first documented isolation of P cepacia in 1988 or 1989, were compared with 18 matched P cepacia-negative controls with cystic fibrosis . To assess potential modes of nosocomial P cepacia transmission, 14 cases with a hospitalization(s) between their last P cepacia-negative culture and first P cepacia-positive culture were compared with 14 hospitalized P cepacia-negative controls with cystic fibrosis . METHODS: Handwiping cultures (N = 68) and selective environmental cultures were performed . MAIN RESULTS: Cases tended to be more likely than controls to have been hospitalized at the cystic fibrosis center in the 3 months before their first P cepacia-positive culture (P = .08) . In addition, cases tended to be more likely than hospitalized controls with cystic fibrosis to have had a P cepacia-positive roommate (P = .06) before becoming colonized with P cepacia organisms . Pseudomonas cepacia was cultured from the hands of two individuals: a P cepacia-colonized patient who had just undergone chest physiotherapy and consequent coughing and the investigator who shook the P cepacia-positive patient's hand after the patient's procedure . CONCLUSIONS: These results suggest that in this cystic fibrosis center, hospitalization is a risk factor for P cepacia acquisition and that person-to-person transmission of P cepacia may occur in the hospital via hand contact.

Biochem Biophys Res Commun, 1994 Jul 29, 202(2), 850 - 6
Identification of the bphA and bphB genes of Pseudomonas sp . strains KKS102 involved in degradation of biphenyl and polychlorinated biphenyls; Fukuda M et al.; The nucleotide sequence of the upstream region of the bphC gene from Pseudomonas sp . strain KKS102 was determined . Four genes were found in this region . Deduced amino acid sequences of the first, second, third and fourth genes showed significant homology with a large subunit of iron-sulfur protein, a small subunit of iron-sulfur protein, ferredoxin and dihydrodiol dehydrogenase, respectively, from other bacteria which degrade biphenyl/polychlorinated biphenyls, toluene and benzene . E . coli, in which the four genes, bphC and the gene for ferredoxin reductase from benzene degrading bacterium were expressed, was able to produce meta-cleavage compounds from chlorinated biphenyls . These results show that these gene products are functional in both biphenyl and polychlorinated biphenyls degradation.

Proc Natl Acad Sci U S A, 1994 Jul 19, 91(15), 6889 - 93
A circularly permuted recombinant interleukin 4 toxin with increased activity; Kreitman RJ et al.; Fusion of ligands such as growth factors to other proteins often dramatically reduces the affinity of the ligand for its receptor . With recombinant DNA techniques, the attachment point between the two proteins has until now been restricted to either the amino or the carboxyl terminus of the ligand . However, binding may be greatly compromised if both ends are close to the site at which the ligand binds to its receptor . To construct a single-chain growth factor fusion protein with the connection at a new site on the growth factor, we constructed a DNA fragment encoding circularly permuted interleukin 4 (IL4), termed IL4(38-37) . This was accomplished by placing a start codon before position 38, connecting codons 1 and 129 with a sequence encoding a peptide linker, and placing a stop codon after codon 37 of IL4 . IL4(38-37) was fused via its new carboxyl terminus, Lys37, to a truncated form of Pseudomonas exotoxin . The purified circularly permuted IL4-toxin bound to the IL4 receptor with 10-fold higher affinity than an IL4-toxin in which the toxin was fused to the carboxyl terminus of IL4 . Circular permuteins of growth factors can improve the effectiveness of recombinant fusion proteins, because the junction can be moved to a site on the growth factor which allows it to bind with higher affinity.

J Biol Chem, 1994 Jul 15, 269(28), 18327 - 31
Improved binding and antitumor activity of a recombinant anti-erbB2 immunotoxin by disulfide stabilization of the Fv fragment; Reiter Y et al.; e23(dsFv)-PE38KDEL is a recombinant immunotoxin composed of the Fv region of anti-erbB2 monoclonal antibody e23 connected to a truncated form of Pseudomonas exotoxin (PE38KDEL), in which the inherently unstable Fv heterodimer (composed of VH and VL) is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL . We have now found that e23(dsFv)-PE38KDEL is considerably more cytotoxic to antigen-positive cell lines than the corresponding single-chain immunotoxin . The basis for the enhanced cytotoxic activity is that the e23 dsFv-immunotoxin binds to erbB2 with greater affinity than the single-chain counterpart . The dsFv-immunotoxin had 4-fold increased binding compared to the scFv and almost identical to the binding affinity of e23 Fab . e23(dsFv)-PE38KDEL was also considerably more stable at 37 degrees C than the single-chain immunotoxin . The therapeutic potential of the disulfide-stabilized immunotoxin was compared with its single-chain counterpart using two animal models of immunodeficient mice bearing subcutaneous tumor xenografts of human gastric tumor N87 cells or human A431 epidermoid carcinoma cells . The antitumor effect of e23(dsFv)-PE38KDEL was significantly better than that of the single-chain immunotoxin . e23(dsFv)-PE38KDEL caused complete regression of tumors at doses which caused no toxic effects in mice, whereas the single-chain immunotoxin did not cause complete regressions at the same doses.

J Biol Chem, 1994 Jul 15, 269(28), 18303 - 6
The relationship between low density lipoprotein-related protein/alpha 2-macroglobulin (alpha 2M) receptors and the newly described alpha 2M signaling receptor; Misra UK et al.; alpha 2-Macroglobulin (alpha 2M)-methylamine binding to macrophages appears to involve two receptors . Binding of alpha 2M-methylamine to low density lipoprotein-related protein (LRP) results in cellular uptake and degradation, while binding to a newly described alpha 2M signaling receptor elevates intracellular calcium ({Ca2+}i) and inositol phosphates . We now demonstrate that binding of lactoferrin, Pseudomonas exotoxin A, and lipoprotein lipase to LRP on macrophages results in increased {Ca2+}i and inositol 1,4,5-triphosphate . Receptor-associated protein, which binds to LRP but not the alpha 2M signaling receptor, blocks the lactoferrin signal but has no effect on alpha 2M-methylamine signaling . The latter observation supports our hypothesis that a distinct signaling receptor binds alpha 2M-methylamine . We further demonstrate that the signaling events induced by lactoferrin may involve a pertussis toxin-sensitive G protein, while the alpha 2M signaling receptor appears to be coupled to a pertussis toxin-insensitive G protein.

J Biol Chem, 1994 Jul 8, 269(27), 18167 - 76
Cleavage of pseudomonas exotoxin and diphtheria toxin by a furin-like enzyme prepared from beef liver; Chiron MF et al.; Pseudomonas exotoxin (PE) is cleaved within mammalian cells between Arg279 and Gly280 to generate an enzymatically active COOH-terminal fragment of 37 kDa which translocates to the cytosol and ADP-ribosylates elongation factor 2 . A protease, with toxin cleaving activity, was prepared from beef liver and subsequently characterized . After achieving a 500-fold enrichment in several chromatographic steps, a soluble form of this protease was identified as a furin-like enzyme . It cleaved PE on the COOH-terminal side of the sequence of RQPR (amino acids 276-279) producing the same fragments as those generated within cells . Cleavage had a pH optimum of 5.0-5.5, was inhibited by EDTA or p-hydroxymercuribenzoate but not by O-phenanthroline,N-ethylmaleimide, trans-epoxysuccinyl-L-leukcylamido-(4-guanidino)-butane, or PMSF (or other well known inhibitors of serine proteases) . The beef protease cleaved PE with an apparent Km of 7 microM . A mutant form of PE, PEala281, was cleaved at the same site, with the same pH optimum, a similar Km (9 microM) but with a Vmax 150 times faster than was seen with the native toxin . Mutational analysis of the amino acids located just before the site of cleavage, confirmed the importance of arginines at P-1 and P-4 . It was also noted that the introduction of a dibasic pair at 278-279 did not increase toxicity or appreciably improve the rate of cleavage . Unnicked diphtheria toxin (DT) was also cleaved by the beef protease; cleavage was on the COOH-terminal side of the sequence RVRR (amino acids 190-193), was seen at pH values ranging from 5.5 to 8.5 and had an optimum at pH 8.0 . Recombinant furin cleaved PE, PEala281, and DT with the same characteristics as the beef protease . In addition, Western blot analysis revealed that anti-furin antibodies reacted specifically with components in the beef protease preparation . Immunodepletion experiments showed that all toxin-cleavage activity could be removed from the beef protease using anti-furin antibodies . The relevance of furin-mediated cleavage was further assessed by adding nicked toxins to intact cells . Nicked PE and DT both killed cells at a faster rate than their unnicked counterparts.

Microbiology, 1994 Jul, 140 ( Pt 7), 1585 - 93
Effect on lipopolysaccharide structure of aeration during growth of a plum isolate of Pseudomonas syringae pv . morsprunorum; Smith AR et al.; The composition of lipopolysaccharide (LPS) extracted with aqueous phenol from a virulent English plum isolate of Pseudomonas syringae pv . morsprunorum varied according to the partial pressure of oxygen (pO2) in the culture medium at the time of harvest . When pO2 was low, the organism grew slowly and produced smooth LPS bearing rhamnan sidechains . As pO2 was raised, the rate of growth increased and smooth LPS was replaced by a rough species deficient in rhamnose, which co-extracted with a D-glucan . Organization of rhamnose and glucose into separate polymers was shown by the selective susceptibility of the rhamnose-containing polymer to hydrolysis by rhamnanase of the phage A7 . By methylation analysis, GC-MS, and 1H- and 13C-NMR spectroscopy, the glucan was shown to consist of alpha (1-->4)-linked residues with alpha (1-->4,6)-branch points and non-reducing terminal residues in the approximate ratio 4:1:1, resembling glycogen in composition . A glucan which co-extracted with LPS using phenol/water from an avirulent plum isolate that was resistant to lysis by phages A1 and A7 was shown by methylation analysis to have a similar structure . Whether the effect on LPS composition was due directly to pO2, or was dependent on the rate of growth, has not been established . It is suggested that, because epiphytic growth would entail exposure to high pO2, English plum isolates growing on the surfaces of host plants might be unable to produce smooth LPS . Since cell surface composition affects virulence in plant-pathogenic pseudomonads, this effect could account for the observed failure of the English plum isolates to enter the host via leaf scars.

Appl Environ Microbiol, 1994 Jul, 60(7), 2389 - 93
Purification and characterization of thermostable and nonthermostable 2-haloacid dehalogenases with different stereospecificities from Pseudomonas sp . strain YL; Liu JQ et al.; Two novel hydrolytic dehalogenases, thermostable L-2-haloacid dehalogenase (L-DEX) inducibly synthesized by 2-chloropropionate (2-CPA) and nonthermostable DL-2-haloacid dehalogenase (DL-DEX) induced by 2-chloroacrylate, were purified to homogeneity from Pseudomonas sp . strain YL . DL-DEX consisted of a monomer with a molecular weight of about 36,000 and catalyzed the dehalogenation of L and D isomers of 2-CPA to produce D- and L-lactates, respectively . It acted on 2-haloalkanoic acids with a carbon chain length of 2 to 4 . The maximum activity on DL-2-CPA was found at pH 10.5 and 45 degrees C . L-DEX, composed of two subunits with identical molecular weights of 27,000, catalyzes the dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids . The enzyme acts not only on short-carbon-chain 2-haloacids such as monochloroacetate and monoiodoacetate in aqueous solution but also on long-carbon-chain 2-haloacids such as 2-bromohexadecanoate in n-heptane . L-DEX is thermostable: it retained its full activity upon heating at 60 degrees C for 30 min . The pH and temperature optima for dehalogenation of L-2-CPA were 9.5 and 65 degrees C, respectively . L-DEX was strongly inhibited by modification of carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and Woodward reagent K, but DL-DEX was not.

Plant Cell, 1994 Jul, 6(7), 927 - 33
A disease resistance gene in Arabidopsis with specificity for two different pathogen avirulence genes; Bisgrove SR et al.; The RPS3 and RPM1 disease resistance loci of Arabidopsis confer resistance to Pseudomonas syringae strains that carry the avirulence genes avrB and avrRpm1, respectively . We have previously shown that RPS3 and RPM1 are closely linked genetically . Here, we show that RPS3 and RPM1 are in fact the same gene . We screened a mutagenized Arabidopsis population with a P . syringae strain carrying avrB and found 12 susceptible mutants . All 12 mutants were also susceptible to an isogenic strain carrying avrRpm1, indicating a loss of both RPS3 and RPM1 functions . No mutants were recovered that lost only RPS3 function . Genetic analysis of four independent mutants revealed that the lesions were in RPS3 . Thus, a single gene in Arabidopsis confers resistance that is specific to two distinct pathogen avirulence genes--a gene-for-genes interaction . This observation suggests that the RPS3/RPM1 gene product can bind multiple pathogen ligands, or alternatively, that it does not function as a receptor.

Int J Syst Bacteriol, 1994 Jul, 44(3), 499 - 510
Classification of Pseudomonas diminuta Leifson and Hugh 1954 and Pseudomonas vesicularis Büsing, Döll, and Freytag 1953 in Brevundimonas gen . nov . as Brevundimonas diminuta comb . nov . and Brevundimonas vesicularis comb . nov., respectively; Segers P et al.; The taxonomic positions of strains previously assigned to Pseudomonas diminuta and Pseudomonas vesicularis were investigated by a polyphasic approach . The results of DNA-rRNA hybridization studies indicated that these two species belong to a separate genus in the alpha subclass (rRNA superfamily IV) of the Proteobacteria, for which the name Brevundimonas is proposed . Genus delineation and species delineation were determined by comparing the results of numerical analyses of whole-cell protein patterns, fatty acid compositions, and phenotypic characteristics and by measuring DNA base ratios and degrees of DNA relatedness . Taxonomic characteristics of Brevundimonas diminuta and Brevundimonas vesicularis strains were compared with characteristics of reference strains belonging to the following phylogenetically related taxa: a group of organisms gathered in Enevold Falsen group 21, the genera Sphingomonas and Rhizomonas, and the generically misclassified organisms {Pseudomonas} echinoides and "{Pseudomonas} riboflavina."

Eur J Biochem, 1994 Jul 1, 223(1), 135 - 9
Pseudothionin-St1, a potato peptide active against potato pathogens; Moreno M et al.; A 5-kDa polypeptide, pseudothionin Solanum tuberosum 1 (Pth-St1), which was active against Clavibacter michiganensis subspecies sepedonicus, a bacterial pathogen of potatoes, has been purified from the buffer-insoluble fraction of potato tubers by salt extraction and HPCL . Pth-St1 was also active against other potato pathogens tested (Pseudomonas solanacearum and Fusarium solani) . The N-terminal amino acid sequence of this peptide was identical (except for a N/H substitution at position 2) to that deduced from a previously reported cDNA sequence (EMBL accession number X-13180), which had been misclassified as a Browman-Birk protease inhibitor . Pth-St1 did not inhibit either trypsin or insect alpha-amylase activities, and, in contrast with true thionins, did not affect cell-free protein synthesis or beta-glucuronidase activity . Northern-blot and tissue-print analyses showed that steady-state mRNA levels were highest in flowers (especially in petals), followed by tubers (especially in the epidermal cell layers and in leaf primordia), stems and leaves . Infection of leaves with a bacterial pathogen suspended in 10 mM MgCl2 switched off the gene, whereas mock inoculation with 10 mM MgCl2 alone induced higher mRNA levels.

Eur J Immunol, 1994 Jul, 24(7), 1590 - 6
Presentation of an exogenous antigen by major histocompatibility complex class I molecules; Ulmer JB et al.; Cytotoxic T lymphocytes (CTL) generally recognize peptides derived from endogenously expressed proteins in association with nascent major histocompatibility complex (MHC) class I molecules . In contrast, peptides derived from exogenous proteins associate with MHC class II following endocytosis to an endosomal compartment . However, we have recently demonstrated that exogenous fusion proteins consisting of the binding and translocating domains of Pseudomonas exotoxin (PE) fused with CTL epitopes derived from either influenza matrix protein (PEMa) or nucleoprotein are internalized, processed, targeted to and presented by MHC class I (Donnelly et al . 1993, Proc . Natl . Acad . Sci . USA 1993 . 90: 3530) . PE is known to be internalized, processed in endosomes, and translocated to the cytosol during intoxication of cells . However, our present studies demonstrate that, unlike PE, PEMa does not require translocation to the cytosol to exert its effect . First, two inhibitors of PE toxicity that exert their effects at steps subsequent to endosomal processing had no effect on the sensitization of target cells for CTL-mediated lysis by PEMa . NH4Cl, which inhibits PE by raising endosomal pH, and brefeldin A, which inhibits PE by disrupting the Golgi complex, did not inhibit sensitization of targets cells by PEMa . Second, PEMa was capable of sensitizing for lysis T2 mutant cells, which are defective in transport of peptides from the cytosol to the lumen of the endoplasmic reticulum for presentation by MHC class I . These results suggest that PEMa is proteolytically processed in endosomes, and association with MHC class I does not require nascent MHC molecules . Such a process may involve internalized MHC class I, and subsequent expression of the peptide-MHC complexes on the cell surface would then lead to recognition by CTL.

J Biol Chem, 1994 Jul 1, 269(26), 17490 - 4
The role of spontaneous cap domain mutations in haloalkane dehalogenase specificity and evolution; Pries F et al.; The first step in the utilization of the xenobiotic chlorinated hydrocarbon 1,2-dichloroethane by Xanthobacter autotrophicus is catalyzed by haloalkane dehalogenase (Dh1A) . The enzyme hydrolyses 1-haloalkanes to the corresponding alcohols . This allows the organism to grow also on short-chain (C2-C4) 1-chloro-n-alkanes . We have expressed Dh1A in a strain of Pseudomonas that grows on long-chain alcohols and have selected 12 independent mutants that utilize 1-chlorohexane . Six different mutant enzymes with improved Km or Vmax values with 1-chlorohexane were obtained . The sequences of the mutated dh1A genes showed that several mutants had the same 11-amino acid deletion, two mutants carried a different point mutation, and three mutants had different tandem repeats . All mutations occurred in a region encoding the N-terminal part of the cap domain of Dh1A, and it is concluded that this part of the protein is involved in the evolution of activity toward xenobiotic substrates.

J Bacteriol, 1994 Jul, 176(14), 4269 - 76
Nucleotide sequence and functional analysis of the meta-cleavage pathway involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp . strain KKS102; Kikuchi Y et al.; Pseudomonas sp . strain KKS102 is able to degrade biphenyl and polychlorinated biphenyls via the meta-cleavage pathway . We sequenced the upstream region of the bphA1A2A3BCD (open reading frame 1 {ORF1}) A4 and found four ORFs in this region . As the deduced amino acid sequences of the first, second, and third ORFs are homologous to the meta-cleavage enzymes from Pseudomonas sp . strain CF600 (V . Shingler, J . Powlowski, and U . Marklund, J . Bacteriol . 174:711-724, 1992), these ORFs have been named bphE, bphG, and bphF, respectively . The fourth ORF (ORF4) showed homology with ORF3 from Pseudomonas pseudoalcaligenes KF707 (K . Taira, J . Hirose, S . Hayashida, and K . Furukawa, J . Biol . Chem . 267:4844-4853, 1992), whose function is unknown . The functions of meta-cleavage enzymes (BphE, BphG, and BphF) were analyzed by using crude extracts of Escherichia coli which expressed the encoding genes . The results showed that bphE, bphG, and bphF encode 2-hydroxypenta-2,4-dienoate hydratase, acetaldehyde dehydrogenase (acylating), and 4-hydroxy-2-oxovalerate aldolase, respectively . The biphenyl and polychlorinated biphenyl degradation pathway of KKS102 is encoded by 12 genes in the order bphEGF (ORF4)A1A2A3BCD (ORF1)A4 . The functions of ORF1 and ORF4 are unknown . The features of this bph gene cluster are discussed.

J Bacteriol, 1994 Jul, 176(14), 4243 - 9
Phosphorylation of lipopolysaccharides in the Antarctic psychrotroph Pseudomonas syringae: a possible role in temperature adaptation; Ray MK et al.; Phosphorylation of lipopolysaccharide (LPS) from a psychrotrophic bacterium, Pseudomonas syringae, from Antarctica was studied by using sucrose gradient-separated membrane fractions . The bacterium was found to possess an LPS kinase which could phosphorylate more LPS postsynthetically at higher temperatures . The phosphorylation was low at a lower temperature and was also found to occur in vivo . After phosphorylation of LPS in vitro, it was found that the major part of the radioactivity (> 85%) was associated with the core oligosaccharide region of the LPS . The phosphate groups of this region are probably involved in the binding of metal ions, which could be removed by EDTA . The cells grown at the lower temperature probably contained fewer divalent cations because of the smaller amount of phosphate and thereby were more sensitive to EDTA . The cells were also more sensitive to cationic antibiotics at the lower temperature . A possible role of this differential phosphorylation of LPS in modulating the function of the outer membrane as a permeability barrier in the psychrotroph is discussed.

J Bacteriol, 1994 Jul, 176(13), 4124 - 32
Plasmid-directed assembly of the lipid-containing membrane of bacteriophage phi 6; Johnson MD 3rd et al.; The nucleocapsid of bacteriophage phi 6 is enveloped within a lipid-containing membrane . The membrane is composed of proteins P3, P6, P9, P10, and P13 and phospholipids . The relationship between membrane protein P9 and morphogenetic protein P12 was studied in the absence of phage infection . cDNA copies of genes 9 and 12 were expressed on plasmids in Pseudomonas syringae pv . phaseolicola . Immunoblotting demonstrated the presence of protein P9 in strains carrying both gene 9 and gene 12 but not in strains with gene 9 alone . In the absence of P12, P9 was found to be unstable . Simultaneous synthesis of proteins P9 and P12 led to the formation of a low-density P9 particle having a buoyant density similar to that of precursor structures composed of phospholipid and proteins isolated from phi 6-infected cells . These results are consistent with results of previous genetic experiments suggesting that P9 and P12 are necessary and sufficient for the formation of the phi 6 envelope . Extensions of P9 at the C terminus do not impair particle formation; however, N-terminal extensions or C-terminal deletions that extend into the hydrophobic region of P9 do impair particle formation.

J Bacteriol, 1994 Jul, 176(13), 4017 - 24
Effect of growth temperatures on the protein levels in a psychrotrophic bacterium, Pseudomonas fragi; Hebraud M et al.; Pseudomonas fragi has the ability to grow between 0 and 35 degrees C and grows optimally at 30 degrees C . Cellular proteins from mid-log-phase cells growing from 4 to 34 degrees C were labeled with L-{35S}methionine during 1 generation time and analyzed by two-dimensional gel electrophoresis . The electrophoretic patterns revealed differences in the patterns of protein synthesis over this temperature span . A qualitative comparison of cellular proteins led to their separation into five thermal classes . The first class contained proteins whose relative rates of synthesis were unaffected by the growth temperature . Three other classes included proteins with optimal expression at 4 to 10, 15 to 20, and 25 to 30 degrees C . A fifth class contained proteins which were more specifically synthesized at a supraoptimal growth temperature (34 degrees C) . Two low-molecular-mass proteins, designated C7.0 and C8.0, were highly concentrated at 4 to 10 degrees C, and their relative rates of synthesis steadily increased with decreasing temperature . Polyclonal antibodies were separately raised against these two proteins . Immunological analyses revealed cross-reaction between these two proteins and between two additional low-molecular-mass proteins which were maximally produced at elevated temperatures . Antisera directed against C8.0 recognized the major cold shock protein of Escherichia coli, CspA, indicating the presence of similarities between these proteins.

Int J Cancer, 1994 Jul 1, 58(1), 142 - 9
Cytotoxic and antitumor activity of a recombinant immunotoxin composed of disulfide-stabilized anti-Tac Fv fragment and truncated Pseudomonas exotoxin; Reiter Y et al.; Disulfide-stabilized Fv (dsFv)-immunotoxins are recombinant immunotoxins in which the inherently unstable Fv moiety, composed of the VH-VL heterodimer, is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL . Anti-Tac(dsFv)-PE38KDEL is composed of such a dsFv, directed to the alpha subunit of the IL2 receptor (IL2R), and containing a truncated form of Pseudomonas exotoxin (PE38KDEL) . We have found this new type of immunotoxin to be indistinguishable in its in vitro activity and specificity from its single-chain immunotoxin counterpart, anti-Tac(Fv)-PE38KDEL . We have now examined the therapeutically relevant factors, including stability, pharmacokinetics, and antitumor activity of this new disulfide-stabilized Fv-immunotoxin . We found that anti-Tac(dsFv)-PE38KDEL was specifically cytotoxic to human activated T-lymphocytes in addition to IL2R bearing cell lines . Anti-Tac(dsFv)-PE38KDEL was considerably more stable at 37 degrees C in human serum and in buffered saline than the single-chain immunotoxin, anti-Tac(Fv)-PE38KDEL . The half-life in blood was similar for both immunotoxins (approx . 20 min) . The therapeutic potential of the disulfide-stabilized immunotoxin was evaluated using an animal model of immunodeficient mice bearing subcutaneous tumor xenografts of human IL2R-bearing cells . Anti-Tac(dsFv)-PE38KDEL caused complete regression of tumors with no toxic effects in mice . Because dsFv-immunotoxins are more stable and can be produced with significantly improved yields compared to scFv-immunotoxins, dsFv-immunotoxin may be more useful for therapeutic applications.

Cancer Res, 1994 Jul 1, 54(13), 3460 - 7
B3(Fab)-PE38M: a recombinant immunotoxin in which a mutant form of Pseudomonas exotoxin is fused to the Fab fragment of monoclonal antibody B3; Choe M et al.; Recombinant immunotoxins were made by fusing the Fab domain of monoclonal antibody (MAb) B3 to PE38M, a truncated mutant form of Pseudomonas exotoxin (PE) . The recombinant toxins were made in Escherichia coli by fusing genes encoding the antibody domains to a gene encoding the mutant form of PE . MAb B3 binds to a carbohydrate antigen found on many kinds of carcinomas . Immunotoxins in which MAb B3 has been chemically coupled to recombinant forms of PE have been shown to be very active cytotoxic agents . PE has also been targeted to tumor cells by replacing the cell-binding domain of PE (domain I) with a single-chain antibody to make a single-chain immunotoxin . In the current study, PE38M, a mutant form of PE, with a deletion of the cell-binding domain (amino acids 1-252) as well as mutations in domain III and some nonessential sequences in domain Ib (amino acids 365-380), was fused to the light chain of MAb B3 . This protein was renatured in the presence of the Fd fragment of MAb B3 to produce a Fab-toxin fusion protein . Alternatively, the Fd fragment of MAb B3 was fused to PE38M and combined with the light chain . Both types of B3(Fab)-PE38M were just as active on target cells as previously described single-chain immunotoxins . Furthermore, the B3(Fab)-PE38M produced complete remissions of human tumor xenografts growing in nude mice . B3(Fab)-PE38M has two advantages over single-chain immunotoxins . One is that the yield of recombinant Fab-toxin is very high, with 10-22% of the starting protein recovered as cytotoxically active immunotoxin after chromatographic purification . The second is that the B3(Fab)-PE38M has a much longer survival in the circulation of mice with a t1/2 beta of approximately 5 h.

Bioconjug Chem, 1994 Jul-Aug, 5(4), 321 - 6
Mutations of two lysine residues in the CDR loops of a recombinant immunotoxin that reduce its sensitivity to chemical derivatization; Benhar I et al.; B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv region of monoclonal antibody B3 is connected to a truncated form of Pseudomonas exotoxin . It would be desirable to use the lysine residues of the molecule for chemical modification so that it can be derivatized with poly(ethylene glycol) to achieve reduced immunogenicity or with the Bolton-Hunter reagent for biodistribution studies . We found that derivatizing lysine residues of B3(Fv)-PE38 causes a marked loss of specific target cell cytotoxicity and/or immunoreactivity . Here we show that two lysine residues in the antibody-combining region of B3(Fv)-PE38 can be replaced with arginines, with only a small loss of cytotoxicity and no change in specificity . This mutant molecule is 3-fold more resistant to inactivation by derivatization with succinimidyl 4-(N-maleimidomethyl)cyclohexane 1-carboxylate (SMCC) or Bolton-Hunter reagent.

Microb Releases, 1994 Jul, 2(4), 247 - 54
Pseudomonas aureofaciens in soil: survival and recovery efficiency; Angle JS et al.; Efficient methods for the recovery of genetically engineered organisms (GEM) added to soil are critical if the safety of potential releases is to be evaluated and the actual release is to be monitored . Pseudomonas aureofaciens strain 3732 RN-L11 (lacZY) was added to 10 g sieved soil microcosms and incubated for 5 and 28 days . Various diluents, shaking methods, and settling of soil were examined to determine the optimum method for recovery of the GEM from the soil . Of the diluents examined, 0.1% agar gave significantly lower numbers than distilled water, 1.0% sodium metaphosphate, 1% peptone, and phosphate-buffered water . After 5 days of incubation, shaking for 10 min with glass beads and shaking for 30 min without glass beads resulted in the highest recovery of the GEM from soil, while sonification resulted in the lowest recovery . After 28 days of incubation, sonification produced significantly lower numbers than any of the other treatments . The addition of 1% CaCl2 to enhance settling significantly increased recovery efficiency . Although the use of CaCl2 in distilled water and shaking for 10 min was an effective method for recovering P . aureofaciens from a Maryland soil, when the same extraction procedure was compared with a standard technique (dd H2O, shaking for 10 min) for eight divergent soils, neither extraction method was consistently better than the other . Statistical analysis of the data showed the need for log transformation of the raw data . Four microcosm and two plate replicates for each dilution provided the greatest ability to detect differences between treatment means while maximizing experimental efficiency.

Mol Plant Microbe Interact, 1994 Jul-Aug, 7(4), 464 - 71
Genetic dissection of oligogenic resistance to bacterial wilt in tomato; Danesh D et al.; To study resistance to bacterial wilt (caused by Pseudomonas solanacearum) in tomato, we analyzed 71 F2 individuals from a cross between a resistant and a susceptible parent with 79 DNA markers . F2 plants were inoculated by two methods: bacteria were injected into shoots of cuttings or poured into soil surrounding wounded roots . Disease responses were scored on a scale of 0 to 5 . Statistical comparisons between DNA marker genotypes and disease phenotypes identified three genomic regions correlated with resistance . In plants inoculated through roots, genomic regions on chromosomes 6 and 10 were correlated with resistance . In plants inoculated through shoots, a region on chromosome 7 was significant, as were the regions on chromosomes 6 and 10 . The relative impact of resistance loci on disease response differed between shoot and root inoculations . To confirm the existence of a partial resistance gene on chromosome 6, an F2 individual homozygous for the resistant parent's alleles on chromosomes 7 and 10, but heterozygous for markers on chromosome 6, was selfed . Analysis of the F3 progeny confirmed that a partial resistance locus was located on chromosome 6, very close to CT184 . The presence of a partial resistance locus on chromosome 10 was similarly confirmed by analysis of progeny of another F2 plant chosen on the basis of its marker phenotype.

Biosci Biotechnol Biochem, 1994 Jul, 58(7), 1306 - 8
Efficient transformation of pseudomonas strains with pNI vectors by electroporation; Itoh N et al.; The optimum conditions for electro-transformation of some Pseudomonas strains including P . putida, P . fluorescens, and P . flavida, were defined using the pNI105 vector, resulting in 100-10,000 fold increases in transformation efficiency compared with conventional chemical transformation with MgCl2 . The growth phase of the cultured cells and the field strength were important in obtaining high transformation efficiency . Under optimal conditions, 3.2 x 10(7) transformants per 1 microgram DNA were obtained using a combination of pNI105 and P . fluorescens IAM12022 . This value is the highest ever obtained for a Pseudomonas species . In addition, the procedures were applicable to the effective transformation of fluorescent Pseudomonas strains with pNI vectors.

Pathology, 1994 Jul, 26(3), 315 - 7
Pseudomonas cepacia in the sputum of cystic fibrosis patients; Taylor PC et al.; Colonization of sputum by Pseudomonas cepacia has been associated with progressive respiratory deterioration and a worsening prognosis for some cystic fibrosis patients . After laboratory methods were changed and a selective medium introduced, P . cepacia was isolated from the sputa of 16 out of 82 patients with cystic fibrosis attending this hospital: a prevalence rate of 20% . P . cepacia was found in the first sputum examined of 12 patients after the new methods were introduced, indicating that colonization was not necessarily a recent event . All isolates were satisfactorily identified using conventional media but half were identified with only a low level of confidence using the API 20NE kit . Further studies using adequate laboratory methods are required to determine the prevalence and significance of this organism in Australia.

J Infect, 1994 Jul, 29(1), 87 - 90
Fatal melioidosis in a tourist returning from Thailand; Wilks D et al.; Severe infections with Pseudomonas pseudomallei, the causative agent of melioidosis, is a common cause of community acquired septicaemia in South East Asia and parts of Northern Australia, but infection in travellers returning to the United Kingdom is extremely rare . We describe the case of a tourist who acquired melioidosis in Thailand . Despite intensive intravenous therapy with antibiotics to which the organism was sensitive, the patient's infection proved fatal . Ps . pseudomallei was isolated from blood cultures at a late stage in his illness by use of a commercial blood culture system that included an antibiotic removal device (Bac/TAlert, Organon-Technica).

Int J Syst Bacteriol, 1994 Jul, 44(3), 410 - 5
Pseudomonas flavescens sp . nov., isolated from walnut blight cankers; Hildebrand DC et al.; Two pseudomonad strains that produce a yellow cellular pigment, in addition to a diffusible fluorescent pigment on Kings medium B, were isolated from cankers on walnut trees . Biochemical properties, such as a positive oxidase reaction, a negative arginine dihydrolase reaction, and the production of a fluorescent pigment, in addition to the results of an extensive nutritional characterization study and DNA-DNA hybridization experiments, indicated that these strains belong to a new Pseudomonas rRNA group I species . This conclusion was supported by the results of a determination of the sequence of the PCR-amplified 16S rRNA gene and a comparison with the 16S rRNA genes of other bacterial species . The genomic DNAs of the strains had a base composition of 63 mol% G+C . The name Pseudomonas flavescens sp . nov . is proposed . Strain B62 (= NCPPB 3063) is the type strain of the species.

J Bacteriol, 1994 Jul, 176(13), 4034 - 42
Multiple replicons constituting the genome of Pseudomonas cepacia 17616; Cheng HP et al.; Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction fragments containing rare restriction enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 megabases (Mb) . Inclusion of the 170-kb cryptic plasmid present in this strain gave an overall estimate of genome size of 7 Mb . Other Southern hybridization experiments indicated that the three large replicons contained rRNA genes as well as insertion sequence elements identified previously in this strain . The distribution of SwaI, PacI, and PmeI sites on the three replicons was determined . A derivative of Tn5-751 carrying a SwaI site was used to inactivate and map genes on the 2.5- and 3.4-Mb replicons . Mutants were isolated in which the 2.5- and 0.9-Mb replicons had been reduced in size to 1.8 and 0.65 Mb, respectively . The loss of DNA from the 2.5-Mb replicon was associated with lysine auxotrophy, beta-lactamase deficiency, and failure to utilize ribitol and trehalose as carbon and energy sources . DNA fragments corresponding in size to randomly linearized forms of the different replicons were detected in unrestricted DNA by pulsed-field gel electrophoresis . The results provide a framework for further genetic analysis of strain 17616 and for evaluation of the genomic complexities of other P . cepacia isolates.

Gene, 1994 Jun 24, 144(1), 9 - 16
The biphenyl/polychlorinated biphenyl-degradation locus (bph) of Pseudomonas sp . LB400 encodes four additional metabolic enzymes; Hofer B et al.; The bph locus of Pseudomonas sp . LB400, encoding biphenyl/polychlorinated biphenyl (PCB) degradation, contains a region of about 3.5 kb of hitherto unknown function, between bphC and bphD . This DNA segment has now been characterized . Four structural genes have been located and identified by a combination of expression cloning, enzyme activity tests and DNA sequencing . The region contains four closely spaced cistrons (bphKHJI) encoding a glutathione S-transferase (GST), a 2-hydroxypenta-2,4-dienoate hydratase, an acetaldehyde dehydrogenase (acylating) and a 4-hydroxy-2-oxovalerate aldolase, respectively . The latter three are enzymes required for conversion of the aliphatic end product of bphABCD-encoded catabolism of biphenyls to Krebs cycle intermediates . The discovery of these genes provides a rationale for growth of the strain on chlorinated biphenyls which yield chlorinated benzoates as dead-end metabolites . The sequences of the enzymes involved are 54-71% identical to those of homologous enzymes encoded by the dmp and xyl operons . The role of the GST in the degradation of biphenyls is less clear, but since it was found to contain, in the putative xenobiotic substrate-binding domain, a region which shares about 29% of identical amino acids with a bacterial tetrachlorohydroquinone dehalogenase, it may be involved in dehalogenation of PCB-degradative intermediates.

Gene, 1994 Jun 24, 144(1), 139 - 40
Cloning and characterisation of the gene encoding cytochrome c4 from Pseudomonas stutzeri; Christensen HE; By use of a degenerate oligodeoxyribonucleotide probe corresponding to the N-terminal amino acid (aa) sequence and Southern blot analysis, the gene encoding the di-heme cytochrome c4 (CC4) from Pseudomonas stutzeri has been cloned . The aa sequence deduced from the nucleotide sequence shows a 20-aa signal peptide and a 190-aa mature protein with 79% identity to the Azotobacter vinelandii CC4 sequence, which had earlier been determined by aa sequencing.

Int J Cancer, 1994 Jun 15, 57(6), 856 - 64
Anti-Tac(Fab)-PE40, a recombinant double-chain immunotoxin which kills interleukin-2-receptor-bearing cells and induces complete remission in an in vivo tumor model; Kreitman RJ et al.; We have produced a single plasmid encoding both the heavy chain Fd domain (VH + CH1) of the anti-interleukin-2 receptor (IL2R) monoclonal antibody anti-Tac, and the anti-Tac light chain fused to PE40, a truncated derivative of Pseudomonas exotoxin . The active immunotoxin anti-Tac(Fab)-PE40 could be recovered from E . coli from either periplasm or renatured inclusion bodies . The double-chain immunotoxin was very cytotoxic toward IL2R-bearing cell lines, human activated T cells and fresh adult-T-cell-leukemia cells . The cytotoxicity was similar to that of anti-Tac(Fv)-PE40, the single-chain recombinant toxin containing only the variable domains of anti-Tac . IL2R-binding affinity was also equivalent to that of anti-Tac(Fv)-PE40, which is one-third that of anti-Tac . The serum half-life in mice was significantly prolonged as compared with anti-Tac(Fv)-PE40, with a beta phase of 430 vs . 57 minutes, but the LD50s were equivalent when the immunotoxins were administered in 3 daily doses . Anti-Tac(Fab)-PE40 was very cytotoxic in vitro toward transfected ATAC-4 carcinoma cells which express IL2Rs . In mice bearing ATAC-4 tumors, anti-Tac(Fab)-PE40 showed significant anti-tumor activity, inducing complete remissions in 80 and 100% of treated animals at approximately 7 and 14% respectively of the LD50 . Anti-Tac(Fab)-PE40 was much more effective in vitro and in vivo than chemical conjugates between anti-Tac and truncated PE molecules . The recombinant Fab toxin should be studied further as potential treatment for IL2R-related malignancies, particularly if smaller recombinant immunotoxins have insufficient half-life in humans.

Biochemistry, 1994 Jun 14, 33(23), 7415 - 22
Characterization of the three tyrosine residues of delta 5-3-ketosteroid isomerase by time-resolved fluorescence and circular dichroism; Wu P et al.; delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni converts delta 5-3-ketosteroids to delta 4-3-ketosteroids via an enolic intermediate . Site-specific mutagenesis has identified Tyr-14 and Asp-38 as the catalytically essential general acid and base, respectively . Three tyrosine residues (Tyr-14, Tyr-55, and Tyr-88) are the only significant fluorophores in the wild-type isomerase . Recent studies of the steady-state fluorescence of the wild-type enzyme and all six mutant enzymes in which one or two tyrosine residues have been mutated to phenylalanine show that the fluorescence intensity of Tyr-14 is very high, that of Tyr-88 is very low, and that of Tyr-55 is intermediate and comparable to that of N-acetyltyrosine amide in solution (Li, Y.-K., Kuliopulos, A., Mildvan, A.S., & Talalay, P . (1993) Biochemistry 32, 1816-1824) . Extension of these experiments by time-resolved fluorescence and fluorescence anisotropy measurements demonstrates that Tyr-14, which is in a hydrophobic environment, has an unusually long fluorescence lifetime (4.6 ns) as compared to Tyr-55 (2.0 ns) or Tyr-88 (0.8 ns) and to most protein tyrosine residues (0.2-2 ns) . The Forster distances obtained from the absorption and emission of these tyrosines predict that total quenching of Tyr-14 fluorescence by Tyr-55, and to a lesser degree by Tyr-88, would occur if their orientations were favorable.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1994 Jun 3, 243(5), 515 - 24
Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp . strain M114; Adams C et al.; An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp . strain M114 has been identified . A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated . The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 bp was identified . This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC . PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli . These enzymes are believed to act via ATP-dependent binding of AMP to their substrate . Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain . The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114 . The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined -16 to -25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence . It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.

J Bacteriol, 1994 Jun, 176(12), 3749 - 56
A novel toluene-3-monooxygenase pathway cloned from Pseudomonas pickettii PKO1; Olsen RH et al.; Plasmid pRO1957, which contains a 26.5-kb fragment from the chromosome of Pseudomonas pickettii PKO1, allows P . aeruginosa PAO1 to grow on toluene or benzene as a sole carbon and energy source . A subclone of pRO1957, designated pRO1966, when present in P . aeruginosa PAO1 grown in lactate-toluene medium, accumulates m-cresol in the medium, indicating that m-cresol is an intermediate of toluene catabolism . Moreover, incubation of such cells in the presence of 18O2 followed by gas chromatography-mass spectrometry analysis of m-cresol extracts showed that the oxygen in m-cresol was derived from molecular oxygen . Accordingly, this suggests that toluene-3-monooxygenation is the first step in the degradative pathway . Toluene-3-monooxygenase activity is positively regulated from a locus designated tbuT . Induction of the toluene-3-monooxygenase is mediated by either toluene, benzene, ethylbenzene, or m-cresol . Moreover, toluene-3-monooxygenase activity induced by these effectors also metabolizes benzene and ethylbenzene to phenol and 3-ethylphenol, respectively, and also after induction, o-xylene, m-xylene, and p-xylene are metabolized to 3,4-dimethylphenol, 2,4-dimethylphenol, and 2,5-dimethylphenol, respectively, although the xylene substrates are not effectors . Styrene and phenylacetylene are transformed into more polar products.

J Cell Physiol, 1994 Jun, 159(3), 495 - 505
Characterization of 3'-azido-3'-deoxythymidine inhibition of ricin and Pseudomonas exotoxin A toxicity in CHO and Vero cells; Wellner RB et al.; Ricin (RIC), modeccin (MOD), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are protein toxins that enter cells by receptor-mediated endocytosis . After intracellular transport and membrane translocation to the cytosol, these toxins inhibit protein synthesis by enzymatically removing a specific adenine residue from ribosomal RNA (RIC, MOD), or by ADP-ribosylation of elongation factor-2 (PE, DT) . Recently, Thompson and Pace (1992) reported that AZT (3'-azido-3'-deoxythymidine) inhibited RIC toxicity in Vero cells, and this inhibition was not due to a block of RIC enzymatic activity . This paper extends these findings and examines the effects of AZT treatment on the toxicities of other protein toxins in Chinese hamster ovary (CHO) and Vero cell lines . AZT treatment did not significantly alter the toxicity of DT or MOD in either cell line, but it markedly reduced RIC and PE toxicity in both cell lines . The ID50 values (concentration of toxin required to inhibit protein synthesis by 50%) for RIC and PE in CHO cells increased approximately 6.5- and 12.5-fold, respectively; while in Vero cells the ID50 values increased ca . 8.5- and 4.5-fold, respectively . Results of further studies revealed differences in the mechanisms by which AZT inhibited RIC and PE toxicity . Results of cell-free translation indicated that, unlike its effects on RIC, AZT blocked the ability of PE to perform its enzymatic activity . As AZT did not block RIC enzymatic activity, we examined the effects of AZT on earlier steps in the RIC intoxication process . AZT treatment did not inhibit cell-surface binding or internalization of {125I}-RIC . Results of kinetic studies showed that when AZT was incubated with cells at the time of RIC exposure, it caused no major change in the lag phase, during which RIC reaches the site of translocation . However, it clearly reduced the subsequent first-order reduction in the rate of protein synthesis, suggesting an effect on translocation . Monensin (an ionophore that perturbs intracellular trafficking and increases the toxicities of RIC and PE) reduced AZT protection against both toxins . Nocodazole and colchicine (agents that disrupt microtubules and some routes of intracellular trafficking) reduced the ability of AZT to inhibit RIC, but not PE, toxicity . In summary, our results suggest that (1) AZT acts within the cytosol to inhibit (directly or indirectly) the enzymatic action of PE, and (2) the AZT inhibition of RIC cytotoxicity does not involve perturbations of RIC cell-surface binding, internalization, or enzymatic activity but might result from an alteration in RIC translocation.

Pediatr Pulmonol, 1994 Jun, 17(6), 390 - 2
Cerebral abscess as a complication of cystic fibrosis; Cooper DM et al.; Two male patients with cystic fibrosis (CF), both 18 years old, developed frontal lobe brain abscesses . Both patients presented with histories of intermittent headache over several days and occasional vomiting . Headache was not more evident in the mornings and not associated with visual disturbance in either patient . Neither was hypertensive nor had visual disturbance . Both patients had documented pansinusitis and nasal polyposis . Both men had had few admissions for pulmonary exacerbations, and neither was significantly malnourished . The abscess in neither patient grew Pseudomonas species.

Biotechnol Appl Biochem, 1994 Jun, 19 ( Pt 3), 271 - 84
Expression and post-translational processing of a broad-spectrum organophosphorus-neurotoxin-degrading enzyme in insect tissue culture; Dave KI et al.; A recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), has been utilized to express the opd (organophosphate-degrading) gene from Pseudomonas diminuta in insect tissue-culture cells (Sf9) of the fall armyworm (Spodoptera frugiperda) . The broad-spectrum organophosphate hydrolase (EC 3.1.8.1) encoded by this gene is a member of a general class of enzymes {organophosphate (OP) anhydrolases} that include parathion hydrolases, di-isopropyl-fluorophosphatases (DFPases), somanases, and OP phosphotriesterases . This particular enzyme possesses the ability to hydrolyse paraoxon (P-O bond), DFP, sarin (P-F bond), VX (P-S bond) and tabun (P-CN bond), as well as a number of other extensively used organophosphorus pesticides . The enzyme produced in infected Sf9 cells is post-translationally processed and resembles the mature form of the enzyme expressed in various bacterial cells as identified by immunoprecipitation on Western blots . N-terminal sequence analysis of enzyme expressed in insect cells revealed Gly-29 as the terminal residue, whereas expression in Escherichia coli removes this residue, exposing Ser-30 at the N-terminus . Conditions for optimal expression of the enzyme in this system are described . Furthermore, hydrolytic efficiency of some OPs with purified enzyme from this system is discussed in relation to the in situ activity of Pseudomonas diminuta MG cells.

Appl Environ Microbiol, 1994 Jun, 60(6), 1805 - 9
Biochemical characterization of a glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas strain BL072; Binder R et al.; Pseudomonas strain BL072 produces an acylase enzyme active in hydrolyzing glutaryl-7-aminocephalosporanic acid to 7-aminocephalosporanic acid . This acylase was purified by column chromatography and gel electrophoresis . The native acylase was composed of two subunits of approximately 65 and 24 kDa, though some heterogeneity was seen in both the native acylase and its small subunit . The isoelectric point of the acylase is approximately 8.5, and it has Km of 1.6 mM for glutaryl desacetoxy aminocephalosporanic acid . The acylase hydrolyzes the desacetoxy and desacetyl derivatives of glutaryl-7-aminocephalosporanic acid at rates similar to that of glutaryl-7-aminocephalosporanic acid . Cephalosporin C was hydrolyzed at a reduced rate . The pH optimum was found to be 8.0, and an activation energy of 9 kcal/mol (ca . 38 kJ/mol) was observed . The acylase has transacylase activity 10 times that of its hydrolytic activity . Eupergit C-immobilized acylase had a half-life of greater than 400 h.

Eur J Biochem, 1994 Jun 1, 222(2), 293 - 303
Nitric oxide reductase from Pseudomonas stutzeri, a novel cytochrome bc complex . Phospholipid requirement, electron paramagnetic resonance and redox properties; Kastrau DH et al.; The nitric oxide reductase (NOR) from Pseudomonas stutzeri is a cytochrome bc complex which shows on SDS/PAGE two subunits with apparent molecular masses of 17 kDa and 38 kDa . Two other species of approximately 45 kDa and 74-78 kDa represent the undissociated enzyme complex and an aggregate of the cytochrome b subunit, respectively . The cytochrome b subunit is highly hydrophobic and results in aberrant electrophoretic mobility . The stability of the enzyme in various detergents and at different pH was investigated . The highest specific activity of 60 mumol NO min-1 mg-1 protein was obtained after electrophoresis in the presence of laurylpropanediol-3-phosphorylcholine ether . Purified NOR contained cardiolipin, phosphatidylglycerol, and phosphatidylethanolamine, the latter as the major component . A phospholipid was required for high catalytic activity with either cardiolipin or phosphatidylglycerol increasing the activity of the enzyme as isolated by a factor of up to 5 . Free fatty acids inhibited NOR, with cis-9-octadecenoic acid (oleic acid) showing the most pronounced effect . Certain detergents substituted for the phospholipid requirement of NOR . The enzyme, as isolated, in 0.1% Triton X-100, 20 mM Tris/HCl pH 8.5, exhibited a complex set of EPR resonances at low magnetic field, with a prominent peak at g 6.34 resulting from Fe(III) high-spin cytochrome b . The second prominent feature arose from a low-spin Fe(III) heme center with strong lines at apparent g values of 3.02 and 2.29, and a broad resonance at g approximately 1.5 which we assigned to the cytochrome c component of the enzyme . From spin quantitation and computer simulations of the various EPR signals a ratio close to 1:1 for the low-spin/high-spin heme centers in NOR was estimated . Shifting the pH from 8.5 to 5.0, replacing Triton X-100 by other detergents, or adding soybean phospholipids to the protein, led to pronounced changes of the EPR signals in the g = 6 region . In contrast, the strong inhibitor oleic acid did not cause significant spectral changes . NOR which had been reduced by L-ascorbate/phenazine methosulfate prior to incubation with its substrate NO gave the characteristic Fe(II) nitrosyl triplet centered at g approximately 2.01, with a hyperfine splitting of 1.70 mT . In the absence of dioxygen, NOR was quantitatively reduced by either sodium dithionite, or photochemically with deazaflavin and oxalate; the enzyme was reoxidizable by ferricyanide in a fully reversible reaction . Spectroelectrochemical oxidoreductive titrations gave E'o (versus standard hydrogen electrode) = +322 mV for the cytochrome b and +280 mV for the cytochrome c component.

Mol Microbiol, 1994 Jun, 12(6), 941 - 50
Two native plasmids of Pseudomonas syringae pathovar tomato strain PT23 share a large amount of repeated DNA, including replication sequences; Murillo J et al.; Strain PT23 of Pseudomonas syringae pv . tomato contains four native plasmids, designated A, B, C, and D . By DNA hybridization of genomic and plasmid DNA digests from the wild type and a plasmid-cured strain, we determined that c . 61 kb (c . 74%) of pPT23B is repeated in pPT23 A and only c . 17 kb (c . 21%) is in single copy in strain PT23 . pPT23B also contains DNA repeated in the chromosome that occurs in three DNA fragments of 0.6, 4.6, and 9.6 kb that might be transposable elements . Additionally, the 9.6 kb fragment also shares sequences with the three other plasmids of strain PT23 . By DNA hybridization with the origin of replication from a native plasmid of P . syringae pv . syringae and in vivo replication tests, we identified the origins of replication of plasmids A, B, and D and showed that they cross-hybridize . The putative par region from pPT23 A has also been identified and is not conserved in the other three native plasmids from strain PT23 . By using the defined minimal origin of replication from pPT23 A as a probe, we showed that it is highly conserved in 14 strains belonging to nine different pathovars of P . syringae and that as many as five different native plasmids with closely related origins of replication coexist in the same cell . The duplication and reorganization of plasmids might therefore occur at high frequency and could be responsible for the existence of large numbers of native plasmids in P . syringae strains.

Biosci Biotechnol Biochem, 1994 Jun, 58(6), 1023 - 7
Role of the gene encoding lipase activator from Pseudomonas sp . strain KWI-56 in in vitro activation of lipase; Iizumi T et al.; The function of the gene, act, which encodes lipase activator and that is downstream from the gene that encodes lipase, lip, in Pseudomonas sp . strain KWI-56, was studied with Escherichia coli as the host organism . E . coli carrying both the lip and act genes in either cis or trans produced an active lipase, but E . coli carrying only the lip gene produced an equal amount of inactive lipase protein . The active and inactive lipases had the same molecular weight and the same N-terminal amino acid sequence, so their primary structures were the same . The inactive lipase was not activated spontaneously, but it was activated in vitro by the addition of a crude cell extract of E . coli carrying an expression plasmid of the act gene . Lipase from Pseudomonas sp . strain KWI-56 was completely without enzymatic activity when denatured, but regained activity in vitro upon addition of the crude cell extract . These results indicate that the act gene affects the activation involved in the conformational change of the lipase protein to its active form.

Am J Respir Crit Care Med, 1994 Jun, 149(6), 1601 - 7
Infectious complications of lung transplantation . Impact of cystic fibrosis; Flume PA et al.; It has been suggested that the presence of airway pathogens prior to lung transplantation (LT) in patients with cystic fibrosis (CF) may place these patients at a higher risk for infectious complications after LT . There is particular concern regarding patients colonized with multiresistant Pseudomonas, including P . cepacia, and fungi, including Aspergillus . We report our experience with LT for patients with CF and compare the results with those of patients with LT for other indications . Between January 1990 and March 1993, we performed LT for 27 patients with CF and 32 without CF . Nearly all (89%) of the patients with CF were colonized with P . aeruginosa; many were cultured with P . cepacia (19%) and Aspergillus (63%) . The non-CF group rarely had organisms identified pre-LT . No patients with CF underwent pre-LT sinus drainage or received pre-LT treatment for Aspergillus . All of the patients received perioperative antibiotics and a standard regimen of immunosuppression and prophylactic antibiotics . The incidence of infectious complications was the same in the two groups; however, there was an association between obliterative bronchiolitis and pulmonary infections . One of the patients with CF with P . cepacia died as a result of this organism . None of the patients with CF required treatment for Aspergillus post-transplant . We conclude that patients with CF, despite the presence of airway pathogens, are at no greater risk of infectious complications after LT than are other patients.

J Bacteriol, 1994 Jun, 176(11), 3117 - 25
Cloning and sequencing of a 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase gene involved in the degradation of gamma-hexachlorocyclohexane in Pseudomonas paucimobilis; Nagata Y et al.; In Pseudomonas paucimobilis UT26, gamma-hexachlorocyclohexane (gamma-HCH) is converted to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL), which is then metabolized to 2,5-dichlorohydroquinone . Here, we isolated from the genomic library of UT26 two genes which expressed 2,5-DDOL dehydrogenase activity when they were transformed into P . putida and Escherichia coli . Both gene products had an apparent molecular size of 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The first gene, named linC, located separately from the two genes (linA and linB) which we had already cloned as genes involved in the gamma-HCH degradation . The other, named linX, located about 1 kb upstream of the linA gene encoding gamma-HCH dehydrochlorinase . A gamma-HCH degradation-negative mutant, named UT72, which lacked the whole linC gene but had the intact linX gene was isolated . The linC gene given in a plasmid could complement UT72 . These results strongly suggest that the linC gene but not the linX gene is essential for the assimilation of gamma-HCH in UT26 . Deduced amino acid sequences of LinC and LinX show homology to those of members of the short-chain alcohol dehydrogenase family.

Biochemistry, 1994 May 31, 33(21), 6424 - 32
An NMR-derived model for the solution structure of oxidized putidaredoxin, a 2-Fe, 2-S ferredoxin from Pseudomonas; Pochapsky TC et al.; A model for the solution structure of oxidized putidaredoxin (Pdx), a 106-residue globular protein containing a Fe2S2 cluster, has been determined using homonuclear NMR methods . Pdx is the first of the class of Fe2S2Cys4 ferredoxins which act as electron-transfer partners for P-450 monooxygenases to be structurally characterized, and no crystal structure has been determined for Pdx or for any closely homologous protein . Pdx is the physiological redox partner of cytochrome P-450cam . A total of 878 NOE distance constraints, 66 phi angular constraints derived from NH-C alpha H coupling constants, and five paramagnetic broadening constraints were used in simulated annealing structural refinements to obtain a family of structures with pairwise rms deviations of 1.14 A for backbone atoms and 1.80 A for all non-hydrogen atoms . Paramagnetic broadening of resonances within a ca . 8-A radius of the metal cluster prevents the use of NMR-derived constraints in this region of the protein; structural constraints used to model the environment of the metal cluster were obtained from site-directed mutagenesis and model compounds and by comparison with known ferredoxin structures . Pdx retains a similar folding topology to other structurally characterized Fe2S2Cys4 ferredoxins but differs from the other ferredoxins in containing a significantly more compact structure in the C-terminal half of the protein.

Gene, 1994 May 27, 143(1), 67 - 71
Recombinant Escherichia coli strains synthesize active forms of naphthalene dioxygenase and its individual alpha and beta subunits; Suen WC et al.; Pseudomonas sp . strain NCIB 9816-4 utilizes naphthalene dioxygenase (NDO), a multicomponent enzyme system, to initiate naphthalene degradation . The terminal component of NDO is an iron-sulfur protein (ISPNAP) with an alpha 2 beta 2 subunit composition . The structural genes encoding the alpha (nahAc) and beta (nahAd) subunits were cloned separately and together into expression vectors where transcription is under the control of the T7 promoter . The recombinant plasmids were transformed into Escherichia coli JM109{pGP1-2} and the synthesis of ISPNAP and its alpha and beta subunits was determined by SDS-PAGE . Low expression of nahAd was shown to be due to inefficient initiation of translation, but a sixfold increase in the amount of beta subunit synthesized was achieved in a coupled translation system . Inclusion bodies were found in all recombinants . Increased levels of soluble active proteins were obtained when E . coli JM109(DE3), used as the host strain for recombinant plasmid, was grown at 25 degrees C . ISPNAP from JM109(DE3){pDTG121} was purified to homogeneity and shown to have the same properties as those determined for the enzyme purified from NCIB 9816-4 . Active ISPNAP was also obtained by mixing cell extracts from separate strains that synthesized the alpha and beta subunits . The availability of large amounts of purified ISPNAP and its alpha and beta subunits will facilitate future studies on the mechanism of oxygen fixation by NDO.

Biochemistry, 1994 May 24, 33(20), 6235 - 43
Increased cytotoxicity of interleukin 2-pseudomonas exotoxin (IL2-PE) chimeric proteins containing a targeting signal for lysosomal membranes; Fishman A et al.; IL2-PE40 is a chimeric protein composed of human interleukin 2 (IL2) genetically fused to the amino terminus of a truncated form of pseudomonas exotoxin lacking its cell recognition domain (PE40) . IL2-PE40 is extremely cytotoxic to IL2 receptor positive cells . This chimeric protein was found to be an effective and selective immunosuppressive agent for IL2 receptor targeted therapy in many models of disorders of the immune response where activated T-cells play a crucial role . In an attempt to produce an improved IL2-PE40 chimeric protein, we constructed new IL2-PE derivatives . This was done by inserting defined DNA sequences within the chimeric gene encoding IL2-PE40 . Inserted sequences represent motifs of other proteins known to be targeted and/or sorted to specific compartments inside or outside the cell . One of the proteins, IL2-PE40(LAP+DUP), containing a targeted signal for lysosomal membrane, was 2-3-fold more active than IL2-PE40 . The insertion of the LAP sequence also increased the cytotoxicity of another IL2-PE derivative, IL2-PE664Glu . Our results suggest that a selective targeting of IL2-PE chimeric proteins to lysosomes may enable the proteins to reach the cytosol more efficiently, thus improving its specific cytotoxicity . The LAP (lysosomal alkaline phosphatase) sequence may be used as a common motif for increasing the cytotoxicity of other chimeric proteins to be used for targeted immunotherapy.

FEBS Lett, 1994 May 23, 345(1), 9 - 13
The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling molecules of higher plants; Weiler EW et al.; The phytotoxic principle, coronatine, which is present in several pathovars of the plant pathogen, Pseudomonas syringae was shown to be highly active in completely different, jasmonate-selective bioassays . At nanomolar to micromolar concentrations, coronatine induced the accumulation of defense-related secondary metabolites in several plant cell cultures, induced transcript accumulation of the elicitor-responsive gene encoding the berberine bridge enzyme of Eschscholtzia californica, as well as the coiling response of Bryonia dioica tendrils . Biological activity critically depended upon the structure of coronatine, and slight modifications, such as methylation of the carboxyl moiety or reduction of the carbonyl group, rendered the molecules almost inactive . Coronafacic acid, obtained by hydrolysis of coronatine, was also nearly inactive . Coronatine did not elicit the accumulation of endogenous jasmonic acid in the systems analyzed . While coronafacic acid is similar in structure to jasmonic acid, we found coronatine to be a close structural analogue of the cyclic C18-precursor of jasmonic acid, 12-oxo-phytodienoic acid . The phytotoxic symptoms produced by coronatine can now be understood on the basis of the toxin's action as a mimic of the octadecanoid signalling molecules of higher plants.

Biochemistry, 1994 May 17, 33(19), 5894 - 900
Domain II of Pseudomonas exotoxin A arrests the transfer of translocating nascent chains into mammalian microsomes; Theuer C et al.; The translocation of PE from the extracytosolic compartment to the cytosol during the intoxication of mammalian cells is mediated by domain II of the toxin . We have shown previously that within domain II amino acids 280-313 of PE promote their own export from mammalian microsomes following signal sequence-directed membrane insertion . In this study, we attempted to target full-length PE into mammalian microsomes using the preprocecropin signal sequence, but found that translocation was arrested to generate a transmembrane protein . "Stop transfer" required the presence of amino acids 280-313 of PE, and the first 313 amino acids of PE were sufficient to generate a transmembrane protein (N-terminus-in/C-terminus-out) . The mechanism of stop transfer appears to be different from that described previously because amino acids 280-313 of PE are not highly hydrophobic and do contain many charged residues . In addition, the transmembrane segment appeared to be influenced by the cytoplasmic domain of the transmembrane proteins.

Cancer Res, 1994 May 15, 54(10), 2714 - 8
Antitumor activity and pharmacokinetics in mice of a recombinant immunotoxin containing a disulfide-stabilized Fv fragment; Reiter Y et al.; Disulfide-stabilized Fvs (dsFv) are recombinant Fv fragments of antibodies in which the inherently unstable VH-VL heterodimer is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL . We have recently described a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin (PE38KDEL) . This disulfide-stabilized immunotoxin is indistinguishable in activity and specificity from its single-chain immunotoxin counterpart (Brinkmann et al., Proc . Natl . Acad . Sci . USA, 90: 7538-7542, 1993) . We have now constructed and evaluated the stability, pharmacokinetics, and antitumor effect of a very similar disulfide-stabilized immunotoxin B3(dsFv)-PE38 . This immunotoxin is specifically cytotoxic to human cancer cell lines such as A431 that express the B3 antigen on their surface . In addition, the dsFv-immunotoxin is more stable at 37 degrees C in human serum than the corresponding single-chain immunotoxin B3(Fv)-PE38 . The survival of the disulfide-stabilized immunotoxin in the circulation of mice was determined by a bioassay on cultured A431 cells after administering the immunotoxin i.v . The half-life in blood was 23 min . To determine the therapeutic effects of the disulfide-stabilized immunotoxin, it was given i.v . to immunodeficient mice bearing s.c . human epidermoid carcinomas . The dsFv-immunotoxin caused complete regression of tumors with no toxic effect on mice . The antitumor effect was similar to that reported for the single-chain Fv-immunotoxin . Our data show that dsFv-immunotoxins retain full in vitro as well as in vivo activity when compared to scFv-immunotoxins . Because dsFv-immunotoxins have full activity, are more stable, and can be produced with significantly improved yields compared to scFv-immunotoxins, the dsFv-immunotoxins may be more useful for therapeutic applications than scFv-immunotoxins.

Anal Biochem, 1994 May 15, 219(1), 37 - 42
Analysis of dicamba degradation by Pseudomonas maltophilia using high-performance capillary electrophoresis; Yang J et al.; A method based on high-performance capillary electrophoresis (HPCE) was developed for the simultaneous analysis of dicamba and its metabolites in media containing the bacterium Pseudomonas maltophilia . Dicamba, 3,6-dichlorosalicylic acid (DCSA), and related products were extracted from media samples using ether under acidic conditions and injected onto an HPCE system containing a pH 10.0 running buffer . Baseline resolution between these compounds was obtained at 30 kV with a total run time of 6 min on a 50-microns i.d . x 50-cm capillary . The linear range for dicamba and DCSA detected at 274 nm extended from 0 to 100 mg/liter and the dynamic range for both compounds extended to 2000 mg/liter . The within-run precision was +/- 5% or less throughout the entire concentration range studied . The limits of detection for dicamba and DCSA in the media samples were 6 and 2 mg/liter . This corresponded to detection limits of 0.3 and 0.1 ng, respectively, in the injected extracts . With this method it was possible to conduct preliminary studies examining the kinetics of dicamba metabolism in P . maltophilia.

FEMS Microbiol Lett, 1994 May 15, 118(3), 279 - 82
Quantitative determination of intracellular depolymerase activity in Pseudomonas oleovorans inclusions containing poly-3-hydroxyalkanoates with long alkyl substituents; Foster LJ et al.; Research regarding the accurate, quantitative degradation of novel poly-3-hydroxyalkanoates has been restricted by the absence of an appropriate monitoring technique . The calibration of a gas chromatograph to poly-3-hydroxyoctanoate reveals a linear relationship between the area under gas chromatograph tracings and polymer weight . With this new method, poly-3-hydroxy-octanoate granules isolated from Pseudomonas oleovorans, which were incubated at 30 degrees C in an alkaline buffer, exhibited a linear degradation rate . Degradation was inhibited by the presence of Triton X-100 and phenylmethylsulfonyl fluoride . The depolymerase was demonstrated to be associated with the polymer granule complex and most likely possessed serine residues at its active site.

Biochemistry, 1994 May 10, 33(18), 5451 - 9
Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions; Reiter Y et al.; Disulfide-stabilized Fv's (dsFv's) are recombinant Fv fragments of antibodies in which the unstable variable heavy (VH) and variable light (VL) heterodimers are stabilized by disulfide bonds engineered at specific sites that lie between structurally conserved framework positions of VH and VL . We have recently described one example of a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin {Brinkmann, U., Reiter, Y., Jung, S.-H., Lee, B., & Pastan, I . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 7538-7542} . This disulfide-stabilized immunotoxin has the same cytotoxic activity and specificity as its single-chain immunotoxin counterpart . To determine whether the stabilization of Fv's by disulfides at these positions is generally applicable, we made and analyzed two other dsFv-containing immunotoxins . One is made from the e23 antibody, which binds to the carcinoma-associated antigen erbB2; the other is made from the anti-Tac antibody, which binds to the p55 subunit of the IL-2 receptor . Comparison of the specificity and activity of these immunotoxins with those of their scFv counterparts revealed that e23(dsFv)-PE38KDEL was considerably more active than e23(Fv)-PE38KDEL, whereas anti-Tac(dsFv)-PE38KDEL was only somewhat more active than its single-chain counterpart . These results suggest that dsFv's have at least the same binding properties as scFv's, and in some cases they may have better binding . Thus, it should be feasible to use the positions we have identified in the conserved framework region to disulfide-stabilize many different Fv's.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1994 May 6, 269(18), 13398 - 404
Pseudomonas exotoxin A mutants . Replacement of surface-exposed residues in domain III with cysteine residues that can be modified with polyethylene glycol in a site-specific manner; Benhar I et al.; Pseudomonas exotoxin A (PE) is composed of three structural and functional domains . Domain Ia is responsible for cell recognition, domain II for translocation of PE across the cell membrane, and domain III for ADP-ribosylation of elongation factor 2 . To investigate the role of the amino acids exposed on the surface of domain III, we replaced 15 of these, generating 29 different mutants at positions 412, 416, 418, 490, 513, 516, 522, 551, 576, 590, 599, 604, 606, 607 and 608 . All but one mutant retained substantial ADP-ribosylation and cytotoxic activities . Modification of proteins with monomethoxy-polyethylene glycol (mPEG) prolongs their circulation in the blood stream and reduces their immunogenicity . Unlike PEGylated enzymes acting on small molecule substrates, PEGylated toxins may lose those functions that are based on macromolecular interactions . Therefore, we selectively PEGylated mutant PEs at positions 490, 513, 516, 522, 604, and 606 . Most PEs modified by a 5-kDa mPEG via a disulfide or a thioether bond retained high cytotoxic activity . However, when a 20-kDa mPEG was used there was a decrease in cytotoxic activity with the disulfide-bonded molecules being more active . Positions 522 and 604 are good sites for PEGylation, but 490 is not . We also found that PEGylation of PE 522C prolonged its in vivo circulation time in mice.

Gene, 1994 May 3, 142(1), 73 - 8
Synthesis and expression of a gene encoding a 48-residue repeat in the Pseudomonas syringae ice nucleation protein; Hine AV et al.; The aim of this work was to construct short analogues of the repetitive water-binding domain of the Pseudomonas syringae ice nucleation protein, InaZ . Structural analysis of these analogues might provide data pertaining to the protein-water contacts that underlie ice nucleation . An artificial gene coding for a 48-mer repeat sequence from InaZ was synthesized from four oligodeoxyribonucleotides and ligated into the expression vector, pGEX2T . The recombinant vector was cloned in Escherichia coli and a glutathione S-transferase fusion protein obtained . This fusion protein displayed a low level of ice-nucleating activity when tested by a droplet freezing assay . The fusion protein could be cleaved with thrombin, providing a means for future recovery of the 48-mer peptide in amounts suitable for structural analysis by nuclear magnetic resonance spectroscopy.

J Bacteriol, 1994 May, 176(10), 3089 - 91
A single promoter sequence recognized by a newly identified alternate sigma factor directs expression of pathogenicity and host range determinants in Pseudomonas syringae; Xiao Y et al.; A conserved sequence motif associated with transcription of avr genes was identified in the promoter regions of six Pseudomonas syringae pv . syringae Pss61 hrp operons . A 34-bp fragment carrying this motif was cloned from the HrpZ promoter region and was shown to confer HrpL-dependent promoter activity . Expression of pathogenicity and host range determinants in P . syringae strains is thus directed by the apparent alternate sigma factor HrpL.

Protein Eng, 1994 May, 7(5), 697 - 704
Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv; Reiter Y et al.; Using molecular modeling technology, we have recently identified two positions in conserved framework regions of antibody Fv fragments (Fvs) that are distant from CDRs, and potentially can be used to make recombinant Fv fragments in which the unstable VH and VL heterodimer is stabilized by an interchain disulfide bond inserted between structurally conserved framework positions . A disulfide bond has been introduced at one of these positions, VH44-VL105, and shown to stabilize various Fvs that retain full binding and specificity . Recombinant immunotoxins, e.g . B3(dsFv)-PE38KDEL in which this disulfide-stabilized Fv moiety is connected to a truncated form of Pseudomonas exotoxin (PE; PE38KDEL) which contains the translocation and ADP ribosylation domains, are indistinguishable in binding and specificity from its single-chain immunotoxin counterparts . We have now analyzed the alternative position, (VH111-VL48), predicted by the modeling methodology, for disulfide stabilization of mAb B3(Fv) by producing a recombinant immunotoxin with such disulfide-stabilized (ds) Fv . This immunotoxin was also very active and retained full specificity to B3 antigen-positive cells . However, it was 2- to 3-fold less active than the VH44-VL105 dsFv-molecule . We also tested various biochemical features of VH44-VL105 and VH111-VL48 dsFv immunotoxins and compared them with the corresponding single-chain immunotoxin . We found the dsFv immunotoxins were more stable in human serum and more resistant to thermal and chemical denaturation than the single chain (sc) Fv immunotoxin . Because dsFv immunotoxins and dsFvs have full activity and specificity and improved stability, they may be more useful than scFv immunotoxins as therapeutic and diagnostic agents.

Proteins, 1994 May, 19(1), 35 - 47
Design of interchain disulfide bonds in the framework region of the Fv fragment of the monoclonal antibody B3; Jung SH et al.; The Fv fragments are the smallest units of antibodies that retain the specific antigen binding characteristics of the whole molecule and are being used for the diagnosis and therapy of human diseases . These are noncovalently associated heterodimers of the heavy (VH) and the light (VL) chain variable domains, which, without modification, tend to dissociate, unfold, and/or nonspecifically aggregate . The fragment is usually stabilized by producing it as a single chain recombinant molecule in which the two chains are linked by means of a short polypeptide linker . An alternative strategy is to connect the two chains by means of an interchain disulfide bond . We used molecular graphics and other modeling tools to identify two possible interchain disulfide bond sites in the framework region of the Fv fragment of the monoclonal mouse antibody (mAb) B3 . The mAb B3 binds to many human cancer cells and is being used in the development of a new anticancer agent . The two sites identified are VH44-VL105 and VH111-VL48 . (VH44-VL100 and VH105-VL43 in the numbering scheme of Kabat et al., "Sequence of Proteins of Immunological Interest," U.S . DHHS, NIH publication No . 91-3242, 1991) . This design was recently tested using the chimeric protein composed of a truncated form of Pseudomonas exotoxin and the Fv fragment of mAb B3 with the engineered disulfide bond at VH44-VL105 (Brinkmann et al., Proc . Natl . Acad . Sci . U.S.A . 90:7538, 1993) . The chimeric toxin was found to be just as active as the corresponding single chain counterpart and considerably more stable . Because these disulfide bond sites are in the framework region, they can be located from sequence alignment alone . We expect that the disulfide bond at these sites will stabilize the Fv fragment of most antibodies and the antigen-specific portion of the T-cell receptors, which are homologous.

Plasmid, 1994 May, 31(3), 275 - 87
Characterization of pPT23B, the plasmid involved in syringolide production by Pseudomonas syringae pv . tomato PT23; Murillo J et al.; Avirulence gene D (avrD) in strain PT23 of Pseudomonas syringae pv . tomato (Pst) specifies the production of syringolides, which are elicitors of plant defense reactions . An 83-kb indigenous plasmid (pPT23B) that carries avrD has been mapped and characterized and a putative par region was identified . pPT23B contains a large amount of DNA that is repeated in other native plasmids in PT23 . A putative mobile insertion element that occurs on plasmid pPT23A as well as on the chromosome was also identified in strain PT23 . New broad-host-range expression vectors that functioned in Pst were constructed for overexpression of the cloned avrD gene and high-level production of the syringolides . Introduction of an avrD overexpression plasmid into PT23 or plasmid-cured strains led to identical syringolide peaks on HPLC with no new peaks observed . These results suggested that neither pPT23B nor other indigenous plasmids in Pst carry additional genes required for syringolide production or metabolism . Pst strains lacking pPT23B were not impaired in virulence on tomato plants.

Mol Plant Microbe Interact, 1994 May-Jun, 7(3), 391 - 400
Molecular characterization of two gene loci required for production of the key pathogenicity factor pectate lyase in Pseudomonas viridiflava; Liao CH et al.; Four pleiotropic mutants of Pseudomonas viridiflava strain PJ-08-6A that were deficient in production of both pectate lyase (Pel) and protease (Prt) were isolated following transposon mutagenesis . Unlike secretion-defective (Out-) mutants, these four showed no accumulation of enzymes within the cells . Southern hybridization analysis revealed that each mutant had Tn5 inserted in one of two EcoRI genomic fragments . These EcoRI fragments (5.2- and 6.3-kb) appeared to contain two distinct gene loci, designated repA and repB, which were required for production of extracellular enzymes in this bacterium . Cosmid clones carrying the functional repA and repB DNA fragments were identified in a genomic library of strain PJ-08-6A . After analysis of repA+ plasmids by restriction mapping and marker-exchange mutagenesis, the repA gene was located in a joint region between the 1.8-kb EcoRI-HindIII and 2.8-kb EcoRI fragments cloned . Nucleotide sequence analysis of the repA region revealed the presence of an open reading frame consisting of 2,790 bases . The RepA protein predicted from the DNA sequence showed 93% similarity in amino acid sequence to the LemA protein of P . syringae pv . syringae, which was previously identified as a member of a two-component global regulatory system . A plasmid carrying the lemA gene of P . syringae pv . syringae was capable of complementing the RepA- mutation in P . viridiflava . The functions of the repA and lemA genes thus appear to be similar and interchangeable . Mutants of P . viridiflava strain SF312A deficient in production of Pel, Prt, and the exopolysaccharide alginate also were identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibiot Khimioter, 1994 May, 39(5), 33 - 7
{Sensitivity of Pseudomonas mallei to tetracyclines and their effectiveness in experimental glanders}; Batmanov VP; MICs and MBcCs of minocycline, doxycycline, methacycline and chlortetracycline for 8 strains of Pseudomonas mallei were determined . The chemotherapeutic efficacy of minocycline and doxycycline was studied on golden hamsters and their efficacy indices were estimated in comparison with those of chlortetracycline in the prophylaxis and treatment of experimental malleus . Minocycline was shown to be the most efficient drug in the treatment of malleus . Doxycycline in a dose of 0.25 mg/kg practically had the same efficacy as chlortetracycline in a dose of 25 mg/kg . Methacycline was inefficient.

Biosci Biotechnol Biochem, 1994 May, 58(5), 889 - 94
Purification and properties of bile acid sulfate sulfatase from Pseudomonas testosteroni; Tazuke Y et al.; The bile acid sulfate sulfatase (BSS) produced by Pseudomonas testosteroni was purified and characterized . Chromatofocusing behavior and amino acid sequence over twelve amino acid residues from N-terminus of the enzyme indicated that BSS was composed of two isoforms of which molecular weights were 125,000 and 103,000 . Each isoform was a homodimer of a subunit of which molecular weight was 53,000 or 51,000, respectively . The optimum pH was 8.5 and BSS was stable at pH 5.8-8.0 . The thermostability above 32 degrees C was improved by the addition of polyols, such as sorbitol, sucrose, and glycerol . BSS was a Mn(2+)-dependent enzyme and contained 1-2 atoms of manganese in its own protein molecule . All 3 alpha-sulfate esters of the bile acids routinely appearing in human serum were hydrolyzed by BSS to 3 beta-hydroxyl iso-compounds corresponding to each bile acid and sulfuric acid . We tentatively named this novel enzyme BSS (bile acid 3 alpha-sulfate sulfohydrolase).

Mikrobiologiia, 1994 May-Jun, 63(3), 537 - 44
{Identification of the bacterium Pseudomonas mallei using Pseudomonas pseudomallei bacteriophages}; Manzeniuk OIu et al.; Phage production by Pseudomonas pseudomallei and Pseudomonas mallei strains has been studied . 32 P . pseudomallei bacteriophages have been isolated . Their spectrum of lytic action against P . pseudomallei, P . mallei and other Pseudomonas sp . has been defined . It has been shown that P . pseudomallei bacteriophages PP19, PP23, PP33 may be used for identification P . mallei among related Pseudomonas.

Zh Mikrobiol Epidemiol Immunobiol, 1994 May-Jun, (3), 69 - 73
{The immunodiagnosis of melioidosis by a solid-phase variant of radioimmunological analysis (RIA)}; Piven' NN et al.; A variant of the solid-phase radioimmunoassay (RIA) has been developed for the detection of specific immunoglobulins in laboratory animals infected with Pseudomonas pseudomallei . The proposed method has advantages over the indirect hemagglutination test and the enzyme immunoassay in its sensitivity and specificity . The newly developed RIA variant, based on group-specific antigens 6 + d, makes it possible to classify the strain causing the disease with the Asian or Australian serovar of P . pseudomallei according to the composition of detected immunoglobulins.

J Clin Microbiol, 1994 May, 32(5), 1326 - 32
Detection of Pseudomonas pseudomallei by PCR and hybridization; Lew AE et al.; A molecular method for the detection of Pseudomonas pseudomallei was developed on the basis of the differences in the 23S rRNA sequences of related species of the genus Pseudomonas . An 18-base oligonucleotide probe, designed following partial sequencing of 23s ribosomal DNA (rDNA), was used for the identification and detection of P . pseudomallei either by hybridization or by direct PCR . Optimal detection was obtained by hybridization of the probe with PCR-amplified rDNA rather than with total genomic DNA or colony blots . One nanogram of template DNA amplified in a PCR mixture containing 14% glycerol could be detected in slot blots hybridized with the digoxigenin-labelled probe and the lumigen PPD detection system . Amplified rDNA sequences from 41 P . pseudomallei strains of various origins hybridized with the probe . The probe also hybridized with three Pseudomonas mallei reference strains under conditions of high stringency but failed to hybridize with amplified rDNA sequences from other closely related Pseudomonas spp . PCR with a conserved primer and the 18-base oligonucleotide probe (direct PCR) specifically amplified P . pseudomallei and P . mallei . By using these methods, approximately 10(4) P . pseudomallei cells per ml could be detected in artificially inoculated blood samples and in blood dried on filter paper following Chelex extraction . The detection limit in blood was increased to 10(2) cells per ml by concentration of bacteria from 0.5 ml of blood or by a 24-h blood culture enrichment prior to PCR . Approximately 10(3) cells per ml were detected in seeded sputum samples . The detection times by direct PCR and indirect PCR and then probe hybridization were approximately 5 h and 24 h, respectively . These results indicate that amplification of conserved rDNA sequences by PCR directly or by hybridization with a probe to PCR fragments offers promise for the detection of P . pseudomallei and P . mallei.

J Pediatr, 1994 May, 124(5 Pt 1), 694 - 702
Acquisition of Pseudomonas cepacia at summer camps for patients with cystic fibrosis . Summer Camp Study Group; Pegues DA et al.; To assess the risk of acquisition of Pseudomonas cepacia by person-to-person transmission at cystic fibrosis summer camps, we conducted in 1990 a study at three camps attended by patients with cystic fibrosis who had P . cepacia infection and patients without P . cepacia infection but who were considered susceptible to infection . We obtained sputum or throat cultures from campers on their arrival at, weekly during, at the end of, and 14 to 30 days after camp . We compared the incidence of sputum conversion of patients at camp with that of patients outside camp by culturing specimens from noncamper control subjects with cystic fibrosis who were known not to be infected < or = 2 weeks before and 4 to 6 weeks after camp . We also determined the risk factors for P . cepacia acquisition by determining the relative risk of acquisition between campers who were exposed versus campers who were not exposed to campers known to be infected or to potential environmental sources of P . cepacia at camp . The ribotype of P . cepacia isolates from campers with sputum conversion was compared with that of isolates from other campers and from an environmental source . The cumulative incidence of sputum conversion during the study period was 6.1% (11/181) among campers compared with no incidence (0/92) among noncampers (p = 0.02, Fisher Exact Test) . The incidence of sputum conversion at camp varied according to the prevalence of campers with known infection (p < 0.001, chi-square test for trend) . The rate of sputum conversion was higher in the camp with longer duration (relative risk = 12.0; 95% confidence interval = 2.7 to 53.5) . Ribotyping showed that P . cepacia isolates from all 11 campers with sputum conversion were identical or similar (1 to 2 band difference) to isolates of other P . cepacia-infected campers including co-converters . These results suggest that P . cepacia can be acquired by patients with cystic fibrosis who are attending summer camp for such patients, possibly through person-to-person transmission, and that the risk increases with the prevalence of P . cepacia-infected campers and the duration of camp.

FEMS Microbiol Lett, 1994 Apr 15, 117(3), 327 - 32
Escherichia coli ferric uptake regulator (Fur) can mediate regulation of a pseudomonad iron-regulated promoter; O'Sullivan DJ et al.; Introduction of a Pseudomonas iron-regulated promoter lacZ fusion (SP1) and a Pseudomonas transcriptional factor into Escherichia coli allowed expression of the promoter in this heterologous host . Evaluation of this promoter in wild-type and fur mutants of E . coli, by measuring beta-galactosidase activity, indicated that E . coli Fur can regulate the Pseudomonas promoter in response to iron starvation . Gel retardation assays suggested that purified Fur protein could interact with the SP1 promoter upstream of the transcriptional start . DNase I footprinting analysis established that Fur protected a primary 58-bp region (-50 to -106 bp) . These protein/DNA interactions correlate with the observed in vivo regulation of the SP1 promoter in E . coli and indicate that Fur can functionally regulate a Pseudomonas iron-regulated promoter.

Cancer Res, 1994 Apr 15, 54(8), 2146 - 50
Targeted therapy with immunotoxins in a nude rat model for leptomeningeal growth of human small cell lung cancer; Myklebust AT et al.; Metastasis to the central nervous system in patients with small cell lung cancer is not uncommon, and a fraction of the cases have leptomeningeal disease for which no effective therapy is available . To establish an experimental model for evaluation of new therapeutic approaches for such tumor lesions, 1 x 10(6) human H-146 cells were injected directly into the cerebrospinal fluid in the cisterna magna of nude rats . Small, superficial leptomeningeal tumors developed, consistently resulting in symptoms of central nervous system involvement after a mean latency of 20 days . The model was used to study the efficacy of intrathecal targeted therapy with immunotoxins . The monoclonal anti-carcinoma antibodies MOC-31 and NrLu10 and the growth factor transferrin were conjugated to Pseudomonas exotoxin A (PE), and 1 day after tumor cell inoculation instilled in the cisterna magna as a single bolus dose of 1.5 micrograms . The antibody conjugates, which were highly cytotoxic to target cells in a protein synthesis inhibition assay in vitro, increased the symptom-free latency by 35-46% . PE had no effect, reflecting a lower in vitro cytotoxicity and possibly also a down-regulation of transferrin-receptor expression in the meningeal H-146 tumors . Delayed or repeated treatment with MOC-31-PE was less effective than day 1 administration, whereas the addition of 10% glycerol to the injection solution increased the symptom-free period to 72% . The efficacy of MOC-31-PE is superior to reported effects obtained in similar models with other therapies, and the results support the development of this immunotoxin towards clinical evaluation in small cell lung cancer patients with leptomeningeal carcinomatosis.

J Biol Chem, 1994 Apr 15, 269(15), 11254 - 60
Purification and characterization of isoquinoline 1-oxidoreductase from Pseudomonas diminuta 7, a novel molybdenum-containing hydroxylase; Lehmann M et al.; Isoquinoline 1-oxidoreductase, which catalyzes the hydroxylation of isoquinoline to 1-oxo-1,2-dihydroisoquinoline with concomitant reduction of a suitable electron acceptor, was purified from the isoquinoline degrading bacterium Pseudomonas diminuta 7 to apparent homogeneity . The native enzyme was a heterodimer with a molecular mass of 95 kDa consisting of a 16- and a 80-kDa subunit . It contained 0.85 g atom molybdenum, 3.95 g atom iron, 3.9 g atom acid-labile sulfur, 2.1 mol of phosphate, and 1 mol of CMP/mol of enzyme . CMP and phosphate are suggested to originate from molybdopterin cytosine dinucleotide of the pterin molybdenum cofactor . It is assumed that the iron and the acid-labile sulfur are arranged in two (2Fe-2S) clusters . The isoelectric point of the isoquinoline 1-oxidoreductase was within the range of pH 6.2 to 6.8 . Cytochrome c, ferricyanide, and several non-physiological electron acceptors served as oxidizing substrates, whereas O2 and NAD were not used . Isoquinoline 1-oxidoreductase revealed a high specificity toward the reducing substrates isoquinoline, 5-hydroxyisoquinoline, quinazoline, and phthalazine . Isoquinoline 1-oxidoreductase was inactivated by methanol, arsenite, p-hydroxymercuribenzoate, 1,10-phenanthroline, and cyanide . Additionally, the enzyme was inactivated upon incubation with its substrates isoquinoline, which slowly inhibited the enzyme in the absence of an electron acceptor, and 5-hydroxy-isoquinoline, which rapidly and very effectively inactivated the enzyme in the presence as well as in the absence of the electron acceptors iodonitrotetrazolium chloride, phenazine methosulfate, or ferricyanide.

Biol Mass Spectrom, 1994 Apr, 23(4), 179 - 85
High-performance thin-layer chromatography/fast atom bombardment (tandem) mass spectrometry of Pseudomonas rhamnolipids; de Koster CG et al.; Several rhamnolipid preparations from Pseudomonas strains were studied by thin-layer chromatography/fast atom bombardment (TLC/FAB) mass spectrometry and TLC/FAB tandem mass spectrometry (MS/MS) . The preparations were separated with normal-phase (Silica 60) and reversed-phase (RP-8) chromatography . Silica 60 plates appeared to be very useful in the separation of rhamnolipids according to the number of monosaccharide residues present . Spectra which show characteristic fragment ions could be obtained from components of mixtures with a total sample size of less than 200 ng . Chromatography on RP-8 plates gave a good separation of the rhamnolipids based on the length of the fatty acid alkyl chain . MS/MS of the sodium cationized molecules gave information about the sequence of the building blocks . Particularly, heterogeneity in beta-hydroxy fatty acid composition was determined for the principal as well as minor components present in natural rhamnolipid mixtures.

J Trauma, 1994 Apr, 36(4), 486 - 90
Dose dependency of granulocyte-macrophage colony stimulating factor for improving survival following burn wound infection; O'Reilly M et al.; Infections remain a serious problem following injury . Immune modulation offers an additional strategy for the treatment of infections . We evaluated the ability of a multilineage hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), to improve survival following burn injury with a superimposed burn wound infection . Groups of 12 BDF1 mice received a 15% total body surface area (TBSA) thermal injury by immersion in 100 degrees C water; 6 x 10(3) Pseudomonas was then applied to the burn wound . The GM-CSF was injected subcutaneously B.I.D . for 7 days . Mice receiving the 10-ng dose of GM-CSF had significantly improved survival compared with the controls; other doses had no significant effect on survival . Clinical trials to assess the ability of GM-CSF to reduce infectious complications following burn injury are underway and these data suggest selecting a specific dose may be critical in achieving maximal benefit.

J Bacteriol, 1994 Apr, 176(8), 2339 - 47
Chloroperoxidase from Streptomyces lividans: isolation and characterization of the enzyme and the corresponding gene; Bantleon R et al.; For the first time, a halogenating enzyme which is not known to produce halogenated metabolites has been isolated from a bacterial strain . The gene encoding the nonheme chloroperoxidase (CPO-L) from Streptomyces lividans TK64 was cloned, and its gene product was characterized . S . lividans TK64 produced only very small amounts of the enzyme . After cloning of the gene into Streptomyces aureofaciens Tu24-88, the enzyme was overexpressed up to 3,000-fold . Based on the overexpression, a simple purification procedure using acid precipitation and hydrophobic interaction chromatography was developed . Thus, 54 mg of homogeneous CPO-L could be obtained from 27 g (wet weight) of mycelium . The native enzyme has a molecular weight of 64,000 and consists of two identical subunits . The enzyme does not exhibit an absorption peak in the Soret region of the optical spectrum . X-ray fluorescence spectroscopy revealed that the enzyme does not contain any metal ions in equimolar amounts . CPO-L showed cross-reaction with antibodies raised against the nonheme chloroperoxidase from Pseudomonas pyrrocinia but not with antibodies raised against CPO-T from S . aureofaciens Tu24 . CPO-L exhibits substrate specificity only for chlorination, not for bromination . Therefore, monochlorodimedone is only brominated by CPO-L, whereas indole is brominated and chlorinated . The functional chloroperoxidase gene was located on a 1.9-kb SalI DNA fragment . DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide of 276 amino acids . The overall identity of the amino acid sequence to that of chloroperoxidase from P . pyrrocinia was 71%, whereas that to bromoperoxidase BPO-A2 from S . aureofaciens ATCC 10762 was only 42%.

J Immunol, 1994 Apr 1, 152(7), 3693 - 700
A chimeric protein comprised of IL-4 and Pseudomonas exotoxin is cytotoxic for activated human lymphocytes; Puri RK et al.; IL4-Pseudomonas exotoxin (IL4-PE4E) is a chimeric molecule in which human IL-4 is genetically fused to the mutated binding domain of Pseudomonas exotoxin . This molecule binds specifically to human IL-4 receptor-bearing cells . IL4-PE4E was extremely cytotoxic to highly purified anti-CD3-activated CD8+ T lymphocytes . The cytotoxic activity of this molecule was dependent on the activation state of CD8+ T cells: 3- and 4-day activated T cells were very susceptible to the cytotoxic activity of IL4-PE4E compared with 0- to 2-day activated cells . PHA-activated lymphocytes and PBL activated in mixed lymphocyte reaction were also highly sensitive to IL4-PE4E . CD16+ and/or CD56+ highly purified NK cells or highly purified, IL-2-activated NK cells were also very sensitive to the cytotoxic effect of IL4-PE4E . IL-2-activated LAK cells had little susceptibility after 1 day but were very sensitive to IL4-PE4E after 3 days . The cytotoxic effects of IL4-PE4E were mediated through a ligand receptor interaction because excess rIL-4 abrogated these effects as did a neutralizing Ab to human IL-4 . A chimeric mutant protein that can bind to IL-4 receptors but lacks the ability to inhibit protein synthesis was not cytotoxic to activated lymphocytes . The IL4-PE4E-mediated cytotoxicity of activated T cells correlated with the level of expression of IL-4 receptors on these cells . CD8+ T cells activated for 3 days expressed the highest density of IL-4 receptors compared with 1- or 2-day activated cells . Among two chimeric toxins tested only IL4-PE4E was cytotoxic to 2-day anti-CD3-activated CD8+ T lymphocytes, whereas IL6-PE4E was not active at all . These studies suggest that human IL4 toxin could be a potent agent for the elimination of activated lymphocytes in allograft rejection, some autoimmune diseases, or treatment of lymphomas and leukemias.

J Bacteriol, 1994 Apr, 176(7), 1850 - 6
Analysis of the binding site of the LysR-type transcriptional activator TcbR on the tcbR and tcbC divergent promoter sequences; Leveau JH et al.; The TcbR transcriptional activator protein, which is encoded by the tcbR gene of Pseudomonas sp . strain P51 (J . R . van der Meer, A . C . J . Frijters, J . H . J . Leveau, R . I . L . Eggen, A . J . B . Zehnder, and W . M . de Vos, J . Bacteriol . 173:3700-3708, 1991), was purified from overproducing Escherichia coli cells by using a two-step chromatographic procedure . Subsequent use of TcbR in gel mobility shift assays with progressively shortened portions of a DNA fragment containing the divergent promoter sequences of the tcbR gene and the tcbCDEF operon showed that the direct binding site of TcbR is located between positions -85 to -40 relative to the tcbCDEF transcriptional start site, containing a LysR-type recognition sequence motif (T-N11-A) . DNase I footprinting experiments revealed that TcbR protected an area on both strands of the intercistronic region which was actually larger than this binding site (from positions -74 to -24) . This stretch of protected DNA was interrupted by a region (positions -52 to -37) which became strongly hypersensitive to DNase I digestion upon addition of TcbR, suggesting that TcbR induces a bend in the DNA at this site.

Cell Immunol, 1994 Apr 1, 154(1), 369 - 79
Human renal cell carcinoma cells are sensitive to the cytotoxic effect of a chimeric protein composed of human interleukin-4 and Pseudomonas exotoxin; Puri RK et al.; We have previously demonstrated that functional high-affinity interleukin-4 receptors (IL-4R) are expressed on human renal cell carcinoma (RCC) cells (N . I . Obiri et al., J . Clin . Invest . 91, 88, 1993) . In the present study, we examined the cytotoxic effect (determined by inhibition of protein synthesis) of a chimeric protein composed of human IL-4 and Pseudomonas exotoxin (PE) on human RCC tumor samples obtained from patients undergoing nephrectomy . The chimeric gene encoding hIL4-PE4E was constructed by fusing a cDNA clone for human IL-4 to the 5' end of a mutated cDNA encoding a full-length PE molecule . This gene was expressed in Escherichia coli, and large quantities of this recombinant protein were isolated to more than 95% purity . This chimeric protein, hIL4-PE4E, was highly cytotoxic to all six RCC cell lines examined . The concentration of hIL4-PE4E at which 50% inhibition of protein synthesis was obtained ranged from < 1 ng/ml (12 pM) to 10 ng/ml (120 pM) in five of the six isolates of RCC and 40-70 ng/ml in one other . A mutant chimeric protein which can bind to IL-4R but lacks the ADP ribosylation activity of PE was not cytotoxic to the RCC cells . The cytotoxic effect of hIL4-PE4E was IL-4R mediated because a fourfold molar excess of IL-4 abrogated the cytotoxic effect of hIL4-PE4E . A neutralizing monoclonal antibody to IL-4 also abrogated the cytotoxic effect of hIL4-PE4E . hIL4-PE4E showed very little cytotoxic activity to a normal human umbilical vein endothelial cell line (ID50 = 1000 ng/ml) and a human fibroblast cell line (ID50 approximately 400 ng/ml) . Nonactivated human peripheral blood lymphocytes (PBL) were also insensitive to hIL4-PE4E (ID50, approximately 500 ng/ml), whereas phytohemagglutinin-activated PBL were highly susceptible to the cytotoxic effect of hIL4-PE4E (ID50, approximately 4 ng/ml) . These data indicate that hIL4-PE4E may be a useful agent for the treatment of human RCC without affecting normal and resting immune cells.

Clin Biochem, 1994 Apr, 27(2), 93 - 7
A sensitive determination of uric acid in serum using uricase/catalase/formaldehyde dehydrogenase coupled with formate dehydrogenase; Kayamori Y et al.; We developed and evaluated an assay for serum uric acid based on the uricase (EC 1.7.3.3)-catalase (EC 1.11.1.6)-formaldehyde dehydrogenase (FADH, EC 1.2.1.46) method coupled with formate dehydrogenase (formate:NAD oxidoreductase, FDH, EC 1.2.1.2) . Formate dehydrogenase from Pseudomonas oxalaticus catalyzes the formation of NADH from formate produced by FADH . Owing to the NADH and formate oxidase activity of the FDH itself, the full reaction curve is not linear, but gradually decreases . The formation of NADH is not stoichiometric with formate removal, but is strictly proportional to it . To overcome this decrease of extinction, we added hydroxylamine hydrochloride to the FDH . The sensitivity of the full reaction in the presence of FDH was about 1.8 times that without FDH . Analysis with a Cobas Bio centrifugal analyzer revealed a linearity of up to 3.56 mmol/L . The uricase-catalase-alcohol dehydrogenase method correlated well with the uricase-peroxidase-chromogen method . Our method is more sensitive than other methods.

Circ Shock, 1994 Apr, 42(4), 174 - 82
Pseudomonas sepsis does not cause more severe cardiovascular dysfunction in patients than non-Pseudomonas sepsis; Pilz G et al.; To evaluate the clinical relevance of the experimental findings of a more severe cardiac depression in Pseudomonas (P.) than in non-P . sepsis, we retrospectively compared the hemodynamic data in 26 patients with P . sepsis (20 cases, single pathogen; six cases, more positive cultures with P . than with non-P . species), and 102 with non-P . sepsis . As in other studies, the left ventricular stroke work index (LVSWI) was used to assess cardiac performance . The two groups (all numbers are means) had a similar disease and sepsis severity profile (P . vs . non-P: septic shock, 81% vs . 87%; APACHE II scores, 29.1 vs . 29.2; Elebute sepsis scores, 18.1 vs . 18.1; mortality, 58% vs . 62%) . Preload (pulmonary capillary wedge pressure 15.0 vs . 16.3 mm Hg) and systemic vascular resistance (588 vs 572 dyn.cm-5.sec) were comparable . Cardiac performance displayed no significant difference (LVSWI, 42.8 vs . 38.3 g.m/m2), a result reproduced in the subgroups with culture-proven bacteremia, with or without preexisting cardiovascular disease or septic shock . Thus, our data suggest that there is no difference in the degree of cardiovascular dysfunction in patients with Pseudomonas compared to non-Pseudomonas sepsis of otherwise equivalent disease severity.

CLAO J, 1994 Apr, 20(2), 109 - 13
Testing hypotheses for risk factors for contact lens-associated infectious keratitis in an animal model; Solomon OD et al.; Various risk factors contributing to contact lens related infectious corneal ulcers were tested . These factors included the presence of infectious organisms, loss of corneal surface integrity, and corneal hypoxia . High concentrations of Pseudomonas bacteria were applied to rabbit corneas under the following conditions: normal, intact corneal surface; "mild" corneal hypoxia with extended-wear soft contact lens and eyes closed; "moderate" corneal hypoxia with daily wear soft contact lens and eyes closed; "severe" corneal hypoxia with daily wear aphakic soft contact lens and eyes closed; corneal abrasion and eyes open; corneal abrasion and eyes closed; corneal abrasion with soft contact lens and closed eyes . No infectious ulcers occurred in normal intact corneas . The frequency of infectious ulcers increased with increasing degrees of corneal swelling . No ulcers were found in corneas with a mean swelling of 10%, whereas a mean swelling of 20% resulted in ulcers in half the group, and a mean swelling of 43% resulted in ulcers in all eyes . The three groups of abraded corneas resulted in infectious ulcers at a 20 to 30% frequency . The presence of infectious organisms alone does not lead to infectious corneal ulcers in rabbits . Corneal abrasions are a risk factor for infectious ulcers, but moderate and severe degrees of hypoxia are more significant.

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